蔗糖异构酶PalⅠ在枯草芽孢杆菌中的异源表达及应用

Heterologous expression and application of sucrose isomerase PalⅠin Bacillus subtilis

目的构建表达蔗糖异构酶PalⅠ的枯草芽孢杆菌B. subtilis WB800重组表达菌,并对其催化蔗糖转化的产物进行分析。方法采用RF-clone方法扩增蔗糖异构酶PalⅠ基因(NCBI:AY040843.1),克隆至pHT43载体,构建重组质粒pHT43-PalⅠ,转化至枯草芽孢杆菌B. subtilis WB800中,构建菌株pHT43-PalⅠ/B. subtilis WB800,IPTG诱导表达,SDS-PAGE分析目的蛋白蔗糖异构酶PalⅠ的表达情况。在含2%、4%、8%、12%蔗糖的培养基中发酵pHT43-PalⅠ/B. subtilis WB800,HPLC分析发酵液中蔗糖异构酶PalⅠ催化蔗糖转化的产物。结果经PCR鉴定,重组质粒p HT43-PalⅠ及重组菌株pHT43-PalⅠ/B. subtilis WB800均构建正确。蔗糖异构酶PalⅠ在B. subtilis WB800中成功分泌表达,其相对分子质量约为66 400。在含12%蔗糖的培养基中,发酵液中蔗糖异构酶PalⅠ可催化蔗糖转化为以异麦芽酮糖为主的二糖产物,其与单糖副产物的最大比值可达92∶1。结论成功构建了表达蔗糖异构酶PalⅠ的枯草芽孢杆菌表达菌株,其可利用重组菌在含蔗糖培养基中直接发酵催化蔗糖的转化,降低副产物的含量。

Objective To construct the recombinant Bacillus subtilits WB800 strain for expression of sucrose isomerase Pal Ⅰ and analyze its activity in catalyzing sucrose conversion. Methods The Pal Ⅰ gene(NCBI:AY040843. 1) was amplified by RF-clone PCR and cloned into vector pHT43. The constructed recombinant shuttle plasmid pHT43-PalⅠwas transferred to B. subtilis WB800 and induced with IPTG. The expressed PalⅠwas analyzed by SDS-PAGE. Recombinant bacterial strain pHT43-PalⅠ/B. subtilis WB800 was fermented in the media containing 2%,4%,8% and 12% sucrose respectively,and the sucrose conversion product catalyzed by Pal Ⅰ in the fermentation broth was analyzed by HPLC.Results PCR proved that recombinant plasmid pHT43-Pal Ⅰ/B. subtilis WB800 was constructed correctly. Sucrose isomerase Pal Ⅰ,with a relative molecular mass of about 66 400,was expressed in B. subtilis WB800. In a medium containing 12% sucrose,PalⅠcatalyzed the sucrose to disaccharide which was mainly isomaltulose,and the maximum ratio of disaccha-ride to monosaccharide by-products was 92 ∶ 1. Conclusion Recombinant B. subtilis WB800 for expression of sucrose isomerase PalⅠwas constructed,which directly catalyzed the conversion of sucrose by fer-mentation in a sucrose-contai-ning medium to decrease the content of by-products.