食品科学 ›› 2019, Vol. 40 ›› Issue (6): 55-61.doi: 10.7506/spkx1002-6630-20180131-430

• 生物工程 • 上一篇    下一篇

酿酒酵母胱硫醚β-裂解酶的异源表达及催化生成糠硫醇

扎木苏1,杨华青1,王 鑫1,孙宝国1,王成涛1,*,卢 松2   

  1. 1.北京食品营养与人类健康高精尖创新中心,北京市食品添加剂工程技术研究中心,北京工商大学,北京 100048;2.阜丰集团内蒙古阜丰生物科技有限公司,内蒙古 呼和浩特 010030
  • 出版日期:2019-03-25 发布日期:2019-04-02
  • 基金资助:
    国家自然科学基金面上项目(31571801);“十三五”国家重点研发计划重点专项(2016YFD0400802;2016YFD0400502);北京市科技创新服务能力建设-科技成果转化-提升计划项目(PXM2018-014213-000033);北京市科技计划项目(Z171100002217019)

Heterologous Expression of Recombinant Cystathionine β-Lyase from Saccharomyces cerevisiae and Catalytic Synthesis of 2-Furfurylthiol

ZHA Musu1, YANG Huaqing1, WANG Xin1, SUN Baoguo1, WANG Chengtao1,*, LU Song2   

  1. 1. Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology & Business University (BTBU), Beijing 100048, China; 2. Neimenggu Fufeng Biotechnologies Co. Ltd., Hohhot 010030, China
  • Online:2019-03-25 Published:2019-04-02

摘要: 为明晰糠硫醇生物转化机制,将来源于酿酒酵母(Saccharomyces cerevisiae)G20的胱硫醚β-裂解酶(cystathionine β-lyase,Str3p)在大肠杆菌(Escherichia coli)中实现异源表达,并验证其催化性质。正交设计试验优化大肠杆菌基因工程菌蛋白表达的诱导条件,其最适诱导表达条件为诱导温度20 ℃、异丙基硫代半乳糖苷浓度0.5 mmol/L、诱导时间13 h。该条件下获得的菌体经超声破碎和镍柱亲和层析,纯化得到的可溶性Str3p分子质量约为52 kDa,其表达量达1.26 mg/mL,较优化前的表达量和酶活力分别提高41.7%和38.6%。实验发现Str3p能够催化裂解半胱氨酸-糠醛加合物生成糠硫醇,且催化过程受pH值影响较大,在pH 8.0时糠硫醇产量达到47 μmol/L。本研究确定Str3p在E.coli BL21中的异源表达及催化特性,为生物转化合成天然糠硫醇提供新的参考途径。

关键词: 酿酒酵母, 胱硫醚β-裂解酶, 异源表达, 体外催化, 糠硫醇

Abstract: In order to clarify the bioconversion mechanism of 2-furfurylthiol, we cloned the cystathionine β-lyase gene (Str3) from Saccharomyces cerevisiae G20 and expressed it in Escherichia coli BL21, and we further evaluated the catalytic activity of the recombinant enzyme. With the use of orthogonal array design, the optimized induction conditions for the recombinant strain were determined as follows: temperature 20 ℃, IPTG concentration 0.5 mmol/L, and time 13 h. The recombinant Str3p was harvested after ultrasonic disruption of the cells and purified by Ni column affinity chromatography and its molecular mass was measured to be about 52 kDa. The content of soluble Str3p was up to 1.26 mg/mL, the expression level and the enzymatic activity were increased by about 41.7% and 38.6%, respectively, compared with those before optimization. The recombinant Str3p was capable of catalyzing the degradation of cysteine-furfural conjugate into 2-furfurylthiol. The process was greatly influenced by pH value, and the maximum yield of 2-furfurylthiol of 47 μmol/L was obtained at pH 8.0. This study can provide useful information on biosynthesis of 2-furfurylthiol.

Key words: Saccharomyces cerevisiae, cystathionine β-lyase, heterologous expression, in vitro catalysis, 2-furfurylthiol

中图分类号: