D-甘露糖异构酶的酶学性质探究和热稳定性改造  

Characterization and thermostability modification of D-mannose isomerase

作　　者：沈玲 赵丽婷 沈昱 陈磊 李军训 顾正华 李由然[1,3] 石贵阳 丁重阳[1,2,3] SHEN Ling;ZHAO Liting;SHEN Yu;CHEN Lei;LI Junxun;GU Zhenghua;LI Youran;SHI Guiyang;DING Zhongyang(National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing,Jiangnan University,Wuxi 214122,China;Key laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China;Schoo1 of Biotechnology,Jiangnan University,Wuxi 214122,China;Shandong Taishan Shengliyuan Group Co.Ltd.,Taian 271000,China)

机构地区：[1]江南大学,粮食发酵与食品生物制造国家工程研究中心,江苏无锡214122 [2]江南大学,糖化学与生物技术教育部重点实验室,江苏无锡214122 [3]江南大学生物工程学院,江苏无锡214122 [4]山东泰山生力源集团股份有限公司,山东泰安271000

出　　处：《食品与发酵工业》2023年第24期103-110,124,共9页Food and Fermentation Industries

基　　金：轻工业技术与工程国家一流学科计划项目(LITE2018-22)。

摘　　要：D-甘露糖异构酶可以催化D-果糖与D-甘露糖之间的相互转化,在D-甘露糖的生物酶法制备中具有应用前景,但是目前报道的D-甘露糖异构酶热稳定性较差,无法满足工业生产高温的需求。该研究将大肠杆菌(Escherichia coli)Dh5α来源的D-甘露糖异构酶异源表达后进行酶学性质研究,并对其进行热稳定性改造。研究结果显示D-甘露糖异构酶的最适反应温度为30℃,最适反应pH值为6.0~7.0,K_(m)、k_(cat)和k_(cat)/K_(m)分别为963.4 mmol/L、1.38 s^(-1)、1.4×10^(-3)L/(mmol·s)。通过理性改造该研究获得了一株热稳定性显著提高的突变体A241P/A116V/G253A/Q379P,其最适反应温度提高了15℃,在50℃条件下,半衰期为野生型的33倍,相对酶活力比野生型高60%。分子动力学模拟分析表明脯氨酸的引入和疏水作用的增强,提高了蛋白质的刚性,从而使热稳定性提高。该研究通过对主流来源的D-甘露糖异构酶进行酶学性质探究及热稳定性改造,为生物酶法高效制备D-甘露糖提供了理论依据,为D-甘露糖异构酶结构-稳定性的研究奠定基础。D-mannose isomerase can catalyze the mutual conversion between D-fructose and D-mannose,which has application prospects in the biological enzymatic preparation of D-mannose.However,most of the reported D-mannose isomerases have poor thermal stability that affects its application in industry.In this study,D-mannose isomerase from Escherichia coli Dh5αwas heterologously expressed and its enzymatic properties were studied.Then,its thermostability was modified.The results indicated that the optimum reaction temperature of D-mannose isomerase was 30℃,the optimum reaction pH was 6.0-7.0,K_(m),k_(cat)and k_(cat)/K_(m)were 963.4 mmol/L,1.38 s^(-1),and 1.4×10^(-3)L/(mmol·s),respectively.Through rational modification,a mutant A241P/A116V/G253A/Q379P with significantly improved thermal stability was obtained.Its optimum reaction temperature was increased by 15℃.At 50℃,its half-life was 33 times that of the wild type,and its relative enzyme activity was 60%higher than that of the wild type.Molecular dynamics simulation analysis showed that the introduction of proline and the enhancement of hydrophobic improve the rigidity of the protein,thus improving the thermal stability.In this study,the enzymatic properties and thermal stability of D-mannose isomerase from mainstream sources were investigated,which provides a theoretical basis for the efficient preparation of D-mannose by bio-enzyme method.It also lays a foundation for the study of the structure-stability of D-mannose isomerase.

关 键 词：D-甘露糖异构酶 异源表达 酶学性质 热稳定性改造 分子动力学模拟 

分 类 号：TS201.2[轻工技术与工程—食品科学]