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Gene sequence, modeling, and enzymatic characterization of α‐amylase AmyLa from the thermophile Laceyella sp. DS3
Author(s) -
ElSayed Ahmed K. A.,
AbouDobara Mohamed I.,
ElFallal Amira A.,
Omar Noha F.
Publication year - 2017
Publication title -
starch ‐ stärke
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.62
H-Index - 82
eISSN - 1521-379X
pISSN - 0038-9056
DOI - 10.1002/star.201600255
Subject(s) - ammonium sulfate precipitation , open reading frame , amylase , phylogenetic tree , biology , peptide sequence , amino acid , biochemistry , gene , microbiology and biotechnology , enzyme , thermophile , gel electrophoresis , size exclusion chromatography , chemistry
α‐Amylase (AmyLa) from Laceyella sp. DS3 was purified 13.8‐fold by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. The purified AmyLa showed an optimum pH of 7 and an optimum temperature 55°C. It had activation energy, K m , and V max of 196.1 kJ, 0.81 mg/mL, and 2.85 mg/min, respectively. Co 2+ , Cu 2+ , Hg 2+ , and Na + were strong inhibitors of AmyLa whereas Cl − was an activator. Although AmyLa is a Ca 2+ independent α‐amylase, it was activated by Ca 2+ . The AmyLa gene encoding α‐amylase activity was sequenced. It revealed putative triple promoters and an open reading frame of 1449 bp (483 amino acids) including a 29 amino acid putative signal peptide. AmyLa was shown to possess four conserved sequence regions of the GH13 family. Polyacrylamide gel electrophoresis identified 69.2 and 72.5 kDa proteins whose α‐amylase activities were confirmed by native gel staining. Modeling of AmyLa revealed three domains A–C with domain A containing a catalytic (β/α) 8 barrel. Phylogenetic and evolutionary tree analysis suggests that AmyLa might be have been transferred from fungi to an ancient thermoactinomycete ancestor, then adapted to bacterial genome features.