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

The Korean Society of Food Science and Nutrition (한국식품영양과학회)
권호 표지
DOI QR코드

DOI QR Code

Purification and Characterization of an Invertase Produced with Saccharomyces cerevisiae JS59 Isolated from Home-made Wine

포도주에서 분리한 Saccharomyces cerevisiae JS59가 생성하는 Invertase의 정제 및 특성

  • Yoo, Ji-Soo (Dept. of Applied Biology and Chemistry, Konkuk University) ;
  • Paik, Hyun-Dong (Dept. of Food Science and Biotechnology of Animal Resources, Konkuk University) ;
  • Kim, Soo-Young (Dept. of Biological Science, Konkuk University) ;
  • Lee, Si-Kyung (Dept. of Applied Biology and Chemistry, Konkuk University)
  • 유지수 (건국대학교 응용생물화학과) ;
  • 백현동 (건국대학교 축산식품생물공학과) ;
  • 김수영 (건국대학교 생명과학과) ;
  • 이시경 (건국대학교 응용생물화학과)
  • Received : 2010.12.14
  • Accepted : 2011.01.10
  • Published : 2011.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

The microorganism producing an invertase (E.C. 3.2.1.26) was isolated from wine and tentatively identified as Saccharomyces cerevisiae by cellular fatty acid analysis. The invertase was purified to homogeneity by ammonium sulfate precipitant, dialysis, ion-exchange chromatography on DEAE-Sephadex A-50, and gel chromatography on Sephadex G-200 from the culture supernatant of Saccharomyces cerevisiae JS59. The specific activity and the purification fold of the purified invertase were 7620.9 unit/mg protein and 13.9, respectively. The molecular weight of the purified invertase was estimated to be 38.5 kDa by SDS-PAGE. The optimum pH and temperature for the invertase activity were pH 5 and $55^{\circ}C$, respectively. The invertase activity was relatively stable at pH 4~6 and temperature $55^{\circ}C$. The activity of invertase was inhibited by $Ag^{2+}$ and $Hg^{2+}$, but on the contrary, activated by $Co^{2+}$ and $Mn^{2+}$. Michaelis constant ($K_m$) for invertase reaction in sucrose solution was 11.5 mM. TLC analysis of the products produced in sucrose solution during invertase reaction showed the progressive presence of glucose and fructose in accordance with sucrose hydrolysis.

전화당(invert sugar)을 생산하기 위하여 포도주로부터 분리한 효모가 생산하는 invertase의 특성을 조사한 결과는 다음과 같다. 포도주로부터 분리한 효모는 지방산 분석을 통하여 Saccharomyces cerevisiae JS59로 잠정 동정되었다. 본 균주가 생성하는 invertase를 ammonium sulfate 침전, DEAE-Sephadex A-50, Sephadex G-200 column chromatography 법으로 정제하였을 때 단일성을 보였으며, specific activity가 7620.9 unit/mg, 최종 회수율은 13.9로 약 14배 정제된 효소를 얻었다. 본 효소의 $K_m$ 값은 11.5 mM이었다. SDS-PAGE로부터 분자량은 38.5 kDa으로 나타났다. 정제된 invertase의 최적 pH는 5였고, pH 4에서도 94%의 높은 효소활성을 나타냈으며 4에서 6까지의 pH영역에서 안정하였고, $55^{\circ}C$에서 최적 활성을 나타내었으며 $50^{\circ}C$까지는 안정하였다. $Ag^{2+}$와 $Hg^{2+}$에 의해서 저해를 받았고, $Co^{2+}$, $Mn^{2+}$에 의해서는 효소활성이 증가되었으며, 기질과 효소 반응물을 thin layer chromatography로 분석한 결과, 본 효소는 기질인 sucrose를 완전히 분해하여 환원당을 생성함이 확인되었다.

