seminar 2013-10-14

Effect of different Trichoderma reesei

mutated strains and culture method on the

cellulase activity

Speaker: Huo, Yi-Hua

Instructor: Chen, Kuo-Lung

October 14, 2013

Abstract

• Cellulase is an important commercial enzyme, widely used in

food, animal feed, grain alcohol fermentation, starch

processing, malting and brewing industries. Filamentous fungi

Trichoderma reesei are considered to be one of the most

efficient hyper producers of cellulase that is used in industry.

• The methods used to improve fungal enzyme production,

activity or stability, one is mutagenisis and the other one is co-

culturing.

• The aim of this study was to investigate the influence of

different mutated T. reesei strains and culture method to the

cellulase activity.2

• Using T. reesei QM6a, QM9414, RUT C30 and QM9414 MG5

strains, the medium pH was maintained at 3-9. Results showed

that T. reesei QM6a has the best cellulose degradation at pH 3

(69.3%) and its mutants QM9414, RUT C30 and QM9414

MG5 has the best cellulose degradation at pH 6 (64.9、60.3

and 58.0%).

• In specific activity, mutants QM9414 has the best

endoglucanase (5.22mg/min/g protein) at pH 4, and mutants

QM9414MG5 has the best exoglucanase and beta-

glucosidase(0.87 and 0.80 mg/min/g protein).

3

• Using solid state fermentation, co-culture T. reesei LM-

UC4 and LM-UC4E1 with Aspergillus niger.

• Results showed that coculture LM-UC4E1 with A.niger

has the best dry biomass (0.27 g/g), filter paper activity

(15.5 IU/g) and endoglucanase activity (129 IU/g).

4

• Using solid state fermentation, co-culture T. reesei LM-UC4

and LM-UC4E1 with Aspergillus niger. Results showed that

coculture LM-UC4E1 with A.niger has the best dry biomass

(0.27 g/g), filter paper activity (15.5 IU/g) and endoglucanase

activity (129 IU/g).

• Using T. reesei RUT C30 with Aspergillus niger LMA co-

culture, compare with their monocultures respectively.

5

• The results of mixed culture experiments exhibited a highly

significant increase in the production of volumetric enzyme

activity (98.4 U/L), filter paper activity (7.1 U/mL),

carboxymethyl cellulase activity (4.7 U/mL), and dry biomass

(21.4 g/ L).

• In conclusion, even if mutants, it can’t able to produce high

levels of the enzymes at the same time while monoculture, the

mutant’s performance can be improved by co-culturing with A.

niger.

6

Outline

• Introduction

• Compare the Trichoderma reesei wild with its mutant strains

on their cellulase activity

• Compare the culture method of monoculture with mixed

culture on its cellulase activity

• Conclusion

7

Introduction

• Cellulase is an important commercial enzyme, widely used in

food, animal feed, textile, pulp and paper, grain alcohol

fermentation, starch processing, pharmaceutical, malting and

brewing industries.

• Filamentous fungi Trichoderma reesei are considered to be one

of the most efficient hyper producers of cellulase that is used

in industry.

8

(Aftab and Patrick, 2008a)

• The application of different strains and processes which are

selected on the basis of the biomass residues used make

comparisons difficult, if not impossible.

• Nevertheless, the most recent and important improvements in

production/activity of fungal enzymes using different

techniques such as mutagenesis, co-culturing and heterologous

gene expression of cellulases are discussed.

9

(Dashtban et al., 2009)

Table 1 Some methods which have been used to improve fungal lignocellulolytic

activity or stability

10

(Dashtban et al., 2009)

Purpose

• The aim of this study was to investigate the influence of

different mutated T. reesei strains and culture method on the

cellulase activity.

11

Material and methods

12

(Sunil et al., 2011)

Trichoderma reesei QM6a

T. reesei QM9414

T. reesei QM9123

T. reesei RUT C30

T. reesei QM9414MG5

Linear accelerator

High-voltage electrons

Fig. 1 The mechanism of cellulase.

13

(Somers et al., 1989)

Results and discussion

14

Table 2 Specific activities of cellulytic enzymes and cellulose degradation by T.

reesei wild and its mutant strains at different pHs

15

(Sunil et al., 2011)

Brief 1

16

• Results showed that T. reesei QM6a has the best cellulose

degradation at pH 3 (69.3%) and its mutants QM9414,

Rut C30 and QM9414MG5 has the best cellulose

degradation at pH 6 (64.9、60.3 and 58.0%).

• In specific activity, mutants QM9414 has the best

endoglucanase (5.22mg/min/g protein) at pH 4, and

mutants QM9414MG5 has the best exoglucanase and

beta-glucosidase(0.87 and 0.80 mg/min/g protein).

• Even if mutants, it can not able to produce high levels of

the enzymes at the same time.


