烟曲霉脯氨酰内肽酶在巴斯德毕赤酵母中的分泌表达与重组酶性质

Secreted expression of prolyl endopeptidase from Aspergillus fumigatus in Pichia pastoris and characterization of the recombinant enzyme

摘要:

【目的】研究烟曲霉脯氨酰内肽酶cDNA基因的异源表达及重组酶性质。【方法】以烟曲霉CICIM F0044总RNA为模板, 反转录合成cDNA; 再以cDNA为模板, 通过PCR扩增去除自身信号肽的脯氨酰内肽酶基因, 构建表达载体pPIC9K-PEP; 电转化酵母宿主菌Pichia pastoris GS115, 获得重组菌PEP-09; 纯化并分析重组酶性质。【结果】重组菌摇瓶发酵酶活力最高可达647.3 U/L。表达产物纯化后的分子量为63 kD左右。重组酶最适反应温度为65 °C, 有较好的温度稳定性, 在55 °C保温8 h能保留90%以上的酶活力。该酶最适pH为5.5, 在pH 3.0?9.0范围内有很好稳定性, 在pH 6.0?8.0的缓冲液中37 °C保温10 d酶活没有明显变化。【结论】烟曲霉脯氨酰内肽酶cDNA基因在巴斯德毕赤酵母中实现了分泌表达, 重组酶活性稳定, 有一定的应用潜力。

Abstract:

[Objective] The study aims to heterologously express Aspergillus fumigatus prolyl endopeptidase cDNA and to characterize the recombinant enzyme. [Methods] The cDNA from A. fumigatus CICIM F0044 was obtained by reverse transcription using the total RNA as the template. The PEP gene that encodes the mature prolyl endopeptidase was amplified using polymerase chain reaction with the cDNA as the template. The recombinant expression vector pPIC9K-PEP was constructed by inserting the PEP gene into pPIC9K, which was then transformed into Pichia pastoris GS115 by electroporation. The resulting recombinant enzyme was purified and characterized. [Results] A maximum yield of 647.3 U/L enzyme activity was obtained from the recombinant yeast. The molecular weight of the purified recombinant enzyme was approximately 63 kD. The optimal reaction temperature of the recombinant enzyme was 65 °C. The enzyme is highly thermostable, retaining 90% of enzyme activity after 8 h of exposure to temperatures 55 °C. The recombinant enzyme exhibited an optimal reaction pH of 5.5 and its activity was highly stable from pH 3.0 to 9.0. No decrease in enzyme activity was observed after 10 days of exposure to pH ranging from 6.0 to 8.0 at 37 °C. [Conclusion] The A. fumigatus prolyl endopeptidase cDNA was expressed in P. pastoris. The activity of the recombinant enzyme was stable, which indicates that the recombinant yeast has potential value in industrial applications.