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Characterization of a pH and detergent-tolerant, cold-adapted type I pullulanase from Exiguobacterium sp. SH3

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Abstract

A pullulanase-encoding gene from psychrotrophic Exiguobacterium sp. SH3 was cloned and expressed in both E. coli and Bacillus subtilis. The functional recombinant enzyme (Pul-SH3) was purified as a His-tagged protein. Pul-SH3 was characterized to be a cold-adapted type I pullulanase with maximum activity at 45 °C. Using fluorescence spectroscopy, the melting temperature of Pul-SH3 was determined to be about 52 °C. The enzyme was able to hydrolyze pullulan, soluble starch, potato starch, and rice flour. It was exceptionally tolerant in the pH range of 4–11, exhibiting maximum activity at pH 8.5 and more than 60 % of the activity in the pH range of 5–11. Being a detergent-tolerant pullulanase, Pul-SH3 retained 99, 89, and 54 % of its activity at 10 % concentration of Triton-X100, Tween 20, and SDS, respectively. The enzyme also exhibited an activity of 80.4 and 93.7 % in the presence of two commercial detergents, Rika (7.5 % v/v) and Fadisheh (2.5 % w/v), respectively. The enzyme was even able to remain active by 54.5 and 85 % after 10-day holding with the commercial detergents. Thermal stability of the enzyme could w on silica. Pul-SH3 with several industrially important characteristics seems desirable for cold hydrolysis of starch.

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References

  • Ashjari M, Mohammadi M, Badri R (2015) Chemical amination of Rhizopus oryzae lipase for multipoint covalent immobilization on epoxy-functionalized supports: modulation of stability and selectivity. J Mol Catal B Enzym 115:128–134

    Article  CAS  Google Scholar 

  • Ayadi DZ, Ali MB, Jemli S, Mabrouk SB, Mezghani M, Messaoud EB, Bejar S (2008) Heterologous expression, secretion and characterization of the Geobacillus thermoleovorans US105 type I pullulanase. Appl Microbiol Biotechnol 78:473–481

    Article  Google Scholar 

  • Bertoldo C, Duffner F, Jorgensen PL, Antranikian G (1999) Pullulanase type I from Fervidobacterium pennavorans Ven5: cloning, sequencing, and expression of the gene and biochemical characterization of the recombinant enzyme. Appl Environ Microbiol 65:2084–2091

    PubMed Central  CAS  PubMed  Google Scholar 

  • Bertoldo C, Armbrecht M, Becker F, Schäfer T, Antranikian G, Liebl W (2004) Cloning, sequencing, and characterization of a heat- and alkali-stable type I pullulanase from Anaerobranca gottschalkii. Appl Environ Microb 70:3407–3416

    Article  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  PubMed  Google Scholar 

  • Chung HJ, Lee EJ, Lim ST (2002) Comparison in glass transition and enthalpy relaxation between native and gelatinized rice starches. Carbohydr Polym 48:287–298

    Article  CAS  Google Scholar 

  • Cinelli BA, Castilho LR, Freire DMG, Castro AM (2015) A brief review on the emerging technology of ethanol production by cold hydrolysis of raw starch. Fuel 150:721–729

    Article  CAS  Google Scholar 

  • Clark JH, Budarin V, Deswarte FEI, Hardy JJE, Kerton FM, Hunt AJ, Luque R, Macquarrie DJ, Milkowski K, Rodriguez A, Samuel O, Tavener SJ, White RJ, Wilson AJ (2006) Green chemistry and the biorefinery: a partnership for a sustainable future. Green Chem 8:853–860

    Article  CAS  Google Scholar 

  • Corre DL, Bras J, Dufresne A (2010) Starch nanoparticles: a review. Biomacromolecules 11:1139–1153

    Article  PubMed  Google Scholar 

  • Elleuche S, Qoura FM, Lorenz U, Rehn T, Brück T, Antranikian G (2015) Cloning, expression and characterization of the recombinant cold-active type-I pullulanase from Shewanella arctica. J Mol Catal B Enzym 116:70–77

    Article  CAS  Google Scholar 

  • Emampour M, Noghabi KA, Zahiri HS (2015) Molecular cloning and biochemical characterization of a novel cold-adapted alpha-amylase with multiple extremozyme characteristics. J Mol Catal B Enzym 111:79–86

    Article  CAS  Google Scholar 

  • Feller G (2003) Molecular adaptations to cold in psychrophilic enzymes. Cell Mol Life Sci 60:648–662

    Article  CAS  PubMed  Google Scholar 

  • Garstka MA, Fish A, Celie PHN, Joosten RP, Janssen GMC, Berlin I, Hoppes R, Stadnik M, Janssen L, Ovaa H, Veelen PAV, Perrakis A, Neefjes J (2015) The first step of peptide selection in antigen presentation by MHC class I molecules. PNAS 112:1505–1510

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Hii SL, Tan JS, Ling TC, Ariff AB (2012) Pullulanase: role in starch hydrolysis and potential industrial applications. Enzyme Res 2012:1–14

    Article  Google Scholar 

  • Hong Y, Liu G, Gu Z (2015) Preparation and characterization of hydrophilic debranched starch modified by pullulanase on swollen granule starch. Food Res Int 67:212–218

