Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Isolation and Characterization of Acid Protease Produced by Staphylococcus sp. CB2-3 from Digestive Organ of Harmonia axyridis

무당벌레 소화기관으로부터 산성 단백질 분해효소를 생산하는 Staphylococcus sp. CB2-3의 분리 및 특성

  • Kim, Se-Jong (Department of Microbial & Nano Materials, Mokwon University) ;
  • Whang, Kyung-Sook (Department of Microbial & Nano Materials, Mokwon University)
  • 김세종 (목원대학교 미생물나노소재학과) ;
  • 황경숙 (목원대학교 미생물나노소재학과)
  • Received : 2011.09.15
  • Accepted : 2011.09.28
  • Published : 2011.09.30
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Abstract

Six protein-degrading bacteria were isolated from digestive organ of Harmonia axyridis. These isolates were categorized as Staphylococcus sciuri subsp. sciuri (3 strains), Bacillus subtilis (1 strain), and Bacillus thuringiensis (2 strains) by 16S rRNA gene sequence analysis. The Staphylococcus sp. CB2-3 was selected as a protease-producing bacterium which showed the highest protease activity of 58.5 U/ml at the pH 5.0 medium. The optimal pH and temperature of protease activity were pH 5.0 and $40^{\circ}C$, respectively. This acid protease had a relatively high stability of 80% between $30-50^{\circ}C$ at broad temperature range. The opimal medium compositions of carbon, nitrogen and mineral source for cell growth and protease activity were investigated. When sorbitol (0.5%) was used as carbon source, enzyme activity was increased about 2 times than that of the basal medium. When skim milk (0.5%) was used as nitrogen source, activity was increased about 2.5 times than that of the control. Cell growth and enzyme activity were increased by mineral source such as KCl, $K_2HPO_4$, $FeSO_4$, but was completely inhibited by divalent ions such as $Co^{2+}$, $Zn^{2+}$, $Mn^{2+}$, $Cu^{2+}$.

무당벌레 소화기관으로부터 단백질 분해 우수세균 6균주를 분리하였다. 단백질 분해세균의 16S rRNA 유전자 염기서열을 해석하여 계통학적 특성을 검토한 결과, Staphylococcus sciuri subsp. sciuri (3균주), Bacillus subtilis (1균주), Bacillus thuringiensis (2균주)로 확인되었다. 이들 균주 중 pH 5.0 배지에서 58.5 U/ml의 높은 효소 활성을 나타내는 Staphylococcus sp. CB2-3을 최종 선발하였다. 효소의 특성을 조사한 결과, pH 4.0-6.0에서 높은 활성을 나타내어 산성 단백질 분해효소임이 확인되었다. 효소의 최적 반응 온도는 $40^{\circ}C$ 이었으며, $30-50^{\circ}C$의 범위에서 80% 이상의 효소 활성을 유지하였다. Staphylococcus sp. CB2-3 균주의 생육과 효소 활성을 위한 최적의 배지성분을 조사하였다. 탄소원으로 0.5% 솔비톨을 첨가하였을 때 효소 활성이 2배로 증가되었으며, 질소원으로 0.5% 탈지유를 첨가한 경우 효소 활성이 2.5배 증가되는 것으로 나타났다. 무기염류로는 KCl, $K_2HPO_4$, $FeSO_4$를 첨가하였을 때 효소 활성이 가장 높은 반면에 2가 금속이온인 $Co^{2+}$, $Zn^{2+}$, $Mn^{2+}$, $Cu^{2+}$를 첨가하였을 때는 균의 성장과 효소 활성이 심하게 저해되는 것으로 나타났다.

