Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Characterization of Extracellular Protease Secreted from Chryseobacterium sp. JK1

Chryseobacterium sp. JK1이 분비하는 세포외 단백질분해효소 특성

  • Lee, Yu-Kyong (Department of Microbiology, Chungbuk National University) ;
  • Oh, Ji-Sung (Department of Microbiology, Chungbuk National University) ;
  • Roh, Dong-Hyun (Department of Microbiology, Chungbuk National University)
  • 이유경 (충북대학교 자연과학대학 미생물학과) ;
  • 오지성 (충북대학교 자연과학대학 미생물학과) ;
  • 노동현 (충북대학교 자연과학대학 미생물학과)
  • Received : 2013.02.27
  • Accepted : 2013.03.21
  • Published : 2013.03.31
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Abstract

A novel Chryseobacterium sp. JK1 strain isolated from soil had been reported that this isolate produced large amount of extracellular protease at mesophilic temperature in previous study. The optimal temperature and pH of extracellular protease were $40^{\circ}C$ and 7.0, respectively, showing narrow range of optimal temperature and relatively broad activity from pH 6.0 to 9.0. In addition, the protease showed greatest activity against skim milk and lowest against bovine serum albumin (BSA). The protease strongly inhibited by ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA) or phenylmethylsulfonyl fluoride (PMSF), and addition of cation $Ag^+$ or $Cu^{2+}$, and slightly inhibited by $Al^{3+}$. No significant inhibition was found with pepstatin, and addition of cation, $K^+$, $Ca^{2+}$, $Na^+$, $Fe^{2+}$ or $Mg^{2+}$. On the contrary, protease was enhanced by addition of divalent cation $Mn^{2+}$ (5 mM). Zymography analysis of concentrated culture supernatant revealed two major bands at 67 and 145 kDa. These results suggest that Chryseobacterium sp. JK1 strain produced extracellular neutral serine proteases which could apply in food industry.

이전의 연구에서 토양으로부터 많은 양의 세포외 단백질분해 효소를 생산하는 신종 중온세균 Chryseobacterium sp. JK1를 분리하였다. 이 균주가 생산하는 단백질 분해효소의 특성조사 결과 최적반응온도와 pH는 각각 $40^{\circ}C$와 7.0이였으며, 좁은 최적온도 구간과 비교적 넓은 pH 구간인 pH 6.0-9.0에서 높은 활성을 보여주었다. 그리고 단백질 분해효소는 EDTA 또는 EGTA, PMSF와 금속이온 $Ag^+$ 또는 $Cu^{2+}$의 첨가에 의해 강하게 저해 되었으며, $Al^{3+}$의 첨가에 의해 약하게 저해되었다. Pepstatin과 금 속이온 $K^+$, $Ca^{2+}$, $Na^+$, $Fe^{2+}$ 또는 $Mg^{2+}$의 첨가는 저해에 큰 영향을 주지 않았다. 이와 반대로 단백질분해효소는 이가 금속이온인 $Mn^{2+}$ (5 mM)의 첨가에 의해 효소활성이 향상되었다. 농축된 배양 상등액의 활성염색 분석으로 67과 145 kDa 크기의 주요 밴드 두 개가 관찰되었다. 이러한 결과들로 Chryseobacterium sp. JK1 균주가 식품산업에 응용 가능한 세포외 중성의 serine 단백질 분해효소를 생산한다는 것을 알 수 있었다.

