Bacillus amyloliquefaciens 35 M can exclusively produce and secrete proteases when cultured in soybean-meal-based medium.

Some microbial strains are ideal producers of extracellular enzymes that can be used in various industries. However, in many fields, especially in the pharmaceutical field, these enzymes need to be recovered and purified through multistep processes and tedious procedures before they can be used. The recovery process is difficult and increases the cost of enzyme production. Therefore, reducing purification steps will greatly benefit the utilization of microbial enzymes. The 35 M strain of Bacillus amyloliquefaciens, which has high extracellular protease production, was isolated from a phosphate mine. When cultured in a medium with soybean meal as the main component, the maximum activity of extracellular protease reached 16,992 U/mL. SDS-PAGE showed that there were two main proteins in the fermentation supernatant, with a paucity of other defined protein bands. Mass spectrometry and zymogram analysis showed that the two main bands were two proteases, corresponding to alkaline protease (AprM) and neutral protease (NprM), respectively. Gene cloning, sequencing, and further comparisons were used to confirm AprM and NprM correspond to these proteases from B. amyloliquefaciens. Notably, SDS-PAGE and zymogram analysis showed that NprM had obviously higher catalytic efficiency toward casein than did AprM. Strain 35 M is a promising protease producer with great potential for applications in industrial protease production. Additionally, this study demonstrates strain 35 M may be particularly well suited to use in degrading anti-nutritional factors in soybean meal, so as to improve the nutritional value of soybean meal.