Characterization of Alternaria alternata ${\alpha}-Amylase$

Alternaria alternata ${\alpha}-Amylase$의 특성에 관한 연구

  • Chung, Sang-Jin (Department of Gentic Engineering, Chongju University) ;
  • Hwang, Baik (Department of Biology, Chonnam National University)
  • 정상진 (청주대학교 유전공학과) ;
  • 황백 (전남대학교 생물학과)
  • Published : 1996.03.30

Abstract

The ${\alpha}-amylase$ of Alternaria alternata was purified through ammonium sulfate precipitation, dialysis and Sephadex G-100 column chromatography. One single band was obtained in SDS-polyacrylamide gel electrophoresis. The optimum pH for enzyme activity was 5.0 and the enzyme activity was maintained at $3.6{\sim}7.0$pH range. The optimum temperature for ${\alpha}-amylase$ activity was $40^{\circ}C$ and 71% of the activity was still maintained until 30 min after heating at $80^{\circ}C$. The ${\alpha}-amylase$ was slightly activated by $Mn^{2+},\;Zn^{2+}\;and\;Sn^{2+}$, but inhibited by $Ba^{2+},\;Pb^{2+},\;Co^{2+}\;and\;Ag^{1+}$. The $Hg^{2+}\;and\;Ag^{2+}$ slightly inhibited the activity of the enzyme at concentrations of $10^{-3}\;and\;10^{-4}M$. The Michaelis constant $(K_m)$ to soluble starch was $6.50{\times}10^{-2}M$ and inhibition constant $(K_i)$ by the 1mM EDTA was $8.0{\times}10^{-2}M$. The inhibition of this enzyme by EDTA was competitive one.

토양에서 분리된 A. alternata의 조효소액은 ammonium sulfate 염석, 투석 Sephadex G-100 column chromatography를 통하여 정제한 후 SDS-polyacrylamide gel 전기영동에 의해 단일 band의 ${\alpha}-amylase$를 얻었다. 이 효소의 물리화학적 특성을 분석한 결과는 다음과 같다. 1. 정제 효소의 최적 활성 pH는 5.0이었고 pH안정성 범위는 pH$3.6{\sim}7.0$으로 비교적 산성에서 안정성을 보이는 내산성 효소로 나타났다. 2. 효소의 최적 활성 온도는 $40^{\circ}C$, 열에 대한 안정성 범위는 $20{\sim}60^{\circ}C$로 나타났으며 $80^{\circ}C$에서 30분 열처리시 잔존 활성이 71%로 세균류와 거의 동일한 내열성을 보였다. 3. 본 효소는 $Mn^{2+},\;Zn^{2+}$$Sn^{2+}$의해 활성이 촉진되었으나 $Ba^{2+},\;Pb^{2+},\;Co^{2+},\;Ag^{1+}$에 의해 저해효과를 나타냈으며 또한 $Hg^{2+},\;Mn^{2+}$$10^{-3}M$$10^{-4}M$ 농도에서 저해작용을 나타냈다. 그러나 최대 활성 저해시 잔존 활성이 83%로 본 효소는 전반적으로 금속 이온의 영향을 크게 받지 않았다. 4. 본 효소의 soluble starch에 대한 $K_m$ 값은 $6.50{\times}10^{-2}$ M이었으며 1mM EDTA에 대한 $K_i$ 값은 $8{\times}10^{-2}M$로서 경쟁적 저해작용을 하였다.

