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New ThermophilicEnzyme Systems for

Biorefineries

Detmold, APR 15, 2008

Dr. Jari VehmaanperäAB Enzymes GmbH / Roal Oy

&Marika Alapuranen, Terhi Puranen,

Sanni Voutilainen, Anu Koivula, Matti Siika-aho and Liisa Viikari

100 Years Of Enzyme Innovation

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ABF Organisation

Vegetable OilsMazola, Capullo

ACH Food Companies

Hot BeveragesTwinings, Ovaltine

Sugars & SweetenersSilver Spoon, Billingtons

Bread, Crispbread etcRyvita, Kingsmill

Allinson, SpeedibakeTip Top, Weston Milling

Packaged GroceriesBlue Dragon, Pataks

Rajah, Lotus

Meat & DairyChapmans, DonsMelosi, Watsonia

Herbs & SpicesTone's, Spice Islands

Durkee

Grocery

SugarBritish Sugar, Illovo

Bo Tian (JV)

Animal FeedsAB Agri

Seed ProcessingAB Agri,

Germains Technology Grp

Primary Foods& Agriculture

UK RetailPrimark

Republic of Ireland RetailPenneys

Retail

Bakery Ingredients/Yeast

AB Mauri

EnzymesAB Enzymes

Yeast ExtractsOhly

ProteinsProtient

Lipid TechnologyABITEC Corp

Cereal SpecialtiesPGP International

ABF Ingredients

Ingredients

ABF

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Associated British Foods – Quick Facts

Incorporated in 1935

Sales over £6 billion (€9 billion) in 2006

More than 75,000 employees in 46 countries

Listed on London Stock Exchange

Manufacturing operations worldwide

Key milestones for 2006/7:

Acquisition of 51% Illovo Africa’s largest sugar companyOpening of 27 Primark storesCollaboration with BP and DuPont on biofuelsAcquisition of Pataks, leading brand in Indian cuisine

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AB Enzymes At A Glance

Established 1907

People > 150

Revenues > €60 million

Growth rate 1999 – 2007: 10% p.a.

Focus on 4 core businesses:

! Baking, Beverage, Feed and Textiles

New focus area in Bio-Fuels/Bio-Ethanol

Patents: > 200 owned and licensed patents

R&D investment: 10% of revenues

Primary technology focus by Roal Oy: Trichoderma

Other strengths: Aspergillus and Bacillus

100% owned by ABF

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AB Enzymes: Markets

• We are active in the following applications worldwide:

" The marketing for Technical Enzymes is done from Finland, whereasFood, Feed and Speciality Enzymes are marketed from Germany

" We develop, distribute and market bio-based solutions" Seminars and technical support are important tools of our marketing approach

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Proud to partner with Roal Oy

" Located in Rajamäki, Finland, near Helsinki" 50/50 joint venture between ABF and Altia, a

Finnish Alcohol Beverage company" Modern fermentation facility with downstream,

drying and mixing capabilities" Research, process and product development, pilot

plant all in one location" Patented production technology based on

Trichoderma -and responsible for Trichodermasales through AB Enzymes, its sole distributor

" Producing non-Trichoderma products for all business fields of AB Enzymes

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TECHNOLOGICAL IMPROVEMENT OF ETHANOL PRODUCTION FROM LIGNOCELLULOSE (TIME)

• Duration: • 1.11.2002-28.2.2006

• Goals: • To reduce the overall production costs of

ethanol from lignocellulose by 10-20% through new developments

• To improve the performance of the key steps in the lignocellulose-to-ethanol process (pretreatment, enzymatic hydrolysis and increased process and energy integration)

• To develop a HTHC process concept• To evaluate the production costs and to assess

the environmental impacts by LCA

• Coordinator:• VTT Biotechnology (Liisa Viikari)

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TIME PARTICIPANTS

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PROCESS CONCEPTS FOR THERMOPHILIC CELLULASES IN BIOETHANOL PRODUCTION

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FACTORS AFFECTING THE EFFICIENCY AND ECONOMICS OF ENZYMATIC HYDROLYSIS

• Composition and accessibility of substrate (cellulose, hemicellulose, lignin), improved by pre-treatment

• Properties of enzymes: specific activity, stability, end-productinhibition, unproductive binding, role of CBD’s etc.

• Composition of enzyme mixtures (cellulase mixtures for optimalsynergy), role of additional enzymes (hemicellulases, ligninmodifying etc.)

