Functional expression of a thermophilic glucuronoyl esterase from Sporotrichum thermophile: identification of the nucleophilic serine
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- Topakas, E., Moukouli, M., Dimarogona, M. et al. Appl Microbiol Biotechnol (2010) 87: 1765. doi:10.1007/s00253-010-2655-7
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A glucuronoyl esterase (GE) from the thermophilic fungus Sporotrichum thermophile, belonging to the carbohydrate esterase family 15 (CE-15), was functionally expressed in the methylotrophic yeast Pichia pastoris. The putative GE gene ge2 from the genomic DNA was successfully cloned in frame with the sequence for the Saccharomyces cerevisiae α-factor secretion signal under the transcriptional control of the alcohol oxidase (AOX1) promoter and integrated in P. pastoris X-33 to confirm that the encoded enzyme StGE2 exhibits esterase activity. The enzyme was active on substrates containing glucuronic acid methyl ester, showing optimal activity at pH 7.0 and 55°C. The esterase displayed broad pH range stability between 4–10 and temperature stability up to 50°C, rendering StGE2 a strong candidate for future biotechnological applications that require robust biocatalysts. ClustalW alignment of StGE2 with characterized GEs and selected homologous sequences, members of CE-15 family, revealed a novel consensus sequence G-C-S-R-X-G that features the characteristic serine residue involved in the generally conserved catalytic mechanism of the esterase family. The putative serine has been mutated, and the corresponding enzyme has been expressed in P. pastoris to prove that the candidate nucleophilic residue is responsible for catalyzing the enzymatic reaction.
KeywordsGlucuronoyl esteraseNucleophilic serineActive siteLignin–carbohydrate complexSporotrichum thermophilePichia pastoris
One of the recently described carbohydrate esterases (CE) which participates in the plant cell wall degradation is glucuronoyl esterase (GE) which was first discovered in the wood-rot fungus Schizophyllum commune (Špániková and Biely 2006). This esterase might play a significant role in biomass degradation, as it is considered to disconnect hemicellulose from lignin through the hydrolysis of the ester bond between 4-O-methyl-d-glucuronic acid residues of glucuronoxylans and aromatic alcohols of lignin (Špániková and Biely 2006). Since the discovery of this new class of enzymes, the first reported amino acid sequence was revealed in Cip2, a GE from Hypocrea jecorina (Li et al. 2007). The primary sequence data was used to search for homologous genes in several filamentous fungi and bacteria leading to the emergence of a new family of CE-15 on the continually updated carbohydrate-active enzymes (CAZy) database (http://www.cazy.org/; Cantarel et al. 2009), which currently contains 49 members. Based on this bioinformatics analysis, two GEs from the filamentous basidiomycete Phanerochaete chrysosporium (Pc1 and Pc2) were recently cloned and expressed in three different eukaryotic hosts confirming their functionality (Ďuranová et al. 2009a). These enzymes were purified and characterized together with other known GEs using a series of new synthetic substrates that consisted of methyl esters of uronic acids and their glycosides (Ďuranová et al. 2009b). The data obtained indicated that the enzymes hydrolyzed efficiently not only esters of 4-O-methyl-d-glucuronic acid, but also methyl esters of d-glucuronic acid carrying a 4-nitrophenyl aglycon. Moreover, the fact that the enzymes did not recognize the 4-epimers of these compounds, the d-galacturonic acid derivatives, supports the hypothesis that these CEs attack ester linkages between 4-O-methyl-d-glucuronic acid of glucuronoxylan and lignin alcohols.
