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Characterization of an exo-acting intracellular α-amylase from the hyperthermophilic bacterium Thermotoga neapolitana

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Abstract

We cloned and expressed the gene for an intracellular α-amylase, designated AmyB, from the hyperthermophilic bacterium Thermotoga neapolitana in Escherichia coli. The putative intracellular amylolytic enzyme contained four regions that are highly conserved among glycoside hydrolase family (GH) 13 α-amylases. AmyB exhibited maximum activity at pH 6.5 and 75°C, and its thermostability was slightly enhanced by Ca2+. However, Ca2+ was not required for the activity of AmyB as EDTA had no effect on enzyme activity. AmyB hydrolyzed the typical substrates for α-amylase, including soluble starch, amylose, amylopectin, and glycogen, to liberate maltose and minor amount of glucose. The hydrolytic pattern of AmyB is most similar to those of maltogenic amylases (EC 3.2.1.133) among GH 13 α-amylases; however, it can be distinguished by its inability to hydrolyze pullulan and β-cyclodextrin. AmyB enzymatic activity was negligible when acarbose, a maltotetraose analog in which a maltose residue at the nonreducing end was replaced by acarviosine, was present, indicating that AmyB cleaves maltose units from the nonreducing end of maltooligosaccharides. These results indicate that AmyB is a new type exo-acting intracellular α-amylase possessing distinct characteristics that distinguish it from typical α-amylase and cyclodextrin-/pullulan-hydrolyzing enzymes.

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References

  • Ballschmiter M, Fütterer O, Liebl W (2006) Identification and characterization of a novel intracellular alkaline α-amylase from the hyperthermophilic bacterium Thermotoga maritima MSB8. Appl Environ Microbiol 72:2206–2211

    Article  CAS  Google Scholar 

  • Bibel M, Brettl C, Gosslar U, Kriegshauser G, Liebl W (1998) Isolation and analysis of genes for amylolytic enzymes of the hyperthermophilic bacterium Thermotoga maritima. FEMS Microbiol Lett 158:9–15

    Article  CAS  Google Scholar 

  • Boel E, Brady L, Brzozowski AM, Derewenda Z, Dodson GG, Jensen VJ, Petersen SB, Swift H, Thim L, Woldike HF (1990) Calcium binding in α-amylases: an X-ray diffraction study at 2.1-Å resolution of two enzymes from Aspergillus. Biochemistry 29:6244–6249

    Article  CAS  Google Scholar 

  • Bok J-D, Yernool DA, Eveleigh DE (1998) Purification, characterization, and molecular analysis of thermostable cellulases CelA and CelB from Thermotoga neapolitana. Appl Environ Microbiol 64:4774–4781

    CAS  Google Scholar 

  • Bradford M (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  • Bronnenmeier KA, Kern A, Liebl W, Staudenbauer WL (1995) Purification of Thermotoga maritima enzymes for the degradation of cellulosic materials. Appl Environ Microbiol 61:1399–1407

    CAS  Google Scholar 

  • Cha HJ, Yoon HG, Kim YW, Lee HS, Kim JW, Kweon KS, Oh BH, Park KH (1998) Molecular and enzymatic characterization of a maltogenic amylase that hydrolyzes and transglycosylates acarbose. Eur J Biochem 253:251–262

    Article  CAS  Google Scholar 

  • Chhabra S, Parker KN, Lam D, Callen W, Snead MA, Mathur EJ, Short JM, Kelly RM (2001) β-mannanases from Thermotoga species. Methods Enzymol 330:224–238

    Article  CAS  Google Scholar 

  • Chhabra SR, Shockley KR, Ward DE, Kelly RM (2002) Regulation of endo-acting glycosyl hydrolases in the hyperthermophilic bacterium Thermotoga maritima grown on glucan- and mannan-based polysaccharides. Appl Environ Microbiol 68:545–554

    Article  CAS  Google Scholar 

  • Chhabra SR, Shockley KR, Conners SB, Scott KL, Wolfinger RD, Kelly RM (2003) Carbohydrate-induced differential gene expression patterns in the hyperthermophilic bacterium Thermotoga maritima. J Biol Chem 278:7540–7552

    Article  CAS  Google Scholar 

  • Conners SB, Montero CI, Comfort DA, Shockley KR, Johnson MR, Chhabra SP, Kelly RM (2005) An expression-driven approach to the prediction of carbohydrate transport and utilization regulons in the hyperthermophilic bacterium Thermotoga maritima. J Bacteriol 187:7267–7282

