Fungal Glucuronoyl and Feruloyl Esterases for Wood Processing...
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INDUSTRIAL BIOTECHNOLOGY Introduc9on Feruloyl esterases (FAEs, E.C. 3.1.1.73, CAZy family CE1) and glucuronoyl esterases (GEs, E.C. 3.1.1., CAZy family CE15) are involved in the degrada9on of plant biomass by hydrolysing ester linkages in plant cell walls, and thus have poten9al use in biofuel produc9on from lignocellulosic materials and in biorefinery applica9ons with the aim of developing new wood based compounds [1, 2]. GEs and FAEs are present in the genomes of a wide range of fungi and bacteria. Under condi9ons of low water content, these enzymes can also carry out (trans)esterifica9on reac9ons, making them promising biocatalysts for the modifica9on of compounds with applica9ons in the food, cosme9c and pharmaceu9cal industry. Compared to the chemical process, enzyma9c synthesis can be carried out under lower process temperatures (5060°C) and results in fewer side products, thus reducing the environmental impact. Cita9ons [1] Topakas, E., Vafiadi, C., and Christakopoulos, P. (2007). Microbial produc9on, characteriza9on and applica9ons of feruloyl esterases. Process Biochemistry, 42(4), 497–509. doi: 10.1016/j.procbio.2007.01.007 [2] Spániková, S., and Biely, P. (2006). Glucuronoyl esterase Novel carbohydrate esterase produced by Schizophyllum commune. FEBS Le9ers, 580(19), 4597–601. doi:10.1016/ j.febslet.2006.07.033 [3] Chávez, R., Bull, P., & Eyzaguirre, J. (2006). The xylanoly9c enzyme system from the genus Penicillium. Journal of Biotechnology, 123(4), 413–433. doi:10.1016/j.jbiotec.2005.12.036 [4] Wood, S. J., Li, X.L., Coka, M. a, Biely, P., Duke, N. E. C., Schiffer, M., & Pokkuluri, P. R. (2008). Crystalliza9on and preliminary Xray diffrac9on analysis of the glucuronoyl esterase cataly9c domain from Hypocrea jecorina. Acta Crystallographica. Sec9on F, Structural Biology and Crystalliza9on Communica9ons, 64(Pt 4), 255–7. doi:10.1107/S1744309108004594 [5] Thörn, C., Gustafsson, H., & Olsson, L. (2011). Immobiliza9on of feruloyl esterases in mesoporous materials leads to improved transesterifica9on yield. Journal of Molecular Catalysis B: Enzyma9c, 72(12), 57–64. doi:10.1016/j.molcatb.2011.05.002 Conclusions We characterised new FAE and GE enzymes from mesophilic, thermophilic and coldtolerant filamentous fungi produced in Pichia pastoris. The enzymes were characterised for both their hydroly9c abili9es on various model substrates (methyl ferulate, pNPferulate) for poten9al applica9ons in deconstruc9on of lignocellulosic materials and extrac9on of valuable compounds as well as for their biosynthe9c capaci9es. We tested and op9mised the FAEs’ transesterifica9on capabili9es on ferulate esters in a 1butanolbuffer system, with the aim of using the most promising candidates for the produc9on of an9oxidant compounds with improved hydrophobic or hydrophilic proper9es, such as prenyl ferulate, prenyl caffeate, glyceryl ferulate and 5O(transferuloyl) arabinofuranose. Chávez et al., 2006 Feruloyl esterases (FAEs) Glucuronoyl esterases (GEs) Figure 1. Schema’c representa’on of secondary cell wall polymers in wood (courtesy of Gunnar Henriksson, WWSC). Figure 2. The structure of xylan and site of ac’on of the enzymes of the xylanase complex. 