Document Type : Original Article
Authors
1
Bioinformatics Unit, Cairo university research park, Cairo University
2
Department of Microbiology, Faculty of Agriculture, Cairo University, Giza, Egypt
3
Agricultural Microbiology Department National Research Center (NRC), Cairo, Egypt
4
Microbiology Department, National Research Centre.
5
Agricultural Microbiology Department National Research Center (NRC)
Abstract
Geothermal water samples collected from Dakhla Oasis, Kharga Oasis, Pharaoh Baths and Ras Sedr hot springs in Egypt were explored for the isolation of industrially efficient thermostable amylase-, protease- and lipase- producers. Of 170 enzyme-producing isolates secured from colonies developed on agar media, 12 superior ones were subjected to morphological characteristics and biochemical profiles. Cells appeared cocci and spiral with the majority as bacilli. Adopting the Diagnostics GN/GP 24 (Ref. 1001, 1002), the tested candidates successfully utilized various substrates as carbon and nitrogen sources besides their abilities to produce a number of exoenzymes. Six potent amylase-, protease- and lipase-producing thermophiles. two for either were further identified by 16S rRNA gene sequencing. The amylase-producers were identified as Aneurinibacillus thermoaerophilus and Bacillus licheniformis with respective similarity percentages of 99.48 and 100. Genetic analysis of protease-producers showed the similarity to B. licheniformis and B. sonorensis (90.02 and 98.14 % identities). Both lipase-producing isolates described as B. licheniformis with similarity percentages of 98.80 and 100). The Plackett-Burman multifactorial Design was implemented to screen the limiting components for growth and subsequent use of Central Composite Design to tailor a suitable medium that supports exponential growth and consequently the protease production of the tested thermophilic bacterium. For optimization of protease production by Bacillus licheniformis (isolate DO24), the applied Placket-Burman methodology screened eleven effective factors including skim milk, peptone, yeast extract, CaCl2, MgSO4.7H2O, NaCl, KH2PO4, inoculums size, pH, temperature and incubation time. Among those; skim milk, yeast extract, inoculum size and incubation time deemed the most influential factors. Adopting the Central Composite Design, the optimized protease activity was achieved at the respective component records of 40 ml L-1, 4.0 g L-1, 40 ml L-1 and 24 h. This study proves that Egyptian hot springs are beneficial reservoirs for thermostable enzyme-producing microbiota of great importance for various bio-industrial applications.
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