密码子优化后的柞蚕溶菌酶在酵母中的表达及活性测定

Expression and activity determination of codon optimized Antheraea pernyi lysozyme in Pichia pastoris

【目的】提高柞蚕溶菌酶在毕赤酵母中的表达量,对柞蚕溶菌酶活性进行测定。【方法】根据毕赤酵母密码子偏爱性对柞蚕溶菌酶基因进行优化,并在目的基因3′端加上6个组氨酸标签,优化后的基因克隆至表达p PIC9K载体中,并通过电转化的方法导入毕赤酵母GS115中。利用不同浓度梯度的G418进行高拷贝转化子的筛选,经甲醇诱导实现分泌表达。通过硫酸铵盐析、镍柱亲和层析等工艺分离纯化柞蚕溶菌酶,利用琼脂扩散方法测定柞蚕溶菌酶对溶壁微球菌、金黄色葡萄球菌、枯草芽孢杆菌、大肠杆菌、苍白杆菌及过氧化醋杆菌的抑菌作用,利用比浊法测定酶活大小。【结果】密码子优化后的柞蚕溶菌酶在诱导温度25°C、甲醇量0.75%和p H 7.0条件下实现了最高表达,表达量达到了2.4 g/L,并且纯化后的柞蚕溶菌酶酶活达到23 970 U/mg。【结论】密码子优化后的柞蚕溶菌酶在毕赤酵母中实现了高效表达,纯化后的柞蚕溶菌酶对溶壁微球菌、金黄色葡萄球菌、枯草杆菌、大肠杆菌、苍白杆菌及过氧化醋杆菌均有抑菌作用。

[Objective] In order to improve the expression of Antheraea pernyi lysozyme in Pichia pastoris and determinate the lysozyme activity. [Methods] According to the Pichia pastoris codon preference, we optimized the codons of Antheraea pernyi lysozyme gene and added six histidine residues at the 3′ terminal of the gene. The optimized gene cloned into p PIC9 K vector. Then we transfered it into Pichia pastoris GS115 through the electroporation method. High copy transformants were screened by using different concentration gradient of G418 and realized the secretory expression by methanol induction. We separated and purificated Antheraea pernyi lysozyme by ammonium sulfate precipitation and Ni-chelating affinity chromatography. Then we determinated the Antheraea pernyi lysozyme activity by turbidimetric method and detected antibacterial activity against Micrococcus lysodeikticus, Staphylococcus aureus, Bacillus subtilis, Ochrobactrum tritici, Escherichia coli and Acetobacter beijerinck by the agar diffusion method. [Results] The expression conditions were optimized, and the highest expression level of Antheraea pernyi lysozyme occurred when the recombinant strain was induced with 0.75% methanol under p H 7.0 at 25 ℃ for 96 h. The expression quantity reached 2.4 g/L and the specific activity of Antheraea pernyi lysozyme towards Micrococcus lysodeikticus was 23 970 U/mg. [Conclusion] Codon optimized Antheraea pernyi lysozyme was successfully produced as a single major secreted protein in Pichia pastoris GS115. Antheraea pernyi lysozyme exhibited antibacterial activity against Micrococcus lysodeikticus, Staphylococcus aureus, Bacillus subtilis, Ochrobactrum tritici, Escherichia coli and Acetobacter beijerinck.