双歧杆菌胆盐水解酶基因的重组表达、纯化与酶学性质

Expression, Purification and Enzymatic Properties Study of the Bile Salt Hydrolase from Bifidobacterium in Escherichia coli

为了提高胆盐水解酶在大肠杆菌中的表达量,研究重组胆盐水解酶的酶学性质。利用大肠杆菌表达系统,对双歧杆菌来源的胆盐水解酶进行了表达。将PCR获得的胆盐水解酶基因克隆至表达载体p ET-22b(+),得到重组质粒p ET-22b(+)-bsh,并转化大肠杆菌BL21(DE3)。经发酵优化,重组菌在20℃、IPTG 1 mmol/L条件下诱导32 h可达最高酶活,对甘氨脱氧胆酸钠和牛磺酸脱氧胆酸钠的酶活分别为135.2 U/m L和121.3 U/m L。采用镍亲和层析柱对重组胆盐水解酶进行纯化,经SDS-PAGE分析,胆盐水解酶相对分子质量(Mr)为35.0×103。酶学性质研究表明,重组胆盐水解酶偏好水解甘氨结合胆盐,对甘氨胆酸钠的催化活性最高,达到220.1 U/mg。酶催化反应的动力学参数经Hill方程拟合,希尔系数大于1,表明底物与酶之间存在协同作用,且不同底物与重组胆盐水解酶之间存在不同的协同作用,表明重组胆盐水解酶属于变构酶。研究结果为其他来源胆盐水解酶在基因工程菌中的表达,和进一步研究胆盐水解酶的结构和催化反应机理提供了参考。

The bile salt hydrolase gene from Bifidobacteriuminfantis KL412 was expressed using Escherichia coli to increase the expression level and study the enzymatic properties of the recombinant bile salt hydrolase. The bile salt hydrolase gene was first amplified by PCR and cloned into the expression vector pET-22b （＋）. The recombinant plasmid pET-22b （-／-）-bsh was the transformed to E. coli BL21 （DE3）. The optimized activity to glycodeoxycholic acid（GDCA） and taurine sodium deoxycholate （TDCA） of the recombinant bile salt hydrolase could reach to 135.2 U/mL and 121.3 U/mL at 20 °C for 32 h with 1 mM IPTG. The molecular weight of bile salt hydrolase was 35.0 xlO3 analyzed by SDS-PAGE after purification through Ni-chelating affinity chromatography. The investigation on enzymatic properties showed that the recombinant bile salt hydrolase had a preference to glycine bile salts and a highest activity was achieved at 220,1 U/mg to sodium glycocholate（GCA）. This study provided a reference for the expression of bile salt hydrolase from different sources in genetically engineered bacteria and the further study of the structure and catalytic mechanism of bile salt hydrolase.