dissertação para obtenção do grau de mestre ... - ulisboa · production and purification of new...

Production and Purification of New Microbial Cellul ases

Bárbara Sofia Silva Rodrigues

Dissertação para obtenção do Grau de Mestre em Engenharia Biológica

Júri

Presidente: Professor Duarte Miguel de França Teixeira dos Prazeres (IST)

Orientadores: Professora Maria Manuela Fonseca (IST)

Professor Francesco Molinari (UNIMI)

Vogal: Doutora Maria Teresa Ferreira Cesário (IST)

Novembro 2011

i

Acknowledgments

This thesis was made possible by the support and assistance of a number of people

whom I would like to personally thank.

First and foremost, I would like to express my gratitude to Dr. Diego Romano and my

supervisor Prof. Francesco Molinari, whose expertise and understanding guided me through my

internship, providing useful advice for the improvement of this work.

A special thank also goes to my lab partner, Tiziana Granato, for making experiments

look amusing, even when they were humourless, for sharing her knowledge, and also for the

encouragement and generous help during the internship and writing. Big thanks also go to

Giovanni, Ilaria, Francesca, and Chiara for helping me fit in and feel welcome from the moment

I arrived in DISTAM. A special thanks to Matteo and Mauro for the unlimited patience to explain

me every doubt I had during my internship.

I would also like to acknowledge my supervisor at IST, Prof. Manuela Fonseca, for her

availability to meet me, to review drafts of this document and for her extremely valuable and

detailed comments.

I wish to express my great gratitude to all my Erasmus and ESN friends that contributed

to making this work and my stay in Milan an enjoyable and fulfilling experience.

A special thanks to my great friends Inês Santos, Catarina Gomes and Sandra Ferreira

for the joy, companionship and unlimited support given throughout the writing part of this work.

The last but not the least, I would like to express my gratitude to my Family for the

support they provided me through my studies. This would not have been possible without their

support and understanding.

ii

Abstract

Recently there has been a significant effort on the development of renewable

alternatives to fossil fuels. One approach is to produce a liquid fuel by enzymatically hydrolyzing

carbohydrate polymers from biomass, such as cellulose, to sugars and fermenting them to

ethanol. Cellulases are the enzymes responsible for this phenomenon and despite extensive

research there are major gaps in understanding how they hydrolyze crystalline cellulose and act

synergistically.

To address this matter, experiments were conducted to optimize the production of

cellulases by two strains, A-1 and N-Y, of a fungal source: Aspergillus terreus. On a first

approach, different concentrations of cellulose were tested as the sole carbon source. The

maximum activity achieved by A.terreus A-1 strain was at the concentration of 30 g⋅L-1 of

cellulose, whereas A.terreus N-Y better produced cellulases at 20 g⋅L-1 of cellulose. Both strains

reached maximum cellulasic activity between 72h and 96h of incubation at 30ºC. The

concentration of spores utilized in the inoculum was also optimized and proven to be best at 0.1

OD/mL. Different sources of nitrogen, MgSO4 concentration and optimal pH were further

assayed. The best cellulolytic activities were obtained by using sodium nitrate as nitrogen

source and a concentration 0.5 g·L-1 of MgSO4·7H2O, at pH 6.

Stability tests regarding the effect of temperature and the use of protease inhibitors on

cellulase activity were also performed.

Finally, the release of sugars from the enzymatic reaction between cellulases from

A.terreus A-1 and commercial cellulose was analyzed by High Performance Liquid

Chromatography (HPLC).

Keywords : Production; Cellulases; Cellulose; Aspergillus terreus; Ethanol

iii

Resumo

Nos últimos tempos têm sido dirigidos esforços no sentido de desenvolver alternativas

energéticas sustentáveis aos combustíveis fósseis. Uma alternativa possível será a produção

de um combustível líquido a partir da hidrólise enzimática de polímeros de carbohidratos

existentes na biomassa, tais como a celulose, a açúcares e a sua posterior fermentação a

etanol. As celulases são as enzimas responsáveis por este fenómeno e, apesar da intensa

pesquisa já realizada, existem lacunas no conhecimento de como estas interagem

sinergisticamente para hidrolisar a celulose.

A fim de colmatar estas lacunas, foram desenvolvidas experiências de forma a

optimizar a produção de celulases a partir de duas estirpes, A-1 e N-Y, do fungo Aspergillus

terreus. Numa primeira abordagem, foram testadas diferentes concentrações de celulose como

única fonte de carbono. A actividade enzimática máxima das celulases produzidas pela estirpe

A-1 foi obtida utilizando uma concentração de celulose de 30 g⋅L-1, enquanto a estirpe N-Y

produziu maior quantidade de celulases usando uma concentração de 20 g⋅L-1 de celulose.

Ambas as estirpes registaram maior actividade celulásica entre as 72h e 96h de incubação do

fungo a 30ºC. A concentração de esporos a utilizar no inóculo foi optimizada, tendo-se

registado os melhores resultados para uma concentração de 0.1 OD/mL. Dois meios de cultura

com diferentes fontes de azoto, concentração de MgSO4 e pH óptimos foram também testados.

As melhores actividades enzimáticas foram obtidas aquando da utilização de nitrato de sódio

como fonte de azoto, e uma concentração de 0.5 g·L-1 de MgSO4·7H2O ao pH 6.

Foram ainda realizados testes de estabilidade de modo a avaliar o efeito da

temperatura e o uso de inibidores de proteases na actividade enzimática das celulases.

Por fim, a produção de açúcares a partir da reacção enzimática entre as celulases

produzidas pela estirpe A-1 de Aspergillus terreus e celulose foi analisada por Cromatografia

Líquida de Alta Eficiência (HPLC).

Palavras-chave : Produção; Celulases; Celulose; Aspergillus terreus; Etanol

iv

Table of Contents

Acknowledgments .................................................................................. i

Abstract .................................................................................................. ii

Resumo ................................................................................................. iii

List of Figures ....................................................................................... vi

List of Tables ....................................................................................... xii

Abbreviations ...................................................................................... xiii

1. Introduction ...................................................................................... 1

1.1 Motivation and Background: Cellulases and Second Generation

Biofuels ................................................................................................. 1

1.1.1 Economic Analysis of Bioethanol Production ................................. 2

1.2 Aspergillus ...................................................................................... 3

1.3 Cellulose ......................................................................................... 4

1.4 Cellulases ....................................................................................... 6

1.4.1 Characterization and Properties .................................................... 7

1.4.2 Cellulase Production ..................................................................... 9

1.4.3 Applications................................................................................. 10

1.4.4 The Challenges in Cellulase Research – Future Perspectives .... 11

1.5 Aim of this Study........................................................................... 12

2. Materials and Methods ................................................................... 14

2.1 Microorganism ............................................................................. 14

2.2 Culture Media ............................................................................... 14

2.3 Maintenance and Growth Conditions............................................ 14

2.4 Characterization of Cellulases ...................................................... 15

2.4.1 Harvest and Separation of Enzymes ........................................... 15

2.4.2 Cellulase Activity Assay .............................................................. 15

2.4.3 Protein Assay – Bradford Method ................................................ 17

v

2.5 Recovery and Concentration of Cellulases ................................... 18

2.5.1 Vacuum Filtration ........................................................................ 18

2.5.2 Ultrafiltration ................................................................................ 18

2.6 Validation Methods of Cellulasic Activity ...................................... 18

2.6.1 Enzymatic Reaction with Commercial Cellulose .......................... 18

2.6.2 HPLC Analysis ............................................................................ 19

2.7 Stability Tests ............................................................................... 19

2.7.1 Effect of Temperature on Cellulase Activity and Stability ............. 19

2.7.2 Effect of EDTA and a Protease Inhibitor Cocktail on Cellulase

Activity and Stability ............................................................................. 20

3. Results and Discussion .................................................................. 21

3.1 Factors Affecting Cellulase Production ......................................... 21

3.1.1 Effect of Cellulose Concentration on Cellulase Activity ................ 21

3.1.2 Effect of Initializing Fungal Growth at Different Spore

Concentration (OD/mL) on Cellulase Activity ........................................ 23

3.1.3 Effect of Medium Composition on Cellulase Activity .................... 25

3.1.4 Effect of Temperature on Cellulase Activity and Stability ............. 29

3.1.5 Effect of EDTA and a Protease Inhibitor Cocktail on Cellulase

Activity and Stability ............................................................................. 30

3.2 Evaluation of the Cellulase System .............................................. 33

3.3 Identification of Sugars Released from the Enzymatic Hydrolysis of

Cellulose ............................................................................................ 35

4. Conclusions and Future Perspectives ........................................... 47

5. References ....................................................................................... 50

Appendix I ............................................................................................ 54

Appendix II ........................................................................................... 62

vi

List of Figures

Figure 1 – Structure and composition of cellulose fibers. The glucose chains are held together

by hydrogen bonds and form crystalline cellulose that is arranged in microfibrils. These are

linked by hemicellulose and lignin, forming the recalcitrant cellulose fibers (Goldschmidt, 2008).

....................................................................................................................................................... 6

Figure 2 - Schematic view of the biodegradation of cellulose. Cellobiohydrolases act on the

nonreducing or reducing termini of cellulose fibers to processively release cellobiose. Endo-β-

1,4-glucanases randomly cleave cellulose chains. Β-glucosidases hydrolyze cellobiose or cello-

oligomers to glucose from nonreducing ends (Watanabe, et al., 2009)........................................ 8

Figure 3 – Aspergillus terreus endo-Cellulase calibration curve performed by using commercial

SIGMA cellulase. Measurements were carried out by using a commercial soluble substrate

(AZOCM-CELLULOSE, Megazyme). Assay conditions: T=40ºC; Incubation time= 30 min. ...... 22

Figure 4 – Effect of different concentrations of cellulose on the enzymatic activity of cellulases

produced by Aspergillus terreus A-1 strain during 9 days of incubation at 30ºC. ....................... 22

Figure 5 - Effect of different concentrations of cellulose on the enzymatic activity of cellulases

produced by Aspergillus terreus N-Y strain during 9 days of incubation at 30ºC. ...................... 23

Figure 6 - Effect of different concentration of spores (0.1, 0.2, 0.5 OD/mL) on the enzymatic

activity of cellulases produced by Aspergillus terreus A-1 strain, along 10 days of incubation at

30ºC in alternative agitation. ....................................................................................................... 24

Figure 7 - Effect of different concentration of spores (0.1, 0.2, 0.5 OD/mL) on the enzymatic

activity of cellulases produced by Aspergillus terreus N-Y strain, along 10 days of incubation at

30ºC in alternative agitation. ....................................................................................................... 24

Figure 8 - Effect of different medium composition, using a 0.1 OD/mL concentration of spores,

on the enzymatic activity of cellulases produced by Aspergillus terreus A-1 strain, along 9 days

of incubation at 30ºC in alternative agitation. .............................................................................. 26


on the enzymatic activity of cellulases produced by Aspergillus terreus A-1 strain, along 9 days


vii


on the enzymatic activity of cellulases produced by Aspergillus terreus N-Y strain, along 9 days



on the enzymatic activity of cellulases produced by Aspergillus terreus N-Y strain, along 9 days


Figure 12 - Enzymatic activity (total Units) of cellulases before ultrafiltration (unconcentrated

cellulases) and recovered in the concentrate and in the permeate after ultrafiltration, using a

PES 50-kDa MWCO membrane in a stirred-cell apparatus. ....................................................... 29

Figure 13 – Effect of temperature on the activity of A. terreus A-1 cellulases, recovered in the

concentrate, after 48h at 25ºC and -20ºC. The control (samples at 25ºC) was taken as having

100% activity. .............................................................................................................................. 30

Figure 14 – Effect of a protease inhibitor cocktail and EDTA on the activity of cellulases from

Aspergillus terreus A-1 strain after 48h at 25ºC. ......................................................................... 31

Figure 15 - Effect of EDTA (2mM) on the activity of cellulases produced by Aspergillus terreus

A-1 after 96h of cultivation at 30ºC in basal media 1 and 2. After 96h of growth cultures were

vacuum filtered and ultrafiltrated, and kept at 25ºC in a room for the next 11 days. .................. 32

Figure 16 - Effect of EDTA (2mM) on the activity of cellulases produced after 96h of cultivation

at 30ºC of Aspergillus terreus A-1 cultures in Medium 1. After 96h of growth cultures were

vacuum filtered and ultrafiltrated, and left at 25ºC for the next 7 days. ...................................... 33

Figure 17 – Activity of cellulases produced by Aspergillus terreus A-1, using different substrates

(Azo-CMcellulose, NPG and NPA), different media (1 and 2), and with or without the addition of

EDTA (at 0h and at 96h of incubation at 30ºC). Samples were conserved at -20ºC and taken

from assays 8, 9 and 10. ............................................................................................................. 34

Figure 18 - HPLC chromatogram for 24h of reaction between commercial cellulose and the

enzyme mixture present in the concentrate solution, recovered from ultrafiltration of cellulases

produced by A. terreus A-1 growing on Medium 1. The identified peaks correspond to:

Cellobiose (12. 8 min); Glucose (15.4 min). ................................................................................ 36



viii

produced by A. terreus A-1 growing on Medium 2. The identified peaks correspond to:

Cellobiose (12.9 min) and Xylose (16.4 min). ............................................................................. 37

Figure 20 - HPLC chromatogram for a 10 g·L-1 mixed sugar solution, showing elution times of

Glucose (6.3 min) , Xylose (10.0 min) and Cellobiose (22.6 min) with a mobile flow rate of 1

mL/min at 23ºC. Column: Lichrospher 100 NH2 ; Mobile phase: 85%Acetonitrile/15%Water. .. 38

Figure 21 – HPLC chromatogram for 24h of reaction between commercial cellulose (10 g·L-1)

and the enzyme mixture present in the concentrate solution, recovered from ultrafiltration of

cellulases produced by A. terreus A-1 growing on Medium 1 (“Control”). Column: Lichrospher

100 NH2 ; Mobile phase: 85%Acetonitrile/15%Water ; Flow rate 1 mL/min at 23ºC. The

identified peaks correspond to: Millipore Water (3.2 min) and Xylose (11.2 min). ...................... 39

Figure 22 - HPLC chromatogram for 48h of reaction between commercial cellulose (10 g·L-1)


cellulases produced by A. terreus A-1 growing on Medium 1 (“Control”). Column: Lichrospher

100 NH2 ; Mobile phase: 85%Acetonitrile/15%Water ; Flow rate 1 mL/min at 23ºC. The

identified peaks correspond to: Millipore Water (3.2 min) and Xylose (11.4 min). ...................... 40


and the enzyme mixture present in the concentrate solution recovered from ultrafiltration, which

contained EDTA (2mM), added at 96h of cultivation of A. terreus A-1 growing on Medium 1.

