Journal of the Korean Society of Food Science and Nutrition (한국식품영양과학회지)

The Korean Society of Food Science and Nutrition (한국식품영양과학회)
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Method for Increasing the Stability and Activity of Polygalacturonase and Its Application to the Production of Vegetable Single Cell

Polygalacturonase의 활성 증진 및 이를 이용한 식물 단세포 제조 방법

  • Kim, Hyuk-Hwa (Dept. of Bio-Food Technology, Korea Bio Polytechnic College)
  • 김혁화 (한국폴리텍 바이오대학 바이오식품과)
  • Published : 2007.12.31
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Abstract

This study was carried out to enhance the stability and activity of polygalacturonase (PGase) purified from Kluyveromyces marxianus IFO 0288. Gums such as xanthan gum, guar gum, and locust bean gum were capable of increasing the catalytic stability and activity of the PGase. At $30^{\circ}C$, the rate constants for the inactivation of the PGase with xanthan gum, guar gum, and locust bean gum were estimated to be $0.0003min^{-1}$, below $0.0001min^{-1},\;and\;0.0001min^{-1}$ respectively, whereas control was estimated to be $0.0082min^{-1}$. The yield of the maceration reaction catalyzed by the PGase for the production of carrot single cells increased by 13% in the presence of guar gum, where the relative enzyme activity supplemented with guar gum was two-fold greater than that of the PGase alone.

본 연구에서는 효소의 활성을 증진시킬 수 있는 새로운 방법의 개발을 위하여 미생물로부터 분리, 정제된 polygalacturonase(PGase)에 xanthan gum, guar gum, locust bean gum 등과 같은 교질물질을 첨가함으로써 효소의 안정성 외에 활성을 특이적으로 향상시킬 수 있는 새로운 방법 조사하였다. 정제된 PGase를 0.2%의 상술한 교질물질을 함유하는 동일한 완충용액에 잘 혼합시켜 $30^{\circ}C$에서 배양하여 효소의 불활성화에 대한 일차반응 속도상수 k값을 구한 결과, 대조군의 k값이 $0.0082min^{-1}$인데 반해 xanthan gum 첨가 시는 $0.0003min^{-1}$, guar gum 첨가 시는 $0.0001min^{-1}$이하, locust bean gum 첨가 시는 $0.0001min^{-1}$로서 교질물질 첨가에 의해 효소의 안정성이 현저히 증가하였다. 또한 대조군에 비해 xanthan gum 첨가 시는 PGase의 상대활성이 89%가 증가되었으며, guar gum 첨가 시는 97%, locust bean gum 첨가 시는 90%가 증가되어 상술한 교질물질들이 효소의 활성 촉진제로서의 기능이 있음을 확인할 수 있었다. Guar gum 처리에 의해 약 2배의 활성이 증진된 상태로 당근 단세포 생성 반응을 수행한 결과 모든 반응시간에서 guar gum을 가했을 때가 PGase만을 가하여 단세포화 반응을 수행한 경우보다 높은 수율을 보였으며, 단세포화 반응 2시간 경과 후에 대조군 대비 13%의 가장 높은 수율 향상을 보였다.

Keywords

References

  1. Maldonado MC, Saad AM. 1998. Production of pectinester-ase and polygalacturonase by Aspergillus niger in submerged and solid state systems. J Ind Microbiol Biotechnol 20: 34-38 https://doi.org/10.1038/sj.jim.2900470
  2. Martel MB, Letoublon R, Fevre M. 1998. Purification and characterization of two endopolygalacturonases secreted during the early stages of the saprophytic growth of Sclerotinia scerotiorum. PFEMS Microbiol Lett 158: 133-138 https://doi.org/10.1111/j.1574-6968.1998.tb12812.x
  3. Takasawa T, Sagisaka K, Yagi K, Uchiyama K, Aoki A, Takaoka K, Yamamoto K. 1997. Polygalacturonase isolated from the culture of the psychrophilic fungus Sclerotinia borealis. Can J Microbiol 43: 417-424 https://doi.org/10.1139/m97-059
  4. Iguchi K, Kishida M, Sakai T. 1996. Purification and characterization of three extra cellular protopectinases with polygalacturonase activity from Trichosporon penicillatum. Biosci Biotechnol Biochem 60: 603-667 https://doi.org/10.1271/bbb.60.603
  5. Gainvors A, Frezier V, Lemaresquire H, Lequart C, Aigle M, Belbarbi A. 1994. Detection of polygalacturonase, pectin-lyase and pectin esterase activities in a Saccharomyces cerevisiae strain. Yeast 10: 1311-1319 https://doi.org/10.1002/yea.320101008
  6. Sakai T, Okushima M, Yoshidake S. 1984. Purification, crystallization, and some properties of endo-polygalacturonase from Kluyveromyces fragilis. Agric Biol Chem 48: 1951-1961 https://doi.org/10.1271/bbb1961.48.1951
  7. Lim JY, Yamasaki Y, Ozawa J. 1980. Multiple forms of endo-polygalacturonase from Saccharomyces fragilis. Agric Biol Chem 44: 473-480 https://doi.org/10.1271/bbb1961.44.473
  8. Gomez-Ruiz L, Garcia-Garibay M, Barana E. 1988. Utilization of endo-polygalacturonase from Kluyveromyces fragilis in the clarification of apple juice. J Food Sci 53: 1236-1240 https://doi.org/10.1111/j.1365-2621.1988.tb13575.x
  9. Call HP, Waler J, Emeis CC. 1985. Maceration activity of an endo- polygalacturonase from Candida macedoniensis. J Food Biochem 9: 325- 348 https://doi.org/10.1111/j.1745-4514.1985.tb00356.x
  10. Mozhaev VV, Melik-Nuvarov NS, Levitsky VY, Siksnis VA, Martnek K. 1992. High stability to irreversible inactivation at elevated temperatures of enzymes covalently modified by hydrophilic reagent. Biotechnol Bioeng 40: 650-662 https://doi.org/10.1002/bit.260400603
  11. Moreau A, Shareck F, Kluepfel D, Morosoli R. 1994. Increase on catalytic activity and thermostability of the xylanase A of Streptomyces lividans 1362 by site-specific mutagenesis. Enz Microb Technol 16: 420-424 https://doi.org/10.1016/0141-0229(94)90158-9
  12. Lee JH. 1999. Preparation of microbial polygalacturonase and its application to the production of vegetable single cell. PhD Dissertation. Yonsei University, Korea
  13. Honda S, Nishimura Y, Takahashi M, Chiba H, Kakehi K. 1982. A manual method for the spectrophotometric determination of reducing carbohydrates with 2-cyanoacetamide. Anal Biochem 119: 194-199 https://doi.org/10.1016/0003-2697(82)90685-6
  14. Nakamura T, Hours RA, Sakai T. 1995. Enzymatic maceration of vegetables with protopectinases. J Food Sci 60: 468-472 https://doi.org/10.1111/j.1365-2621.1995.tb09805.x
  15. Sakai T, Sakamoto T, Hallaert J, Vandamme EJ. 1993. Pectin, pectinase, and protopectinase: Production, properties, and applications. Adv Appl Microbiol 39: 213-294 https://doi.org/10.1016/S0065-2164(08)70597-5