一种新糖苷酶家族的A.tabescens β-甘露聚糖酶Man78基因克隆及序列分析的研究
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摘要
β-1,4-D-甘露聚糖酶(β-1,4-D-mannan mannohydrolase;Ec.3.2.1.78),简称β—甘露聚糖酶(β-mannanase),是一类能够水解含有β-1,4-D-甘露糖苷键的甘露寡糖、甘露多糖的内切水解酶,它属于半纤维素酶类,广泛存在于动物、植物和微生物中。迄今,所报道的β—甘露聚糖酶均归属糖苷水解酶家族5和26。该酶已在饲料、食品、造纸、石油开采及生物质能等多方面有了广泛的应用。
本研究通过诱导培养本实验室保藏菌种A.tabescens EJLY2098,且对其分泌的β—甘露聚糖酶Man78蛋白进行纯化,获得了电泳纯的β—甘露聚糖酶,利用MS/MS技术分析了其肽段氨基酸序列,并以此获得的肽段氨基酸序列设计简并引物,以基因组DNA为模板进行简并PCR,再通过SMART-RACE技术,获得β—甘露聚糖酶的末端cDNA序列,最后进行成熟肽PCR,获得全长cDNA序列。对获得的全长cDNA序列进行ORF分析,获得蛋白序列。然后对所获得的蛋白序列进行同源性、一级结构、二级结构、三级结构的生物信息学分析。经过分析发现,所获得的A.tabescensβ—甘露聚糖酶Man78与现已报道的属糖苷水解酶家族5和26的β—甘露聚糖酶没有同源性,存在氧化还原酶结构域,推测该酶属于一个新的糖苷水解酶家族。
本研究的完成标志A.tabescens所产的两个组分的β—甘露聚糖酶的分离和基因的全长cDNA克隆全部完成。A.tabecscensβ—甘露聚糖酶Man78可能属一个新的糖苷水解酶家族,深入开展β—甘露聚糖酶Man78的研究对认识β—甘露聚糖酶有十分重要的意义。
β-mannanase is a short name forβ-1,4-D-mannan mannanohydrolase.It is a kind of endohydro-enzyme that can hydrolyze t he substance wit hβ-1,4-D-indican-bond.It universally exits in the nature,including the inferior animals and legume plants,whereas microorganism is the main source forβ-mannanase.All mannanases belong to GHF 5 and GHF26.So,it is a new age for the research and usage of beta—mannanase.
By inducing A.tabescens EJLY2098,and purifing the protein that A.tabescens EJLY2098 secreted,a single band on the SDS-PAGE was obtained,β-mannanase. Basing on this,the purifiedβ-mannanase was enzymolysis in gel,then analyzed the partial peptides in the protein.Three random peptides was obtained.Acording these sequences,we designed degenerate primers to amplify.A 1457bp DNA fragment was amplidied from A.tabescens EJLY2098 with the primers by degenerate PCR with the genome DNA as template.Gene specific primers derived from this partial sequence were used for the amplication of the 3'and 5'ends of this cDNA by RACE method,then splicing to a full-length cDNA sequence.Then the analysis including the ORF,homologous, primary structure,secondary structure,tertiary structure was made with the sequence.
This work means that it has been finished that the seperation and the genes cloning of beta-mannanase Man45 and Man78 which produced by A.tabescens EJLY2098.The beta -mannanase Man 78 maybe a member of another new GHF but of GHF5 and GHF26.The penetrating research of the beta-mannanase Man78 is very important for the knowledge of beta-mannanases.
本研究通过诱导培养本实验室保藏菌种A.tabescens EJLY2098,且对其分泌的β—甘露聚糖酶Man78蛋白进行纯化,获得了电泳纯的β—甘露聚糖酶,利用MS/MS技术分析了其肽段氨基酸序列,并以此获得的肽段氨基酸序列设计简并引物,以基因组DNA为模板进行简并PCR,再通过SMART-RACE技术,获得β—甘露聚糖酶的末端cDNA序列,最后进行成熟肽PCR,获得全长cDNA序列。对获得的全长cDNA序列进行ORF分析,获得蛋白序列。然后对所获得的蛋白序列进行同源性、一级结构、二级结构、三级结构的生物信息学分析。经过分析发现,所获得的A.tabescensβ—甘露聚糖酶Man78与现已报道的属糖苷水解酶家族5和26的β—甘露聚糖酶没有同源性,存在氧化还原酶结构域,推测该酶属于一个新的糖苷水解酶家族。
本研究的完成标志A.tabescens所产的两个组分的β—甘露聚糖酶的分离和基因的全长cDNA克隆全部完成。A.tabecscensβ—甘露聚糖酶Man78可能属一个新的糖苷水解酶家族,深入开展β—甘露聚糖酶Man78的研究对认识β—甘露聚糖酶有十分重要的意义。
β-mannanase is a short name forβ-1,4-D-mannan mannanohydrolase.It is a kind of endohydro-enzyme that can hydrolyze t he substance wit hβ-1,4-D-indican-bond.It universally exits in the nature,including the inferior animals and legume plants,whereas microorganism is the main source forβ-mannanase.All mannanases belong to GHF 5 and GHF26.So,it is a new age for the research and usage of beta—mannanase.
