Second generation fuel ethanol:

the role of enzymes

CBM8, Ischia, May 11 2009

David van Eylen, Jan de Bont and Mirjam Kabel

3

Royal Nedalco

• Subsidiary of Royal Cosun.

• Producer of alcohol since 1899

with production sites in the

Netherlands, the UK and

Germany.

• Strong focus on residue stream

valorisation (molasses and c-

starch).

• Producer of high quality natural

alcohol and small scale fuel

ethanol producer since 2005.

Bergen op Zoom

Head Office & Production

Manchester

Production (2007)

Sas van Gent

Production

Heilbronn

Sales & Rectification

4

High end natural alcohol

• Nedalco is market leader in the

high end natural alcohol market.

• In the high end spirits market

Nedalco has a leading share of

20% → vodka’s and other

premium global brands.

• In the high end technical market

(pharmacy) we want to gain

ground with natural alcohol.

• In the overall North-West

European alcohol market Nedalco

is in 2nd position (13%) after

market leader Tereos (26%).

Share in the North-West

European market

Spirits 20%

Pharmacy 15%

Industrial 20%

Cosmetic 9%

5

Sas van Gent (2005)

6

Terminology

Traditional product:

• Alcohol

For cars:

• Bioethanol

• Fuel ethanol

Ethanol from non-food feedstocks:

• Second generation ethanol

• Cellulosic ethanol

7

Nedalco towards Fuel Ethanol

• The short term driver behind cellulosic ethanol is political

support for reasons of energy independence (US) and

sustainability (EU).

• Driven by public and private investments the US ‘R&D market’ is

developing fast.

• EU market is lagging behind but shifting focus towards ‘second

generation biofuels’ could alter this environment.

8

Ethanol plant locations

9

Market size

US fuel ethanol demand

0

5.000

10.000

15.000

20.000

25.000

30.000

35.000

40.000

1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 2016 2018 2020 2022

x 1

.000.0

00 g

allo

ns

imports (to date)

corn ethanol

cellulosic biofuel

advanced biofuel

source: RFA, Energy

Independance and Security

Act 2007

16 billion

gallons

10

DOE-sponsored Projects

http://www.energy.gov/media/DOE_Coverage_Map_Jan_29_2008.pdf

11

Cellulosic Feedstocks

12

Molasses

Sugar cane,

Beet sugar

Wheat,

Corn

Starch-containing

wheat or corn

by-products

Wheat bran,

Corn fiber

Pretreatment

Enzymes

Special C5-yeast

Wheat straw,

Corn stover

Woody

materials

Time 2008

Complexity

First generation

feedstocks

Second

generation

feedstocks

Feedstocks and complexity

Beet pulp

Pulp / Paper

sludges

13

Physical

and/or

chemical

pretreatment

Enzymatic

hydolysis

Yeast

fermentation Distillation

Ethanol Co-products

Second

Generation

Feedstock

Industrial yeast

fermenting both

C5 and C6 sugars

Enzyme cocktails

hydrolyzing

(hemi)cellulose

Mild and effective

pretreatment

technologies

Second Generation Ethanol

14

Physical

and/or

chemical

pretreatment

Enzymatic

hydolysis

Yeast

fermentation Distillation

Ethanol Co-products

Second

Generation

Feedstock

Industrial yeast

fermenting both

C5 and C6 sugars

Enzyme cocktails

hydrolyzing

(hemi)cellulose

Mild and effective

pretreatment

technologies

Second Generation Ethanol

Collaborations

Key technology Nedalco

15

Starch

Cellulose

Arabinoxylan

(xylose and arabinose)

other sugars (galactose,

glucuronic acid, mannose)

Lignin

Protein

Lipid

Ash

wheat wheat bran

corn corn fiber

Wheat and Corn, and Co-products

16

www.itis.gov

Kingdom Plantae

Tracheobionta (vascular plants)‏

Coniferophyta (gymnosperms)‏

Magnoliophyta (angiosperms)‏

others

others

Pinopsida others

Quercus (Oak)‏

Hard-wood

Liliopsida (monocotyledons)‏ Magnoliopsida (dicotyledons)‏

Commelinidae others

Hamamelidae others

Cyperales others

etc.

