A novel cold-active xylanase gene from the environmental DNA of goat rumen contents: direct cloning, expression and enzyme characterization

A xylanase-coding gene, xynGR40, was cloned directly from the environmental DNA of goat rumen contents and expressed in Escherichia coli BL21 (DE3). The 1446-bp full-length gene encodes a 481-residue polypeptide (XynGR40) containing a catalytic domain belonging to glycosyl hydrolase (GH) family 10. Phylogenetic analysis indicated that XynGR40 was closely related with microbial xylanases of gastrointestinal source. Purified recombinant XynGR40 exhibited high activity at low temperatures, and remained active (～10% of the activity) even at 0°C. The optimal temperature of XynGR40 was 30°C, much lower than other xylanases from rumen. Compared with mesophilic and thermophilic counterparts, XynGR40 had fewer hydrogen bonds and salt bridges, and lengthened loops in the catalytic domain. The enzyme also had relatively better stability at mesophilic temperatures and a higher catalytic efficiency than other known GH 10 cold active xylanases. These properties suggest that XynGR40 is a novel cold active xylanase and has great potential for basic research and industrial applications.     

A novel thermostable sulfite oxidase from Thermus thermophilus: characterization of the enzyme, gene cloning and expression in Escherichia coli

A novel sulfite oxidase has been identified from Thermus thermophilus AT62. Despite this enzyme showing significant amino-acid sequence homology to several bacterial and eukaryal putative and identified sulfite oxidases, the kinetic analysis, performed following the oxidation of sulfite and with ferricyanide as the electron acceptor, already pointed out major differences from representatives of bacterial and eukaryal sources. Sulfite oxidase from T. thermophilus, purified to homogeneity, is a monomeric enzyme with an apparent molecular mass of 39.1 kDa and is almost exclusively located in the periplasm fraction. The enzyme showed sulfite oxidase activity only when ferricyanide was used as electron acceptor, which is different from most of sulfite-oxidizing enzymes from several sources that use cytochrome c as co-substrate. Spectroscopic studies demonstrated that the purified sulfite oxidase has no cytochrome like domain, normally present in homologous enzymes from eukaryotic and prokaryotic sources, and for this particular feature it is similar to homologous enzyme from Arabidopsis thaliana. The identified gene was PCR amplified on T. thermophilus AT62 genome, expressed in Escherichia coli and the recombinant protein identified and characterized.     

Molecular cloning and characterization of a novel thermostable xylanase from Paenibacillus campinasensis BL11

An open reading frame (XylX) with 1131 nucleotides from Paenibacillus campinasensis BL11 was cloned and expressed in E. coli. It encodes a family 11 endoxylanase, designated as XylX, of 41 kDa. The homology of the amino acid sequence deduced from XylX is only 73% identical to the next closest sequence. XylX contains a family 11 catalytic domain of the glycoside hydrolase and a family 6 cellulose-binding module. The recombinant xylanase was fused to a His-tag for affinity purification. The XylX activity was 2392 IU/mg, with a K_m of 6.78 mg/ml and a V_max of 4953 mol/min/mg under optimal conditions (pH 7, 60 °C). At pH 11, 60 °C, the activity was still as high as 517 IU/mg. Xylanase activities at 60 °C under pH 5 to pH 9 remained at more than 69.4% of the initial activity level for 8 h. The addition of Hg²⁺ at 5 mM almost completely inhibited xylanase activity, whereas the addition of tris-(2-carboxyethyl)-phosphine (TCEP) and 2-mercaptoethanol stimulated xylanase activity. No relative activities for Avicel, CMC and d-(+)-cellobiose were found. Xylotriose constitutes the majority of the hydrolyzed products from oat spelt and birchwood xylan. Broad pH and temperature stability shows its application potentials for biomass conversion, food and pulp/paper industries.     

