食品科学 ›› 2019, Vol. 40 ›› Issue (14): 107-114.doi: 10.7506/spkx1002-6630-20180917-178

• 生物工程 • 上一篇    下一篇

南极假丝酵母脂肪酶B在黑曲霉中的分泌表达及其硅藻土固定化应用

张玲敏,王 斌,潘 力   

  1. 华南理工大学生物科学与工程学院,广东省发酵与酶工程重点实验室,广东 广州 510006
  • 出版日期:2019-07-25 发布日期:2019-07-23
  • 基金资助:
    国家自然科学基金面上项目(31870024;31871736);广东省自然科学基金项目(2017A030313097);广东省科技计划项目(2016A050503016;2016A010105004)

Secretory Expression of Candida antarctica Lipase B in Aspergillus niger and Its Application in Diatomite Immobilization

ZHANG Lingmin, WANG Bin, PAN Li   

  1. Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China
  • Online:2019-07-25 Published:2019-07-23

摘要: 为提高南极假丝酵母脂肪酶B(Candida antarctica lipase B,CALB)表达量,根据黑曲霉(Aspergillus niger)密码子偏好性设计合成CALB基因,以PnaII/tpi为启动子构建CALB表达载体并转化到黑曲霉HL-1中表达,摇瓶发酵120 h后上清液中重组CALB活力为171 U/mL。研究重组CALB的酶学性质,最适反应温度和pH值分别为50 ℃和8.0,在pH 6.0~9.0和45 ℃以下具有较高的稳定性,10 mmol/L Cu2+、Zn2+和0.1 g/100 mL十二烷基硫酸钠强烈抑制酶活力,而1 mmol/L Ca2+、0.1 g/100 mL山梨醇对酶活力具有非常明显的激活作用。此外,以硅藻土为载体固定CALB,固定化酶活力为187 U/g。将制备的硅藻土-CALB固定化酶用于生物合成己酸乙酯,经反应条件优化后在无溶剂体系中己酸乙酯产率达91%。

关键词: 南极假丝酵母脂肪酶B, 黑曲霉, 分泌表达, 酶学性质, 固定化, 酯化

Abstract: In order to increase the expression level of Candida antarctica lipase B (CALB), the CALB gene was designed and synthesized according to the codon preference of Aspergillus niger, and then the CALB expression vector was constructed with PnaII/tpi as promoter and transformed into A. niger HL-1. The enzymatic activity of recombinant CALB was 171 U/mL after shaking flask fermentation for 120 h. Then, the enzymatic properties of recombinant CALB were studied. The optimum reaction temperature and pH for the enzyme were 50 ℃ and 8.0, respectively. The enzyme was very stable at pH 6.0–9.0 and temperature below 45 ℃. Its activity was strongly inhibited by 10 mmol/L Cu2+, Zn2+ and 0.1 g/100 mL SDS but was potently activated by 1 mmol/L Ca2+ and 0.1 g/100 mL sorbitol. Furthermore, the CALB was immobilized using diatomite as the carrier, and the activity of immobilized enzyme was 187 U/g under the optimal immobilization conditions. The yield of ethyl hexanoate in a solventless system was up to 91% when biosynthesized using immobilized CALB under the optimized reaction conditions.

Key words: Candida antarctica lipase B (CALB), Aspergillus niger, secretory expression, enzymatic properties, immobilization, esterification

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