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

逯付之; 徐炜; 吴昊; 张文立; 光翠娥; 沐万孟

doi:10.13386/j.issn1002-0306.2020.19.022

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

江南大学食品科学与技术国家重点实验室, 江苏无锡 214122

基金项目:

国家自然基金青年基金(31801583)

江南大学基本科研计划青年基金(JUSRP11966)。

江苏省自然科学青年基金(BK20180607)

详细信息

作者简介:
逯付之(1991-),男,硕士研究生,研究方向:食品生物技术,E-mail:2696258760@qq.com。

通讯作者:
光翠娥(1976-),女,博士,副教授,研究方向:食品酶工程,E-mail:guang1226@jiangnan.edu.cn。

中图分类号: TS201.3
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出版历程
- 收稿日期: 2019-12-26
- 网络出版日期: 2020-11-12
- 刊出日期: 2020-09-30

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

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China

摘要

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Zn²⁺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。
- 甘露醇 /
- 甘露醇脱氢酶(MDH) /
- P. bacterium 1109 /
- 热稳定性 /
- 大肠杆菌BL21(DE3)
Abstract: 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(K_m)and catalytic efficiency(k_cat/K_m)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.
- mannitol /
- mannitol dehydrogenase(MDH) /
- P. bacterium 1109 /
- thermostable /
- E. coli BL21(DE3)

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