Pinus L. (Pine)‏

Soft-wood

etc.

Cyperaceae Poaceae (gramineae, grasses)‏

others Zea L. (corn)‏

Triticum (wheat)‏

Pennisetum (grass)‏

arabinoxylans

Plants and hemicelluloses

17

Wheat Bran

18

Starch (endosperm)

Corn fiber

(outer layers)

Corn Cobs

(after removal of kernels)

Corn Stover

(remaining plant

material on land)

Corn fibers

(R&D Nedalco)

Corn cobs

(Kabel et al. (2002))

Corn Stover

(NREL_1)

Compound (g/kg) (g/kg) (g/kg)

Protein 67 30 31

Lignin 100 100 180

Ash 30 50 52

Acetic acid 28 30 29

Starch 200 0 0

Cellulose 118 330 370

Hemicellulose (AX) 398 360 270

Other polymers 37 20 40

Composition of feedstock

Corn and co-products

19

Plant cell wall architecture

Rosette (cellulose synthase enzymes)

Lignin (Lignification in secondary wall)

Protein

Hemicellulose

Cellulose

Middle lamella

Primary wall

Secondary wall

Plasma

membrane

Sticklen, M.B. (2008). "Plant genetic engineering for biofuel production: towards affordable cellulosic ethanol." Nature Reviews 9(Genetics): 433-443

In grasses:

Arabinoxylans (AX)

20

-1,4-endo-xylanases (EC 3.2.1.8)‏

O O O O O O O O O O O O

O

Feruloyl esterase (EC 3.1.1.73)‏

xylan-acetylesterase (EC 3.1.1.72)‏

-(4-O-methyl)glucuronosidases (EC 3.2.1.131)

O

1,3-xylosidase?

-1,5-arabinofuranosidase?

galactosidase + xylosidase?

O

O

O

O

O

O

O

Corn arabinoxylans and

enzymes

O O

-xylosidase (EC 3.2.1.37)‏

O

Depolymerizing activities

Debranching activities

-L-arabinofuranosidase (EC 3.2.1.55)

-L-arabinofuranosidase (EC 3.2.1.55)

xylose O

arabinose

O glucuronic acid

O-acetyl

O galactose

O

O-feruloyl

Lignin

21

-1,4-endo-xylanases (EC 3.2.1.8)‏

O O O O O O O O O O O O

O

Feruloyl esterase (EC 3.1.1.73)‏

xylan-acetylesterase (EC 3.1.1.72)‏

-(4-O-methyl)glucuronosidases (EC 3.2.1.131)

O

1,3-xylosidase?

-1,5-arabinofuranosidase?

galactosidase + xylosidase?

O

O

O

O

O

O

O

Corn arabinoxylans and

enzymes

O O

-xylosidase (EC 3.2.1.37)‏

O

Depolymerizing activities

Debranching activities

-L-arabinofuranosidase (EC 3.2.1.55)

-L-arabinofuranosidase (EC 3.2.1.55)

xylose O

arabinose

O glucuronic acid

O-acetyl

O galactose

O

O-feruloyl

Lignin

Present in commercial enzyme preparations

22

Pretreatment

37 °C, Low pH

On Site

Enzyme production

Efficiency hydrolysis

approx. 80 %

Remaining

polymers

Pretreatment & enzymatic

hydrolysis

23

Major Technological Issues

1. Industrial yeast capable of C5 fermentations

2. Pretreatment

3. Hemicellulolytic enzymes

24

Xylose Arabinose

Glucose

Fructose

Starch

Cellulose Arabinoxylan

Sucrose

Fermentation of Sugars

by Saccharomyces cerevisiae

25

Major Technological Issues

1. Industrial yeast capable of C5 fermentations

2. Pretreatment

3. Hemicellulolytic enzymes

26

1 L reactor

Electric heating, but also steam (20 bar)! -> heating up time: within 5 min to 180 °C

steam (20 bar)‏

through spiral

Wheat bran heat-pretreatment

lab-scale

27

Pretreatments (20 Experiments):

