Armillariella tabescens EJLY2098 β-甘露聚糖酶的克隆、表达及性质分析

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

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1481 bp, 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72 h时重组酶活可达到1.067 U/mL, 蛋白含量为440 mg/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。

Abstract:

We used reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) techniques to obtain the full-length cDNA of b-mannanase (EC 3.2.1.78) from Armillariella tabescens EJLY2098 (an edible fungus). Sequence analysis of the 1481 bp full-length cDNA 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 b-mannanase domain. Thus, we classified this gene as a member of glycoside hydrolase family 5. Next, we cloned a 1308 bp fragment encoding the b-mannanase mature peptide (re-atMAN47) into the expression vector pPICZaA 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 60oC and an optimal pH of 5.5. It manifested broad thermostability from 30oC~65oC, and was stable between pH 4.5~7.0. This study represents the ?rst 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.