Advertisement

Applied Microbiology and Biotechnology

, Volume 98, Issue 5, pp 2121–2131 | Cite as

Maltose-forming α-amylase from the hyperthermophilic archaeon Pyrococcus sp. ST04

  • Jong-Hyun Jung
  • Dong-Ho Seo
  • James F. Holden
  • Cheon-Seok Park
Biotechnologically relevant enzymes and proteins

Abstract

The deduced amino acid sequence from a gene of the hyperthermophilic archaeon Pyrococcus sp. ST04 (Py04_0872) contained a conserved glycoside hydrolase family 57 (GH57) motif, but showed <13 % sequence identity with other known Pyrococcus GH57 enzymes, such as 4-α-glucanotransferase (EC 2.4.1.25), amylopullulanase (EC 3.2.1.41), and branching enzyme (EC 2.4.1.18). This gene was cloned and expressed in Escherichia coli, and the recombinant product (P yrococcus sp. ST04 maltose-forming α-amylase, PSMA) was a novel 70-kDa maltose-forming α-amylase. PSMA only recognized maltose (G2) units with α-1,4 and α-1,6 linkages in polysaccharides (e.g., starch, amylopectin, and glycogen) and hydrolyzed pullulan very poorly. G2 was the primary end product of hydrolysis. Branched cyclodextrin (CD) was only hydrolyzed along its branched maltooligosaccharides. 6-O-glucosyl-β-cyclodextrin (G1-β-CD) and β-cyclodextrin (β-CD) were resistant to PSMA suggesting that PSMA is an exo-type glucan hydrolase with α-1,4- and α-1,6-glucan hydrolytic activities. The half-saturation value (K m) for the α-1,4 linkage of maltotriose (G3) was 8.4 mM while that of the α-1,6 linkage of 6-O-maltosyl-β-cyclodextrin (G2-β-CD) was 0.3 mM. The k cat values were 381.0 min−1 for G3 and 1,545.0 min−1 for G2-β-CD. The enzyme was inhibited competitively by the reaction product G2, and the K i constant was 0.7 mM. PSMA bridges the gap between amylases that hydrolyze larger maltodextrins and α-glucosidase that feeds G2 into glycolysis by hydrolyzing smaller glucans into G2 units.

Keywords

Pyrococcus sp. ST04 Hyperthermophile Maltose-forming α-amylase Maltose inhibition 

Notes

Acknowledgments

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST; no. 2012–0005289).

Supplementary material

253_2013_5068_MOESM1_ESM.docx (430 kb)
ESM 1 (DOCX 429 kb)