常压室温等离子体快速诱变绿色糖单孢菌筛选 木聚糖酶高产菌株及其酶学性质研究

[目的]利用常压室温等离子体快速诱变绿色糖单孢菌,筛选耐热耐碱木聚糖酶高产菌株,并对其进行酶学性质分析,确保其适用于生物制浆漂白工艺.[方法]采用刚果红平板水解圈法结合摇瓶发酵胞外酶测定法进行菌株筛选,并通过DNS木聚糖酶活性测定等方法对来源于不同突变株的木聚糖酶进行酶学性质分析对比.[结果]筛选出遗传稳定性良好的两株木聚糖酶高产菌株AT24和AT22-2,以麦草浆为诱导底物的粗酶液中,突变株AT24及AT22-2所产的木聚糖酶活性分别为512.74、552.70U/mL,分别为原始菌株S.v的16和17倍的.来源于突变株AT22-2的木聚糖酶的最适反应pH为9.5,最适反应温度为90℃,在50℃-90℃温度范围内具有良好的热稳定性,在100℃条件下处理30 min剩余酶活仍为68％;突变株AT24所产木聚糖酶的最适反应温度为60℃,最适pH为10.0,在60℃-80℃的高温环境下,突变株AT24所产的木聚糖酶具有良好的热稳定性.[结论]突变株AT22-2所产具有耐碱耐高温性质的木聚糖酶,在应用领域尤其在纸浆造纸行业具有较大的潜在应用价值.     

A novel cold-active xylanase from the cellulolytic myxobacterium Sorangium cellulosum So9733-1: gene cloning, expression, and enzymatic characterization

The cellulolytic myxobacterium Sorangium cellulosum is able to efficiently degrade many kinds of polysaccharides, but none of the enzymes involved have been characterized. In this paper, a xylanase gene (xynA) was cloned from S. cellulosum So9733-1 using thermal asymmetric interlaced PCR. The gene is composed of 1,209 bp and has only 52.27% G + C content, which is much lower than that of most myxobacterial DNA reported (67–72%). Gene xynA encodes a 402 amino acid protein that contains a single catalytic domain belonging to the glycoside hydrolase family 10. The novel xylanase gene, xynA, was expressed in Escherichia coli BL21 (DE3) and the recombinant protein (r-XynA) was purified by Ni-affinity chromatography. The r-XynA had the optimum temperature of 30–35°C and exhibited 33.3% activity at 5°C and 13.7% activity at 0°C. Approximately 80% activity was lost after 20-min pre-incubation at 50°C. These results indicate that r-XynA is a cold-active xylanase with low thermostability. At 30°C, the K _m values of r-XynA on beechwood xylan, birchwood xylan, and oat spelt xylan were 25.77 ± 4.16, 26.52 ± 4.78, and 38.13 ± 5.35 mg/mL, respectively. The purified r-XynA displayed optimum activity at pH 7.0. The activity of r-XynA was enhanced by the presence of Ca²⁺. The r-XynA hydrolyzed beechwood xylan, birchwood xylan, and xylooligosaccharides (xylotriose, xylotetraose, and xylopentose) to produce primarily xylose and xylobiose. To our knowledge, this is the first report on the characterization of a xylanase from S. cellulosum.     

Three forms of thermostable lactose-hydrolase from Thermus sp. IB-21: cloning, expression, and enzyme characterization

Three thermostable lactose-hydrolases, namely, two beta-glycosidases (bglA and bglB) and one beta-galactosidase (bgaA) genes were cloned from the genomic library of Thermus sp. IB-21. The bglA, bglB, and bgaA consisted of 1311 bp (436 amino acid residues), 1296 bp (431 aa), and 1938 bp (645 aa) of nucleotides with predicted molecular masses of 49,066, 48,679, and 72,714 Da, respectively. These enzymes were overexpressed in Escherichia coli BL21(DE3) using pET21b(+) vector system. The recombinant enzymes were purified to homogeneity by a heat precipitation (70 degrees C, 40 min) and a Ni2+-affinity chromatography. The molecular masses of the purified enzymes estimated by SDS-PAGE agreed with their predicted values. All the purified enzymes showed their optimal pH at around 5.0-6.0. In contrast, the temperature profiles for activity and thermostability patterns were different for each enzyme. BglB beta-glycosidase displayed the best lactose hydrolysis activity of the three enzymes without substrate inhibition up to 200 mM lactose at 70 degrees C and pH 7.0. The specific activities (U/mg) of BglA, BglB, and BgaA on 138 mM lactose at 70 degrees C and pH 7.0 were 36.8, 160.3, and 8.5, respectively.     

