肌酐酶在枯草芽孢杆菌中的异源表达及分泌机制

Secretion exploration and heterologous expression of creatininase in Bacillus subtilis

肌酐酶是用于临床检测肾小球滤过功能的关键酶之一,但目前国内肌酐酶的生产量较低无法满足市场需求,多依赖于国外进口。为了解决这一问题,本研究将恶臭假单胞杆菌肌酐酶基因克隆至原核表达载体pMA5实现肌酐酶在枯草芽孢杆菌1A751中的异源表达。随后通过启动子优化,使肌酐酶的蛋白表达量显著提高到1.08mg/mL,并且胞外肌酐酶的比酶活力达到238U/mg。同时发现肌酐酶可不依赖于信号肽即可实现胞外分泌,因此对肌酐酶在枯草芽孢杆菌中的分泌机制进行研究。通过对经典分泌途径和Holin途径的分析排除、利用Calcein-AM/PI双染色法鉴定表达菌株1AGC的细胞膜不完整,最后通过电子显微镜观察结果表明表达菌株表面存在潜在的泄漏位点,因此证实肌酐酶在枯草芽孢杆菌中通过细胞泄漏的方式,释放到胞外培养基中。本文构建了一株基于细胞泄漏的肌酐酶高产菌株,为肌酐酶的表达和潜在工业化应用提供理论基础,同时也为枯草芽孢杆菌泄漏表达系统提供了一定的研究基础。

Creatininase is one of the key enzymes used to evaluate glomerular filtration function.At present,the domestic creatininase production cannot meet the market demand,and it is mostly dependent on import.To solve this problem,our study employed the prokaryotic expression vector pMA5 to achieve the initial expression of creatininase from Pseudomonas putida in Bacillus subtilis 1A751.Subsequently,the promoter optimization successfully increased the creatininase expression level to 1.08 mg/mL,and the extracellular specific enzyme activity was improved to 238 U/mg.Meanwhile,we found that the creatininase without any signal peptides could be secreted into the extracellular medium directly,so the potential secretion mechanism of creatininase in B.subtilis was investigated.The classical secretion pathway and Holin pathway were excluded by experiments,while the membrane damage of strain 1AGC was detected by the Calcein-AM/PI double staining assay.Also,the scanning and transmission electron microscopy analysis revealed that the potential leakage sites were present on the surface of the expressing bacteria.Therefore,we speculated that the creatininase was secreted into extracellular medium by cell leakage in B.subtilis.Overall,our study constructed a strain with high-level creatininase extracellular production based on cell leakage strategy,which provided the theoretical basis for the expression and potential industrial application of creatininase.Moreover,it could help us in establishing a leakage-based expression system in B.subtilis.