References

  1. Blesák K, Janeček Š (2012) Sequence fingerprints of enzyme specificities from the glycoside hydrolase family GH57. Extremophiles 16(3):497–506PubMedCrossRefGoogle Scholar
  2. Brown SH, Kelly RM (1993) Characterization of amylolytic enzymes, having both α-1,4 and α-1,6 hydrolytic activity, from the thermophilic archaea Pyrococcus furiosus and Thermococcus litoralis. Appl Environ Microbiol 59(8):2614–2621PubMedCentralPubMedGoogle Scholar
  3. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res 37:D233–238PubMedCentralPubMedCrossRefGoogle Scholar
  4. Choi HC, Seo DH, Jung JH, Ha SJ, Kim MJ, Lee JH, Chang PS, Kim HY, Park CS (2011) Development of new assay for sucrose phosphorylase and its application to the characterization of Bifidobacterium longum SJ32 sucrose phosphorylase. Food Sci Biotechnol 20(2):513–518. doi: 10.1007/s10068-011-0071-0 CrossRefGoogle Scholar
  5. Comfort DA, Chou CJ, Conners SB, VanFossen AL, Kelly RM (2008) Functional-genomics-based identification and characterization of open reading frames encoding α-glucoside-processing enzymes in the hyperthermophilic archaeon Pyrococcus furiosus. Appl Environ Microbiol 74(4):1281–1283PubMedCentralPubMedCrossRefGoogle Scholar
  6. Dickmanns A, Ballschmiter M, Liebl W, Ficner R (2006) Structure of the novel α-amylase AmyC from Thermotoga maritima. Acta Crystallogr D: Biol Crystallogr 62:262–270CrossRefGoogle Scholar
  7. Dong G, Vieille C, Zeikus JG (1997) Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme. Appl Environ Microbiol 63(9):3577–3584PubMedCentralPubMedGoogle Scholar
  8. Gruyer S, Legin E, Bliard C, Ball S, Duchiron F (2002) The endopolysaccharide metabolism of the hyperthermophilic archeon Thermococcus hydrothermalis: polymer structure and biosynthesis. Curr Microbiol 44(3):206–211PubMedCrossRefGoogle Scholar
  9. Janeček Š, Blesák K (2011) Sequence-structural features and evolutionary relationships of family GH57 α-amylases and their putative α-amylase-like homologues. Protein J 30(6):429–435PubMedCrossRefGoogle Scholar
  10. Jiao YL, Wang SJ, Lv MS, Xu JL, Fang YW, Liu S (2011) A GH57 family amylopullulanase from deep-sea Thermococcus siculi: expression of the gene and characterization of the recombinant enzyme. Curr Microbiol 62(1):222–228PubMedCrossRefGoogle Scholar
  11. Jung JH, Jung TY, Seo DH, Yoon SM, Choi HC, Park BC, Park CS, Woo EJ (2011) Structural and functional analysis of substrate recognition by the 250s loop in amylomaltase from Thermus brockianus. Proteins 79(2):633–644PubMedCrossRefGoogle Scholar
  12. Jung JH, Lee JH, Holden JF, Seo DH, Shin H, Kim HY, Kim W, Ryu S, Park CS (2012) Complete genome sequence of the hyperthermophilic archaeon Pyrococcus sp. strain ST04, isolated from a deep-sea hydrothermal sulfide chimney on the Juan de Fuca Ridge. J Bacteriol 194(16):4434–4435PubMedCentralPubMedCrossRefGoogle Scholar
  13. Kaper T, Talik B, Ettema TJ, Bos H, van der Maarel MJ, Dijkhuizen L (2005) Amylomaltase of Pyrobaculum aerophilum IM2 produces thermoreversible starch gels. Appl Environ Microbiol 71(9):5098–5106PubMedCentralPubMedCrossRefGoogle Scholar
  14. Koga S, Yoshioka I, Sakuraba H, Takahashi M, Sakasegawa S, Shimizu S, Ohshima T (2000) Biochemical characterization, cloning, and sequencing of ADP-dependent (AMP-forming) glucokinase from two hyperthermophilic archaea, Pyrococcus furiosus and Thermococcus litoralis. J Biochem 128(6):1079–1085PubMedCrossRefGoogle Scholar
  15. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23(21):2947–2948PubMedCrossRefGoogle Scholar
  16. Lee HS, Shockley KR, Schut GJ, Conners SB, Montero CI, Johnson MR, Chou CJ, Bridger SL, Wigner N, Brehm SD, Jenney FE Jr, Comfort DA, Kelly RM, Adams MW (2006) Transcriptional and biochemical analysis of starch metabolism in the hyperthermophilic archaeon Pyrococcus furiosus. J Bacteriol 188(6):2115–2125PubMedCentralPubMedCrossRefGoogle Scholar
  17. Lee JH, Karamychev VN, Kozyavkin SA, Mills D, Pavlov AR, Pavlova NV, Polouchine NN, Richardson PM, Shakhova VV, Slesarev AI, Weimer B, O’Sullivan DJ (2008) Comparative genomic analysis of the gut bacterium Bifidobacterium longum reveals loci susceptible to deletion during pure culture growth. BMC Genomics 9:247PubMedCentralPubMedCrossRefGoogle Scholar
