Functional Expression and Characterization of Acetyl Xylan Esterases CE Family 7 from Lactobacillus antri and Bacillus halodurans.

Acetyl xylan esterase (AXE; E.C. 3.1.1.72) is one of the accessory enzymes for xylan degradation, which can remove the terminal acetate residues from xylan polymers. In this study, two genes encoding putative AXEs (LaAXE and BhAXE) were cloned from Lactobacillus antri DSM 16041 and Bacillus halodurans C-125, and constitutively expressed in Escherichia coli. They possess considerable activities towards various substrates such as p-nitrophenyl acetate, 4-methylumbelliferyl acetate, glucose pentaacetate, and 7-amino cephalosporanic acid. LaAXE and BhAXE showed the highest activities at pH 7.0 and 8.0 at 50°C, respectively. These enzymes are AXE members of carbohydrate esterase (CE) family 7 with the cephalosporine-C deacetylase activity for the production of antibiotics precursors. The simultaneous treatment of LaAXE with Thermotoga neapolitana ß-xylanase showed 1.44-fold higher synergistic degradation of beechwood xylan than the single treatment of xylanase, whereas BhAXE showed no significant synergism. It was suggested that LaAXE can deacetylate beechwood xylan and enhance the successive accessibility of xylanase towards the resulting substrates. The novel LaAXE originated from a lactic acid bacterium will be utilized for the enzymatic production of D-xylose and xylooligosaccharides.

Identification and characterization of an acetyl esterase from Paenibacillus sp. XW-6-66 and its novel function in 7-aminocephalosporanic acid deacetylation.

OBJECTIVES: To obtain a new acetyl esterase from Paenibacillus sp. XW-6-66 and apply the enzyme to 7-aminocephalosporanic acid (7-ACA) deacetylation. RESULTS: The acetyl esterase AesZY was identified from Paenibacillus sp. XW-6-66, and its enzymatic properties were investigated. With the putative catalytic triad Ser114-Asp203-His235, AesZY belongs to the Acetyl esterase (Aes) family which is included in the α/ß hydrolase superfamily and contains the consensus Gly-X-Ser-X-Gly motif. The maximum activity of AesZY was detected at pH 8.0 and 40 °C. AesZY was stable at different pH values ranging from 5.0 to 12.0, and was tolerant to several metal ions. Furthermore, the deacetylation activity of AesZY toward 7-ACA was approximately 7.5 U/mg, and the Kcat/Km value was 2.04 s-1 mM-1. CONCLUSIONS: Our results demonstrate the characterization of a new acetyl esterase belonging to the Aes family with potential biotechnological applications.

Two Novel Acetylesterases from Pantoea dispersa: Recombinant Expression, Purification, and Characterization.

Two novel acetylesterases from Pantoea dispersa, with low amino acid sequence identity between them, were expressed in Escherichia coli with a carboxyl-His6 tail given by the expression plasmid, purified, and characterized. The purified proteins, named Est-1 and Est-2, had a molecular mass of 33 kDa and 37 kDa, respectively. Both proteins presented a modeled structure of homodimers with monomers presenting the α/ß-hydrolase fold, with the catalytic triad Ser-Asp-His present in the active site. The KM for p-nitrophenyl acetate and Vmax values found for Est-1 were of 1.4 ± 0.2 mM and 8.66 ± 0.59 µmol/min and for Est-2 were of 0.36 ± 0.077 mM and 6.13 ± 0.56 µmol/min, respectively. Both enzymes presented an optimum pH of 7.0. The optimum temperature for Est-1 was 40 °C and for Est-2 was 50 °C. The temperatures in which the enzymes Est-1 and Est-2 lost half of their activity (T50) were 44.1 and 58.9 °C, respectively. SDS, EDTA, and PMSF significantly inhibited the enzymes. The two purified enzymes also presented activity against triacetin and were able to deacetylate the carbohydrates pectin and xylan, with higher activity against pectin. Thus, they could be considered as carbohydrate esterases.

