用GAP启动子在Pichia pastoris GS115中组成型表达鼠灰链霉菌腺苷酸脱氨酶

Constitutive expression of AMP deaminase from Streptomyces murinus in Pichia pastoris GS115 using the GAP promoter

摘要:

【目的】构建产AMP脱氨酶的重组毕赤酵母(Pichia pastoris GS115)菌株，并初步优化其发酵条件。【方法】以鼠灰链霉菌(Streptomyces murinus)基因组为模板PCR扩增获得腺苷酸脱氨酶基因AMPD，以pGAP9K为载体构建重组表达质粒pGAP9K-AMPD并通过电转化法转入Pichia pastoris GS115，筛选转化子对其酶活进行测定，并初步优化其发酵条件。【结果】构建了毕赤酵母重组菌，通过分光光度法测定，显示重组菌有明显的酶活；初步优化发酵条件为：该重组菌最适发酵培养基为：甘油2%，蛋白胨2%，酵母膏1%，KH2PO4 0.5%，MgSO4·7H2O 0.05%，pH 6.0；发酵条件为：接种龄24 h，转接量3%，30 °C﹑200 r/min培养96 h，取发酵上清液测定酶活，重组菌腺苷酸脱氨酶酶活达到2 230±60 U/mL。【结论】构建了一株产AMP脱氨酶活性较高的重组毕赤酵母菌株，并通过优化发酵条件使其酶活达到2 230±60 U/mL。为AMP脱氨酶工业化生产奠定了一定的基础。

Abstract:

[Objective] We constructed a recombinant Pichia pastoris GS115 strain to produce AMP deaminase and optimized the fermentation conditions. [Methods] The AMPD gene was amplified from Streptomyces murinus and cloned into the expression plasmid pGAP9K, the recombinant expression plasmid was transformed into Pichia pastoris GS115 by electrotransformation. Furthermore, we determined the AMP deaminase activity of positive transformants. Finally, we optimized the fermentation conditions. [Results] We constructed a recombinant P. pastoris GS115 strain (P. pastoris GS115/pGAP9K-AMPD) that showed AMP deaminase activity. The fermentation conditions of the recombinant strain were studied. The results showed that the optimum fermentation medium of the recombinant strain contains 2% glycerin, 2% peptone, 1% yeast extract, 0.5% KH2PO4 and 0.05% MgSO4·7H2O (pH 6.0). And the recombinant enzyme activity of fermentation supernatant was 2 230±60 U/mL with 3% of inoculation amount, 24 hours of seed time, 96 hours for 200 r/min at temperature of 30 °C. [Conclusion] We constructed a recombinant P. pastoris GS115 strain that showed AMP deaminase activity about 2 230±60 U/mL under optimized fermentation conditions. This research is helpful to advance the industrial production of AMP deaminase.