蔗糖异构酶的GST融合表达和酶学性质  

Expression and enzymatic properties of recombinant sucrose isomerase fused with GST-tag

作　　者：刘羽欣 高向红 孙佳明 王从纲 李宪臻[1] LIU Yuxin;GAO Xianghong;SUN Jiaming;WANG Conggang;LI Xianzhen(School of Biological Engineering,Dalian Polytechnic University,Dalian 116034,China)

机构地区：[1]大连工业大学生物工程学院,辽宁大连116034

出　　处：《大连工业大学学报》2025年第1期14-19,共6页Journal of Dalian Polytechnic University

基　　金：辽宁省教育厅基本科研项目(LJKFZ20220213)。

摘　　要：为提高重组蔗糖异构酶的可溶性表达量并改善其酶学性质,通过融合GST标签技术对源自Klebsiella sp.LX3的蔗糖异构酶SIase进行表达和改性。以大肠杆菌BL21(DE3)为表达宿主,分别对N/C端融合GST标签的融合酶进行诱导表达获得可溶性蛋白,利用亲和层析技术对重组融合酶进行分离纯化获得纯酶。对融合酶进行酶学性质表征,结果显示,GST-SIase和SIase-GST纯酶的最适反应温度均为40℃,最适反应pH均为6.0。动力学结果显示,GST-SIase对底物蔗糖的K_(m)为(137.6±5.7)mmol/L,k_(cat)/K_(m)为(6.41±0.15)L/(mmol·s);SIase-GST对底物蔗糖的K_(m)为(159.5±11.8)mmol/L,k_(cat)/K_(m)为(7.06±0.56)L/(mmol·s)。产物特异性分析结果显示,融合酶转化蔗糖的主产物均为异麦芽酮糖,随着转化反应温度的提高,产物中海藻酮糖比例降低,单糖比例提高。利用融合GST标签技术提高了重组SIase的可溶性表达,有利于其在工业上的规模化制备和应用。To enhance the soluble expression of recombinant SIase and improve its enzymatic properties,a SIase from Klebsiella sp.LX3 was expressed and modified using a fusion tag GST.The recombinant SIases fused with GST-tag were expressed in E.coli BL21(DE3),then purified using affinity chromatography and characterized.The results indicated that GST-SIase and SIase-GST exhibited maximum activity at 40℃and pH 6.0.K_(m)of GST-SIase for sucrose was(137.6±5.7)mmol/L and K_(cat)/K_(m)was(6.41±0.15)L/(mmol·s).K_(m)of SIase-GST for sucrose was(159.5±11.8)mmol/L and K_(cat)/K_(m)was(7.06±0.56)L/(mmol·s).The result of product specificity showed that the main product of fusion enzymes was isomaltulose.The proportion of trehalulose in the product decreased with increasing reaction temperature,while the proportion of monosaccharides increased.The use of GST tag fusion technology has improved the soluble expression of recombinant SIase,which is beneficial for its large-scale preparation and application in industry.

关 键 词：蔗糖异构酶 GST标签 异麦芽酮糖 

分 类 号：TS201.1[轻工技术与工程—食品科学]