来源于P.bacterium 1109的甘露醇脱氢酶的重组纯化及酶学性质研究  

Purification and Characterization of Recombinant Mannitol Dehydrogenase from P.bacterium 1109

作　　者：逯付之 徐炜 吴昊 张文立 光翠娥[1] 沐万孟[1] LU Fu-zhi;XU Wei;WU Hao;ZHANG Wen-li;GUANG Cui-e;MU Wan-meng(State Key Laboratory of Food Science and Technology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学食品科学与技术国家重点实验室,江苏无锡214122

出　　处：《食品工业科技》2020年第19期137-143,165,共8页Science and Technology of Food Industry

基　　金：国家自然基金青年基金(31801583);江苏省自然科学青年基金(BK20180607);江南大学基本科研计划青年基金(JUSRP11966)。

摘　　要：本文将来自P.bacterium 1109的甘露糖醇脱氢酶(MDH)表达并提取纯化,研究了该重组酶的酶学性质及其在甘露醇生产中的工艺条件。结果显示重组MDH是一个相对分子量为37 kDa的四聚体。氨基酸序列比对发现其与大多数MDHs的同源性小于40%。该酶的最适pH和温度分别为8.5和80℃,且当金属离子Zn^(2+)存在时,重组MDH的活力提高到对照组的260%。此外,重组MDH在75℃孵育6 h后仍可保留超过85%的残留活性,热稳定性较高,比大多数MDHs的活性高。底物特异性研究表明其对D-果糖具有较高的专一性。重组MDH催化D-果糖的米氏常数(K_m)和催化效率(k_(cat)/K_m)分别为20 mmol/L和7.5 L/(mmol·min)。重组MDH在以400 mmol/L的D-果糖为底物的反应系统中,可将80%以上的D-果糖转化为甘露糖醇。通过对反应条件的优化,为后续工业化生产制备甘露醇奠定了基础。the mannitol dehydrogenase(MDH)from P.bacterium 1109 was expressed and purified.Enzymatic properties of the recombinant enzyme and the process conditions to produce mannitol were studied.Results showed that recombinant enzyme was a tetramer with molecular weight of 37 kDa.Amino acid sequence alignment showed that its homology with most MDHs was less than 40%.The optimum pH and temperature of the enzyme were 8.5 and 80℃,respectively.The activity of recombinant MDH could increase to 260%of control group with the presence of metal ion Zn2+.In addition,the recombinant MDH could retain more than 85%of residual activity after incubation at 75℃for 6 h,indicating a higher thermal stability than most MDHs.Substrate specificity studies showed that it had a high specificity for D-fructose.The Michaelis constant(Km)and catalytic efficiency(kcat/Km)of D-fructose catalyzed by recombinant MDH were 20 mmol/L and 7.5 L/(mmol·min),respectively.The recombinant MDH could produce more than 80%mannitol in the reaction system composed of 400 mmol/L of D-fructose substrate.The optimization of the reaction conditions laid the foundation for the subsequent industrial production of mannitol.

关 键 词：甘露醇 甘露醇脱氢酶(MDH) P.bacterium 1109 热稳定性 大肠杆菌BL21(DE3) 

分 类 号：TS201.3[轻工技术与工程—食品科学]