Column :Lichrospher 100 NH2 ; Mobile phase: 85%Acetonitrile/15%Water ; Flow rate 1 mL/min

at 23ºC The identified peaks correspond to: Millipore Water (3.3 min) and Xylose (9.8 min). ... 41


and the enzyme mixture (20 times more concentrated) present in the concentrate solution,

recovered from ultrafiltration of cellulases produced by A. terreus A-1 growing on Medium 1

(“Control”). Column :Lichrospher 100 NH2 ; Mobile phase: 85%Acetonitrile/15%Water ; Flow

rate 1 mL/min at 23ºC. The identified peaks correspond to: Millipore Water (3.2 min) and

Cellobiose (23.5 min). ................................................................................................................. 42




(“Control”). Column :Lichrospher 100 NH2 ; Mobile phase: 85%Acetonitrile/15%Water ; Flow

ix

rate 1 mL/min at 23ºC. The identified peaks correspond to: Millipore Water (3.4 min) and

Cellobiose (23.8 min). ................................................................................................................. 43


and the enzyme mixture (20 times more concentrated) present in the concentrate solution after

ultrafiltration, which contained EDTA (2mM), added at 96h of cultivation of A. terreus A-1

growing on Medium 1. Column :Lichrospher 100 NH2 ; Mobile phase:

85%Acetonitrile/15%Water ; Flow rate 1 mL/min at 23ºC. The identified peaks correspond to:

Millipore Water (3.4 min) and Cellobiose (23.1 min). ................................................................. 44


and the enzyme mixture (20 times more concentrated) present in the concentrate solution after

ultrafiltration, which contained EDTA (2mM), added at 96h of cultivation of A. terreus A-1

growing on Medium 1. Column: Lichrospher 100 NH2 ; Mobile phase:

85%Acetonitrile/15%Water ; Flow rate 1 mL/min at 23ºC. The identified peaks correspond to:

Millipore Water (2.9 min) and Cellobiose (23.1 min). .................................................................. 45

Figure 28 - HPLC chromatogram for a standard solution of Cellobiose (10 g·L-1). Column:

Prepacked Column RT Polyspher-OA HY (300 x 6.5 mm) with a H2SO4 0.0025 M mobile phase.

Flow rate: 0.3 mL/min ; P=23 bar ; T=65ºC. ................................................................................ 54

Figure 29 - HPLC chromatogram for a standard solution of Glucose (10 g·L-1). Column:



Figure 30 - HPLC chromatogram for a standard solution of Xylose (10 g·L-1). Column:



Figure 31 - HPLC chromatogram for a standard solution of Arabinose (10 g·L-1). Column:





produced by A. terreus A-1 growing on Medium 1. ..................................................................... 57

x






produced by A. terreus A-1 growing on Medium 1. The peaks identified for Cellobiose (12.8)

and Glucose (15.4) were superimposed. .................................................................................... 59










Figure 38 – HPLC chromatogram for 0h of reaction between commercial cellulose (10 g·L-1)


cellulases produced by A. terreus A-1 growing on Medium 1 (“Control”). The identified peak

corresponds to: Millipore Water (3.6 min) ................................................................................... 62

Figure 39 - HPLC chromatogram for 2h of reaction between commercial cellulose (10 g·L-1) and

the enzyme mixture present in the concentrate solution, recovered from ultrafiltration of

cellulases produced by A. terreus A-1 growing on Medium 1 (“Control”). The identified peak

corresponds to: Millipore Water (3.4 min) ................................................................................... 63


the enzyme mixture present in the concentrate solution recovered from ultrafiltration, which

contained EDTA (2mM), added at 96h of cultivation of A. terreus A-1 growing on Medium 1. The

identified peak corresponds to: Millipore Water (3.6 min) ........................................................... 64


the enzyme mixture present in the concentrate solution recovered from ultrafiltration, which

xi

contained EDTA (2mM), added at 96h of cultivation of A. terreus A-1 growing on Medium 1. The

identified peak corresponds to: Millipore Water (3.6 min). .......................................................... 65




(“Control”). The identified peaks correspond to: Millipore Water (3.3 min) and Cellobiose (23.5

min). ............................................................................................................................................. 66

xii

List of Tables

Table 1 – Cellulase enzyme production by different fungi in a solid state fermentation (Jahromi,

et al., 2011). ................................................................................................................................... 4

Table 2 – Specific activities (U/mg of protein) obtained for cellulases produced by Aspergillus

terreus A-1 growing on media 1 and 2. The enzyme assay was specific for the endo-1,4-β-D-

glucanase activity and was performed using Azo-CMcellulose as substrate. ............................ 35

Table 3 – HPLC retention times obtained for a 10 g·L-1 mixed sugar solution containing glucose,

xylose and cellobiose. The column used was Lichrospher 100 NH2 with a

85%Acetonitrile/15%Water mobile phase at a 1 mL/min flow rate and 23ºC. ............................ 38

xiii

Abbreviations

A.aculeatus Aspergillus aculeatus

A.carbonarius Aspergillus carbonarius

A.ellipticus Aspergillus ellipticus

A.foetidus Aspergillus foetidus

A.heteromorphus Aspergillus heteromorphus

A.japonicus Aspergillus japonicus

A.nidulans Aspergillus nidulans

A.niger Aspergillus niger

A.terreus Aspergillus terreus

A.tubingensis Aspergillus tubingensis

CBH Cellobiohydrolase

CMC Carboxymethyl cellulose

CMCase Carboxymethyl cellulase

DOE U.S. Department of Energy

EG Endoglucanase

EDTA Ethylene diamine tetra acetate

FPase Filter Paper activity for cellulases

HPLC High Performance Liquid Chromatography

MWCO Molecular weight cut-off

NPA 4-nitrophenyl-α-arabinofuranoside

NPG 4-nitrophenyl-β-glucopyranoside

NREL National Renewable Energy Laboratory

OD Optical Density

PES Polyethersulfone

T.reesei Trichoderma reesei

Tris hydroxymethylaminomethane

UF Ultrafiltration

1

1. Introduction

1.1 Motivation and Background: Cellulases and Secon d

Generation Biofuels

Rising oil prices in the last few years and environmental concerns due to climate

change have led to an increasing interest in biofuels. Biofuels are renewable, can substitute

fossil fuels, reduce fossil greenhouse gas emissions and they can be produced, where they are

needed, to reduce the dependence on oil producing countries. First generation biofuels, such as

bioethanol produced from sugarcane and maize, and biodiesel, produced from soybeans, are

the only ones used on an industrial scale at the time.

Because first generation biofuels have had some drawbacks due to environmental and

social impacts from food crops, the bioethanol that is generated from cellulosic materials, so

called second generation biofuel, has become an object of interest. (Goldschmidt, 2008) The

majority of fermentable sugars from biomass feedstocks, with greatest interest for second-

generation ethanol production, are in the form of cellulose.

Organisms with cellulase systems that are capable of converting biomass to alcohol

directly are already reported. But none of these systems described are effective alone to yield a

commercially viable process. The strategy employed currently in bioethanol production from

lignocellulosic residues is a multi-step process involving pre-treatment of the residue to remove

lignin and hemicellulose fraction, cellulose treatment at 50ºC to hydrolyze the cellulosic residue

to generate fermentable sugars, and finally use of a fermentative microorganism to produce

alcohol from the hydrolyzed cellulosic material (Sukumaran, et al., 2005). The cellulose

preparation needed for the bioethanol plant is prepared using a lignocellulosic residue as

substrate, and the organism employed is almost always T.reesei, although other fungi like

Humicola, Penicillium and Aspergillus have also the ability to yield high levels of extracellular

cellulases (Sukumaran, et al., 2005).

To develop efficient technologies for biofuel production, significant research has been

directed towards the identification of efficient cellulase systems and process conditions, as well

as studies have been directed at the biochemical and genetic improvement of the existing

organisms utilized in the process.

The use of pure enzymes in the conversion of biomass to ethanol or to fermentation

products is currently uneconomical due to the high cost of commercial cellulases. Cellulosic

biomass is an attractive resource that can serve as substrate for the production of value added

metabolites and cellulases as such. Again, the most critical part in producing products from

2

lignocellulosic materials is the enzyme production cost. In early process designs, enzymes

accounted for more than 50% of the ethanol production cost. (Cleantech, 2010) Regardless of

the type of cellulosic feedstock, the cost and hydrolytic efficiency of enzymes are major factors

that restrict the commercialization of the biomass bioconversion processes. (Gusakov, et al.,

2007) Improving the effectiveness of enzymes, while reducing the quantity needed, is thus one

of the key challenges that must be overcome to make second-generation ethanol commercially

viable.

1.1.1 Economic analysis of bioethanol production:

About 15 years ago the cost of ethanol production from cellulosic substrates was

US$4.0/gallon (Verma, et al., 2011).

In 2009 the U.S. Department of Energy (DOE), who promotes the production of ethanol

and other liquid fuels from lignocellulosic biomass feedstocks by funding research, estimated

that the enzyme cost contribution to a bioethanol production plant should be $0.35/gal (2007$)

and should decrease to $0.12/gal ethanol by 2012. (Humbird, et al., 2011) By February 2010

the major enzyme manufacturers, Genencor and Novozymes, announced new commercial-

grade cellulase enzyme preparations capable of higher performance at lower loadings.

However, both cited an enzyme cost contribution of approximately $0.50/gal of ethanol

(Humbird, et al., 2011).

Earlier this year (2011), the National Renewable Energy Laboratory (NREL)

investigated the production economics of ethanol using lignocellulosic biomass feedstocks. A

biorefinery using a process design that converts corn stover to ethanol by dilute-acid

pretreatment, enzymatic saccharification, and co-fermentation, processing 2.205 dry ton/day at

76% theoretical ethanol yield (79 gal/dry ton), was described to have an ethanol selling price of

$2.15/gal in 2007$. However, the predicted cost of enzymes to the ethanol plant is $0.34/gal of

ethanol (Humbird, et al., 2011).

If enzymatic processing and biomass improvement together are met, the projected cost

would be as low as US $ 0.20 per liter by 2015 (Verma, et al., 2011).

3

1.2 Aspergillus

Aspergillus species are highly aerobic and are found in almost all oxygen-rich

environments, where they commonly grow as molds on the surface of a substrate, as a result of

the high oxygen tension. Molds derive energy from the organic matter in which they live.

Typically, molds secrete hydrolytic enzymes, mainly from the hyphal tips. These enzymes

degrade complex biopolymers such as starch, cellulose and lignin into simpler substances

which can be absorbed by the hyphae. In this way, molds play a major role in causing

decomposition of organic material, enabling the recycling of nutrients throughout ecosystems.

The genus Aspergillus is group of filamentous fungi with a large number of species. In

1926 a first classification of these fungi was proposed describing 11 groups within the genus

(DeVries, et al., 2001). A re-examination of the groups was performed by Thom and Raper and

14 distinct groups were identified (DeVries, et al., 2001). In addition to the morphological

techniques traditionally applied, new molecular and biochemical techniques have been used in

the reclassification of this group of aspergilli. These analyses resulted in the clear distinction of

eight groups of black aspergilli (A. niger, A. tubingensis, A. foetidus, A. carbonarius,

A. japonicus, A. aculeatus, A. heteromorphus, and A. ellipticus), which are important for

industrial applications. The black aspergilli have a number of characteristics which make them

ideal organisms for industrial applications, such as good fermentation capabilities and high

levels of protein secretion; ability to assimilate various organic substrates; suppress the

development of other microorganisms; and high sporulation capacity (DeVries, et al., 2001).

The majority of Aspergillus species are accepted to be mitosporic, without any known

sexual spore production, although some species have been described to have a teleomorphic

state. Around 20 species have so far been reported as causative agents of opportunistic

infections in man, although Aspergillus terreus is among the ones less commonly isolated as

opportunistic pathogens (Kassebullah, 2006).

Filamentous fungi, such as Aspergillus, have the ability to produce enzymes involved in

the degradation of plant cell wall materials (cellulases, hemicellulases, pectinases,

glycosidases, phytases), starch (amylases), lipids (lipases) and proteins (proteases and

peptidases) and the oxidation of phenolic compounds (laccases) (MacCabe, et al., 2002).

Strains of Aspergillus terreus produce neutral and alkaline type of proteases (Hussain, et al.,

2010).

Industrial applications of cellulases have mainly focused on fungal enzymes. Aspergillus

terreus is a fungus used for producing cellulolytic enzymes. It is a thermoacidophilic fungus

commonly isolated from soil, plant debris, and indoor air environment. Different studies have

reported the ability of A. terreus for the production of FPase (exoglucanase), CMCase

(endoglucanase) and β-glucosidase, xylanase and amyloglucosidase, which are important in

the process of cell wall degradation of biomass (Jahromi, et al., 2011). Aspergillus strains are

4

also known for their ability to produce β–glucosidase with significantly higher yields than

Trichoderma species (Damisa, et al., 2011).

Abilities of different fungi for the production of cellulases from literature are shown in

Table 1 (Jahromi, et al., 2011).

Table 1 – Cellulase enzyme production by different fungi in a solid state fermentation (Jahromi, et

al., 2011).

Microorganism Carbon

source

Enzyme activity (U/g DM)

CMCase Fpase β-Glucosidase

Fusarium

oxysporum Corn Stover 304 0.14

Trichoderma reesei

MGG77 Rice brain 2.31

Trichoderma reesei

ZU-02 Corncob 158

Aspergillus niger

KK2 Rice straw 129 19.5 100

A. terreus M11 Corn stover 581 243 128

1.3 Cellulose

Most carbohydrates in plants are in the form of lignocellulose, which can be converted

into products that are of commercial interest, such as ethanol. Lignocellulosic biomass makes

about 50% of the total biomass in the world with an estimated annual production of 10-50 billion

tons. (Goldschmidt, 2008) The major polysaccharides comprising lignocellulosic residues are

cellulose (30 to 56%) and hemicellulose (10 to 27%) (Emtiazi, et al., 2001).

Cellulose is the most common organic polymer, representing about 1.5x1012 tons of the

total annual biomass production through photosynthesis, and is considered to be an almost

inexhaustible source of raw material for different products. It is the most abundant and

renewable biopolymer on earth and the dominating waste material from agriculture (Sukumaran,

et al., 2005).

