Browse > Article
http://dx.doi.org/10.5352/JLS.2013.23.2.273

Production and Characterization of Alkaline Protease of Micrococcus sp. PS-1 Isolated from Seawater  

Jin, Young-Rang (Department of Microbiology and Immunology, Pusan National University School of Medicine)
Yu, Sun-Nyoung (Department of Microbiology and Immunology, Pusan National University School of Medicine)
Kim, Kwang-Youn (Department of Microbiology and Immunology, Pusan National University School of Medicine)
Kim, Sang-Hun (Department of Microbiology and Immunology, Pusan National University School of Medicine)
Park, Seul-Ki (Department of Microbiology and Immunology, Pusan National University School of Medicine)
Kim, Hyeun-Kyeung (Medical Research Institute, Pusan National University)
Lee, Yong-Seok (Department of Biotechnology, Dong-A University)
Choi, Yong-Lark (Department of Biotechnology, Dong-A University)
Ji, Jae Hoon (Genome Instability Research Center, Ajou University School of Medicine)
Ahn, Soon-Cheol (Department of Microbiology and Immunology, Pusan National University School of Medicine)
Publication Information
Journal of Life Science / v.23, no.2, 2013 , pp. 273-281 More about this Journal
Abstract
The purpose of this research was to investigate the production and characterization of alkaline protease from Micrococcus sp. PS-1 newly isolated from seawater. Micrococcus sp. PS-1 was grown in Luria-Bertani (LB) medium. Its optimal temperature and pH for growth were $30^{\circ}C$ and 7.0, respectively. The effect of nitrogen sources was investigated on optimal enzyme production. A high level of alkaline protease production occurred in LB broth containing 2% skimmed milk. The protease was purified in a 3-step procedure involving ultrafiltration, acetone precipitation, and dialysis. The procedure yielded a 16.43-purification fold, with a yield of 54.25%. SDS-PAGE showed that the enzyme had molecular weights of 35.0 and 37.5 kDa. Its maximum protease activity was exhibited at pH 9.0 and $37^{\circ}C$, and its activity was stable at pH 8.0-11.0 and $25-37^{\circ}C$. The protease activity was strongly inhibited by PMSF, EDTA, and EGTA. Taken together, the results demonstrate that the protease enzyme from Micrococcus sp. PS-1 probably belongs to a subclass of alkaline metallo-serine proteases.
Keywords
Alkaline protease; Micrococcus sp.; seawater; characterization; skim milk;
Citations & Related Records
Times Cited By KSCI : 3  (Citation Analysis)
연도 인용수 순위
1 Cavicchioli, R., Siddiqui, K. S., Andrews, D. and Sowers, K. R. 2002. Low-temperature extremophiles and their applications. Curr Opin Biotechnol 13, 253-261.   DOI   ScienceOn
2 Clark, D. J., Hawrylik, S. J., Kavanagh, E. and Opheim, D. J. 2000. Purification and characterization of a unique alkaline elastase from Micrococcus luteus. Protein Expr Purif 18, 46-55.   DOI   ScienceOn
3 Choi, N. S., Choi, J. H., Kim, B. H., Han, Y. J., Kim, J. S., Lee, S. G. and Song, J. J. 2009. Mixed-substrate (glycerol tributyrate and fibrin) zymography for simultaneous detection of lipolytic and proteolytic enzymes on a single gel. Electrophoresis 12, 2234-2237.
4 Choi, N. S., Hahm, J. H., Maeng, P. J. and Kim, S. H. 2005. Comparative study of enzyme activity and stability of bovine and human plasmins in electrophoretic reagents, ${\beta}$-mercaptoethanol, DTT, SDS, Triton X-100, and Urea. J Biochem Mol Biol 38, 177-181.   과학기술학회마을   DOI   ScienceOn
5 Choi, N. S., Kim, B. Y., Lee, J. Y., Yoon, K. S., K. Han, Y. and Kim, S. H. 2002. Relationship between acrylamide concentration and enzymatic activity in an improved single fibrin zymogram gel system. J Biochem Mol Biol 35, 236-238.   과학기술학회마을   DOI   ScienceOn
6 Cristobal, H. A., Schmidt, A., Kothe, E., Breccia, J. and Abate, C. M. 2009. Characterization of inducible cold-active ${\beta}$-glucosidases from the psychrotolerant bacterium Shewanella sp. G5 isolated from a sub-antarctic ecosystem. Enzyme Microb Technol 45, 498-506.   DOI   ScienceOn
7 Cristobal, H. A., Breccia, J. D. and Abate, C. M. 2008. Isolation and molecular characterization of Shewanella sp. G5, a producer of cold-active ${\beta}$-D-glucosidases. J Basic Microbiol 48, 16-25.   DOI   ScienceOn
8 Fernandez, J., Mohedano, A. F., Polanco, M. J., Medina, M. and Nunez, M. 1996. Purification and characterization of an extracellular cysteine proteinase produced by Micrococcus sp. INIA 528. J Appl Bacteriol 81, 27-34.   DOI   ScienceOn
9 Amoozegar, M. A., Fatemi, A. Z., Karbalaei-Heidarib, H. R. and Razavi, M. R. 2007. Production of an extracellular alkaline metallo protease from a newly isolated, moderately halophile, Salinivibrio sp. strain AF-2004. Microbiol Res 4, 369-377.
