Display of Thermotoga maritima MSB8 nitrilase on the spore surface of Bacillus subtilis using out coat protein CotG as the fusion partner

Abstract

In the present study, probiotic Bacillus spores were used as a matrix for enzyme immobilization, because of their inherent resistance to extreme temperatures, UV irradiation, solvents and drying. An Escherichia coli– Bacillus subtilis shuttle vector (pHS-CotG-nit) was constructed for the spore surface display of the nitrilase from hyperthermophilic bacterium Thermotoga maritima MSB8. The successful display of CotG-nit fusion protein on the spore surface of B. subtilis was verified by western blot analysis and activity measurement. The optimal temperature and pH of the spore surface-dispalyed nitrilase were observed to be 50°C and pH 8.0, respectively, which were higher than the free nitrilase (45°C and pH 7.5). The analysis of thermal and pH stability indicated that the spore surface-displayed nitrilase retained 79% and 97% at 75°C and pH 8.0 after 1h of incubation, whereas it were 32% and 52%, respectively, for free nitrilase. Furthermore, the reusability experiments indicated that the activity of the spore surface-displayed nitrilase was not significantly decreased throughout the reusability process, which still retained 83% of the initial activity at the fifth cycle. Above all, these results suggested that surface display of enzymes on the spore of B. subtilis might be an effective method for enzyme immobilization and help to meet the ever-increasing industrial demand for preparation and stabilization of biocatalysts.