By inducing A.tabescens EJLY2098,and purifing the protein that A.tabescens EJLY2098 secreted,a single band on the SDS-PAGE was obtained,β-mannanase. Basing on this,the purifiedβ-mannanase was enzymolysis in gel,then analyzed the partial peptides in the protein.Three random peptides was obtained.Acording these sequences,we designed degenerate primers to amplify.A 1457bp DNA fragment was amplidied from A.tabescens EJLY2098 with the primers by degenerate PCR with the genome DNA as template.Gene specific primers derived from this partial sequence were used for the amplication of the 3'and 5'ends of this cDNA by RACE method,then splicing to a full-length cDNA sequence.Then the analysis including the ORF,homologous, primary structure,secondary structure,tertiary structure was made with the sequence.
This work means that it has been finished that the seperation and the genes cloning of beta-mannanase Man45 and Man78 which produced by A.tabescens EJLY2098.The beta -mannanase Man 78 maybe a member of another new GHF but of GHF5 and GHF26.The penetrating research of the beta-mannanase Man78 is very important for the knowledge of beta-mannanases.
引文
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[2]蒋燕军,程立中.枯草芽孢杆菌B 2甘露聚糖酶的三种纯化方法研究[J].云南大学学报(自然科学版),1998,20(3):200-202.
[3]吴襟,何秉旺.诺卡氏菌形放线菌β2甘露聚糖酶的化学修饰及活性中心的研究[J].中国生物化学与分子生物学报,2000,16(2):227-230.
[4]张庆民.产β-甘露聚糖酶NK-27菌株基因文库的构建[D].天津:南开大学,2000,13(1):117-122.
[5]马延和,周培瑾,田新玉,等.碱性β2甘露聚糖酶发酵工艺的研究[J].微生物学通报,1992,29(1):13-17.
[6]陈小兵,丁红标,乔 宇,等.甘露寡糖的益生作用免疫机制与应用技术[J].营养与饲养,2005,(8):6-8.
[7]田亚平,金其荣.黑曲霉β2甘露聚糖酶的纯化及基本性质[J].无锡轻工大学学报,1998,17(2):16-21.
[8]马建华,高扬,牛秀田,等.枯草芽孢杆菌中性β2甘露聚糖酶的纯化及性质研究[J].中国生物化学与分子生物学报,1999,15(1):79-82.
[9]Johnson K G.Exocelluar β-mannanase from hemicelluiolytic fungi [J].Microbiol Biotechnol,1990,60(2):209-217.
[10]李江华,房 峻,邬显章.黑曲霉酸性β2甘露聚糖酶的摇瓶发酵条件[J]江苏食品与发酵,2002,4(4):16-18.
[11]Politz O,Krah M,Thomsen K K,et al.A highly thermostable endo-(1,4)-beta-mannanase from the marine bacterium Rhodothermus marinus(J).Appl Microbiol Biotechnol,2000,53(6):715-721.
[12]Gibbs M D,Reeves R A,Sunna A,et al.Sequencing and expression of a beta-mannanase gene from the extreme themophile Dictyoglomus thermophilum Rt468B.1,and characteristics of the recombinant enzyme[J].Curr Microbiol 1999,39(6):351-357.
[13]Christgau S,Kauppinen S,Vind J,et al.Expression cloning,purification and characterization of a beta-1,4-mannanase from Aspergillus aculeatus[J].Biochem Mol Biol Int,1994,33(5):917-925.
[14]Ether N,Talbot G,Syguzch J.Gene Cloning,DNA Sequencing,and Expression of Themostable-Mannanase from Bacillus sterothermophillus[J].Appl.Envir.Microbiol.1998,649(ii):4428-4432.
[15]Bewley J D,Burton R A,Morohashi Y,et al.Molecular cloning of a cDNA encoding a(1-4>)-beta-mannan endohydrolase from the seeds of germinated tomato(Lycopersicon esculentum)[J]Planta,1997,203(4):454-459.
[16]Nonogaki,H,Morohashi Y.An endo-β-mannanase develops exclusively in the micropylar endosperm of tomato seed prior to radlicle emergence.Plant Physiol.1996,110:555-559.
[17]Samonte,J.L.Mendoza,E.M.T.Ilag,L.L.et al.Galactomannan degrading enzymes in maturing normal and makapuno and germinating normal coconut endosperm.Phytochemistry.1989,28(9):2269-2273.
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