Industrial applications of biocatalysts

Mirjana Gelo-PujicSOLVAY, Research and Innovation Center Lyon

ENZINOV: Enzymes Innovations Industries27-28 October 2014

EUROPE

15,800     49            8

ASIA PACIFIC &

REST OF THE WORLD  

6,000         28         4

LATIN AMERICA

3,900         7          1

NORTH AMERICA

3,700         33         2

2

Employees      Sites        R&I center

33% sales31% sales 25% sales

11% sales90% of sales in businesses among the top 3 global leaders

A balanced portfolio of activities, directed at growth regions

A diversified offer serving numerous end markets

Solvay way ‐ a culture of  sustainability,

Solvay ‐ Historical strength in fast‐growing regions

% of Group net sales (2013)

Automotive& Aeronautics17%

Consumer Goods& Healthcare  25%

Agro, Feed  & Food12%

Energy & Environment11%

Building & Construction10%

Electrical & Electronics6%

IndustrialApplications19%

117 IND SITES29400 employees in 55 countries

117 IND SITES29400 employees in 55 countries

15 MAJOR R&I 1950 employees15 MAJOR R&I 1950 employees

€9.9 bnNET SALES€9.9 bnNET SALES

R&I ‐ Opening up Innovation

Academic partnerships a link between fundamental and applied researchExploratory partnerships with start‐ups and venture capital fundsPartnership with market key players (customers, suppliers)

252new patents in 2013

280 m€R&I effort

2. Biotechnologies for an Industrial

Use of living organisms or substances obtained from living material to make products of value from renewable carbon sources using eco‐friendly bioprocesses.

• living organism ‐ essentially of microbial origin• substances obtained from living material – enzymes• products of value ‐ chemicals, materials and biofuels

Biocatalysis

enzyme-catalyzed reaction

Fermentation & Biotransformation

growing / resting cell bioconversions • Catalyze a wide variety of organic reactions

• Very efficient catalysts• Chiral catalysts  selectivity• Act under mild conditions• Environmentally “friendly”• Can be easily prepared by fermentation• etc. etc.

Advantages of using biocatalysts

Biosynthesis

“de novo” synthesis synthetic biology

A Strain or an Enzyme ?

Enzyme is preferred when:‐ commercially available or easy to produce‐ one or two‐step reaction‐ no need for cofactors ‐ good stability‐ label “natural” (e.g. flavors)

Bacteria (Bacillus) Yeasts (Saccharomyces)Fungi (Penicillium)Pyrococcus abyssi  nirtilase

Strain is preferred when:‐multi‐step reactions ‐whole pathway design‐ enzyme not commercially available‐ label “natural” (e.g. flavors)

Bio‐process block

Enzyme / Strain / Library (ex. metagenomic…) screeninggenetic constructions, engineering …

Process for product recovery (DSP)

Process forBiotransformation / fermentationBiocatalysis (bio‐chemistry)

Strain 7 Strain 8 Strain 9 Strain 10Strain 11

Control

Strain 1 Strain 2 Strain 3 Strain 4 Strain 5 Strain 6

A

H

1 12

Enzymatic & microbial screening

Screening of enzymes, wt & recombinant strains

Enzymatic & microbial screening

Bacteria (Bacillus) Yeasts (Saccharomyces)Fungi (Penicillium)Pyrococcus abyssi  nirtilase

Our experiences & examples

Biocatalysis in the synthesis of antioxidant derivatives for dermo-cosmetic applications and their controlled release by skin enzymes

Antioxidants and anti‐aging compounds lack solubility and stability in cosmetic preparations.

Aim:• synthesis of new & original molecules combination of vitamins and anti‐oxidans: Resveratrol, 

Luteolin, Tetrahydrocurcumin, Lipoic acid, Vitamins (A & E), Ferulic acid, L‐Carnosine• stable precursors of bioactive compounds • their controlled release by the action of extracellular skin enzymes

Solvay (Rhodia) - Chanel partnershipWO2006/134282; International Journal of Cosmetic Science, 2008, 30, 195

Resveratrol‐lipoate derivative

Enzymatic step is required to selectively remove an acetate group from Res(Ac)3 precursor

Substrate concentration:        200  g/l Enzyme load: 1 % w/wProductivity:  15 g/l/hIsolated yield:            95 %Selectivity:                                   99 % di‐Acetate (1% mono‐Acetate)

Solvay (Rhodia) ‐ Chanel partnershipWO2006/134282; International Journal of Cosmetic Science, 2008, 30, 195

Key step ‐ selective de‐acetylation of resveratrol triacetate

Res(Ac)2Lipo

Res(Ac)2

ResOH

Res(Ac)

Lipoic acid

6h

8h

Stratum corneumAcO

OAc

OO

SS

HO

OH

OH

HOO

SS+ +

Acetate

In vitro hydrolysis with skin enzymes 

 