Purification and characterization of xylanase from alkali-tolerant Aspergillus fischeri Fxn1

Abstract Alkali-tolerant Aspergillus fischeri Fxn1 produced two extracellular xylanases. The major xylanase ( M _r 31000) was purified to electrophoretic homogeneity by ammonium sulfate precipitation, anion exchange chromatography and preparatory PAGE. Xylose was the major hydrolysis product from oat spelt and birch wood xylans. It was completely free of cellulolytic activities. The optimum pH and temperature were 6.0 and 60 °C, respectively. pH stability ranged from 5 to 9.5 and the t_{1 / 2} at 50 °C was 490 min. It had a K _m of 4.88 mg ml⁻¹and a V _max of 588 μmol min⁻¹ mg⁻¹. The activity was inhibited (95%) by AlCl₃ (10 mM). This enzyme appears to be novel and will be useful for studies on the mechanism of hydrolysis of xylan by xylanolytic enzymes.     

A novel thermostable phytase from the fungus Aspergillus aculeatus RCEF 4894: gene cloning and expression in Pichia pastoris

A novel gene of thermostable phytase, phyA, was isolated by polymerase chain reaction (PCR) techniques from Aspergillus aculeatus RCEF 4894. The full-length phyA gene comprises 1,404 bp and encodes 467 amino-acid residues, including a 19-residue putative N-terminal signal peptide. The phytase of A. aculeatus was a novel addition to the histidine-acid phosphatase family, as evidenced by both the conserved motifs RHGXRXP and HD in the amino-acid sequence, and 3D structure models. The recombinant phytase was overexpressed in Pichia pastoris, and its specific activity reached 3,000 U mL⁻¹ at the optimum pH of 5.5. This recombinant, thermostable phytase was able to withstand temperatures of up to 90 °C for 10 min, with a loss of only 13.9% of initial enzymatic activity, and showed high activity with phytic-acid sodium salt at a pH range of 2.5–6.5. The broad pH optima and high thermostability of the phytase makes it a promising candidate for feed-pelleting applications.     

A novel esterase from Ralstonia sp. M1: gene cloning, sequencing, high-level expression and characterization

A newly isolated gene from Ralstonia sp. M1, encoding an esterase, was cloned in Escherichia coli and its nucleotide sequence determined. The 1.6kb insert revealed one complete open reading frame, predicted to encode an esterase (320 aa, 34.1kDa) with a pI of 9.86. EstR contained a putative oxyanion hole H36G37, a conserved pentapeptide G103HSLG107 and a conserved catalytic His265 and Asp237. The EstR sequence shared 64-70 and 44-48% identity with the hydrolases/acyltransferases from Burkholderia strains and from Ralstonia strains, respectively, 44 and 38% identity with the lactone-specific esterase from Pseudomonas fluorescens and Mesorhizobium loti, respectively. The esterase EstR was expressed with a high level of 41mg/g wet cells. The Ni-NTA-purified esterase EstR showed an optimal activity in the temperature range 60-65 degrees C and pH range 7.5-9.0 towards p-nitrophenyl caproate. The enzyme was found to be highly resistant to many organic solvents especially induced by ethanolamine. Metal ions showed slight effect on esterase activity. The inhibitor phenylmethanesulfonyl fluoride inhibited strongly the esterase. Triton X-45 induced the activation of EstR, but other detergents slightly to strongly decreased or completely inhibited. Among tested p-NP esters, caproate was the most preferential substrate of this esterase.     

A novel halotolerant xylanase from marine isolate Bacillus subtilis cho40: gene cloning and sequencing

Although several xylanases have been studied, only few xylanases from marine micro-organisms have been reported. We report here a novel halotolerant xylanase from marine bacterium Bacillus subtilis cho40 isolated from Chorao island of mandovi estuary Goa, India. Extracellular xylanase was produced by using agricultural residue such as wheat bran as carbon source under solid-state fermentation (SSF). The optimal pH and temperature of xylanase were reported to be 6.0 and 60°C, respectively. Xyn40 was highly salt-tolerant, and showed highest activity at 0.5M NaCl. Xylanase activity was greatly induced (140%) when pre-incubated with 0.5M NaCl for 4h. The xylanase gene, xyn40, from marine bacterium B. subtilis cho40 was cloned, and expressed in Escherichia coli. The xylanase gene was 645 bp long and had a 215 amino acid ORF protein with a molecular mass of 22.9 kDa. It had all features of xylanase enzyme and showed homology to xylanases reported from B. subtilis. It differs from the earlier reported xylanase sequences by the presence of more serine residues compared to threonine and also by the presence of polar (hydrophilic) amino acids in higher abundance (61%) than non-polar amino acids (39%). The novel xylanase, reported in this study is a halotolerant enzyme from marine isolate and can play a very important role in bioethanol production from marine seaweeds.     

