DEVELOPMENT of INDUSTRIAL ENZYMES Laboratory … seminar 2014/Sinitsyn Zorov.pdf · DEVELOPMENT of...
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DEVELOPMENT of INDUSTRIAL ENZYMES Laboratory of Enzymes Biotechnology A.N. Bach Institute of Biochemistry (INBI) Russian Academy of Sciences Moscow State University, Dept of Enzyme Chemistry Prof. Arkady P. Sinitsyn Dr. Ivan N. Zorov Dr. Alexandra M. Rozhkova Russian-German Workshop December 5, 2014 Moscow
Transcript of DEVELOPMENT of INDUSTRIAL ENZYMES Laboratory … seminar 2014/Sinitsyn Zorov.pdf · DEVELOPMENT of...
DEVELOPMENT of INDUSTRIAL ENZYMESLaboratory of Enzymes Biotechnology
A.N. Bach Institute of Biochemistry (INBI) Russian Academy of Sciences
Moscow State University, Dept of Enzyme Chemistry
Prof. Arkady P. SinitsynDr. Ivan N. Zorov
Dr. Alexandra M. Rozhkova
Russian-German WorkshopDecember 5, 2014
Moscow
Possibilities of Laboratory•Renewable lignocellulosic biomass bioconversion (BIOREFINERY), discovery of a new key individual cellulases (carbohydrases) and carbohydrases complexes for highly efficient biomass degradation
•Design and optimization of enzymatic preparations composition for commercial application using genetic engineering and microbiology tools
•Development of strains – producers of industrial enzymes (creation of recombinant strains based on Penicillium host fungal systems, classical mutagenesis of different fungal strains)
•Enzymology and biochemistry of industrial enzymes, purification of different enzymes (analytical and large scale, by LC and FPLC), investigation of the basic properties of individual enzymes
•Enzymes application in different areas, application trials, development of mini-and micro-scale application assays to mimic “real life large scale application”
December 5, 2014
Most important fields of industrial application of enzymes
Food industry – improvement of food quality, increasing of the yield offinal product, improvement of processing conditions, bioconversionof plant feedstocks
Feed additives (carbohydrases for hydrolysis of non-starch polysaccharides and phytic acid) - Improvement of feed quality
Biorefinery – effective bioconversion of renewable cellulosic feedstocks (wood and agricultural wastes)
Textile industry - improvement of fibers and fabric quality, denim color changes, improvement of exploitation characteristics
Pulp and paper industry - improvement of cellulose defibrillation & yield, energy saving, secondary cellulose recycling, decreasing of wastes
December 5, 2014
We use two strategies for creation of recombinant enzymatic preparations
I. Way one: Classic strategy for creation of producer strains
II. Way two: Genetic engineering approach
Selection of unique enzyme producersamong wide range of fungal strainsIncreasing of target enzymes productionby UV-induced mutagenesis
Fermentation process optimization
Classic strategy Common problems
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Unstable strains
Strain obtaining is time consuming
Very difficult to predict compositionof secreted enzymatic complex
1. Stable integration into fungal chromosome(5 and more generations are stable)
2. Reproducible time to generate new strain(2 months from gene cloning to enzyme preparation)
3. Rational design of secreted complexes(purpose dependent complex design)
4. Possibility of simultaneous expression of 2 and more genes (fine tuning of secreted complex composition)
5. The same approach can be used for any ascomycetes(Aspergillus, Trichoderma etc)
Advantages of genetic engineering approaches in design of fungal strains
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Penicillium verruculosumPenicillium canescens
Productivity- 10-12 g of protein/L CF Productivity- 30-40 g of protein/L CF
Xylanolytical strain:Basal enzymes- xylanases, arabinasesand β-galactosidases
Hydrolytical strain:Basal enzymes- cellulases
Some enzyme preparationshad approved by food industry
Possibility of multienzymatic complexes obtaining in frame of one strain
Very simple and chip fermentation media → sugar beet pulp
The partial withdrawal of glucoserepression → fed-batch → more protein
Two «wheelhorses» in Lab
December 5, 2014
Expression systems in Penicillium strains
constitutive expression(histone promoter)
inducible expression(cbhI, xylA, bgaS, abfA)
MC
Xylanase A
December 5, 2014
CBH 2 (30%)
CBH 1 (35%)EGs (20%)
CBH 2 (20%)
CBH 1 (51%)XylA (6%)EGs (11%)
CBH 2 (15%)CBH 1 (25%)
XylA (30%)
EGs (18%)
Target activities in recombinant strainsPreparation Substrate Targ.activity, U/ml Increase,fold
Abf6 p-NPh-α-L-Ara 50-60 >10AglA33 p-NPh-α-Gal 1200-1400 >500AglC4 p-NPh-α-Gal 140-160 >15FAE9 p-NPh–butyrate 50-70 >30XG9 Xyloglucan 60-80 >20PhPl29 Phytin, pectin 220, 180 >10, >90PhPlAgl9 Phytin, pectin, p-NPh-α-Gal 280, 120, 325 >15, >60, >400Phy215 Phytin 400 >150PEC23 Pectin from citrus 190 ~100Eg2 Carbohymethylcellulose 1200-1500 >200pBGL-32 p-NPh-β-Glc 800-1000 >500CBHI Avicel 7-10 >5CBHII Avicel 7-10 >5INU1 Inulin 2500-3000 >800MANB Galactomannan 35 >30XYLIII Birch xylan 500-600 >10
