草酸青霉外切葡聚糖酶基因(cbh1)克隆及其在毕赤酵母中的表达和重组外切葡聚糖酶特性分析

摘要

Most of the Trichoderma strains are deficient in the p-glucosidase production, results in end product inhibition, and the cellulase from Trichoderma shows relatively low growth rates, Thus, cellulase from Penicillium has attracted renewed attention. The complete gene of exo-3-l,4-D-glucanases (cbhl) was cloned and expressed in Pichia pastoris to characterize the recombinant enzyme. The gene of cbhl (GenBank Accession No. HQ843504), cloned from M strain of Penicillium oxalicum by the modified thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was 1 641 bp without intron and encoding 547 amino acids. Because its Glu236, Asp238 and Glu241 were the classical catalytic residues, we predicted cbhl belongs to glycoside hydrolase family 7. The recombinant plasmid pPIC9-c6/i7 was constructed by cloning the gene into vector pPIC9 and then was transformed into Pichia pastoris strain GS115 by electroporation. The sequencing and activity analysis of the positive recombinant showed pPC9-cbhl could successfully express cbhl in P. pastoris. To our knowledge, it was the first report that exo-P-l,4-D-glucanases from Penicillium oxalicum successfully expressed in P. pastoris. The enzyme activity of the recombinant reached up to 56 IU/mg. The optimum pH and temperature of the recombinant enzyme were 6.0 and 55 °C respectively, and the stability of pH and temperature were both good. The results suggest that cloning of the cbhl enriches the gene resources of cellulases, and its successful expression in P. pastoris provides the fundament for the construction of gene engineering strains.%木霉的纤维素酶系中通常缺少β-葡萄糖苷酶,导致了终产物抑制,木霉生长速度明显没有青霉快,因此,来源于青霉的纤维素酶受到了越来越多的重视.通过热不对称交错PCR(TAIL-PCR)技术从草酸青霉(Penicillium oxalicum)M菌株中克隆得到外切葡聚糖酶基因cbh1(GenBank登录号:HQ843504).该基因全长为1 641 bp,无内含子序列,编码547个氨基酸,具有Glu236,Asp238和Glu241典型催化残基,推测属于糖基水解酶第7家族.将cbh1克隆到毕赤酵母表达载体pPIC9中,构建重组表达质粒pPIC9-cbh1,电击转入毕赤酵母(Pichia pastoris )GS 115菌株.阳性重组子经测序及活性分析表明,cbh1基因成功分泌表达,表明来源于草酸青霉的外切葡聚糖酶基因首次在毕赤酵母中获得成功表达.重组酶活性分析表明,其活性可达56IU/mg,最适反应pH、温度分别为6.0和55℃,且pH和温度稳定性均较好.研究结果认为,cbh1基因的克隆丰富了纤维素酶的基因资源,其在毕赤酵母中的成功表达也为该类纤维素酶基因高效工程菌株的构建提供了基础.