生物学杂志

生物学杂志 ›› 2022, Vol. 39 ›› Issue (6): 47-.doi: 10.3969/j.issn.2095-1736.2022.06.047

• 研究报告 • 上一篇    下一篇

漆酶Lcc9在灰盖鬼伞菌中的同源过表达

  

  1. 安徽大学生命科学学院现代生物制造安徽省重点实验室
    安徽省微生物与生物催化工程技术研究中心，合肥230601
  • 出版日期:2022-12-18 发布日期:2022-12-12
  • 通讯作者: 方泽民，博士，教授，研究方向为微生物学，E-mail：zemin_fang@ahu.edu.cn
  • 作者简介:张晶娜，硕士研究生，主要研究方向为微生物酶学，E-mail：z15205607610@163.com
  • 基金资助:
    国家自然科学基金项目（31870068）；安徽省自然科学基金杰出青年基金项目（2008085J12）

Homologous overexpression of laccase Lcc9 in Coprinopsis cinerea

  1. Anhui Key Laboratory of Modern Biomanufacturing， Anhui Provincial Engineering Technology Research Center of
    Microorganisms and Biocatalysis， School of Life Sciences， Anhui University， Hefei 230601， China
  • Online:2022-12-18 Published:2022-12-12

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摘要： 为了高效制备漆酶Lcc9，克隆了Lcc9编码基因，利用酿酒酵母同源重组技术构建lcc9过表达载体并在Corprinopsis cinerea Okayama 7#130（OK7）和FA2222中进行同源过表达。经原生质体共转化、再生培养基初筛、最简培养基复筛，分别获得46株OK7和29株FA2222阳性转化子。摇瓶发酵结果显示，在Kjalke培养基中接种5%（体积分数），经37 ℃培养6~7d后，OK7阳性菌株漆酶活力达到5.1~21.1U/mL，FA2222阳性菌株漆酶活力达到3.7~12.3U/mL，分别为野生型表达量的19.2和241.2倍。在3L发酵罐中对FA2222阳性菌株进行漆酶的发酵制备，结果显示，经37 ℃、50r/min培养108h后菌株漆酶活力达59U/mL，相比普通摇瓶发酵，漆酶活力进一步提高了4.8倍。

关键词: 漆酶, Lcc9, 灰盖鬼伞, 同源过表达, 高密度发酵

Abstract: In order to prepare a large amount of Lcc9, the gene lcc9 was cloned and homologously overexpressed in Corprinopsis cinerea Okayama 7#130 (OK7) and FA2222. A total of 46 OK7-lcc9 and 29 FA2222-lcc9 positive transformants were obtained, respectively, based on the primary screening in regeneration medium and re-screening in minimal medium. After inoculation of 5% (V/V) seed cultures in Kjalke medium and cultured at 37 ℃ for 6-7 d, laccase activity of OK7-lcc9 and FA2222-lcc9 in shaking flasks reached 5.1-21.1 U/mL and 3.7-12.3U/mL, respectively, 19.2 and 241.2 times higher than that of the wild type strains. One of the FA2222 positive strains was cultured in a 3 L fermenter for laccase preparation. Results showed that the laccase activity of the strain reached 59 U/mL after incubation at 37 ℃ and 50 r/min for 108 h, which was 4.8 times higher than that in the liquid shake flask cultures.

Key words: Laccase, Lcc9, Coprinopsis cinerea, homologous overexpression, high-density fermentation

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