  18. Li X, Li D, Park KH (2013) An extremely thermostable amylopullulanase from Staphylothermus marinus displays both pullulan- and cyclodextrin-degrading activities. Appl Microbiol Biotechnol 97(12):5359–5369Google Scholar
  19. Lin HY, Chuang HH, Lin FP (2008) Biochemical characterization of engineered amylopullulanase from Thermoanaerobacter ethanolicus 39E-implicating the non-necessity of its 100 C-terminal amino acid residues. Extremophiles 12(5):641–650PubMedCrossRefGoogle Scholar
  20. Mukund S, Adams MW (1995) Glyceraldehyde-3-phosphate ferredoxin oxidoreductase, a novel tungsten-containing enzyme with a potential glycolytic role in the hyperthermophilic archaeon Pyrococcus furiosus. J Biol Chem 270(15):8389–8392PubMedCrossRefGoogle Scholar
  21. Murakami T, Kanai T, Takata H, Kuriki T, Imanaka T (2006) A novel branching enzyme of the GH-57 family in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. J Bacteriol 188(16):5915–5924PubMedCentralPubMedCrossRefGoogle Scholar
  22. Niehaus F, Bertoldo C, Kahler M, Antranikian G (1999) Extremophiles as a source of novel enzymes for industrial application. Appl Microbiol Biotechnol 51(6):711–729PubMedCrossRefGoogle Scholar
  23. Oslowski DM, Jung JH, Seo DH, Park CS, Holden JF (2011) Production of hydrogen from α-1,4- and β-1,4-linked saccharides by marine hyperthermophilic Archaea. Appl Environ Microbiol 77(10):3169–3173PubMedCentralPubMedCrossRefGoogle Scholar
  24. Palomo M, Pijning T, Booiman T, Dobruchowska JM, van der Vlist J, Kralj S, Planas A, Loos K, Kamerling JP, Dijkstra BW, van der Maarel MJ, Dijkhuizen L, Leemhuis H (2011) Thermus thermophilus glycoside hydrolase family 57 branching enzyme: crystal structure, mechanism of action, and products formed. J Biol Chem 286(5):3520–3530PubMedCrossRefGoogle Scholar
  25. Park KM, Jun SY, Choi KH, Park KH, Park CS, Cha J (2010) Characterization of an exo-acting intracellular α-amylase from the hyperthermophilic bacterium Thermotoga neapolitana. Appl Microbiol Biotechnol 86(2):555–566PubMedCrossRefGoogle Scholar
  26. Santos CR, Tonoli CC, Trindade DM, Betzel C, Takata H, Kuriki T, Kanai T, Imanaka T, Arni RK, Murakami MT (2011) Structural basis for branching-enzyme activity of glycoside hydrolase family 57: structure and stability studies of a novel branching enzyme from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Proteins 79(2):547–557PubMedCrossRefGoogle Scholar
  27. Stetter KO (1999) Extremophiles and their adaptation to hot environments. FEBS Lett 452(1–2):22–25PubMedCrossRefGoogle Scholar
  28. Tachibana Y, Takaha T, Fujiwara S, Takagi M, Imanaka T (2000) Acceptor specificity of 4-α-glucanotransferase from Pyrococcus kodakaraensis KOD1, and synthesis of cycloamylose. J Biosci Bioeng 90(4):406–409PubMedGoogle Scholar
  29. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24(8):1596–1599PubMedCrossRefGoogle Scholar
  30. Tang SY, Yang SJ, Cha H, Woo EJ, Park C, Park KH (2006) Contribution of W229 to the transglycosylation activity of 4-α-glucanotransferase from Pyrococcus furiosus. Biochim Biophys Acta 1764(10):1633–1638PubMedCrossRefGoogle Scholar
  31. Woo EJ, Lee S, Cha H, Park JT, Yoon SM, Song HN, Park KH (2008) Structural insight into the bifunctional mechanism of the glycogen-debranching enzyme TreX from the archaeon Sulfolobus solfataricus. J Biol Chem 283(42):28641–28648PubMedCrossRefGoogle Scholar
  32. Xavier KB, Peist R, Kossmann M, Boos W, Santos H (1999) Maltose metabolism in the hyperthermophilic archaeon Thermococcus litoralis: purification and characterization of key enzymes. J Bacteriol 181(11):3358–3367PubMedCentralPubMedGoogle Scholar
  33. Zona R, Chang-Pi-Hin F, O’Donohue MJ, Janeček Š (2004) Bioinformatics of the glycoside hydrolase family 57 and identification of catalytic residues in amylopullulanase from Thermococcus hydrothermalis. Eur J Biochem 271(14):2863–2872PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Jong-Hyun Jung
    • 1
  • Dong-Ho Seo
    • 1
  • James F. Holden
    • 2
  • Cheon-Seok Park
    • 1
    • 3
  1. 1.Graduate School of Biotechnology and Institute of Life Science and ResourcesKyung Hee UniversityYonginSouth Korea
  2. 2.Department of MicrobiologyUniversity of MassachusettsAmherstUSA
  3. 3.Department of Food Science and Biotechnology and Institute of Life Science and ResourcesKyung Hee UniversityYonginSouth Korea

Personalised recommendations