    Article  CAS  Google Scholar 

  • Kang J, Park KM, Choi KH, Park CS, Kim GE, Kim D, Cha J (2011) Molecular cloning and biochemical characterization of a heat-stable type I pullulanase from Thermotoga neapolitana. Enzyme Microb Technol 48:260–266

    Article  CAS  PubMed  Google Scholar 

  • Kar S, Ray RC, Mohapatra UB (2012) Purification, characterization and application of thermostable amylopullulanase from Streptomyces erumpens MTCC 7317 under submerged fermentation. Ann Microbiol 62:931–937

    Article  CAS  Google Scholar 

  • Kennedy JF, Cabalda VM, White CA (1988) Enzymic starch utilization and genetic engineering. Trends Biotechnol 6:184–189

    Article  CAS  Google Scholar 

  • Kim CH, Choi HI, Lee DS (1993) Pullulanases of alkaline and broad pH range from a newly isolated alkalophilic Bacillus sp. S-1 and a Micrococcus sp. Y-1. J Ind Microbiol 12:48–57

    Article  CAS  Google Scholar 

  • Li Y, Zhang L, Niu D, Wang Z, Shi G (2012) Cloning, expression, characterization, and biocatalytic investigation of a novel bacilli thermostable type I pullulanase from Bacillus sp. CICIM 263. J Agric Food Chem 60:11164–11172

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Mojallali L, Zahiri HS, Rajaei S, Noghabi KA, Haghbeen K (2014) A novel ~34-kDa α-amylase from psychrotroph Exiguobacterium sp. SH3: Production, purification, and characterization. Biotechnol Appl Biochem 61:118–125

    Article  CAS  PubMed  Google Scholar 

  • Nguyen QD, Rezessy-Szabó JM, Claeyssens M, Stals I, Hoschke Á (2002) Purification and characterisation of amylolytic enzymes from thermophilic fungus Thermomyces lanuginosus strain ATCC 34626. Enzyme Microb Technol 31:345–352

    Article  CAS  Google Scholar 

  • Plant AR, Clemens RM, Daniel RM, Morgan HW (1987) Purification and properties of a thermoactive and thermostable pullulanase from Thermococcus hydrothermalis, a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent. Appl Microbiol Biotechnol 26:427–433

    Article  CAS  Google Scholar 

  • Qiao Y, Peng Q, Yan J, Wang H, Ding H, Shi B (2015) Gene cloning and enzymatic characterization of alkalitolerant type I pullulanase from Exiguobacterium acetylicum. Lett Appl Microbiol 60:52–59

    Article  CAS  PubMed  Google Scholar 

  • Qoura F, Elleuche S, Brueck T, Antranikian G (2014) Purification and characterization of a cold-adapted pullulanase from a psychrophilic bacterial isolate. Extremophiles 18:1095–1102

    Article  CAS  PubMed  Google Scholar 

  • Rajaei S, Heidari R, Zahiri HS, Sharifzadeh S, Torktaz I, Noghabi KA (2014) A novel cold-adapted pullulanase from Exiguobacterium sp. SH3: Production optimization, purification, and characterization. Starch 66:225–234

    Article  CAS  Google Scholar 

  • Roy I, Gupta MN (2004) Hydrolysis of starch by a mixture of glucoamylase and pullulanase entrapped individually in calcium alginate beads. Enzyme Microb Tech 34:26–32

    Article  CAS  Google Scholar 

  • Saha BC, Mathupala SP, Zeikus JG (1988) Purification and characterization of a highly thermostable novel pullulanase from Clostridium thermohydrosulfuricum. Biochem J 252:343–348

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Sapińska E, Balcerek M, Pielech-Przybylska K, Stanisz M (2014) The impact of treatment of cereal mashes with hydrolases of non-starch polysaccharides and pullulanase on the chemical composition of the obtained distillates. J Inst Brew 120:105–110

    Article  Google Scholar 

  • Wu H, Yu X, Chen L, Wu G (2014) Cloning, overexpression and characterization of a thermostable pullulanase from Thermus thermophilus HB27. Protein Expres Purif 95:22–27

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Iran National Science Foundation (Grant No. 91004311).

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Authors and Affiliations

  1. Department of Energy and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Pajoohesh Blvd., Tehran-Karaj Highway, Tehran, P.O. Box 14155-6343, Iran

    Sarah Rajaei, Kambiz Akbari Noghabi & Hossein Shahbani Zahiri

  2. Department of Genetics, Faculty of Biological Science, Tarbiat Modares University, Tehran, P.O. Box 14115-154, Iran

    Sarah Rajaei & Majid Sadeghizadeh

Authors
  1. Sarah Rajaei
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  2. Kambiz Akbari Noghabi
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  3. Majid Sadeghizadeh
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  4. Hossein Shahbani Zahiri
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Corresponding author

Correspondence to Hossein Shahbani Zahiri.

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Communicated by L. Huang.

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Rajaei, S., Noghabi, K.A., Sadeghizadeh, M. et al. Characterization of a pH and detergent-tolerant, cold-adapted type I pullulanase from Exiguobacterium sp. SH3. Extremophiles 19, 1145–1155 (2015). https://doi.org/10.1007/s00792-015-0786-6

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  • DOI: https://doi.org/10.1007/s00792-015-0786-6

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