الفهرس 
Molecular characterization of ?-amylase producing strains, enzyme characterization and improvement of productivity by different techniques including gamma radiation
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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.