A thermostable <Emphasis Type="SmallCaps">L</Emphasis>-aminoacylase from<Emphasis Type="Italic"> Thermococcus litoralis</Emphasis>: cloning,overexpression, characterization,and applications in biotransformations

A thermostable L-aminoacylase from Thermococcus litoralis was cloned, sequenced, and overexpressed in Escherichia coli. The enzyme is a homotetramer of 43 kDa monomers and has an 82% sequence identity to an aminoacylase from Pyrococcus horikoshii and 45% sequence identity to a carboxypeptidase from Sulfolobus solfataricus. It contains one cysteine residue that is highly conserved among aminoacylases. Cell-free extracts of the recombinant enzyme were characterized and were found to have optimal activity at 85 degrees C in Tris-HCl at pH 8.0. The recombinant enzyme is thermostable, with a half-life of 25 h at 70 degrees C. Aminoacylase inhibitors, such as mono-tert-butyl malonate, had only a slight effect on activity. The enzyme was partially inhibited by EDTA and p-hydroxymercuribenzoate, suggesting that the cysteine residue and a metal ion are important, but not essential, for activity. Addition of Zn2+ and Co2+ to the apoenzyme increased the enzyme activity, whereas Sn4+ and Cu2+ almost completely abolished enzyme activity. The enzyme was most specific for substrates containing N-benzoyl- or N-chloroacetyl-amino acids. preferring substrates containing hydrophobic, uncharged, or weakly charged amino acids such as phenylalanine, methionine, and cysteine.     

pH stability of penicillin acylase from <Emphasis Type="Italic">Escherichia coli</Emphasis>

The inactivation kinetics of penicillin acylase from Escherichia coli have been investigated over a wide pH range at 25 and 50 degrees C. The enzyme was very stable in neutral solutions and quickly lost its catalytic activity in acidic and alkaline solutions. In all cases, the inactivation proceeded according to first order reaction kinetics. Analysis of the pH dependence of enzyme stability provides evidence that stable penicillin acylase conformation is maintained by salt bridges. Destruction of the salt bridges due to protonation/deprotonation of the amino acid residues forming these ion pairs causes inactivation by formation of the unstable "acidic" EH(4)(3+), EH(3)(2+), EH(2)(+) and "alkaline" E(-) enzyme forms. At temperatures above 35 degrees C penicillin acylase apparently undergoes a conformational change that is accompanied by destruction of one of these salt bridges and change in the catalytic properties.     

A cold-adapted esterase from psychrotrophic <Emphasis Type="Italic">Pseudoalteromas</Emphasis> sp. strain 643A

A psychrotrophic bacterium producing a cold-adapted esterase upon growth at low temperatures was isolated from the alimentary tract of Antarctic krill Euphasia superba Dana, and classified as Pseudoalteromonas sp. strain 643A. A genomic DNA library of strain 643A was introduced into Escherichia coli TOP10F', and screening on tributyrin-containing agar plates led to the isolation of esterase gene. The esterase gene (estA, 621 bp) encoded a protein (EstA) of 207 amino acid residues with molecular mass of 23,036 Da. Analysis of the amino acid sequence of EstA suggests that it is a member of the GDSL-lipolytic enzymes family. The purification and characterization of native EstA esterase were performed. The enzyme displayed 20-50% of maximum activity at 0-20 degrees C. The optimal temperature for EstA was 35 degrees C. EstA was stable between pH 9 and 11.5. The enzyme showed activity for esters of short- to medium-chain (C(4) and C(10)) fatty acids, and exhibited no activity for long-chain fatty acid esters like that of palmitate and stearate. EstA was strongly inhibited by phenylmethylsulfonyl fluoride, 2-mercaptoethanol, dithiothreitol and glutathione. Addition of selected divalent ions e.g. Mg(2+), Co(2+) and Cu(2+) led to the reduction of enzymatic activity and the enzyme was slightly activated ( approximately 30%) by Ca(2+) ions.     

