诺卡氏菌腈水合酶基因在重组毕赤酵母中的表达

为从基因水平上改造腈水合酶，进行了诺卡氏菌腈水合酶基因的外源表达研究。在重组大肠杆菌表达系统内，腈水合酶的α亚基几乎不能正常表达，在重组E. coli BL21(DE3) (pET32aNHBAX)中，腈水合酶活性仅为0.04U/mg。构建重组毕赤酵母表达质粒pPIC3.5kNHBAX，采用电穿孔转化法将其转入宿主菌P. pastoris GS115中，经过菌株培养和腈水合酶的诱导表达，筛选获得了优选菌株P. pastoris NH4。对P. pastoris NH4的细胞培养和腈水合酶的诱导表达条件进行优化，结果表明，重组腈水合酶在毕赤酵母中的表达水平可以达到0.52U/mg，但不能稳定积累。

Heterogenous expression of nitrile hydratase gene of Nocardia sp. in recombinant Pichia pastoris

Heterogenous expression of active nitrile hydratase (NHase) was focused for its great potential in genetically evolution of the operational stability of NHase. Two recombinant Escherichia coli strains, E. coli JM105 (pUC18-NHBAX) and E. coli BL21 (DE3) (pET32a-NHBAX), were first constructed and used for heterogenous expression of a NHase gene cloned from Nocardia YS-2002. It was found that the alpha subunit of NHase can not be effectively expressed in both recombinant E. coli, which results in as low as 0.04U/mg (dry cell weight) activity of NHase in E. coli BL21 (DE3) (pET32a-NHBAX), and no activity in JM105 (pUC18-NHBAX) at all. Therefore, the recombinant and active expression of NHase in Pichia pastoris was especially interested. A new plasmid, pPIC3.5k-NHBAX, was constructed by insertion of the NHase gene, and then successfully transformed into the cell of Pichia pastoris GS115 by electroporation. A novel superior strain, P. pastoris NH4, was selected from 6 target-clones and used for optimization of cell culture and NHase expression. The final activity of NHase expressed in P. pastoris NH4 under optimal conditions is 0.52U/mg (dry cell weight), which is 13-fold higher than that in recombinant E. coli. However, the activity of NHase expressed in recombinant P. pastoris NH4 can not be stably maintained longer than 6 h.