    Article  CAS  Google Scholar 

  • Conners SB, Mongodin EF, Johnson MR, Montero CI, Nelson KE, Kelly RM (2006) Microbial biochemistry, physiology, and biotechnology of hyperthermophilic Thermotoga species. FEMS Microbiol Rev 30:872–905

    Article  CAS  Google Scholar 

  • Dipple R, Boos W (2005) The maltodextrin system of Escherichia coli: metabolism and transport. J Bacteriol 187:8322–8331

    Article  Google Scholar 

  • Duffaud GD, McCutchen CM, Leduc P, Parker KN, Kelly RM (1997) Purification and characterization of extremely thermostable β-mannanase, β-mannosidase, and α-galactosidase from the hyperthermophilic eubacterium Thermotoga neapolitana 5068. Appl Environ Microbiol 63:169–177

    CAS  Google Scholar 

  • Fox JD, Robyt JF (1991) Miniaturization of three carbohydrate analyses using a microsample plate reader. Anal Biochem 195:93–96

    Article  CAS  Google Scholar 

  • Gabelsberger J, Liebl W, Schleifer KH (1993) Cloning and characterization of β-galactoside and β-glucoside hydrolyzing enzymes of Thermotoga maritima. FEMS Microbiol Lett 109:131–137

    CAS  Google Scholar 

  • Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280:309–316

    CAS  Google Scholar 

  • Janecek S (1994) Parallel β/α-barrels of alpha-amylase, cyclodextrin glycosyltransferase and oligo-1, 6-glucosidase versus the barrel of β-amylase: evolutionary distance is a reflection of unrelated sequences. FEBS Lett 353:119–123

    Article  CAS  Google Scholar 

  • Janecek S (1995) Close evolutionary relatedness among functionally distantly related members of the (α/β)8-barrel glycosyl hydrolases suggested by the similarity of their fifth conserved sequence region. FEBS Lett 377:6–8

    Article  CAS  Google Scholar 

  • Janecek S (2002) How many conserved sequence regions are there in the α-amylase family? Biologia 57(Suppl 11):29–41

    CAS  Google Scholar 

  • Kim TJ, Kim MJ, Kim BC, Kim JC, Cheong TK, Kim JW, Park KH (1999) 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 Microbiol 65:1644–1651

    CAS  Google Scholar 

  • Kim TJ, Nguyen VD, Lee HS, Kim MJ, Cho HY, Kim YW, Moon TW, Park CS, Kim JW, Oh BH, Lee SB, Svensson B, Park KH (2001) Modulation of the multisubstrate specificity of Thermus maltogenic amylase by truncation of the N-terminal domain and by a salt-induced shift of the monomer/dimmer equilibrium. Biochemistry 40:14182–14190

    Article  CAS  Google Scholar 

  • Lee HS, Kim MS, Cho HS, Kim JI, Kim TJ, Choi JH, Park C, Lee HS, Oh BH, Park KH (2002a) Cyclomaltodextrinase, neopullulanase, and maltogenic amylase are nearly indistinguishable from each other. J Biol Chem 277:21891–21897

    Article  CAS  Google Scholar 

  • Lee MH, Kim YW, Kim TJ, Park CS, Lim JW, Moon TW, Park KH (2002b) A novel amylolytic enzyme from Thermotoga maritima, resembling cyclodextrinase and α-glucosidase, that liberates glucose from the reducing end of the substrates. Biochem Biophys Res Commun 295:818–825

    Article  CAS  Google Scholar 

  • Liebl W (2001) Cellulolytic enzymes from Thermotoga species. Methods Enzymol 330:290–300

    Article  CAS  Google Scholar 

  • Liebl W, Stemplinger I, Ruile P (1997) Properties and gene structure of the Thermotoga maritima α-amylase AmyA, a putative lipoprotein of a hyperthermophilic bacterium. J Bacteriol 179:941–948

    CAS  Google Scholar 

  • Lim WJ, Park SR, An CL, Lee JY, Hong SY, Shin EC, Kim EJ, Kim JO, Kim H, Yun HD (2003) Cloning and characterization of a thermostable intracellular α-amylase gene from the hyperthermophilic bacterium Thermotoga maritima MSB8. Res Microbiol 154:681–687

