短小芽孢杆菌耐碱性β-甘露聚糖酶基因的异源表达及其酶学特性

Alkaline-adapted beta-mannanase of Bacillus pumilus:gene heterologous expression and enzyme characterization

摘要:

摘要:【目的】本研究报道了一种新颖的耐碱性β-甘露聚糖酶基因的异源表达并研究其酶学特性，为其工业应用奠定基础。【方法】通过基因同源性分析以及染色体步移技术，从短小芽孢杆菌Nsic-2中克隆得到甘露聚糖酶基因(manB)。然后分别将manB基因在大肠杆菌BL21(DE3)和枯草芽胞杆菌WB800N中进行表达，并研究其酶学特性。【结果】克隆得到的manB基因序列有一个含1104 bp的开放阅读框，编码一种含有367个氨基酸的甘露聚糖酶(ManB)。经预测其蛋白序列N末端有一个含有31个氨基酸的信号肽。将ManB的氨基酸序列进行同源性分析，发现其与来源于短小芽孢杆菌CCAM080065的甘露聚糖酶具有很高的一致性，可以推测ManB属于糖苷水解酶家族26。manB基因在大肠杆菌BL21(DE3)中成功地表达，得到甘露聚糖酶最高酶活为11021.3 U/mL。与其它甘露聚糖酶比较，ManB在碱性条件下表现出较高的稳定性，在pH6.0－9.0之间酶活相对稳定。纯化后的ManB比活可达4191±107 U/mg。酶反应动力学参数Km和Vmax分别为35.7 mg/mL和14.9 μmol/(mL·min)。同时，在枯草芽胞杆菌WB800N中也成功地实现了重组蛋白ManB的分泌表达。【结论】β-甘露聚糖酶基因成功实现异源表达，并得到其酶学性质。本文是首次报道从臭豆腐卤液中分离菌株，克隆表达甘露聚糖酶，并描述其酶学特性。ManB在碱性条件下的酶活稳定性，使得其在工业应用中具备较高的潜在应用价值。

Abstract:

Abstract:［Objective］We expressed a novel alkaline-adapted beta-mannanase gene and characterized the enzyme for potential industrial applications.［Methods］We obtained a mannanase gene (named manB) from Bacillus pumilus Nsic-2 and expressed the gene manB in Escherichia coli and Bacillus subtilis． Furthermore，we characterized the enzyme.［Results］The gene manB had an open reading frame of 1104 bp that encoded a polypeptide of 367-amino-acid betamannanase (ManB)．The protein sequence showed the highest identity with the beta-mannanase from B．pumilus CCAM080065． We expressed the gene manB in E.coli BL21 (DE3) with the enzyme activity of 11021. 3 U/mL．Compared with other mannanases，ManB showed higher stability under alkaline conditions and was stable at pH6.0－9.0．The specific activity of purified ManB was 4191±107 U/mg．The Km and Vmax values of purified ManBwere 35.7 mg/mL and 14.9 μmol/(mL·min)，respectively．Meanwhile，we achieved recombinant protein secretion expression in B．subtilis WB800N.［Conclusion］ We achieved heterologous expression of the gene manB and characterized its enzyme．The alkaline-adapted ManBshowed potential value in industrial applications due to its pH stability.