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Easier said than done

Why/What enzymes ??$$$$$

What fibre ?

Balancing act:

Economic progress

Environmental care

Social responsibility

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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 Interest in hemp production increasing (28% increase in licensed area in 2004) Converting 5X more CO 2 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 Hemp ?

(Quinte, ON )

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Why (Not) Hemp?

Photo credit: The Gazette

$10 billion cash crop (3xannual revenue of wheat)

Bloomberg News, Oct. 1, 2005

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CelluloseHemi-

cellulosePectin Lignin Wax Protein Ash

Bastfibre

bundle55 16 18 4 1 2 4

Woody

core(hurd) 44 18 4 28 1 3 2

Chemical analysisof hemp (% w/w)

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

Bast fibre represents 20-30% of the whole plant

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Present chemical process (NaOH) dirty and fibre-damaging

Glycosidases (pectinases & others) potentially capable toclean fibre without damaging durability

The green advantage of enzymes

environmental saving (less waste, reduced energy

consumption)

Need for Enzyme Processing

From one end of Canada to the other, bio-composite projects areemerging; and the ability to thoroughly clean and yet preserve thestrength and durability of natural fibres will be critical. John Baker

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Processed hemp fibre Unprocessed fibre

IBS Laboratory Results Patent pending

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Pectinase is a family of enzymes

Endo-polygalacturonaseExo-polygalacturonase

Pectate lyase

Pectin (methyl) esterasePectin lyase

www.rcsb.org/pdb

www.ncbe.reading.ac.uk

polygalacturonate

(pectin main chain)

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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 andtemperature

Strategy : Two approaches -(1) protein engineering of a known pectinase,

(2) genomic screening of effective pectinases frommicrobial diversity and creating genetic improvement (e.g.via directed evolution)

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The name of the game is to find the right

catalyst. You can do this by mutation, or you cando 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 .

Biocatalyst Business

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EnzymesMade-to-Measure

Changes in catalytic properties

Increase in V max

Decrease in K m Change in pH optimum

Alteration of substrate specificity

Changes in structural properties Thermostability

Stability in organic solvents

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Erwinia endopolygalacturonase

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Erwinia carotovora endopolygalacturonase

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Genome Mining of Potential Pectinase-encoding Sequences

Mining of microbial genomedatabases an economicalapproach to new biocatalyst

discovery Bait a known bacterialpectinase sequence

Catches Multiple; selected 9 representing the three subclasses of pectinases for cloning and expression (5 putative lyases, 2polygalacturonase and 2 methylesterases from Xanthomonas campestris andStreptomyces coelicolor )

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Phylogram Tree

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Activity Assay

Polygalacturonic acidassay

Three goodcandidates - two lyases(from Sc and Xc) andone hydrolase (from

Xc)

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Thermostability

Effect of 1hr Incubation at Temperature

0

20

40

60

80

100

120

20 30 40 50 60 70

temperature (C)

%

r e m a

i n i n g a c

t i v

i t y

pectate lyase fromS. coelicolor

polygalacturonasefrom X. campestris

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Characterization of Pectinases

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Further characterization of cloned pectinases

Engineering of pectinases with optimal activity at elevatedtemperature and alkaline pH

Recombination of individual pectinase enzymes for anoptimal efficiency of fibre extraction.

Preparation of pectinase combination without other enzymecontaminants (cellulase, etc.) which may attack the integrityof the fibre (A winning synergistic cocktail)

Ongoing Investigation

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Availability of enzyme reagents produced and supplied inindustrial scale

Economics price (environmental saving)

Optimal activity associated energy usage

Quality of the finished fibre (endurance, brightness, softness,strength)

Viability and Challengesof an Enzymatic Process

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Impact Category

ChemicalProcess

EnzymeProcess

Change

Energy consumption

(MJ LHV)5450 3810 1640

Global warming(kg CO 2 eqv.)

382 281 101

Acidification

(g SO 2 eqv.)1100 792 308

Nutrient enrichment(g PO 4 eqv.)

3480 1200 2280

Summer smog

(g ethylene eqv.)331 238 93Source: Novozyme

Scourzyme Application

Scourzyme

is a pectatelyase thatremovesimpurities innaturalcotton

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Scourzyme Application The Environmental

Impact

Enzymatic vs Conventional

Primary energydemand 75 % D

Resourceconsumption 73 % O

Emissions towater 40 % WTotal treatmentcost 70 % N

Source:

Novozyme

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Flexing Our Muscles

for a Flux of Flax Activities

Green Catalyst / Chemistry and Advanced Technologies

Natural Fibres Initiativefor Biochemicals &Biomaterials

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

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Natural Fibres Initiative for Biochemicals & Biomaterials

High/Improved

Fibre

Characterization

Biocomposites

Feedstock

Flax

Shive

Fibre

Bound FA

free FA

Other phenolics

Biochemicals

(SEM

NIRNMR )

IBSBRI

EnzymeDiscovery/development

IMI

AAFCExtraction

AAFCExtraction

BRI

AAFCICPETBRI

Vanillin

Vinylguaiacol

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A Blue Field of Dreams

Photo Courtesy of R. Marleau ,

Biolin Research Inc.

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Thank You Stay tuned (Whats hempening ? )*