1: endoxylanases; 2: αlarabinofuranosidases; 3: glucuronidases; 4: feruloyl and coumaroyl esterases; 5: acetyl xylan esterases. Adapted from Chávez et al., 2006 [3]. Figure 3. Proposed crosslink structure between lignin and glucuronoxylan in plant cell walls. MeGlcA = 4OmethylDglucuronic acid. The arrow indicates the ester bond hydrolyzed by GE. Adapted from Wood et al., 2008 [4]. Hydroly’c assays GEs MFA FA FAE pNPF pNP FAE BenzD GlcA GlcA GE Methods & Results FAEs For screening of they hydroly9c ac9vity of FAEs and GEs, spectrophotometric assays were used Immobilisa’on on mesoporous silica par’cles (SBA15) Figure 8. Enzyme loading on mesoporous par’cles at different pH values. Immobilisa9on of enzymes can improve the stability and reusability of the biocatalyst, and can also change the substrate specificity [5]. SBA15 (Santa Barbara Amorphous material) is a wellordered, hexagonal mesoporous silica (MPS) matrix with a controllable pore size. It is an akrac9ve support material, as it has a very large surface area that can be func9onalized if desired, and proper9es such as pore structure can be adjusted to individual needs. Immobilisa9on on MPS is done by simple adsorp9on. The enzyme loading is es9mated by measuring protein concentra9on in the supernatant aser immobilisa9on. Adsorp9on is mediated mostly by electrosta9c and hydrophobic interac9on, thus the pH of the used buffer is very important for immobilisa9on efficiency. wash (5x) add enzyme incubate overnight wash (3x) dry & store add MPS 0 0.002 0.004 0.006 0.008 0.01 0.012 0.014 0.016 0.018 pH 3 pH 4 pH 5 pH 6 pH 7 pH 8 pH 9 Enzyme loading (mg Engzyme / mg MPS) 0.1 M citrate phosphate 0.1 M TrisHCl Genome mining Produc’on of fungal enzymes in Pichia pastoris Homologues of known FAEs were found in the genomes of two coldtolerant Aspergillus strains (A. glaucus and A. zonatus), several candidate genes were cloned and subsequently expressed in Pichia pastoris. Ini’al screening of 19 fungal strains (mesophilic and thermophilic) isolated from soil, wood and agricultural compost in Vietnam 0 10 20 30 40 50 60 FEC 40 FEC42 FEC93 FEC108 FEC110 FEC128 FEC130 FEC161 FEC162 FEC258 FEC385 FCH 5.2 FCH 5.4 FCH 5.5 FCH 5.7 FCH 9.4 FCH 10.4 FCH 10.5 mU 0.1M MOPS pH 6.0, 37°C, 30 min 0.1M MOPS pH 6.0, 45°C, 30 min 0 10 20 30 40 50 60 70 80 FEC40 FEC42 FEC93 FEC108 FEC110 FEC128 FEC130 FEC156 FEC161 FEC162 FEC258 FEC385 FCH5.2 FCH5.4 FCH5.5 FCH5.7 FCH9.4 FCH10.4 FCH10.5 mU 0.1 M K3phosphate buffer, pH 6.0, 45°C, 30 min Selec’on of novel FAEs/GEs DNA & RNA sequencing Figure 4. Screening of mesophilic (FEC) and thermophilic (FCH) fungal strains for FAE ac’vity on methyl ferulate (MFA). Figure 5. Screening of mesophilic (FEC) and thermophilic (FCH) fungal strains for GE ac’vity on benzylDglucuronate (BenzDGlcA). Two thermophilic fungal strains were chosen for DNA and RNA sequencing. Mycelium was grown for 48h at 50°C on glucose, washed thoroughly and subsequently divided between shake flasks containing 1% glucose, 1% wheat bran, 1% beechwood xylan or 1% wheat bran + 0.01% ferulic acid as the sole carbon source. Samples were taken at different 9me points, RNA extracted and sequenced. Comparing the expression pakerns of genes on different (inducing and noninducing) media will allow the iden9fica9on of novel FAEs and GEs. These enzymes will then be cloned and overexpressed in the heterologous host Pichia pastoris. condition 1 (e.g. growth on glucose) condition 2 (e.g. growth on wheat bran/xylan)) Transesterifica’on reac’on Figure 6. Example of a chromatogram obtained from HPLC analysis of the products of the transesterifica’on reac’on methyl ferulate (MFA) to butyl ferulate (BFA) in a reac’on system with 92.5% 1 butanol and 7.5% buffer. In a reac9on system with very low water content, FAEs preferably carry out a (trans)esterifica9on reac9on. We used a Myceliophthora thermophila FAE to produce butyl ferulate (BFA) from methyl ferulate (MFA) in a binary reac9on system consis9ng of 1butanol and 7.5% buffer, pH 6.5. At 30°C, aser 69h more than 55% of MFA is converted to BFA, while only 14% of MFA is hydrolysed to ferulic acid (FA). Figure 7. Ra’os of BFA, FA and MFA present in the reac’on at different ’me points. BFA FA MFA MFA + butanol ( ( BFA FA FAE FAE 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0h 4h 6h 23h 27h 48h 69h % BFA % FA % MFA Cellulose Glucomannan Xylan Lignin Cellulose
Transcript of Fungal Glucuronoyl and Feruloyl Esterases for Wood Processing...
