海洋链霉菌Streptomyces olivaceus FXJ7.023来源多功能几丁质酶的克隆、表达及鉴定(英文)

Isolation,Expression and Identification of Multifunctional Chitosanase from Marine Streptomyces olivaceus FXJ7. 023

碳水化合物水解酶家族在自然界碳素循环及农业废弃物中几丁质、纤维素等碳水化合物的生物质转化利用中发挥了重要作用。通过PCR技术从海洋链霉菌Streptomyces olivaceus strain FXJ 7.023的fosmid基因组文库中成功克隆得到1个全长885bp的编码295个氨基酸残基的包含1个19个氨基酸残基的N-末端信号肽的壳聚糖酶完全编码区。系统进化分析表明该基因编码蛋白与已报道的Streptomyces sp.SirexAA-E来源壳聚糖酶csnA同源性为71%,与Streptomyces coelicolor A3(2)来源的csn46A同源性为70%。将该编码区重组入原核表达质粒载体pET32a并转化大肠杆菌表达菌株BL21(DE3)plysS,添加IPTG在18℃条件下振荡诱导该蛋白表达,Ni2+-NTA亲和纯化获得分子量为50.3 kDa融合表达蛋白TrxA-SoCsn。该融合重组蛋白在最适反应条件下对底物胶体壳聚糖和羧甲基纤维素的最大酶活分别为3.673U/mg和1.302U/mg,最适反应温度分别为37℃和50℃,最适反应pH分别为pH5.0和pH6.0。TrxA-SoCsn相关的研究结果表明该酶在农业废弃物生物质转化等方面具有一定的应用潜力。

A predicted chitosanase was cloned from a fosmid genomic library of Streptomyces olivaceus strain FXJ 7. 023 by PCR. The coding fragment of 885 bp encodes of 295 amino acids was fused to the expression vector pET32a（ ＋） and transformed into Escherischia coli strains BL21（ DE3） plysS. A novel fusion protein with molecular weight of 50. 3 kDa was obtained by induced the engineered strain with 1mmol /L IPTG under18℃ and purified with the affinity chromatography of Ni^2 ＋-NTA. The recombinant chitosanase showed multifunctional catalytic activity of hydrolyze colloidal chitosan and carboxymethylcellulose with the maximal catalytic activity of 3. 673 U /mg and 1. 302 U /mg respectively. Due to it has multifunctional catalytic activity,such protein may have a potential application for the recycling of the carbohydrates and sugars in the waste.