DOI QR코드

DOI QR Code

Purification and Characterization of a Chitinase in Culture Media of Cordyceps militaris(Linn.) Link.

Cordyceps militaris 배양액으로부터 키틴분해효소의 분리 정제 및 그 특성 분석

  • 이강협 (동국대학교 이과대학 화학과) ;
  • 민태진 (동국대학교 이과대학 화학과)
  • Published : 2003.12.30

Abstract

In this study, Cordyceps militaris was grown in a liquid medium containing colloidal chitin. A chitinase was purified from the supernatant or cultured medium by ammonium sulfate fractionation, DEAE-Sephadex A-25 and Sephadex G-50 column chromatography. Optimum temperature and pH of this enzyme were $35^{\circ}C$ and 5.5, respectively. The molecular weight of the chitinase was estimated to be 48.5 kDa by SDS-PAGE and its Km value was 0.57 mM. The activity of this enzyme was inhibited by $Cu^{2+},\;Mn^{2+},\;Hg^{2+},\;Zn^{2+},\;CO_{3}^{2-},\;SO_4^{2-},\;CN^-,\;ion,\;and\;OCN^-$ maleic anhydride, acetic anhydride or N-bromo succinimide, especially strongly inhibited by sodium cyanate for 84.0 percentage. But its activity wag slightly stimulated by $Mg^{2+}\;and\;K^+$ ion, respectively. The products formed during hydrolysis of the hexa-N-acetylchitohexaose with this enzyme were N,N'-diacetylchitobiose and N,N',N'-triacetylchitotriose. These results imply that this purified enzyme may be an endo-chitinase.

C. militaris 균사를 콜로이달 키틴이 첨가된 액체 배지에서 배양한 후 황산암모늄 분별침전, 이온 교환 및 겔 여과 크로마토그래피를 이용하여 배양액 중의 chitianse를 분리 정제하였다. 이 효소의 최적 pH와 온도는 각각 5.5와 $35^{\circ}C$이었으며 겉보기 분자량은 48.5 kDa이었고, 그 Km 값은 0.57 mM이었다. 이 효소는 $Cu^{2+},\;Mn^{2+},\;Hg^{2+},\;Zn^{2+},\;CO_{3}^{2-},\;SO_4^{2-},\;CN^-$OCN^-$ 이온에 의하여 활성이 억제되었으나, $Mg^{2+}$$K^+$ 이온에 의하여 활성이 약간 증가되었다. 또한 효소 단백질의 아미노산 잔기와 선택적으로 반응하는 무수말레인산, 무수아세트산 및 N-bromo succinimide에 의하여 84.0% 활성이 억제되어 카르복실기를 가진 아미노산 잔기가 이 효소의 활성 부위에 중요한 역할을 함을 알았다. NAG6에 의한 기질 분해 특이성 실험을 통하여 이 효소는 endo-형태의 chitinase임을 알았다.