Cloning,expression, and characterization of serine protease from thermophilic fungus <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> var. <Emphasis Type="Italic">levisporus</Emphasis>

The serine protease gene from a thermophilic fungus Thermoascus aurantiacus var. levisporus, was cloned, sequenced, and expressed in Pichia pastoris and the recombinant protein was characterized. The full-length cDNA of 2,592 bp contains an ORF of 1,482 bp encoding 494 amino acids. Sequence analysis of the deduced amino acid sequence revealed high homology with subtilisin serine proteases. The putative enzyme contained catalytic domain with active sites formed by three residues of Aspl83, His215, and Ser384. The molecular mass of the recombinant enzyme was estimated to be 59.1 kDa after overexpression in P. pastoris. The activity of recombinant protein was 115.58 U/mg. The protease exhibited its maximal activity at 50°C and pH 8.0 and kept thermostable at 60°C, and retained 60% activity after 60 min at 70° C. The protease activity was found to be inhibited by PMSF, but not by DTT or EDTA. The enzyme has broad substrate specificity such as gelatin, casein and pure milk, and exhibiting highest activity towards casein.     

Characterization and large-scale production of recombinant <Emphasis Type="Italic">Streptoverticillium platensis</Emphasis> transglutaminase

Recombinant Streptomyces platensis transglutaminase (MtgA) produced by the Streptomyces lividans transformant 25-2 was purified by ammonium sulfate fractionation, followed by CM-Sepharose CL-6B fast flow, and blue-Sepharose fast flow chromatography. The purification factor was ~33.2-fold, and the yield was 65%. The molecular weight of the purified recombinant MtgA was 40.0 KDa as estimated by SDS-PAGE. The optimal pH and the temperature for the enzyme activity were 6.0 and 55 degrees C, respectively, and the enzyme was stable at pH 5.0-6.0 and at temperature 45-55 degrees C. Enzyme activity was not affected by Ca(2+), Li(+), Mn(2+), Na(+), Fe(3+), K(+), Mg(2+), Al(3+), Ba(2+), Co(2+), EDTA, or IAA but was inhibited by Fe(2+), Pb(2+), Zn(2+), Cu(2+), Hg(2+), PCMB, NEM, and PMSF. Optimization of the fermentation medium resulted in a twofold increase of recombinant MtgA activity in both flasks (5.78 U/ml) and 5-l fermenters (5.39 U/ml). Large-scale productions of the recombinant MtgA in a 30-l air-lift fermenter and a 250-l stirred-tank fermenter were fulfilled with maximal activities of 5.36 and 2.54 U/ml, respectively.     

Purification and characterization of agarases from a marine bacterium <Emphasis Type="Italic">Vibrio</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> F-6

Marine bacterium Vibrio sp. F-6, utilizing agarose as a carbon source to produce agarases, was isolated from seawater samples taken from Qingdao, China. Two agarases (AG-a and AG-b) were purified to a homogeneity from the cultural supernatant of Vibrio sp. F-6 through ammonium sulfate precipitation, Q-Sepharose FF chromatography, and Sephacryl S-100 gel filtration. Molecular weights of agarases were estimated to be 54.0 kDa (AG-a) and 34.5 kDa (AG-b) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH values for AG-a and AG-b were about 7.0 and 9.0, respectively. AG-a was stable in the pH range of 4.0-9.0 and AG-b was stable in the pH range of 4.0-10.0. The optimum temperatures of AG-a and AG-b were 40 and 55 degrees C, respectively. AG-a was stable at temperature below 50 degrees C. AG-b was stable at temperature below 60 degrees C. Zn(2+), Mg(2+) or Ca(2+) increased AG-a activity, while Mn(2+), Cu(2+) or Ca(2+) increased AG-b activity. However, Ag(+), Hg(2+), Fe(3+), EDTA and SDS inhibited AG-a and AG-b activities. The main hydrolysates of agarose by AG-a were neoagarotetraose and neoagarohexaose. The main hydrolysates of agarose by AG-b were neoagarooctaose and neoagarohexaose. When the mixture of AG-a and AG-b were used, agarose was mainly degraded into neoagarobiose.     

