食品科学 ›› 2020, Vol. 41 ›› Issue (6): 79-85.doi: 10.7506/spkx1002-6630-20181229-362

• 生物工程 • 上一篇    下一篇

葡萄糖氧化酶在无孢黑曲霉中的重组表达及酶学性质

林晓彤,潘力,罗时渝,王斌   

  1. (1.华南理工大学生物科学与工程学院,广东 广州 510006;2.广东省发酵与酶工程重点实验室,广东 广州 510006)
  • 出版日期:2020-03-25 发布日期:2020-03-23
  • 基金资助:
    国家自然科学基金面上项目(31871736;31870024);广东省自然科学基金项目(2017A030313097); 广东省调味食品生物发酵先进技术企业重点实验室开放基金项目(2017B030302002)

Recombinant Expression and Enzymatic Characterization of Glucose Oxidase in Aconidial Aspergillus niger Strain

LIN Xiaotong, PAN Li, LUO Shiyu, WANG Bin   

  1. (1. School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China; 2. Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, Guangzhou 510006, China)
  • Online:2020-03-25 Published:2020-03-23

摘要: 针对目前食品级葡萄糖氧化酶的工业生产产量不高,选择黑曲霉CBS513.88的葡萄糖氧化酶基因goxC,通过密码子优化、信号肽替换对其编码区进行改造,并利用强启动子PglaA、杂合启动子Pna2/tpi及营养缺陷标记pyrG构建表达载体,旨在构建高活力的葡萄糖氧化酶表达工业菌株。goxC表达载体转化低蛋白背景的黑曲霉SH2原生质体后,经营养缺陷标记pyrG筛选获得重组菌株,邻联茴香胺显色和十二烷基硫酸钠-聚丙烯酰氨凝胶电泳结果显示,在SH2宿主中成功表达葡萄糖氧化酶,蛋白质大小约为80 kDa。500 mL摇瓶发酵在第7天时酶活力可达563.8 U/mL;50 L发酵罐发酵在179 h时酶活力最高,达到1 128 U/mL,相比摇瓶发酵提高了1 倍多,在目前曲霉表达系统的相关表达研究中,本研究葡萄糖氧化酶表达量已达到较高的水平。酶学性质研究发现,重组葡萄糖氧化酶最适pH值为5.5,最适温度为45 ℃。综上所述,本研究成功实现了葡萄糖氧化酶在黑曲霉中的高效重组表达。

关键词: 葡萄糖氧化酶, 黑曲霉, 高效表达, 酶学性质

Abstract: Considering that currently glucose oxidase (GOD), a food-grade enzyme, is industrially produced in low yield, in this study, we chose the glucose oxidase gene goxC from Aspergillus niger CBS513.88 for modification by codon optimization and signal peptide replacement to construct a recombinant strain with high GOD activity. Aconidial A. niger strain SH2, low-background ofsecreted proteins, was used as the host, carrying the high efficient promoter PglaA. Positive transformants were selected using the auxotroph marker pyrG. Catalytic assay of o-dianisidine and SDS-PAGE results showed that the recombinant glucose oxidase with a molecular mass of 80 kDa was successfully expressed in A. niger SH2. The enzymatic activity reached 563.8 U/mL on the 7th day of fermentation in a 500-mL shake flask. In the 50-L tank fermentation, the enzymatic activity attained 1 128 U/mL at 179 h, which was over twice as much as that achieved in the shaking flask fermentation. In the current study, the expression of glucose oxidase was higher than previous literature values in A. niger expression system. This study showed that the optimum temperature and pH of the recombinant glucose oxidase were 45 ℃ and 5.5, respectively. Overall, a recombinant A. niger strain highly effective expression of goxC has been constructed.

Key words: glucose oxidase, Aspergillus niger, high-level expression, enzymatic characteristics

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