IN
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STRIAL B
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TECHN
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Introduc9on Feruloyl esterases (FAEs, E.C. 3.1.1.73, CAZy family CE1) and glucuronoyl esterases (GEs, E.C. 3.1.1.-‐, CAZy family CE15) are involved in the degrada9on of plant biomass by hydrolysing ester linkages in plant cell walls, and thus have poten9al use in biofuel produc9on from lignocellulosic materials and in biorefinery applica9ons with the aim of developing new wood-‐based compounds [1, 2]. GEs and FAEs are present in the genomes of a wide range of fungi and bacteria. Under condi9ons of low water content, these enzymes can also carry out (trans)esterifica9on reac9ons, making them promising biocatalysts for the modifica9on of compounds with applica9ons in the food, cosme9c and pharmaceu9cal industry. Compared to the chemical process, enzyma9c synthesis can be carried out under lower process temperatures (50-‐60°C) and results in fewer side products, thus reducing the environmental impact.
Cita9ons [1] Topakas, E., Vafiadi, C., and Christakopoulos, P. (2007). Microbial produc9on, characteriza9on and applica9ons of feruloyl esterases. Process Biochemistry, 42(4), 497–509. doi:10.1016/j.procbio.2007.01.007 [2] Spániková, S., and Biely, P. (2006). Glucuronoyl esterase -‐ Novel carbohydrate esterase produced by Schizophyllum commune. FEBS Le9ers, 580(19), 4597–601. doi:10.1016/j.febslet.2006.07.033 [3] Chávez, R., Bull, P., & Eyzaguirre, J. (2006). The xylanoly9c enzyme system from the genus Penicillium. Journal of Biotechnology, 123(4), 413–433. doi:10.1016/j.jbiotec.2005.12.036 [4] Wood, S. J., Li, X.-‐L., Coka, M. a, Biely, P., Duke, N. E. C., Schiffer, M., & Pokkuluri, P. R. (2008). Crystalliza9on and preliminary X-‐ray diffrac9on analysis of the glucuronoyl esterase cataly9c domain from Hypocrea jecorina. Acta Crystallographica. Sec9on F, Structural Biology and Crystalliza9on Communica9ons, 64(Pt 4), 255–7. doi:10.1107/S1744309108004594 [5] Thörn, C., Gustafsson, H., & Olsson, L. (2011). Immobiliza9on of feruloyl esterases in mesoporous materials leads to improved transesterifica9on yield. Journal of Molecular Catalysis B: Enzyma9c, 72(1-‐2), 57–64. doi:10.1016/j.molcatb.2011.05.002
Conclusions We characterised new FAE and GE enzymes from mesophilic, thermophilic and cold-‐tolerant filamentous fungi produced in Pichia pastoris. The enzymes were characterised for both their hydroly9c abili9es on various model substrates (methyl ferulate, pNP-‐ferulate) -‐ for poten9al applica9ons in deconstruc9on of lignocellulosic materials and extrac9on of valuable compounds -‐ as well as for their biosynthe9c capaci9es. We tested and op9mised the FAEs’ transesterifica9on capabili9es on ferulate esters in a 1-‐butanol-‐buffer system, with the aim of using the most promising candidates for the produc9on of an9oxidant compounds with improved hydrophobic or hydrophilic proper9es, such as prenyl ferulate, prenyl caffeate, glyceryl ferulate and 5-‐O-‐(trans-‐feruloyl)-‐arabinofuranose.
Chávez et al., 2006
Feruloyl esterases (FAEs)
Glucuronoyl esterases (GEs)
Figure 1. Schema'c representa'on of secondary cell wall polymers in wood (courtesy of Gunnar Henriksson, WWSC).
Figure 2. The structure of xylan and site of ac'on of the enzymes of the xylanase complex. 1: endoxylanases; 2: α-‐l-‐arabinofuranosidases; 3: glucuronidases; 4: feruloyl and coumaroyl esterases; 5: acetyl xylan esterases. Adapted from Chávez et al., 2006 [3].
Figure 3. Proposed cross-‐link structure between lignin and glucuronoxylan in plant cell walls. MeGlcA = 4-‐O-‐methyl-‐D-‐glucuronic acid. The arrow indicates the ester bond hydrolyzed by GE. Adapted from Wood et al., 2008 [4].
Hydroly'c assays
GEs
MFA FA FAE
pNPF pNP FAE
Benz-‐D-‐GlcA GlcA
GE
Methods & Results
FAEs
For screening of they hydroly9c ac9vity of FAEs and GEs, spectrophotometric assays were used
Immobilisa'on on mesoporous silica par'cles (SBA-‐15)
Figure 8. Enzyme loading on mesoporous par'cles at different pH values.
