高等学校化学学报 ›› 2012, Vol. 33 ›› Issue (07): 1498-1504.doi: 10.3969/j.issn.0251-0790.2012.07.025

• 生物化学 • 上一篇    下一篇

棘孢曲霉β-葡萄糖苷酶Ⅰ在毕赤酵母中的表达及烷基糖苷的催化合成

韦斌如, 刘端玉, 韩双艳, 林影, 郑穗平   

  1. 华南理工大学生物科学与工程学院, 广州 510006
  • 收稿日期:2011-08-05 出版日期:2012-07-10 发布日期:2012-07-10
  • 通讯作者: 郑穗平, 男, 博士, 副教授, 主要从事酶工程研究. E-mail: spzheng@scut.edu.cn E-mail:silvio_zheng@163.com
  • 基金资助:

    国家自然科学基金(批准号: 31171633)和广东省工业科技攻关项目(批准号: 2010B011000004)资助.

Expression of Aspergillus aculeatus β-Glucosidase Ⅰ Gene in Pichia pastoris and Its Application on the Synthsis of Alkyl Glucoside

WEI Bin-Ru, LIU Duan-Yu, HAN Shuang-Yan, LIN Ying, ZHENG Sui-Ping   

  1. School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China
  • Received:2011-08-05 Online:2012-07-10 Published:2012-07-10

摘要: 将来自棘孢曲霉(Aspergillus aculeatus NO. F-50)的β-葡萄糖苷酶Ⅰ在毕赤酵母中分泌表达. 初步研究表明, 目的蛋白得到较好表达, 以对硝基酚-β-D-葡萄糖苷(4-Nitrophenyl-β-D-glucopyranoside, pNPG)为底物, 重组β-葡萄糖苷酶Ⅰ酶促反应的最适温度为65 ℃, 最适pH为5.0, 50 ℃下反应发酵上清液中的酶活力可达33.8 U/mL, 蛋白表达量最高可达0.388 mg/mL. 该重组酶可通过逆水解或转糖苷反应催化合成烷基糖苷. 在有机-水双相反应体系中, 初步优化了pH 值、 含水量、 葡萄糖浓度及酶量等条件. 结果表明, 在优化的反应条件下, 丁基、 己基、 辛基和癸基葡萄糖苷最大产率分别为51.4%, 28.8%, 6.9%和3.0%.

关键词: 棘孢曲霉, β-葡萄糖苷酶, 毕赤酵母, 分泌表达, 烷基糖苷

Abstract: β-Glucosidases, as a specific group of glycosyl hydrolase could catalyze the hydrolysis of β-1,4-glycosidic bond presented in short-chain oligosaccharides(containing 2-6 monosaccharides), are widely used in the degradation of cellulose, the improvement of food flavor and so on. The gene of Aspergillus aculeatus NO. F-50 β-glucosidase Ⅰ was cloned and integrated into Pichia pastoris GS115, in which β-glucosidase Ⅰ can be expressed and secreted in an active form. The recombinant β-glucosidase Ⅰ had an optimum pH of 5.0 and an optimum temperature of 65 ℃ using 4-nitrophenyl-β-D-glucopyranoside(pNPG) as substrate. The highest hydrolytic activity and protein expression level at 50 ℃ in the culture supernatant of recombinant strain were up to 33.8 U/mL and 0.388 mg/mL, respectively. The recombinant β-glucosidase Ⅰ was found to be able to catalyze the synthesis of alkyl glucoside through reverse-hydrolysis or trans-glycosylation reaction. Some major influential factors in water/organic two-phase system such as pH value, water content, overall concentration of glucose and enzyme concentration were optimized. The yield of butyl glucoside, hexyl glucoside, octyl glucoside and decyl glucoside was 51.4%, 28.8%, 6.9% and 3.0%, respectivly.

Key words: Aspergillus aculeatus, β-Glucosidase, Pichia pastoris, Secretory expression, Alkyl glucoside

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