假密环菌Armillariella tabescens EJLY2098 β-甘露聚糖酶的克隆、表达及性质分析(英文)  

Cloning,expression and characterization of mannanase from Armillariella tabescens EJLY2098 in Pichia pastoris

作　　者：王艳凤[1] 王衡馨[1] 黄小葵[1] 刘大岭[1] 姚冬生[1] 

机构地区：[1]暨南大学微生物生物技术研究所,广州510632

出　　处：《生物工程学报》2009年第6期920-926,共7页Chinese Journal of Biotechnology

基　　金：Supported by:Guangdong Province Science&Technology Program(No.2005B200601004)~~

摘　　要：本研究利用RT-PCR和RACE技术从Armillariella tabescens EJLY2098(一种食用真菌)中克隆出了β-甘露聚糖酶的全长cDNA,构建到pPICZaA载体上,并在毕赤酵母GS115中表达了含His标签的β-甘露聚糖酶(re-atMAN47)。该酶的全长cDNA共1481bp,编码445个氨基酸,序列分析表明该序列除含有β-甘露聚糖酶结构域外,还含有CBD和GHF5的结构域,因此可被归为糖苷水解酶家族5的一个新成员。诱导培养72h时重组酶活可达到1.067U/mL,蛋白含量为440mg/L。重组酶的最适反应温度为60°C,在30°C～65°C比较稳定;酶促最适pH为5.5、4.5～7.0之间比较稳定。这是首次关于Armillariella. tabescens EJLY2098产β-甘露聚糖酶的报道,得到了一个有较好热稳定性、pH稳定性和生物安全性的糖苷水解酶,将在饲料、食品、药物生产等方面有广泛的应用。We used reverse transcriptase polymerase chain reaction （RT-PCR） and rapid amplification of eDNA end （RACE） techniques to obtain the full-length cDNA of β-mannanase （EC 3.2.1.78） from ArmillarieUa tabescens EJLY2098 （an edible fungus）. Sequence analysis of the 1481 bp full-length eDNA encoding 445 amino acid residues indicated that the gene contained two structural domains, cellulose-binding domains （CBD） and glycoside hydrolase family 5 （GHF5） domains, other than the conserved β-marmanase domain. Thus, we classified this gene as a member of glycoside hydrolase family 5. Next, we cloned a 1308 bp fragment encoding the β-mannanase mature peptide （re-atMAN47） into the expression vector pPICZαA and expressed it in Pichia pastoris. The yield was 440 mg/L. Enzyme activity reached a maximum of 1.067 IU/mL after 72 h of methanol induction. The re-atMAN47 had an optimal temperature of 60℃ and an optimal pH of 5.5. It manifested broad thermostability from 30℃-65℃, and was stable between pH 4.5-7.0. This study represents the first record of a β-mannanase from Armillariella tabescens EJLY2098 and provides a new source of carbohydrate hydrolysis enzyme with good biosafety, thermostability and wide pH stability. It is a good approach for the industrial needs of feed, food and pharmaceutical manufacturers.

关 键 词：β-1 4-甘露聚糖酶 Armillariella tabescens EJLY2098 酶学性质 GHF5 

分 类 号：Q93[生物学—微生物学]