• Hydrolysis technologies: separate/simultaneous/stepwise, temperature, mixing (affecting e.g. diffusion), recycling of enzymes

• Enzyme prize; efficient production systems

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POTENTIAL ADVANTAGES OF THERMOSTABLE ENZYMES IN LIGNOCELLULOSE HYDROLYSIS

• Higher specific activity in high temperature, i.e. smaller enzyme demand

• Allow more flexibility in the process configuration

• May allow process with improved integration in terms of heat recovery and recycling of process streams

• Allow increase in dry matter content due to lower viscosity at high temperature

• Smaller risk of contamination in high temperature stages

• Higher stability of HT enzymes in general

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DEVELOPMENT STEPS OF THERMOPHILIC HYDROLYSIS MIXTURES - TIME APPROACH

• Screening and characterization of potential thermophilic proteins(CBH's, endoglucanases, betaglucosidases)

• Cloning the most potential candidates• Overproducing the candidate proteins• Purification of the candidate enzymes for hydrolysis studies and

further characterization• Evaluation of the most applicable proteins by hydrolysis studies

using characterized mixtures of purified proteins on technicalsubstrates

• Composing the mixtures of the most promising enzymes and evaluation of them to find the best performance for each substrate

• Evaluation of the thermophilic mixtures in hydrolysis (and SSF)

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Summary table / TIME Expressed EnzymespH, temperature optima (60 min assays) and Tm

pH opt Topt Tm

CBH Thermoascus CBH / Cel7A 5,0 75 ºC+CBD_Tr 5,0 74 ºC+CBD_Ch 5,0 75 ºC

Acremonium CBH / Cel7A 5.0-5.5 69 ºCChaetomium CBH / Cel7A 5.0-5.5 75 ºC

EG Thermoascus EG28 / Cel5A 6.0 75 ºC+CBD_Ch 6.0 75 ºC

Acremonium EG40 / Cel45A 5.0-5.5 75 ºCEG40 / Cel45B 5.0-5.5 60 ºC

Chaetomium EG54 / Cel7B 5.5-6.5 65 ºC

XYN Thermoascus XYN30 /Xyn10 4.5-5.0 75 ºC

βG Thermoascus βG81 /Cel3A 4.5 75 ºCAcremonium βG101 /Cel3A 4.5 70 ºCChaetomium βG76 /Cel3A 5.5-6.0 65 ºC

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Purified T.reesei produced CBHI proteins

10% SDS-PAGE; Lane 1, Thermoascus; Lane 2, Thermoascus + Ct CBD; Lane 3, Thermoascus + Tr CBD; Lane 4, Acremonium; Lane 5, Chaetomium; Lane 6, Chaetomium core (papain); Lane 7, Trichoderma Cel7A; Lane 8 Trichoderma Cel7A core (papain); Lane 9, Melanocarpus Cel7B; Lane 10 Melanocarpus Cel7B + Tr CBD

LMW 1 2 3 4 5 6 7 8 9 10 LMW

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Thermostability2-module versions of the Cel7 enzymes

The temperature induced unfolding curves were measured by Circular Dichroism (CD) spectroscopy at 202 nm

190 200 210 220 230 240

-12-10

-8-6-4-202468

1012

CD spectrum of folded Cel7A CD spectrum of unfolded Cel7A

mde

g

wavelenght nm30 40 50 60 70 80 90

0.0

0.2

0.4

0.6

0.8

1.0

Enzyme Tm (°C) At Cel7A 69.0 ±1Ct Cel7A 75.0 ±1Ta Cel7A + Ct CBM 75.0 ±1Ta Cel7A + Tr CBM 75.0 ±1Tr Cel7A 65.0 ±1

Frac

tion

fold

ed

Temperature (°C)

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Temperature optimum curves

Methylumbelliferyl lactoside (MULac) used as a substrate

At Cel7A (■) Ct Cel7A (□)Ta Cel7A (▲) T. reesei Cel7A (●)

Results:• Topt ≥ 65 oC for Ct Cel7A and TaCel7A, and ≥ 60 oC for At Cel7A and ~ 60 oC for Tr Cel7A• Ct Cel7A clearly the most active cellobiohydrolase (already at lower temperatures).

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Microcrystalline cellulose (Avicel) hydrolysis at 70 ºC2-module versions of the cellobiohydrolases

The time-course of Avicel hydrolysis was followed for 24 hours bymeasuring soluble reducing sugars.