Structural and functional analysis of de-N-acetylase PgaB from periodontopathogen Aggregatibacter actinomycetemcomitans.

The oral pathogen Aggregatibacter actinomycetemcomitans uses pga gene locus for the production of an exopolysaccharide made up of a linear homopolymer of ß-1,6-N-acetyl-d-glucosamine (PGA). An enzyme encoded by the pgaB of the pga operon in A. actinomycetemcomitans is a de-N-acetylase, which is used to alter the PGA. The full length enzyme (AaPgaB) and the N-terminal catalytic domain (residues 25-290, AaPgaBN) from A. actinomycetemcomitans were cloned, expressed and purified. The enzymatic activities of the AaPgaB enzymes were determined using 7-acetoxycoumarin-3-carboxylic acid as the substrate. The AaPgaB enzymes displayed significantly lower de-N-acetylase activity compared with the activity of the deacetylase PdaA from Bacillus subtilis, a member of the CE4 family of enzymes. To delineate the differences in the activity and the active site architecture, the structure of AaPgaBN was determined. The AaPgaBN structure has two metal ions in the active site instead of one found in other CE4 enzymes. Based on the crystal structure comparisons among the various CE4 enzymes, two residues, Q51 and R271, were identified in AaPgaB, which could potentially affect the enzyme activity. Of the two mutants generated, Q51E and R271K, the variant Q51E showed enhanced activity compared with AaPgaB, validating the requirement that an activating aspartate residue in the active site is essential for higher activity. In summary, our study provides the first structural evidence for a di-nuclear metal site at the active site of a member of the CE4 family of enzymes, evidence that AaPgaBN is catalytically active and that mutant Q51E exhibits higher de-N-acetylase activity.

Cloning, Purification and Characterization of Acetyl Xylane Esterase from Anoxybacillus flavithermus DSM 2641(T) with Activity on Low Molecular-Weight Acetates.

Family 4 carbohydrate esterases (CE-4) have deacetylate different forms of acetylated poly/oligosaccharides in nature. This family is recognized with a specific polysaccharide deacetylase domain assigned as NodB homology domain in their secondary structure. Most family 4 carbohydrate esterases have been structurally and biochemically characterized. However, this is the first study about the enzymological function of pdaB-like CE4s from thermophilic bacterium Anoxybacillus flavithermus DSM 2641(T). A. flavithermus WK1 genome harbors five putative CE4 family genes. One of them is 762 bp long and encodes a protein of 253 amino acids in length and it was used as reference sequence in this study. It was described as acetyl xylane esterase (AXE) in genome project and this AfAXE gene was amplified without signal sequence and cloned. The recombinant protein was expressed in E. coli BL21 (DE3), purified by nickel affinity chromatography and its purity was visualized on SDS-PAGE. The activity of the recombinant enzyme was shown by zymogram analysis with α-naphtyl acetate as a substrate. The enzyme was characterized spectrophotometrically using chromogenic p-nitrophenyl acetate. Optimum temperature and pH were determined as 50 °C and 7.5, respectively. Km and Vmax were determined as 0.43 mM and 3333.33 U/mg, respectively under optimum conditions. To our knowledge this is the first enzymological characterization of a pdaB-like family 4 carbohydrate esterase from the members of Anoxybacillus genus.

Hydrolysis of wheat arabinoxylan by two acetyl xylan esterases from Chaetomium thermophilum.

