Production, purification, and characterization of a novel milk-clotting metalloproteinase produced by Bacillus velezensis YH-1 isolated from traditional Jiuqu

Bacillus velezensis YH-1 isolated and identified from conventional rice wine starter (Jiuqu) was shown to produce a milk-clotting enzyme (MCE) with remarkable milk-clotting activity (MCA) (642.96 SU/mL). The MCE was produced and purified with a specific 5762.7 SU/mg activity. LC-MS/MS combined with 3D structural modeling was used for the identification of MCE and this analysis indicated that it was a Zn2+ metalloproteinase. The MCE was shown to be relatively stable between pH 6-7 at temperatures below 50 degrees C. Certain parameters (Km and Vmax are 8.58 mg/mL and 15.65 U/min) were calculated for MCE that could cleave casein at the Lys21-Ile22 and Tyr30-Val31 links. The purpose is to destabilize casein structure and allow coagulation. Whole-genome sequencing and Liquid Chromatograph Mass Spectrometer/Mass Spectrometer (LC-MS/MS) analysis determined the MCE zymogen-producing gene from B.velezensis YH-1 to be 1566 bp long, yielding a protein that is 56.8 kDa in mass and containing 521 amino acid residues. According to an additional structural simulation study, the MCE was hydrophilic and included more-helices, contributing to its structural stability. The signal peptide was in the first 27 amino acids of the MCE. When compared to commercial rennet, MCE was superior in its ability to increase the milk curd's flexibility and viscosity and speed up the coagulation process. This novel study document the presence of a metalloproteinase milk-clotting factor from B. velezensis, the MCE has the potential to replace chymosin in cheese production.

Automated summary

Bacillus velezensis YH-1 isolated from rice wine starter was found to produce a milk-clotting enzyme (MCE) with remarkable activity. The MCE was identified as a Zn2+ metalloproteinase and showed superior performance compared to commercial rennet in cheese production.