H2SO4 (%): 0, 1 or 2 %w/w (based on dry weight feedstock) Temp (°C): 120, 140, 160, 180 °C

Time (min): 5, 10, 15 minutes

All: 12% dry weight wheat bran in water

Wheat bran: pretreatment

28

% H2SO4

T (°C)‏

t (min)‏

Furfural

HMF

0

0.5

1

1.5

2

2.5

0 10 10 5 10 15 5 10 5 10 5 10 5 10 15 5 10 5 10 5 10

0 120 180 120 140 160 180 120 140 160 180

0 1 2

Am

ou

nt

(g/L

‏(

confidential

Twenty pretreatments:

HMF and furfural

29

Enzymes

• Cellulases

• Arabinoxylanases

30

Commercial Cellulases

http://bioenergy.novozymes.com/files/documents/2009-03043-01.pdf

http://www.genencor.com/cms/resources/file/eb93d70b32b8f51

/ACCELLERASE%201500%20Brochure.pdf

31

General aspects arabinoxylans and enzymes

• Arabinoxylans from wheat bran

• Arabinoxylans from corn fiber

Arabinoxylans and enzymes

32

Arabinoxylanases

• Many scientific papers

• Few or no commercial preparations

33

Pubmed hits

Enzyme 2008/2009 Total

-L-arabinofuranosidase 23 282

-1,5-arabinofuranosidase 4 13

acetyl xylan esterase 2 78

ferulic acid esterase 20 156

-glucuronosidase 4 51

-xylosidase 33 1072

34

Nature R&D Reports Arabinoxylanases

• Focus mostly on characterization enzymes

– at DNA level (sequences and their comparison, cloning)

– at protein level (active site, structures)

• Limited information on relation enzyme-substrate

– activity on real AX (e.g. soluble oligomers)

– which AX side groups hinder enzymes

– which structures are left after incubating AX with the enzymes

• Literature on real substrates

– focus on liberation monosugars

– limited information on AX structures not broken down by enzymes

• Difficult to deduce which additional enzyme activities are necessary to obtain a better overall AX degradation

35

Enzymatic Hydrolysis of Wheat Arabinoxylan by a Recombinant “Minimal”

Enzyme Cocktail Containing β-Xylosidase and Novel endo-1,4-β-Xylanase and

α-L-Arabinofuranosidase Activities

Hanne R. Sørensen, Sven Pedersen, Christel T. Jørgensen, and Anne S. Meyer

Novozymes A/S, Krogshøjvej 36, DK-2880 Bagsværd, Denmark, and Department of Chemical

Engineering, Technical University of Denmark, DK- 2800 Kgs. Lyngby, Denmark

American Chemical Society and American Institute of Chemical Engineers

Published on Web 01/09/2007

Example paper “real substrates”

36

Test both commercial and experimental enzymes on

Nedalco substrates

Detect effectiveness; especially as based on analysis of

remaining degradation products

Select best enzyme or devise cocktail of enzymes

Iterative process with enzyme companies

Enzymes strategy at Nedalco

37

Collaboration with Wageningen University

Sugar composition (Complete hydrolysis into monosaccharides (H2SO4);

quantitative analysis of monosaccharides)

• Dumas: quantitative analysis of N (protein = N *6.25)

• HPSEC (High Performance Size Exclusion Chromatography):

Separation based on hydrodynamic volume (Mw 1-100kDa)

• HPAEC (High Performance Anion Exchange Chromatography):

Highly specific separation of mono- and oligosaccharides

(~Mw 0.1-3 kDa) (Dionex-system)

• MALDI-TOF MS (Matrix Assisted Laser Desorption-Time of Flight Mass Spectrometry):

Mass determination on a MALDI-sample plate;

accuracy up to 1 Da (Mw 0.3-50 kDa)