The basic structure of cellulose are 1,4-β-glycosidic linked D-glucose molecules that

form un-branched chains, consisting of several thousands of glucose molecules. The number

of glucose units in the cellulose molecules varies and the degree of polymerization ranges from

250 to well over 10000, depending on the source and treatment method (Sukumaran, et al.,

5

2005). Though lignocellulosic biomass is generally recalcitrant to microbial action, suitable

pretreatments resulting in the disruption of lignin structure and increase accessibility of enzymes

have been shown to increase the rate of its biodegradation (Sukumaran, et al., 2005).

Cellulose is a crystalline polymer, an unusual feature among biopolymers. Cellulose

chains in the crystals are stiffened by inter and intra chain hydrogen bonds and the adjacent

sheets which overlie one another are held by weak Van-der Waals forces. The result of this

process is a crystalline structure which is very difficult to degrade. However, cellulose fibers are

not completely crystalline, but contain several types of irregularities, such as micropores or

kinks, with amorphous character. These regions are the points of attack for the cellulolytic

enzymes. When the cellulolytic enzymes cleave the glucose chains, the results are called

cellodextrins, which are short glucose chains of various lengths. The shortest ones, i.e. glucose

dimers, are called cellobiose and are sometimes not included in the cellodextrin classification

(Goldschmidt, 2008).

In nature, cellulose is present in nearly pure state in a few instances, while in most

cases the cellulose fibers are embedded in a matrix of other structural biopolymers, primarily

hemicelluloses and lignin (Sukumaran, et al., 2005). Hemicellulose is a mixture of short linear

and branched polymers consisting of different pentose and hexose sugars. 20-35% of the plant

dry weight consists of hemicellulose, thus it is the second most abundant polymer in the world

(Goldschmidt, 2008). It mostly consists of xylose, a pentose sugar, which implies a problem for

bioethanol production, because not all microbes can metabolize xylose. If it is not possible to

overcome this problem, a large fraction of the sugars in the cellulosic biomass will not be

available for fermentation (Goldschmidt, 2008).

6

Figure 1 – Structure and composition of cellulose fi bers. The glucose chains are held together by

hydrogen bonds and form crystalline cellulose that is arranged in microfibrils. These are linked by

hemicellulose and lignin, forming the recalcitrant cellulose fibers (Goldschmidt, 2008) .

1.4 Cellulases

Cellulases have been the target of active research for over five decades, and are

currently the third largest industrial enzyme worldwide (by dollar volume) because of their use in

cotton processing, paper recycling, as detergent enzymes, in juice extraction, and as animal

feed additives (Wilson, 2009). Thus, if ethanol (or another fermentation product of sugars),

produced from biomass by enzymes, becomes a major transportation fuel, cellulases will

become the largest volume industrial enzyme (Wilson, 2009).

The enzymatic hydrolysis of cellulose releases soluble sugars including glucose, xylose,

and other hexoses and pentoses. To make the sugar monomers available for fermentation, the

cellulose and hemicellulose chains have to be hydrolyzed. The hydrolysis of hemicelluloses are

catalyzed by xylanases, together with other accessory enzymes (α-L-arabinofuranosidases,

feruloyl and acetylxylan esterases, β-xylosidases, etc), while the hydrolysis of cellulose can be

undertaken by microorganisms that produce enzymes known as the cellulase systems

(Gusakov, et al., 2007).

7

Cellulolytic microbes are primarily carbohydrate degraders and are generally unable to

use proteins or lipids as energy sources for growth (Sukumaran, et al., 2005). Cellulases used

for current industrial applications are mainly produced from aerobic cellulolytic fungi, such as

Trichoderma reesei and Aspergillus species primarily due to efficiencies in fungal enzyme

secretion (Wilson, 2009). In fact these organisms produce a complex mix of enzymes at high

productivity and catalytic efficiency, both of which are required for low-cost enzyme supply.

The cellulolytic enzymes can be either secreted into the substrate or attached to the cell

wall of the microorganism (Goldschmidt, 2008). Unlike most bacteria, fungal cellulases are

typically secreted into the growth medium, allowing easy harvest and consequent cost-efficient

separation of the active enzymes in a liquid form.

Furthermore, it is possible to genetically modify these fungal strains to tailor the set of

enzymes they produce, so as to give optimal activity for specific uses (Wilson, 2009).

1.4.1 Characterization and Properties

Cellulases are often modular, containing a catalytic core, a linker and a carbohydrate-

binding module (Teter, et al., 2005). These enzymes are classified into glycosyl hydrolase

families considering the degree of sequence identity. Based on mechanism, cellulases can be

divided into three groups based on their enzymatic activities: endoglucanases; exoglucanases

and β-glucosidases. They all have in common the ability of hydrolyzing the 1,4-β-glycosidic

bond between the D-glucose molecules, but they differ in their starting point and substrate when

hydrolyzing (Goldschmidt, 2008).

Endoglucanases (EG), also known as 1,4-β-D glucan-4-glucanohydrolases, attach to

the cellulose at arbitrary internal amorphous sites and cleave the polysaccharide chain by

inserting a water molecule in the 1,4-β bond. The results are oligosaccharides of various lengths

with a reducing and a non-reducing end (Goldschmidt, 2008). The molecular weight of an endo-

β-1,4-glucanase from a thermoacidophilic fungus, Aspergillus terreus M11 was found to be 25

kDa (Gao, et al., 2007). EGs activities can be measured using a soluble cellulose derivative with

a high degree of polymerization such as carboxymethylcellulose (CMC). In early studies

regarding cellulases the available enzyme preparations would scarcely hydrolyze insoluble

cellulose, although they often broke down soluble derivatives such as carboxymethyl cellulose

(CMC) readily. The reason for this was that they consisted chiefly of endo-β-glucanases and

lacked the exo-β-glucanases. Thus, for cellulase preparations derived from organisms such as

Aspergillus niger, carboxymethylcellulose is used as substrate (Mandels, et al., 1976).

The exoglucanases start at either the reducing or non-reducing end of the

oligosaccharide chains, resultant from EGs action, and release either glucose or a cellobiose

dimer. The glucose releasing enzymes are called glucanases and the cellobiose releasing

8

enzymes cellobiohydrolases (CBH). The exoglucanases can also work autonomously and peel

cellulose chains from microcrystalline cellulose. Finally, the β-glucosidases hydrolyze the

glucose dimers (cellobiose) and the cellodextrins of various lengths to glucose (Goldschmidt,

2008). These fundamentally different catalytic mechanisms allow different types of cellulases to

interact synergistically (Figure 2). At high concentrations, cellobiose inhibits cellobiohydrolase

activity. Hence, β-glucosidase, which converts cellobiose into glucose, is often required for

optimal cellulose performance in conditions where cellobiose accumulates, preventing end-

product inhibition.

Figure 2 - Schematic view of the biodegradation of c ellulose. Cellobiohydrolases act on the nonreducing or reducing termini of cellulose fibers to processively release cellobiose. Endo- β-1,4-

glucanases randomly cleave cellulose chains. Β-glucosidases hydrolyze cellobiose or cello-oligomers to glucose from nonreducing ends (Watanab e, et al., 2009).

1.4.1.1 Physical and Chemical Properties

Most cellulases studied have similar pH optima, solubility and amino acid composition.

Thermal stability and exact substrate specificity may vary. However, it should be taken into

account that cellulase preparations generally contain other enzymatic activities besides

cellulolytic activity, and these may also affect the properties of the preparations.

9

• Optimum pH: Cellulase preparations are effective between pH 3 and 7. The

optimum pH generally lies between 4 and 5 (GmbH, 2011);

• Optimum temperature : The optimum temperature for cellulolytic activities of A.

niger was 30 ºC and 35 ºC for A. nidulans. (Usama, et al., 2008) A maximum yield of

cellulases from A. terreus QTC 828 was achieved at 40 ºC (Ali, et al., 1991).

Temperature is a cardinal factor affecting the extent and rate of growth of an organism

and the increasing temperature has the general effect of increasing enzyme activity, but

the enzyme begins to suffer thermal inactivation at higher temperatures.

• Inhibitors: Cellulase is inhibited by its reaction products i.e. glucose,

cellobiose. Moreover, Hg inhibits cellulases completely, whereas Mn, Ag,Cu and Zn

ions are only slightly inhibitory (GmbH, 2011);

• Stability and storage: The activity of cellulase preparations were found to be

completely destroyed after 10-15 minutes at 80 °C. Solutions of cellulase at pH 5-7 are

stable for 24 hours at 4°C. These products should b e stored at 4 °C, in a dry place in

tightly-closed containers. If stored in this manner, lyophilized preparations are stable for

several months without significant loss of activity (GmbH, 2011).

1.4.2 Cellulase Production

Commercial production of cellulases on a commercial scale is induced by growing the

fungus on solid cellulose or by culturing the organism in the presence of a disaccharide inducer,

such as lactose. However, on an industrial scale, both methods of induction result in high costs.

Since the enzymes are inducible by cellulose, it is possible to use cellulose containing media for

production, although the process is controlled by the dynamics of induction and repression. At

low concentrations of cellulose, glucose production may be too slow to meet the metabolic

needs of active cell growth and function. On the other hand, cellulase synthesis can be stopped

by glucose repression when glucose generation is faster than consumption. Thus, expensive

process control schemes are required to provide slow substrate addition and monitoring of

glucose concentration (Sukumaran, et al., 2005).

In vivo cellulase production is associated to growth and is influenced by various factors.

Interactions amongst these factors can affect cellulase productivity (Sukumaran, et al., 2005).

Because cellulases are inducible extracellular enzymes, their rate of production is greatly

influenced by nutrient medium composition. In the majority of commercial cellulase

fermentations the carbon sources are cellulosic biomass including straw, spent hulls of cereals,

rice or wheat bran, bagasse, paper industry waste and various other lignocellulosic residues

(Sukumaran, et al., 2005). Though fermentation conditions for the development of economically

10

feasible bioprocesses are still being sought, combinatorial interactions of medium components

with the production of the desired compounds are numerous, and optimum processes may be

developed using an effective experimental design method.

The challenges in cellulase production involve developing suitable bioprocesses and

media for cellulase fermentation, besides identification of cheaper substrates and inducers.

Genetic modification of the cellulase producers to improve cellulase activity has gone a long

way to give better producers with high enzyme titers, but still cellulase production economics

needs further improvement for commercial production of ethanol from biomass (Sukumaran, et

al., 2005).

1.4.3 Applications

Cellulases are used in the textile industry, in detergents, pulp and paper industry,

improving digestibility of animal feeds, in food industry, and these enzymes account for a

significant share of the world enzyme market (Sukumaran, et al., 2005).

Cellulases were initially investigated several decades back for the bioconversion of

biomass which gave way to research in the industrial applications of the enzymes in animal

feed, food, textiles and detergents and in the paper industry (Sukumaran, et al., 2005). With the

shortage of fossil fuels and the arising need to find alternative source for renewable energy and

fuels, there is a renewal of interest in the bioconversion of lignocellulosic biomass using

cellulases and other enzymes.

Perhaps the most important application currently being investigated is in the utilization

of lignocellulosic wastes for the production of biofuel (Sukumaran, et al., 2005). The

lignocellulosic residues represent the most abundant renewable resource available to mankind,

but their use is limited due to lack of cost effective technologies. A potential application of

cellulase is the conversion of cellulosic materials to glucose and other fermentable sugars,

which in turn can be used as microbial substrates for the production of single cell proteins or a

variety of fermentation products like ethanol.

11

1.4.4 The Challenges in Cellulase Research – Future Perspectives

Lignocellulose is the potential source of biofuels, bio-fertilizers, animal feed and

chemicals, besides being the raw material for paper industry. (Sukumaran, et al., 2005)

Exploitation of this renewable resource needs either chemical or biological treatment of the

material and, in the latter context, cellulases have gained wide popularity over the past several

decades. Research has shed light into the mechanisms of microbial cellulase production and

has led to the development of technologies for production and applications of cellulose

degrading enzymes. However, there is no single process, which is cost effective, and efficient in

the conversion of the natural lignocellulosic materials for production of useful metabolites or

biofuel. The use of the current commercial preparations of cellulose for bioconversion of

lignocellulosic waste is economically not feasible (Sukumaran, et al., 2005).

The major goals for future cellulase research would be the reduction in the cost of its

production and the improvement of the performance of cellulases to make them more effective,

so that less enzyme is needed. The former task may include such measures such as optimizing

growth conditions or processes, while the latter requires directed efforts in protein engineering

and microbial genetics to improve the properties of the enzymes (Sukumaran, et al., 2005).

Optimization of growth conditions and processes has been attempted to a large extent

in improving cellulase production. For instance, empirical optimization of process variables to

improve productivity has been the focus of many of the works using fermentation for the

production of cellulases (Sukumaran, et al., 2005). Many of the current commercial production

technologies utilize submerged fermentation technology and employ hyper producing mutants.

Despite several efforts directed at generating hyper producers by directed evolution, the cost of

enzymes has remained high. Alternative strategies in cellulase production include mainly solid

substrate fermentation on lignocellulosic biomass, particularly by using host/substrate specific

microorganisms. There are several reports on such use of filamentous fungi in production of

optimal enzyme complex for the degradation of host lignocellulose (Sukumaran, et al., 2005).

Feedback inhibition of cellulase biosynthesis by the end products, glucose and

cellobiose, generated by endogenous cellulolytic activity on the substrate is another major

problem encountered in cellulase production. Cellobiose is an extremely potent inhibitor of the

CBH and EG biosynthesis. Trichoderma and the other cellulase-producing microbes make very

little β-glucosidase compared to other cellulolytic enzymes. The low amount of β-glucosidase

results in a shortage of capacity to hydrolyze the cellobiose to glucose, resulting in a feedback

inhibition of enzyme production and, in the particular case of biomass conversion applications,

in the inhibition of cellulases. This issue has been addressed by various means such as the

12

addition of exogenous β-glucosidases to remove the cellobiose and engineering β-glucosidase

genes into the organism so that it is overproduced (Sukumaran, et al., 2005). In the future,

developments in process design and medium formulations will no longer be enough and

controlled genetic interventions into the physiology of cellulase producers to improve production

will be key to make the cellulase production process more cost effective. The major tasks ahead

include overriding the feedback control by glucose and development of integrated bioprocesses

for the production of cellulases.

Improvements in cellulase activities or giving of desired features to enzymes by protein

engineering are probably other areas where cellulase research has to advance. Active site

modifications can be imparted through site directed mutagenesis and the mutant proteins can

be used for understanding the mechanisms of action as well as for altering the substrate

specificities or improving the activities (Sukumaran, et al., 2005).

