Applied Microbiology and Biotechnology

, Volume 87, Issue 1, pp 225–233

Cloning, expression, and characterization of a thermostable glucoamylase from Thermoanaerobacter tengcongensis MB4

Biotechnologically Relevant Enzymes and Proteins

Abstract

A thermostable glucoamylase (TtcGA) from Thermoanaerobacter tengcongensis MB4 was successfully expressed in Escherichia coli. The full-length gene (2112 bp) encodes a 703-amino acid polypeptide including a predicted signal peptide of 21 residues. The recombinant mature protein was partially purified to 30-fold homogeneity by heat treatment and gel filtration chromatography. The mature protein is a monomer with the molecular weight of 77 kD. The recombinant enzyme showed maximum activity at 75 °C and pH 5.0. It is the most thermostable bacterial glucoamylase described to date with nearly no activity loss after incubation at 75 °C for 6 h. TtcGA can hydrolyze both α-1, 4- and α-1, 6-glycosidic linkages in various α-glucans. It showed preference for maltooligosaccharides over polysaccharides with specific activity of 80 U/mg towards maltose. Kinetic studies revealed that TtcGA had the highest activity on maltooligosaccharide with four monosaccharide units. The cations Ca2+, Mn2+, Co2+, Mg2+, and reducing agent DTT showed no obvious effects on the action of TtcGA. In contrast, the enzyme was inactivated by Zn2+, Pb2+, Cu2+, and EDTA.

Keywords

Glucoamylase Thermoanaerobacter tengcongensis Recombinant expression Thermostable 

