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Purification and Characterization of a New Metallo-Neutral Protease for Beer Brewing from Bacillus amyloliquefaciens SYB-001

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Abstract

The increased additive amount of adjuncts in the raw materials of Chinese beer requires the usage of protease to release more water-soluble proteins. Here, a metallo-neutral protease suited for brewing industry was purified from Bacillus amyloliquefaciens SYB-001. A 5.6-fold purification of the neutral protease was achieved with a 4-step procedure including ammonium sulfate precipitation, ion-exchange, hydrophobic interaction, and gel-filtration chromatography. The molecular mass of the enzyme was estimated to be 36.8 kDa. The protease was active and stable at a wide range of pH from 6.0–10.0 with an optimum at pH 7.0. The highest activity of the purified enzyme was found at 50 °C. The existence of manganese ion would specifically enhance the protease activity. Comparing with other commercial neutral proteases in China, adding the new neutral protease during mashing process would release more amino acids from wort such as aspartic acid, arginine, methione, and histidine, resulting in a better amino acid profile in wort. Moreover, the wort processed with the new neutral protease had a higher α-amino nitrogen concentration, which would ensure a vigorous yeast growth and better flavor. The study of the enzyme could lay a foundation for its industrial application and further research.

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References

  1. GU, G. X. (1996). Brewing technology. Beijing: China Light Industry Press.

    Google Scholar 

  2. Steiner, E., Auer, A., Becker, T., & Gastl, M. (2012). Comparison of beer quality attributes between beers brewed with 100 % barley malt and 100 % barley raw material. Journal of the Science of Food and Agriculture, 92, 803–813.

    Article  CAS  Google Scholar 

  3. Wang, J. J., Wang, Z. Y., He, X. P., & Zhang, B. R. (2012). Integrated expression of the alpha-amylase, dextranase and glutathione gene in an industrial brewer’s yeast strain. World Journal of Microbiology and Biotechnology, 28, 223–231.

    Article  CAS  Google Scholar 

  4. Choi H, Mussar K (1991) Foam stabilizing proteinase. US Patent 5,035,902. 30 July 1991

  5. Liu, X. F., Wang, Z. Y., Wang, J. J., Lu, Y., He, X. P., & Zhang, B. R. (2009). Expression of GAI gene and disruption of PEP4 gene in an industrial brewer’s yeast strain. Letters in Applied Microbiology, 49, 117–123.

    Article  Google Scholar 

  6. Kasana, R. C., Salwan, R., & Yadav, S. K. (2011). Microbial proteases: detection, production, and genetic improvement. Critical Reviews in Microbiology, 37, 262–276.

    Article  CAS  Google Scholar 

  7. Matsubara, H., & Feder, J. (1971). Other bacterial mold and yeast proteases. In P. D. Boyer (Ed.), The enzymes (vol 3 3rd ed., pp. 721–795). New York: Acadamic Press.

    Google Scholar 

  8. Wang, J. L., & Han, H. (2009). Application of enzyme preparations in beer brewing. Liquor-Making Science and Technology, 11, 76–79.

    Google Scholar 

  9. Oliveira, M. G. A., De Simone, S. G., Xavier, L. P., & Guedes, R. N. C. (2005). Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis. Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology, 140, 369–380.

    Article  CAS  Google Scholar 

  10. Hadj-Ali, N. E., Agrebi, R., Ghorbel-Frikha, B., Sellami-Kamoun, A., Kanoun, S., & Nasri, M. (2007). Biochemical and molecular characterization of a detergent stable alkaline serine-protease from a newly isolated Bacillus licheniformis NH1. Enzyme and Microbial Technology, 40, 515–523.

    Article  Google Scholar 

  11. Haddar, A., Bougatef, A., Agrebi, R., Sellami-Kamoun, A., & Nasri, M. (2009). A novel surfactant-stable alkaline serine-protease from a newly isolated Bacillus mojavensis A21. Purification and characterization. Process Biochemistry, 44, 29–35.

    Article  CAS  Google Scholar 

  12. Shrinivas, D., & Naik, G. R. (2011). Characterization of alkaline thermostable keratinolytic protease from thermoalkalophilic Bacillus halodurans JB 99 exhibiting dehairing activity. International Biodeterioration and Biodegradation, 65, 29–35.

