漆酶Lcc9在灰盖鬼伞菌中的同源过表达  

Homologous overexpression of laccase Lcc9 in Coprinopsis cinerea

作　　者：张晶娜 周刚 刘娟娟[1] 方泽民[1] 肖亚中[1] ZHANG Jingna;ZHOU Gang;LIU Juanjuan;FANG Zemin;XIAO Yazhong(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)

机构地区：[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　　处：《生物学杂志》2022年第6期47-51,共5页Journal of Biology

基　　金：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)。

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6~7 d后,OK7阳性菌株漆酶活力达到5.1~21.1 U/mL,FA2222阳性菌株漆酶活力达到3.7~12.3 U/mL,分别为野生型表达量的19.2和241.2倍。在3 L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℃、50 r/min培养108 h后菌株漆酶活力达59 U/mL,相比普通摇瓶发酵,漆酶活力进一步提高了4.8倍。In order to prepare a large amount of Lcc9,the genelcc9 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 3.7-12.3 U/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.

关 键 词：漆酶 Lcc9 灰盖鬼伞 同源过表达 高密度发酵 

分 类 号：Q936[生物学—微生物学]