乳糖诱导E. coli发酵生产FAD为辅基的葡萄糖脱氢酶

doi:10.16085/j.issn.1000-6613.2018-1256

摘要/Abstract

摘要：

黄素腺嘌呤二核苷酸（FAD）为辅基的葡萄糖脱氢酶（FAD-GDH，EC1.1.5.9），具有辅基结合紧密、催化效率高的优点，可替代目前诊断用葡萄糖氧化酶应用于血糖指标的临床生化检测。本文选取Burkholderia cepacia的葡萄糖脱氢酶基因（gdh）构建表达质粒pTrc99a-gdh，转化E. coli BL21(DE3)。异丙基硫代半乳糖苷（IPTG）诱导发酵，通过酶活测定及聚丙烯酰胺凝胶电泳分析，获得可溶性表达的FAD-GDH，分子量约为60000。利用乳糖替代IPTG作为诱导剂，摇瓶水平初步探索诱导条件，酶活达到994U/L。7.5L发酵罐放大培养该菌，采用梯度流加补料策略，分阶段温度控制，并采用不同的乳糖流加速率进行诱导。当采用0.3mL/min的乳糖流加速率时，酶活达22200U/L，菌体量达69.48g/L。经过镍柱层析，最终获得纯酶的比酶活104.5U/mg。为医用诊断原料用酶葡萄糖脱氢酶新酶种的工业化生产提供借鉴。

关键词: 黄素腺嘌呤二核苷酸, 葡萄糖脱氢酶, 乳糖诱导, 发酵优化, 层析分离

Abstract:

FAD-conjugated glucose dehydrogenase (FAD-GDH, EC1.1.5.9) has the advantages of tightly binding of prosthetic groups and high catalytic efficiency. The glucose dehydrogenase gene (gdh) of the bacterial source Burkholderia cepacia was selected to construct the expression plasmid pTrc99a-gdh. In order to obtain high-yield FAD-GDH, the recombinant E. coli BL21 (DE3) was induced using IPTG. The supernatant was analyzed by enzyme activity and SDS-PAGE electrophoretogram, which indicated` that soluble expression was obtained. Using lactose instead of IPTG as the inducer, the shake flask level was initially explored for inducing conditions, and the enzyme activity reached 994U/L. The gradient feed fermentation of E. coli was carried out to produce FAD-GDH in 7.5L fermenter under stepped temperature control strategy. Under the condition of lactose addition rate of 0.3mL/min, enzyme activity and dry cell weight reached 22200U/L and 69.48g/L, respectively. After nickel column chromatography, it was found that the enzyme had a specific activity of 104.5U/mg. The study provides a certain reference for industrial production of the new glucose dehydrogenase.

Key words: FAD, glucose dehydrogenase, lactose induction, fermentation optimization, chromatographic separation

中图分类号:

张玲, 宋祖坤, 林荣, 王男, 杨海麟. 乳糖诱导E. coli发酵生产FAD为辅基的葡萄糖脱氢酶[J]. 化工进展, 2019, 38(04): 1879-1886.

Ling ZHANG, Zukun SONG, Rong LIN, Nan WANG, Hailin YANG. Lactose induces fermentation of E. coli to produce FAD-assisted glucose dehydrogenase[J]. Chemical Industry and Engineering Progress, 2019, 38(04): 1879-1886.

图/表 12

参考文献 20

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来源	表达宿主	培养方式	培养时间/h	诱导剂	表达水平/U·L^-1	参考文献
Penicillium sp.	—	10L罐	64	—	—	[16]
Aspergillus oryzae	—	摇瓶	96	—	1.97×10^-3	[17]
Mucor sp.	—	30L罐	72	—	9.9×10^-3	[18]
Aspergillus flavus	Escherichia coli	摇瓶	24	乳糖	200	[4]
Aspergillus niger	Escherichia coli	摇瓶	24	乳糖	93
Aspergillus terreus	Escherichia coli	5L罐	—	IPTG	23883	[19]
Glomerella cingulata	Escherichia coli	摇瓶	—	阿拉伯糖	10	[20]
	Pichia pastoris	7L罐	50.5	甲醇	48000
Aspergillus terreus	Pichia pastoris	15L罐	84	甲醇	260000	[5]

来源	表达宿主	培养方式	培养时间/h	诱导剂	表达水平/U·L^-1	参考文献
Penicillium sp.	—	10L罐	64	—	—	[16]
Aspergillus oryzae	—	摇瓶	96	—	1.97×10^-3	[17]
Mucor sp.	—	30L罐	72	—	9.9×10^-3	[18]
Aspergillus flavus	Escherichia coli	摇瓶	24	乳糖	200	[4]
Aspergillus niger	Escherichia coli	摇瓶	24	乳糖	93
Aspergillus terreus	Escherichia coli	5L罐	—	IPTG	23883	[19]
Glomerella cingulata	Escherichia coli	摇瓶	—	阿拉伯糖	10	[20]
	Pichia pastoris	7L罐	50.5	甲醇	48000
Aspergillus terreus	Pichia pastoris	15L罐	84	甲醇	260000	[5]

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