Identification and characterization of novel thermostable ?-amylase from Geobacillus sp. GS33
Özet
In this study, the heterologous expression and biochemical characterization of a thermostable ?-amylase from Geobacillus sp. GS33 was investigated. The recombinant ?-amylase was overexpressed in Escherichia coli BL21 (?DE) and purified via anion exchange and size-exclusion chromatography. The purified ?-amylase had a molecular weight of about 60 kDa, and was active in a broad range of pH 3–10 and temperature (40–90 °C) with maximum activity at pH 7–8 and 60 °C. The enzyme retained 50% residual activity at 65 °C, but only 20% at 85 °C after 16 h. At pH 9 and pH 7, the residual activity at 65 °C was 50% and 30%, respectively. The enzyme was remarkably activated by Co2+, Ca2+, Mg2+, PMSF, DTT, and Triton X-100, but partially inhibited by Cu2+, methanol, hexane, ethanol, acetone, SDS, and Tween 20. A molecular phylogeny analysis showed that the enzyme's amino acid sequence had the closest connection with an ?-amylase from Geobacillus thermoleovorans subsp. stromboliensis nov. 3D-structure-based amino acid sequence alignments revealed that the three catalytic residues (D217, E246, D314) and the four Ca2+ ion coordination residues (N143, E177, D186, H221) were conserved in ?-amylase from Geobacillus sp. GS33. The temperature stability and neutral pH optimum suggest that the enzyme may be useful for industrial applications. © 2020 Elsevier B.V.
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