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LULEÅ UNIVERSITY OF TECHNOLOGY DIVISION OF CHEMICAL ENGINEERING BIOCHEMICAL PROCESS ENGINEERING
Biocatalytic applications of feruloyl and glucuronoyl
esterases P. Christakopoulos
2nd Lund Symposium on lignin and
hemicellulose valorisation
NOVEMBER 3-4, 2015
Covalent linkages between lignin and hemicelluloses
p-coumaric or ferulic acid, linked etherically to lignin, and esterically to hemicellulose sugars (feruloyl esterases)
Covalent linkages between lignin and hemicelluloses
ether linkages between OH-groups of saccharides and lignin alcohols (benzyl-ethers) (hemicellulose:lignin etherase – not identified yet) (Softwood)
Covalent linkages between lignin and hemicelluloses
ester linkages between 4-O-methyl-D-glucuronic acid (MeGlcA) or D-glucuronic acid residues of glucuronoxylans and hydroxyl groups of lignin alcohols benzyl-esters (glucuronoyl esterases) (Softwood)
Should be clarified in natural substrates
Carbohydrate and lignin based biorefinery
Carbohydrate and lignin based biorefinery
Substrate Specificity of FAEs Feruloyl
esterase type:
-A-
-B-
-C-
-D- Hydrolysis of
methyl esters of: MSA MFA
MpCA MCA MFA
MFA MCA MSA MpCA
MFA MSA MCA MpCA
Release of diferulic acid:
Yes (5,5’)
No No Yes (5,5’)
Crepin et al (2004)
Tyr80, that is responsible for interaction with the hydroxyl group on the phenol ring and the oxygen in the methoxy
side group.
Potential applications of FAEs
FAEs Hydrolysis of ester bond Synthesis of ester bond
Modification of Hydroxycinnamates using FAEs
Aliphatic alcohols (Increase Lipophilicity-allow their
application in oil-based processes)
Mono/oligo saccharides (Increase Hydrophilicity-allow their
application in water-based processes-antitumor activity)
Esterification with (FAEs)
Phenolic (sugar) ester Synthesis
Surfactantless microemulsions as a reaction system for various phenolic acids catalyzed by
feruloyl esterases
Modification to Alkyl Hydroxycinnamates
R1 R2 R3 Ferulic acid (FA) OCH3 OH H
p-Coumaric acid (pCA) H OH H
Caffeic acid (CA) OH OH H
Sinapinic acid (SA) OCH3 OH OCH3
Reaction System: n-hexane:1-butanol:water
The synthetic activity pattern of esterases is similar to that of hydrolytic action of the enzyme against various methyl esters of cinnamic acids
n=0 arabinose n=1 ~biose n=2 ~triose n=3 ~tetraose n=4 ~pentaose n=5 ~exaose
Synthesis of phenolic sugar esters
Esterified on the primary hydroxyl group situated on the non-reducing arabinofuranose ring
The enzymatic esterification of glycerol with SA catalysed by AnFaeA was the first example of activity of FAEs in ILs. Esterified glycerol has a satisfactory antioxidant activity against LDL oxidation in vitro and therefore expands the use of SA as an antioxidant in adequate processes.
The synthetic reaction was optimised in hexafluorophosphate anion-containing ionic liquids [C2OHmim][PF6] and the highest conversion yield was 72.5 ± 2.1%,
AnFaeA was immobilised according to the CLEAs methodology, using ethanol as precipitant and 100 mM glutaraldehyde
Crystals of FoFAEC
Identification of the catalytic Ser in glucuronoyl esterases, members of family CE-15
Identification of a new fingerprint motif G-C-S-R-X-G which does not fit the general consensus sequence G-X-S-X-G
StG
E2 S
213 A
• StGE2 structure determined to
1.55 Å resolution
• 2nd structure of CE15 family of
CAZy database
• 3-layer αβα sandwich
architecture, similar to Hypocrea
jecorina GE
• Catalytic triad involving Ser213,
Glu236, His346
Structural studies of a Myceliophthora thermophila glucuronate esterase, StGE2
Structure of S213A mutant in complex with methyl 4-O-methyl-β-D-glucuronate (MCU)
• The methoxy group enhances binding via van der Waals interactions
Acta Crystallographica (2013)
Enzymatic synthesis of D-glucuronoyl esters TLC separation
Substrate Km (mM) kcat (min-1) kcat/ Km (mM-1∙min-1)
StGE2 PaGE1 StGE2 PaGE1 StGE2 PaGE1 IV 3.63 (0.6) 2.66 (0.5) 115.9 (7.7) 315.3 (40.4) 31.9 (6.1) 118.6 (29.0)
V 7.24 (3.3) 0.94 (0.1) 166.4 (62.4) 46.5 (2.7) 23 (13.4) 49.4 (7.8)
VI n.d. 1.34 (0.4) n.d. 11.4 (1.5) n.d. 8.5 (2.6)
Purification Identification (NMR)
Substrate specificity for StGE2 & PaGE1
Reaction
Higher affinity towards substrate IV (cinnamyl ester)
Enzymatic synthesis of novel esters recognized by GEs
Appl Microbiol Biotechnol (2014) simple ester LCC mimics comprising glucuronoyl esters of alkyl and arylalkyl alcohols,
GE Screening and Characterization Assays Utilizing
Benzyl Glucuronate
Molecules (2015)
OPTIBIOCAT is a four-year project funded by the 7th Framework Programme (FP7) The AIM of OPTIBIOCAT is to replace chemical processes currently used for the production of cosmetics with cost-effective, energy-efficient and eco-friendly bioconversions. These bioconversions are based on transesterication reactions catalyzed by feruloyl esterases (FAEs) and glucuronoyl esterases (GEs) for the production of molecules with antioxidant activity belonging to the classes of phenolic fatty and sugar esters.