17

(Gutierrez-Correa et al., 1999)

T. reesei LM-UC4

Aspergillus niger ATCC 10864

T. reesei LM-UC4E1

7 days 1.5-2 days 1.5-3aysC:N

= 10:1

Fig. 2 Growth kinetics of T.

reesei LM-UC4 (□,■ ) or T.

reesei LM-UC4E1 (△,▲)

with A. niger (○) in single

(open symbols) and mixed

(closed symbols) culture

SSF on bagasse

supplemented with

ammonium sulfate and

urea (A) or soymeal (B). 18


□ LM-UC4

■ LM-UC4+ A. niger

△ LM-UC4E1

▲ LM-UC4E1+ A. niger

○ A. niger

Fig. 3 Time course

profiles of enzyme

production by single and

mixed culture SSF on

bagasse supplemented

with ammonium sulfate

and urea (A) or soymeal

(B). 19


□ LM-UC4


△ LM-UC4E1


○ A. niger

Fig. 4 Time course

profiles of enzyme

production by single

and mixed culture SSF

on bagasse

supplemented with

ammonium sulfate and

urea (A) or soymeal (B). 20


□ LM-UC4


△ LM-UC4E1


○ A. niger

Fig. 5 Time course profiles of enzyme production by single and mixed culture SSF on bagasse supplemented with ammonium sulfate and urea (A) or soymeal (B). 21


□ LM-UC4


△ LM-UC4E1


○ A. niger

Table 3 Maximum growth and enzyme activities in mixed culture

solid substrate fermentation on sugar cane bagasse

22


Table 4 Comparison of maximum growth and enzyme activities in mixed

culture solid substrate fermentation on sugar cane bagasse

23


Brief 2

• Mixed culturing is advantageous in nutrient-limited conditions,

where symbiotic associations may overcome such limitations.

• In practical terms, it means that cheaper media may be used for

enzyme production by mixed cultures than by single cultures,

without sacrificing enzyme yields.

24


25


Aspergillus niger LMA

T. reesei RUT C30

T. reesei RUT C30 + Aspergillus niger LMA

Stirred tank

bioreactor

Table 5 The extent of cellulolytic enzyme production in T. reesei RUT-C30

strain grown in different culture media: cellulose–yeast extract (A); corn

steep–glucose (B); cellulose–yeast extract–peptone (C); and cellulose–yeast

nitrogen base–CMC (D) with lactose fed at regular intervals in a 7 L New

Brunswick stirred tank bioreactor maintained at 30 ◦C, an agitation of 250

rpm, and culture pH of 4.8

26

(Aftab and Patrick, 2008a)

Table 6 Comparison of growth and cellulase enzyme productivity from mono-culture (A.

niger) and mixed culture (T. reesei and A. niger) strains grown in a Cellulose–Yeast extract

culturemedium with lactose fed at regular intervals in a 3 L New Brunswick stirred tank

bioreactormaintained at 30 ◦C, an agitation of 250 rpm, and culture pH of 4.8

27

(Aftab and Patrick, 2008b)

Table 7 Comparison of growth and cellulase enzyme productivity from mono-

culture (T. reesei RUT-C30 and A. niger) and mixed culture (T. reesei and A.

niger) strains grown in a Cellulose–Yeast extract culturemedium with lactose

fed at regular intervals in a 3 L New Brunswick stirred tank bioreactor

maintained at 30 ◦C, an agitation of 250 rpm, and culture pH of 4.8

28

(Aftab and Patrick, 2008a; Aftab and Patrick, 2008b )

Mono culture

T. reesei RUT-C30 A. niger

Brief 3

• Mixed culture can increase in the production of volumetric

enzyme activity, filter paper activity, carboxymethyl cellulase

activity and dry biomass than monoculture.

29

Conclusion

• Even if mutants, it can not able to produce high levels of

the enzymes at the same time while monoculture, the

mutant’s performance can be improved by co-culturing

with A. niger.

30

Thank you for your attention!

31

Questions & AnswersOct. 21, 2013

Question 1 What is heterologous expression of cellulases? (東隴)

• Answer

Heterologous expression is a powerful technique to improve

production yield of enzymes, as well as activity. In order to make

a robust lignocellulolytic fungal strain, many different fungal

cellulases with higher and/or specific activity based on the need

for a functional cellulase system in the organism have been

cloned and expressed.

For example, thermostable β-glucosidase (cel3a) from

thermophilic fungus T. emersonii was expressed in T. reesei

RUT-C30 using a strong T. reesei cbh1 promoter. The expressed

enzyme has been shown to be highly thermostable (optimum

temperature at 71.5 °C) with high specific activity.33

Question 2 Why the unit is mg/min/g protein?

(連老師)

• Answer

34

Question 3 How much is strain? (耀庭)

• Answer

35

Question 4 What is the principle of Linear

Accelerator? (群翔)

• Answer

37

https://www.youtube.com/watch?v=e-LVl4xuGeM




seminar 2013-10-14

Technology