To date, there is no information on 3-D structures of any CE-15 member, and therefore nothing is known about the catalytic site of GEs. In this respect, the catalytic residues have not been fully identified in members of the CE-15 family. The catalytic domain of Cip2 of H. jecorina was crystallized by Wood et al. (2008), and it awaits solution of its 3-D structure. Moreover, the physiological function of GEs has not yet been established, since there is no direct evidence that this enzyme cleaves the ester linkage between uronic acid and aromatic alcohols, both being cell wall components. Overexpression of recombinant GEs is required for the investigation of the biotechnological potential and probably for the elucidation of their physiological role in plant cell wall degradation. Supporting this idea, our group has identified and purified the first thermophilic GE from the culture filtrate of the fungus Sporotrichum thermophile (Vafiadi et al. 2009). The available genome sequence for an ever-increasing number of microorganisms allows the development of a complementary powerful tool for enzyme discovery. The ability of S. thermophile to produce GEs together with the recently published (June 2009) genome sequence (http://genome.jgi-psf.org/Spoth1/Spoth1.home.html, v1.0, DOE Joint Genome Institute) raised our interest for discovering novel GEs with remarkable properties. Various protein expression systems (Aspergillus vadensis, Pycnoporus cinnabarinus, S. commune) have been explored for the heterologous expression of GEs (Ďuranová et al. 2009a). The methylotrophic yeast Pichia pastoris expression system, as a eukaryotic expression system, has been a favorite system for expressing heterologous proteins due to its many advantages such as protein processing, protein folding, and post-translational modification. Most importantly, the yeast can be grown in inexpensive media, making it an important candidate expression host in industrial biotechnology.
In the present work, we report the successful cloning of the complete genomic DNA sequence of S. thermophile GE gene and its expression in the methylotrophic yeast P. pastoris. ClustalW alignment of characterized GEs and selected homologous sequences, members of CE-15 family, revealed a novel consensus sequence G-C-S-R-X-G that features the characteristic serine residue involved in the generally conserved catalytic mechanism of the esterase family. The putative serine has been mutated, and the corresponding enzyme has been expressed in P. pastoris to prove that the candidate nucleophilic residue is responsible for catalyzing the enzymatic reaction.
Materials and methods
Chemicals and reagents
VentR® DNA Polymerase was purchased from New England Biolabs (Beverly, MA). Perfectprep Gel Cleanup and Nucleospin Plasmid Kits were purchased by Eppendorf (Germany) and Macherey Nagel (Germany), respectively. Zero Blunt® PCR Cloning Kit, pPICZα vectors, and EasySelect™ Pichia Expression Kit were purchased from Invitrogen (USA) while restriction enzymes were purchased from TAKARA (Japan). Methyl esters of 4-O-methyl-α-d-glucopyranuronate (Me-GlcA-1; Hirsch et al. 2005) and 4-nitrophenyl 2-O-(methyl-4-O-methyl-α-d-glucopyranosyluronate)-β-d-xylopyranoside (Me-GlcA-2; Hirsch et al. 1998) were generous gifts from Dr. Ján Hirsch and Dr. Peter Biely (Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia). All other chemicals were of analytical grade.
Strains, vectors, and media
For the cloning of the GE gene from S. thermophile, Escherichia coli One Shot® Top10 (Invitrogen, USA) and Zero Blunt® PCR Cloning Kit (Invitrogen, USA) were used as the host–vector system. P. pastoris host strain X-33 and pPICZαC (Invitrogen, USA) were used for protein expression. The wild type (WT) S. thermophile strain ATCC 42464 was used in the present investigation. The stock culture was maintained on 1.5% malt–peptone–agar slants at 4°C.
E. coli was grown at 37°C in Luria–Bertani medium containing 50 µg kanamycin per milliliter for selection of clones transformed with the Zero Blunt® PCR vector. P. pastoris was routinely grown in shaking flasks at 30°C, in a rich medium containing 1% (w/v) yeast extract, 2% (w/v) peptone, 0.1 M potassium phosphate buffer, pH 6.0, 1.34% (w/v) yeast nitrogen base, 4 × 10−5 biotin, and 1% (v/v) glycerol (BMGY) before induction, or 0.5% (v/v) methanol (BMMY) for induction. For maintaining cultures and plates, 1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) dextrose (YPD) medium was used, and for selection of transformants, YPD plates containing 1 M sorbitol (YPDS) and 100 µg ml−1 Zeocin™ at final concentration were used.
DNA isolation and manipulation
For the production of S. thermophile biomass, the fungus was grown on sucrose (1%, w/v) and yeast extract (1%, w/v) for 2 days at 47°C in a mineral medium previously reported (Topakas et al. 2003). Freeze-dried cells (0.1 g, dry weight) were powdered in a mortar, and total cellular DNA was isolated according to the GenElute™ Plant Genomic Miniprep Kit (Sigma-Aldrich, St. Louis, MO, USA). DNA manipulations were performed according to the standard methodology (Sambrook et al. 1989).