Keywords

References

  1. Pusan Industrial Univ. Thesis Coll v.2 Studies on the amylase produced by Actinomycetes J-9525s. Part II. Culture conditions for amylase production by Actinomycetes J-9525s Bak, H.S.
  2. Methods of Enzymatic Analysis, Vol. 2 Bergmeyer, H.D.
  3. Methods in Enzymolgy Bernfeld, P.;Colowick, S.P.;Kaplan, N.O.
  4. Methods in Microbiology, Vol. 4 Booth, C.
  5. Kor. J. Appl. Microbial. Bioeng v.10 no.2 Studies on ${\alpha}-amylase$ of Bacillus circulans F-2. Part II. Enzymatic characteristics of the purified ${\alpha}-amylase$ Chung, M.J.;Taniguchi, H.;Maruyama, Y.;Lee, M.J.
  6. Appl. Environ. Microbiol Purificasion and characterization of extracellular amylolytic enzymes from the Yeast Filobasidium capsuligenum De Mot, R.;Verachtert, H.
  7. Difco Mannual Difco
  8. Compendium of Soil Fingi, Vol. 1 Domsch, K.H.;Gams, W.;Anderson, T.H.
  9. Enzyme Microbiol. Technol v.8 Extracellular amylase synthesis by Streptomyces limosus Fairbairn, D.A.;Priest, F.G.;Stark, J.R.
  10. Helv. Chim. Act. v.40 Purification and crystallization of ${\alpha}-amylase$ from Bacillus subtilis (Strain Takamine) amylolytic enzyme 23 Fellig, J.;Stein, E.A.;Fisher, E.H.
  11. Enzymes v.4 ${\alpha}-amylase$ Fisher, E.H.;Stein, E.A.
  12. Developments in microbial extracellular Enzymes;Topics in Enzyme and Fermentation Biotechnology, 3 Fogarty, W.M.;Kelly, C.T.
  13. Transactions of the British Mycolog. Soci v.38 A Method for studying active mycelia on living roots and other surface in the soil Harley, J.L.;Waid, J.S.
  14. Jin Ju Education Coll. Thesis. Coll v.13 Study on the development of fermentation food;1: Effects of several carbon sources on amylases production in Rhizopus spp Hur, J.H.
  15. Appl. Environ. Microbiol v.45 Production and characterization of amylase from Calvatea gigantea Kekos, D.;Macris, B.J.
  16. Studies on the producing amylase by the Aspergillus sp Kim, Y.S.
  17. Arch. Biochem. Biophys Two forms of glucoamylase of Aspergillus niger Lineback, D.R.;Russell, I.J.;Rasmussen, C.
  18. J. Biol. Chem. v.193 Protein measurement with the Folin-phenol reagent Lowry, O.H.;Rosebrough, N.J.;Farr, A.L.;Randall, R.J.
  19. Mannual of Industrial Microbiology and Biotechnology Miller, T.L.;Churchill, B.W.
  20. Kor. J. Appl. Microbial. Bioeng v.9 no.2 Studies on the production of thermostable amylase Oh, D.H.;Lee, K.P.;Pyun, Y.R.;Yu, J.H.
  21. Biochem. J. v.209 The purification of a novel amylase from B. subtilis and its inhibition by wheat proteins Orlando, A.R.;Ade, P.;Di Maggio, D.;Fanelli, C.;Vittozzi, L.
  22. Starch Robyt, J.F.
  23. Agric. Biol. Chem. v.47 no.8 Purification of ${\alpha}-maltotetraose$ forming exo-amylase of Pseudomonas stutzeri-Two forms of the amylase and their enzymatic properties Sakano, Y.;Kashiyama, E.;Kobayashi, T.
  24. Kyung Buk Univ. Thesis Coll v.11 Studies on acid stable ${\alpha}-amylase$ producting by black Aspergili Seu, J.H.;Kim, J.K.
  25. J. Biol. Chem. v.244 no.16 The reliability of molecular weight determinations by dodecyl sulfate polyacrylamide gel electrophoresis Weber, K.;Osborn, M.
  26. Agri. Biol. Chem. v.49 no.12 Purification and properties of an amylase from Bacillus cereus NY-14 Yoshigi, N.;Chikano, T.;Kamimura, M.
  27. J. Ferm. Technol v.61 no.1 Purification and Characterization of acid stable $\{alpha}-amylase$ from Paecilomyces sp Zenin, C.T.;Park, Y.K.