The present study primarily deals with the identification of substrate-binding site and elucidati... more The present study primarily deals with the identification of substrate-binding site and elucidation of catalytic residue of the phytase from Bacillus sp. (Genbank Accession No. EF536824) employing molecular modeling and site-directed mutagenesis. Homology-based modeling of the Bacillus phytase revealed propeller structure with twelve active-site aminoacid residues, viz., D75, R77, Y78, H138, Q140, D189, D190, E191, Y238, Y239, N346 and R348. Docking of substrate Ins(1,2,3,4,5,6)hexakisphosphate with the phytase model disclosed interaction of Y78 residue with the sixth position phosphate, while D75 and R77 residues revealed hydrogen bonding with the fifth position phosphate of the phytate. Analysis of hydrolysis products of phytate indicated the sequential removal of alternate phosphates, resulting in the formation of final product Ins triphosphate. Mutant phytases Y78A/F, derived from site-directed mutagenesis, exhibited complete loss of enzyme activity despite substrate binding, thereby suggesting the intrinsic role of Y78 residue in the catalytic activity. The Bacillus mutant phytases can be used to generate enzyme crystals complexed with phytate and lower Ins phosphates for indepth analysis of substrate binding and catalytic activity of the enzyme.
Staphylococcus aureus can undergo phenotypic switching between a normal colony phenotype (NCP) an... more Staphylococcus aureus can undergo phenotypic switching between a normal colony phenotype (NCP) and a small colony variant (SCV). The SCV phenotype confers increased antibiotic resistance and the capacity to persist within human tissues and cells, and because these cells can revert back to the NCP they cause chronic and/or recurrent infections that are very difficult to treat. A complete picture of the genetic events that can lead to phenotypic switching in S. aureus is currently lacking. We describe the selection of an SCV with a previously unreported genetic alteration leading to an ochre mutation of aroD. In addition to the known mechanisms of phenotypic switching between the SCV and the NCP we describe a previously unreported mechanism involving tRNA ochre suppressors arising. The ochre suppressor strains had wild-type growth rates and restored antibiotic sensitivity, similar to the wild-type strain. However, whilst they had increased virulence compared to the SCV parent strain, ...
European Journal of Biology and Biotechnology, 2021
Background: Microbial contamination continues to be one of the leading risks to food safety. Cont... more Background: Microbial contamination continues to be one of the leading risks to food safety. Contaminated leafy green vegetables are the primary cause of infection among children, elderly, and immunocompromised people. The purposes of this work were to isolate and identify of Salmonella spp. in fresh leafy vegetables collected from Jeddah Central Market, Jeddah district, western area, kingdom of Saudi Arabia, estimated of the number and percentage of isolated Salmonella spp and determined the antimicrobial susceptibility of the isolated Salmonella spp. Methods: Five-hundred samples were examined for the presence of Salmonella spp, by using standard microbiological and biochemical tests. Further, detection of Salmonella spp. was done by PCR with the primers targeting invA gene, a key factor for entry of Salmonella into epithelial cells. Susceptibility of the isolated Salmonella spp was done toward thirteen different antibiotics. Results: The percentage of isolation of Salmonella spp was 1.2 % (06/500). It was isolated as (0.40%, 02/500) from Basil, (0.20%, 01/500) from Spinach, Rocket, Parsley and Chards. Two isolates (2/6, 33.3%) showed positive Salmonella invA gene (244 bp). All isolated Salmonella showed resistance to Cephalexin (30 µg/disc), Metronidazole (5 µg/disc) and Methicillin (5 µg/disc).
Background L-asparaginase produced by plant and bacteria can be used in the pharmaceutical and fo... more Background L-asparaginase produced by plant and bacteria can be used in the pharmaceutical and food industry. Unlike the bacterial counterparts, fungal L-asparaginase has more stability, more activity, and less adverse effects. Central composite design (CCD) was used to optimize temperature, pH, incubation time, and carbon-to-nitrogen ratio for L-asparaginase production by Cladosporium tenuissimum via submerged fermentation. CCD reduces the number of tests and time for optimization. Objective To optimize the culture conditions, such as temperature, pH, production time, and the ratio between concentration of carbon and nitrogen sources, for the production of L-asparaginase by isolated C. tenuissimum via submerged fermentation. Materials and methods Primarily, four significant parameters (temperature, pH, incubation period, and carbon-to-nitrogen ratio) were identified that affect the production process of L-asparaginase via submerged fermentation using the modified Czapek Dox medium. CCD was used to optimize the selected parameters concurrently, and their results were compared. Results and conclusions The highest L-asparaginase enzyme activity obtained was 2.6471 U/ml at 37°C, pH 6.2, incubation time 72 h, and 2 : 1 carbon-to-nitrogen ratio. The P value of interaction between every two factors was only significant for the interaction between temperature and incubation period (P<0.0281). The most significant factor was temperature followed by pH (P<0.0154) and carbon-to-nitrogen ratio (P<0.0346). Incubation period has no major effect on the production of Lasparaginase, but it has a quadratic effect (P<0.0001). Our results showed the significant role of culture conditions (temperature, pH, incubation period, and carbon-to-nitrogen ratio) in L-asparaginase production and confirmed the need for optimization.
