الفهرس | Only 14 pages are availabe for public view |
Abstract Alpha-amylase, a starch-degrading amylolytic enzyme, has many industrial applications. We aimed to isolate α-amylase producers from Egyptian soil samples, optimize the production, and improve characteristics by gamma-irradiation. We also aimed to immobilize the partially purified α-amylase on chitosan-loaded barium ferrite nanoparticles (CLBFNPS). Alpha amylase producers were isolated from soil samples on starch agar plates and further confirmed their activity using the dinitrosalicylic acid (DNS) method. Identification was established using phenotypic methods, 16S-rRNA sequencing, and phylogenetic mapping. Sequential optimization of α-amylase production involved the use of two statistical designs: Plackett Burman design (P-BD) and central composite design (CCD), and exposing the α-amylase producer to different doses of gamma-irradiation. Partially purified alpha-amylase was immobilized on CLBFNPS, and the nanocomposite was characterized by X-ray diffraction (XRD), Fourier transform infrared (FTIR), and scanning electron microscopy with energy dispersive analysis of X-ray (SEM/EDAX) analysis. Forty-five α-amylase producers, including 23 bacterial and 22 fungal isolates, were recovered from hundred of soil samples. The highest activity for α-amylase (177.12±6.12 U/mg) was demonstrated by MS009 isolate, identified as Bacillus paramycoide, which was used in further experiments. An increase in α-amylase activity (222.3±5.07 U/mg) upon using the optimum culture conditions obtained from the analysis of the PB-D results. The activity further increased to 232.456 ± 5.98 U/mg when using the optimum culture conditions obtained from the analysis of the CCD results, which consisted of a high concentration of peptone 0.45 g%, culture-to-flask volume ratio 75:250 mL, CaCl2 1.5 g%, and an incubation period of 84 h. A further increase in activity (319.45±4.91 U/mg) was detected upon exposing the culture to a Gamma irradiation dose of 6 kGy. Immobilization of α-amylase on CLBFNPS resulted in higher activity (246.85±6.76 U/mg) compared to the free α-amylase (222.254±4.89 U/mg), besides retaining its activity for up to five cycles of usage. Gammairradiation enhanced the activity of α-amylase. Immobilization of α-amylase on CLBFNPS facilitated enzyme recovery from the medium using magnetic force, enabled its repetitive use, localized the interaction, and prevented product contamination. |