5651
Ahmed Fikry El-Sayed Mustafa Rizk
Genetic and molecular studies on some β-glucosidase producing bacterial isolates
cellulose, β-glucosidase, SDS-PAGE, ISSR, 16 s rDNA, mutation, bgl gene and Cloning.
The main objective of the present study is isolation, screening, identification and optimization of strains with high β-glucosidase activity from Egyptian soil, then genetic improvement through different genetic methods to get highly producing strains that have industrial properties and studying their molecular characteristics. All experiments of this investigation were conducted in Microbial genetics laboratory, National Research Center. The period study was conducted during (2017 – 2019). Among eight isolated strains, isolate NRC4 showed the highest β-glucosidase, Endo-glucanase activity and Protein Content 37.24 U ml-1, 1.89 U ml-1 and 220 μg/ml, respectively. Morphologically, it was Gram-ve, motile, rod, bacilli in cell shape, blue in colony color. Biochemically it was positive for Catalase, Oxidase, Voges Proskauer, Citrate, nitrate, Gelatin, H2S production and esculin, and it was negative for starch and Casein hydrolysis. Physiologically, it was grown at wide range of pH (5-9), NaCl concentration (1-5) % and temperature range (20-40°C). In addition, the efficient β-glucosidases producing NRC4 was identified genetically by 16s rRNA gene sequence as Pseudomonas aeruginosa and submitted on GenBank with accession number LC455963.1. Phylogenetic tree was determined the relationship of strain NRC4 with different strains of genus Pseudomonas spp. Optimized conditions for β-glucosidase production were characterized as 1% inoculum size and pH 7 at 45℃ for 48 hours of incubation using cellobiose (1%w/v) as carbon source. Also, Ammonium chloride at (0.5% w/v) was optimum for β-glucosidase production. Genetic improvement was carried out to improve β-glucosidase production using selected strain P. aeruginosa: firstly, induction of mutation by UV, three mutants (20%) as result of UV mutagenesis produced were selected and showed 1.2-fold 1.6-fold and 1.5-foldincrease of β-glucosidase activity higher than W.T. secondly, induction of mutation by EMS, Survival (%) for EMS mutagenesis was determined as 7.1, 2.4, 1 and 0% at different concentrations (25, 50, 75 and 100 μl.ml-1). results showed that, the mutant PA.50.E6 showed 1.9 -fold increase in β-glucosidase production over W.T.
2020
Ph.d
Al-Azhar
Science