Cloning,expression, and characterization of thermostable Manganese superoxide dismutase from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> var. <Emphasis Type="Italic">levisporus</Emphasis>

A superoxide dismutase (SOD) gene of Thermoascus aurantiacus var. levisporus, a thermophilic fungus, was cloned, sequenced, and expressed in Pichia pastoris and its gene product was characterized. The coding sequence predicted a 231 residues protein with a unique 35 amino acids extension at the N-terminus indicating a mitochondrial-targeting sequence. The content of Mn was 2.46 μg/mg of protein and Fe was not detected in the purified enzyme. The enzyme was found to be inhibited by NaN₃, but not by KCN or H₂O₂. These results suggested that the SOD in Thermoascus aurantiacus var. levisporus was the manganese superoxide dismutase type. In comparison with other MnSODs, all manganese-binding sites were also conserved in the sequence (H88, H136, D222, H226). The molecular mass of a single band of the enzyme was estimated to be 21.7 kDa. The protein was expressed in tetramer form with molecular weight of 68.0 kDa. The activity of purified protein was 2,324 U/mg. The optimum temperature of the enzyme was 55°C and it exhibited maximal activity at pH 7.5. The enzyme was thermostable at 50 and 60°C and the half-life at 80°C was approximately 40 min.     

A Thermostable phytase from <Emphasis Type="Italic">Neosartorya spinosa</Emphasis> BCC 41923 and its expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A phytase gene was cloned from Neosartorya spinosa BCC 41923. The gene was 1,455 bp in size, and the mature protein contained a polypeptide of 439 amino acids. The deduced amino acid sequence contains the consensus motif (RHGXRXP) which is conserved among phytases and acid phosphatases. Five possible disulfide bonds and seven potential N-glycosylation sites have been predicted. The gene was expressed in Pichia pastoris KM71 as an extracellular enzyme. The purified enzyme had specific activity of 30.95 U/mg at 37°C and 38.62 U/mg at 42°C. Molecular weight of the deglycosylated recombinant phytase, determined by SDS-PAGE, was approximately 52 kDa. The optimum pH and temperature for activity were pH 5.5 and 50°C. The residual phytase activity remained over 80% of initial activity after the enzyme was stored in pH 3.0 to 7.0 for 1 h, and at 60% of initial activity after heating at 90°C for 20 min. The enzyme exhibited broad substrate specificity, with phytic acid as the most preferred substrate. Its K _m and V _max for sodium phytate were 1.39 mM and 434.78 U/mg, respectively. The enzyme was highly resistant to most metal ions tested, including Fe²⁺, Fe³⁺, and Al³⁺. When incubated with pepsin at a pepsin/phytase ratio of 0.02 (U/U) at 37°C for 2 h, 92% of its initial activity was retained. However, the enzyme was very sensitive to trypsin, as 5% of its initial activity was recovered after treating with trypsin at a trypsin/phytase ratio of 0.01 (U/U).     

An activated by cobalt alkaline aminopeptidase from <Emphasis Type="Italic">Bacillus mycoides</Emphasis>

