Methods for New Biocatalyst Discovery: Sources, Techniques, and Applications, Slides of Biochemistry

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Module 2: Methods for newbi^

t^ l^ t dibiocatalyst discovery

Lecture2: Methods for newbiocatalyst discovery

Biocatalysis^ and

Biotransformation

Introduction^ Main^ components^ #^ Plant/animal,

microbial^ and

recombinant

enzymes

#^ Screening^ for

new^ enzymes

(from^ nature)

with^ novel

g^

y^ (^

catalytic^ properties#^ Development

of^ efficient^ assay

for^ biomaterials

p^

y

#^ Finding^ the

natural/promiscuous

activity^ in^ biocatalyst

How^ to^ find

new^ biocatalyst?

Plant/animal tissue homogenate

Culture collection

Recombinant methods

Commercial sources

“You get what you screen for”You^ get

what^ you

screen^ for

•^ Plant taxonomyPlant^ taxonomy •^ Microorganism

taxonomy,^

enrichment

and

acclimatization techniquesacclimatization

techniques

•^ Microbial

genetics

•^ Recombinant

DNA^ technology

Plant^ enzymes Examples:^ Papain^

,^ bromelain^ and^ ficin

like^ proteases. Lipoxygenase^ from

soyabean^ and^ peroxidase

from^ horseradish^

are^ examples^ of

typical^ plant^ enzymes.Hydroxynitrile^ lyase

(HNL)^ from^ cyanogenic

plant^ is^ an^ important

plant^ enzyme.

h^ d f

l^

l k^ d^ l

d

They^ are^ extracted

from^ various^ plant

tissues^ like^ seeds,

roots,^ leaves^ etc^ and

collected^ as^ liquid

homogenate. Method:^ Tissue^ materials

are^ crushed^ in^ a^ homogenizer

using^ a^ suitable^ buffer.

Centrifugation and ultrafiltration affords concentrated enzyme solution TheCentrifugation^ and

ultrafiltration^ affords

concentrated^ enzyme

solution.^ The

enzymes^ can^ be^ purified

or^ partially^ purified

prior^ to^ use. Homogenizer

Animal^ enzymes *^ Porcine^ liver^ esterase

(PLE),^ porcine^ pancreatic^ lipase

(PPL)^ and (^ ), p^ p

p^

(^ )

arginase^ (calf^ liver)

are^ well‐known

biocatalysts^ isolated

from

animal^ tissues.* All are very cheap and can be isolated from the respective animalAll^ are^ very^ cheap

and^ can^ be^ isolated

from^ the^ respective

animal

tissue^ by^ standard

homogenization

technique. *The^ use^ of^ animal

enzymes^ in^ near

future^ seems^ to

be^ gradually

decreasing^ because

of^ disease^ and

a^ variable^ supply.

Important collections of microorganisms in the public domain American type culture collection (ATCC), USACentraalbureau voor Schimmelcultures (CBS), NetherlandNational collection of Industrial and Marine Bacterial Ltd. (NCIMB), UKD^ t^ h^ S^ l

Mik^

i^ d Z llk lt

GMbH

Deutsche Sammlung von Mikroorganismen und Zellkulturen GMbH(DSM-Z), GermanyNational Biotechnological Resource center (NBRC), japan*Microbial type culture collection (MTCC), Indiayp^

(^ ),

Northern Regional Research Laboratory (NRRL), USA All Union Collection of Microorganisms (VKM), Russia

Standard microbiological practices for BSL-

1. Access to the laboratory limited or restricted at the discretion of the laboratory director when experiments or workwith cultures and specimens are in progress.2. Persons wash their hands after they handle viable materials, after removing gloves, and before leaving thelaboratory.3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use are notpermitted in the work areas. Persons who wear contact lenses in laboratories should also wear goggles or a faceshield. Food is stored outside the work area in cabinets or refrigerators designated and used for this purpose only.4. Mouth pipetting is prohibited; mechanical pipetting devices are used.p p^ g^ p^

;^ p p^ g

5. Policies for the safe handling of sharps are instituted.6. All procedures are performed carefully to minimize the creation of splashes or aerosols.7 W^ k^ f^ d^

t^ i^ t d^ t l^ t^

d^ d^ ft^ ill^ f^

i bl^ t^ i l

7. Work surfaces are decontaminated at least once a day and after any spill of viable material.8. All cultures, stocks, and other wastes are decontaminated before disposal by an approved decontamination methodsuch as autoclaving. Materials to be decontaminated outside of the immediate laboratory are to be placed in adurable, leak proof container and closed for transport from the laboratory. Materials to be decontaminated outside ofthe immediate laboratory are packaged in accordance with applicable local state and federal regulations beforethe immediate laboratory are packaged in accordance with applicable local, state and federal regulations beforeremoval from the facility.9. A biohazard sign can be posted at the entrance to the laboratory whenever infectious agents are present. The signmay include the name of the agent(s) in use the name and phone number of the investigator.10. An insect and rodent control program is very much needed.

Common production process for industrial extracellular enzymes Preparation of fermentation medium....(starch, sugar, yeast extract, minerals, inducers etc)DissolutionSterilizationInoculationInoculationFermentation in flask (100-500mL)Fermentation in jar (3-30L)^

Partial inoculation (between each steps) Fermentation in seed tank (1000-3000L)Fermentation in mainj tank (>3000L)Broth outBroth-outFiltrationConcentration by ultrafiltrationPrecise filtrationSolvent precipitationFiltration or centrifugationDryingCrude enzymedifferent purification methodsPure enzyme

Common production process for special intracellular enzymes Preparation of fermentation medium....(starch, sugar, yeast extract, minerals, inducers etc)DissolutionSterilizationSterilizationInoculationFermentation in flask to in main tankCentrifugation or filtrationDisruption and extractionFiltrationConcentration by ultrafiltrationCrude enzymediff^ t^ ifi^ ti

th d different purification methodsPure enzyme

Enzyme suppliers for biotransformationCompany^

Country Altus Biologics Inc.^

USA Amano Enzyme Inc.^

JAPAN Asahi Chemical Co.^

JAPAN i^ iBiocatalysis Ltd.^

UK Biozyme Labs Ltd.^

UK Calbiochem Corp.^

USA, Denmark Diversa^

USA Fl k^ Ch^ i^ l^ L d

G^ UK Fluka Chemicals Ltd.

Germany, UK Genecor Int.^

Finland, USA Genzyme Ltd.^

UK DSM^

Holland M i^ S^ C^

JAPAN Meito Sangyo Co.^

JAPAN Novo Nordisk AS^

Denmark Oriental Yeast Co.^

JAPAN Roche Diagnostics GmbH

GERMANY R h^ G^ bH^

GERMANY Rohm GmbH^

GERMANY Sigma^

USA

Ad^ t^ d di^ d

t^ i^ i^ l t d

h l^ ll bi

t l^ t

Nature of enzymes^

Advantages^

Disadvantages

Advantages^ and^ disadvantages

using^ isolated^ enzymes

or^ whole^ cell^ biocatalysts

Nature^ of enzymes^

Advantages^

Disadvantages Isolated enzymes^

High catalyst concentrationpossible.No side reaction.

Limited stabilityCofactor regeneration needed. Simple product recovery.No transport limitation.Multienzymatic reactionspossible.possible. Whole cell biocatalysis

Unlimited availabilityexploiting growth.Cofactor recycling by cellular

Side reactions.Transport limitation.Complex product recovery. machinery itself.Multistep conversionpossible.

Many enzymes in the reactionmixture.