Structural and functional insights of starch processing α-amylase from hyperthermophilic archaeon Pyrococcusabyssi

Carbohydr Res. 2024 May:539:109122. doi: 10.1016/j.carres.2024.109122. Epub 2024 Apr 22.

Abstract

The genomic screening of hyper-thermophilic Pyrococcus abyssi showed uncharacterized novel α-amylase sequences. Homology modelling analysis revealed that the α-amylase from P. abyssi consists of an N-terminal GH57 catalytic domain, α-amylase central, and C-terminal domain. Current studies emphasize in-silico structural and functional analysis, recombinant expression, characterization, structural studies through CD spectroscopy, and ligand binding studies of the novel α-amylase from P. abyssi. The soluble expression of PaAFG was observed in the E. coli Rosetta™ (DE3) pLysS strain upon incubation overnight at 18 °C in an orbital shaker. The optimum temperature and pH of the PaAFG were observed at 90 °C in 50 mM phosphate buffer pH 6. The Km value for PaAFG against wheat starch was determined as 0.20 ± 0.053 mg while the corresponding Vmax value was 25.00 ± 0.67 μmol min-1 mg-1 in the presence of 2 mM CaCl2 and 12.5 % glycerol. The temperature ramping experiments through CD spectroscopy reveal no significant change in the secondary structures and positive and negative ellipticities of the CD spectra showing the proper folding and optimal temperature of PaAFG protein. The RMSD and RMSF of the PaAFG enzyme determined through molecular dynamic simulation show the significant protein's stability and mobility. The soluble production, thermostability and broad substrate specificity make this enzyme a promising choice for various industrial applications.

Keywords: CD spectroscopy; Hyperthermophile; MD simulation; Molecular docking; Pyrococcus abyssi; α-Amylase.

MeSH terms

  • Enzyme Stability
  • Hydrogen-Ion Concentration
  • Models, Molecular
  • Pyrococcus abyssi* / enzymology
  • Starch* / chemistry
  • Starch* / metabolism
  • Temperature
  • alpha-Amylases* / chemistry
  • alpha-Amylases* / genetics
  • alpha-Amylases* / metabolism