Chiang Mai Journal of Science

This study aimed to investigate the expression of the collagenase gene of Bacillus cereus in Escherichia coli BL21 and determine the optimal conditions for the activity of the recombinant protease ColM13. B. cereus MBL13-U was used as the original strain to degrade bone collagen. The whole genome of B. cereus MBL13-U was sequenced, and the collagenase (ColM13) gene was cloned. The target gene was ligated with the E. coli expression vector pET30a and transferred into the host E. coli strain BL21 to obtain the engineered strain pET30a-ColM13/BL21 that degrades bone collagen. The molecular weight of ColM13 was determined via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). The effects of different induction times and concentrations on ColM13 enzyme activity were determined. Results showed that 37°C and 6 h of induction with 0.6% isopropyl β-D-thiogalactoside (IPTG, 100 mmol/L) were the best conditions. The highest enzyme activity after optimization was 64.99 U/mL, which was 4.24 times higher than that before optimization. The results of hydrolysis circle test, UV spectroscopy, and scanning electron microscopy showed that the engineered bacteria had been constructed successfully. This engineered strain has great potential in the hydrolysis of animal bone waste. This work provided a new research idea for the deep development and comprehensive utilization of livestock and poultry bone resources in China.