An intracellular arginine—specific aminopeptidase synthesized by Bacillus mycoides was purified and characterized. The purification procedure for studied aminopeptidase consisted of ammonium sulphate precipitation and three chromatographic steps: anion exchange chromatography and gel permeation chromatography. A molecular weight of ∼50 kDa was estimated for the aminopeptidase by gel permeation chromatography and SDS-PAGE. The optimal activity of the enzyme on arginyl-β-naphthylamide as a substrate was at 37°C and pH 9.0. The enzyme showed maximum specificity for basic amino acids: such as Arg and Lys but was also able to hydrolyze aromatic amino acids: Trp, Tyr, and Phe. Co²⁺ ions activated the enzyme, while Zn²⁺, Cu²⁺, Hg²⁺ and Mn²⁺ inhibited it. The enzyme is a metalloaminopeptidase whose activity is inhibited by typical metalloaminopeptidase inhibitors: EDTA and 1,10-phenanthroline. Analysis of fragments of the amino acid sequence of the purified enzyme demonstrated high similarity to Amp S of Bacillus cereus and APII of B. thuringensis.     

Purification and characterization of <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-tagatose

l-arabinose isomerase (EC5.3.1.4. AI) mediates the isomerization of d-galactose into d-tagatose as well as the conversion of l-arabinose into l-ribulose. The AI from Lactobacillus plantarum SK-2 was purified to an apparent homogeneity giving a single band on SDS–PAGE with a molecular mass of 59.6 kDa. Optimum activity was observed at 50°C and pH 7.0. The enzyme was stable at 50°C for 2 h and held between pH 4.5 and 8.5 for 1 h. AI activity was stimulated by Mn²⁺, Fe³⁺, Fe²⁺, Ca²⁺ and inhibited by Cu²⁺, Ag⁺, Hg²⁺, Pb²⁺. d-galactose and l-arabinose as substrates were isomerized with high activity. l-arabitol was the strongest competitive inhibitor of AI. The apparent Michaelis–Menten constant (K _m), for galactose, was 119 mM. The first ten N-terminal amino acids of the enzyme were determined as MLSVPDYEFW, which is identical to L. plantarum (Q88S84). Using the purified AI, 390 mg tagatose could be converted from 1,000 mg galactose in 96 h, and this production corresponds to a 39% equilibrium.     

A thermostable feruloyl-esterase from the hemicellulolytic bacterium <Emphasis Type="Italic">Thermobacillus xylanilyticus</Emphasis> releases phenolic acids from non-pretreated plant cell walls

A gene (Tx-est1) encoding a thermostable feruloyl-esterase was isolated from the genome of the Gram-positive hemicellulolytic thermophilic bacterium Thermobacillus xylanilyticus. This gene contains an open reading frame of 1,020 bp encoding a protein with molecular mass of 37.4 kDa, similar to feruloyl-esterases from cellulolytic bacteria and fungi. The recombinant enzyme Tx-Est1 was expressed and produced in Escherichia coli. Tx-Est1 contains the conserved putative lipase residues Ser 202, Asp 287, and His 322 which act as catalytic triad in its C-terminus part. Purified Tx-Est1 was active against phenolic acid derivatives and stable at high temperatures. Optimal activity was observed at 65 °C and the optimal pH was around 8.5. The kinetic parameters of the esterase were determined on various substrates. The enzyme displayed activity against methyl esters of hydrocinnamic acids and feruloylated arabino-xylotetraose, exhibiting high specificity and affinity for the latter. Our results showed that Tx-Est1 is a thermostable feruloyl-esterase which could be useful to hydrolyze arabinoxylans from graminaceous plant cell walls as the enzyme is able to release phenolic acids from a lignocellulose biomass.     

Substrate specificity of a peptidyl-aminoacyl-<Emphasis Type="SmallCaps">l</Emphasis>/<Emphasis Type="SmallCaps">d</Emphasis>-isomerase from frog skin