Keywords

References

  1. Kim JW, Park JS. 1997. Industrially useful microorganism (III). Kor J Microbiol Biotechnol 10: 26-33.
  2. Cha SK, Choi HS, Kim WJ, Yoon SH, Kim YB. 1996. Screening of yeast cell wall lytic enzyme producing microorganism and optimization of enzyme production. Kor J Appl Microbiol Biotechnol 24: 143-148.
  3. Cipan A, Alkan S, Toppare L, Hepuzer Y, Yagci Y. 2003. Immobilization of invertase in conducting copolymers of 3-methylthienyl methacrylate. Bioelectrochem 59: 29-33. https://doi.org/10.1016/S1567-5394(02)00186-X
  4. Myrback K. 1960. Invertase. In The Enzyme. Boyer PD, Lardy H, Myrback K, eds. Academic Press Inc., New York, NY, USA. Vol 4, p 379-396.
  5. Ester JT, Michele V. 2007. Production of high-fructose syrup using immobilized invertase in a membrane reactor. J Food Eng 80: 662-667. https://doi.org/10.1016/j.jfoodeng.2006.07.002
  6. Santiao JA, Vásquez JM. 2006. Direct immobilization of a recombinant invertase to Avicel by E. coli overexpression of a fusion protein containing the extracellular invertase from Zymomonas mobilis and the carbohydrate-binding domain DBDcex from Cellulomonas fimi. Enz Microb Technol 40: 172-176. https://doi.org/10.1016/j.enzmictec.2005.10.052
  7. Arnold WN. 1974. Expression of cryptic $\beta$-fructofuranosidase in Sacch. rouxii. J Bacteriol 120: 887-894.
  8. Arnold WN. 1972. The structure of the yeast cell wall. Solubilization of a marker enzyme, $\beta$-fructofuranosidase by the autolytic enzyme system. J Biol Chem 247: 1161-1169.
  9. Gascon S, Lampen JO. 1968. Purification of internal invertase of yeast. J Biol Chem 243: 1567-1572.
  10. Babczinski P. 1980. Partial purification, characterization and localization of the membrane-associated invertase of yeast. Biochem Biophys Acta 614: 121-133. https://doi.org/10.1016/0005-2744(80)90173-4
  11. Onyezili FN, Onitiri AC. 1981. Immobilization of invertase on modified nylon tubes. Anal Biochem 113: 203-206. https://doi.org/10.1016/0003-2697(81)90067-1
  12. Metzenberg RL. 1962. A gene affecting the repression of invertase and trehalase in Neurospora. Arch Biochem Biophys 96: 468-474. https://doi.org/10.1016/0003-9861(62)90322-3
  13. Ottolenghi P. 1971. A comparison of five genetically distinct invertase from Saccharomyces sp. Eur J Biochem Biophys 18: 544-552. https://doi.org/10.1111/j.1432-1033.1971.tb01275.x
  14. Welch DF. 1991. Applications of cellular fatty acid analysis. Cli Microbiol Rev 4: 422-438. https://doi.org/10.1128/CMR.4.4.422
  15. Miller GL. 1959. Use of dinitrosalycylic acid reagent for determination of reducing sugar. Anal Chem 31: 426-428. https://doi.org/10.1021/ac60147a030
  16. Lowry OH, Rosebrough NJ. 1951. Protein measurement with the folin phenol reagent. J Biol Chem 193: 265-275.
  17. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacterophage T4. Nature 227: 680-685. https://doi.org/10.1038/227680a0
  18. Lineweaver H, Burke D. 1934. Determination of enzyme dissociation constants. J Am Chem Soc 56: 658-666. https://doi.org/10.1021/ja01318a036
  19. Hay GW, Lewis BA, Smith F. 1963. Thin-film chromatography in the study of carbohydrates. J Chromat 11: 479-486. https://doi.org/10.1016/S0021-9673(01)80949-3
  20. Hiroshi H, Koji T. 2003. Purification and characterization of two soluble acid invertase isozyme from Japanese pear fruit. Phytochemistry 63: 125-129. https://doi.org/10.1016/S0031-9422(03)00107-9
  21. Lamia LH, Zhang W. 2000. Purification and partial characterization of fructosyltransferase and invertase from Aspergillus niger AS0023. J Biotechnol 81: 73-84. https://doi.org/10.1016/S0168-1656(00)00277-7
  22. Maria CR, Rosa R. 2002. Invertase from a strain of Rhodotorula glutinis. Phytochemistry 61: 605-609. https://doi.org/10.1016/S0031-9422(02)00336-9
  23. Quang DN, Judit MR. 2005. Purification and some properties of $\beta$-fructofruanosidase from Aspergillus niger IMI 303386. Process Biochem 40: 2461-2466. https://doi.org/10.1016/j.procbio.2004.09.012
  24. Kim NM, Kim DW, Kim JH. 2002. Biosynthetic regulation of invertase from thermophilic and alkalophilic Bacillus sp. TA-11. Korean J Food Nutr 15: 126-130.
  25. Elina L, Laine C. 2003. Purification and characterization of Aspergillus $\beta$-D-galactanases acting on $\beta$-1,4- and $\beta$-1, 3/6-linked arabinogalactans. Carbohydr Polym 53: 155-168. https://doi.org/10.1016/S0144-8617(02)00303-X
  26. Muramatsu M, Kakuki T. 2005. Enzymatic synthesis of novel fructosyl and oligofructosyl trehaloses by Aspergillus sydowi $\beta$-fructofurandsidase. Biosci Biotechnol Biochem 59: 208-212.
  27. Hong JM, Lee KA. 1990. Production and properties of invertase from Aspergillus niger. J Korean Soc Food Nutr 19: 577-582.
  28. Sayago JE, Vattuone MA. 2002. Proteinaceous inhibitor versus fructose as modulators of Pteris deflexa invertase activity. J Enz Inhib Medicinal Chem 17: 123-130. https://doi.org/10.1080/14756360290026450
  29. Ester JT, Michele V. 2006. Catalytic performance of invertase immobilized by adsorption on anionic exchange resin. Process Biochem 41: 1325-1331. https://doi.org/10.1016/j.procbio.2006.01.004
  30. Sanjay G, Sugunan S. 2005. Enhanced pH and thermal stabilities of invertase immobilized on montmorillonite K-10. Food Chem 94: 573-579.
  31. Choi YJ, Lee JS. 1995. Production of $\beta$-fructofuranosidase from alkalophilic, thermophilic Bacillus sp. TA-11. Kor J Appl Microbiol Biotechnol 23: 197-202.
  32. Emel E, Sibel S. 2006. Polyacrylamide-gelatine carrier system used for invertase immobilization. Food Chem 97: 591-597. https://doi.org/10.1016/j.foodchem.2005.05.017
  33. Bazar T, Tuncel A. 2004. Concanavalin A carring reactive beads for yeast invertase purification. Reactiv Function Polym 61: 203-210. https://doi.org/10.1016/j.reactfunctpolym.2004.05.006
  34. Liu CC, Huang LC. 2006. Purification and characterization of soluble invertases from suspension-cultured bamboo (Bambusaedulis) cells. Food Chem 96: 621-631. https://doi.org/10.1016/j.foodchem.2005.02.044
  35. Madyastha KM, Ganguli AR. 1987. Extracellular invertase from Aspergillus athecius: isolation and immobilization. Biotechnol Lett 9: 555-560. https://doi.org/10.1007/BF01026660
  36. Haq NB, Asgher M, Abbas A. 2006. Studies on kinetics and thermostability of a novel acid invertase from Fusarium solani. J Agric Food Chem 54: 4617-4623. https://doi.org/10.1021/jf053194g