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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

Copyright © 2012

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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

6

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

8/7/2017 Internal Use Only 9

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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