In the skin of fire-bellied toads (Bombina species), an aminoacyl-l/d-isomerase activity is present which catalyses the post-translational isomerization of the l- to the d-form of the second residue of its substrate peptides. Previously, this new type of enzyme was studied in some detail and genes potentially coding for similar polypeptides were found to exist in several vertebrate species including man. Here, we present our studies to the substrate specificity of this isomerase using fluorescence-labeled variants of the natural substrate bombinin H with different amino acids at positions 1, 2 or 3. Surprisingly, this enzyme has a rather low selectivity for residues at position 2 where the change of chirality at the alpha-carbon takes place. In contrast, a hydrophobic amino acid at position 1 and a small one at position 3 of the substrate are essential. Interestingly, some peptides containing a Phe at position 3 also were substrates. Furthermore, we investigated the role of the amino-terminus for substrate recognition. In view of the rather broad specificity of the frog isomerase, we made a databank search for potential substrates of such an enzyme. Indeed, numerous peptides of amphibia and mammals were found which fulfill the requirements determined in this study. Expression of isomerases with similar characteristics in other species can therefore be expected to catalyze the formation of peptides containing d-amino acids.     

<Emphasis Type="Italic">APHO1</Emphasis> from the yeast <Emphasis Type="Italic">Arxula adeninivorans</Emphasis> encodes an acid phosphatase of broad substrate specificity

The extracellular acid phosphatase-encoding Arxula adeninivorans APHO1 gene was isolated using degenerated specific oligonucleotide primers in a PCR screening approach. The gene harbours an ORF of 1449 bp encoding a protein of 483 amino acids with a calculated molecular mass of 52.4 kDa. The sequence includes an N-terminal secretion sequence of 17 amino acids. The deduced amino acid sequence exhibits 54% identity to phytases from Aspergillus awamori, Asp. niger and Asp. ficuum and a more distant relationship to phytases of the yeasts Candida albicans and Debaryomyces hansenii (36–39% identity). The sequence contains the phosphohistidine signature and the conserved active site sequence of acid phosphatases. APHO1 expression is induced under conditions of phosphate limitation. Enzyme isolates from wild and recombinant strains with the APHO1 gene expressed under control of the strong A. adeninivorans-derived TEF1 promoter were characterized. For both proteins, a molecular mass of approx. 350 kDa, corresponding to a hexameric structure, a pH optimum of pH 4.8 and a temperature optimum of 60°C were determined. The preferred substrates include p-nitrophenyl-phosphate, pyridoxal-5-phosphate, 3-indoxyl-phosphate, 1-naphthylphosphate, ADP, glucose-6-phosphate, sodium-pyrophosphate, and phytic acid. Thus the enzyme is a secretory acid phosphatase with phytase activity and not a phytase as suggested by strong homology to such enzymes.     

Cloning and characterization of a heat-stable CMP-<Emphasis Type="Italic">N</Emphasis>-acylneuraminic acid synthetase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

In this study, we report the cloning, recombinant expression, and biochemical characterization of a heat-stable CMP-N-acylneuraminic acid (NeuAc) synthetase from Clostridium thermocellum ATCC 27405. A high throughput electrospray ionization mass spectrometry (ESI-MS)-based assay demonstrates that the enzyme has an absolute requirement for a divalent cation for activity and reaches maximum activity in the presence of 10 mM Mn²⁺. The enzyme is active at pH 8–13 in Tris–HCl buffer and at 37–60 °C, and maximum activity is observed at pH 9.5 and 50 °C in the presence of 0.2 mM dithiothreitol. In addition to NeuAc, the enzyme also accepts the analog N-glycolylneuraminic acid (NeuGc) as a substrate. The apparent Michaelis constants for cytidine triphosphate and NeuAc or NeuGc are 240 ± 20, 130 ± 10, and 160 ± 10 μM, respectively, with corresponding turnover numbers of 3.33, 2.25, and 1.66 s⁻¹, respectively. An initial velocity study of the enzymatic reaction indicates an ordered bi–bi catalytic mechanism. In addition to demonstration of a thermostable and substrate-tolerant enzyme, confirmation of the biochemical function of a gene for CMP-NeuAc synthetase in C. thermocellum also opens the question of the biological function of CMP-NeuAc in such nonpathogenic microorganisms.     