Cloning of ge2 of S. thermophile
An E. coli/P. pastoris vector, pPICZαC, was used to achieve secreted expression of the recombinant GE from S. thermophile. pPICZαC contains the tightly regulated AOX1 promoter and the sequence for the Saccharomyces cerevisiae α-factor secretion signal located immediately upstream of the multiple cloning site (Higgins et al. 1998). The gene for the hypothetical protein StGE2 was PCR-amplified from genomic DNA using primers designed based upon the available ge2 gene sequence located in scaffold_6:2604359-2605552 (S. thermophile v1.0, DOE Joint Genome Institute; http://genome.jgi-psf.org/Spoth1/Spoth1.info.html). The two specific PCR primers were designed as follows: 5′-CCATCGATGGCGCCCATGAACCACATCTTTG-3′ with the ClaI site (underlined) and 5′-CGTCTAGAGCCAGAGTCGGGGCGCCG-3′ with the XbaI site (underlined). A high-fidelity VentR® DNA polymerase producing blunt ends was used for the DNA amplification which was carried out with 35 cycles of denaturation (90 s at 95°C), annealing (90 s at 58°C), and extension (90 s at 72°C), followed by 6 min of further extension at 72°C. In order to determine the DNA sequence, the PCR product was cloned into the pCR-Blunt® vector according to the method described by the Zero Blunt® PCR Cloning Kit. The pCR-Blunt® carrying the ge2 gene was digested with ClaI and XbaI, and the produced fragment was gel-purified before cloning into the pPICZαC vector resulting in the recombinant pPICZαC–ge2 which was amplified in E. coli TOP10F′, and the transformants were selected by scoring for Zeocin™ resistance (25 μg ml−1). The recombinant vector pPICZαC/ge2 was confirmed by restriction analysis and DNA sequencing and finally transformed into P. pastoris.
Transformation of P. pastoris and screening of recombinant transformants
The recombinant plasmid pPICZαC/ge2 was linearized with SacI, and then transformation of P. pastoris and cultivation in shaken flasks were performed according to the EasySelectTMPichia Expression Kit. High-level expression transformants were screened from the YPDS plates containing ZeocinTM at a final concentration of 100 μg ml−1. The presence of the ge2 gene in the transformants was confirmed by PCR using yeast genomic DNA as template.
To screen the P. pastoris transformants for GE expression, ten colonies were grown in 50 ml BMGY medium for 24 h and induced for another 24 h in BMMY medium in shaking flasks at 30°C. GE activity was measured using a semi-quantitative assay on Me-GlcA-1 described in the “Enzyme characterization” paragraph.
Production and purification of recombinant GE
The recombinant P. pastoris harboring the ge2 gene was grown at 30°C in 25 ml BMGY medium in a shaking incubator until the cell density reached an OD600 of 4. Cells were harvested aseptically by centrifugation (5,000×g, 5 min). The cells were then resuspended in 150 ml BMMY medium in a 500-ml flask to an OD600 1 to start the induction. The culture was kept in a shaking incubator at 30°C for 9 days (200 rpm) with the addition of 0.75 ml methanol once a day to maintain induction (0.5% v/v).
For the purification of the recombinant GE, 500 ml of culture broth were centrifuged and concentrated 25-fold using an Amicon ultrafiltration apparatus (Amicon chamber 8400 with membrane Diaflo PM-10, exclusion size 10 kDa, Millipore, Billerica, USA). The concentrate was dialyzed overnight at 4°C against a 20 mM Tris–HCl buffer containing 300 mM NaCl (pH 8.0) and loaded onto a immobilized metal-ion affinity chromatography (IMAC) column (Talon, Clontech; 1.0 cm i.d., 15 cm length) equilibrated with the same buffer. The column was first washed with 60 ml buffer, then a linear gradient from 0 to 100 mM imidazole in 20 mM Tris–HCl buffer containing 300 mM NaCl (60 ml, pH 8.0) was applied at a flow rate of 2 ml min−1. Fractions (2 ml) containing GE activity were concentrated and dialyzed against a 20 mM Tris–HCl buffer pH 8, while the homogeneity was checked by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).
