Korean Journal of Food Science and Technology (한국식품과학회지)

Korean Society of Food Science and Technology (한국식품과학회)
권호 표지
DOI QR코드

DOI QR Code

Molecular Cloning and Characterization of Maltogenic Amylase from Deinococcus geothermalis

Deinococcus geothermalis 유래 maltogenic amylase의 유전자 발현 및 특성확인

  • Jung, Jin-Woo (Department of Food Science & Biotechnology and Institute of Life Sciences & Resources, Kyung Hee University) ;
  • Jung, Jong-Hyun (Department of Food Science & Biotechnology and Institute of Life Sciences & Resources, Kyung Hee University) ;
  • Seo, Dong-Ho (Department of Food Science & Biotechnology and Institute of Life Sciences & Resources, Kyung Hee University) ;
  • Kim, Byung-Yong (Department of Food Science & Biotechnology and Institute of Life Sciences & Resources, Kyung Hee University) ;
  • Park, Cheon-Seok (Department of Food Science & Biotechnology and Institute of Life Sciences & Resources, Kyung Hee University)
  • 정진우 (경희대학교 식품생명공학과 및 생명자원과학연구원) ;
  • 정종현 (경희대학교 식품생명공학과 및 생명자원과학연구원) ;
  • 서동호 (경희대학교 식품생명공학과 및 생명자원과학연구원) ;
  • 김병용 (경희대학교 식품생명공학과 및 생명자원과학연구원) ;
  • 박천석 (경희대학교 식품생명공학과 및 생명자원과학연구원)
  • Received : 2011.03.05
  • Accepted : 2011.04.14
  • Published : 2011.06.30
Download PDF
⟨ Previous Next ⟩

Abstract

A putative maltogenic amylase gene (DGMA) was cloned from the Deinococcus geothermalis DSM 11300 genome using the polymerase chain reaction. The gene encoded 608 amino acids with a predicted molecular mass of 68,704 Da. The recombinant DGMA was constitutively expressed using the pHCXHD plasmid. As expected, the recombinant DGMA hydrolyzed cyclodextrins and starch to maltose and pullulan to panose by cleaving the ${\alpha}$-(1,4)-glycosidic linkages, as observed for typical maltogenic amylases. Characterization of the recombinant DGMA revealed that the highest maltogenic amylase activity occurred at $40^{\circ}C$ and pH 6.0. The half-life of catalytic activity at $65^{\circ}C$ and $55^{\circ}C$ were 8.2 min and 187.4 min, respectively. DGMA mainly hydrolyzed ${\beta}$-cyclodextrin, soluble starch, and pullulan and its efficient ratio of those substrates was 9:4.5:1.

D. geothermalis의 dgeo_0475 유전자로부터 만들어지는 효소를 정제하여 그 특성을 확인하였다. DGMA는 분자량이 약 68 kDa 크기의 효소로서 ${\beta}$-CD, soluble starch 및 pullulan을 가수분해하는 CD 분해 효소임을 확인하였다. 효소의 최적 온도는 $40^{\circ}C$ 최적 pH 는 6.0이며 대부분의 기질들을 glucose와 maltose로 가수분해 하였고 pullulan 및 soluble starch로부터 미량의 panose를 생성하였다. ${\beta}$-CD를 가장 잘 가수분해하나 기질간 상대적 활성차이는 다른 CD 분해효소에 비하여 크지 않았다.

