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

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 enzymatic 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 encoding 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 highest 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 temperature and pH of rLac were 55 ℃ and 3.0，respectively. The enzyme was stable at pH 4~10 and 20~40 ℃. rLac can effectively decolorize indigo，azo and triphenylmethane dyes