Hydrolase mutants were prepared employing the PCR-based QuikChange mutagenesis kit (Stratagene), according to the vendor's instructions, using plasmid pPICZαC/ge2 as template and primers designed as follows: 5′-TGACGGGCTGCGCGCGCAACGGC-3′ and 5′-GCCGTTGCGCGCGCAGCCCGTCA-3′ (codons altered are in bold). The mutation (S213A) on vector pPICZαC/ge2 was confirmed by DNA sequencing and finally transformed into P. pastoris X-33 by electroporation. Mutant proteins were further produced and purified using the same protocol as described previously.
For the qualitative detection of esterase activity, an assay based on TLC analysis for the hydrolysis of Me-GlcA-1 was used as described previously (Špániková and Biely 2006), changing the incubation temperature from 30°C to 50°C.
Quantitative GE assay was based on the measurement of the decrease of Me-GlcA-2 concentration due to deesterification, as described by Vafiadi et al. (2009). The same substrate and similar reaction conditions were used when the effect on the enzyme activity of phenylmethanesulfonyl fluoride (PMSF) was evaluated. One unit of GE activity is defined as the amount of the enzyme deesterifying 1 µmol of Me-GlcA-2 in 1 min at 50°C. Protein concentration was determined by measuring A280 (Stoscheck 1990) using a molar extinction coefficient of 50,795 M−1 cm−1.
The molecular mass of the purified GE was estimated by SDS-PAGE using unstained protein molecular weight markers from Fermentas. SDS-PAGE was performed according to the method of Laemmli (1970), using a 12.5% polyacrylamide gel.
The effect of pH and temperature on enzyme activity and stability was measured on Me-GlcA-2 (2 mM) using HPLC for detection of the free acid. The optimum pH was determined by measuring the activity at 50°C after incubation for 15 min over the pH region 3.0–11.0 using the buffers 0.1 M citrate–phosphate (pH 3.0–7.0), 0.1 M Tris–HCl (pH 7.0–9.0), and 0.1 M glycine–NaOH (pH 9.0–11). The stability at different pH was determined after incubating the enzyme in the above buffers at 25°C for 24 h and by measuring the residual activity at 50°C and pH 6. The optimum temperature was determined by assaying the enzyme activity at various temperatures (30–70°C) for 15 min in 0.1 M citrate–phosphate buffer, pH 6.0. The thermostability was determined by measuring the residual activity at 50°C and pH 7, after incubation of the purified esterase between 30°C and 70°C and pH 7.0, for different periods of time.
Identification of genes for family 15 esterases in S. thermophile genome
In the last 3 years, the evidence for the occurrence of the GE gene(s) in genomes of a series of fungi led to emergence of a new family of CE-15 (Li et al. 2007; Ďuranová et al. 2009a). However, all protein primary sequences do not provide any evidence of the catalytic mechanism of this new family of esterases which might lead us to the discrimination of their physiological role for the decomposition of plant cell wall.
Protein translations of gene sequences present in the S. thermophile genomic database were searched for sequence identity to known GEs. The sequences identified in the S. thermophile genome were then conversely compared to sequences available in the general databank (National Center for Biotechnology Information, NCBI) using the BLAST program at NCBI (Altschul et al. 1997). The translation of two open reading frames (ORFs) from the S. thermophile genome database shows significant primary sequence identity with GEs. One ORF located at scaffold_6:2604359-2605552 (Model ID: 55568) encodes putative GE showing high sequence identity with characterized GE genes, such as Cg2 (EAQ83419) from Chaetomium globosum CBS 148.51 (82%), Nc1 (EAA29361) from Neurospora crassa OR74A (72%), Pn1 (EAT83498) from Phaeosphaeria nodorum SN15 (58%), and Cip2 (AAP57749) from H. jecorina (55%) (Li et al. 2007). The second ORF located in the same scaffold_6:4254590-4256052 (Model ID: 96309) encodes a putative GE showing high homology with the unknown hypothetical protein EAQ93859 from C. globosum CBS 148.51 (84%) and lower sequence identity with products of known and proposed GE genes, such as CAP59671.1 from Podospora anserina (78%), EAU83544.1 from Coprinopsis cinerea (60%), Cg1 (EAQ82956) from C. globosum CBS 148.51 (34%), and Cip2 (AAP57749) from H. jecorina (33%) (Li et al. 2007). The sequence identity of the two translated ORFs (IDs 55568 and 96309) from the S. thermophile genome is relatively low (30%), while 45% of both sequences consist of amino acids with similar physicochemical properties. The hypothetical protein represented by Model ID: 55568 that shows the higher sequence identity with family CE-15 GEs was selected as a candidate GE from S. thermophile. The corresponding gene which was provisionally named ge2 had no introns based on the bioinformatics analysis provided by the S. thermophile genome database, therefore leading us to the cloning, expression, and characterization of the encoding enzyme named StGE2.
