齿毛菌漆酶的基因克隆、异源表达及脱色研究  

Gene cloning,heterologous expression and decolorization study of a novel laccase gene from Cerrena sp.

作　　者：郑邦晓[1] 余湘萍[1] 叶秀云[1] 林娟[1] 杨捷[1] 

机构地区：[1]福州大学生物科学与工程学院,福建省海洋酶工程重点实验室,福建福州350116

出　　处：《福州大学学报（自然科学版）》2015年第2期285-292,共8页Journal of Fuzhou University(Natural Science Edition)

基　　金：国家自然科学基金资助项目(41306120);海洋公益性行业科研专项(201305015);污染控制与资源化研究国家重点实验室开放课题资助项目(PCRRF12011);国家大学生创新性实验计划资助项目(201310386003)

摘　　要：通过简并PCR、TAIL-PCR、SiteFinding PCR等方法从实验室前期筛选得到的一株齿毛菌中获得漆酶Lac基因（包括启动子）序列,将其cDNA转入Pichia pastoris X33中表达,以ABTS（2,2-azinobis-（3-ethylbenzothiazoline-6-sulfonic acid））为底物测定温度和pH对酶活及其稳定性的影响,并探究重组漆酶的脱色效果.结果表明,Lac DNA长3 129 bp,含有10个内含子,编码的Lac蛋白共有542个氨基酸,与已知漆酶氨基酸序列的相似性最高为60％,在5’端950 bp序列内发现多个可能的启动子元件;成功分泌表达重组漆酶rLac,在含1 mmol·L-1铜离子的YP培养基中28℃发酵15 d达到酶活最高峰2.89 U·mL-1;酶学性质研究发现,rLac的最适作用温度和pH值分别为55℃和3.0,且在pH 4～10及20 ～ 40℃有较好的稳定性;rLac对靛类、偶氮类、三苯甲烷类等多种染料都有脱色效果.To obtain a novel laccase gene from a white - rot fungal strain previously isolated in our laboratory, the Lac gene and its promoter sequence were cloned by degenerate PCR, TAIL -PCR and SiteFinding PCR. The Lac coding sequence was transformed into Pichia pastoris X33, and the enzy- matic properties and decolorization efficiencies of the recombinant enzyme were analyzed. The results showed that the cloned Lac genetic sequence was 3 129 bp in length, containing 10 introns and enco- ding 542 amino acids; the highest identity against known laccase sequence is 60%, multiple putative transcription regulatory elements were identified in 950 bp promoter sequence. A recombinant laccase （rLac） was successfully expressed in P. pastoris and secreted into the culture medium, and the high- est enzyme activity of 2.89 U mL-1 was achieved after fermentation at 28 ℃ for 15 d in YP medium supplemented with 1 mmol L-1 copper. The optimal 3.0, respectively. The enzyme was stable at pH 4 - 10 temperature and pH of rLac were 55 ℃ and and 20 -40℃. rLac can effectively decolorize indigo, azo and triphenylmethane dyes.

关 键 词：齿毛菌 漆酶 克隆 异源表达 酶学性质 脱色 

分 类 号：Q814.9[生物学—生物工程]