Protein engineering has been successfully employed to improve the stability of a

cellulase from Humicola in the presence of detergents; better the thermostability of a mesophilic

endo-1,4-β-glucanase from alkaliphilic Bacillus sp., as well as altering the pH profile of a

cellobiohydrolase and, more recently, endoglucanase from T. reesei (Sukumaran, et al., 2005).

Such modifications affecting the enzyme properties may be useful for a better overall

performance of cellulases, as well as understanding their mode of action, which will therefore

enable the use of such enzymes for biomass conversion. More basic research is needed to

make designer enzymes suited for specific applications.

1.5 Aim of this study

The goal of this project is to study the use of new cellulases, produced by Aspergillus

terreus, as catalysts for the hydrolysis of cellulose from plant biomass, for further use in

energetic applications.

With this objective in mind, work is done in order to optimize the production of cellulases

from Aspergillus terreus. The experimental conditions studied are close to recent works found in

literature (Usama, et al., 2008), (Nour, et al., 2011). Different Aspergillus terreus strains, various

concentration of spores utilized to prepare the inoculum, different cellulose concentrations

supplemented to growth media, as well as the use of another basal medium (with another pH

and nitrogen and Mg2+ sources) are tested for optimum cellulase activity. It should be noted that

almost all the research is focused on an enzyme activity assay specific for an endo-cellulase

(endo-β1,4-glucanase) and not for total enzyme activity. However, experiments are also

conducted to investigate and compare the activity of β-glucosidase in the enzyme mixtures.

13

Furthermore, stability tests are performed to analyze the influence of temperature in the

enzymatic activity of cellulases, as well as the impact of a protease inhibitor cocktail and EDTA

(a metalloprotease inhibitor) on cellulasic activity.

Finally, after selecting the best performing enzyme preparation, cellulases are tested by

HPLC for their capability to hydrolyze commercial cellulose to their precursor sugars, such as

glucose, xylose and cellobiose.

14

2. Materials and Methods

2.1 Microorganism

Applied fungus was Aspergillus terreus strains named A-1 and N-Y, isolated from soil.

2.2 Culture Media

The basal medium (without carbon source) utilized for cellulase production (Medium 1)

had the following composition (g⋅L-1):

o NaNO3 2.0

o KH2PO4 1.0

o MgSO4⋅7H2O 0.5

o KCl 0.5

o FeSO4 0.01

o pH 6.5

In order to optimize the production of cellulases, trials were made changing the

nitrogen source, magnesium concentration and the pH. The medium used (Medium 2) had the

following composition (g⋅L-1):

o Peptone 6.0

o KH2PO4 1.0

o MgSO4⋅7H2O 1.72

o KCl 0.5

o FeSO4 0.01

o pH 5.5

The media were sterilized in the autoclave at 116ºC for 20 min.

2.3 Maintenance and Growth Conditions

Strains were routinely maintained on agar-malt at 30ºC until sporulation occurred and

conserved at 4ºC. The fungi were grown in 1 L Erlenmeyer baffled flasks containing 150 mL of

the basal medium supplemented with different concentrations of cellulose (Sigma Aldrich) or

sucrose. Cellular growth regards the following phases:

15

a) Pre-inoculum

On a first approach, spores from 10-days old cultures on malt slants were re-suspended

with 6 mL of sterilized water and the obtained suspension was utilized to prepare the inoculum

for the flasks.

On a second approach, spores from slants were re-suspended with 6 mL of sterilized

water and filtered using sterile glass-whole filters. The amount of spores was determined by

reading the O.D. of the suspension at 595 nm in a UV/Visible spectrophotometer (Pharmacia

Biotech, Ultrospec 1000). The filtered solution was used to make the inoculum of the flasks.

b) Growth in Flasks

On the first approach, each flask was inoculated with 10 mL of spore suspension. On

the second approach, the O.D. of the inoculum was read and the flasks inoculated with either

0.1 ; 0.2 or 0.5 OD/mL concentrations of spores.

In both cases, the cultures were incubated at 30ºC under alternative agitation (120 spm)

up to 216h.

2.4 Characterization of Cellulases

2.4.1 Harvest and Enzyme Separation

Samples of 6 mL were collected from each flask, under sterile conditions, on a 24h

basis, up to 216 h. These were then centrifuged at 5000 rpm for 15 minutes and the

supernatant was collected to 10 mL sterile tubes and stored at -20ºC for further use in enzyme

assays.

2.4.2 Cellulase Activity Assay

a) endo-1,4-β-D-glucanase activity

The method for the enzyme assay used in most of the work is specific for the endo-1,4-

β-D-glucanase activity (endo-cellulase) present in cellulose preparations.

16

• Substrate

The substrate Azo-Alpha Cellulose (Megazyme) utilized for the enzyme assays was

prepared by Megazyme by dyeing CM-Cellulose 4M with Remazolbrilliant Blue dye, to a dye

content of approx. one dye molecule per 20 sugar residues. Alpha-cellulose is an amorphous

alkali-resistant cellulose and is a potential substrate for the assay of endo-1,4-β-D-glucanase

(endo-cellulase).

• Solutions

The solutions used during the cellulase activity assays were the following:

• Sodium Acetate buffer

Sodium Acetate Trihydrate 2 M in demineralized water. The pH was adjusted to 4.5 with

HCl 0.5 N.

• Substrate solution

Two grams of powdered substrate were added to 80 mL of boiling and vigorously stirred

water on a hot-plate with a magnetic stirrer. The heat was turned off and was carried out with

stirring until the solution was homogeneous (approx. 20 min). A volume of 5 mL of sodium

acetate buffer (2 M; pH 4.5) was added and the solution was cooled at room temperature. The

pH was adjusted to 4.5 and the volume to 100 mL. The solution was stored at 4ºC for posterior

use.

• Precipitant solution

Forty grams of sodium acetate trihydrate and four grams of zinc acetate were dissolved

in 150 mL of demineralized water. The pH was adjusted to 5.0 with 5 M HCl and the volume to

200 mL with demineralized water. Two hundred milliliters of this solution was added to 800 mL

of ethanol (95%), mixed well and stored at room temperature in a well-sealed bottle.

The procedure used for the enzyme assay is specific for the endo-1,4-β-D-glucanase

activity (endo-cellulase) present in cellulose preparations. When incubating dyed CMC with

cellulase, the substrate is depolymerized by an endo-mechanism to produce low molecular

weight dyed fragments, which remain in solution after the addition of a precipitant solution to the

reaction mixture. High-molecular material is removed by centrifugation and the color of the

supernatant is measured.

17

• Assay Procedure

An aliquot of 0.1 mL of a pre-equilibrated substrate solution was added to 0.1 mL of

enzyme solution (pre-equilibrated at 40ºC) and stirred on a vortex mixer. The mixture was

incubated at 40ºC for exactly 30 minutes and the reaction was then terminated by the addition

of 0.5 mL of precipitant solution (the high-molecular weight unhydrolysed substrate was

precipitated). The reaction tubes were equilibrated to room temperature for 10 min and

vigorously stirred for 10 sec on a vortex mixer. The tubes were then centrifuged (Eppendorf

Centrifuge 5415D) at 12000 rpm for 10 min. The supernatant solution was poured directly into a

spectrophotometer cuvette and the absorbance of the reaction solutions was measured

(Microplate Reader BIO RAD Model 3550) at 595 nm against the reaction blank.

b) β-glucosidase activity

In order to evaluate other eventual cellulase activities, β-glucosidase and α-

arabinosidase were also assayed. The substrates used were NPG (4-nitrophenyl-β-

glucopyranoside) and NPA (4-nitrophenyl-α-arabinofuranoside) from Sigma-Aldrich. An aliquot

(0.1 mL) of diluted enzyme was added to 0.9 mL of a 5 mM solution of the appropriate glycoside

(NPG or NPA). Buffer was 50 mM citrate, adjusted at the appropriate pHopt for each activity (3.6

for α-arabinosidase and 4.8 for β-glucosidase) . After 5 min at 25ºC, the reaction was stopped

by adding 1 mL of 200 mM sodium carbonate. The absorbance of the reaction solutions was

measured at 400 nm against a reaction blank, on a UV/Visible Ultrospec 1100 Pro

Spectrophotometer (Amersham Biosciences).

2.4.3 Protein Assay - Bradford Method

Protein content was measured according to the method of (Bradford, 1976) using

bovine serum albumin (BSA, Sigma) as a standard. This method analyzes the interaction

between the proteins’ aminoacids and the colorant Coomassie Blue G-250 on an acid solution.

An aliquot of 100 µL of sample was added to 1 mL of standard and the reading was made at

595 nm on a UV/Visible Ultrospec 1100 Pro Spectrophotometer (Amersham Biosciences). The

values of absorbance were compared with a standard curve for bovine serum albumin. Each

assay was performed in duplicate.

18

2.5 Recovery and Concentration of Cellulases

2.5.1 Vacuum Filtration

After incubation for 96h, mycelia from Erlenmeyer flasks were harvested by vacuum

filtration using a Buckner filter. Vacuum filtration was run until filtrate was no longer being

collected (about 5 min), and the resulting filter cake was scraped off. The filtrate was used in

subsequent experiments.

2.5.2 Ultrafiltration

Ultrafiltration was carried out to concentrate the cellulase solution by recovering any

soluble enzymes present in the culture filtrate.

On a first approach, a 4.45-cm-diameter polyethersulfone membrane disk from

Millipore, with a 50-kDa molecular weight cut-off was used in a stirred-cell apparatus

(Ultrafiltration Cell Model 8050, AMICON Corp.). The stirred-cell was pressurized with nitrogen

to a maximum pressure of 1.5 bar. After ultrafiltration the stirred-cell was two times washed with

Tris-HCl 10 mM (pH=7.4) buffer. Finally, a volume of 150 mL that remained in the stirred cell

(concentrate) was collected. The concentrate was maintained at 4ºC. Protein transmission

during vacuum filtration and protein rejection during ultrafiltration were measured by

spectrophotometry using the previously described enzyme assay for the endo-1,4-β-D-

glucanase activity.

A different approach to concentrate cellulases was made using VIVASPIN 2

Concentrator (Viva Science) ultrafiltration devices containing a polyethersulfone membrane with

10 kDa molecular weight cut-off. A volume of 4 mL from the vacuum filtrate was centrifuged at

5000 rpm for 15 minutes. The devices were then washed two times with Tris-HCl 2 mM

(pH=7.4) buffer and a volume of 0.5 mL was collected as the concentrate.

2.6 Validation Methods of Cellulasic Activity

2.6.1 Enzymatic reaction with commercial cellulose

19

To evaluate the capability of cellulases produced by Aspergillus terreus to release

fermentable sugars (such as glucose, xylose, cellobiose and arabinose), the reaction of the

enzyme mixture with cellulose was assayed.

The reaction was carried out in glass tubes with a screw cap, in a water bath at 30ºC

and under magnetic agitation. Each tube contained:

o 1 mL of a 10 g⋅L-1 solution of commercial cellulose (Sigma Aldrich)

o 500 µL of enzyme recovered from ultrafiltration

An aliquot of 100 µL from each reaction tube was taken at different times and

centrifuged at 13000 rpm for 3 minutes. The sample was then injected in an HPLC system.

2.6.2 HPLC analysis

Hydrolytic products released from cellulose were analyzed by high performance liquid

chromatography (HPLC). On a first approach, soluble products were separated by using a

Prepacked Column RT Polyspher-OA HY (300 x 6.5 mm). The column was washed with

Millipore water and then eluted with a solution of H2SO4 0.0025 M at a flow rate of 0.3 ml/min

and temperature of 65ºC. The analysis of the hydrolysis products was performed using a

refractive index detector (BIO-RAD Refractive Index Monitor).

A second HPLC analytical assay was performed was performed on a Lichrospher

100 NH2 column (4 x 250 mm) 5 µm + Pre-Column (with the same stationary phase) with a

degassed 85:15 (v/v) acetonitrile/water (Sigma-Aldrich/Milli-Q) eluent system at a 1 mL/min flow

rate, at room temperature (23ºC). The analysis of the hydrolysis products was performed using

the same refractive index detector.

2.7 Stability Tests

2.7.1 Effect of Temperature on Cellulase Activity a nd Stability

Temperature effects on enzyme activity were assayed at the following temperatures:

-20ºC and 25ºC. For the measurement of thermal stability, samples (in duplicate) of the

concentrate, recovered from ultrafiltration, were kept at temperatures -20ºC and 25ºC for 2

20

days. The cellulase activity was determined using the standard enzyme (endo-1,4-β-D-

glucanase) assay conditions. The control (sample at 25ºC) was taken as having 100% activity.

2.7.2 Effect of EDTA and a Protease Inhibitor Cockt ail on

Cellulase Activity and Stability

This experiment was carried out to test the effect of ethylene diamine tetra acetate

(EDTA) and of a protease inhibitor cocktail (Sigma-Aldrich), attempting at the stabilization of

cellulases present in the concentrate solution after ultrafiltration. The activity of cellulases was

assayed under standard enzyme (endo-1,4-β-D-glucanase) conditions, in the presence of EDTA

(2mM) and a protease inhibitor cocktail after 2 days at 25ºC. Samples were assayed in

duplicate and the control (without any additive) was taken as having 100% activity.

21

3. Results and Discussion

3.1 Factors Affecting Cellulase Production

3.1.2 Effect of Cellulose Concentration on Cellulas e Activity

Aspergillus terreus A-1 and N-Y strains were grown in Erlenmeyer flasks containing

basal Medium 1 supplemented with different concentrations of cellulose (the sole carbon

source), in order to study the effect on enzyme production. Flasks containing sucrose as the

sole carbon source were used as control, as in this case no cellulolytic activity is required for

growth of the microorganism. (DISMA Biochemistry Lab., 2010)

The enzymatic activity of cellulases produced by the fungi was measured according to

the procedure described for the endo-1,4-β-D-glucanase. Activity is expressed in equivalent

U/mL calculated on the basis of a calibration curve performed by using commercial Sigma-

Aldrich cellulase (Figure 3). Measurements were carried out by using a commercial soluble

chromogenic substrate (AZO-CM-CELLULOSE; Megazyme).

Since no assays to evaluate the variation of Absorbance (595 nm) during the reaction

time course were performed due to a tight schedule, throughout this work the enzyme activity is

expressed in relation to the reaction time (30 min), instead of in relation to the initial reaction

rates. Also, no calibration curve was established between the Absorbance595nm and the amount

of glucose released from the action of endo-cellulases on the substrate (CMC-4M). Under these

circumstances, one “Unit of activity” is defined as micrograms of substrate which give rise to

one unit of Absorbance595nm at pH 4.5 and 40°C.

