Mining of alkaline proteases from Bacillus altitudinis W3 for desensitization of milk proteins: Their heterologous expression, purification, and characterization

Abstract

In this study, three active alkaline proteases (AprEs) (BaApr1, BaApr2, and BaApr9) from Bacillus altitudinis W3 were obtained through bioinformatics analysis and verification. Multiple sequence alignment showed low identity of 64.60% and suggested that the three AprEs belonged to the S8A subfamily of serine proteases. They showed maximal activity with pH of 9.5 at 55 °C, 8.5 at 50 °C, and 10.5 at 45 °C, respectively. They were stable at alkaline condition and below 50 °C. In the presence of Ca²⁺, the optimal temperatures and thermostability of them were significantly improved. They were activated by Ca²⁺ and Mg²⁺ but inhibited by ethylenediaminetetraacetic acid (EDTA) and phenylmethanesulfonyl fluoride (PMSF). Surfactants had little effect on them, but most organic solvents had some inhibitory effect except for n-hexane. They were effective in hydrolyzing natural proteins such as casein and NON-fat powdered milk. BaApr1 exhibited the highest catalytic efficiency towards casein and showed an excellent effect on the desensitization of milk proteins. The present study reveals some useful characteristics of the three AprEs, and indicates that AprEs have potential application values in the desensitization process of milk proteins.

Keywords: Alkaline protease; Bacillus altitudinis W3; Bioinformatics analysis; Desensitization; Milk proteins.