A cold-adapted esterase of a novel marine isolate, Pseudoalteromonas arctica: gene cloning, enzyme purification and characterization

A gene encoding an esterase (estO) was identified and sequenced from a gene library screen of the psychrotolerant bacterium Pseudoalteromonas arctica. Analysis of the 1,203 bp coding region revealed that the deduced peptide sequence is composed of 400 amino acids with a predicted molecular mass of 44.1 kDa. EstO contains a N-terminal esterase domain and an additional OsmC domain at the C-terminus (osmotically induced family of proteins). The highly conserved five-residue motif typical for all α/β hydrolases (G × S × G) was detected from position 104 to 108 together with a putative catalytic triad consisting of Ser¹⁰⁶, Asp¹⁹⁶, and His²²⁵. Sequence comparison showed that EstO exhibits 90% amino acid identity with hypothetical proteins containing similar esterase and OsmC domains but only around 10% identity to the amino acid sequences of known esterases. EstO variants with and without the OsmC domain were produced and purified as His-tag fusion proteins in E. coli. EstO displayed an optimum pH of 7.5 and optimum temperature of 25°C with more than 50% retained activity at the freezing point of water. The thermostability of EstO (50% activity after 5 h at 40°C) dramatically increased in the truncated variant (50% activity after 2.5 h at 90°C). Furthermore, the esterase displays broad substrate specificity for esters of short-chain fatty acids (C₂–C₈).     

A novel thermostable arylesterase from the archaeon Sulfolobus solfataricus P1: purification, characterization, and expression

A novel thermostable arylesterase, a 35-kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 94°C and 7.0, respectively. The enzyme displayed remarkable thermostability: it retained 52% of its activity after 50 h of incubation at 90°C. In addition, the purified enzyme showed high stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity besides showing an arylesterase activity toward aromatic esters: it exhibits not only carboxylesterase activity toward tributyrin and p-nitrophenyl esters containing unsubstituted fatty acids from butyrate (C₄) to palmitate (C₁₆), but also paraoxonase activity toward organophosphates such as p-nitrophenylphosphate, paraoxon, and methylparaoxon. The k_cat/K_m ratios of the enzyme for phenyl acetate and paraoxon, the two most preferable substrates among all tested, were 30.6 and 119.4 s⁻¹·μM⁻¹, respectively. The arylesterase gene consists of 918 bp corresponding to 306 amino acid residues. The deduced amino acid sequence shares 34% identity with that of arylesterase from Acinetobacter sp. strain ADP1. Furthermore, we successfully expressed active recombinant S. solfataricus arylesterase in Escherichia coli. Together, our results show that the enzyme is a serine esterase belonging to the A-esterases and contains a catalytic triad composed of Ser156, Asp251, and His281 in the active site.     

Molecular cloning, characterization and expression of a novel jasmonate-dependent defensin gene from Ginkgo biloba

A novel defensin gene was isolated from Ginkgo biloba. The full-length cDNA of G. biloba defensin (designated as Gbd) was 534bp. The cDNA contained a 240-bp open reading frame encoding an 80-amino acid protein of 5.68 kDa with a potential 30 aa signal peptide. The putative GbD mature protein showed striking similarity to other plant defensins, representing low molecular size antimicrobial polypeptides. Eight cysteine sites conserved in plant defensins were also found in GbD at similar positions. Three-dimensional structure modeling showed that GbD strongly resembled defensin from tobacco (NaD1) and consisted of an alpha-helix and a triple-strand antiparallel beta-sheet that were stabilized by four intramolecular disulfide bonds, implying GbD may have functions similar to NaD1. The genomic DNA gel blot indicated that Gbd belonged to a multigene family. Expression analysis revealed that Gbd was up-regulated by wounding and methyl jasmonate treatments, suggesting that Gbd is potentially involved in plant resistance or tolerance to pathogens during wounding.     

Purification and characterization of a thermostable xylanase from Fomitopsis pinicola

An extracellular xylanase was purified to homogeneity by sequential chromatography of Fomitopsis pinicola culture supernatants on a DEAE-sepharose column, a gel filtration column, and then on a MonoQ column with fast protein liquid chromatography. The relative molecular weight of F. pinicola xylanase was determined to be 58 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis and by size exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the xylanase had a pH optimum of 4.5 and a temperature optimum of 70 degreesC. The enzyme showed t(1/2) value of 33 h at 70 degrees C and catalytic efficiency (k(cat) = 77.4 s?1, k(cat)/K(m) = 22.7 mg/ml/s) for oatspelt xylan. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase (GH) family 10, indicating that the F. pinicola xylanase is a member of GH family 10.     