Producers of enzymes for Industrial Biotechnologycellulases, β-glucanases,xylanases, pectinases, acid proteases, phytase
pectinases, cellobiohydrolases,endoglucanase, β-glucosidase,α-galactosidase, β-galactosidase,Inulinases, xyloglucanase
endo-1,4-β-glucanase
endo-1,4-β-glucanaseendo-1,4-β-xylanase
cellulases, β-glucosidase,hemicellulases, α-amylase, glucoamylase
Agriculture/Animal feeds: Feeds additives
Food Industry and Processing:Clarification of fruit juices,Food Industry by-products treatment
Textile:Fabric polishing, Denim washing
Pulp-and-paper:Cellulose defibrillation, bio-bleaching, nano-cellulose
Biorefinery:G6, G5 sugars from starch and lignocellulose, biofuel, org.acids
December 5, 2014
RT Biotechprom – the subsidiary of Rostechnologia State Enterprise
M.V. Lomonosov Moscow State UniversityDepartment of Enzyme Chemistry
A.N. Bach Institute of Biochemistry RAS
Biorefinery Pilot Plant Project – main contributors
The project is supported by the Russian Ministry of Education and Science
VOGELBUSCH Biocommodities TRIS Biotech
Engineering partners:
December 5, 2014
Production of Enzyme prepa‐rations with high hydrolytic ability
Enzymatic treatment
Diluted acid pretreatment
• Acid concentration 0.4‐2% • Temperature 140‐180oC• 15 – 30 minutes
• Enzymatic complex with enhanced β‐glucosidase activity for high glucose yield
• T=50‐55oC, pH = 4,5+0.5• 16 – 36 hours depending on
enzyme dosage
Woodymaterials
> 55% yield of sugars from wood
Pretreated substrate
Lignin
C6 sugars(glucose**),C5 sugars
Further chemical / microbial
transformationsteps
Energy / additives to the construction materials
C5 sugars *(xylose, arabinose)
* ‐ > 90% of xylose and arabinose (C5 sugars); < 10% of glucose, traces of mannose and galactose (C6 sugars);** ‐ > 80% of glucose (C6 sugar)
Concept of the Biorefinery Pilot Plant
December 5, 2014
Production of Enzyme prepa‐rations with high hydrolytic ability
Enzymatic treatment
> 55% yield of sugars from wood
Pretreated substrateC6 sugars(glucose**),C5 sugars
Responsibility of INBI in the Project
December 5, 2014
ЦБГ I 55 кДа
ЦБГ I 66 кДа
ЦБГ II 50 кДа
ЦБГ II 60 кДа
β-ГЛ 116 кДа (собственная)
КГ 70 кДа ЭГ
Ксил 32 кДа
Ксил 23 кДаEnzyme
preparation
CMCase Avicelase Xylanase bGluc
BCCP 15 0.6 15 1.2
β-GP 10 0.4 4 45
Two recombinant strains producers of cellulases were created
General Scheme of the Pilot Plant
December 5, 2014
Biorefinery Pilot Plant - general view
December 5, 2014
Enzyme production unit 50-500-2000-10000 L fermenters
December 5, 2014
Enzymes in Food IndustryAPPLICATION TO RAW MATERIAL-) increasing of the juice yield-) clarification of juices-) improving of the product quality-) reducing of the maintain costs
BIOCONVERSION OF FEEDSTOCKS
-) increasing of the reducing sugar yield-) reducing of the stress on the ecological system
December 5, 2014
Structure of the plant cell wall
Cellulose
Hemicellulose
Pectin
Multienzymatic compex had to contain pectinases, hemicellulases and cellulases
December 5, 2014
New pectinases and cellulasespreparations for food application produced by novel Penicillium strains
others44,4%
XYL15,2%
PEL11%
BG11,8%
EG17,6%
BG11%
CBHs33%
EG13%
others16% PEL
27%
PB5 C9
December 5, 2014
Juice clarification and decreasing of strawberry juice viscosity pectinases, β-glucanase, arabinases
Con
trolPB5
Yield of strawberry juice for 15 min after enzymatic treatment for 3 h and its viscosity
0
1
2
3
4
5
6
7
8
PB5 C9 control PB5 C9 control
ml,
cPs
Yield of juice, ml
Viscosity, cPs
CONDITIONS: EP=0,05%, 20 g of wet substrate
25 C 40 C
Dec 5 2014
Juice clarification and decreasing of apple juice viscosity pectinases, β-glucanase, arabinases
commercial preps
lab preps No
enzy
mes
YEILD OF OUTCOMING APPLE JUICE
CONDITIONS:0,05% EP6 hourspH=5T=40C20g minced apples___________
Time of experiment15 minC
9
PB5
host
December 5, 2014
Conversion fructooligosaccharides to fructose and glucose
Jerusalem artichoke syrup processing
245
250
255
260
265
270
275
280
0 5 10 15 20 25 30
Exoinulinase
244
246
248
250
252
254
256
258
260
0 3 6 9
FOS DP 3 - 42
Fructose
SucroseGlucose
Glucose
Fructose
30 min,40 С
Inulinases for food industry (exoinulinase)
December 5, 2014
nC
time, min
0 min
30 min
3h
INULIN before hydrolysis
INULIN after 30 minhydrolysis
INULIN after 3 hhydrolysis
FOS DP 3-7
Inulinases for food industry (endoinulinase)
[EP]=0,5 mg/g S[S]=100g/l (DW)pH=5, T=50C
December 5, 2014
Enzymes in Animal FeedsImproved digestibility and
energy value of feedsReduction of «antinutritional»
factors in feedsImprovement of the
physiological state of animalsGrowth and productivity
acceleration
Feed components and enzymes used:
Wheat, rye, barley, corn – xylanases,β-glucanases, phytaseSoy, peas, lupin – a-galactosidases, protease
Sunflower – protease, hemicellulasesProtein-containing substrates (feather, trimming flour) - (endo)protease
December 5, 2014
“Agroferment Co.” a new enzyme producing plant use our technologies to produce feed enzymes
December 4, 2014
