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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