Abstract
In this study, a strain of Bacillus amyloliquefaciens D1, with a notably high production of neutral protease, was isolated from Morchella crassipes. The protease was purified to 10.4-fold with a specific activity of 4542.9 U/mg and 2.7% recovery. The enzyme was purified by 70% (NH4)2SO4 and DEAE-Cellulose-52 column. The estimated molecular mass of the purified protease obtained by SDS-PAGE was approximately 40 kDa. The enzyme was optimally active at pH 6.0 and 50 °C. Furthermore, the maximum hydrolysis rate (Vmax) and apparent Michaelis–Menten constant (Km) values of the purified protease were 8.2 mg/mL and 65.7 µg/(min mL). The enzymatic properties and rapid and efficient purification of Bacillus amyloliquefaciens D1 provide the basis for its potential commercialization and industrial development. Moreover, more essential amino acids, such as isoleucine, leucine, and phenylalanine, would be released when the strain fermented soybean milk, and then a better amino acid profile would be formed in soybean milk. Results suggest that this strain exhibits great potential in fermented soybean milk, and the enzyme could lay a foundation for its industrial application and further research.
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Funding
This work was financially supported by the Key-Area Research and Development Program of Guangdong Province (2020B020226008, 2018B020206001) and the National Natural Science Foundation of China (NSFC31171673).
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Liyu Du performed all the experiments and wrote the manuscript along with Zhenlin Liao. Jie Wang supervised the work and approved the final draft for submission. Data collection were performed by Weizhe Chen. The analysis and interpretation of the data were performed by Junjie Chen and Qikai Zheng. Edited and reviewed by Xiang Fang. All authors commented on previous versions of the manuscript.
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Du, L., Wang, J., Chen, W. et al. Isolation and Purification of Bacillus amyloliquefaciens D1 Protease and Its Application in the Fermentation of Soybean Milk to Produce Large Amounts of Free Amino Acids. Appl Biochem Biotechnol 195, 451–466 (2023). https://doi.org/10.1007/s12010-022-04133-x
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DOI: https://doi.org/10.1007/s12010-022-04133-x