Analytical tools

38

Tools to study degradation of AX

39

• General aspects arabinoxylans and enzymes

Arabinoxylans from wheat bran

• Arabinoxylans from corn fiber

Arabinoxylans and enzymes

40

Twenty Pretreatments:

Enzymes and Release Xylose

% H2SO4

T (°C)

t (min)

0

2

4

6

8

10

12

14

16

18

20

0 10 10 5 10 15 5 10 5 10 5 10 5 10 15 5 10 5 10 5 10

0 120 180 120 140 160 180 120 140 160 180

0 1 2

Xylo

se m

on

om

er

(g/L

)

Maximum release possible as analyzed after acid hydrolysis

Released after Pretreatment (Pt)

Released after Pt + Exp. enzymes + Celluclast + Novozyme188 + AMG + amylase

Released after Pt + same enzymes PLUS protease

41

• General aspects arabinoxylans and enzymes

• Arabinoxylans from wheat bran

Arabinoxylans from corn fiber

Arabinoxylans and enzymes

42

0

5

10

15

20

25

30

35

40

g/l

Arabinose

Galactose

Glucose

Xylose

Pretreatment Corn Fiber

1% H2SO4 (w/w) 0% 2%

t (min)

T (ºC) 120 140 140 140 120 120

60 10 30 30 60 10 30 60 30 60 10 30 10

140

5%

60 max

60

6%

120 160

43

Pretreatment plus Enzymes

H2SO4 (w/w)

t (min)

T (ºC)

1% 0% 2%

120 140 140 140 120 120

60 60 30 60 10 30 60 30 60 10 30 10

140

5%

60 max

160

30

0

5

10

15

20

25

30

35

40

45

su

gar

co

ncen

trati

on

(g

/l)

Arabinose Galactose Glucose Xylose

Rest of the sugars?

Same enzyme cocktail as with wheat bran

44

• Comparison in situations of almost no pretreatment

• Experimental enzymes + Celluclast + Novozyme188

Release of monomers (%):

Corn fiber Wheat bran

Xylose 18 68

Why these differences?

Corn fiber versus wheat bran

45

O O O O O O O O O O O O

O O

O

O

O

O

O

O

O

O O O O O O O O O O O O

O O

O

O

O

O

O

Corn hemicellulose structure:

Wheat bran hemicellulose structure:

Calculated from molar composition:

Per 100 xyloses: - 66 Arabinoses

- 20 GlcA(me)‏

- 24 Acetyl

- 14 Galactoses

Calculated from molar composition:

Per 100 xyloses: - 58 Arabinoses

- 7 GlcA(me)‏

- 7 Acetyl

- 5 Galactoses

Corn versus wheat bran

46

Pretreatment plus Enzymes

H2SO4 (w/w)

t (min)

T (ºC)

1% 0% 2%

120 140 140 140 120 120

60 60 30 60 10 30 60 30 60 10 30 10

140

5%

60 max

160

30

0

5

10

15

20

25

30

35

40

45

su

gar

co

ncen

trati

on

(g

/l)

Arabinose Galactose Glucose Xylose

Rest of the sugars?

Same enzyme cocktail as with wheat bran

47

Mass balances

• Goal:

– Sugars can be present as

• Monomer

• Oligomer (soluble)

• Residue (insoluble)

– In which form are the sugars present after

pretreatment?

48

Analysis of:

- Monomeric sugars

- Oligomeric sugars

- Molecular size distribution of oligomers

- Resistant structures in oligomers

Resistant oligomers?

Corn Fiber

Pretreatment Centrifuge +

Liquid Residue

With or without enzymes (AMG / cellulases / arabinoxylanases

49

Pretreatment

1% H2SO4 (w/w) 2%

t (min)

T (ºC) 140 140 140 120

10 30 30 30 30

5%

160

0%

PT enz PT enz PT enz PT enz PT enz

Xylose

0

20

40

60

80

100 %

of

tota

l

mono

oligo

furfural

unaccouted

residue

Not monomer

2%

and Enzymes

50

Oligomers and Enzymes

• Not all xylose present as soluble oligomers is monomerized by

enzymes

• Enzyme accessibility is not the only problem

• Some enzyme activities are missing

Nature of the oligomeric compounds?

