The xylitol dehydrogenase(XDH)is a crucial enzyme involved in the xylose utilization in pentose⁃catabolizing yeasts and fungi.In addition to producing xylulose,XDH can also be employed to develop a biosensor for monit...The xylitol dehydrogenase(XDH)is a crucial enzyme involved in the xylose utilization in pentose⁃catabolizing yeasts and fungi.In addition to producing xylulose,XDH can also be employed to develop a biosensor for monitoring xylitol concentration.In this study,the gene encoding the thermophilic fungus Talaromyces emersonii XDH(TeXDH)was heterologously expressed in Escherichia coli BL21(DE3)at 16℃in the soluble form.Recombinant TeXDH with high purity was purified by using a Ni⁃NTA affinity column.Size⁃exclusion chromatography and SDS⁃PAGE analysis demonstrated that the puri⁃fied recombinant TeXDH exists as a native trimer with a molecular mass of approximately 116 kD,and is composed of three identical subunits,each with a molecular weight of around 39 kD.The TeXDH strictly preferred NAD^(+)as a coenzyme to NADP^(+).The optimal temperature and pH of the TeXDH were 40℃and 10.0,respectively.After EDTA treatment,the enzyme activity of TeXDH decreased to 43.26%of the initial enzyme activity,while the divalent metal ions Mg^(2+)or Ca^(2+)could recover the enzyme activity of TeXDH,reaching 103.32%and 110.69%of the initial enzyme activity,respectively,making them the optimal divalent metal ion cofactors for TeXDH enzyme.However,the divalent metal ions of Mn^(2+),Ni^(2+),Cu^(2+),Zn^(2+),Co^(2+),and Cd^(2+)significantly inhibited the activity of TeXDH.ICP⁃MS and molecular doc⁃king studies revealed that 1 mol/L of TeXDH bound 2 mol/L Zn^(2+)ions and 1 mol/L Mg^(2+)ion.Further⁃more,TeXDH exhibited a high specificity for xylitol,laying the foundation for the development of future xylitol biosensors.展开更多
An agar-degrading bacterium, designated as Pseudoalteromonas sp. NJ21, was isolated from an Antarctic sediment sample. The agarase gene aga1161 from Pseudoalteromonas sp. NJ21 consisting of a 2 382-bp coding region wa...An agar-degrading bacterium, designated as Pseudoalteromonas sp. NJ21, was isolated from an Antarctic sediment sample. The agarase gene aga1161 from Pseudoalteromonas sp. NJ21 consisting of a 2 382-bp coding region was cloned. The gene encodes a 793-amino acids protein and was found to possess characteristic features of the Glyco_hydro 42 family. The recombinant agarase （rAgal 161） was overexpressed in Escherichia coli and purified as a fusion protein. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were 30--40℃ and 8.0, respectively. rAga 1161 was found to maintain as much as 80% of its maximum activity at 10℃, which is typical of a cold- adapted enzyme. The pattern of agar hydrolysis demonstrated that the enzyme is an β-agarase, producing neoagarobiose （NA2） as the final main product. Furthermore, this work is the first proof of an agarolytic activity in Antarctic bacteria and these results indicate the potential for the Antarctic agarase as a catalyst in medicine, food and cosmetic industries.展开更多
The development of industrial biotechnology has created an increasing demand for alkaline lipolytic enzymes with functional diversity. In this study, an alkaline soil metagenomic library was constructed to search for ...The development of industrial biotechnology has created an increasing demand for alkaline lipolytic enzymes with functional diversity. In this study, an alkaline soil metagenomic library was constructed to search for new lipolytic enzymes. Two novel gene encoded alkaline esterases（designated as estA and estB） were isolated by functional screening from the library. The estA gene consisted of 834 bp and coded for 277 amino acids with a molecular mass of 29998. Amino acid sequence homology analysis indicates that EstA belongs to α/β hydrolase fold family 4.4（abH4.4）, with EstA being the smallest member of that family yet reported. The estB gene consisted of 1185 bp and encoded 394 amino acids with a theoretical molecular mass of 40090. Its conserved domain analysis shows that EstB belongs to the GDSL hydrolase superfamily. Both EstA and EstB exhibit only moderate identity（〈38%） in amino acid sequence to the known lipolytic enzyme genes in the database. The two genes were respectively expressed in Escherichia coli and the protein products were purified for functional characterization. While the expressed EstA did not exhibit the functional properties that were superior to those of other esterases previously reported, the EstB was stable at temperature up to 45 ℃ and its maximum activity was measured to be 53.6 U/mg at pH=10. Both the en- zymes have further enriched the diversity of the lipolytic enzymes database and also appear to be promising biocatalysts for potential biotechnological application.展开更多