OPTIBIOCAT
The consortium: University of Naples (Coordinator) BIOCOM AG CBS-KNAW Chalmers University of Technology CLEA Technologies Dyadic NL INRA KORRES Lulea University of Technology NZYTech ProteoNic Service XS SUPREN GmbH Taros Chemicals University of Helsinki Westfälische Wilhelms-Universität Münster
OPTIBIOCAT objectives An inventory of novel fully characterized recombinant FAEs and GEs: - 50 novel esterases from fungi - 500 novel esterases from bacteria - 25 rationally designed mutants - 20 best directed evolved mutants
Optimized biocatalysts based on FAEs and GEs exhibiting higher operational stability, higher thermo-resistance, higher yield, higher productivity.
A library of 60 novel compounds belonging to the classes of phenolic fatty esters and phenolic sugar esters fully characterized for their antioxidant activity
.
Scale-up of production of at least four FAEs- and GEs- biocatalysts
Four new chemical entities (leads) for the cosmetic industry
Techno-economic viability of the developed processes, within their supply/value chain and applying life cycle thinking (LCA), with demonstration of a significant improvement of the economic efficiency and environmental performance of existing and future biorefineries
Enhanced awareness about OPTIBIOCAT biocatalysts, bioconversions and product of among the stakeholders.
Six main targeted biological active compounds -prenyl ferulate -prenyl caffeate -arabinose ferulate -glyceryl ferulate -benzyl D-glucuronate and -prenyl D-glucuronate
Our aim Efficient synthesis of 6 main targeted biological active compounds: prenyl ferulate prenyl caffeate glyceryl ferulate arabinose ferulate benzyl D-glucuronate and prenyl D-glucuronate
Task 5.2. Evaluation of synthetic abilities of FAEs and GEs
Task 5.3. Optimization of reaction conditions
OHO
OHOH
OO
OH
OH
OO
O
OH
OHO
O
OH
OO
O OHOH
OHO
OHOH
OO
OH
OH
OO
O
OH
OO
O
O OHOH
OH
Prenyl ferulate
Prenyl caffeate
Glyceryl ferulaten-Butyl ferulate
Prenyl D-glucuronate
5-O-trans-feruloyl-L-arabinose
Benzyl D-glucuronate
Step by step optimization:
Optimization of reaction conditions
Water content Donor concentration (vinyl ferulate)
Acceptor concentration (prenol)
Enzyme concentration
Factors:
pH
(ongoing)
Temperature /reaction time
Organic solvents offering higher donor solubility
40oC, pH 6, 8 h of incubation, hexane :t-butanol: MOPS-NaOH
Optimization of reaction conditions
Enzyme TIMES INCREASE comparing to initial
conditions
Conversion to prenyl ferulate
Total conversion
Synthesis: hydrolysis ratio
C1FaeA1 6.51 1.87 3.48
C1FaeA2 5.17 1.34 3.73
C1FaeB1 3.53 1.96 1.80
C1FaeB2 3.16 1.87 1.69
MtFae1a 2.23 1.20 1.86
Acceptor and enzyme concentration are crucial factors for improving the ratio between synthesis and hydrolysis
C1FaeB2 is the best enzyme for the synthesis of prenyl ferulate, although it exhibited moderate
improvement after optimization, comparing to type A FAEs. MtFae1a has lower substrate specificity and higher tolerance to prenol comparing to C1FaeB2
Enzyme Conversion to prenyl ferulate (%)
Total conversion (%)
Synthesis: hydrolysis ratio (%)
C1FaeA1 30.3 : 69.7
C1FaeA2 12.3 : 87.7
C1FaeB1 48.3 : 51.7
C1FaeB2 67.2 : 32.8
MtFae1a 48.6 : 51.4
At optimal conditions:
Indications of StGE2 synthetic potential
Isolation of zone II product: StGE2 catalyzed esterification of GlcA with Bu (water content 3.2%)
Zones III & IV: might represent ring opening/closing isomers
Identification of butyl D-glucuronate by MS (m/z 271.2)
(W) 2%, 3.2% & 5%: GlcA & butanol (Bu) or heptanol (H)
Structural studies of FoFaeC • Belongs to the fungal tannase
superfamily of ESTHER database
• One member with determined crystal
structure: FaeB from Aspergillus
oryzae (AoFaeB)
• FoFaeC structure determined to 2.3 Å
resolution using AoFaeB as starting
model
• Very similar overall fold
• Novel lid domain with a Ca2+ binding
site
• CS-D-HC motif: disulphide bond next
to catalytic triad
FoFaeC AoFaeB
MSA activity is probably an artefact due to the small methyl group that allows a flipped orientation (for MSA, no correct orientations were found)
Small molecule docking experiments of FAE substrates to
FoFaeC
Binding of MSA methoxy groups into body of FoFaeC
Comparison of FoFaeC and AoFaeB active site for FA (left) and SA(right)
Is within the functionally active distance
FoFaeC mutant compared with WD with docked MFA (a) of MSA (b)
Opens up
the right side of the pocket allowing the methoxy to fit
Under this orientation the methyl group is not involved in the binding and could be replaced by sugar
Docking of GA and MGA
WT G238N/L311H
Conclusions
Understanding structure-function relationships can help in optimizing the reaction conditions of enzyme based biocatalytic applications. The correlation of synthetic and hydrolytic
activity is a big challenge for both enzymes
Acknowledgement
• Evangelos Topakas • Christina Vafiadi • Io Antwnopoulou • Cameron Hunt • Maria Moukouli • Marianna Charavgi • Maria Dimarogona • Peter Biely • Lisbeth Olsson • Hampus Sunner