异甘露聚糖酶基因的克隆表达及酶学性质分析

Cloning and expression of isomannanase gene and characterization of the enzyme

摘要: 以pET-30（a）为表达载体,将来源于枯草芽孢杆菌K-6（Bacillus subtilis K-6）的异甘露聚糖酶基因（isoman K-6）在Escherichia coli BL21（DE3）中进行了表达。Isoman K-6登录号为HQ902141,基因全长为1083bp,共编码360个氨基酸,工程菌酶活为415.9U/mL,SDS-PAGE电泳表明,工程酶ISOMAN K-6分子量约为45000u,与预期分子量相符。经IDAHisBind树脂柱纯化的纯酶酶学性质研究表明,该重组酶为金属酶,Al3+和Ba2+对其有很强的激活作用,其最适反应温度为55℃、最适pH为6.0,且在pH4.5⁷之间有着较好的稳定性,以槐豆胶为底物时,Vmax和Km分别为1.3U/mL和7.3mg/mL。

Abstract: A gene (isoman K-6) encoding an isomannanase of Bacillus subtilis K-6 was cloned using the plasmid vector pET-30 (a) and expressed in Escherichia coli BL21 (DE3) . Genbank accession number of isoman K-6 was HQ902141, full length of isoman K6 was 1083bp, encoding 360 amino acid residues, activity of recombinant strain was 415.9U/mL. SDS-PAGE demonstrated the molecular mass of recombinant enzyme was 45000u. The isomannanase was purified by IDA HisBind resin column, enzymatic characteristics analysis showed that the enzyme could be activated by Al3+ and Ba2+, the optimum temperature was 55℃, the optimum pH for the enzyme was 6.0 and was stable from pH4.5 to 7, The apparent Km and Vmax values for locust bean gum were 1.3U/mg and 7.3mg/mL, respectively.