Abstract

The wider application of creatinase, an enzyme used to determine renal function, is limited by the yield problem and current production cannot meet the needs of society. Therefore, the work constructed an efficient B. subtilis expression strain for creatinase production based on cell leakage. Initial expression of creatinase in B. subtilis was achieved using the native promoter P hpa Ⅱ . Subsequently, the promoter was optimized, and the efficient expression of creatinase was realized by using the maltose-induced promoter P glv , which increased the enzyme activity 5-fold and the properties of creatinase were analyzed. Meanwhile, it was found that creatinase without a signal peptide could be secreted into the extracellular medium, so the secretion of creatinase in B. subtilis was investigated. Disabling the classical secretory pathways didn’t have an effect on the expression level of extracellular creatinase, indicating that the excretion of creatinase didn’t depend on these pathways. Calcein-AM/PI double staining results showed that the membrane of the expression strain was damaged, while electron microscopy images revealed that leakage sites were present on the surface of the expressing bacteria, which led to the passive leakage of creatinase from B. subtilis . Finally, the enzyme activity was further improved to 141.9 U/mL in 5-L high-density fermentation.