51

15 16 17 18 19

D3 s4

Liquid of pretreated

Corn fiber

Time (min)

RI

180

(Dextrins (Da))

4000 10000

+ amyloglucosidase

+ amyloglucosidase

+ cellobiase

+ hemicellulases

HPSEC of oligomeric fraction

Resistant oligomers

52 Fraction I

Reversed Phase Chromatography of Liquid (Detection by using UV and ELSD)

0.0 10.0 20.0 30.0 40.0 50.0

Time (min)

4

3

2

1 ELSD

UV 325nm

UV 205nm

UV 280nm

Specific for ferulic

acid and

coumaric acid

Evaporative Light Scattering

Protein

Oligomers in Liquid

Reversed Phase HPLC

Inte

nsity

II III IV V

53

Reversed Phase Chromatography of Fraction I (Detection by using UV and ELSD)

0.0 10.0 20.0 30.0 40.0 50.0

Time (min)

ELSD

UV 325nm

UV 205nm

UV 280nm

Specific for ferulic

acid and

coumaric acid

Evaporative Light Scattering

Protein

Oligomers in Fraction I

Reversed Phase HPLC

Inte

nsity

54

46

7.4

49

7.3

52

3.3

43

7.5

53

9.3

48

3.3

511

.3

62

9.4

74

3.5

56

9.4

59

9.4

54

9.2

45

3.4

70

1.5

75

9.5

65

9.4

10

93

.6

64

5.4

58

5.4

73

1.5

85

1.6

611

.4

11

09.6

77

3.5

10

13

.6

68

9.4

71

7.4

78

9.5

67

1.4

86

7.5

90

5.6

98

3.6

93

5.6

10

29

.6

10

51

.6

95

3.6

92

1.5

10

67

.6

89

1.5

83

7.5

99

9.6

10

85

.7

500 600 700 800 900 1000 1100 m/z

Inte

nsity

P3

P4

P4Ac

P5

P5Ac

P4GlcAme

P5GlcAme

HP2 Fraction I:

Many pentose containing oligomers

enriched with O-acetyl and/or Glucuronic acid side groups

Oligomers in Liquid

Maldi-TOF MS Fraction I

H = hexose

P = pentose

Ac = O-acetyl

GlcAme= 4-O-methyl-Glucuronic acid

55

General Conclusions

Arabinoxylanases

• Detailed fundamental knowledge available on many but

not on all required enzymes

• Limited knowledge available in terms of the application

of the enzymes as cocktails in the hydrolysis of complex

feedstocks such as wheat bran and corn fiber

56

General Conclusions

• Second generation bioethanol would benefit from

arabinoxylanases

• Timeline of commercial arabinoxylanases?

• Cost of commercial arabinoxylanases?

• Equally perfect location next meeting!

57

58

Twenty Pretreatments:

Enzymes and Release Arabinose

% H2SO4

T (°C)

t (min)

Maximum release possible as analyzed after acid hydrolysis

Released after Pretreatment (Pt)

Released after Pt + Exp. enzymes + Celluclast + Novozyme188 + AMG + amylase

Released after Pt + same enzymes PLUS protease

0

2

4

6

8

10

12

14

0 10 10 5 10 15 5 10 5 10 5 10 5 10 15 5 10 5 10 5 10

0 120 180 120 140 160 180 120 140 160 180

0 1 2

Ara

bin

ose m

on

om

er

(g/L

)

59

1. Can be done at high acid concentrations

2. Milder pretreatment only if new and better enzymes

available

Conlusions Liberation of Xylose

from Corn

60

Literature

• Example: Literature on -L-arabinofuranosidase 2008-2009

• Characterization on DNA level (5/23):

– Differential expression of alpha-l-arabinofuranosidase and alpha-l-arabinofuranosidase/beta-

d-xylosidase genes during peach growth and ripening. Carolina Di Santo M, Pagano EA,

Sozzi GO. Plant Physiol Biochem. 2009.