Two highly thermostable paralogous single-stranded DNA-binding proteins from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis>

The thermophilic bacterium Thermoanaerobacter tengcongensis has two single-stranded DNA-binding (SSB) proteins, designated TteSSB2 and TteSSB3. In a SSB complementation assay in Escherichia coli, only TteSSB3 took over the in vivo function of EcoSSB. We have cloned the ssb genes obtained by PCR and have developed E. coli overexpression systems. The TteSSB2 and TteSSB3 consist of 153 and 150 amino acids with a calculated molecular mass of 17.29 and 16.96 kDa, respectively. They are the smallest known bacterial SSB proteins. The homology between amino acid sequences of these proteins is 40% identity and 53% similarity. They are functional as homotetramers, with each monomer encoding one single-stranded DNA binding domain (OB-fold). In fluorescence titrations with poly(dT), both proteins bind single-stranded DNA with a binding site size of about 40 nt per homotetramer. Thermostability with half-life of about 30 s at 95 degrees C makes TteSSB3 similar to the known SSB of Thermus aquaticus (TaqSSB). The TteSSB2 was fully active even after 6 h incubation at 100 degrees C. Here, we show for the first time paralogous thermostable homotetrameric SSBs, which could be an attractive alternative for known homodimeric thermostable SSB proteins in their applications for molecular biology methods and analytical purposes.     

Biochemical characterization of an ATP-dependent DNA ligase from the hyperthermophilic crenarchaeon <Emphasis Type="Italic">Sulfolobus shibatae</Emphasis>

A gene encoding a putative ATP-dependent DNA ligase was identified in the genome of the hyperthermophilic archaeon Sulfolobus shibatae and expressed in Escherichia coli. The 601 amino acid recombinant polypeptide was a monomeric protein capable of strand joining on a singly nicked DNA substrate in the presence of ATP ( K(m)=34 micro mu) and a divalent cation (Mn(2+), Mg(2+), or Ca(2+)). dATP was partially active in supporting ligation catalyzed by the protein, but GTP, CTP, UTP, dGTP, dCTP, dTTP, and NAD(+) were inactive. The cloned Ssh ligase showed an unusual metal cofactor requirement; it was significantly more active in the presence of Mn(2+) than in the presence of Mg(2+) or Ca(2+). Unexpectedly, the native Ssh ligase preferred Mg(2+) and Ca(2+) rather than Mn(2+). Both native and recombinant enzymes displayed optimal nick-joining activity at 60-80 degrees C. Ssh ligase discriminated against substrates containing mismatches on the 3'-side of nick junction and was more tolerant of mismatches at the 5'-end than of those at the penultimate 5'-end. The enzyme showed little activity on a 1-nucleotide gapped substrate. This is the first biochemical study of a DNA ligase from the crenarchaeotal branch of the archaea domain.     

An amine: hydroxyacetone aminotransferase from <Emphasis Type="Italic">Moraxella lacunata</Emphasis> WZ34 for alaninol synthesis

An amine:hydroxyacetone aminotransferase from an isolated soil bacterium, Moraxella lacunata WZ34, was employed to synthesize alaninol in the presence of hydroxyacetone and isopropylamine in this study. The optimal carbon and nitrogen sources were glycerol and beef extract, respectively. A wide range of amino donor specificity was detected with the aminotransferase, which exhibited a relative high activity (9.83 U mL(-1)) in the presence of isopropylamine. The enzyme was the most active at pH 8.5, and showed relatively higher activity at alkaline than acidic pH. Maximum activity was achieved at 30 degrees C, and the enzyme had good thermal stability below 60 degrees C. Metal ions such as Mg(2+) had positive effect (132.6%) on the enzyme, and (aminooxy)acetic acid, a typical aminotransferase inhibitor, significantly inhibited its activity. The enzyme activity was enhanced by the addition of 0.05 mM pyridoxal-5'-phosphate (PLP).     