Keywords

References

  1. Anson, M.L. 1938. The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. J. Gen. Physiol. 22, 79-89. https://doi.org/10.1085/jgp.22.1.79
  2. Arvidson, S. 1973. Studies on extracellular proteolytic enzymes from Staphylococcus aureus. II. Isolation and characterization of an EDTA-sensitive protease. Biochim. Biophys. Acta. 302, 149-157. https://doi.org/10.1016/0005-2744(73)90017-X
  3. Ayora, S. and F. Gotz. 1994. Genetic and biochemical properties of an extracellular neutral metalloprotease from Staphylococcus hyicus subsp. hyicus. Mol. Gen. Genet. 242, 421-430.
  4. Ayora, S, P.E. Lindgren, and F. Gotz. 1994. Biochemical properties of a novel metalloprotease from Staphylococcus hyicus subsp. hyicus involved in extracellular lipase processing. J. Bacteriol. 176, 3218-3223. https://doi.org/10.1128/jb.176.11.3218-3223.1994
  5. Bae, K.S. and H.Y. Park. 2004. Biochemical characterization of an extracellular protease in Serratia proteamaculans isolated from a spider. Kor. J. Microbiol. 40, 269-274.
  6. Bersanetti, P., H.Y. Park, K.S. Bae, K.H. Son, D.H. Shin, I.Y. Hirata, M.A. Juliano, A.K. Carmona, and L. Juliano. 2005. Characterization of arazyme, and exocellular metalloprotease isolated from Serratia proteamaculans culture medium. Enzyme Microb. Technol. 37, 574-581. https://doi.org/10.1016/j.enzmictec.2005.01.041
  7. Bjorklind, A. and H. Jornvall. 1974. Substrate specificity of three different extracellular proteolytic enzymes for Staphylococcus aureus. Biochim. Biophys. Acta. 370, 524-529. https://doi.org/10.1016/0005-2744(74)90113-2
  8. Breznak, J.A. and A. Brune. 1994. Role of microorganisms in the digestion of lignocellulose by termites. Annu. Rev. Entomol. 39, 453-487. https://doi.org/10.1146/annurev.en.39.010194.002321
  9. Broderick, N.A., K.F. Raffa, R.M. Goodman, and J. Handelsman. 2004. Gensus of the bacterial community of the gypsy moth larval midgut by using culturing and cultureindependent methods. Appl. Environ. Microbiol. 70, 293-300. https://doi.org/10.1128/AEM.70.1.293-300.2004
  10. Chapman, C.F. 1985. Coordination of digestion. In Comprehensive Insect Physiology, Biochemistry and Pharmacology (Edited by Kerkut G. A. and Gilbert L.I.). Pergamon Press, New York. 4, 213-240.
  11. Christopher, M.S.M. and S. Mathavan. 1985. Regulation of digestive enzyme activity in the larvae of Catopsilia crocale (Lepidoptera). J. Insect Physiol. 31, 217-221. https://doi.org/10.1016/0022-1910(85)90122-2
  12. Desmazeaud, M.J. and J.H. Hernier. 1971. Specificite de la protease neutre de Micrococcus caseolyticus. Eur. J. Biochem. 19, 51-55. https://doi.org/10.1111/j.1432-1033.1971.tb01286.x
  13. Drapeau, G.R. 1978. Role of a metalloprotease in activation of the precursor of staphylococcal protease. J. Bacteriol. 136, 607-613.
  14. Egert, M., B. Wagner, T. Lemke, A. Brune, and M. Friedrich. 2003. Microbial community structure in the midgut and hindgut of the humus-feeding larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae). Appl. Environ. Microbiol. 69, 6659-6668. https://doi.org/10.1128/AEM.69.11.6659-6668.2003
  15. Eutick, M.L., R.W. O′Brien, and M. Slaytor. 1978. Bacteria from the gut of Australian termites. Appl. Environ. Microbiol. 35, 823-828.
  16. French, J.R.J., G.L. Turner, and J.F. Bradbury. 1976. Nitrogen fixation by bacteria from the hindgut of termites. J. Gen. Microbiol. 95, 202-206. https://doi.org/10.1099/00221287-95-2-202
  17. Giesecke, U.E., G. Bierbaum, H. Rudde, U. Spohn, and C. Wandrey. 1991. Production of alkaline protease with Bacillus licheniformis in a controlled fed-batch process. Appl. Microbiol. Biotechnol. 35, 720-724.
  18. Godfrey, T. and S. West. 1996. Industial enzymology. 2nd ed. New York, N.Y., Macmillan Publishers Inc. p.3.
  19. Hagen, K.S. 1962. Biology and ecology of predaceous Coccinellidae. Annu. Rev. Entomol. 7, 289-326. https://doi.org/10.1146/annurev.en.07.010162.001445
  20. Heo, S., J. Kwak, H.W. Oh, D.S. Park, K.S. Bae, D.H. Shin, and H.Y. Park. 2006. Characterization of an extracellular xylanase in Paenibacillus sp. HY-8 isolated from an herbivorous longicorn beetle. J. Microbiol. Biotechnol. 16, 1753-1759.
  21. Hukusima, S. and S. Takeda. 1975. Artificial diets for larvae of Harmonia axyridis Pallas (Coleoptera: Coccinellidae), an insect predator of aphids and scale insect. Res. Bull. Agr. Gifu Univ. 38, 49-53.
  22. Iperti, G. 1999. Biodiversity of predacious coccinellidae in relation to bioindication and economic importance. Agricul. Ecosys. Environ. 74, 323-342. https://doi.org/10.1016/S0167-8809(99)00041-9