An extracellular lipase from Aureobasidium pullulans was obtained and purified with a specific activity of 17.7 U/mg of protein using ultrafiltration and a DEAE-Sepharose Fast Flow column. Characterization of the lipa...An extracellular lipase from Aureobasidium pullulans was obtained and purified with a specific activity of 17.7 U/mg of protein using ultrafiltration and a DEAE-Sepharose Fast Flow column. Characterization of the lipase indicated that it is a novel finding from the species A. pullulans. The molecular weight of the lipase was 39.5 kDa, determined by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis(SDS-PAGE). The enzyme exhibited its optimum activity at 40 °C and pH of 7. It also showed a remarkable stability in some organic solutions(30%, v/v) including n-propanol, isopropanol, dimethyl sulfoxide(DMSO), and hexane. The catalytic activity of the lipase was enhanced by Ca2+ and was slightly inhibited by Mn2+ and Zn2+ at a concentration of 10 mmol/L. The lipase was activated by the anionic surfactant SDS and the non-ionic surfactants Tween 20, Tween 80, and Triton X-100, but it was drastically inhibited by the cationic surfactant cetyl trimethyl ammonium bromide(CTAB). Furthermore, the lipase was able to hydrolyze a wide variety of edible oils, such as peanut oil, corn oil, sunflower seed oil, sesame oil, and olive oil. Our study indicated that the lipase we obtained is a potential biocatalyst for industrial use.展开更多
A superoxide dismutase was purified from Enteromorpha linza using a simple and safe procedure, which comprised phosphate buffer extraction, ammonium sulphate precipitation, ion exchange chromatography on Q-sepharose c...A superoxide dismutase was purified from Enteromorpha linza using a simple and safe procedure, which comprised phosphate buffer extraction, ammonium sulphate precipitation, ion exchange chromatography on Q-sepharose column, and gel filtration chromatography on Superdex 200 10/300GL. The E. linza superoxide dismutase （E/SOD） was purified 103.6-fold, and a yield of 19.1% and a specific activity of 1 750 U/rag protein were obtained. The SDS-PAGE exhibited E/SOD a single band near 23 kDa and the gel filtration study showed E/SOD＇s molecular weight is near 46 kDa in nondenatured condition, indicating it＇s a homodimeric protein. E/SOD is an iron-cofactored superoxide dismutase （Fe-SOD） because it was inhibited by hydrogen peroxide, insensitive to potassium cyanide. The optimal temperature for its maximal enzyme activity was 35℃, and it still had 29.8% relative activity at 0℃, then E/SOD can be classified as a cold-adapted enzyme. E/SOD was stable when temperature was below 40℃ or the pH was within the range of 5 10. The first 11 N-terminal amino acids orE/SOD were ALELKAPPYEL, comparison of its N-terminal sequence with other Fe-SOD N-terminal sequences at the same position suggests it is possibly a chloroplastic Fe-SOD.展开更多
[Objective] This study aimed to investigating properties of alkaline protease produced by strain Ⅰ 13.[Method] Crude enzyme of alkaline protease was obtained from alkaline protease produced by strain I 13,while effec...[Objective] This study aimed to investigating properties of alkaline protease produced by strain Ⅰ 13.[Method] Crude enzyme of alkaline protease was obtained from alkaline protease produced by strain I 13,while effects of temperature and pH value on enzyme activity were also investigated in this study.[Result] The optimal temperature of alkaline protease produced by strain Ⅰ 13 was 40 ℃,while enzyme activity maintains a higher level from 30 to 60 ℃ and over 40% of the largest enzyme activity still maintained within the range from 20 to 30 ℃.The optimal pH value was 10.5,and over 90% of the largest enzyme activity still maintained within the range from 8.0 to 11.0,which had broader pH value spectrum.[Conclusion] This alkaline protease has huge potential to be developed into washing-powder additive.展开更多
基金湖南省教育厅基金优秀青年项目(No.22B0482)湖南科技大学博士启动基金(No.E51992 and E51993)资助。
