分子伴侣共表达对重组蔗糖异构酶异源可溶性表达的影响
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Q786

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Effect of Molecular Chaperone Co-Expression on Heterologous Soluble Expression of Recombinant Sucrose Isomerase
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    摘要:

    蔗糖异构酶(sucrose isomerase,SIase)在异麦芽酮糖的酶法制备中发挥着重要作用。为了提高重组蔗糖异构酶的可溶性表达,作者利用分子伴侣共表达策略将源自Klebsiella sp. LX3的基因SIase进行异源表达并研究了重组酶的酶学性质。利用酶切连接技术构建重组质粒pET-24b-SIase并转入大肠杆菌E.coli BL21(DE3)中进行表达,发现其主要表达为包涵体。分别将携带4种分子伴侣蛋白基因的质粒(pKJE7、pGro7、pG-Tf2、pTf16)与含有目的基因的重组质粒在大肠杆菌E.coli BL21(DE3)中共表达,筛选到最佳分子伴侣质粒pGro7且提高了重组SIase的可溶性表达水平,其酶活力为14.8 U/mL,比未进行共表达的工程菌发酵酶活力(3.5 U/mL)有明显提高,进一步利用金属离子螯合层析技术纯化到SIase纯酶,酶学性质研究显示其最适反应温度为40 ℃,最适反应pH为6.0。动力学常数Km为(179.10±20.65) mmol/L、kcat/Km为(5.44±0.72) L/(mmol·s)。产物特异性研究结果显示,随反应温度的提高,产物中异麦芽酮糖和海藻酮糖比例下降,单糖比例提高。利用分子伴侣共表达策略能够提高重组SIase的可溶性表达水平,促进SIase的规模化制备和应用。

    Abstract:

    Sucrose isomerase (SIase) plays an important role in the enzymatic preparation of isomaltulose. In this study, a SIase gene from Klebsiella sp. LX3 was expressed in E. coli by using the molecular chaperone co-expression system, and then purified and characterized. The SIase gene was cloned into pET-24b to yield the construct pET-24b-SIase, which was introduced into E. coli BL21(DE3) for expression. Unfortunately, the enzyme was expressed mainly as inclusion bodies. In order to enhance the soluble form of the enzyme, SIase was coexpressed with a molecular chaperone. Four plasmids (pKJE7, pGro7, pG-Tf2, pTf16), each carrying a molecular chaperone gene, were separately introduced into E. coli BL21(DE3) carrying pET-24b-SIase and the expression of SIase was evaluated. The plasmid pGro7 was the most effective at improving the expression of recombinant SIase as a soluble enzyme, yielding a specific activity of 14.8 U/mL for the crude cell extract compared with a mere 3.5 U/mL for the crude cell extract derived from cells harbouring only pET-24b-SIase. The expressed SIase was purified by affinity chromatography using Ni-NTA. The purified enzyme exhibited the maximum activity at 40 ℃ and pH 6.0. Its Km for sucrose was(179.10±20.65) mmol/L and the kcat/Km was (5.44±0.72) L/(mmol·s). The results of the product specificity study showed that the proportion of isomaltulose and trehalulose in the products decreased and the proportion of monosaccharides increased with increasing reaction temperature. The results of this study showed that the level of soluble SIase expressed in E. coli could be enhanced by coexpressing the enzyme with a molecular chaperone, suggesting a potential strategy for the large-scale production of SIase and its applications.

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孙佳明,庞焦,高向红,李明玉,王从纲,李宪臻.分子伴侣共表达对重组蔗糖异构酶异源可溶性表达的影响[J].食品与生物技术学报,2022,41(10):104-112.

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  • 在线发布日期: 2022-11-16
  • 出版日期: 2022-10-25

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