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第 36 卷第 7 期 精 细 化 工 Vol.36, No.7
201 9 年 7 月 FINE CHEMICALS J u l y 2019

生物工程
NMN 转移酶和乙醇脱氢酶共固定化及其动力学特性

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余带兵 ，李红梅 ，杨 娟 ，高露姣 ，孙十凡
（1. 上海理工大学 医疗器械与食品学院，上海 200093；2. 东海水产研究所，上海 200093）
摘要：采用磁性纳米颗粒对烟酰胺单核苷酸（NMN）转移酶和乙醇脱氢酶进行共价共固定，用以生产 NADH（烟
酰胺腺嘌呤二核苷酸），并对共固定化条件、共固定化双酶酶学性质及其动力学进行了考察。结果显示：双酶最
佳共固定化反应条件为：NMN 转移酶和乙醇脱氢酶添加量分别为 6.5 U/mg 和 10.3 U/ mg，体系 pH 控制在
5.0~7.0，25 ℃固定化 2 h，NADH 产率可达到 87%，与游离酶的催化产率 73%相比提高了 14%。所得共固定化
双酶在连续使用 11 次后，剩余酶活仍保留在 61.1%左右，表明共固定化酶具有较好的操作稳定性。双酶动力学
方程推导与验证结果表明：共固定化双酶反应体系符合米氏方程，其动力学反应速率取决于固定化乙醇脱氢酶
的反应速率。
关键词：NADH；磁性纳米颗粒；NMN 转移酶；乙醇脱氢酶；共固定化；生物工程
中图分类号：Q814.2 文献标识码：A 文章编号：1003-5214 (2019) 07-1346-06

Co-immobilization of NMN Transferase and Alcohol
Dehydrogenase and Its Kinetics

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YU Dai-bing , LI Hong-mei , YANG Juan , GAO Lu-jiao , SUN Shi-fan
（1. School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai
200093, China; 2. East China Sea Fisheries Research Institute, Shanghai 200093, China）
Abstract: Nicotinamide mononucleotide (NMN) transferase and ethanol dehydrogenase were covalently
immobilized with magnetic nanoparticles to produce nicotinamide adenine dinucleotide (NADH). The
conditions of co-immobilization, the properties and kinetics of co-immobilized enzyme were investigated.
The optimized immobilization conditions were obtained as follows: addition amount of NMN transferase
6.5 U/mg, addition amount of alcohol dehydrogenase 10.3 U/mg, system pH being controlled at 5.0~7.0,
immobilization temperature 25 ℃ and immobilization time 2 h. Under these conditions, NADH yield
reached 87%, which was an improvement of 14% than that catalyzed by free enzyme. After 11 reuses of the
co-immobilized double enzyme, the residual enzyme activity still remained about 61.1%, which showed
that the co-immobilized double enzyme had a good operational stabilization. The deduction and validation
of the kinetic equation of two enzymes revealed that the reaction system of co-immobilized double enzymes
was accorded with Michaelis equation, and its kinetic reaction rate depended on the reaction rate of
immobilized ethanol dehydrogenase.
Key words: nicotinamide adenine dinucleotide; magnetic nanoparticles; nicotinamide mononucleotide
transferase; alcohol dehydrogenase; co-immobilization; biological engineering

NADH（nicotinamide adenine dinucleotide）是 但还原型辅酶 NADH 不仅价格昂贵，而且稳定性差，
细胞能量代谢以及大多数氧化还原酶行使催化功能 难以重复利用，因此，实现 NADH 高效生产是氧化
所必需的一种还原型辅酶，参与微生物细胞内包括 还原酶工业化应用的关键。早期 NADH 的研究主要
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糖、脂、蛋白质代谢等 300 多个氧化还原反应 [1-2] 。 侧重于细胞筛选和 NAD 与 NADH 在生物催化过程

收稿日期：2018-10-11; 定用日期：2019-03-12; DOI: 10.13550/j.jxhg. 20180749
作者简介：余带兵（1991—），男，硕士生，E-mail：834426058@qq.com。联系人：李红梅（1975—），女，博士，硕士生导师，电话：
021-55271117，E-mail：sunnysand@126.corn。