The present study primarily deals with the identification of substrate-binding site and elucidati... more The present study primarily deals with the identification of substrate-binding site and elucidation of catalytic residue of the phytase from Bacillus sp. (Genbank Accession No. EF536824) employing molecular modeling and site-directed mutagenesis. Homology-based modeling of the Bacillus phytase revealed propeller structure with twelve active-site aminoacid residues, viz., D75, R77, Y78, H138, Q140, D189, D190, E191, Y238, Y239, N346 and R348. Docking of substrate Ins(1,2,3,4,5,6)hexakisphosphate with the phytase model disclosed interaction of Y78 residue with the sixth position phosphate, while D75 and R77 residues revealed hydrogen bonding with the fifth position phosphate of the phytate. Analysis of hydrolysis products of phytate indicated the sequential removal of alternate phosphates, resulting in the formation of final product Ins triphosphate. Mutant phytases Y78A/F, derived from site-directed mutagenesis, exhibited complete loss of enzyme activity despite substrate binding, thereby suggesting the intrinsic role of Y78 residue in the catalytic activity. The Bacillus mutant phytases can be used to generate enzyme crystals complexed with phytate and lower Ins phosphates for indepth analysis of substrate binding and catalytic activity of the enzyme.
Staphylococcus aureus can undergo phenotypic switching between a normal colony phenotype (NCP) an... more Staphylococcus aureus can undergo phenotypic switching between a normal colony phenotype (NCP) and a small colony variant (SCV). The SCV phenotype confers increased antibiotic resistance and the capacity to persist within human tissues and cells, and because these cells can revert back to the NCP they cause chronic and/or recurrent infections that are very difficult to treat. A complete picture of the genetic events that can lead to phenotypic switching in S. aureus is currently lacking. We describe the selection of an SCV with a previously unreported genetic alteration leading to an ochre mutation of aroD. In addition to the known mechanisms of phenotypic switching between the SCV and the NCP we describe a previously unreported mechanism involving tRNA ochre suppressors arising. The ochre suppressor strains had wild-type growth rates and restored antibiotic sensitivity, similar to the wild-type strain. However, whilst they had increased virulence compared to the SCV parent strain, their virulence was not restored to that of the NCP parental strain. These findings establish that phenotypic switching between the NCP and SCV states can give rise to strains with different pathogenic potential.
IOP Conference Series: Earth and Environmental Science, 2022
Phosphorus (P) shortage in the soil is a key problem in agricultural output. Phytate (Myo-inosito... more Phosphorus (P) shortage in the soil is a key problem in agricultural output. Phytate (Myo-inositol phosphate), which is stable and unavailable to plants, is abundant in soils, accounting for up to 50% of all organic P. The conversion of phytate to Myo-inositol and phosphate is catalyzed by phytases. Microorganisms that solubilize P, biofertilizers, are common in soils and may play a major role in providing P to plants. Secreted microbial phytases enhancing plant development in terms of phosphorus availability. This study aimed to identify possible phytate degrading microorganisms from the rhizosphere, animal dung, soil, and livestock farms samples collected from farms in Khartoum, Sudan. Modified Luria Bertani and phytase screening media supplemented with wheat bran as a source of phytate were used as selection media. Each sample was serially diluted 10 times, and 100 µl from dilution 10 3 was distributed onto each of two screening medium plates, cultured aerobically at 37°C for 1 to 4 days, A putative phytate degrading bacterium was detected in isolates with a clear zone around their growing colonies. A total of 17 isolates were obtained, with 15/17 (88%) being bacterial isolates and 2/17 being fungal isolates. Phosphate solubilizing activity was performed on Pikovskaya's agar plate, and plates were incubated at 37°C for 7 days. The plates were examined for the presence of a halo zone around each isolated culture. out of the 17 isolates, only one Gram-positive Bacilli (isolate AB13) was found to have phosphate solubilizing activity and the ability to liberate P from phytate, suggesting that it could be used as a biofertilizer.
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Papers by Ahmed A Osman