Keywords

References

  1. Lee HS, Kim MS, Cho HS, Kim JI, Kim TJ, Choi JH, Park C, Oh BH, Park KH. Cyclomaltodextrinase, neopullulanase, and maltogenic amylase are nearly indistinguishable from each other. J. Biol. Chem. 277: 21891-21897 (2002) https://doi.org/10.1074/jbc.M201623200
  2. Park KH, Kim TJ, Cheong TK, Kim JW, Oh BH, Svensson B. Structure, specificity, and function of cyclomaltodextrinase, a multispecific enzyme of the $\alpha$-amylase family. Biochim. Biophys. Acta 1478: 165-185 (2000) https://doi.org/10.1016/S0167-4838(00)00041-8
  3. Kim JS, Cha SS, Kim HJ, Kim TJ, Ha NC, Oh ST, Cho HS, Cho MJ, Kim MJ, Lee HS, Kim JW, Choi KY, Park KH, Oh BH. Crystal structure of a maltogenic amylase provides insights into a catalytic versatility. J. Biol. Chem. 274: 26279-26286 (1999) https://doi.org/10.1074/jbc.274.37.26279
  4. Smith KA, Salyers AA. Characterization of a neopullulanase and an alpha-glucosidase from Bacteroides thetaiotaomicron 95-1. J.Bacteriol. 173: 2962-2968 (1991)
  5. Igarashi K, Ara K, Saeki K, Ozaki K, Kawai S, Ito S. Nucleotide sequence of the gene that encodes a neopullulanase from an alkalophilic Bacillus. Biosci. Biotech. Bioch. 56: 514-516 (1992) https://doi.org/10.1271/bbb.56.514
  6. Yebra MJ, Blasco A, Sanz P. Expression and secretion of Bacillus polymyxa neopullulanase in Saccharomyces cerevisiae. FEMS Microbiol. Lett. 170: 41-49 (1999) https://doi.org/10.1111/j.1574-6968.1999.tb13353.x
  7. Takata H, Kuriki T, Okada S, Takesada Y, Iizuka M, Minamiura N, Imanaka T. Action of neopullulanase. Neopullulanase catalyzes both hydrolysis and transglycosylation at$\alpha$-(1$\rightarrow$4)- and $\alpha$-(1$\rightarrow$6)- glucosidic linkages. J. Biol. Chem. 267: 18447-18452 (1992)
  8. Kamasaka H, Sugimoto K, Takata H, Nishimura T, Kuriki T. Bacillus stearothermophilus neopullulanase selective hydrolysis of amylose to maltose in the presence of amylopectin. Appl. Environ. Microb. 68: 1658-1664 (2002) https://doi.org/10.1128/AEM.68.4.1658-1664.2002
  9. Kim TJ, Shin JH, Oh JH, Kim MJ, Lee SB, Ryu S, Kwon K, Kim JW, Choi EH, Robyt JF, Park KH. Analysis of the gene encoding cyclomaltodextrinase from alkalophilic Bacillus sp. I-5 and characterization of enzymatic properties. Arch. Biochem. Biophys. 353: 221-227 (1998) https://doi.org/10.1006/abbi.1998.0639
  10. Kim IC, Cha JH, Kim JR, Jang SY, Seo BC, Cheong TK, Lee DS, Choi YD, Park KH. Catalytic properties of the cloned amylase from Bacillus licheniformis. J. Biol. Chem. 267: 22108- 22114 (1992)
  11. Makarova KS, Omelchenko MV, Gaidamakova EK, Matrosova VY, Vasilenko A, Zhai M, Lapidus A, Copeland A, Kim E, Land M, Mavrommatis K, Pitluck S, Richardson PM, Detter C, Brettin T, Saunders E, Lai B, Ravel B, Kemner KM, Wolf YI, Sorokin A, Gerasimova AV, Gelfand MS, Fredrickson JK, Koonin EV, Daly MJ. Deinococcus geothermalis: The pool of extreme radiation resistance genes shrinks. PLoS One 2: e955 (2007) https://doi.org/10.1371/journal.pone.0000955
  12. Ferreira AC, Nobre MF, Rainey FA, Silva MT, Wait R, Burghardt J, Chung AP, da Costa MS. Deinococcus geothermalis sp. nov. and Deinococcus murrayi sp. nov., two extremely radiation-resistant and slightly thermophilic species from hot springs. Int. J. Syst. Bacteriol. 47: 939-947 (1997) https://doi.org/10.1099/00207713-47-4-939