The thermophilic filamentous ascomycete Chaetomium thermophilum produces functionally diverse hemicellulases when grown on hemicellulose as carbon source. Acetyl xylan esterase (EC 3.1.1.72) is an important accessory enzyme in hemicellulose biodegradation. Although the genome of C. thermophilum has been sequenced, its carbohydrate esterases are not annotated yet. We applied peptide pattern recognition (PPR) tool for sequence analysis of the C. thermophilum genome, and 11 carbohydrate esterase genes were discovered. Furthermore, we cloned and heterologously expressed two putative acetyl xylan esterase genes, CtAxeA and CtAxeB, in Pichia pastoris. The recombinant proteins, rCtAxeA and rCtAxeB, released acetic acids from p-nitrophenyl acetate and water-insoluble wheat arabinoxylan. These results indicate that CtAxeA and CtAxeB are true acetyl xylan esterases. For both recombinant esterases, over 93 % of the initial activity was retained after 24 h of incubation at temperatures up to 60 °C, and over 90 % of the initial activity was retained after 24 h of incubation in different buffers from pH 4.0 to 9.0 at 4 and 50 °C. The overall xylose yield from wheat arabinoxylan hydrolysis was 8 % with xylanase treatment and increased to 34 % when xylanase was combined with rCtAxeA and rCtAxeB. In sum, the present study first report the biochemical characterization of two acetyl xylan esterases from C. thermophilum, which are efficient in hydrolyzing hemicellulose with potential application in biomass bioconversion to high value chemicals or biofuels.

A novel cephalosporin deacetylating acetyl xylan esterase from Bacillus subtilis with high activity toward cephalosporin C and 7-aminocephalosporanic acid.

A cephalosporin deacetylating acetyl xylan esterase was cloned from the genomic DNA of Bacillus subtilis CICC 20034 and functionally expressed in Escherichia coli. Its gene contained an open reading frame of 957 bp encoding 318 amino acids with a calculated mass of 35,607 Da, and it displayed significant identity to acetyl xylan esterases from Bacillus sp. 916, B. subtilis 168, and Bacillus pumilus Cect5072. The enzyme was a native homohexamer but a trimer under the condition of 1% sodium dodecyl sulfate (SDS); both forms were active and could transit to each other by incubating in or removing SDS. The enzyme belongs to carbohydrate esterase family 7 and had a double specificity on both the acetylated oligosaccharide and cephalosporin C (CPC) and 7-aminocephalosporanic acid (7-ACA). The activity of this purified enzyme toward CPC and 7-ACA was highest among all the acetyl xylan esterase from CE family 7, which were 484 and 888 U/mg, respectively, and endowed itself with great industrial interest on semi-synthetic ß-lactam antibiotics. The optimum pH of the purified enzyme was 8.0, and the optimum temperature was 50 °C, and the enzyme had high thermal stability, broad range of pH tolerance, and extremely organic solvent tolerance.

Characterization of SfPgdA, a Shigella flexneri peptidoglycan deacetylase required for bacterial persistence within polymorphonuclear neutrophils.

Peptidoglycan deacetylases protect the Gram-positive bacteria cell wall from host lysozymes by deacetylating peptidoglycan. Sequence analysis of the genome of Shigella flexneri predicts a putative polysaccharide deacetylase encoded by the plasmidic gene orf185, renamed here SfpgdA. We demonstrated a peptidoglycan deacetylase (PGD) activity with the purified SfPgdA in vitro. To investigate the role SfPgdA in virulence, we constructed a SfpgdA mutant and studied its phenotype in vitro. The mutant showed an increased sensitivity to lysozyme compared to the parental strain. Moreover, the mutant was rapidly killed by polymorphonuclear neutrophils (PMNs). Specific substitution of histidines residues 120 and 125, located within the PGD catalytic domain, by phenylalanine abolished SfPgdA function. SfPgdA expression is controlled by PhoP. Mutation of phoP increases sensitivity to lysozyme compared to the SfpgdA mutant. Here, we confirmed that SfPgdA expression is enhanced under low magnesium concentration and not produced by the phoP mutant. Ectopic expression of SfPgdA in the phoP mutant restored lysozyme resistance and parental bacterial persistence within PMNs. Together our results indicate that PG deacetylation mechanism likely contributes to Shigella persistence by subverting detection by the host immune system.

Characterization and mode of action of two acetyl xylan esterases from Chrysosporium lucknowense C1 active towards acetylated xylans.

