Gene Cloning, Expression, and Characterization of an Exo-inulinase from Paenibacillus polymyxa ZJ-9
Gene Cloning, Expression, and Characterization of an Exo-inulinase from Paenibacillus polymyxa ZJ-9
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多粘类芽孢杆菌 ZJ-9 外切菊粉酶的基因克隆、表达和表征
DOI：
10.1007/s12010-014-0950-y
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发表时间：
2014-07-01
影响因子：
3
通讯作者：
Feng, Xiao-Hai
中科院分区：
文献类型：
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作者：
Gao, Jian;Xu, You-Yong;Feng, Xiao-Hai
从自然界中筛选到一株产菊粉酶的多粘类芽孢杆菌（Paenibacillus polymyxa）ZJ-9，以耶路撒冷为原料，经一步发酵生产R，R-2，3-丁二醇。克隆了多粘毕赤酵母ZJ-9的菊粉酶基因，并在大肠杆菌BL 21（DE 3）中进行了高效表达，经SDS-PAGE和凝胶过滤层析鉴定，重组菊粉酶的分子量约为56 kDa。这一结果表明菊粉酶的活性形式可能是单体。菊粉的末端水解果糖单位表明酶是外切菊粉酶。纯化的重组酶在25 ℃和pH6.0时显示出最大活性，这表明其非常适合于工业应用。Zn ~（2+）、Fe ~（2+）、Mg ~（2+）对纯化的酶活性有促进作用，Co ~（2+）、Cu ~（2+）、Ni ~（2+）对酶活性有抑制作用。菊糖水解的K（m）和V（max）值分别为1.72 mM和21.69 μ mol min（-1）mg（-1）蛋白质。针对蔗糖的相同参数分别为41.09 mM和78.7 μ mol min（-1）mg（-1）蛋白质。考虑到菊粉酶的底物特异性和其他酶学特性，我们认为该基因可以转化到其他特殊菌株中，通过一步发酵将菊粉转化为其他生物化学品和生物能源。
An inulinase-producing strain, Paenibacillus polymyxa ZJ-9, was isolated from natural sources to produce R,R-2,3-butanediol via one-step fermentation of raw inulin extracted from Jerusalem artichoke tubers. The inulinase gene from P. polymyxa ZJ-9 was cloned and overexpressed in Escherichia coli BL21 (DE3), and the purified recombinant inulinase was estimated to be approximately 56 kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. This result suggests that the active form of the inulinase is probably a monomer. Terminal hydrolysis fructose units from the inulin indicate that enzymes are exo-inulinase. The purified recombinant enzyme showed maximum activity at 25 A degrees C and pH 6.0, which indicate its extreme suitability for industrial applications. Zn2+, Fe2+, and Mg2+ stimulated the activity of the purified enzyme, whereas Co2+, Cu2+, and Ni2+ inhibited enzyme activity. The K (m) and V (max) values for inulin hydrolysis were 1.72 mM and 21.69 mu mol min(-1) mg(-1) protein, respectively. The same parameters toward sucrose were 41.09 mM and 78.7 mu mol min(-1) mg(-1) protein, respectively. Considering its substrate specificity and other enzymatic characteristics, we believe that this inulinase gene from P. polymyxa ZJ-9 could be transformed into other special bacterial strains to allow inulin conversion to other biochemicals and bioenergy through one-step fermentation.