Purification, characterization and cloning of a thermotolerant isoamylase produced from Bacillus sp. CICIM 304

  • Youran Li
  • Dandan Niu
  • Liang Zhang
  • Zhengxiang Wang
  • Guiyang Shi
Biocatalysis

Abstract

A novel thermostable isoamylase, IAM, was purified to homogeneity from the newly isolated thermophilic bacterium Bacillus sp. CICIM 304. The purified monomeric protein with an estimated molecular mass of 100 kDa displayed its optimal temperature and pH at 70 °C and 6.0, respectively, with excellent thermostability between 30 and 70 °C and pH values from 5.5 to 9.0. Under the conditions of temperature 50 °C and pH 6.0, the K m and V max on glycogen were 0.403 ± 0.018 mg/mg and 0.018 ± 0.001 mg/(min mg), respectively. Gene encoding IAM, BsIam was identified from genomic DNA sequence with inverse PCRs. The open reading frame of the BsIam gene was 2,655 base pairs long and encoded a polypeptide of 885 amino acids with a calculated molecular mass of 101,155 Da. The deduced amino acid sequence of IAM shared less than 40 % homology with that of microbial isoamylase ever reported, which indicated it was a novel isoamylase. This enzyme showed its obvious superiority in the industrial starch conversion process.

Keywords

Bacillus sp. Thermostable isoamylase Purification Enzyme properties Cloning 

Notes

Acknowledgments

This study was granted by China-South Africa Joint project (2009DFA31300), the ‘863’ program (2011AA100905), Program for New Century Excellent Talents in University (NCET-11-0665), Innovative Research Team of Jiangsu Province, the Priority Academic Program Development of Jiangsu Higher Education Institutions and the 111 Project (No. 111-2-06).

Supplementary material

10295_2013_1249_MOESM1_ESM.doc (329 kb)
Supplementary material 1 (DOC 329 kb)

