摘要
[目的]改善D-来苏糖异构酶在酸性环境下的活性。[方法]通过对来源于Caldanaerobius polysaccharolyticus菌株的D-来苏糖异构酶进行表面残基的溶剂可及表面生物信息学分析,并设计得到突变位点K2D、K2E、K8D、K8E、K18D和K18E。通过构建突变体,实现其在大肠杆菌中的重组诱导异源表达,利用镍离子亲和层析柱分离纯化得到重组突变体酶,并开展体外重组突变体酶在酸性pH 5.5和近中性pH 6.5下催化转化D-果糖制备D-甘露糖的差异比较试验。[结果]重组突变体酶K8D和K8E显著提升了D-果糖的催化转化率,最优条件(pH 6.5)下的转化率达140%;而在pH 5.5时,转化率较野生酶提高了1.26倍。[结论]通过分子改造手段成功提高了D-来苏糖异构酶突变体在酸性条件下的反应活性。
[Objective]To improve the activity of D-lyxose isomerase under acidic conditions.[Methods]Solvent-accessible surface bioinformatics analysis was performed on surface residues of D-lyxose isomerase derived from Caldanaerobius polysaccharolyticus,and six mutation sites,i.e.,K2D,K2E,K8D,K8E,K18D,and K18E,were designed.Mutants were constructed and subjected to recombinant inducible heterologous expression in Escherichia coli.The recombinant mutant enzymes were isolated and purified using a nickel affinity chromatography column.In vitro comparative experiments were conducted to evaluate the catalytic conversion of D-fructose to D-mannose by the recombinant mutant enzymes at acidic pH 5.5 and near-neutral pH 6.5.[Results]The recombinant mutant enzymes K8D and K8E showed significantly improved catalytic conversion rates of D-fructose,with the conversion rate reaching 140%under optimal conditions(pH 6.5).At pH 5.5,the conversion rate was 1.26 times higher than that of the wild-type enzyme.[Conclusion]Molecular modification successfully enhanced the catalytic activity of D-lyxose isomerase mutants under acidic conditions.
作者
李巧玲
胡阳
周慧灵
喻勋
文李
吴昊
LI Qiaoling;HU Yang;ZHOU Huiling;YU Xun;WEN Li;WU Hao(School of Food Science and Bioengineering,Changsha University of Science&Technology,Changsha,Hunan 410114,China)
出处
《食品与机械》
北大核心
2025年第4期1-7,共7页
Food and Machinery
基金
湖南省自然科学基金青年项目(编号:2023JJ40018)
国家自然科学基金青年项目(编号:32201963)
湖南省科技创新计划资助(编号:2023RC3137)
国家级大学生创新创业训练计划项目(编号:S202310536041)。
关键词
D-甘露糖
D-来苏糖异构酶
分子改造
生物酶法
酸稳定性
D-mannose
D-lyxose isomerase
molecular modification
biological enzyme method
acid stability
