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细菌麦芽糖淀粉酶在枯草芽孢杆菌中的诱导型异源表达
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国家自然科学基金青年基金项目(No. 31401674);江南大学自主重点项目(No. JUSRP51503)


Inducible heterogenous expression of bacterial maltogenic amylase in Bacillus subtilis
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    摘要:

    【目的】实现地衣芽孢杆菌麦芽糖淀粉酶在枯草芽孢杆菌中的高效异源表达,并研究该重组酶的酶学性质。【方法】克隆巨大芽孢杆菌木糖异构酶基因的启动子区域及其调控蛋白,构建一个大肠杆菌/芽孢杆菌穿梭型诱导表达质粒,使用该诱导型启动子介导麦芽糖淀粉酶编码基因,实现其在枯草芽孢杆菌中的功能表达。对重组枯草芽孢杆菌的诱导条件进行优化,提高麦芽糖淀粉酶的产量。【结果】获得了诱导表达麦芽糖淀粉酶基因的重组枯草芽孢杆菌菌株。最适诱导温度为45 °C,最适诱导剂添加浓度为1%,最适添加诱导剂时间为接种培养9 h后。重组酶蛋白分子量大小为67 kD,对该酶的酶学性质研究发现,以可溶性淀粉为底物,反应生成麦芽糖和葡萄糖,其中麦芽糖含量为60.42%。重组酶最适作用温度为45 °C,最适作用pH为6.5,Ca2+、Co2+、EDTA对该重组麦芽糖淀粉酶具有激活作用。【结论】通过木糖诱导表达系统可以实现麦芽糖淀粉酶在枯草芽孢杆菌中的高效诱导型表达,酶活最高可达296.64 U/mL发酵液,在工业上有着较好的应用前景。

    Abstract:

    [Objective] To achieve the efficient heterogenous expression of a bacterial maltogenic amylase in Bacillus subtilis and investigate the characterization of the recombinant enzyme. [Methods] We cloned the genes of xylose isomerase promoter region and its regulatory protein from Bacillus megatherium into an Escherichia coli / Bacillus sp. shuttle expression vector, and constructed an recombinant expression plasmid which harbored an encoding gene of maltogenic amylase from Bacillus licheniformis. Then we transformed the expression plasmid into Bacillus subtilis and optimized the induction conditions of the transformant to increase the production of the recombinant enzyme. [Results] The recombinant Bacillus subtilis which inductively expressed the maltogenic amylase was obtained. The optimal induction condition was as follow, that 1% inducer was added into fermentation medium after 9 h-incubation at 45 °C. The recombinant maltogenic amylase had a molecular size of 67 kD. From the study of the enzymatic properties of the enzyme, it was found that, with soluble starch as substrate, the reaction products were 60.42% maltose and few glucose. Furthermore, the recombinant enzyme showed an optimal activity at 45 °C with pH 6.5. Ca2+、Co2+ and EDTA could improve the efficiency of enzyme reaction. [Conclusion] With induction of xylose, the bacterial maltogenic amylase was efficiently expressed in the recombinant Bacillus subtilis, and the maximal yield reached 296.64 U/mL, which showed good application prospect in industry.

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杨韵霏,李由然,张梁,李赢,顾正华,丁重阳,石贵阳. 细菌麦芽糖淀粉酶在枯草芽孢杆菌中的诱导型异源表达[J]. 微生物学通报, 2017, 44(2): 263-273

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  • 在线发布日期: 2017-02-10
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