الفهرس 
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Abstract
This study has offered an α-amylase from a novel bacterial species for biotechnological applications. The new α-amylase producing bacterium was classified on the basis of polyphasic approach including both phenotypic and genotypic methods. The name Laceyella aegyptius was proposed to this novel species which represented by the type strain Laceyella aegyptius DS3.
The produced α-amylase by Laceyella aegyptius DS3 was purified 13.8-fold. SDS-PAGE technique for the purified protein identified 69.2 kDa and 72.5 kDa proteins whose α-amylase activities were confirmed by native gel staining. The purified α-amylase showed an optimum pH of 7.0 and an optimum temperature of 55°C. It had activation energy, Km, and Vmax 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 this α-amylase, whereas Cl- was an activator. Although this α-amylase is a Ca+2 independent α-amylase, it was activated by Ca+2.
The AmyLa gene encoding α-amylase activity was sequenced. 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.
The AmyLa gene was heterologous expressed in E. coli BL21 under T7 promoter. SDS-PAGE identified 51 kDa recombinant protein whose α-amylase activity was confirmed by native gel staining. The recombinant AmyLa was purified 2.19-fold.
The purified recombinant AmyLa was immobilized on different carriers (DEAE-cellulose, calcium alginate as adsorbent, calcium alginate as a trap and Chitosan). Its immobilization on chitosan by covalent binding method showed the best efficiency in addition to activate the enzyme. The immobilized recombinant AmyLa could be reused with its full activity for 7 cycles.