Identification and characterization of novel thermostable ?-amylase from Geobacillus sp. GS33
Yükleniyor...
Dosyalar
Tarih
2020
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Elsevier B.V.
Erişim Hakkı
info:eu-repo/semantics/openAccess
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.
Açıklama
Anahtar Kelimeler
Geobacillus, Thermostability, ?-Amylase, acetone, alcohol, amylase, benzylsulfonyl fluoride, calcium ion, cobalt, copper ion, dithiothreitol, dodecyl sulfate sodium, hexane, magnesium ion, methanol, polysorbate 20, triton x 100, amylase, bacterial protein, amino acid sequence, anion exchange chromatography, Article, controlled study, enzyme activation, enzyme active site, enzyme activity, enzyme inhibition, enzyme stability, enzyme structure, Geobacillus, Geobacillus thermoleovorans, molecular phylogeny, molecular weight, nonhuman, nucleotide sequence, pH, sequence alignment, size exclusion chromatography, temperature, thermostability, chemistry, enzymology, genetics, Geobacillus, heat, ion exchange chromatography, metabolism, molecular cloning, molecular evolution, molecular model, phylogeny, procedures, protein conformation, thermodynamics, alpha-Amylases, Amino Acid Sequence, Bacterial Proteins, Chromatography, Gel, Chromatography, Ion Exchange, Cloning, Molecular, Enzyme Stability, Evolution, Molecular, Geobacillus, Hot Temperature, Hydrogen-Ion Concentration, Models, Molecular, Molecular Weight, Phylogeny, Protein Conformation, Thermodynamics
Kaynak
International Journal of Biological Macromolecules
WoS Q Değeri
Q1
Scopus Q Değeri
Q1
Cilt
164