  23. Kim, K.K., S.I. Han, C.W. Moon, Y.M. Yu, and K.S. Whang. 2011. Biodiversity and isolation of gut microbes from digestive organs of Harmonia axyridis. Kor. J. Microbiol. 47, 66-73.
  24. Kim, K.D., D.S. Park, D.H. Shin, B.N. Han, H.W. Oh, Y.N. Youn, and H.Y. Park. 2006. Characterization of a ligninase producing strain, Serratia marcescens HY-5 isolated from Sympetrum depressiusculum. Kor. J. Appl. Entomol. 45, 301-307.
  25. Kinya. K., S. Kozaki, and M. Sakuranaga. 1998. Degradation of lignin compounds by bacteria from termite guts. Biotechnol. Lett. 20, 459-462. https://doi.org/10.1023/A:1005432027603
  26. Kohei, O., S. Masafumi, F. Kenichi, and M. Sawao. 1987. Purification and properties of a pepstatin-insensitive carboxylproteinase from a gram negative bacterium. Biochim. Biophys. Acta. 923, 463-469. https://doi.org/10.1016/0304-4165(87)90055-9
  27. Kwak, J., D.H. Lee, Y.D. Park, S.B. Kim, J.S. Maeng, H.W. Oh, H.Y. Park, and K.S. Bae. 2006. Polyphasic assignment of a highly proteolytic bacterium isolated from a spider to Serratia proteamaculans. J. Microbiol. Biotechnol. 16, 1537-1543.
  28. Lee, K.E., C.H. Kim, H.J. Kwon, J.Y. Kwak, D.H. Shin, D.S. Park, K.S. Bae, and H.Y Park. 2004. Biochemical characterization of an extracellular protease in Serratia proteamaculans isolated from a spider. Kor. J. Microbiol. 40, 269-274.
  29. Lin, X.L. and J. Tang. 1990. Purification, characterization, and gene cloning of thermopsin, a thermostable acid protease from Sulfolobus acidocaldarius. J. Biol. Chem. 265, 1490-1495.
  30. Mark, P., P. Keith, and D. Roy M. 1995. Characterization of a thermostable pepstatin-insensitive acid proteinase from a Bacillus sp. Int. J. Biochem. 27, 729-739. https://doi.org/10.1016/1357-2725(95)00032-K
  31. Matsuka, M. and I. Okada. 1975. Nutritional studies of an aphidophagous coccinellid, Harmonia axyridis (I) Examination of artificial diets for the larval growth with special reference to drone honeybee powder. Bull. Fac. Arg. Tamagawa Univ. 15, 1-9.
  32. Moon, C.W., K.K. Kim, K.S Whang, M.J. Seo, Y.N. Youn, and Y.M. Yu. 2011. Characteristics of enterobacteria from Harmonia axyridis and effects of Staphylococcus spp. on development of H. axyridis. Kor. J. Appl. Entomol. 50, 157-165. https://doi.org/10.5656/KSAE.2011.06.0.30
  33. Moon, E.Y., H.W. Oh, P.J Maeng, and K.S. Bae. 2001. Identification of enteric bacteria from Nephila clavata. Kor. J. Microbiol. 37, 1-8.
  34. Moro, C.V., J. Thioulouse, C. Chauve, P. Normand, and L. Zenner. 2009. Bacterial taxa associated with the hematophagous mite Dermanyssus gallinae detected by 16S rRNA PCR amplification and TTGE fingerprinting. Res. Microbiol. 160, 63-70. https://doi.org/10.1016/j.resmic.2008.10.006
  35. Park, H.C. 1993. Systematics and ecology of Coccinellidae (Insecta: Coleoptera) in Korea. Ph. D. Thesis. Korea University.
  36. Park, K.T. and Y.C. Park. 1994. Survey on the aphidivorous predators for biological control agents. RDA. J. Agri. Sci. 36, 109-118.
  37. Park, J.S., K.S. Whang, and J.S. Cheon. 2005. Procedure of microbial classification and identification, pp. 58-103. World Science Korea.
  38. Rao, M.B., A.M. Tanksale, M.S. Ghatge, and V.V. Deshpande. 1998. Molecular and biotechnological aspects of microbial proteases. Microbiol. Mol. Biol. Rev. 62, 597-635.
  39. Scharf, M.E. and A. Tartar. 2008. Termite digestomes as sources for novel lignocellulases. Biofuels. Bioprod. Bioref. 2, 540-552. https://doi.org/10.1002/bbb.107
  40. Semra, K. and O. Hatice. 2007. An extracellular-Pepstatin insensitive acid protease produced by Thermoplasma volcanium. Biores. Technol. 98, 112-117. https://doi.org/10.1016/j.biortech.2005.11.016
  41. Teufel, P. and F. Gotz. 1993. Characterization of an extracellular metalloprotease with elastase activity from Staphylococcus epidermidis. J. Bacteriol. 175, 4218-4224. https://doi.org/10.1128/jb.175.13.4218-4224.1993
  42. van der Hoeven, R., G. Betrabet, and S. Forst. 2008. Characterization of the gut bacterial community in Manduca sexta and effect of antibiotics on bacterial diversity and nematode reproduction. FEMS. Microbiol. Lett. 286, 249-256. https://doi.org/10.1111/j.1574-6968.2008.01277.x
  43. Ward, O.P. 1985. Proteolytic enzymes, pp. 789-818. In M.Y. Murray (ed.), Comprehensive Biotechonology : The principles, applications and regulations of biotechonology in industry, agriculture and medicine, vol. 3. Pergamon press Inc., Maxwell House, Fairview Park, Elmsford, New York, USA.
  44. Wheeler, M.M., M.R. Tarver, M.R. Coy, and M.E Scharf. 2010. Characterization of four esterase genes and esterase activity from the gut of the termite Reticulitermes flavipes. Arch. Ins. Biochem. Physiol. 73, 30-48.