Two novel acetyl xylan esterases, Axe2 and Axe3, from Chrysosporium lucknowense (C1), belonging to the carbohydrate esterase families 5 and 1, respectively, were purified and biochemically characterized. Axe2 and Axe3 are able to hydrolyze acetyl groups both from simple acetylated xylo-oligosaccharides and complex non-soluble acetylglucuronoxylan. Both enzymes performed optimally at pH 7.0 and 40 °C. Axe2 has a clear preference for acetylated xylo-oligosaccharides (AcXOS) with a high degree of substitution and Axe3 does not show such preference. Axe3 has a preference for large AcXOS (DP 9-12) when compared to smaller AcXOS (especially DP 4-7) while for Axe2 the size of the oligomer is irrelevant. Even though there is difference in substrate affinity towards acetylated xylooligosaccharides from Eucalyptus wood, the final hydrolysis products are the same for Axe2 and Axe3: xylo-oligosaccharides containing one acetyl group located at the non-reducing xylose residue remain as examined using MALDI-TOF MS, CE-LIF and the application of an endo-xylanase (GH 10).

Molecular cloning and characterization of a bifunctional xylanolytic enzyme from Neocallimastix patriciarum.

A cDNA encoding a bifunctional acetylxylan esterase/xylanase, XynS20E, was cloned from the ruminal fungus Neocallimastix patriciarum. A putative conserved domain of carbohydrate esterase family 1 was observed at the N-terminus and a putative conserved domain of glycosyl hydrolase family 11 was detected at the C-terminus of XynS20E. To examine the enzyme activities, XynS20E was expressed in Escherichia coli as a recombinant His(6) fusion protein and purified by immobilized metal ion-affinity chromatography. Response surface modeling combined with central composite design and regression analysis was then applied to determine the optimal temperature and pH conditions of the recombinant XynS20E. The optimal conditions for the highest xylanase activity of the recombinant XynS20E were observed at a temperature of 49 degrees C and a pH of 5.8, while those for the highest carbohydrate esterase activity were observed at a temperature of 58 degrees C and a pH of 8.2. Under the optimal conditions for the enzyme activity, the xylanase and acetylxylan esterase specific activities of the recombinant XynS20E toward birchwood xylan were 128.7 and 873.1 U mg(-1), respectively. To our knowledge, this is the first report of a bifunctional xylanolytic enzyme with acetylxylan esterase and xylanase activities from rumen fungus.

A proteomic approach to study Escherichia coli. Acetyl esterase interactors unveil a sequence motif involved in protein-protein interaction.

E. coli acetyl esterase (Aes) and beta-cysthationase (MalY) interact, probably with the same mechanism, with the N-terminus of transcriptional activator of maltose regulon (MalT). In order to investigate the basic mechanism of this interaction, we used both a proteomic and a bioinformatic approach. Affinity-based mass spectrometry experiments with purified Aes protein as bait allowed to fish twenty-three, apparently specific, interactors from crude extracts of E. coli cells grown in different conditions. The group of interactors appeared quite heterogeneous, comprising Aes itself, some molecular chaperons, metabolic enzymes, and several proteins of unknown function. Among the identified proteins, two are in some way related to the maltose metabolism and two are related to the lipopolysaccharide metabolism. By superposing the structures of the Alicyclobacillus acidocaldarius EST2, an Aes homolog, and MalY, a region of structural similarity was discovered that allowed detecting a short stretch of nine residues with sequence similarity among EST2, AES and MalY. Degenerated sequence consensuses derived from the alignment were used to analyse the E. coli proteome in the Swiss Prot database and permitted to retrieve sequences of Aes interactors already known or detected in this study. Most of these interactors (14 out of 25) contain the expected consensus. A site-directed mutant of Aes R179A made in the consensus sequence resulted in complete loss of interaction. Based on the analysis of the available three-dimensional structures and mutagenic and structural data inferred from literature, we predict a role of this motif in protein-protein interaction.

Molecular cloning, and characterization of a modular acetyl xylan esterase from the edible straw mushroom Volvariella volvacea.