Reference

  1. Aleshin AE, Golubev A, Firsov LM, Honzatko RB (1992) Crystal structure of glucoamylase from Aspergillus awamori var. X100 to 2.2-A resolution. J Biol Chem 267(27):19291–19298Google Scholar
  2. Aleshin AE, Feng PH, Honzatko RB, Reilly PJ (2003) Crystal structure and evolution of a prokaryotic glucoamylase. J Mol Biol 327:61–73CrossRefGoogle Scholar
  3. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410Google Scholar
  4. Aquino ACMM, Jorge JA, Terenzi HF, Polizeli MLT (2001) Thermostable glucose-tolerant glucoamylase produced by the thermophilic fungus Scytalidium thermophilum. Folia Microbiol 46(1):11–16CrossRefGoogle Scholar
  5. Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISSMODEL workspace: a web-based environment for protein structure homology modeling. Bioinformatics 22:195–201CrossRefGoogle Scholar
  6. Bao Q, Tian Y, Li W, Xu Z, Xuan Z, Hu S, Dong W, Yang J, Chen Y, Xue Y, Xu Y, Lai X, Huang L, Dong X, Ma Y, Ling L, Tan H, Chen R, Wang J, Yu J, Yang H (2002) A complete sequence of the T. tengcongensis genome. Genome Res 12:689–700CrossRefGoogle Scholar
  7. Bender H (1981) A bacterial glucoamylase degrading cyclodextrins. Partial purification and properties of the enzyme from a Flavobacteikn species. Eur J Biochem 115:287–291CrossRefGoogle Scholar
  8. Bendtsen JD, Nielsen H, Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795CrossRefGoogle Scholar
  9. Coutinho PM, Reilly PJ (1994) Structure-function relationships in the catalytic and starch binding domains of glucoamylase. Protein Eng 7(3):393–400CrossRefGoogle Scholar
  10. Dock C, Hess M, Antranikian G (2008) A thermoactive glucoamylase with biotechnological relevance from the thermoacidophilic euryarchaeon Thermoplasma acidophilum. Appl Microbiol Biotechnol 78:105–114CrossRefGoogle Scholar
  11. Ducki A, Grundmann O, Konermann L, Mayer F, Hoppert M (1998) Glucoamylase from Thermoanaerobacterium thermosaccharolyticum: sequence studies and analysis of the macromolecular architecture of the enzyme. J Gen Appl Microbiol 44:327–335CrossRefGoogle Scholar
  12. Fagerstrom T, Kalkkinen N (1995) Characterization, subsite mapping and partial amino acid sequence of glucoamylase from the filamentous fungus Trichoderma reesei. Biotechnol Appl Bioc 21:223–231Google Scholar
  13. Feng P, Berensmeier S, Buchholz K, Reilly PJ (2002) Production, purification, and characterization of Thermoanaerobacterium thermosaccharolyticum glucoamylase. Starch 54:328–337CrossRefGoogle Scholar
  14. Fogarty WM, Benson CP (1983) Purification and properties of a thermophilic amyloglucosidase from Aspergillus niger. Eur J Appl Microbiol Biotechnol 19:271–278CrossRefGoogle Scholar
  15. Hyun HH, Zeikus JG (1985) General biochemical characterization of thermostable pullulanase and glucoamylase from Clostridium thermohydrosulfuricum. Appl Environ Microbiol 49(5):1168–1173Google Scholar
  16. James JA, Berger JL, Lee BH (1997) Purification of glucoamylase from Lactobacillus amylovorus ATCC 33621. Curr Microbiol 34:186–191CrossRefGoogle Scholar
  17. Kim MS, Park JT, Kim YW, Lee HS, Nyawira R, Shin HS, Park CS, Yoo SH, Kim YR, Moon TW, Park KH (2004) Properties of a novel thermostable glucoamylase from the hyperthermophilic archaeon Sulfolobus solfataricus in relation to starch processing. Appl Environ Microbiol 70(7):3933–3940CrossRefGoogle Scholar
  18. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J App Cryst 26:283–291CrossRefGoogle Scholar
  19. Li Y, Coutinho PM, Ford C (1998) Effect on thermostability and catalytic activity of introducing disulfide bonds into Aspergillus awamori glucoamylase. Protein Eng 11:661–667CrossRefGoogle Scholar
  20. Liu H, Wang W (2003) Protein engineering to improve the thermostability of glucoamylase from Aspergillus awamori based on molecular dynamics simulations. Protein Eng 16:19–25CrossRefGoogle Scholar
  21. Liu H, Doleyres Y, Coutinho PM, Ford C, Reilly PJ (2000) Replacement and deletion mutations in the catalytic domain and belt region of Aspergillus awamori glucoamylase to enhance thermostability. Protein Eng 13:655–659CrossRefGoogle Scholar
  22. Morris AL, MacArthur MW, Hutchinson EG, Thornton JM (1992) Stereochemical quality of protein structure coordinates. Proteins 12:345–364CrossRefGoogle Scholar
  23. Nielsen H, Engelbrecht J, Brunak J, Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6CrossRefGoogle Scholar
  24. Ohnishi H, Sakai H, Ohta T (1991) Purification and some properties of a glucoamylase from Clostridium sp. G0005. Agric Biol Chem 55(7):1901–1902Google Scholar
  25. Ohnishi H, Kitamura H, Minowa T, Sakai H, Ohta T (1992) Molecular cloning of a glucoamylase gene from a thermophilic Clostridium and kinetics of the cloned enzyme. Eur J Biochem 207:413–418CrossRefGoogle Scholar
  26. Oren A (1983) A thermophilic amyloglucosidase from Halobacterium sodomense, a halophilic bacterium from the Dead Sea. Curr Microbiol 8:225–230CrossRefGoogle Scholar
  27. Serour E, Antranikian G (2002) Novel thermoactive glucoamylases from the thermoacidophilic Archaea Thermoplasma acidophilum, Picrophilus torridus and Picrophilus oshimae. Anton Leeuw Int J G 81:73–83CrossRefGoogle Scholar
  28. Specka U, Mayer F, Antranikian G (1991) Purification and properties of a thermoactive glucoamylase from Clostridium thermosaccharolyticum. Appl Environ Microbiol 57(8):2317–2323Google Scholar
  29. Spinelli LBB, Polizeli ML, Terenzi HF, Jorge JA (1996) Biochemical characterization of glucoamylase from the hyperproducer exo-1 mutant strain of Neurospora crassa. FEMS Microbiol Lett 138:173–177CrossRefGoogle Scholar
  30. Srivastava RAK (1984) Studies on extracellular and intracellular purified amylases from a thermophilic Bacillus stearothermophilus. Enzyme Microb Tech 6(9):422–426CrossRefGoogle Scholar
  31. Studier FW (2005) Protein production by auto-induction in high-density shaking cultures. Protein Expres Purif 41:207–234CrossRefGoogle Scholar
  32. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882CrossRefGoogle Scholar
  33. Uotsu-Tomita R, Tonozuka T, Sakai H, Sakano Y (2001) Novel glucoamylase-type enzymes from Thermoactinomyces vulgaris and Methanococcus jannaschii whose genes are found in the flanking region of the α-amylase genes. Appl Microbiol Biotechnol 56:465–473CrossRefGoogle Scholar
  34. Xue Y, Xu Y, Liu Y, Ma Y, Zhou P (2001) Thermoanaerobacter tengcongensis sp. nov., a novel anaerobic, saccharolytic, thermophilic bacterium isolated from a hot spring in Tengcong, China. Int J Syst Evol Micr 51:1335–1341Google Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  1. 1.State Key Laboratory of Microbial Resources, Institute of MicrobiologyChinese Academy of SciencesBeijingChina
  2. 2.The Graduate SchoolChinese Academy of SciencesBeijingChina
  3. 3.Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesTianjinChina
  4. 4.Lehrstuhl für BiotechnologieRWTH Aachen UniversityAachenGermany

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