Biofibre Industry Advancement Workshop Saskatoon, Oct 11-12, 2005

Enzyme Development and Retting Hemp

Peter C.K. Lau
Group Head, Bioconversion & Sustainable Development (CBIN T&I project on Superior Pectinase for Processing Industrial Hemp and Other Agro-Fibres in collaboration with Dr. W. L. Sung, IBS)

$$$$$

Why/What Easier said than done enzymes ??

What fibre ?

Balancing act: Economic progress Environmental care Social responsibility

Why Hemp ?
In 03/1998, the Industrial Hemp Regulations enabled commercial cultivation of industrial hemp which produces little or no THC. Grower associations in almost all provinces (Quinte, ON) Interest in hemp production increasing (28% increase in licensed area in 2004) Converting 5X more CO2 to biomass vs. forestry, without herbicide or pesticide Strongest and most durable among natural fibres Advanced products: priced hemp garments, automobile panels, windmill blade Renewed interest for medical, pulp and fibre purposes Feasibility of hemp uniforms for Canadas 2010 Olympic team (Hemptown, Van)

Why (Not) Hemp?

Photo credit: The Gazette

$10 billion cash crop (3x annual revenue of wheat)

Bloomberg News, Oct. 1, 2005

Chemical analysis of hemp (% w/w)

Hemicellulose Protei n

Cellulose Bast fibre bundle Woody core (hurd)

Pectin Lignin Wax

Ash

55 44

16 18

18 4

4 28

1 1

2 3

4 2

Bast fibre represents 20-30% of the whole plant

Ref: Vignon et al, (1996) Bioresource Technol. 58:203-215

Need for Enzyme Processing

! Present chemical process (NaOH) dirty and fibre-damaging ! Glycosidases (pectinases & others) potentially capable to clean fibre without damaging durability ! The green advantage of enzymes environmental saving (less waste, reduced energy consumption)
From one end of Canada to the other, bio-composite projects are emerging; and the ability to thoroughly clean and yet preserve the strength and durability of natural fibres will be critical. John Baker

IBS Laboratory Results

Patent pending

Processed hemp fibre

Unprocessed fibre

Pectinase is a family of enzymes

Endo-polygalacturonase Exo-polygalacturonase Pectate lyase Pectin (methyl) esterase Pectin lyase polygalacturonate (pectin main chain)

www.rcsb.org/pdb www.ncbe.reading.ac.uk

Novel and Engineered Pectinases

Overall objective: The production and use of superior biocatalyst for the processing of plant fibre Goal: The synthesis of superior pectinase compatible to operation requirement neutral, or high pH and temperature Strategy: Two approaches (1) protein engineering of a known pectinase, (2) genomic screening of effective pectinases from microbial diversity and creating genetic improvement (e.g. via directed evolution)

Biocatalyst Business

The name of the game is to find the right catalyst. You can do this by mutation, or you can do it by finding the perfect enzyme in nature. But you have to do it quickly and efficiently.
Frances H. Arnold, Professor of Chemical Engineering and Biochemistry, Caltech. In Innovations, Chemistry & Biology, 12:503-505, 2005.

Enzymes Made-to-Measure
Changes in catalytic properties Increase in Vmax Decrease in Km Change in pH optimum Alteration of substrate specificity Changes in structural properties Thermostability Stability in organic solvents

Erwinia endopolygalacturonase

Erwinia carotovora endopolygalacturonase

Genome Mining of Potential Pectinase-encoding Sequences Mining of microbial genome databases an economical approach to new biocatalyst discovery Bait a known bacterial pectinase sequence Catches Multiple; selected 9 representing the three subclasses of pectinases for cloning and expression (5 putative lyases, 2
polygalacturonase and 2 methylesterases from Xanthomonas campestris and Streptomyces coelicolor)

Phylogram Tree

Protein Expression and Activity Assay

Over-expression of 3 Lyases in Rosetta cells (E. coli BL21/pRARE)
kDa

1. 2. 3. 4.

Control Sc 626147 47.7 kDa Sc 627050 53.3 kDa Xc 638163 37.4 kDa

Substrate: citrus pectin 30% esteried Clearing zone indicates activity

Activity Assay

Polygalacturonic acid assay Three good candidates - two lyases (from Sc and Xc) and one hydrolase (from Xc)

Thermostability
Effect of 1hr Incubation at Temperature
120 % remaining activity 100 80 60 40 20 0 20 30 40 50 60 70 temperature (C) pectate lyase from S. coelicolor polygalacturonase from X. campestris

Characterization of Pectinases

Ongoing Investigation
! ! ! ! Further characterization of cloned pectinases Engineering of pectinases with optimal activity at elevated temperature and alkaline pH Recombination of individual pectinase enzymes for an optimal efficiency of fibre extraction. Preparation of pectinase combination without other enzyme contaminants (cellulase, etc.) which may attack the integrity of the fibre (A winning synergistic cocktail)

Viability and Challenges of an Enzymatic Process

! ! ! ! Availability of enzyme reagents produced and supplied in industrial scale Economics price (environmental saving) Optimal activity associated energy usage Quality of the finished fibre (endurance, brightness, softness, strength)

Scourzyme Application
Chemical Process 5450 382 1100 3480 331 Enzyme Process 3810 281 792 1200 238 Change

Impact Category

Scourzyme is a pectate lyase that removes impurities in natural cotton

Energy consumption (MJ LHV) Global warming (kg CO2 eqv.) Acidification (g SO2 eqv.) Nutrient enrichment (g PO4 eqv.) Summer smog

1640 101 308 2280 93

Source: Novozyme

(g ethylene eqv.)

Scourzyme Application The Environmental Impact

Enzymatic vs Conventional Primary energy 75 % D demand Resource consumption Emissions to water

Source: Novozyme

73 % 40 % 70 %

O W N

Total treatment cost

Flexing Our Muscles for a Flux of Flax Activities

Natural Fibres Initiative for Biochemicals & Biomaterials

(CBIN T&I Project Theme: Integrated Biorefining [agricultural fibre] )

Green Catalyst / Chemistry and Advanced Technologies

Natural Fibres Initiative for Biochemicals & Biomaterials Feedstock

High/ Improved Fibre
Enzyme Discovery/ development IBS BRI AAFC Extraction

Bound FA
BRI

Flax Shive Fibre

AAFC Extraction

Vinyl guaiacol

free FA Vanillin Other phenolics

AAFC ICPET BRI

Characterization
(SEM NIR NMR)

Biocomposites
IMI

Biochemicals

A Blue Field of Dreams

Photo Courtesy of R. Marleau, Biolin Research Inc.

Thank You
Stay tuned (Whats hempening ?)*