A new Volvariella volvacea gene encoding an acetyl xylan esterase (designated as Vvaxe1) was cloned and expressed in Pichia pastoris. The cDNA contained an ORF of 1047 bp encoding 349 amino acids with a calculated mass of 39 990 Da. VvAXE1 is a modular enzyme consisting of an N-terminal signal peptide, a catalytic domain, and a cellulose-binding domain. The amino acid sequence of the enzyme exhibited a high degree of similarity to cinnamoyl esterase B from Penicillium funiculosum, and acetyl xylan esterases from Aspergillus oryzae, Penicillium purpurogenum, and Aspergillus ficuum. Recombinant acetyl xylan esterase released acetate from several acetylated substrates including beta-d-xylose tetraacetate and acetylated xylan. No activity was detectable on p-nitrophenyl acetate. Enzyme-catalyzed hydrolysis of 4-methylumbelliferyl acetate was maximal at pH 8.0 and 60 degrees C, and reciprocal plots revealed an apparent K(m) value of 307.7 microM and a V(max) value of 24 733 IU micromol(-1) protein. ReAXE1 also exhibited a capacity to bind to Avicel and H(3)PO(4) acid-swollen cellulose.

Purification and partial characterization of acetic acid esterase from malted finger millet (Eleusine coracana, Indaf-15).

Acetic acid esterase (EC 3.1.1.6) cleaves the acetyl groups substituted at O-2/O-3 of the xylan backbone of arabinoxylans and is known to modulate their functional properties. To date, this enzyme from cereals has not received much attention. In the present study, acetic acid esterase from 72 h ragi malt was isolated and purified to apparent homogeneity by a four-step purification, i.e., ammonium sulfate precipitation, DEAE-cellulose, Sephacryl S-200, and phenyl-Sepharose column chromatography, with a recovery of 0.36% and a fold purification of 34. The products liberated from alpha-NA and PNPA by the action of purified ragi acetic acid esterase were authenticated by ESI-MS and 1H NMR. The pH and temperature optima of the enzyme were found to be 7.5 and 45 degrees C, respectively. The enzyme is stable in the pH range of 6.0-9.0 and temperature range of 30-40 degrees C. The activation energy of the enzymatic reaction was found to be 7.29 kJ mol-1. The apparent Km and Vmax of the purified acetic acid esterase for alpha-NA were 0.04 microM and 0.175 microM min-1 mL-1, respectively. The molecular weight of the native enzyme was found to be 79.4 kDa by GPC whereas the denatured enzyme was found to be 19.7 kDa on SDS, indicating it to be a tetramer. EDTA, citric acid, and metal ions such as Fe+3 and Cu+2 increased the activity while Ni+2, Ca+2, Co+2, Ba+2, Mg+2, Mn+2, Zn+2, and Al+3 reduced the activity. Group-specific reagents such as eserine and PCMB at 25 mM concentration completely inhibited the enzyme while iodoacetamide did not have any effect. Eserine was found to be a competitive inhibitor.

Determination of enzymes from Colletotrichum sp. AHU9748 essential for lepidimoide production from okra polysaccharide.

The allelopathic substance lepidimoide (Lp), which exhibits multiple functions in the growth and development of plants, was produced by Colletotrichum sp. AHU9748 from okra polysaccharide. Okra polysaccharide has the repeating structure (1-->4)-O-alpha-(d-galactopyranosyluronic acid)-(1-->2)-O-alpha-l-rhamnopyranose in its hexasaccharide repeating unit of its main chain. To determine the enzymes essential for Lp production, the supernatant of a culture broth was fractionated by repeated column chromatographies to identify two serial fractions responsible for Lp production and non-Lp production by measuring Lp production together with beta-galactosidase (beta-gal), rhamnogalacturonan lyase (RG-lyase) and acetylesterase (AE) activities, which we hypothesized to be necessary for Lp production from the structure of Lp. We confirmed the presence of these three enzymatic activities in the highest-Lp-producing fraction. The addition of purified RG-lyase to fractions producing no or a small amount of Lp demonstrated that beta-gal and RG-lyase activities are necessary for Lp production. The N-terminal amino acid sequences of the three separated proteins on SDS-PAGE confirmed the presence of enzymes identical to beta-gal, RG-lyase and AE in the Lp-producing fractions.