  13. Palomo M, Kralj S, van der Maarel MJ, Dijkhuizen L. The unique branching patterns of Deinococcus glycogen branching enzymes are determined by their N-terminal domains. Appl. Environ. Microb. 75: 1355-1362 (2009) https://doi.org/10.1128/AEM.02141-08
  14. Jung JH, Seo DH, Ha SJ, Song MC, Cha J, Yoo SH, Kim TJ, Baek NI, Baik MY, Park CS. Enzymatic synthesis of salicin glycosides through transglycosylation catalyzed by amylosucrases from Deinococcus geothermalis and Neisseria polysaccharea. Carbohyd. Res. 344: 1612-1619 (2009) https://doi.org/10.1016/j.carres.2009.04.019
  15. Park JM, Han NS, Kim TJ. Rapid detection and isolation of known and putative $\alpha$-L-arabinofuranosidase genes using degenerate PCR primers. J. Microbiol. Biotech. 17: 481-489 (2007)
  16. Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426-428 (1959) https://doi.org/10.1021/ac60147a030
  17. Tonozuka T, Mogi S, Shimura Y, Ibuka A, Sakai H, Matsuzawa H, Sakano Y, Ohta T. Comparison of primary structures and substrate specificities of two pullulan-hydrolyzing $\alpha$-amylases, TVA I and TVA II, from Thermoactinomyces vulgaris R-47. Biochim. Biophys. Acta 1252: 35-42 (1995) https://doi.org/10.1016/0167-4838(95)00101-Y
  18. Hondoh H, Kuriki T, Matsuura Y. Three-dimensional structure and substrate binding of Bacillus stearothermophilus neopullulanase. J. Mol. Biol. 326: 177-188 (2003) https://doi.org/10.1016/S0022-2836(02)01402-X
  19. Kim TJ, Kim MJ, Kim BC, Kim JC, Cheong TK, Kim JW, Park KH. Modes of action of acarbose hydrolysis and transglycosylation catalyzed by a thermostable maltogenic amylase, the gene for which was cloned from a Thermus strain. Appl. Environ. Microb. 65: 1644-1651 (1999)
  20. Cha HJ, Yoon HG, Kim YW, Lee HS, Kim JW, Kweon KS, Oh BH, Park KH. Molecular and enzymatic characterization of a maltogenic amylase that hydrolyzes and transglycosylates acarbose. Eur. J. Biochem. 253: 251-262 (1998) https://doi.org/10.1046/j.1432-1327.1998.2530251.x
  21. Oh KW, Kim MJ, Kim HY, Kim BY, Baik MY, Auh JH, Park CS. Enzymatic characterization of a maltogenic amylase from Lactobacillus gasseri ATCC 33323 expressed in Escherichia coli. FEMS Microbiol. Lett. 252: 175-181 (2005) https://doi.org/10.1016/j.femsle.2005.08.050
  22. Cheong KA, Kim TJ, Yoon JW, Park CS, Lee TS, Kim YB, Park KH, Kim JW. Catalytic activities of intracellular dimeric neopullulanase on cyclodextrin, acarbose and maltose. Biotechnol. Appl. Bioc. 35: 27-34 (2002) https://doi.org/10.1042/BA20010052
  23. Cho HY, Kim YW, Kim TJ, Lee HS, Kim DY, Kim JW, Lee YW, Leed S, Park KH. Molecular characterization of a dimeric intracellular maltogenic amylase of Bacillus subtilis SUH4-2. Biochim. Biophys. Acta 1478: 333-340 (2000) https://doi.org/10.1016/S0167-4838(00)00037-6
  24. Ibuka A, Tonozuka T, Matsuzawa H, Sakai H. Conversion of neopullulanase-$\alpha$-amylase from Thermoactinomyces vulgaris R-47 into an amylopullulanse-type enzyme. J. Biochem. 123: 275-282 (1998) https://doi.org/10.1093/oxfordjournals.jbchem.a021933
  25. Turner P, Labes A, Fridjonsson OH, Hreggvidson GO, Schonheit P, Kristjansson JK, Holst O, Karlsson EN. Two novel cyclodextrin- degrading enzymes isolated from thermophilic bacteria have similar domain structures but differ in oligomeric state and activity profile. J. Biosci. Bioeng. 100: 380-390 (2005) https://doi.org/10.1263/jbb.100.380