Keywords

References

  1. Methods Enzymol. v.161 Chitinase from Neurospora crass Begovich, A.A. https://doi.org/10.1016/0076-6879(88)61060-3
  2. Beauveria bassiana. Can. J. Microbiol. v.39 Partial purification and characterisation of two extracellular N-acety1-D-g1ucosaminidases produced by the entomopathogenic fungus Bidochka, M.J.;Khachatourians, G.G. https://doi.org/10.1139/m93-006
  3. Methods Enzymol. v.161 Physical methods for the determination of chitin structure and conformation Blackwell, J. https://doi.org/10.1016/0076-6879(88)61053-6
  4. Methods Enzynwl. v.161 Chitinase from Phaseolus vulgaris leave Boller, T.;Gehri, A.;Mauch, F.;Vogeli, U. https://doi.org/10.1016/0076-6879(88)61062-7
  5. J. Am. Chem. Soc. v.120 Substrate distortion to a boat conformation at subsite -1 is critical in the machnism of family 18 chitinase Brameld, K.A.;Goddard, W.A. III, https://doi.org/10.1021/ja972282h
  6. Methods Enzymol. v.25 Maleylation of amino groups Butler, P.J.G.;Hartley, B.S. https://doi.org/10.1016/S0076-6879(72)25016-9
  7. Kor. J. Mycol. v.27 The condition of production of artificial fruiting body of Cordyceps militaris Choi, I.Y.;Choi, J.S.;Lee, W.H.;Yu, Y.J.;Joung, G.T.;Ju, I.O.;Choi, Y.K.
  8. Environ. Biotech. v.9 The molecular biology of chitin digestion Cohen-Kupies, R.;Chet, I. https://doi.org/10.1016/S0958-1669(98)80058-X
  9. Int. J. Biochem. v.7 Chitinolytic enzymes of the gastric mucosa of Perodicticus potto (Primate prosimian): Purification and enzyme specifificity Cornelius, C.;Dandrifosse, G.;Jeuniaux, C.H. https://doi.org/10.1016/0020-711X(76)90043-4
  10. Comp. Biochem. Physiol. v.79 Levels of chitinolytic activity during development of three entomopathogenic fungi Coudron, T.A.;Kroha, M.J.;Ignoffo, C.M. https://doi.org/10.1016/0300-9629(84)90523-1
  11. Appl. Microbiol. Biotechnol. v.51 Regulation and cloning of microbial chitinase gene Felse, P.A.;Panda, T. https://doi.org/10.1007/s002530051374
  12. Bioprocess Engineering v.23 Production of microbial chitinases-A revisit Felse, P.A.;Panda, T. https://doi.org/10.1007/PL00009117
  13. Ann. Rev. Entomol. v.23 Biological control of insect pests by entomogenous fungi Ferron, P. https://doi.org/10.1146/annurev.en.23.010178.002205
  14. Ecology of entomopathogenic fungi.;Handbook of Applied Mycology 2 Glare, T.R.;Milner, R.J.;Arora, D.K.(ed.);Ajello, L.(ed.);Muketji, K.G.(ed.)
  15. Bioprocess Engineering v.23 Characterization and application of chitinases from Trichiderma harzianum-a review Gokul, B.;Lee, J.H.;Song, K.B.;Rhee, S.K.;Kim, C.H.;Panda, T. https://doi.org/10.1007/s004499900138
  16. Gel electrophoresis of protein Hames, B.D.;Rickwood, D.
  17. J. Biochem. v.105 Purification and characterization of chitinase produced by Strptomyces erythraeus Hara, S.;Yamamura, Y.;Fujii, Y.;Mega, T.;Ikenaka, T.
  18. J. Invertebr. Pathol. v.61 Induction and purification of Beauveria bassiana chitinolytic enzymes Havukkala, I.;Mitarnura, C.;Hara, S.;Hiratae, K.;Nishizawa, Y.;Hibi, T. https://doi.org/10.1006/jipa.1993.1017
  19. Classification of chitinases modules;Chitin and Chitinases Henrissat, B.;Jolles, P.(ed.);Muzzarelli, R.A.(ed.)
  20. Appl. Microbiol. v.29 Powdered chitin agar as a selective medium for enumeration of actinomycetes in water and soil Hsu, S.C.;Lockwood, J.L.
  21. Methods Enzymol. v.8 Chitinase Jeuniaux, C. https://doi.org/10.1016/0076-6879(66)08117-5
  22. Biochim. Biophys. Acta. v.1473 Physiological aspects of chitin catabolism in marine bacteria Keyhani, N.O.;Roseman, S. https://doi.org/10.1016/S0304-4165(99)00172-5
  23. Biochemistry of chitinase;Chitin and Chitinases Koga, D.;Mitsutomi, M.;Kono, M.;Matsumiya, M.;Jolles, P.(ed.);Muzzarelli, R.A.(ed.)