Penicillium purpurogenum produces a family 1 acetyl xylan esterase containing a carbohydrate-binding module: characterization of the protein and its gene.

At least three acetyl xylan esterases (AXE I, II and III) are secreted by Penicillium purpurogenum. This publication describes more detailed work on AXE I and its gene. AXE I binds cellulose but not xylan; it is glycosylated and inactivated by phenylmethylsulphonyl fluoride, showing that it is a serine esterase. The axe1 gene presents an open reading frame of 1278 bp, including two introns of 68 and 61 bp; it codes for a signal peptide of 31 residues and a mature protein of 351 amino acids (molecular weight 36,693). AXE I has a modular structure: a catalytic module at the amino terminus belonging to family 1 of the carbohydrate esterases, a linker rich in serines and threonines, and a family 1 carboxy terminal carbohydrate binding module (CBM). The CBM is similar to that of AXE from Trichoderma reesei, (with a family 5 catalytic module) indicating that the genes for catalytic modules and CBMs have evolved separately, and that they have been linked by gene fusion. The promoter sequence of axe1 contains several putative sequences for binding of gene expression regulators also found in other family 1 esterase gene promoters. It is proposed that AXE I and II act in succession in xylan degradation; first, xylan is attacked by AXE I and other xylanases possessing CBMs (which facilitate binding to lignocellulose), followed by other enzymes acting mainly on soluble substrates.

Characterization of a gene encoding an acetylase required for pyoverdine synthesis in Pseudomonas aeruginosa.

Strains of Pseudomonas aeruginosa secrete one of three pyoverdine siderophores (types I to III). We have characterized a gene, pvdY(II) (for the pvdY gene present in type II P. aeruginosa strains), that is only present in strains that make type II pyoverdine. A mutation in pvdY(II) prevented pyoverdine synthesis. Bioinformatic, genetic, and biochemical approaches indicate that the PvdYII enzyme catalyzes acetylation of hydroxyornithine. Expression of pvdY(II) is repressed by the presence of iron and upregulated by the presence of type II pyoverdine. Characterization of pvdY(II) provides insights into the molecular basis for production of different pyoverdines by different strains of P. aeruginosa.

Identification of catalytically important amino acid residues of Streptomyces lividans acetylxylan esterase A from carbohydrate esterase family 4.

Multiple sequence alignment of Streptomyces lividans acetylxylan esterase A and other carbohydrate esterase family 4 enzymes revealed the following conserved amino acid residues: Asp-12, Asp-13, His-62, His-66, Asp-130, and His-155. These amino acids were mutated in order to investigate a functional role of these residues in catalysis. Replacement of the conserved histidine residues by alanine caused significant reduction of enzymatic activity. Maintenance of ionizable carboxylic group in side chains of amino acids at positions 12, 13, and 130 seems to be necessary for catalytic efficiency. The absence of conserved serine excludes a possibility that the enzyme is a serine esterase, in contrast to acetylxylan esterases of carbohydrate esterase families 1, 5, and 7. On the contrary, total conservation of Asp-12, Asp-13, Asp-130, and His-155 along with dramatic decrease in enzyme activity of mutants of either of these residues lead us to a suggestion that acetylxylan esterase A from Streptomyces lividans and, by inference, other members of carbohydrate esterase family 4 are aspartic deacetylases. We propose that one component of the aspartate dyad/triad functions as a catalytic nucleophile and the other one(s) as a catalytic acid/base. The ester/amide bond cleavage would proceed via a double displacement mechanism through covalently linked acetyl-enzyme intermediate of mixed anhydride type.

An Aspergillus oryzae acetyl xylan esterase: molecular cloning and characteristics of recombinant enzyme expressed in Pichia pastoris.

