Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465.

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  • Additional Information
    • Source:
      Publisher: International Institute of Ecology Country of Publication: Brazil NLM ID: 101129542 Publication Model: eCollection Cited Medium: Internet ISSN: 1678-4375 (Electronic) Linking ISSN: 15196984 NLM ISO Abbreviation: Braz J Biol Subsets: MEDLINE
    • Publication Information:
      Original Publication: São Carlos, SP [Brazil] : International Institute of Ecology, [2000]-
    • Subject Terms:
      Escherichia coli*/genetics ; alpha-Amylases*/genetics ; alpha-Amylases*/metabolism; Cloning, Molecular ; Enzyme Stability ; Geobacillus ; Hydrogen-Ion Concentration ; Temperature
    • Abstract:
      Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM-465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% β-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.
    • Accession Number:
      EC 3.2.1.1 (alpha-Amylases)
    • Subject Terms:
      Geobacillus thermodenitrificans
    • Publication Date:
      Date Created: 20210609 Date Completed: 20210610 Latest Revision: 20210610
    • Publication Date:
      20221213
    • Accession Number:
      10.1590/1519-6984.239449
    • Accession Number:
      34105678