Immobilisa9on of enzymes can improve the stability and reusability of the biocatalyst, and can also change the substrate specificity [5]. SBA-‐15 (Santa Barbara Amorphous material) is a well-‐ordered, hexagonal mesoporous silica (MPS) matrix with a controllable pore size. It is an akrac9ve support material, as it has a very large surface area that can be func9onalized if desired, and proper9es such as pore structure can be adjusted to individual needs. Immobilisa9on on MPS is done by simple adsorp9on. The enzyme loading is es9mated by measuring protein concentra9on in the supernatant aser immobilisa9on. Adsorp9on is mediated mostly by electrosta9c and hydrophobic interac9on, thus the pH of the used buffer is very important for immobilisa9on efficiency.
wash (5x)
add enzyme
incubate overnight
wash (3x)
dry & store
add MPS
0"
0.002"
0.004"
0.006"
0.008"
0.01"
0.012"
0.014"
0.016"
0.018"
pH"3" pH"4" pH"5" pH"6" pH"7" pH"8" pH"9"
Enzyme'load
ing'(m
g'En
gzym
e'/'mg'
MPS)'
0.1"M"citrate"phosphate"
0.1"M"TrisHCl"
Genome mining
Produc'on of fungal enzymes in Pichia pastoris
Homologues of known FAEs were found in the genomes of two cold-‐tolerant Aspergillus strains (A. glaucus and A. zonatus), several candidate genes were cloned and subsequently expressed in Pichia pastoris.
Ini'al screening of 19 fungal strains (mesophilic and thermophilic) isolated from soil, wood and agricultural compost in Vietnam
0"
10"
20"
30"
40"
50"
60"
FEC"40"
FEC42"
FEC93"
FEC108"
FEC110"
FEC128"
FEC130"
FEC161"
FEC162"
FEC258"
FEC385"
FCH"5.2"
FCH"5.4"
FCH"5.5"
FCH"5.7"
FCH"9.4"
FCH"10.4"
FCH"10.5"
mU#
0.1M"MOPS"pH"6.0,"37°C,"30"min"
0.1M"MOPS"pH"6.0,"45°C,"30"min"
0"
10"
20"
30"
40"
50"
60"
70"
80"
FEC40"
FEC42"
FEC93"
FEC108"
FEC110"
FEC128"
FEC130"
FEC156"
FEC161"
FEC162"
FEC258"
FEC385"
FCH5.2"
FCH5.4"
FCH5.5"
FCH5.7"
FCH9.4"
FCH10.4"
FCH10.5"
mU#
0.1"M"K3phosphate"buffer,"pH"6.0,"45°C,"30"min""
Selec'on of novel FAEs/GEs
DNA & RNA sequencing
Figure 4. Screening of mesophilic (FEC) and thermophilic (FCH) fungal strains for FAE ac'vity on methyl ferulate (MFA).
Figure 5. Screening of mesophilic (FEC) and thermophilic (FCH) fungal strains for GE ac'vity on benzyl-‐D-‐glucuronate (Benz-‐D-‐GlcA).
Two thermophilic fungal strains were chosen for DNA and RNA sequencing. Mycelium was grown for 48h at 50°C on glucose, washed thoroughly and subsequently divided between shake flasks containing 1% glucose, 1% wheat bran, 1% beechwood xylan or 1% wheat bran + 0.01% ferulic acid as the sole carbon source. Samples were taken at different 9me points, RNA extracted and sequenced.
Comparing the expression pakerns of genes on different (inducing and non-‐inducing) media will allow the iden9fica9on of novel FAEs and GEs. These enzymes will then be cloned and overexpressed in the heterologous host Pichia pastoris.
condition 1 (e.g. growth on glucose)
condition 2 (e.g. growth
on wheat bran/xylan))
Transesterifica'on reac'on
Figure 6. Example of a chromatogram obtained from HPLC analysis of the products of the transesterifica'on reac'on methyl ferulate (MFA) to butyl ferulate (BFA) in a reac'on system with 92.5% 1-‐butanol and 7.5% buffer.
In a reac9on system with very low water content, FAEs preferably carry out a (trans)esterifica9on reac9on. We used a Myceliophthora thermophila FAE to produce butyl ferulate (BFA) from methyl ferulate (MFA) in a binary reac9on system consis9ng of 1-‐butanol and 7.5% buffer, pH 6.5. At 30°C, aser 69h more than 55% of MFA is converted to BFA, while only 14% of MFA is hydrolysed to ferulic acid (FA).
Figure 7. Ra'os of BFA, FA and MFA present in the reac'on at different 'me points.
BFA
FA
MFA
MFA + butanol
( (
BFA FA FAE FAE
0%#
10%#
20%#
30%#
40%#
50%#
60%#
70%#
80%#
90%#
100%#
0h# 4h# 6h# 23h# 27h# 48h# 69h#
%#BFA#
%#FA#
%#MFA#
Cellulose
Glucomannan
Xylan
Lignin
Cellulose