We screened 20,000 clones of an expressed sequence tag (EST) library from Aspergillus oryzae (http://www.nrib.go.jp/ken/EST/db/index.html) and obtained one cDNA clone encoding a protein with similarity to fungal acetyl xylan esterase. We also cloned the corresponding gene, designated as Aoaxe, from the genomic DNA. The deduced amino acid sequence consisted of a putative signal peptide of 31-amino acids and a mature protein of 276-amino acids. We engineered Aoaxe for heterologous expression in P. pastoris. Recombinant AoAXE (rAoAXE) was secreted by the aid of fused alpha-factor secretion signal peptide and accumulated as an active enzyme in the culture medium to a final level of 190 mg/l after 5 days. Purified rAoAXEA before and after treatment with endoglycosidase H migrated by SDS-PAGE with a molecular mass of 31 and 30 kDa, respectively. Purified rAoAXE displayed the greatest hydrolytic activity toward alpha-naphthylacetate (C2), lower activity toward alpha-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. The recombinant enzyme catalyzed the release of acetic acid from birchwood xylan. No activity was detectable using methyl esters of ferulic, caffeic or sinapic acids. rAoAXE was thermolabile in comparison to other AXEs from Aspergillus.

Biochemical characterization of recombinant acetyl xylan esterase from Aspergillus awamori expressed in Pichia pastoris: mutational analysis of catalytic residues.

We engineered an acetyl xylan esterase (AwaxeA) gene from Aspergillus awamori into a heterologous expression system in Pichia pastoris. Purified recombinant AwAXEA (rAwAXEA) displayed the greatest hydrolytic activity toward alpha-naphthylacetate (C2), lower activity toward alpha-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. Putative catalytic residues, Ser(119), Ser(146), Asp(168) and Asp(202), were substituted for alanine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the four mutant enzymes were examined. The S119A and D202A mutant enzymes were catalytically inactive, whereas S146A and D168A mutants displayed significant hydrolytic activity. These observations indicate that Ser(119) and Asp(202) are important for catalysis. The S146A mutant enzyme showed lower specific activity toward the C2 substrate and higher thermal stability than wild-type enzyme. The lower activity of S146A was due to a combination of increased K(m) and decreased k(cat). The catalytic efficiency of S146A was 41% lower than that of wild-type enzyme. The synthesis of ethyl acetate was >10-fold than that of ethyl n-hexanoate synthesis for the wild-type, S146A and D168A mutant enzymes. However, the D202A showed greater synthetic activity of ethyl n-hexanoate as compared with the wild-type and other mutants.

Protein-protein interaction conferring stability to an extracellular acetyl (xylan) esterase produced by Termitomyces clypeatus.

Acetyl esterase (AE) activity present in the culture filtrate of Termitomyces clypeatus was separated into lower molar mass (LMM) and higher molar mass (HMM) protein fractions during BioGel P-200 gel chromatography. AE was purified as a 30 kDa nonglycosylated protein from LMM fractions by CM-Sepharose ion exchange chromatography and HPGPLC. Although the HMM fraction had a number of enzyme activities (sucrase, beta-xylosidase, beta-glucosidase, and alpha-L-arabinofuranosidase) other than AE, protein present in the fraction was eluted as a single protein peak in HPGPLC and gave a single band in native PAGE. The fraction, subsequently purified by DEAE-Sephadex chromatography, was a SDS-PAGE homogeneous 80 kDa glycoprotein, but with both AE and cellobiase activities. The aggregate dissociated during ConA-Sepharose chromatography and 30 kDa AE and 56 kDa glycosylated cellobiase were purified separately. The dissociation caused significant loss of cellobiase activity but not that of AE. AE purified from both HMM and LMM fractions was characterized to be the same enzyme in terms of molar masses, pI (7.3), and other physicochemical properties. AE as an aggregate with cellobiase showed higher thermostability, temperature optimum, and resistance toward chemical denaturants than those of purified AE. Compared to cellobiase purified earlier from the same fungus, the enzyme present with AE in the aggregate also showed higher catalytic activity, thermostability, and temperature optimum. The study indicated that the formation of such SDS-resistant enzyme aggregate was associated with significant changes in the physicochemical properties of the enzymes, mainly toward improvement of rigidity of enzymes, and sometimes with the improvement of catalytic activity.