木聚糖酶EvXyn11~(TS)N端区域对其耐热性贡献的生物信息学分析
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摘要
系统分析了11家族木聚糖酶EvXyn11~(TS)N端区域对其耐热性的影响。在GenBank数据库中借助BLAST服务器搜寻与EvXyn11~(TS)序列同源性大于55%且温度特性已知的30种木聚糖酶;运用ClustalW2程序和MEGA 5.1软件对31种木聚糖酶进行了多序列比对及进化树构建,从中选择了包含EvXyn11~(TS)在内的8种代表性耐热和中温酶;采用生物学程序软件分析了所选酶在N端的异同点。分析结果表明,EvXyn11~(TS)N端的耐热有利氨基酸含量、β-折叠股A1和氨基酸相互作用等对其耐热性有一定的贡献。
Effect of the N-terminus of a family 11 xylanase EvXyn11~(TS)on its thermostability is systematically analyzed.Thirty xylanases that had the sequence homologies more than 55%aligned with EvXyn11~(TS)and the known temperature characteristics are sought out from the GenBank database using the BLAST server.Then,the multiple alignment of 31 xylanase sequences is performed using the ClustalW2 program,and the phylogenetic tree is constructed using a MEGA 5.1software.Eight representative thermophilic and mesophilic xylanases(including EvXyn11~(TS))are selected from the phylogenetic tree.Finally,the differences and similarities at the N-termini between EvXyn11~(TS)and the other xylanases are analyzed using biology softwares.The analytical results indicate that the contents of amino acids favorable to thermostability,theβ-strand A1 and the interactions between amino acids at the N-terminus of EvXyn11~(TS)have certain contributions to its thermostability.
Effect of the N-terminus of a family 11 xylanase EvXyn11~(TS)on its thermostability is systematically analyzed.Thirty xylanases that had the sequence homologies more than 55%aligned with EvXyn11~(TS)and the known temperature characteristics are sought out from the GenBank database using the BLAST server.Then,the multiple alignment of 31 xylanase sequences is performed using the ClustalW2 program,and the phylogenetic tree is constructed using a MEGA 5.1software.Eight representative thermophilic and mesophilic xylanases(including EvXyn11~(TS))are selected from the phylogenetic tree.Finally,the differences and similarities at the N-termini between EvXyn11~(TS)and the other xylanases are analyzed using biology softwares.The analytical results indicate that the contents of amino acids favorable to thermostability,theβ-strand A1 and the interactions between amino acids at the N-terminus of EvXyn11~(TS)have certain contributions to its thermostability.
引文
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[23]ZHOU C,XUE Y F,MA Y H. Enhancing the thermostability ofα-glucosidase from Thermoanaerobacter tengcongensis MB4 by single proline substitution[J]. Journal of Bioscience and Bioengineering,2010,110(1):12-17.
[24]SILVA I R,LARSEN D M,JERS C,et al. Enhancing RGI lyase thermostability by targeted single point mutants[J]. Applied Microbiology and Biotechnology,2013,97(22):9727-9735.
[2]ZHANG H M,LI J F,WANG J Q,et al. Determinants for the improved thermostability of a mesophilic family 11 xylanase predicted by computational methods[J]. Biotechnology for Biofuels,2014,7:3.
[3]KUMAR S,TSAI C J,NUSSINOV R. Factors enhancing protein thermostability[J]. Protein Engineering,2000,13(3):179-191.
[4]DUMON C,VARVAK A,WALL M A,et al. Engineering hyperthermostability into a GH11 xylanase is mediated by subtle changes to protein structure[J]. The Journal of Biological Chemistry,2008,283(33):22557-22564.
[5]MIN Rou,LI Jianfang,GAO Shujuan,et al. Correlation between thermostability of the xylanase Ev Xyn11TSand its N-terminal disulfide bridge[J]. Acta Microbiologica Sinica,2013,53(4):346-353.(in Chinese)
[6]GAO Shujuan,WAHG Junqing,WU Minchen,et al. Effect of N-terminal disulfide bridge on thermostability of family 11xylanases[J]. Chinese Journal of Biotechnology,2012,28(12):1441-1449.(in Chinese)
[7]LESKINEN S,MANTYLA A,FAGERSTROM R,et al. Thermostable xylanases,Xyn10A and Xyn11A,from the actinomycete Nonomuraea flexuosa:isolation of the genes and characterization of recombinant Xyn11A polypeptides produced in Trichoderma reesei[J]. Applied Microbiology Biotechnology,2005,67:495-505.