Purification and characteristics of an enzyme with both bilirubin oxidase and laccase activities from mycelium of the basidiomycete <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

A homogenous enzyme with both bilirubin oxidase and laccase activities was isolated from a submerged culture of the basidiomycete Pleurotus ostreatus mycelium and characterized. The yield of the enzyme was 127 μg/g dry biomass of the mycelium. The specific activity of the enzyme was 21 and 261 U/mg to bilirubin and to a laccase substrate ABTS, respectively. The intracellular phenol oxidase from the P. ostreatus mycelium was identified as bilirubin oxidase with the amino acid sequence highly homologous to that of the pox2 gene-encoded product. The enzyme displayed the maximal laccase activity at 50–55°C to all substrates examined, whereas the pH optimum was substrate-dependent and changed from 3.0 for ABTS to 7.0 for syringaldazine and guaiacol. The enzyme maintained catalytic activity within a broad pH range but was inactivated at pH 4.0. The enzyme was thermostable but very sensitive to metal chelating inhibitors. Trypan Blue (5 mg/liter) was completely decolorizated upon 3 h of incubation with the bilirubin oxidase (20 mU/ml) at room temperature.     

Cloning of a xylanase gene <Emphasis Type="Italic">xyn2A</Emphasis> from rumen fungus <Emphasis Type="Italic">Neocallimastix</Emphasis> sp. GMLF2 in <Emphasis Type="Italic">Escherichia coli</Emphasis> and its partial characterization

Anaerobic fungi belonging to the family Neocallimastigaceae are native inhabitants in the rumen of the most herbivores, such as cattle, sheep and goats. A member of this unique group, Neocallimastix sp. GMLF2 was isolated from cattle feces and screened for its xylanase encoding gene using polymerase chain reaction. The gene coding for a xylanase (xyn2A) was cloned in Escherichia coli and expression was monitored. To determine the enzyme activity, assays were conducted for both fungal xylanase and cloned xylanase (Xyl2A) for supernatant and cell-associated activities. Optimum pH and temperature of the enzyme were found to be 6.5 and 50°C, respectively. The enzyme was stable at 40°C and 50°C for 20 min but lost most of its activity when temperature reached 60°C for 5-min incubation time. Rumen fungal xylanase was mainly released to the supernatant of culture, while cloned xylanase activity was found as cell-associated. Multiple alignment of the amino acid sequences of Xyl2A with published xylanases from various organisms suggested that Xyl2A belongs to glycoside hydrolase family 11.     

An acid and highly thermostable xylanase from <Emphasis Type="Italic">Phialophora</Emphasis> sp. G5

An endo-β-1,4-xylanase gene, designated xyn10G5, was cloned from Phialophora sp. G5 and expressed in Pichia pastoris. The 1,197-bp full-length gene encodes a polypeptide of 399 amino acids consisting of a putative signal peptide at residues 1–20, a family 10 glycoside hydrolase domain, a short Gly/Thr-rich linker and a family 1 carbohydrate-binding module (CBM). The deduced amino acid sequence of XYN10G5 shares the highest identity (53.4%) with a putative xylanase precursor from Aspergillus terreus NIH2624. The purified recombinant XYN10G5 exhibited the optimal activity at pH 4.0 and 70 °C, remained stable at pH 3.0–9.0 (>70% of the maximal activity), and was highly thermostable at 70 °C (retaining ~90% of the initial activity for 1 h). Substrate specificity studies have shown that XYN10G5 had the highest activity on soluble wheat arabinoxylan (350.6 U mg⁻¹), and moderate activity to various heteroxylans, and low activity on different types of cellulosic substrates. Under simulated gastric conditions, XYN10G5 was stable and released more reducing sugars from soluble wheat arabinoxylan; when combined with a glucanase (CelA4), the viscosity of barley–soybean feed was significantly reduced. These favorable enzymatic properties make XYN10G5 a good candidate for application in the animal feed industry.