22

Figure 3 – Aspergillus terreus endo-Cellulase calibration curve performed by usin g commercial

SIGMA cellulase. Measurements were carried out by us ing a commercial soluble substrate (AZOCM-CELLULOSE, Megazyme). Assay conditions: T=40ºC; Incubation time= 30 min.

As expected, the fungi did not produce cellulases when grown on sucrose (Figure 4

Figure 5). In contrast, cellulases were produced extracellularly by both strains of the fungi when

grown on cellulose. As shown in Figure 4, the activity of cellulases produced by A.terreus A-1

strain had a maximum (13.2 U/mL) after 72h of incubation (30ºC) and at the concentration of 30

g⋅L-1 of cellulose, the sole carbon source. On the other hand, the maximum production of

cellulases by N-Y strain (10.2 U/mL) was shown to be at the concentration of 20 g⋅L-1 of

cellulose, also after 72h of incubation (Figure 5). This value of cellulase enzymatic activity was

higher than that obtained from cellulases produced by A. niger grown on basal medium

supplemented with 20 g⋅L-1 of cellulose (Gautam, et al., 2010).

Figure 4 – Effect of different concentrations of cel lulose on the enzymatic activity of cellulases produced by Aspergillus terreus A-1 strain during 9 days of incubation at 30ºC.

S30 – Concentration of Sucrose 30 g⋅L-1; C20 – Concentration of cellulose 20 g⋅L-1 ; C30 – Concentration of cellulose 30 g⋅L-1 ; C40 – Concentration of cellulose 40 g⋅L-1 ; C50 – Concentration of cellulose 50 g⋅L-1 ;

C60 – Concentration of cellulose 60 g⋅L-1

y = 0,036x + 0,032

R² = 0,9724

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0,4

0,45

0 2 4 6 8 10 12

Ab

sorb

an

ce,

59

5 n

m

µg of substrate (CMC-4M) /0,1 mL

-2

0

2

4

6

8

10

12

14

0 24 48 72 96 120 144 168 192 216 240

U/m

L

Time (hours)

S30

C20

C30

C40

C50

C60

23

Figure 5 - Effect of different concentrations of cel lulose on the enzymatic activity of cellulases produced by Aspergillus terreus N-Y strain during 9 days of incubation at 30ºC.

S30 – Concentration of Sucrose 30 g⋅L-1; C20 – Concentration of cellulose 20 g⋅L-1 ; C30 – Concentration of cellulose 30 g⋅L-1 ; C40 – Concentration of cellulose 40 g⋅L-1 ; C50 – Concentration of cellulose 50 g⋅L-1 ;

C60 – Concentration of cellulose 60 g⋅L-1

For both strains the activity of cellulases decreased abruptly after its maximum value at

72h of incubation, but increased again from 96h until 168h of incubation. However, in both

cases, the activity of cellulases started to decay after 168h (7 days) of incubation of the fungus.

In order to attempt getting more reproducible results, a new approach using a specific amount

of spores was conducted.

3.1.2 Effect of Initializing Fungal Growth at Diffe rent Spore

Concentration (OD/mL) on Cellulase Activity

A second assay was performed by growing Aspergillus terreus A-1 and N-Y strains in

Erlenmeyer flasks containing basal Medium 1, this time supplemented only with a 30 g⋅L-1

concentration of cellulose (sole carbon source). The flasks were inoculated with the following

concentration of spores: 0.1, 0.2, 0.5 OD/mL.

Figure 6 and Figure 7 show the activity of cellulases produced by A.terreus A-1 and N-Y

strains using a 30 g⋅L-1 concentration of cellulose and different concentration of spores along 10

days of incubation at 30ºC, respectively.

-2

0

2

4

6

8

10

12

0 24 48 72 96 120 144 168 192 216 240

U/m

L

Time (hours)

S20

C20

C30

C40

C50

C60

24

Figure 6 - Effect of different concentration of spor es (0.1, 0.2, 0.5 OD/mL) on the enzymatic activity of cellulases produced by Aspergillus terreus A-1 strain, along 10 days of incubation at 30ºC in

alternative agitation.

Figure 7 - Effect of different concentration of spor es (0.1, 0.2, 0.5 OD/mL) on the enzymatic activity of cellulases produced by Aspergillus terreus N-Y strain, along 10 days of incubation at 30ºC in

alternative agitation.

For both strains and each concentration of spores utilized the maximum cellulasic

activity occurs between 72h and 96h of incubation. Subsequently, the enzyme activities

decreased, and in the case of N-Y strain, only about half of the maximum activities could be

detected on the seventh day (168h). These results are in agreement with a similar study on

Aspergillus glaucus XC9 where the optimal cultivation period for cellulase production was 3– 4

days (Chang, et al., 2006). However, the time course required to reach maximum levels of

0

0,5

1

1,5

2

2,5

3

3,5

0 24 48 72 96 120 144 168 192 216 240 264

U/m

L

Time (hours)

A. terreus A-1

0,1 OD/mL 0,2 OD/mL 0,5 OD/mL

-1

0

1

2

3

0 24 48 72 96 120 144 168 192 216 240 264

U/m

L

Time (hours)

A. terreus N-Y

0,1 OD/mL 0,2 OD/mL 0,5 OD/mL

25

activity may be affected by several factors, such as the presence of different ratios of

amorphous to crystalline cellulose (Ogel, et al., 2001).

It is also noticeable that the cultures that received higher concentrated inocula (0.5

OD/mL) showed the highest cellulasic activity (2.8 U/mL for either strains). A previous study on

Aspergillus sp. SU14 showed the importance of inoculum density in cellulase production

(VanHanh, et al., 2010). These authors reported that higher inoculum levels increased the spore

number per gram of substrate, hindering the penetration of oxygen in the medium and leading

to inhibition of fungal growth and cellulase production. However, a lower density may result in

excessive growth of the mycelium and in the delay of enzyme production.

3.1.3 Effect of Medium Composition on Cellulase Act ivity

Because the rate of production of cellulase, an inducible extracellular enzyme, is greatly

influenced by nutrient medium composition, optimum processes may be developed using

effective experimental design methods (Nour, et al., 2011). In order to further optimize the

production of cellulases (obtain higher cellulasic activity), a different basal medium (Medium 2)

was tested, under the previously described conditions. This new medium contained a different

nitrogen source (peptone, at the concentration of 6 g⋅L-1); MgSO4 (7mM); and different pHopt

(5.5), found in literature to be optimal (Nour, et al., 2011). Concentrations of spores of 0.1 and

0.5 OD/mL were used to inoculate the flasks.

Figure 8Figure 11 show the activity of cellulases produced by A.terreus A-1 and N-Y

strains in different media and different concentration of spores (0.1 or 0.5 OD/mL) along 9 days

of incubation at 30ºC, respectively.

26

Figure 8 - Effect of different medium composition, u sing a 0.1 OD/mL concentration of spores, on the enzymatic activity of cellulases produced by Aspergillus terreus A-1 strain, along 9 days of

incubation at 30ºC in alternative agitation.

Figure 9 - Effect of different medium composition, u sing a 0.5 OD/mL concentration of spores, on the enzymatic activity of cellulases produced by Aspergillus terreus A-1 strain, along 9 days of


-2

-1

0

1

2

3

4

5

0 24 48 72 96 120 144 168 192 216 240

U/m

L

Time (hours)

A. terreus A-1, 0.1 OD/mL

Medium 1 Medium 2

0

0,5

1

1,5

2

2,5

3

3,5

0 24 48 72 96 120 144 168 192 216 240

U/m

L

Time (hours)

A. terreus A-1, 0.5 OD/mL

Medium 1 Medium 2

27

Figure 10 - Effect of different medium composition, using a 0.1 OD/mL concentration of spores, on the enzymatic activity of cellulases produced by Aspergillus terreus N-Y strain, along 9 days of


Figure 11 - Effect of different medium composition, using a 0.5 OD/mL concentration of spores, on the enzymatic activity of cellulases produced by Aspergillus terreus N-Y strain, along 9 days of


Comparing the production of cellulases by A.terreus A-1 and N-Y strains when

cultivated in different basal media supplemented with cellulose (30 g⋅L-1), the cellulasic activity

was more remarkable when using Medium 1. These data were in accordance to previous

reports on Aspergillus niger and A. nidulans, where sodium nitrate enhanced cellulolytic

activities (Usama, et al., 2008). Nonetheless, a study on Aspergillus terreus M11 (Gao, et al.,

-1,5

-1

-0,5

0

0,5

1

1,5

2

2,5

3

0 24 48 72 96 120 144 168 192 216 240

U/m

L

Time (hours)

A. terreus N-Y, 0.1 OD/mL

Medium 1 Medium 2

-1

-0,5

0

0,5

1

1,5

2

0 24 48 72 96 120 144 168 192 216 240

U/m

L

Time (hours)

A. terreus N-Y, 0.5 OD/mL

Medium 1 Medium 2

28

2008) showed that enzyme activities were higher using peptone and yeast extract as nitrogen

sources.

The cellulasic activity obtained using a 0.5 OD/mL concentration of spores was similar

or lower than the activity obtained when using a 0.1 OD/mL concentration. For this reason an

inoculum concentration of 0.1 OD/mL was used in the following experiments. Also, because A.

terreus A-1 strain seemed to follow a more regular pattern, it was the chosen to perform the

next trials.

At this point of the experimental work, the optimal conditions found for the production of

cellulases by Aspergillus terreus were:

• Strain (A-1 ; N-Y) : A-1

• Concentration of cellulose (20 - 60 g·L-1) : 30 g·L-1

• Concentration of Spores (0.1 , 0.2 , 0.5 OD/mL) : 0.1 OD/mL

• Time of growth (24h – 216 h) : between 72h and 96h

• Basal medium (Medium 1 ; Medium 2) : Medium 1

A different approach was attempted, this time introducing a vacuum filtration followed by

an ultrafiltration, after cultivation of A. terreus A-1 cultures for 96h at 30ºC. The conditions

chosen for cellulase production were basal media 1 and 2, supplemented with 30 g·L-1 of

cellulose and a 0.1 OD/mL concentration of spores.

The purpose of the use of vacuum filtration was to remove the mycelium, whereas

ultrafiltration was used to concentrate the cellulase solution obtained from the previous filtration.

The utilization of an ultrafiltration membrane with appropriate molecular weight cut-off can keep

the enzymes and cellulose in the concentrate, while low molecular weight molecules, such as

sugars, can pass through the membrane and leave as permeate.

The molecular weight of an endo-1,4-β-glucanase from Aspergillus terreus M11 was

found in literature to be 25 kDa (Gao, et al., 2007). However, due to the lack of membranes in

the laboratory with a lower molecular weight cut-off (MWCO), it was decided, on a first

approach, to use a polyethersulfone membrane disk with a 50-kDa MWCO in a stirred-cell

system.

Due to experimental errors, the results are shown only for cellulases produced by A.

terreus A-1 growing on basal Medium 2. A volume of 23 mL that remained in the stirred cell

(concentrate) was collected and kept at 4ºC for further spectrophotometry measurement at 595

nm. The same was done for the permeate (250 mL) to check eventual cellulase transmission

through the membrane. The enzymatic activity of cellulases was measured according to the

procedure described for the endo-1,4-β-D-glucanase.

29

Figure 12 - Enzymatic activity (total Units) of cell ulases before ultrafiltration (unconcentrated cellulases) and recovered in the concentrate and in the permeate after ultrafiltration, using a PES

50-kDa MWCO membrane in a stirred-cell apparatus.

As can be observed in Figure 12, the activity of cellulases decreased after ultrafiltration.

One possible drawback of cellulase separations using UF membranes is thus cellulase

deactivation. In fact, previous studies reported losses in cellulase activity due to shear

inactivation (Ganesh, et al., 2000).

On the other hand, the enzyme activity found in the permeate was 69% higher than the

activity of cellulases present in the concentrate. This may have occurred due to the fact that the

membrane cut-off was not the most appropriate, i.e. cellulases from Aspergillus terreus A-1

strain with molecular weight lower than 50 kDa may have permeated the ultrafiltration

membrane. A study made on cellulases produced by T.reesei showed that their recovery in the

permeate increased from 2 to 6% for polyethersulfone membranes with 30 and 50-kDa

molecular weight cut-offs, respectively (Mores, et al., 2001).

3.1.4 Effect of Temperature on Enzyme Activity and Stability

Stability is an interesting property of enzymes because (1) it is of great industrial

importance, (2) it is relatively easy to screen for, and (3) the molecular basis of stability relates

closely to contemporary issues in protein science, such as the protein folding problem. Thus,

engineering enzyme stability is of both commercial and scientific interest.

Naturally available enzymes are usually not optimally suited for industrial applications.

This incompatibility often relates to the stability of the enzymes under process conditions. The

stability of an enzyme is affected by many factors, temperature being one of them.

380.0

70.3

119.2

0

50

100

150

200

250

300

350

400

Uto

tal

Unconcentrated cellulases Concentrate (4ºC) Permeate (4º C)

30

Hence, to test the thermal stability of cellulases present in the concentrate solution

recovered from ultrafiltration, samples (in duplicate) were maintained at -20ºC and 25ºC for 48h.

The cellulase activity was determined under standard enzyme (endo-1,4-β-D-glucanase) assay

conditions. The control (samples at 25ºC) was taken as having 100% activity.

Figure 13 – Effect of temperature on the activity of A. terreus A-1 cellulases, recovered in the concentrate, after 48h at 25ºC and -20ºC. The contr ol (samples at 25ºC) was taken as having 100%

activity.

As shown in Figure 13 samples at -20 ºC were deactivated after 48h. The decrease of

cellulase activity after 48h at -20ºC suggests that proteases had some effect on enzyme activity.

For this reason, the effect of protease inhibitors on cellulase activity was tested. Also, it was

decided to perform cellulase activity assays right after the harvest of the cells and separation of

cellulases.

3.1.5 Effect of EDTA and a Protease Inhibitor Cockt ail on

Enzyme Activity and Stability

Protease inhibitors are commonly used with the purpose to protect proteins from

digestion by proteases. Metalloproteases are common and can be readily deactivated by the

addition of EDTA, which binds to the metal ions that the proteases need to function.

Many kinds of cellulases have serine-rich sequences which are very sensitive to

protease if they are not protected in any way (Wang, et al., 2006). Several alkaline proteases

from the different species of Aspergilli have been characterized in detail. For instance, in a

study by Charles, et al., 2008, an isolate of A. nidulans has shown to exhibit the ability to secret

an alkaline protease, whose activity was completely inhibited by the protease inhibitor cocktail.