    Article  CAS  Google Scholar 

  13. Yasuda, M., Aoyama, M., Sakaguchi, M., Nakachi, K., & Kobamoto, N. (1999). Purification and characterization of a soybean-milk-coagulating enzyme from Bacillus pumilus TYO-67. Applied Microbiology and Biotechnology, 51, 474–479.

    Article  CAS  Google Scholar 

  14. Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of head of bacteriophage T4. Nature, 2276, 6.

    Google Scholar 

  15. Neuhoff, V., Arold, N., Taube, D., & Ehrhardt, W. (1988). Electrophoresis, 9, 255–262.

    Article  CAS  Google Scholar 

  16. Garciacarreno, F. L., Dimes, L. E., & Haard, N. F. (1993). Substrate-gel electrophoresis for composition and molecular weight of proteinases or proteinaceous proteinase inhibitors. Analytical Biochemistry, 214, 65–69.

    Article  CAS  Google Scholar 

  17. Guan, D. Y., & Tang, S. W. (2009). Handbook for brewing industry. Beijing: China Light Industry Press.

    Google Scholar 

  18. Lin, X. J., Li, Q., & Gu, G. X. (2005). Screening of Bacillus amyloliquefaciens Mutant 5582 in production beta-glucanase. Journal of Food Science and Biotechnology (Chinese), 24, 57–61.

    CAS  Google Scholar 

  19. Haddar, A., Agrebi, R., Bougatef, A., Hmidet, N., Sellami-Kamoun, A., & Nasri, M. (2009). Two detergent stable alkaline serine-proteases from Bacillus mojavensis A21: purification, characterization and potential application as a laundry detergent additive. Bioresource Technology, 100, 3366–3373.

    Article  CAS  Google Scholar 

  20. Sierecka, J. K. (1998). Purification and partial characterization of a neutral protease from a virulent strain of Bacillus cereus. The International Journal of Biochemistry & Cell Biology, 30, 579–595.

    Article  CAS  Google Scholar 

  21. Shah, K., Mody, K., Keshri, J., & Jha, B. (2010). Purification and characterization of a solvent, detergent and oxidizing agent tolerant protease from Bacillus cereus isolated from the Gulf of Khambhat. Journal of Molecular Catalysis B: Enzymatic, 67, 85–91.

    Article  CAS  Google Scholar 

  22. Jaouadi, B., Abdelmalek, B., Fodil, D., et al. (2010). Purification and characterization of a thermostable keratinolytic serine alkaline proteinase from Streptomyces sp. strain AB1 with high stability in organic solvents. Bioresource Technology, 101, 8361–8369.

    Article  CAS  Google Scholar 

  23. O'Connor-Cox, E. P. J., & Ingledew, W. (1991). Improved ethanol yields through supplementation with excess assimilable nitrogen. Journal of Industrial Microbiology, 8, 45–52.

    Article  Google Scholar 

  24. Piddocke, M. P., Fazio, A., Vongsangnak, W., et al. (2011). Revealing the beneficial effect of protease supplementation to high gravity beer fermentations using "-omics" techniques. Microbial Cell Factories, 10, 27.

    Article  CAS  Google Scholar 

  25. Yu, X. H. (2003). The optimal content of alpha-amino nitrogen in wort. Liquor-Making Science and Technology, 119, 66–70.

    Google Scholar 

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Acknowledgments

This study was financially supported by the program for New Century Excellent Talents in University of China (No. NCET-10-0453), the National High Technology Research and Development Program 863 (No.2012AA021303), and National Science Foundation (No.31271919).

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Authors and Affiliations

  1. Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China

    Jinjing Wang, Ailan Xu, Yansong Wan & Qi Li

  2. School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China

    Qi Li

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  1. Jinjing Wang
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  2. Ailan Xu
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  3. Yansong Wan
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  4. Qi Li
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Correspondence to Qi Li.

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Wang, J., Xu, A., Wan, Y. et al. Purification and Characterization of a New Metallo-Neutral Protease for Beer Brewing from Bacillus amyloliquefaciens SYB-001. Appl Biochem Biotechnol 170, 2021–2033 (2013). https://doi.org/10.1007/s12010-013-0350-8

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  • DOI: https://doi.org/10.1007/s12010-013-0350-8

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