Cloning and expression of StGE2
The uninterrupted ORF of ge2 was introduced in the pPICZαC multiple cloning site, under the control of AOX1 promoter. The resulting expression construct pPICZαC/ge2 and the parent vector were linearized with restriction enzyme SacI to allow gene replacement at the AOX1 locus and were used to transform P. pastoris X-33. All pPICZαC/ge2 transformants produced a major secreted protein product of ca. 43 kDa upon examination of culture supernatants by SDS-PAGE, whereas no protein could be detected with the vector control (data not shown).
To confirm the production of GE activity by the transformants, all ten independent clones were assayed using Me-GlcA-1, a fast method based on TLC analysis detailed in the “Materials and methods” section. The clone showing the highest GE activity was retained for further study. Activity of StGE2 in P. pastoris shaking flasks, using Me-GlcA-2, could be first detected in the medium 1 day after inoculation and peaked at the seventh day with a titer of 5.2 U l−1.
Protein sequence analysis of StGE2: identification of the catalytic nucleophilic serine of family CE-15
The ORF of ge2 encodes a protein of 397 amino acids including a secretion signal peptide of 18 amino acids (MVHLTSALLVAGAAFAAA) based upon the prediction using SignalP, which is a web-based program (Bendtsen et al. 2004). The predicted mass and isoelectric point of the mature protein was 40,056 Da and pH 5.76, respectively, by calculations using the ProtParam tool of ExPASY (Gasteiger et al. 2005). The translated sequence of ge2 gene, in contrast with the partially sequenced StGE1 from S. thermophile ATCC 34628 (Vafiadi et al. 2009), does not feature a recognizable cellulose-binding domain, unlike those observed for the Cip2 from H. jecorina (Li et al. 2007) and GE1 from P. chrysosporium (Ďuranová et al. 2009a). This was further confirmed experimentally, as the StGE2 did not bind to insoluble cellulose (Avicel) and did not demonstrate interaction with soluble derivatized form of cellulose (CMC; data not shown). Moreover, no potential N- and O-glycosylation sites were identified along the whole protein sequence, as predicted by using the NetNGlyc 1.0 and NetOGlyc 1.0 servers (Blom et al. 2004; Julenius et al. 2005).
Effects of pH and temperature on the activity and stability of StGE2
The optimum temperature for the enzyme activity was 55°C; however, the enzyme retained >70% of its activity in the range of 60–70°C (Fig. 4b). After preincubation at pH 7.0 and 55°C for 24 h, the enzyme lost 53% of its activity, while the thermostability of the enzyme was rapidly lost above 60°C. The esterase was relatively stable for temperatures up to 50°C for 24 h, exhibiting half lives of ca. 22.5 and 0.5 h at 55°C and 60°C, respectively.
Genome sequences are rapidly becoming an information source that can be mined to provide clues for novel research. To analyze the GE system of the thermophilic fungus S. thermophile, a BlastP analysis was conducted using amino acid sequences of characterized GE genes against the recently published genome sequence. The gene encoding StGE2 from S. thermophile was expressed in the heterologous host P. pastoris, demonstrating high GE activity, which proves that a bioinformatics-assisted enzyme discovery is highly effective and can identify novel enzymes in an expeditious manner. After induction with methanol, the hydrolytic activity toward Me-GlcA-2 and accumulation of the recombinant enzyme in the culture broth increased significantly up to 5.2 U ml−1 after 7-day culture. This expression yield is 68- and 42-fold higher than the production yield of WT strains of S. thermophile ATCC 34628 and P. chrysosporium ME 446 induced by wheat straw and sugar beet pulp, respectively (Vafiadi et al 2009; Ďuranová et al. 2009a). The P. pastoris expression system has been used successfully to study other recombinant hemicellulolytic esterases, such as feruloyl and acetyl xylan esterases (Moukouli et al. 2008; Koseki et al. 2006), indicating that it is an excellent host for the heterologous expression of robust CEs in the field of Industrial Biotechnology. The molecular mass determined for StGE2 by SDS-PAGE was ca. 43 kDa, which is similar to the other GEs like S. commune esterase (44 kDa; Špániková and Biely 2006), while it differs significantly from CBM-containing GEs reported to date, such as StGE1 from S. thermophile ATCC 34628 (58 kDa; Vafiadi et al. 2009) and Cip2 of H. jecorina (55 kDa; Li et al. 2007). StGE1 identified from the WT strain of S. thermophile ATCC 34628 is a different GE compared to StGE2, due to significant difference in MWs and due to peptides identified in StGE1 by nanoLC-ESI-MS/MS which were found in Cg1 (EAQ82956; MASCOT search) that do no exist in Cg2 (EAQ83419) from C. globosum CBS 148.51, a GE showing the highest homology to StGE2.