100.0

88.5

0

20

40

60

80

100

120

25ºC -20ºC

Re

lati

ve

en

zym

e a

ctiv

ity

(%

)

Effect of temperature on the activity of A.

terreus A-1 cellulases

31

Other protease inhibitors, such as EDTA, inhibited the enzyme activity at 79%. More recently, it

has been shown that strains of Aspergillus terreus also produced neutral and alkaline type of

proteases, the latter type of which was inhibited by serine protease inhibitors and also by EDTA

(Hussain, et al., 2010).

Since it was not known whether cellulases produced from A. terreus A-1 strain were

protease resistant, some experiments were undertaken in order to evaluate the effect of

protease inhibitors on cellulase activity and stability. The experiments were performed based on

the idea that the presence of protease inhibitors could help to stabilize cellulases, in a way that

they would inhibit possible proteases present in the supernatant, and therefore

maintain/increase the activity of cellulases.

In the first set of experiments, a protease inhibitor cocktail and EDTA (2mM) were

added to concentrate solutions recovered from the previously described ultrafiltration. Samples

were assayed in duplicate and the activity of cellulases was determined under standard enzyme

(endo-1,4-β-D-glucanase) conditions. The control (without any additive) was taken as having

100% activity.

Figure 14 – Effect of a protease inhibitor cocktail and EDTA on the activity of cellulases from Aspergillus terreus A-1 strain after 48h at 25ºC.

The addition of a protease inhibitor cocktail, as well as EDTA (2 mM) helped to stabilize

cellulases, as their relative activity is 35.4 and 41%, respectively, higher than the cellulases

present in the control mixture, which did not contain any additive Figure 14. Due to economic

reasons it was chosen to use EDTA (2 mM) over the protease inhibitor cocktail in subsequent

tests on cellulase stability.

100.0

135.4

141.0

0

20

40

60

80

100

120

140

160

Control Protease inhibitor EDTA

Re

lati

ve

act

ivit

y (

%)

32

In the next experiments A. terreus A-1 cultures grown for 96h at 30ºC were vacuum

filtered and ultrafiltrated, this time using VIVASPIN 2 Concentrator (Viva Science) ultrafiltration

devices containing a polyethersulfone membrane with a 10 kDa molecular weight cut-off. The

conditions chosen for cellulase production were basal media 1 and 2, supplemented with 30

g·L-1 of cellulose and a 0.1 OD/mL concentration of spores.

Figure 15 - Effect of EDTA (2mM) on the activity of c ellulases produced by Aspergillus terreus A-1 after 96h of cultivation at 30ºC in basal media 1 a nd 2. After 96h of growth cultures were vacuum

filtered and ultrafiltrated, and kept at 25ºC in a room for the next 11 days.

It is apparent that the addition of EDTA (2mM) to the mixture (after 96h of cultivation at

30ºC) increased the enzyme activity of A. terreus A-1 cultures cultivated in both media (Figure

15). Although the trend of the activity of cellulases was more regular for Medium 2 after the

addition of EDTA at 96h, the enzyme activity was more pronounced for cellulases produced by

A.terreus A-1 when growing on Medium 1.

A similar experiment was undertaken, this time growing A. terreus A-1 cultures only on

basal medium 1, supplemented with 30 g·L-1 of cellulose and with a 0,1 OD/mL concentration of

spores. The cultures grown for 96h at 30ºC were vacuum filtered and ultrafiltrated. The addition

of EDTA (2 mM) to concentrate solutions at 96h was also tested and the activity of cellulases

was compared to a control sample, which did not contain any additive. Samples were assayed

in duplicate and the activity of cellulases was determined under standard enzyme (endo-1,4-β-

D-glucanase) conditions.

0

1

2

3

4

5

0 24 48 72 96 120 144 168 192 216 240 264 288 312 336 360 384

U/m

L

Time (hours)

Effect of the addition of EDTA (at 96h) on

cellulase activity

Medium 1 Medium 2

33

Figure 16 - Effect of EDTA (2mM) on the activity of c ellulases produced after 96h of cultivation at 30ºC of Aspergillus terreus A-1 cultures in Medium 1. After 96h of growth cult ures were vacuum

filtered and ultrafiltrated, and left at 25ºC for t he next 7 days.

As shown on Figure 16, the effect of EDTA (2mM) on cellulase activity was two-edged:

the activity of cellulases from 144h until the end of the monitored time period (264h) remained

almost constant and enzymes were apparently stable, yet the activities achieved were lower

than the ones observed for the control mixture (without the addition of EDTA).

The results described in literature regarding the effect of EDTA on cellulases are

variable. For example, in the case of Aspergillus terreus GN1, EDTA did not have any

significant effect on CMCase activity, suggesting that the enzymes produced were not

dependent on a metallic co-factor at its active site (Garg, et al., 1982). The same result was

obtained by (Elshafei, et al., 2009) in a study using Aspergillus terreus DSM 826. Furthermore,

the addition of EDTA (2 mM) did not have any noteworthy effects on the activity of a protease-

resistant cellulase produced by Aspergillus niger (Akiba, et al., 1995).

On the other hand, partial inhibition was observed by Ajayi, et al., 2007 when adding

various concentrations of EDTA (0 mM, 2 mM, 4 mM,6 mM and 8 mM) to a partially purified

cellulase from tomato fruits deteriorated by Aspergillus flavus. The inhibition was more

pronounced at increased EDTA concentrations.

3.2 Evaluation of the Cellulase System

Cellulases are a mixture of enzymes that act synergistically, (i.e., individual purified

enzymes have little or no action on insoluble cellulose, although an appropriate mixture can

0

1

2

3

4

0 24 48 72 96 120 144 168 192 216 240 264 288

U/m

L

Time (hours)

Effect of the addition of EDTA (at 96h) on

cellulase activity

Control EDTA t=96h

34

totally convert it to glucose) and thus cellulase systems from different microbial sources may

have a different mode of action.

This experiment was carried out to evaluate other eventual cellulase activities, besides

endo-1,4-β-D-glucanase, present in samples collected after 96h of incubation of Aspergillus

terreus A-1, growing on basal media 1 and 2 and supplemented with 30 g·L-1 of cellulose.

Therefore, an alternative enzyme assay, specific for β-glucosidase and α-arabinosidase, was

performed. The substrates used were NPG (4-nitrophenyl-β-glucopyranoside) and NPA (4-

nitrophenyl-α-arabinofuranoside) from Sigma-Aldrich. The absorbance was measured at 400

nm according to standard β-glucosidase assay conditions.

In order to compare the various activities, the same enzyme mixtures were tested

according to the assay specific for the endo-1,4-β-D-glucanase. The substrate here used was

Azo-CMcellulose from Megazyme.

Figure 17 – Activity of cellulases produced by Aspergillus terreus A-1, using different substrates (Azo-CMcellulose, NPG and NPA), different media (1 an d 2), and with or without the addition of EDTA (at 0h and at 96h of incubation at 30ºC). Sample s were conserved at -20ºC and taken from

assays 8, 9 and 10.

As it can be noted on Figure 17, cellulases produced by A.terreus A-1 showed β-

glucosidase and α-arabinosidase activities besides endo-1,4-β-D-glucanase, confirming the

synergism of the cellulase system.

The activity of cellulases growing on Medium 1 was considerably higher compared to

those growing on Medium 2, suggesting that the former medium is, in fact, optimal for the

production of cellulases from A.terreus A-1. In general, the activity of the endo-1,4-β-D-

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

Medium 1

(8)

Medium 2

(8)

Medium 1,

Control (9)

Medium 1,

EDTA t=0h

(9)

Medium 1,

EDTA t=96h

(9)

Medium 1,

Control (10)

Medium 1,

EDTA t=96h

(10)

Ab

sorb

an

ce

Azo-CMcellulose

β-NPG

α-NPA

35

glucanase was higher compared to the β-glucosidase activity. Furthermore, the activity of endo-

1,4-β-D-glucanase was always higher than the activity of α-arabinosidase, which is responsible

for releasing arabinose from hemicellulose. Nevertheless, taking, for instance, the control

sample (without the addition of EDTA) of assay number 9, the activities achieved, using each

kind of substrate, were much alike.

Only a small amount of cellulases was produced when adding EDTA at the beginning of

incubation (t=0h) of A. terreus A-1 cultures growing on basal Medium 1. Moreover, the activities

observed were lower when compared to those achieved by cellulases growing without any

EDTA (Control, assay number 9). It can be inferred that the addition of EDTA at the beginning

of cultivation (t=0h) of A. terreus A-1 may inhibit cellulase production.

The exact protein concentration of cellulases produced by Aspergillus terreus A-1 strain

was determined by the Bradford assay. The obtained values, together with the endo-1,4-β-D-

glucanase activity assay, gave the specific activities shown on Table 2.

Table 2 – Specific activities (U/mg of protein) obta ined for cellulases produced by Aspergillus terreus A-1 growing on media 1 and 2. The enzyme assay was specific for the endo-1,4- β-D-

glucanase activity and was performed using Azo-CMce llulose as substrate.

U/mg

protein

Medium 1 (8) 33,7

Medium 2 (8) 9,13

Medium 1, Control (9) 5,12

Medium 1, EDTA t=96h (9) 11,3

Medium 1, Control (10) 11,4

Medium 1, EDTA t=96h (10) 28,2

3.3 Identification of Sugars Released From the Enzy matic

Hydrolysis of Cellulose

To evaluate the capability of cellulases produced by Aspergillus terreus to release

fermentable sugars (such as glucose, xylose, cellobiose and arabinose), the reaction of the

enzyme mixture on cellulose was assayed.

The products released from the enzymatic reaction with commercial cellulose were

qualitatively analyzed, at different times, by high performance liquid chromatography (HPLC)

36

using refractive index detection. On the first approach adopted it was used a Prepacked Column

RT Polyspher-OA HY (300 x 6.5 mm) with a H2SO4 0.0025 M mobile phase. Once optimum flow

conditions were established (0.3 ml/min), repeated injections of standards were performed to

obtain reproducible elution times. These chromatograms are shown on Appendix I.

Temperature was also adjusted to 65ºC according to the MERCK Chrombook fermentation

standards.

The enzymatic reaction was studied for 24h. The chromatograms for 0h, 1h and 18h are

presented on Appendix I. The results for 24h of reaction between commercial cellulose and

enzymes recovered in the concentrate, after ultrafiltration of cellulases produced by A.terreus A-

1 growing on media 1 and 2, respectively, are shown on Figure 18 and Figure 19.

Figure 18 - HPLC chromatogram for 24h of reaction be tween commercial cellulose and the enzyme mixture present in the concentrate solution, recove red from ultrafiltration of cellulases produced

by A. terreus A-1 growing on Medium 1. The identified peaks corr espond to: Cellobiose (12. 8 min); Glucose (15.4 min).

37

Figure 19 - HPLC chromatogram for 24h of reaction be tween commercial cellulose and the enzyme mixture present in the concentrate solution, recove red from ultrafiltration of cellulases produced by A. terreus A-1 growing on Medium 2. The identified peaks corr espond to: Cellobiose (12.9 min)

and Xylose (16.4 min).

The chromatograms above show that different sugars are released when using

cellulases from A. terreus A-1 growing on media 1 or 2. For instance, xylose is only released

from the reaction between commercial cellulose and the cellulase mixture produced by A.

terreus A-1 growing on Medium 2. However, with this column and these operating conditions it

was not possible to separate both peaks of glucose and xylose, which had similar retention

times.

In order to improve the results and attempt at a better separation of sugars, a second

HPLC analytical assay was performed on a Lichrospher 100 NH2 column (4 x 250 mm)

using a 85:15 (v/v) acetonitrile/water mobile phase. The flow rate was adjusted to 1 mL/min and

the temperature was 23ºC. Again, injections of standards (10 g·L-1) were performed to obtain

reproducible elution times (Figure 20).

38

Figure 20 - HPLC chromatogram for a 10 g·L -1 mixed sugar solution, showing elution times of

Glucose (6.3 min) , Xylose (10.0 min) and Cellobios e (22.6 min) with a mobile flow rate of 1 mL/min at 23ºC. Column: Lichrospher 100 NH 2 ; Mobile phase: 85%Acetonitrile/15%Water.

Table 3 – HPLC retention times obtained for a 10 g ·L-1 mixed sugar solution containing glucose, xylose and cellobiose. The column used was Lichrosp her 100 NH2 with a 85%Acetonitrile/15%Water mobile phase at a 1 mL/min flow rate and 23ºC.

Retention time (min)

Glucose 6.3

Xylose 10

Cellobiose 22.6

In order to identify the sugars produced from the enzymatic reaction between

commercial cellulose (10 g·L-1) and the cellulase mixture present in the concentrate (recovered

from ultrafiltration of cellulases produced by A. terreus A-1 growing on basal Medium 1),

samples were taken along 48h of reaction. The addition of EDTA (2 mM) to concentrate

solutions after 96h of cultivation of A. terreus A-1 was also tested, and the release of sugars in

this case was compared to a control sample, which did not contain any additive.

When analyzing the “Control” samples, no peaks were registered at 0h nor at 2h of

reaction (see Appendix II). The best results were obtained after 24h and 48h from start, when a

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mV

olts

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

mV

olts

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

3,48

5 5

8,69

5 3

9492

368

6,27

3 1

4,22

5 9

5709

96

10,0

38

13,9

02

9354

019

22,5

77

13,1

79

8867

227

UV L-7400mixglucxylcellobiosio10grL_13.07.11

Retention TimeArea PercentArea

39

peak of xylose could be identified at 11 min of retention time (Figure 21 Figure 22). The huge

peak at around 3 minutes represents the water from the eluent and will be present in all HPLC

chromatograms, despite being irrelevant to the present study. As the reaction time increases, so

does the area of the peak of xylose, indicating that increased numbers of hemicellulose chains

are breaking down into smaller sugar units.

Figure 21 – HPLC chromatogram for 24h of reaction be tween commercial cellulose (10 g ·L-1) and the enzyme mixture present in the concentrate solut ion, recovered from ultrafiltration of cellulases

produced by A. terreus A-1 growing on Medium 1 (“Control”). Column: Lichr ospher 100 NH 2 ; Mobile phase: 85%Acetonitrile/15%Water ; Flow rate 1 mL/min at 23ºC. The identified peaks

correspond to: Millipore Water (3.2 min) and Xylose (11.2 min).