– alpha-l-Arabinofuranosidase from strawberry fruit: Cloning of three cDNAs, characterization

of their expression and analysis of enzymatic activity in cultivars with contrasting firmness.

Rosli HG, Civello PM, Martínez GA. Plant Physiol Biochem. 2009; 47(4):272-81.

– Purification and characterization of an extracellular alpha-L-arabinosidase from a novel

isolate Bacillus pumilus ARA and its over-expression in Escherichia coli. Pei J, Shao W. Appl

Microbiol Biotechnol. 2008; 78(1):115-21.

– Cloning, purification, and characterization of a thermostable alpha-L-arabinofuranosidase

from Anoxybacillus kestanbolensis AC26Sari. Canakci S, Kacagan M, Inan K, Belduz AO,

Saha BC. Appl Microbiol Biotechnol. 2008; 81(1):61-8.

– Improved cloning vectors for bifidobacteria, based on the Bifidobacterium catenulatum pBC1

replicon. Alvarez-Martín P, Belén Flórez A, Margolles A, del Solar G, Mayo B. Appl Environ

Microbiol. 2008; 74(15):4656-65.

61

Literature

• Characterization on protein level (6/23) – Purification and properties of an extracellular beta-xylosidase from Aspergillus japonicus and

sequence analysis of the encoding gene. Wakiyama M, Yoshihara K, Hayashi S, Ohta K. J Biosci Bioeng. 2008; 106(4):398-404.

– Structural analysis of a glycoside hydrolase family 43 arabinoxylan arabinofuranohydrolase in complex with xylotetraose reveals a different binding mechanism compared with other members of the same family. Vandermarliere E, Bourgois TM, Winn MD, van Campenhout S, Volckaert G, Delcour JA, Strelkov SV, Rabijns A, Courtin CM. Biochem J. 2009; 418(1):39-47.

– Characterization of a family 54 alpha-L-arabinofuranosidase from Aureobasidium pullulans. de Wet BJ, Matthew MK, Storbeck KH, van Zyl WH, Prior BA. Appl Microbiol Biotechnol. 2008; 77(5):975-83.

– Beta-D-xylosidase from Selenomonas ruminantium: thermodynamics of enzyme-catalyzed and noncatalyzed reactions. Jordan DB, Braker JD. Appl Biochem Biotechnol. 2009; 155(1-3):330-46.

– The structure of the complex between a branched pentasaccharide and Thermobacillus xylanilyticus GH-51 arabinofuranosidase reveals xylan-binding determinants and induced fit. Paës G, Skov LK, O'Donohue MJ, Rémond C, Kastrup JS, Gajhede M, Mirza O. Biochemistry. 2008; 47(28):7441-51.

– A family 51 alpha-l-arabinofuranosidase from Penicillium purpurogenum: purification, properties and amino acid sequence. Fritz M, Ravanal MC, Braet C, Eyzaguirre J. Mycol Res. 2008; 112(Pt 8):933-42.

62

Literature

• Effect enzyme on model substrate (3/23)

– Extraction of water-soluble hemicelluloses from barley husks. Roos AA, Persson T, Krawczyk

H, Zacchi G, Stålbrand H. Bioresour Technol. 2009;100(2):763-9.

– Two distinct arabinofuranosidases contribute to arabino-oligosaccharide degradation in

Bacillus subtilis. Inácio JM, Correia IL, de Sá-Nogueira I. Microbiology. 2008; 154(Pt 9):2719-

29.

– Characterization of a modular enzyme of exo-1,5-alpha-L-arabinofuranosidase and arabinan

binding module from Streptomyces avermitilis NBRC14893. Ichinose H, Yoshida M, Fujimoto

Z, Kaneko S. Appl Microbiol Biotechnol. 2008; 80(3):399-408.

• Real second generation substrate (3/23)

– Enhanced ethanol production from brewer's spent grain by a Fusarium oxysporum consolidated system. Xiros C, Christakopoulos P. Biotechnol Biofuels. 2009; 2(1):4.

