BIO Performance Enzymes - Biotechnology Innovation ... Slide - Default Innovation Center,...
Transcript of BIO Performance Enzymes - Biotechnology Innovation ... Slide - Default Innovation Center,...
Title Slide - Default
Innovation Center,
DEVELOPMENT & COMMERCIALIZATION OF PERFORMANCE ENZYMES FOR FOOD
Vince Sewalt, Senior Director, Product Stewardship & Regulatory
Genencor DuPont Innovation Center, Palo Alto CA 8/7/2017
.
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DUPONT INDUSTRIAL BIOSCIENCES BUSINESS SCOPE
Bioactives Biomaterials Biofuels
Bioprocessing aidsBiofuel technology
BiochemicalsBiomaterials
Enzymes, Proteins
Peptides, Betaine, Probiotics
Key Capabilities to develop, operate and commercialize cell factories
Microbiology
Protein engineeringMetabolic engineering
Strain EngineeringBiochemistry
Safety Assessment
Bioprocess Scale-UpPolymer scienceMolecular Biology
Applying the power of biotechnology to enable business and environmental success
Copyright © 2016 DuPont. All rights reserved.
Bioinformatics
RegulatoryAnalytical Chemistry
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TAILORING ENZYMES BY PROTEIN ENGINEERING & HIGH-THROUGHPUT SCREENING
Copyright © 2012
DuPont. All rights
reserved. 3
Micro-scale screen
development
Library Design
Robust expression
system
Super Screen
Lead Validation
Combina-torials
Product Platform
• Gene discovery
• IP assessment
per week:
• 10,000 variants
• 10 assays per variant
105 data points
• Application
• IP assessment
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STRAIN OPTIMIZATION AND FERMENTATION SCALE-UP
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reserved. 4
Optimize Expression
Host Select Best
CandidatesTest Robustness Plant Transfer
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Innovation Center,
MAKING SURE OUR ENZYMES ARE SAFE(AND THAT OUR METHODS ARE UP-TO-DATE)
.
Sewalt et al. 2016. The Generally Recognized as Safe (GRAS) Process for
Industrial Microbial Enzymes. Industrial Biotechnology 11 (2016): 295-302
http://online.liebertpub.com/doi/pdfplus/10.1089/ind.2016.0011
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ENZYME SAFETY EVALUATION BY PUBLISHED DECISION TREE
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Safety
Margin
Safety
Margin
Safety
Studies
Manufacturing
Process
StrainEnzymeEnzyme
1. Strain genetically modified?
2. Modification by rDNA?
5. Strain well characterized, no
pleiotropic effects?
4. DNA randomly integrated ?
3e. All introduced DNA well
characterized and safe?
3c. TA Free of transferable anti-
biotic resistance gene DNA?
3a. Expressed product
history of safe use ?
Test Article (TA) is accepted
11. Is NOAEL for TA sufficiently
high to ensure safety?
6. Production strain from a Safe
Strain Lineage?
7. Production strain non-
pathogenic?
8. TA free of antibiotics?
9. TA free of oral toxins known
for members of this species?
10. Are the toxin levels in TA
below levels of concern?
Test Article is not accepted
3b. NOAEL of TA OK?
3d. Resistance gene encodes
for antibiotic used in man/animal?
N
Y
Y
Y
Y
Y
N
Y
Y
Y
N
N
N
N
N
YY
N
Y
N
N
Y
N
Y
Y
N
N
N
Y
(Pariza and Johnson, 2001; Pariza and Cook, 2010)
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Innovation Center,
LEADING THE ENZYME INDUSTRY’S DEFENSE OF SOLID REGULATIONS SUCH AS GRAS
.
Sewalt et al. 2017.
Letter to the editor
regarding “GRAS from
the ground up: Review
of the Interim Pilot
Program for GRAS
notification” by Hanlon
et al., 2017. Food and
Chemical Toxicology
xxx (in press).
http://dx.doi.org/10.101
6/j.fct.2017.06.042
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Innovation Center,
DRIVING GLOBAL ADVOCACY FOR SCIENCE BASED REGULATION OF BIOTECHNOLOGY
.
case-by-case evaluation,
‘from scratch’
Knowledge
building as
lever
2 columns: 50%/50%
JUST 1 EXAMPLE – G4 AMYLASE
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G4 amylase chops maltotetraose from starch
Applications:
Baking application for increased softness and shelf life
Sugar syrups with high DP4 (prebiotic)
BAKING APPLICATION – ALTER STARCH RETROGRADATION
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Dough Stage Fresh Bread Reheating Stale Bread
Amorphous amylose Amylose helix Crystalline Gelled
amylopectin amylopectin
Retrograded amylose Amylose Gluten Polar lipid complex
Amylopectin – shortening of side chain reduces retrogradation
AgeingBaking
Modified from Zobel & Kulp, Baked Goods Freshness, eds. Hebeda & Zobel; Marcel Dekker, Inc., 1996
PROPERTIES AND APPLICATIONS OF MALTOTETRAOSE (DP4)
Reference 11
Properties
Stable to heat and acid, low Maillard reactivity,
colorless
Mild sweetness (1/3 of sucrose)
Clean tasting, well-digested
Resistant to retrogradation of starch gel
Higher moisture retainment at low humidity,
lower moisture absorptivity at high humidity than
sucrose
Higher freezing point
Higher viscosity
Inhibits growth of intestinal putrefactive bacteria
Uses
- property enhancer for various foods &
powdering materials
- saccharide for dry milk
- liquid diets for the sick
- a body reinforcement agent for refreshing
drinks.
- Prebiotic
ENZYME ENGINEERING STRATEGIES
Back bone for enzyme engineering:Maltotetraohydrolase (= PS4wt) from Pseudomonassaccharophila
Engineering and directed evolution:ThermostabilityExo-specificity
Assays/high throughput screening systems: HTS on stability: Residual activity at 60-90CHTS on exo-specificity based on activity ratiosFunctionality tests in model baking or regular baking trials
CONCLUSIONS AND PERSPECTIVES
Technology Achievements:
Exo-specificity: increased > 5 times vs. wildtype G4 amylase
Thermostability: increased by >31°C
Product specificity: maltotetraose is main hydrolysis product
Patented
Approved ~globally (most recently in Indonesia and Japan)
Customer gains:
Superior bread & tortilla softening throughout shelf life
DP4 sugar syrups (food texture, energy drinks, prebiotic)
G4
Neg. control
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E. I.du Pont de Nemours and Company or its affiliates.
Nothing contained herein shall be construed as a representation that any recommendations, use or resale of the product or process
described herein is permitted and complies with the rules or regulations of any countries, regions, localities, etc., or does not infringe
upon patents or other intellectual property rights of third parties.
The information provided herein is based on data DuPont believes to be reliable, to the best of its knowledge and is provided at the
request of and without charge to our customers. Accordingly, DuPont does not guarantee or warrant such information and assumes no
liability for its use. If this product literature is translated, the original English version will control and DuPont hereby disclaims
responsibility for any errors caused by translation. This document is subject to change without further notice.
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