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mV

olts

-800

-600

-400

-200

0

200

400

600

800

mV

olts

-800

-600

-400

-200

0

200

400

600

800

3,16

1 9

5,25

2 4

2055

274 11

,199

4,

748

209

6202

UV L-7400tvcontrollo_24h


40

Figure 22 - HPLC chromatogram for 48h of reaction be tween commercial cellulose (10 g·L -1) and the enzyme mixture present in the concentrate solut ion, recovered from ultrafiltration of cellulases

produced by A. terreus A-1 growing on Medium 1 (“Control”). Column: Lichr ospher 100 NH 2 ; Mobile phase: 85%Acetonitrile/15%Water ; Flow rate 1 mL/min at 23ºC. The identified peaks

correspond to: Millipore Water (3.2 min) and Xylose (11.4 min).

Figure 23 displays the chromatogram at 24h of reaction between commercial cellulose

and the enzyme mixture present in the concentrate solution, which contained EDTA (2mM)

added to the cellulase solution produced after 96h of cultivation of A.terreus A-1 growing on

basal Medium 1. Note that, once again, no peaks were registered at 0h and at 2h of reaction

(see Appendix II).

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mV

olts

-800

-700

-600

-500

-400

-300

-200

-100

0

100

200

300

400

500

600

700

800

mV

olts

-800

-700

-600

-500

-400

-300

-200

-100

0

100

200

300

400

500

600

700

800

3,15

8 9

0,82

7 3

8586

879

6,83

8 0

,742

31

5170

11,3

89

8,02

7 3

4100

66

26,1

45

0,40

4 1

7171

8

UV L-7400tvcontrollo_48h


41

Figure 23 - HPLC chromatogram for 24h of reaction be tween commercial cellulose (10 g·L -1) and the enzyme mixture present in the concentrate solut ion recovered from ultrafiltration, which

contained EDTA (2mM), added at 96h of cultivation of A. terreus A-1 growing on Medium 1. Column :Lichrospher 100 NH 2 ; Mobile phase: 85%Acetonitrile/15%Water ; Flow r ate 1 mL/min at 23ºC The

identified peaks correspond to: Millipore Water (3. 3 min) and Xylose (9.8 min).

The chromatogram on Figure 23 showed little variation between sugars produced from

the reaction of cellulases from the concentrate which contained EDTA (2mM) and the ones

produced from the enzyme mixture that did not contain any additive (Figure 21). One was only

able to identify xylose at approximately 10 minutes of retention time, despite the peak was less

noticeable than the one identified for the reaction between cellulose and the enzyme mixture

from the “Control” sample.

Because the peak of xylose had a residual area, and glucose and cellobiose peaks

were not even present, the conditions of the assay had to be improved. It was therefore chosen

to concentrate the enzyme mixture about twenty times more in the ultrafiltration step and also to

change the concentration of commercial cellulose from 10 g·L-1 to 30 g·L-1. Note that at this

point and due to inexperience, the experiments were being conducted on a trial and error basis.

The reaction was studied after 24h, 96h, 186h from start. The results for 96h and 186h

of reaction between commercial cellulose (30 g·L-1) and the cellulase mixture present in the

concentrate, which did not contain any additive (“Control”), are displayed on Figure 24 Figure

25.

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mV

olts

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

mV

olts

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

3,33

8 9

6,74

0 3

1802

229

9,79

6 2

,903

95

4322

21,4

32

0,35

7 1

1733

3

UV L-7400edta22hseconda_13.07.11


42

Figure 24 - HPLC chromatogram for 96h of reaction b etween commercial cellulose (30 g·L -1) and the enzyme mixture (20 times more concentrated) pre sent in the concentrate solution, recovered

from ultrafiltration of cellulases produced by A. terreus A-1 growing on Medium 1 (“Control”). Column :Lichrospher 100 NH 2 ; Mobile phase: 85%Acetonitrile/15%Water ; Flow r ate 1 mL/min at

23ºC. The identified peaks correspond to: Millipore Water (3.2 min) and Cellobiose (23.5 min).

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mV

olts

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

mV

olts

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

3,24

5 9

5,88

9 3

8098

936

23,5

43

4,11

1 1

6334

87

UV L-7400edtapiuconcentrato48h(15.07)_18.07.11


43

Figure 25 - HPLC chromatogram for 186h of reaction b etween commercial cellulose (30 g·L -1) and the enzyme mixture (20 times more concentrated) pre sent in the concentrate solution, recovered

from ultrafiltration of cellulases produced by A. terreus A-1 growing on Medium 1 (“Control”). Column :Lichrospher 100 NH 2 ; Mobile phase: 85%Acetonitrile/15%Water ; Flow r ate 1 mL/min at

23ºC. The identified peaks correspond to: Millipore Water (3.4 min) and Cellobiose (23.8 min).

As shown in Figure 24 and Figure 25, cellobiose was detected (RT=23 min) as the main

product of the reaction between commercial cellulose (30 g·L-1) and the enzyme mixture (20

times more concentrated) present in the “Control”. The peaks were, again, not very noticeable,

however it seems that the intensity of the cellobiose peak is slightly higher after 186h of reaction

than the one at 96h.

The following chromatograms (Figure 26 Figure 27) track the sugars released from the

enzymatic reaction of commercial cellulose (30 g·L-1) with the enzyme mixture (20 times more

concentrated) containing EDTA (2mM) over 96h and 186h of reaction time.

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mV

olts

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

mV

olts

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

3,36

0 9

2,90

1 3

6838

053

23,8

40

7,09

9 2

8151

42

UV L-7400controllopiuconcentrato96h+90h_19.07.11


44

Figure 26 - HPLC chromatogram for 96h of reaction be tween commercial cellulose (30 g·L -1) and

the enzyme mixture (20 times more concentrated) pre sent in the concentrate solution after ultrafiltration, which contained EDTA (2mM), added a t 96h of cultivation of A. terreus A-1 growing

on Medium 1. Column :Lichrospher 100 NH 2 ; Mobile phase: 85%Acetonitrile/15%Water ; Flow r ate 1 mL/min at 23ºC. The identified peaks correspond t o: Millipore Water (3.4 min) and Cellobiose

(23.1 min).

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mV

olts

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

mV

olts

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

3,40

3 9

7,74

1 3

5663

701

23,0

70

2,25

9 8

2442

2

UV L-7400edtapiuconcentrato92h_18.07.11


45

Figure 27 - HPLC chromatogram for 186h of reaction b etween commercial cellulose (30 g·L -1) and the enzyme mixture (20 times more concentrated) pre sent in the concentrate solution after

ultrafiltration, which contained EDTA (2mM), added a t 96h of cultivation of A. terreus A-1 growing on Medium 1. Column: Lichrospher 100 NH 2 ; Mobile phase: 85%Acetonitrile/15%Water ; Flow ra te

1 mL/min at 23ºC. The identified peaks correspond t o: Millipore Water (2.9 min) and Cellobiose (23.1 min).

The results of HPLC analysis shown on Figure 26 and Figure 27 show that cellobiose

was slightly detected at 23 min of retention time, although the results were, again, very

unsatisfactory, since the same samples recovered from ultrafiltration proved to have significant

endo-cellulasic activity. Still, it is worthy of note that while the activities estimated for cellulases

were specific for an endocellulase (the substrate used in the enzyme assay was CM-cellulose),

in this test one did not check only for the individual cellulose polysaccharide chains produced

from the action of endocellulases, but for the end products, such as cellobiose and glucose,

released from all cellulases acting synergistically. Activity tests regarding exocellulasic activity

should have been conducted in order to further analyze this aspect. It would be expected

however, to obtain glucose as an end product of the enzymatic reaction, since the NPG assay

showed β-glucosidase activity (Figure 17).

Comparing the sugars released from the reaction between cellulose and the enzyme

mixture containing EDTA (2mM) in the concentrate and the ones that did not contain an additive

(“Control”), one can observe that in both cases the same sugars are released, although lower

intensities of the peaks were observed when EDTA was added to the concentrate.

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mV

olts

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

mV

olts

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

2,93

8 9

6,92

4 3

4090

919

23,0

79

3,07

6 1

0818

98

UV L-7400edtapiuconcentrato96h+91h_19.07.11


46

All things considered, the results from HPLC analysis confirm the existence of

cellulases in the enzyme mixture produced by Aspergillus terreus A-1 strain, that act

synergistically to release sugars, such as xylose and cellobiose.

It is, however, possible that these results are not very realistic, since the sensitivity of

the refractive index detector was seriously affected and one struggled with adjusting the base

line, as well as with the recurrent presence of air bubbles in the system.

47

4. Conclusions and Future Perspectives

In this work cellulases were successfully produced by two strains, A-1 and N-Y, of

Aspergillus terreus isolated from soil. A. terreus A-1 strain seemed to follow a more regular

pattern and was, therefore, used in most of the experiments.

Different concentrations of cellulose, ranging from 20 g·L-1 to 60 g·L-1, were assayed as

the sole carbon source of the growth medium of A.terreus A-1 and N-Y strains. In this

experiment the activity of cellulases produced by A.terreus A-1 strain had a maximum (13.2

U/mL) at the concentration of 30 g⋅L-1 of cellulose. On the other hand, the maximum production

of cellulases by N-Y strain (10.2 U/mL) was obtained on 20 g⋅L-1 of cellulose.

For both strains the maximum cellulasic activity occurred between 72h and 96h of

incubation at 30ºC. Furthermore, when testing different concentration of spores to prepare the

inoculum, the cellulasic activity obtained using a 0.5 OD/mL concentration of spores was similar

or lower than the activity obtained when using a 0.1 OD/mL concentration.

The rate of production of cellulases, since they are extracellular enzymes, is greatly

influenced by the nutrient medium composition. Accordingly, two media containing different

sources of nitrogen, different MgSO4 concentration and different optimal pH were assayed in

order to achieve the optimal production of cellulases, in an attempt to maximize the cellulasic

activity. The use of sodium nitrate (2 g⋅L-1) instead of peptone (6 g⋅L-1), and a concentration 0.5

g·L-1 of MgSO4·7H2O instead of 1.72 g·L-1 proved to enhance the cellulolytic activities of

A.terreus A-1 and N-Y strains.

A different approach was performed introducing a vacuum filtration followed by an

ultrafiltration, after cultivation of A. terreus A-1 cultures for 96h at 30ºC. The vacuum filtration

successfully removed the mycelium, whereas the cellulase solution obtained from the previous

step was concentrated by ultrafiltration. The results obtained after performing the enzyme

activity assay were, however, unsatisfactory, as the activity of cellulases decreased after

ultrafiltration. This may have occurred due to the use of a membrane with an inappropriate

molecular weight cut-off (50 kDa), and thus cellulases with a molecular weight less than 50 kDa

may have passed to the permeate.

The stability of an enzyme is affected by various factors, such as temperature, which

can make them unsuitable for industrial applications. Hence, experiments were conducted in

order to test the thermal stability of cellulases present in the concentrate solution recovered

from ultrafiltration. The results obtained suggest that proteases had some effect on enzyme

activity, since it decreased after 48h of incubation at -20ºC. Therefore, it was decided to perform

48

cellulase activity assays right after the harvest of the cells and separation of cellulases, in order

to avoid undesirable protease action.

In addition, some experiments were carried out in order to assess the effect of protease

inhibitors on cellulase activity and stability. The results indicate that the protease inhibitor

cocktail and EDTA (2 mM) helped to stabilize cellulases, as their relative activity is 35.4 and

41%, respectively, higher than the activity of cellulases present in the control mixture, which did

not contain any additive. Other trials were performed with the same purpose, showing that the

addition of EDTA (2mM) to the mixture (after 96h of cultivation at 30ºC) increased the enzyme

activity of A. terreus A-1 cultures growing on media 1 and 2. Despite, the enzyme activity was

more pronounced for cellulases produced by A.terreus A-1 when growing on Medium 1. As for

the enzyme mixtures that contained EDTA (2mM) and the ones without an additive, the results

on cellulase activity were two-edged: the activity of cellulases, after the addition of EDTA,

remained almost constant and enzymes were apparently stable, yet the activities achieved were

lower than the ones obtained for the control mixture (without the addition of EDTA).

Moreover, it was investigated whether other cellulase activities existed besides endo-

1,4-β-D-glucanase in the enzyme mixture collected after 96h of incubation of Aspergillus terreus

A-1 cultures. Hence, an enzyme assay specific for β-glucosidase and α-arabinosidase was

conducted. In these experiments, cellulases produced by A.terreus A-1 showed β-glucosidase

and α-arabinosidase activities in addition to endo-1,4-β-D-glucanase, confirming the synergism

of the cellulase system. The activity of cellulases produced from fungi growing on Medium 1

was considerably higher compared to those produced by A.terreus grown on Medium 2,

suggesting that the former medium is optimal for the production of cellulases from A.terreus A-1.

It was also noted that the addition of EDTA (2 mM) at the beginning of cultivation (t=0h) of A.

terreus A-1 inhibited cellulase production, since the activities observed were much lower than

the ones obtained by cellulases growing without any additive.

In order to evaluate the capability of cellulases produced by Aspergillus terreus to

release fermentable sugars when reacting with commercial cellulose, the products of the

reaction were qualitatively analyzed, at different times, by HPLC using refractive index

detection. Overall, the results from HPLC analysis confirm the existence of cellulases in the

enzyme mixture produced by Aspergillus terreus A-1, that act synergistically to release sugars,

such as xylose and cellobiose. Samples containing EDTA (2mM) in the concentrate released

the same sugar as those that did not contain any additive (“Control”), when reacting with

commercial cellulose, although lower intensities of the peaks were observed in the former case.

It should be mentioned, however, that it is possible that these results are not very realistic, since

the sensitivity of the refractive index detector was seriously affected.

49

Future work

Although the results were not as expected, due to the available time and lack of

knowledge on the composition of the cellulases produced from A. terreus A-1 and N-Y strains, it

would be possible, in the future, to initiate the second phase of experiments: the fermentation of

the sugars released from the reaction of the enzyme mixture produced by Aspergillus terreus on

cellulose from plant biomass, using strains such as Saccharomyces cerevisiae, which are

known for their ability to produce ethanol in high concentrations. Because this particular yeast is

unable to ferment pentoses, such as xylose and arabinose, another future prospect would be

the use of non-conventional yeasts, such as Brettanomyces naardenensis, to ferment

substrates obtained in the previous step. The acquisition of information on carbon metabolism

by yeasts capable of fermenting xylose will allow the definition of the most appropriate mode of

the fermentation process (batch, fed-batch, or continuous culture) and the parameters for

chemical/physical optima (pH, temperature, oxygenation) for the development of the production

process of bioethanol on an industrial scale.

