耐热赖氨酸氨肽酶毕赤酵母表达及培养条件优化

为实现耐热耐氨酸氨肽酶的异源表达，通过PCR扩增技术从Pseudomonas aeruginosa NJ -814基因组中克隆获得耐热的赖氨肽酶基因（PLAP），构建重组载体pPIC9K-PLAP，并最终实现在毕赤酵母GS115中的分泌表达。对重组毕赤酵母进行初步筛选，单因素实验优化获得重组毕赤酵母最佳诱导条件如下：诱导培养基初始pH为5.0、甲醇质量浓度1.5 g/dL、诱导温度28 ℃、装液量25 mL/250 mL、Co2+浓度50 μmol/mL、山梨醇添加量9 g/L。在最优条件下，氨肽酶酶活提高了5.2倍，比酶活2.74 U/mg。实验结果表明：PLAP基因已成功转入毕赤酵母GS115并获得表达。
The aim of this study was toachieve heterologous expression of thermo-Stable Lysine Aminopeptidase（PLAP），the PLAP gene thathave been investigated producing a thermo-stable Lysine Aminopeptidasewas cloned from the genomic DNA of Pseudomonas aeruginosa NJ-814 by PCR.The expression vector pPIC9K-PLAP was constructed to express extracellular protein in P. pastoris GS115. After preliminary screening of the recombinant P. pastoris，the optimized fermentation conditions based on single factor experiment wereidentified as follows：induction methanol concentration was 15 g/L，induction pH was 5.0，induction temperature was 28 ℃，the medium volume was 25 mL/250 mL，the amount of Co2+ was 50 μmol/mL，induction sorbitol was 9 g/L. Finally，the enzyme activity was 5.2 times as in original conditions，the specific activitywas 2.74 U/mg. The result showed that PLAP gene has been successfully transferred and expressed in the Pichia pastoris GS115