The pH and temperature optima (pH 7, 55°C) of StGE2 are in the range reported for other GEs from S. commune (Špániková and Biely 2006), H. jecorina (Li et al. 2007), and S. thermophile ATCC 34628 (Vafiadi et al. 2009). The recombinant enzyme showed significant stability over a broad range of temperatures up to 50°C and a broad pH range of 4–10 after 24 h of incubation. The above stability behavior renders StGE2 a strong candidate for future biotechnological applications in which catalytic stability is an important factor.
The amino acid sequences of the identified genes in the other genomes as well as those of characterized GE-encoding genes were used with the ClustalW program to produce a multiple alignment that was employed to identify a conserved sequence G-C-S-R-X-G, containing a candidate catalytic nucleophile serine as component of the catalytic site of CE-15 hydrolases. The combination of mutational analysis and sequence alignment indicated that Ser213 of StGE2 is important for catalysis, which possibly suggests that the reaction mechanism involves a catalytic triad which is typical of serine esterases. This is further reflected by the fact that 10 mM PMSF reduced the catalytic activity of StGE2, in accordance with previous report on H. jecorina Cip2 GE, in which case 5 and 10 mM of PMSF resulted in more than 80% inhibition of the enzyme activity (Li et al. 2007). However, lower PMSF concentration (1 mM) did not inhibit S. commune GE (Špániková and Biely 2006), indicating the different PMSF sensitivity for the covalent binding of the catalytic serine. StGE2 together with all known identified GEs, which are members of the CE-15 family, exhibit a fingerprint motif G-X-S-X-X-G which does not fit the general consensus sequence G-X-S-X-G found in the known mammalian serine hydrolases, i.e., serine peptide hydrolases, serine esterases, and lipases (Brenner 1988). However, the active site serine sequence of GEs belonging in CE family 15 is similar to Xaa-propyl dipeptidyl aminopeptidases (PepX) which contain a motif (G-X-S-Y-X-G) conserved in lactococcal PepX, mammalian DPPIV, and yeast DPAB (Chich et al. 1992; Kabashima et al. 1996; Vesanto et al. 1995). The PepXs have amidase and esterase activities in addition to peptidase activities (Yoshpe-Besancon et al. 1994). Besides PepXs, a cocaine esterase from Rhodococcus sp. strain MB1 (CocE) found to contain the same motif of serine esterase (G-X-S-Y-X-G), showing on average a sequence identity of 39% and a sequence similarity of 50% with PepXs from Lactococcus lactis and Lactobacillus sp. (Bresler et al. 2000). On the other hand, StGE2 and characterized GEs show no sequence similarity and identity with known members of PepXs and the cocaine esterase CocE. The conserved sequence G-C-S-R-X-G can be proposed as a consensus for enzymes with GE activity of CE-15 hydrolases. Further experiments using site-directed mutagenesis in combination with the first 3-D structure would help determining which residues are important for GE activity.
In conclusion, this is the first report to investigate the catalytic nucleophilic serine of GEs belonging in CE family 15 from the thermophilic fungus S. thermophile functionally expressed in P. pastoris. The search of the physiological role in natural substrates together with the structural and functional study will enhance our understanding on the catalytic mechanism of this novel class of enzymes.
The authors thank Dr. Peter Biely from Institute of Chemistry, Slovak Academy of Sciences (Bratislava, Slovakia) who supplied the synthetic substrates for assaying GE activity.