黑曲霉β-甘露聚糖酶基因克隆及在毕赤酵母中的表达

Gene cloning of endo-1,4-β-mannanase from Aspergillus niger and overexpression in Pichia pastoris

采用RT-PCR方法从黑曲霉中克隆得到β-甘露聚糖酶基因(manA)cDNA,按照毕赤酵母密码子优化序列后,构建至毕赤酵母表达载体pPIC9K的Eco R I和Not I酶切位点之间,并与醇氧化酶强启动子序列和α因子分泌肽信号序列融合。构建好的载体进行大肠杆菌转化,提取大量质粒并用Sac I线性化后进行毕赤酵母转化,筛选鉴定获得酵母转化子。重组菌接种到YPD培养基中摇瓶培养24 h后,经甲醇诱导,取发酵上清液经SDS-PAGE检测到预期分子量大小的目标蛋白带,β-甘露聚糖酶最高酶活为125.78 IU/mL,酶的比活力为188.86 IU/mg。结果表明β-甘露聚糖酶基因已在毕赤酵母中成功表达并能有效分泌到细胞外。

The cDNA of β-mannanase gene(manA)was cloned from Aspergillus niger by RT-PCR.After the sequence was optimized according to the codons of Pichia pastoris,it was constructed between Eco R I and Not I sites of P.pastoris expression vector pPIC9K,and fused with ethanol oxidase strong promoter sequence andαfactor secretion peptide signal sequence.Plasmids were extracted and linearized with Sac I,and then transformed into P.pastoris.Yeast transformants were screened and identified.After the recombinant yeast was inoculated into YPD medium and shaken for 24 h,the expected target protein band was detected by SDS-PAGE in the supernatant of fermentation after methanol induction.The highest activity of recombinantβ-mannanase was 125.78 IU/mL,and the specific activity of enzyme was 188.86 U/mg.The results showed thatβ-mannanase gene had been successfully expressed in P.pastoris and could be effectively secreted out of the cells.