10 Arulmani, M., Aparanjini, K., Vasanthi, K., Arumugam, P., Arivuchelvi, M. and Kalaichelvan, T. 2007. Purification and partial characterization of serine protease from thermostable alkalophilic Bacillus laterosporus-AK1. World J Microbiol Biotechnol 23, 475-481.   DOI   ScienceOn
11 Kunitz, M. 1947. Crystalline soyabean trypsin inhibitor II. general properties. J Gen Physiol 30, 291-310.   DOI
12 Hartley, B. S. 1960. Proteolytic enzymes. Annu Rev Biochem 29, 45-72.   DOI   ScienceOn
13 Jonathan, K., Burkhardt, F., Stephen, A. J., David, P. H. L., John, P. M., Fergal, O. G., and Alan, D. W. D. 2010. Marine metagenomics: New tools for the study and exploitation of marine microbial metabolism. Mar Drugs 8, 608-628.   DOI   ScienceOn
14 Joo, H. S., Kumar, C. G., Park, G. C., Paik, S. R. and Chang, C. S. 2003. Oxidant and SDS-stable alkaline protease from Bacillus clausiiI-52: production and some properties. J Appl Microbiol 95, 267-272.   DOI   ScienceOn
15 Lee, J. H., Shin, H. H., Lee, D. S., Kwon, K. K., Kim, S. J., and Lim, H. K. 1999. Bacterial diversity of cultivable isolates from sea water and a marine coral, Plexauridae sp. near Mun-Sum, Cheju-Island. J Microbiol 37, 193-199.
16 Li, H. Zheng, P., H., Yuan, J. L., Fan, H. D., Chen, W., Wang, S. J., Zheng, S. S., Zheng, Z. L. and Zou, G. L. 2007. A novel extracellular protease with fibrinolytic activity from the culture supernatant of Cordyceps sinensis: purification and characterization. Phytother Res 12, 1234-1241.
17 Mala, B. R., Aparna, M. T., Mohini, S. G. and Vasanti, V. D. 1998. Molecular and biotechnological aspect of microbial protease. Microbiol Mol Biol 62, 597-635.
18 Massaki, Y., Kazuo, S. and Mitsuo, M. 1984. Purification and properties of acid protease from Monascus sp. No. 3403. Agric Biol Chem 48, 1637-1639.   DOI
19 Mohsen, F. N. 2005. Potential application of protease isolated from Pseudomonas aeruginosa PD100. Electron J Biotechnol 8, 197-203.   DOI
20 Nicholas, C. P. and Lewis, S. 1999. Fundamentals of Enzymology, pp. 35-36, 3rd eds., Oxford University press, UK.
21 Reis, V. R. and Zydney, A. 2001. Membrane separations in biotechnology. Curr Opin Biotechnol 12, 208-211.   DOI   ScienceOn
22 Park, P. J., Lee, S. H., Byun, H. G., Kim, S. H. and Kim, S. K. 2002. Purification and characterization of a collagenase from the mackerel, Scomber japonicus. J Biochem Mol Biol 35, 576-582.   과학기술학회마을   DOI   ScienceOn
23 Prasad, R., Malik, R. K. and Mathur, D. K. 1986. Purification and characterization of extracellular caseinolytic enzyme of Micrococcus Sp. MCC-315 isolated from Cheddar cheese. J Dairy Sci 69, 633-642.   DOI   ScienceOn
24 Rajesh, P., Mital, D. and Satya, P. S. 2005. Extracellular alkaline protease from a newly isolated haloalkaliphilic Bacillus sp.: Production and optimization. Process Biochem 40, 3569-3575.   DOI   ScienceOn
25 Runying, Z., Rui, Z., Jing Z. and Nianwei, L. 2003. Cold-active serine alkaline protease from the psychrophilic bacterium Pseudomonas strain DY-A: enzyme purification and characterization. Extremophiles 7, 335-337.   DOI   ScienceOn
26 Tarun, B. and Elmer, H. M. 1989. Purification and partial characterization of an aminopeptidase from Micrococcus freudenreichii ATCC 407 systematic and applied microbiology. Syst Appl Microbiol 12, 112-118.   DOI