0

20

40

60

80

100

0 3 6 9 12

t (h)

Res(Ac)2‐LipoateIntermediatesResveratrol

 

Area (%) at 300 nm

New original molecules, stable and non‐toxic precursors of antioxidants

Efficient and easy method for preparation of a human Stratum corneum enzymes

POC of in vitro hydrolysis of different resveratrol precursors with skin enzymes

Toxicology   Local Lymph Node Assay (LLNA) NegativeAmes NegativeAcute oral toxicity Non classified (LD50 > 2000 mg/kg)Skin irritation Non irritantEye irritation Irritant

t (min)

0 10 20 30 40 50 60 70 80

mAU

0

20

40

60

80

100

120

t (min)

0 5 10 15 20 25 30 35 40

mAU

0

20

40

60

80

100

120

140 UV (Sun test)

Blank at t=0

45 °C 45 °CUV Blank

4000 3200 2400 1800 1400 1000 650

cm-1

%T

45 °C

UV (Sun test)

Blank at t=0

4000 3200 2400 1800 1400 1000 650

cm-1

%T

UV (Sun test)

Blank at t=0

45 °C

Stability & sun test

Our experiences & examples

Fermentation / biotransformation in the synthesis of L‐pipecolic acid from L‐Lysine using Streptomyces pristinaespiralis L‐Lysine CycloDeaminase (LCD)

Polyketides from Streptomyces spp

HO

OO

N

O

OO

OOH

O

O

OHR

FK506 R =FK520 R =

NHO

NHO

HN

O O

NO

OHN

O

N

O

O

N

O

N

Pristinamycin PI Rapamycin

HO

O

O O

ON

O

O

O

OH

OO

HO

O

L‐pipecolic acid

Solvay (Rhodia) – Evologic (Virtual genome) collaboration

Synthesis of L-pipecolic acid from L-Lysine using Lysine Cyclo Deaminase

Biochimie vol. 89 issue 5 May, 2007. p. 591-604

• Identification & characterization of Streptomyces pristinaespirales wt LCD 

• pipA gene cloned & expressed as native and His‐tag LCD in E.coli

• Characterization of recombinant LCD enzyme

• Random mutagenesis to increase  LCD performances

0,E+00

4,E+05

8,E+05

1,E+06

2,E+06

0 20 40 60 80

temperature, C

Are

a PP

A p

eak

0,E+00

4,E+05

8,E+05

1,E+06

noNa+NH4+ Al3+Fe2

+Ca2

+Zn2+Mn2+Mg2+

Metal at 3 mM

Are

a PP

A p

eak

pH

4 5 6 7 8 9

PPA

pea

k A

rea

1e+5

2e+5

3e+5

4e+5

5e+5

6e+5

7e+5

1‐7 m3fermenter

0‐70 l fermenter

50‐120 m3fermenter

Ferulic acid preparation tanks

4 ml  cryotube

5 l  flask

1mL cryotube

1L flask

1m3 fermenter

20L fermenter

DSP

Steps: • Growth of recombinant E.coli on C source & biomass production (culture seed in a 1l flask, 20L 

inoculum fermenter, 1m3 production fermenter)• Induction of enzyme production • Cell permeabilisation, lysine addition and biocatalysis• Pipecolic acid isolation and purification

Substrate (L‐Lys) concentration:           120 g/l Productivity:  5  g/l/hIsolated yield:                                             75      %Selectivity:                                                100      %

Synthesis of L‐pipecolic acid from L‐Lysine using  Lysine Cyclo Deaminase

• Highly efficient & selective conversion of L‐Lys  L‐pipecolic• Random mutagenesis of LCD toward novel substrates using XL1‐Red mutator strain• Colorimetric test for a rapid screening of mutants

C5 and C6 amino acid substrates  4‐position can be C, O, N, S, C‐OH

4. Conclusions

The use of enzymes has been expanded by advances in genetic engineering, “omics”, synthetic biology, improved stabilization and immobilization techniques and by understanding of structure-function relationships…

Industrial applications are today in pharmaceutical chemistry & healthcare industry, food and consumer products, biopolymers, bioremediation…

There is an enormous potential among the microbial world with almost no reaction that can not be catalyzed by an enzyme !Enzymes can do what chemists have always been dreamed of and have not been able to realize. You just have to imagine !

Team Financial support

Claude BensoussanFabien BarbiratoVéronique BastianiMarie-Claude Bontoux Corporate ResearchFlorence Foray Rhodia OrganicsMirjana Gelo-Pujic Marie Currie FellowshipCédric MarionErwan Vermel Virginie Neugnot-Roux (postdoctoral fellow)Georgia Tsotsou (postdoctoral fellow)Interns

Acknowledgments

Thanks for your attention