麦芽低聚糖生成酶在枯草芽孢杆菌中的分泌表达及其酶学性质研究

Secretory Expression of a Maltooligosaccharide-forming Amylase in Bacillus subtilis and Its Enzymatic Properties

首先构建了来源于嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)STB04的麦芽低聚糖生成酶(MFA酶)的枯草芽孢杆菌分泌表达系统,并对含表达载体的枯草芽孢杆菌WB600产重组酶的发酵条件进行了优化,发现以TB为发酵培养基、发酵温度为30℃时,有利于重组酶的分泌表达。其次,通过疏水和强阴离子交换色谱对重组酶进行了分离纯化,得到了电泳纯的酶。最后,对重组酶的酶学性质和产物合成进行了分析,结果显示:它在溶液中为单聚体,最适反应温度为45℃,且在该温度下半衰期为30 min;最适反应p H为6.5,且具有很好的p H稳定性;酶活力对金属离子存在一定依赖性并受Fe3+和Sn2+等重金属离子的显著抑制;重组MFA酶的动力学性质可以用米氏方程进行很好的描述,对麦芽糊精的水解速率最高;重组酶作用不同来源原淀粉及麦芽糊精的主要产物均为麦芽三糖、麦芽四糖和麦芽五糖,且以麦芽五糖为最高。

A secretory expression system for the maltooligosaccharide-forming amylase from B.stearothermophilus STB04 was constructed in Bacillus subtilis and the fermentation conditions of B.subtilis WB600 containing the expression vectors for the production of the recombinant enzyme were optimized.The results showed that the secretory expression of recombinant enzyme was optimal in terrific broth（TB） medium as the fermentation medium and at a fermentation temperature of 30 ℃.The recombinant amylase was purified through a combination of hydrophobic interaction and strong anion exchange chromatography to yield electrophoretically pure enzyme.The purified enzyme was characterized with respect to its properties and the biochemical reaction it catalyzes.The results showed that this enzyme was a monomer in solution with an optimum reaction temperature of 45 ℃,and a half-life of approximately 30 min at this temperature.The enzyme functioned optimally at p H 6.5 with high p H stability.The activity of this enzyme was dependent on metal ions to a certain extent and was dramatically inhibited by some heavy metal ions,especially Fe^3＋ and Sn^2＋.The kinetics of the amylase-catalyzed reaction could be fairly well described by the Michaelis-Menten equation,and the maltodextrin hydrolysis rate was the highest compared with other substrates.The main products of the hydrolysis of native starches from different sources and maltodextrin with this recombinant enzyme were maltotriose,maltotetrose,and maltopentose.Maltopentose was predominant among the hydrolysis products.