References

  1. 1.
    Abe A, Tonozuka T, Sakano Y, Kamitori S (2004) Complex structures of Thermoactinomyces vulgaris R-47 [alpha]-amylase 1 with malto-oligosaccharides demonstrate the role of domain N acting as a starch-binding domain. J Mol Biol 335(3):811–822PubMedCrossRefGoogle Scholar
  2. 2.
    Abe J-i, Ushijima C, Hizukuri S (1999) Expression of the isoamylase gene of Flavobacterium odoratum KU in Escherichia coli and identification of essential residues of the enzyme by site-directed mutagenesis. Appl Environ Microbiol 65(9):4163–4170PubMedGoogle Scholar
  3. 3.
    Amemura A, Chakraborty R, Fujita M, Noumi T, Futai M (1988) Cloning and nucleotide sequence of the isoamylase gene from Pseudomonas amyloderamosa SB-15. J Biol Chem 263(19):9271–9275PubMedGoogle Scholar
  4. 4.
    Ara K, Saeki K, Ito S (1993) Purification and characterization of an alkaline isoamylase from an alkalophilic strain of Bacillus. J Gen Microbiol 139(4):781–786Google Scholar
  5. 5.
    Benedict SR (1909) A reagent for the detection of reducing sugars. J Biol Chem 5(5):485–487Google Scholar
  6. 6.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72(1–2):248–254PubMedCrossRefGoogle Scholar
  7. 7.
    Chen JH, Chen ZY, Chow TY, Chen JC, Tan ST, Hsu WH (1090) Nucleotide sequence and expression of the isoamylase gene from an isoamylase-hyperproducing mutant, Pseudomonas amyloderamosa JD210. Biochim Biophys Acta 1087(3):309–315Google Scholar
  8. 8.
    Dauvillée D, Kinderf IS, Li Z, Kosar-Hashemi B, Samuel MS, Rampling L, Ball S, Morell MK (2005) Role of the Escherichia coli glgX gene in glycogen metabolism. J Bacteriol 187(4):1465–1473PubMedCrossRefGoogle Scholar
  9. 9.
    Fang TY, Tseng WC, Yu CJ, Shih TY (2005) Characterization of the thermophilic isoamylase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092. J Mol Catal B-Enzym 33(3–6):99–107CrossRefGoogle Scholar
  10. 10.
    Gomes I, Gomes J, Steiner W (2003) Highly thermostable amylase and pullulanase of the extreme thermophilic eubacterium Rhodothermus marinus: production and partial characterization. Bioresour Technol 90(2):207–214PubMedCrossRefGoogle Scholar
  11. 11.
    Gruber AR, Lorenz R, Bernhart SH, Neuböck R, Hofacker IL (2008) The Vienna RNA Websuite. Nucleic Acids Res 36(suppl 2):W70–W74PubMedCrossRefGoogle Scholar
  12. 12.
    Guzmán-Maldonado H, Paredes-López O, Biliaderis CG (1995) Amylolytic enzymes and products derived from starch: a review. Crit Rev Food Sci Nutri 35(5):373–403CrossRefGoogle Scholar
  13. 13.
    Harada T, Yokobayashi K, Misaki A (1968) Formation of isoamylase by Pseudomonas. Appl Microbiol 16(10):1439–1444PubMedGoogle Scholar
  14. 14.
    Henrissat B, Bairoch A (1993) New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 293(Pt 3):781–788PubMedGoogle Scholar
  15. 15.
    Hudson ER, Pan DA, James J, Lucocq JM, Hawley SA, Green KA, Baba O, Terashima T, Hardie DG (2003) A novel domain in AMP-activated protein kinase causes glycogen storage bodies similar to those seen in hereditary cardiac arrhythmias. Curr Biol 13(10):861–866PubMedCrossRefGoogle Scholar
  16. 16.
    Jeanningros R, Creuzet-Sigal N, Frixon C, Cattaneo J (1976) Purification and properties of a debranching enzyme from Escherichia coli. Biochim Biophys Acta 438(1):186–199PubMedCrossRefGoogle Scholar
  17. 17.
    Kato M (1999) Trehalose production with a new enzymatic system from Sulfolobus solfataricus KM1. J Mol Catal B-Enzym 6(3):223–233CrossRefGoogle Scholar
  18. 18.
    Katsuya Y, Mezaki Y, Kubota M, Matsuura Y (1998) Three-dimensional structure of Pseudomonas isoamylase at 2.2 A resolution. J Mol Biol 281(5):885–897PubMedCrossRefGoogle Scholar
  19. 19.
    Marshall JJ (1973) Inhibition of pullulanase by Schardinger dextrins. FEBS Lett 37(2):269–273PubMedCrossRefGoogle Scholar
  20. 20.
    Maruo B, Kobayashi T (1951) Enzymic scission of the branch links in amylopectin. Nature 167(4250):606–607PubMedCrossRefGoogle Scholar
  21. 21.
    Mikami B, Iwamoto H, Malle D, Yoon H-J, Demirkan-Sarikaya E, Mezaki Y, Katsuya Y (2006) Crystal structure of pullulanase: evidence for parallel binding of oligosaccharides in the active site. J Mol Biol 359(3):690–707PubMedCrossRefGoogle Scholar
  22. 22.
    Polekhina G, Gupta A, Michell BJ, van Denderen B, Murthy S, Feil SC, Jennings IG, Campbell DJ, Witters LA, Parker MW, Kemp BE, Stapleton D (2003) AMPK beta subunit targets metabolic stress sensing to glycogen. Curr Biol 13(10):867–871PubMedCrossRefGoogle Scholar
  23. 23.
    Rodríguez-Sanoja R, Oviedo N, Sánchez S (2005) Microbial starch-binding domain. Curr Opin Microbiol 8(3):260–267PubMedCrossRefGoogle Scholar
  24. 24.
    Rohban R, Amoozegar M, Ventosa A (2009) Screening and isolation of halophilic bacteria producing extracellular hydrolyses from Howz Soltan Lake, Iran. J Ind Microbiol Biotechnol 36(3):333–340PubMedCrossRefGoogle Scholar
  25. 25.
    Romeo T, Kumar A, Preiss J (1988) Analysis of the Escherichia coli glycogen gene cluster suggests that catabolic enzymes are encoded among the biosynthetic genes. Gene 70(2):363–376PubMedCrossRefGoogle Scholar
  26. 26.
    Saha BC, Zeikus JG (1989) Novel highly thermostable pullulanase from thermophiles. Trends Biotechnol 7(9):234–239CrossRefGoogle Scholar
  27. 27.
    Shaw JF, Sheu JR (1992) Production of high-maltose syrup and high-protein flour from rice by an enzymatic method. Biosci Biotech Biochem 56(7):1071–1073CrossRefGoogle Scholar
  28. 28.
    Suzuki Y, Hatagaki K, Oda H (1991) A hyperthermostable pullulanase produced by an extreme thermophile, Bacillus flavocaldarius KP 1228, and evidence for the proline theory of increasing protein thermostability. Appl Microbiol Biotechnol 34(6):707–714PubMedCrossRefGoogle Scholar
  29. 29.
    Urlaub H, Wober G (1975) Identification of isoamylase, a glycogen-debranching enzyme, from Bacillus amyloliquefaciens. FEBS Lett 57(1):1–4PubMedCrossRefGoogle Scholar
  30. 30.
    Väisänen O, Elo S, Marmo S, Salkinoja-Salonen M (1989) Enzymatic characterization of Bacilli from food packaging paper and board machines. J Ind Microbiol 4(6):419–428CrossRefGoogle Scholar
  31. 31.
    Vallee BL, Stein EA, Sumerwell WN, Fischer EH (1959) Metal content of α-amylases of various origins. J Biol Chem 234(11):2901–2905PubMedGoogle Scholar
  32. 32.
    Vihinen MMP (1989) Microbial amylolytic enzymes. Crit Rev Biochem Mol Biol 24(4):329–418PubMedCrossRefGoogle Scholar
  33. 33.
    Violet M, Meunier JC (1989) Kinetic study of the irreversible thermal denaturation of Bacillus licheniformis alpha-amylase. Biochem J 263(3):665–670PubMedGoogle Scholar
  34. 34.
    Wiatrowski HA, van Denderen BJW, Berkey CD, Kemp BE, Stapleton D, Carlson M (2004) Mutations in the Gal83 glycogen-binding domain activate the Snf1/Gal83 kinase pathway by a glycogen-independent mechanism. Mol Cell Biol 24(1):352–361PubMedCrossRefGoogle Scholar
  35. 35.
    Yamada K, Terahara T, Kurata S, Yokomaku T, Tsuneda S, Harayama S (2008) Retrieval of entire genes from environmental DNA by inverse PCR with pre-amplification of target genes using primers containing locked nucleic acids. Environ Microbiol 10(4):978–987PubMedCrossRefGoogle Scholar
  36. 36.
    Yang H, Liu MY, Romeo T (1996) Coordinate genetic regulation of glycogen catabolism and biosynthesis in Escherichia coli via the CsrA gene product. J Bacteriol 178(4):1012–1017PubMedGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2013

Authors and Affiliations

  • Youran Li
    • 1
  • Dandan Niu
    • 1
  • Liang Zhang
    • 1
  • Zhengxiang Wang
    • 1
  • Guiyang Shi
    • 1
  1. 1.Research Center of Bioresource & Bioenergy, School of BiotechnologyJiangnan UniversityWuxiPeople’s Republic of China

Personalised recommendations