A novel xylanase from Streptomyces sp. FA1: Purification,characterization, identification,and heterologous expression

A xylanase (XynA) was purified from the culture medium of Streptomyces sp. FA1, which was previously isolated from a bamboo retting system. XynA had a molecular mass of 43 kDa, displayed maximal activity at pH 5.5, retained 41% of its maximal activity at pH 11.0, and was stable over a wide pH range (3.0 ～ 11.0). Purified XynA was subjected to peptide mass fingerprinting, which led to the cloning of the xynA gene. The xynA gene, which encodes a mature protein of 436 amino acids, was heterologously expressed in E. coli BL21(DE3). The activity in the culture medium could reach 213.5 U/mL, which was 11.2-fold higher than that produced by Streptomyces sp. FA1. BLAST searching revealed that full-length XynA shares less than 90% identity with most of its homologues, whereas amino acids 48-436 of the enzyme share 97% identity with an open reading frame encoding a putative full-length mature xylanase from Streptomyces tendae. The truncated xynA gene, xynA ^48-436 , was cloned and expressed in E. coli, however, no xylanase activity could be detected in the culture medium. Based on these results, it is suggested that XynA is a new member of glycoside hydrolases family10 with exceptional catalytic efficiency at alkaline pH.     

米曲霉耐热木聚糖酶纯化及酶学特性研究

对米曲霉原始发酵液中耐热木聚糖酶进行纯化和酶学特性研究,利用甘蔗渣为碳源培养米曲霉,通过超滤和阴离子交换柱两步纯化得到木聚糖酶XynH1,分子量35．402kDa,利用飞行时间质谱和SDS—PAGE分析,推断XynH1为XylanaseXynF1,分子量为35．402kDa。XynH1属于糖苷水解酶家族10,酶活为442．2IU／nag,最适pH和温度分别为pH6．0和65℃,80℃以下及pH4．0～10．5范围内较稳定。     

N-ethylmaleimide-resistant acyl-coenzyme A oxidase from Arthrobacter ureafaciens NBRC 12140: molecular cloning, gene expression and characterization of the recombinant enzyme

N-ethylmaleimide (NEM)-resistant acyl-coenzyme A oxidase (ACO) has been desired for the determination of free fatty acids (FFAs). In order to meet this demand, we prepared recombinant ACO from Arthrobacter ureafaciens NBRC 12140. The coding region of the gene was 2109, encoding a protein of 703 amino acids with a predicted molecular mass of 76.5 kDa. The heterologous expression level in Escherichia coli was 520-fold higher than that in the native strain. The purified enzyme retained more than 60% activity after incubation in the presence of 10 mM NEM at 37 degrees C for 4 h, while other commercially available ACOs showed only less than 10% activities after the same NEM treatment. We presume that this is due to the presence of only three cysteines in ACO from A. ureafaciens. Site-directed mutagenesis studies and close scrutiny of the three-dimensional structures of other related ACOs suggested that these cysteines were buried in the protein and unreactive to NEM. The recombinant enzyme was used for the colorimetric determination of free fatty acid, which gave a linear calibration.     

A novel thermostable xylanase from Thermomonospora sp.: influence of additives on thermostability

An alkalothermophilic Thermomonospora sp. producing high levels of xylanase was isolated from self-heating compost. The culture produced 125 IU/ml of xylanase when grown in shake flasks at pH 9 and 50 degrees C for 96 h. The culture filtrate also contained cellulase (23 IU/ml), mannanase (1 IU/ml) and beta-xylosidase (0.1 IU/ml) activities. The xylanase was active at a broad range of pH (5-9) and temperature (40-90 degrees C). The optimum pH and temperature were 7 and 70 degrees C, respectively. The enzyme was stable in the pH range 5-8 and was thermostable with half-lives of 8 and 4 h at 60 degrees C and 70 degrees C, respectively, but only 9 min at 80 degrees C. The effects of a variety of compounds to enhance the stability of xylanase at 80 degrees C was studied. Addition of sorbitol, mannitol and glycerol increased the thermostability of xylanase in proportion to the number of hydroxyl groups per polyol molecule. Glycine also offered protection against thermoinactivation. Xylan, trehalose, gelatin and trehalose-gelatin mixture had marginal effect on the thermostability of xylanase at 80 degrees C.