mirjana gelo-pujic solvay, research and innovation center lyon · industrial applications of...
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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