– Hydrolysis and fermentation of brewer's spent grain by Neurospora crassa. Xiros C, Topakas E, Katapodis P, Christakopoulos P. Bioresour Technol. 2008; 99(13):5427-35.

– alpha-L-Arabinofuranosidase from Streptomyces sp. PC22: purification, characterization and

its synergistic action with xylanolytic enzymes in the degradation of xylan and agricultural

residues. Raweesri P, Riangrungrojana P, Pinphanichakarn P. Bioresour Technol. 2008;

99(18):8981-6.

63

Literature

• Relation AX structure-enzyme activity (1/23)

– Simultaneous production of endo-beta-1,4-xylanase and branched xylooligosaccharides by Thermomyces lanuginosus. Puchart V, Biely P. J Biotechnol. 2008; 137(1-4):34-43.

• Other; not related to arabinoxylan degradation (5/23)

– A high molecular arabinogalactan from Ribes nigrum L.: influence on cell physiology of human skin fibroblasts and keratinocytes and internalization into cells via endosomal transport. Zippel J, Deters A, Pappai D, Hensel A. Carbohydr Res. 2009.

– Material properties of films from enzymatically tailored arabinoxylans. Höije A, Sternemalm E, Heikkinen S, Tenkanen M, Gatenholm P. Biomacromolecules. 2008 Jul;9(7):2042-7.

– Fusarium species detected in onychomycosis in Colombia. Castro López N, Casas C, Sopo L, Rojas A, Del Portillo P, Cepero de García MC, Restrepo S. Mycoses. 2008 .

– Cell wall modifications in Arabidopsis plants with altered alpha-L-arabinofuranosidase activity. Chávez Montes RA, Ranocha P, Martinez Y, Minic Z, Jouanin L, Marquis M, Saulnier L, Fulton LM, Cobbett CS, Bitton F, Renou JP, Jauneau A, Goffner D. Plant Physiol. 2008; 147(1):63-77.

– New chromogenic substrates for feruloyl esterases. Marmuse L, Asther M, Fabre E, Navarro D, Lesage-Meessen L, Asther M, O'Donohue M, Fort S, Driguez H. Org Biomol Chem. 2008; 6(7):1208-14.

64

Literature

• Conclusion Literature on -L-arabinofuranosidase 2008-2009

– Recent literature (2008-2009) on -L-arabinofuranosidase focuses

mainly on characterizing the enzyme on DNA/protein level

– Little information about activity of enzyme on real second generation

substrates

– Activity is studied on model substrates, but little information on

remaining structures after enzyme activity

– Only one publication on structures remaining after incubation of real

second generation substrate with enzyme

65

Cellulase Novozymes

• Iets van hun website halen

• http://bioenergy.novozymes.com/

• Emphasis on cellulose, little info on hemicellulose

• Importance of pretreatment is stressed

• New cocktails to replace first-generation Celluclast enzymes

– Cellic CTec - a cellulase complex

– Cellic HTec - a hemicellulase

(http://bioenergy.novozymes.com/files/documents/2009-03043-01.pdf)

• Commercial viability in 2010

66

Cellulase Genencore

• Iets van hun website halen

• http://www.genencor.com/cms/connect/genencor/products_and_services/business_d

evelopment/biorefineries/products/accellerase_product_line_en.htm

• Accellerase® 1500

Accellerase 1500 is a new lower-cost, more effective product available in bulk for

pilot, demo, and commercial-scale use. Launched March 2009

• Accellerase® XY

accessory xylanase enzyme complex enhances both xylan (C5) and glucan (C6)

conversion when blended with other Accellerase® enzyme products.

• Accellerase® XC

accessory xylanase/cellulase enzyme complex contains a broad profile of

hemicellulase and cellulase activities and enhances both xylan (C5) and glucan (C6)

conversion when blended with other Accellerase® enzyme products.

• Accellerase® BG

accessory beta-glucosidase enzyme enhances glucan (C6) conversion when blended

with cellulase products.