一种碱性γ-谷氨酰转肽酶在Bacillus subtilis 168中的分泌表达与酶学性质分析

Homologous Cloning and Characterization of Bacillus subtilis 168 γ-Glutamyltranspeptidase

摘要:

将γ-谷氨酰转肽酶（GGT）在Bacillus subtilis 168同源过量表达，发现该酶能够自动分泌到细胞外，且重组菌酶活比原始菌提高了3 568倍。通过SDS-PAGE分析发酵液上清液发现，该γ-谷氨酰转肽酶是由一个相对分子质量为42 000的大亚基和一个22 000的小亚基组成。通过酶学性质分析发现，该γ-谷氨酰转肽酶是一个耐碱性的酶，最适反应pH为10；同时该酶具有良好的耐热性，最适反应温度达到50 ℃；另外，加入5.0 mmol/L的Mg2+、Ca2+、K+、La3+、Li+和 NH4+对GGT转肽活力具有明显的促进作用，其中添加的NH4+对GGT活力促进作用最为显著，GGT活力提高45%以上，而Cu2+和Zn2+则对其具有抑制作用。

Abstract:

Gammaglutamyltranspeptidase gene from Bacillus subtilis 168 was cloned and overexpressed with pMA5 vector in Bacillus subtilis 168 for overproduction of GGT. GGT was secreted in the extracellular space. The overexpressed enzyme was 3568 fold higher in activity than that from the parent strain and exhibited a specific transpeptidase activity of 49.8 U/mg. The molecular mass of two sub-units was estimated to be 42 000 and 22 000 respectively，by SDS-PAGE. The overexpressed GGT was a very versatile enzyme in alkaline pH. Its optimal temperature was 50 ℃ showing a moderately high thermostability. The effect of ions on purified GGT was also examined. Results showed that NH4+ had the highest activation effect on GGT（>45%），other ions such as Ca2+、K+、Mg2+、Li+ and La3+ had a significant activation effect on the enzyme，whereas Cu2+ and Zn4+ recorded the highest deactivation on GGT activity.