[8]VERMA D,KAWARABAYASI Y,MIYAZAKI K,et al. Cloning,expression and characteristics of a novel alkalistable and thermostable xylanase encoding gene(Mxyl)retrieved from compost-soil metagenome[J]. Plos One,2013,8(1):e52459.
[9]FU X Y,ZHAO W,XIONG A S,et al. High expression of recombinant Streptomyces sp. S38 xylanase in Pichia pastoris by codon optimization and analysis of its biochemical properties[J]. Molecular Biology Reports,2011,38:4991-4997.
[10]MILLWARD-SADLER S J,POOLE D M,HENRISSAT B,et al. Evidence for a general role for high-affinity non-catalytic cellulose binding domains in microbial plant cell wall hydrolases[J]. Molecular Microbiology,1994,11(2):375-382.
[11]RULLER R,ROSA J C,FACA V M,et al. Efficient constitutive expression of Bacillus subtilis xylanase A in Escherichia coli DH5αunder the control of the Bacillus Bs XA promoter[J]. Biotechnology and Applied Biochemistry,2006,43:9-15.
[12]HE J,YU B,ZHANG K Y,et al. Expression of a Trichoderma reeseiβ-xylanase gene in Escherichia coli and activity of the enzyme on fiber-bound substrates[J]. Protein Expression and Purification,2009,67:1-6.
[13]LI J F,GAO S J,LIU X T,et al. Modified pPIC9K vector-mediated expression of a family 11 xylanase gene,Aoxyn11A,from Aspergillus oryzae in Pichia pastoris[J]. Annals of Microbiology,2013,63:1109-1120.
[14]ZHANG Guimin,RAO Ben,YE Jian,et al. Molecular cloning and heterologous expression of a new xylanase gene from Verticillium dahliae[J]. Acta Microbiologica Sinica,2008,48(6):765-771.(in Chinese)
[15]REETZ M T,CARBALLEIRA J D. Iterative saturation mutagenesis(ISM)for rapid directed evolution of functional enzymes[J].Nature Protocols,2007,2(4):891-903.
[16]LU Baisong,WANG Guoli,HUANG Peitang. A comparison of amino acid composition of proteins from thermophiles and mesophiles[J]. Acta Microbiologica Sinica,1998,38(1):20-25.(in Chinese)
[17]CHEN Z F,ZHANG H M,WANG J Q,et al. Engineering the thermostability of a xylanase from Aspergillus oryzae by an enhancement of the interactions between the N-terminus extension and theβ-sheet A2 of the enzyme[J]. Biotechnology Letters,2013,35(12):2073-2079.
[18]RULLER R,DELIBERTO L,FERREIRA T L,et al. Thermostable variants of the recombinant xylanase A from Bacillus subtilis produced by directed evolution show reduced heat capacity changes[J]. Proteins-Structure Function and Bioinformatics,2008,70(4):1280-1293.
[19]CHEN Zhongfa,TANG Cunduo,WANG Junqing,et al. Effect of engineering a disulfide bridge into the N-terminal region of Aspergillus oryzae xylanase on its thermostability[J]. Journal of Food Science and Biotechnology,2013,32(7):666-671.(in Chinese)
[20]ZHANG S,ZHANG K,CHEN X Z,et al. Five mutations in N-terminus confer thermostability on mesophilic xylanase[J].Biochemical and Biophysical Research Communications,2010,395(2):200-206.
[21]黄志坚.氨基酸的构型和性质研究[D].合肥:中国科学技术大学,2006.
[22]SUZUKI Y,OISHI K,NAKANO H. A strong correlation between the increase in number of proline residues and the rise in thermostability of five Bacillus oligo-1,6-glucosdiases[J]. Applied Microbiology and Biotechnology,1987,26:546-551.
[23]ZHOU C,XUE Y F,MA Y H. Enhancing the thermostability ofα-glucosidase from Thermoanaerobacter tengcongensis MB4 by single proline substitution[J]. Journal of Bioscience and Bioengineering,2010,110(1):12-17.
[24]SILVA I R,LARSEN D M,JERS C,et al. Enhancing RGI lyase thermostability by targeted single point mutants[J]. Applied Microbiology and Biotechnology,2013,97(22):9727-9735.