    Article  CAS  Google Scholar 

  • McCutchen CM, Duffaud GD, Leduc P, Petersen ARH, Tayal A, Khan SA, Kelly RM (1996) Characterization of extremely thermostable enzymatic breakers (α-1,6-galactosidase and β-1, 4-mannanase) from the hyperthermophilic bacterium Thermotoga neapolitana 5068 for hydrolysis of guar gum. Biotechnol Bioeng 52:332–339

    Article  CAS  Google Scholar 

  • Miller GL (1959) Use of dinitrosalicylic acid reagent for determination reducing sugar. Anal Chem 31:426–428

    Article  CAS  Google Scholar 

  • Miller ES, Parker KN, Liebl W, Lam D, Callen W, Snead MA, Mathur EJ, Short JM, Kelly RM (2001) α-galactosidases from Thermotoga species. Methods Enzymol 330:246–260

    Article  CAS  Google Scholar 

  • Miwa I, Okudo J, Maeda K, Okuda G (1972) Mutarotase effect on colorimetric determination of blood glucose with d-glucose oxidase. Clin Chim Acta 37:538–540

    Article  CAS  Google Scholar 

  • Nelson KE, Clayton RA, Gill SR, Gwinn ML, Dodson RJ, Haft DH, Hickey EK, Peterson JD, Nelson WC, Ketchum KA, McDonald L, Utterback TR, Malek JA, Linher KD, Garrett MM, Stewart AM, Cotton MD, Pratt MS, Phillips CA, Richardson D, Heidelberg J, Sutton GG, Fleischmann RD, Eisen JA, White O, Salzberg SL, Smith HO, Venter JC, Fraser CM (1999) Evidence for lateral gene transfer between archaea and bacteria from genome sequence of Thermotoga maritima. Nature 399:323–329

    Article  CAS  Google Scholar 

  • Nguyen TN, Borges KM, Romano AH, Noll KM (2001) Differential gene expression in Thermotoga neapolitana in response to growth substrate. FEMS Microbiol Lett 195:79–83

    Article  CAS  Google Scholar 

  • Nielsen JE, Borchert TV (2000) Protein engineering of bacterial α-amylases. Biochim Biophys Acta 1543:253–274

    CAS  Google Scholar 

  • Oslancová A, Janecek S (2002) Oligo-1,6-glucosidase and neopullulanase enzyme subfamilies from the α-amylase family defined by the fifth conserved sequence region. Cell Mol Life Sci 59:1945–1959

    Article  Google Scholar 

  • Park KH, Kim TJ, Cheong TK, Kim JW, Oh BH, Svensson B (2000) Structure, specificity and function of cyclomaltodextrinase, a multispecific enzyme of the α-amylase family. Biochim Biophys Acta 1478:165–185

    CAS  Google Scholar 

  • Park SH, Cha H, Kang HK, Shim JH, Woo EJ, Kim JW, Park KH (2005a) Mutagenesis of Ala290, which modulates substrate subsite affinity at the catalytic interface of dimeric ThMA. Biochim Biophys Acta 1751:170–177

    CAS  Google Scholar 

  • Park TH, Choi KW, Park CS, Lee SB, Kang HY, Shon KJ, Park JS, Cha J (2005b) Substrate specificity and transglycosylation catalyzed by a thermostable β-glucosidase from marine hyperthermophile Thermotoga neapolitana. Appl Microbiol Biotechnol 69:411–422

    Article  CAS  Google Scholar 

  • Parker KN, Chhabra SR, Lam D, Callen W, Duffaud GD, Snead MA, Short JM, Mathur EJ, Kelly RM (2001a) Galactomannanases Man2 and Man5 from Thermotoga species: growth physiology on galactomannans, gene sequence analysis and biochemical properties of recombinant enzymes. Biotechnol Bioeng 75:322–333

    Article  CAS  Google Scholar 

  • Parker KN, Chhabra SR, Lam D, Snead MA, Mathur EJ, Kelly RM (2001b) β-Mannosidase from Thermotoga species. Methods Enzymol 330:238–246

    Article  CAS  Google Scholar 

  • Peist R, Schneider-Fresenius C, Boos W (1996) The MalT-dependent and malZ-encoded maltodextrin glucosidase of Escherichia coli can be converted into a dextrinyltransferase by a single mutation. J Biol Chem 271:10681–10689

    Article  CAS  Google Scholar 

  • Robyt JF, Mukerjea R (1994) Separation and quantitative determination of nanogram quantities of maltodextrins and isomaltodextrins by thin-layer chromatography. Carbohydr Res 251:187–202