The bioprocess aspects regarding cellulosic biomass will become the crux of future

researches involving cellulases and cellulolytic microorganisms. The problems that require

attention are not limited to cellulase production alone, but need a concerted effort in

understanding the basic physiology of cellulolytic microbes. The use of this knowledge coupled

with engineering principles will, hopefully, enable better processing and the utilization of this

most abundant natural resource.

50

5. References

Ajayi and Adejuwon A.O., Awojobi O.K., Olutiola P.O . 2007. Effect of Cations and Chemicals

on the Activity of Partially Purified Cellulase from Tomato (Lycopersicon esculentum Mill) Fruits

Deteriorated by Aspergillus flavus, Pakistan Journal of Nutrition, 2007. pp. 198-200. Vol. 6.

Akiba and Kimura Y., Yamamoto K., Kumagai. 1995. Purification and Characterization of a

Protease-Resistant Cellulase from Aspergillus niger, Journal of Fermentation and

Bioengineering, 1995. pp. 125-130. Vol. 79.

Ali, S. and Sayed A., Sarker R. I., Alam R. 1991. Factors affecting cellulase production by

Aspergillus terreus using water hyacinth, World J. of Microbial and Biotechnol., 1991. pp. 62-66.

Vol. 7.

Babel, et al. 2007. Advances in Biochemical Engineering/Biotechnology: Biofuels, Springer,

2007.

Blondin, B. 1983. Purification and properties of the β-glucosidase of a yeast capable of

fermenting cellobiose to ethanol, Eur. J. Appl. Microb. Biotech., 1983.

Bradford, M. 1976. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities

of Protein Utilizing the Principle of Protein-Dye Binding, ANALYTICAL BIOCHEMISTRY, 1976.

pp. 248-254.

Chang, X. and Minnan L., Xiaobing W., Huijuan X., Z hongan C., Fengzhang Z., Liangshu

X. 2006. Screening and Characterization of the High-Cellulase-Producing Strain Aspergillus

glaucus XC9, Higher Education Press and Springer-Verlag, 2006.

Charles, P. and Devanathan V., Anbu P., Ponnuswamy M. N., Kalaichelvan P. T., Hur B.

2008. Purification, characterization and crystallization of an extracellular alkaline protease from

Aspergillus nidulans HA-10, Journal of Basic Microbiology, 2008. pp. 347–352. Vol. 48 .

Cleantech, Magazine. 2010. Low cost enzymes could catalyse a revolution in ethanol

production. [Online] April 2010. http://www.cleantechinvestor.com/portal/bioenergy/5164-

enzymes.html.

Damisa, D. and Ameh, J.B., Egbe, N.E.L. 2011. Cellulase Production by native Aspergillus

niger obtained from soil environments. 2011.

DeVries, R.P. and J., Visser. 2001. Aspergillus Enzymes Involved in Degradation of Plant Cell

Wall Polysaccharides, American Society for Microbiology, 2001.

DISMA Biochemistry Lab., University of Milan. 2010. Molecular details of the interaction

between cellylolytic enzymes and their substrates. 2010.

51

Elshafei, A. M. and Hassan, M. M., Haroun, B. M., A bdel-Fatah, O. M., Atta, H. M. and

Othman, A. M. 2009. Purification and properties of an endoglucanase of Aspergillus terreus

DSM 826, Journal of Basic Microbiology, 2009. pp. 426–432. Vol. 49.

Emtiazi, G. and Naghavi N., Bordbar A. 2001. Biodegradation of lignocellulosic waste by

Aspergillus terreus. 2001.

Ganesh, K. and Joshi J.B., Sawant S.B. 2000. Cellulase deactivation in a stirred reactor,

Biochemical Engineering Journal 4, 2000. pp. 137-141.

Gao, J. and Weng H., Yu Xi, Daheng Zhu, Shaoyin Han . 2007. Purification and

characterization of a novel endo-b-1,4-glucanase from the thermoacidophilic Aspergillus

terreus, Springer Science, 2007.

Gao, J. and Weng H., Zhu D., Yuan M., Guan F., Xi Y . 2008. Production and characterization

of cellulolytic enzymes from the thermoacidophilic fungal Aspergillus terreus M11 under solid-

state cultivation of corn stover, Bioresource Technology, 2008.

Garg, S. K. and Neelakantan, S. 1982. Studies on the properties of cellulase enzyme from

Aspergillus terreus GN1, Biotechnology and Bioengineering,, 1982. pp. 737–742. Vol. 24.

Gautam, S.P. and Bundela P.S., Pandey A.K., Awasthi M.K., Sarsaiya S. 2010. Effect of

different carbon sources on production of cellulases by Aspergillus niger, Journal of Applied

Sciences in Environmental Sanitation, 2010.

GmbH, SERVA Electrophoresis. 2011. SERVA Electrophoresis GmbH. Cellulases. [Online]

2011. http://www.serva.de/servaWeb/www_root/documents/16419_26.pdf.

Goldschmidt, Felix. 2008. From Cellulose to Ethanol: Engineering Microorganisms to Produce

Biofuel. Institute of Biogeochemistry and Pollutant Dynamics, Department Environmental

Sciences. Zurich, 2008.

Gusakov, A. V. and Salanovich T. N., Antonov A. I., Ustinov B. B., Okunev O. N.,

Burlingame R., Emalfarb M., Baez M., Sinitsyn A. P. 2007. Design of Highly Efficient

Cellulase Mixtures, Wiley InterScience, 2007.

Henrissat, B. et al. 1985. Biotechnology. 1985. pp. 722-6. Vol. 3.

Humbird, D. and Davis R., Tao L.,Kinchin C., Hsu D. , Aden A. 2011. Process Design and

Economics for Biochemical Conversion of Lignocellulosic Biomass to Ethanol (Dilute-Acid

Pretreatment and Enzymatic Hydrolysis of Corn Stover). [Online] National Renewable Energy

Laboratory, May 2011. [Cited: September 16, 2011.]

http://www.nrel.gov/biomass/pdfs/47764.pdf.

52

Hussain, A. and Mannan A., Zubair H., Mirza B. 2010 . Purification and Characterization of

Alkaline Proteases from Aspergillus terreus, J. Chem. Soc. Pak., 2010. Vol. 32.

Jahromi, M. F. and J.B. Liang, M. Rosfarizan, Y.M. Goh, P. Shokryazdan, Y.W. Ho. 2011.

Efficiency of rice straw lignocelluloses degradability by Aspergillus terreus ATCC 74135 in solid

state fermentation. 2011.

Kassebullah, M. N. B. 2006. Production of cellulase enzyme from Aspergillus terreus SUK-1

using sugar cane waste: The effect of substrate concentration and assay temperature. Faculty

of Chemical Engineering and Natural Resources. Malaysia, 2006.

MacCabe, A. P. and Orejas M., Tamayo E.N., Villanue va A., Ramón D. 2002. Improving

extracellular production of food-use enzymes from Aspergillus nidulans. Instituto de

Agroquimica y Tecnologia de Alimentos (CSIC). Valencia, Spain : Journal of Biotechnology,

2002.

Mandels, M. and Andreotti R., Roche C. 1976. Measurement of saccharifying cellulase,

Biotechnol Bioeng Symp, 1976. pp. 21–33.

Mores, W. D. and Knutsen J. S., Davis R. H. 2001. Cellulase Recovery via Membrane

Filtration, Department of Chemical Engineering, University of Colorado, 2001.

Nour, M.S. and Wahab M.N.A., Ariff A., Mohamad R. 2 011. Optimization of Cellulase

Production by Aspergillus Terreus under Submerged Fermentation Using Response Surface

Methodology, Australian Journal of Basic and Applied Sciences, 2011.

Ogel, Z.B. and K. Yarangumeli, H. Du, J. Ifrij. 200 1. Submerged cultivation of Scytalidium

thermophilum on complex lignocellulosic biomass. s.l. : Enzyme Micobiol. Technol. 28, 2001.

pp. 689-695.

Sukumaran, R. K. and Sighania R. R., Pandey A. 2005 . Microbial cellulases - Production,

applications and challenges. s.l. : Journal of Scientific & Industrial Research, 2005.

Teter, S. and Joel, R. Cherry. 2005. Improving Cellulose Hydrolysis with New Cellulase

Compositions. Novozymes, Inc. 2005.

Usama, F. A. and SaadEl-Dein, H. S. 2008. Production and Partial Purification of Cellulase

Complex by Aspergillus niger and A. nidulans Grown on Water Hyacinth Blend, Journal of

Applied Sciences Research, 2008.

VanHanh and Pham T.A., Kim K. 2010. Improvement of Fungal Cellulase Production by

Mutation and Optimization of Solid State Fermentation, Mycobiology, 2010.

53

Verma, N. and Vivek K., Bansal M.C. 2011. Importance of cellulases in bioethanol from

biomass: A review. [Online] 2011. [Cited: September 16, 2011.] www.compete-

bioafrica.net/events/events2/seminar_india/.../5-5-Verma.pdf.

Wang, H. T. and T., Hsu J. 2006. Usage of Enzyme Substrate to Protect the Activities of

Cellulase, Protease and α-Amylase in Simulations of Monogastric Animal and Avian Sequential

Total Tract Digestion, Asian-Aust. J. Anim. Sci., 2006. pp. 1164-1173. Vol. 19 .

Watanabe, H. and Tokuda, G. 2009. Cellulolytic Systems in Insects, Annual Review of

Entomology, 2009.

Wikipedia. 2011. Types of Cellulases. [Online] 2011.

http://en.wikipedia.org/wiki/File:Types_of_Cellulase2.png.

Wilson, D. B. 2009. Cellulases and biofuels. Elsevier, 2009.

54

Appendix I

HPLC Analysis Sugar Standards

Column: Prepacked Column RT Polyspher-OA HY (300 x 6.5 mm) ; M obile Phase: H 2SO4 0.0025 M

Flow rate: 0.3 mL/min ; P=23 bar ; T=65ºC.

Figure 28 - HPLC chromatogram for a standard solutio n of Cellobiose (10 g·L -1). Column:

Prepacked Column RT Polyspher-OA HY (300 x 6.5 mm) wit h a H2SO4 0.0025 M mobile phase. Flow rate: 0.3 mL/min ; P=23 bar ; T=65ºC.

55

Figure 29 - HPLC chromatogram for a standard solutio n of Glucose (10 g·L -1). Column: Prepacked Column RT Polyspher-OA HY (300 x 6.5 mm) with a H 2SO4 0.0025 M mobile phase. Flow rate: 0.3

mL/min ; P=23 bar ; T=65ºC.

Figure 30 - HPLC chromatogram for a standard solutio n of Xylose (10 g·L -1). Column: Prepacked Column RT Polyspher-OA HY (300 x 6.5 mm) with a H 2SO4 0.0025 M mobile phase. Flow rate: 0.3

mL/min ; P=23 bar ; T=65ºC.

56

Figure 31 - HPLC chromatogram for a standard solution of Arabino se (10 g·L -1). Column: Prepacked Column RT Polyspher-OA HY (300 x 6.5 mm) wit h a H2SO4 0.0025 M mobile phase. Flow

rate: 0.3 mL/min ; P=23 bar ; T=65ºC.

57

Concentrate solution, medium 1



Time of reaction: 0h

Figure 32 - HPLC chromatogram for 0h of reaction bet ween commercial cellulose and the enzyme mixture present in the concentrate solution, recove red from ultrafiltration of cellulases produced

by A. terreus A-1 growing on Medium 1.

58




59



by A. terreus A-1 growing on Medium 1. The peaks id entified for Cellobiose (12.8) and Glucose (15.4) were superimposed.

60

Concentrate solution, medium 2






61







62

Appendix II

HPLC Analysis Column: Lichrospher 100 NH 2 column (4 x 250 mm) ; Mobile Phase: 85:15 (v/v) a cetonitrile/water

Flow rate: 1 mL/min ; Temperature: 23ºC.

Control sample, Time of reaction: 0h

Figure 38 – HPLC chromatogram for 0h of reaction bet ween commercial cellulose (10 g·L -1) and the enzyme mixture present in the concentrate solution, recovered from ultrafiltration of cellulases

produced by A. terreus A-1 growing on Medium 1 (“Control”). The identifie d peak corresponds to: Millipore Water (3.6 min)

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mV

olts

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

mV

olts

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

3,65

0 1

00,0

00

3933

5240

UV L-7400controllo0h_12.07.11


63

Control sample, Time of reaction: 2h

Figure 39 - HPLC chromatogram for 2h of reaction bet ween commercial cellulose (10 g·L -1) and the enzyme mixture present in the concentrate solution, recovered from ultrafiltration of cellulases

produced by A. terreus A-1 growing on Medium 1 (“Control”). The identifie d peak corresponds to: Millipore Water (3.4 min)

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mV

olts

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

mV

olts

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

3,36

8 1

00,0

00

4249

9139

UV L-7400controllo2h_12.07.11


64

EDTA t=96h sample, Time of reaction: 0h

Figure 40 - HPLC chromatogram for 0h of reaction bet ween commercial cellulose (10 g·L -1) and the enzyme mixture present in the concentrate solution recovered from ultrafiltration, which contained

EDTA (2mM), added at 96h of cultivation of A. terreus A-1 growing on Medium 1. The identified peak corresponds to: Millipore Water (3.6 min)

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mV

olts

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

mV

olts

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

3,64

8 1

00,0

00

3931

3879

UV L-7400edta0h_12.07.11


65

EDTA t=96h sample, Time of reaction: 2h

Figure 41 - HPLC chromatogram for 2h of reaction bet ween commercial cellulose (10 g·L -1) and the enzyme mixture present in the concentrate solution recovered from ultrafiltration, which contained

EDTA (2mM), added at 96h of cultivation of A. terreus A-1 growing on Medium 1. The identified peak corresponds to: Millipore Water (3.6 min).

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mV

olts

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

mV

olts

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

3,67

0 9

9,45

7 3

9115

402 24,6

75

0,54

3 2

1365

7

UV L-7400edta2h_12.07.11


66

Control sample (20 times more concentrated), Time of reaction: 24h

Figure 42 - HPLC chromatogram for 24h of reaction be tween commercial cellulose (30 g·L -1) and the enzyme mixture (20 times more concentrated) pre sent in the concentrate solution, recovered

from ultrafiltration of cellulases produced by A. terreus A-1 growing on Medium 1 (“Control”). The identified peaks correspond to: Millipore Water (3. 3 min) and Cellobiose (23.5 min).

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mV

olts

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

mV

olts

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

3,24

5 9

5,88

9 3

8098

936

23,5

43

4,11

1 1

6334

87

UV L-7400edtapiuconcentrato48h(15.07)_18.07.11


dissertação para obtenção do grau de mestre ... - ulisboa · production and purification of new...

Documents