文摘The xylitol dehydrogenase(XDH)is a crucial enzyme involved in the xylose utilization in pentose⁃catabolizing yeasts and fungi.In addition to producing xylulose,XDH can also be employed to develop a biosensor for monitoring xylitol concentration.In this study,the gene encoding the thermophilic fungus Talaromyces emersonii XDH(TeXDH)was heterologously expressed in Escherichia coli BL21(DE3)at 16℃in the soluble form.Recombinant TeXDH with high purity was purified by using a Ni⁃NTA affinity column.Size⁃exclusion chromatography and SDS⁃PAGE analysis demonstrated that the puri⁃fied recombinant TeXDH exists as a native trimer with a molecular mass of approximately 116 kD,and is composed of three identical subunits,each with a molecular weight of around 39 kD.The TeXDH strictly preferred NAD^(+)as a coenzyme to NADP^(+).The optimal temperature and pH of the TeXDH were 40℃and 10.0,respectively.After EDTA treatment,the enzyme activity of TeXDH decreased to 43.26%of the initial enzyme activity,while the divalent metal ions Mg^(2+)or Ca^(2+)could recover the enzyme activity of TeXDH,reaching 103.32%and 110.69%of the initial enzyme activity,respectively,making them the optimal divalent metal ion cofactors for TeXDH enzyme.However,the divalent metal ions of Mn^(2+),Ni^(2+),Cu^(2+),Zn^(2+),Co^(2+),and Cd^(2+)significantly inhibited the activity of TeXDH.ICP⁃MS and molecular doc⁃king studies revealed that 1 mol/L of TeXDH bound 2 mol/L Zn^(2+)ions and 1 mol/L Mg^(2+)ion.Further⁃more,TeXDH exhibited a high specificity for xylitol,laying the foundation for the development of future xylitol biosensors.
基金Supported by the Public Science and Technology Research Funds Project of Ocean(No.201105027)the Shandong Province Young and the Middle-Aged Scientists Research Awards Fund(No.DS2010HZ001)the Basic Scientific Research Funds of First Institute of Oceanography,State Oceanic Administration(No.GY02-2011G17)
文摘An agar-degrading bacterium, designated as Pseudoalteromonas sp. NJ21, was isolated from an Antarctic sediment sample. The agarase gene aga1161 from Pseudoalteromonas sp. NJ21 consisting of a 2 382-bp coding region was cloned. The gene encodes a 793-amino acids protein and was found to possess characteristic features of the Glyco_hydro 42 family. The recombinant agarase （rAgal 161） was overexpressed in Escherichia coli and purified as a fusion protein. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were 30--40℃ and 8.0, respectively. rAga 1161 was found to maintain as much as 80% of its maximum activity at 10℃, which is typical of a cold- adapted enzyme. The pattern of agar hydrolysis demonstrated that the enzyme is an β-agarase, producing neoagarobiose （NA2） as the final main product. Furthermore, this work is the first proof of an agarolytic activity in Antarctic bacteria and these results indicate the potential for the Antarctic agarase as a catalyst in medicine, food and cosmetic industries.
基金Supported by the National High-tech Research and Development Program of China(No.2007AA021307)
文摘The development of industrial biotechnology has created an increasing demand for alkaline lipolytic enzymes with functional diversity. In this study, an alkaline soil metagenomic library was constructed to search for new lipolytic enzymes. Two novel gene encoded alkaline esterases（designated as estA and estB） were isolated by functional screening from the library. The estA gene consisted of 834 bp and coded for 277 amino acids with a molecular mass of 29998. Amino acid sequence homology analysis indicates that EstA belongs to α/β hydrolase fold family 4.4（abH4.4）, with EstA being the smallest member of that family yet reported. The estB gene consisted of 1185 bp and encoded 394 amino acids with a theoretical molecular mass of 40090. Its conserved domain analysis shows that EstB belongs to the GDSL hydrolase superfamily. Both EstA and EstB exhibit only moderate identity（〈38%） in amino acid sequence to the known lipolytic enzyme genes in the database. The two genes were respectively expressed in Escherichia coli and the protein products were purified for functional characterization. While the expressed EstA did not exhibit the functional properties that were superior to those of other esterases previously reported, the EstB was stable at temperature up to 45 ℃ and its maximum activity was measured to be 53.6 U/mg at pH=10. Both the en- zymes have further enriched the diversity of the lipolytic enzymes database and also appear to be promising biocatalysts for potential biotechnological application.
