康氏木霉纤维素酶CBHI基因克隆及在毕赤酵母中的表达研究

Study on the Cloning of Trichoderma koningii Cellulase CBHI & Its Expression in Pichia Pastoris

在毕赤酵母中表达康氏木霉(Trichoderma koningii)纤维素酶基因cbhI。提取经诱导的康氏木霉mRNA,通过反转录及PCR反应扩增纤维素酶cbhI基因cDNA。将cbhI基因克隆到毕赤酵母表达载体pPICZαA上,采用电激法将经SacⅠ线性化的重组质粒pPICZαA-cbhI转化毕赤酵母GS115,MD及G418抗性平板筛选cbhI基因高拷贝转化子,并用PCR鉴定转化子。BMMY培养基中加入0.5%甲醇对毕赤酵母进行纤维素酶CBHI诱导表达。SDS-PAGE电泳结果显示,表达的重组蛋白相对分子质量约67 kD,pNPC酶活为24.1 U/L,酶蛋白量为0.22 mg/mL,表明,康氏木霉cbhI可以在毕赤酵母中表达,并具有较高活性。

The purpose of this study is to express cellulase cbhI gene of Trichoderma koningii in Pichia pastoris.Cellulase cbhI gene was amplified by using Trichoderma koningii mRNA as template.Then the amplified cbhI cDNA was cloned into pPICZαA vector and the linearized recombinant plasmid pPICZαA-cbhI was transformed to Pichia pastoris GS115 strain by electroporation.The positive transformants were selected by MD and G418-resistance flat screen,and identified by PCR method.0.5 % methanol were added into the medium to induce the protein expression.SDS-PAGE results showed that the relative molecular weight of expressed product was 67kD,the enzyme activity was 24.1U/L,while AE was 0.22 mg/mL.As a result,recombinant cellulase CBHI of Trichoderma koningii may express in Pichia pastoris and it could display higher emzyme activity.