Keywords

References

  1. Bach, E., Daroit, D.J., Correa, A.P.F., and Brandelli, A. 2011. Production and properties of keratinolytic protease from three novel Gram-negative feather-degrading bacteria isolated from Brazilian soils. Biodegrad. 22, 1191-1201. https://doi.org/10.1007/s10532-011-9474-0
  2. Cha, I.-T., Lim, H.-J., and Roh, D.-H. 2007. Isolation of Pseudoaltero monas sp. HJ47 from deep sea water of East Sea and characterization of its extracellular protease. Kor. J. Life Sci. 17, 272-278. https://doi.org/10.5352/JLS.2007.17.2.272
  3. Cha, I.-T., Oh, Y.-S., Cho, W.-D., Lim, C.-S., Lee, J.-K., Lee, O.-S., and Roh, D.-H. 2009. Production condition and characterization of extracellular protease from Micrococcus sp. HJ-19. Kor. J. Microbiol. 45, 69-73.
  4. Hartley, B.S. 1960. Proteolytic enzymes. Annu. Rev. Biochem. 29, 45-72. https://doi.org/10.1146/annurev.bi.29.070160.000401
  5. Kalisz, H.M. 1988. Microbial proteinases. Adv. Biochem. Eng. Biotechnol. 36, 1-65.
  6. Kasana, R.C., Salwan, R., Yadave, S.K. 2011. Microbial proteases: detection, production, and genetic improvement. Crit. Rev. Microbiol. 37, 262-276. https://doi.org/10.3109/1040841X.2011.577029
  7. Kirk, O., Borchert, T.V., and Fuglsang, C.C. 2002. Industrial enzyme applications. Curr. Opin. Biotechnol. 13, 345-351. https://doi.org/10.1016/S0958-1669(02)00328-2
  8. Kumar, D., Savitri, Thakur, N., Verma R., and Bhalla, T.C. 2008. Microbial proteases and application as laundry detergent additive. Res. J. Microbiol. 3, 661-672. https://doi.org/10.3923/jm.2008.661.672
  9. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. https://doi.org/10.1038/227680a0
  10. Lee, Y.-K., Oh, Y.-S., and Roh, D.-H. 2012. Production properties on extracellular protease from Chryseobacterium novel strain JK1. Kor. J. Microbiol. 48, 48-51. https://doi.org/10.7845/kjm.2012.48.1.048
  11. Lowery, O.H., Rosenbrough, N.J., Farr, A.L., and Randall, R.J. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275.
  12. Ou, J.F. and Zhu, M.-J. 2012. An overview of current and novel approaches for microbial neutral protease improvement. Int. J. Mod. Biol. Med. 2, 1-31.
  13. Pragash, G., Narayanan, M.K.B., Naik, P.R., and Saktivel, N. 2009. Characterization of Chryseobacterium aquaticum strain PUPC1 producing a novel antifungal protease from rice rhizosphere soil. J. Microbiol. Biotechnol. 19, 99-107. https://doi.org/10.4014/jmb.0803.173
  14. Rao, M.B., Tanksale, A.M., Ghatge, M.S., and Deshpande, V.V. 1998. Molecular and biotechnological aspects of microbial proteases. Microbiol. Mol. Biol. Rev. 62, 597-635.
  15. Riffel, A., Brandelli, A., Bellato, C.M., Souza, G. H.M.F., Eberilin, M.N., and Tavares, F.C.A. 2007. Purification and characterization of a keratinolytic metalloprotease from Chryseobacterium sp. kr6. J. Biotech. 128, 693-703. https://doi.org/10.1016/j.jbiotec.2006.11.007
  16. Salwan, R., Gulati, A., and Kasana, R.C. 2010. Phylogenetic diversity of alkaline protease-producing psychrotrophic bacteria from glacier and cold environments of Lahaul and Spiti, India. J. Basic Microbiol. 50, 1-10. https://doi.org/10.1002/jobm.201090001
  17. Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular cloning: a laboratory manual. 2nd ed., p. 18.31. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, N.Y., USA.
  18. Vandamme, P., Bernardet, J.F., Segers, P., Kersters, K., and Holems, B. 1994. New perspectives in the classification of the Flavobacteria: description of Chryseobacterium gen. nov., Bergeyella gen. nov., and Empedobacter nom. rev. Int. J. Syst. Bacteriol. 44, 827-831. https://doi.org/10.1099/00207713-44-4-827
  19. Wang, S.-L., Yang, C.H., Liang, T.-W., and Yen, Y.-H. 2008. Optimization of conditions for protease production by Chryseobacterium taeanense TKU001. Bioresour. Technol. 99, 3700-3707. https://doi.org/10.1016/j.biortech.2007.07.036
  20. Windle, H.J. and Kelleher, D. 1997. Identification and characterization of a metalloprotease activity from Helicobacter pylori. Infect. Immun. 65, 3132-3137.
  21. Yi, H.K., Chun, Y.J., and Kim, H.B. 1999. Characterization of Bacillus cereus SH-7 extracellular protease. J. Microbiol. 37, 213-217.