基金Project supported by the Science&Technology Major Project of Zhejiang Province,China(No.2012C12005-2)
文摘An extracellular lipase from Aureobasidium pullulans was obtained and purified with a specific activity of 17.7 U/mg of protein using ultrafiltration and a DEAE-Sepharose Fast Flow column. Characterization of the lipase indicated that it is a novel finding from the species A. pullulans. The molecular weight of the lipase was 39.5 kDa, determined by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis(SDS-PAGE). The enzyme exhibited its optimum activity at 40 °C and pH of 7. It also showed a remarkable stability in some organic solutions(30%, v/v) including n-propanol, isopropanol, dimethyl sulfoxide(DMSO), and hexane. The catalytic activity of the lipase was enhanced by Ca2+ and was slightly inhibited by Mn2+ and Zn2+ at a concentration of 10 mmol/L. The lipase was activated by the anionic surfactant SDS and the non-ionic surfactants Tween 20, Tween 80, and Triton X-100, but it was drastically inhibited by the cationic surfactant cetyl trimethyl ammonium bromide(CTAB). Furthermore, the lipase was able to hydrolyze a wide variety of edible oils, such as peanut oil, corn oil, sunflower seed oil, sesame oil, and olive oil. Our study indicated that the lipase we obtained is a potential biocatalyst for industrial use.
基金Supported by the National Key Technology R&D Program of China(No.2012BAC07B03)
文摘A superoxide dismutase was purified from Enteromorpha linza using a simple and safe procedure, which comprised phosphate buffer extraction, ammonium sulphate precipitation, ion exchange chromatography on Q-sepharose column, and gel filtration chromatography on Superdex 200 10/300GL. The E. linza superoxide dismutase （E/SOD） was purified 103.6-fold, and a yield of 19.1% and a specific activity of 1 750 U/rag protein were obtained. The SDS-PAGE exhibited E/SOD a single band near 23 kDa and the gel filtration study showed E/SOD＇s molecular weight is near 46 kDa in nondenatured condition, indicating it＇s a homodimeric protein. E/SOD is an iron-cofactored superoxide dismutase （Fe-SOD） because it was inhibited by hydrogen peroxide, insensitive to potassium cyanide. The optimal temperature for its maximal enzyme activity was 35℃, and it still had 29.8% relative activity at 0℃, then E/SOD can be classified as a cold-adapted enzyme. E/SOD was stable when temperature was below 40℃ or the pH was within the range of 5 10. The first 11 N-terminal amino acids orE/SOD were ALELKAPPYEL, comparison of its N-terminal sequence with other Fe-SOD N-terminal sequences at the same position suggests it is possibly a chloroplastic Fe-SOD.
基金Supported by National Natural Science Foundation of China(30670067)~~
文摘[Objective] This study aimed to investigating properties of alkaline protease produced by strain Ⅰ 13.[Method] Crude enzyme of alkaline protease was obtained from alkaline protease produced by strain I 13,while effects of temperature and pH value on enzyme activity were also investigated in this study.[Result] The optimal temperature of alkaline protease produced by strain Ⅰ 13 was 40 ℃,while enzyme activity maintains a higher level from 30 to 60 ℃ and over 40% of the largest enzyme activity still maintained within the range from 20 to 30 ℃.The optimal pH value was 10.5,and over 90% of the largest enzyme activity still maintained within the range from 8.0 to 11.0,which had broader pH value spectrum.[Conclusion] This alkaline protease has huge potential to be developed into washing-powder additive.