At Cel7A (■) Ct Cel7A (□) Ta Cel7A + Ct CBM (▲)Ta Cel7A + Tr CBM (Δ) T. reesei Cel7A (● )

Results:• Ta Cel7A + Ct CBM the most efficient enzyme

1-module (core) versions resulted in about half of the hydrolysis (not shown)

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Kinetic constants and Cellobioseinhibition

Enzyme CNPLac

kcat(min-1)

Km(µM)

kcat/Km (min-1M-1)

Ki (Glc2)(µM)

Type of inhibition

Ct Cel7A 19 ±1 2000 ±200 9.5 x 103 39 ±14 comp.

Ta Cel7A 1.7 ±0.1 990 ±70 1.7 x 103 107 ±14 comp.

At Cel7A 2.8 ±0.1 2100 ±150 1.3x 103 141 ±25 comp.

Tr Cel7A 2.6 ±0.05 520 ±30 5.0 x 103 19± 4 comp.

pH 5.7, 22 oC

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TEMPERATURE OPTIMUM OF THE THERMOPHILIC MIXTURES DETERMINED BY FPU ASSAY

0

50

100

150

200

250

30 35 40 45 50 55 60 65 70 75

Assay temperature

Rel

ativ

e FP

U a

ctiv

ity (F

PU/m

l) Econase+Novozym188Celluclast+Novozym188MIXTURE 2MIXTURE 2XMIXTURE 3X

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SUGARS RELEASED FROM 100 g OF STEAM EXPLODED CORN STOVER IN 72h ENZYME HYDROLYSIS(THEORETICAL MAXIMUM WAS 56.7 g)

0

10

20

30

40

50

60

35°C 45°C 55°C 60°C 35°C 45°C 55°C 60°C

Benchmark cellulase+ beta-Glu

Thermophilic mixture B(MIX3)

Suga

rspr

oduc

ed(g

) GalactoseArabinoseXyloseGlucose

* Benchmark Trichodermacellulase + mesophilic beta-Glu : total dosage 11.5 FPU/g dry matter

* Mixture of thermostableenzymes: total dosage 9.8 FPU/g d.m.

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HYDROLYSIS OF STEAM PRETREATED SPRUCE WITH THE ENZYME MIXTURES

0

10

20

30

40

50

60

70

80

90

100

35°C 45°C 55°C 60°C 35°C 45°C 55°C 60°C

Benchmark cellulase+ beta-Glu

Thermophilicenzymes

Hyd

roly

sis

(% o

f the

or. m

axim

um) 0h

24h48h72h

�Thermophilic enzymes (CBH, EG, β-Glu, XYL): 9.8 FPU/g cellulose�Benchmark Trichoderma cellulase + mesophilic β-Glu 11.5 FPU/g

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CONCLUSIONS - CBH's AND ENZYME MIXTURES

• Several thermophilic CBH enzymes of Family 7 performed better than T. reesei Cel7A in the hydrolysis of technical substrates at 45 ° C

• The performance of Thermoascus aurantiacus Cel7A could be greatly improved by genetically attached CBM of T. reesei Cel7A or C. thermophilum Cel7A

• With the new enzyme mixtures studied the hydrolysis temperature can be increased by up to 10 °C when compared to the present industrial products

• Clearly more efficient hydrolysis per applied FPU unit was obtained in total hydrolysis of lignocellulose

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ACKNOWLEDGEMENTS

• Roal Oy, Finland• Marika Alapuranen• Teemu Halonen• Jarno Kallio• Terhi Puranen• Jari Vehmaanperä

• VTT, Finland• Sanni Voutilainen• Satu Hooman• Arja Lappalainen• Matti Siika-aho• Tapani Reinikainen• Liisa Viikari• Ulla Vornamo• Anu Koivula

Financial support:• European Union: TIME project (ENK6-CT-2002-

00604)• The Academy of Finland (SV)• The Finnish Glycoscience Graduate School (SV)

ReferencesÖhgren K, Vehmaanperä J, Siika-aho M, Galbe M, Viikari

L, Zacchi G. (2007) Enzyme Microb Technol 40(4):607-613.

Viikari, L., Alapuranen, M., Puranen, T., Vehmaanperä, J. and Siika-aho, M. (2007) Thermostable enzymes in lignocellulose hydrolysis. Adv Biochem EnginBiotechnol 108, 121-145

Voutilainen, S., Puranen, T., Siika-aho, M., Lappalainen, A., Alapuranen, M., Kallio, J., Hooman, S., Viikari, L., Vehmaanperä, J. and Koivula, A. (2008) - Accepted for publication in Biotech and Bioeng.