  24. J. BioI. Chem. v.254 An endochitinase from wheat germ: Activity on nascent and preformed chitin Molano, J.;Polacheck, I.;Duran, A.;Cabib, E.
  25. Methods Enzymol. v.161 Viscosmetric assay for chitinase Ohtakara, A.;Mitsutomi, M. https://doi.org/10.1016/0076-6879(88)61056-1
  26. J. Biol. Chem. v.181 A submicrodetermination of glucose Park, J.T.;Johnson, M.J.
  27. Methods Enzymol. v.161 Chitinase from Verticillium albo-atum Pego, G.F. https://doi.org/10.1016/0076-6879(88)61061-5
  28. Methods Enzymol. v.25 Acetylation Riordan, J.F.;Vallee, B.L. https://doi.org/10.1016/S0076-6879(72)25045-5
  29. J. Biol. Chem. v.263 Cloning and expression of a Streptomyces plicatus chitinase (chitinase-63) in Escherichia coli. Robbins, P.W.;Albright, C.;Benfield, B.
  30. Anal. Biochem. v.127 Serratia marcescens chitinase: One-step purification and use for the determination of chitin Roberts, R.L.;Cabib, E. https://doi.org/10.1016/0003-2697(82)90194-4
  31. Arch. Biochem. Biophys. v.111 Dissolution of fungal cell walls by a streptomycete chitinase and ${\beta}-(1-3)$ glucanase Skujins, J.J.;Potgieter, H.J.;Alexander, M. https://doi.org/10.1016/0003-9861(65)90197-9
  32. J. lnvertebr. Pathol. v.42 Chitinase is an inducible enzyme in Beauveria bassiana Smith, R.J.;Grula, E.A. https://doi.org/10.1016/0022-2011(83)90170-2
  33. J. lnvertebr. Pathol. v.38 Requirement for sequential enzymatic activities for penetration of the integument of the com earworm (Heliothis zea) Smith, R.J.;Pekrul, S.;Grula, E.A. https://doi.org/10.1016/0022-2011(81)90099-9
  34. Methods Enzymol. v.11 Tryptophan involvement in binding sites of proteins and in enzyme-inhibitor complexes as determined by oxidation with N-bromosuccinimide Spande, T.F.;Witkop, B. https://doi.org/10.1016/S0076-6879(67)11062-8
  35. Methods Enzymol. v.11 Modification of proteins with cyanate Stark, G.R. https://doi.org/10.1016/S0076-6879(67)11074-4
  36. J. Gen. Microbiol. v.132 Cutiledegrading enzymes of entomopathogenic fungi: Regulation of production of chitinolytic enzymes Leger, R.J.;Cooper, R.M.;Charnley, A.K. https://doi.org/10.1099/00221287-132-6-1509
  37. J. lnvertebr. Pathol. v.47 Cuticle-degrading enzymes of entomopathogenic fungi: Cuticle degradation in vitro by enzymes from entomopathogens Leger, R.J.;Cooper, R.M.;Charnley, A.K. https://doi.org/10.1016/0022-2011(86)90043-1
  38. Kor. J. Mycol. v.21 Studies on distribution and utilization of Cardyceps militaris and C. nutans Sung, J.M.;Kim, C.H.;Yang, K.J.;Lee, H.K.;Kim, Y.S.
  39. Kor. J. Mycol. v.25 Distribution and taxonomy of entomopathogenic fungal species from Korea Sung, J.M.;Lee, H.K.;Choi, Y.S.;Kim, Y.Y.;Kim, S.H.;Sung, G.H.
  40. Kor. J. Mycol. v.26 Classification of Cordyceps species based on protein anding pattern Sung, J.M.;Lee, H.K.;Yoo, Y.J.;Choi, Y.S.;Kim, S.H.;Kim, Y.O.;Sung, G.H.
  41. Biochem. J. v.61 Chitinase in some Basidiomycetes Tracey, M.V.
  42. J. Gen. Microbiol. v.137 Regulation of chitinase synthesis in Trichoderma harzianum Ulhoa, C.J.;Peberdy, J.F. https://doi.org/10.1099/00221287-137-9-2163
  43. J. BioI. Chem. v.268 Identification of glutamic acid204 and aspartic acid 200 in chitinase Al of Bacillus circulans WL-12 as essential residues for chitinase activity Watanabe, T.;Kobori, K.;Miyashita, K.;Fujii, T.;Sakai, H.;Uchida, M.;Tanaka, H.

Cited by

  1. Pharmacological and therapeutic potential of Cordyceps with special reference to Cordycepin vol.4, pp.1, 2014, https://doi.org/10.1007/s13205-013-0121-9
  2. Selection of entomopathogenic fungus for biological control of chili anthracnose disease caused by Colletotrichum spp. vol.146, pp.3, 2016, https://doi.org/10.1007/s10658-016-0941-7