    Article  CAS  Google Scholar 

  • Ruile P, Winterhalter C, Liebl W (1997) Isolation and analysis of a gene encoding α-glucuronidase, an enzyme with a novel primary structure involved in the breakdown of xylan. Mol Microbiol 23:267–279

    Article  CAS  Google Scholar 

  • Schumann J, Wrba A, Jaenicke R, Stetter KO (1991) Topographical and enzymatic characterization of amylases from the extremely thermophilic eubacterium Thermotoga maritima. FEBS Lett 282:122–126

    Article  CAS  Google Scholar 

  • Selig M, Xavier KB, Santos H, Schönheit P (1997) Comparative analysis of Embden-Meyerhof and Entner-Doudoroff glycolytic pathways in hyperthermophilic archaea and the bacterium Thermotoga. Arch Microbiol 167:217–232

    CAS  Google Scholar 

  • Stam MR, Danchin EG, Rancurel C, Coutinho PM, Henrissat B (2006) Dividing the large glycoside hydrolase family 13 into subfamilies: towards improved functional annotations of α-amylase-related proteins. Protein Eng Des Sel 19:555–562

    Article  CAS  Google Scholar 

  • Tang SY, Le QT, Shim JH, Yang SJ, Auh JH, Park C, Park KH (2006) Enhancing thermostability of maltogenic amylase from Bacillus thermoalkalophilus ET2 by DNA shuffling. FEBS J 273:3335–3345

    Article  CAS  Google Scholar 

  • Thompson JD, Higgins FG, Gibson TJ (1994) CLSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4763–4680

    Article  Google Scholar 

  • Veith B, Zvwelov VV, Lunina NA, Berezina OV, Raasch C, Velikodvorskaya GA, Liebl W (2003) Comparative analysis of the recombinant α-d-glucosidases from the Thermotoga neapolitana and Thermotoga maritima maltodextrin utilization gene clusters. Biocatal Biotransform 21:147–158

    Article  CAS  Google Scholar 

  • Wassenberg D, Schurig H, Liebl W, Jaenicke R (1997) Xylanase XynA from the hyperthermophilic bacterium Thermotoga maritima: structure and stability of the recombinant enzyme and its isolated cellulose-binding domain. Protein Sci 6:1718–1726

    Article  CAS  Google Scholar 

  • Yang SJ, Lee HS, Park CS, Kim YR, Moon TW, Park KH (2004) Enzymatic analysis of an amylolytic enzyme from the hyperthermophilic archaeon Pyrococcus furiosus reveals its novel catalytic properties as both an α-amylase and a cyclodextrin-hydrolyzing enzyme. Appl Environ Microbiol 70:5988–5995

    Article  CAS  Google Scholar 

  • Yernool DA, McCarthy JK, Eveleigh DE, Bok JD (2000) Cloning and characterization of the glucooligosaccharide catabolic pathway β-glucan glucohydrolase and cellobiose phosphorylase in the marine hyperthermophile Thermotoga neapolitana. J Bacteriol 182:5172–5179

    Article  CAS  Google Scholar 

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Acknowledgments

This study was supported, in part, by the Marine and Extreme Genome Research Center Program of the Ministry of Land, Transportation and Maritime Affairs and by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD; KRF-2007-521-F00056), Republic of Korea.

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Authors and Affiliations

  1. Department of Microbiology, College of Natural Sciences, Pusan National University, San 30, Jangjeon-dong, Geumjeong-gu, Busan, 609-735, Korea

    Kyung-Min Park, So-Young Jun, Kyoung-Hwa Choi & Jaeho Cha

  2. Department of Biology, University of Incheon, Incheon, 402-749, Korea

    Kwan-Hwa Park

  3. Graduate School of Biotechnology, and Institute of Life Science and Resources, Kyung Hee University, Yongin, 446-701, Korea

    Cheon-Seok Park

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  1. Kyung-Min Park
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  2. So-Young Jun
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Correspondence to Jaeho Cha.

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Kyung-Min Park and So-Young Jun contributed equally to the work.

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Park, KM., Jun, SY., Choi, KH. et al. Characterization of an exo-acting intracellular α-amylase from the hyperthermophilic bacterium Thermotoga neapolitana . Appl Microbiol Biotechnol 86, 555–566 (2010). https://doi.org/10.1007/s00253-009-2284-1

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