الفهرس 
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Abstract
Thermophilic bacteria are economically important source of a large variety of novel bioactive components of biotechnological importance in agriculture, mining, nanotechnology, chemical and industrial fields at elevated temperatures. Thermozymes are the most important bioactive products produced by thermophilic microorganisms. In this study, soil samples were collected from Pharaoh bath, South Sinai, Egypt. The temperature and pH of the sample sites were 60 - 69°C and 6.7 - 6.9, respectively. A total of 57 bacterial isolates were recovered from the soil samples. The isolates were characterized phenotypically based on cultural characteristics and Gram staining. All isolates were Gram positive, rods, endospore former, off white color and the size of colonies varied from pinpoint to large colonies. According to the optimum temperature of growth of the isolates, 45 bacterial isolates (79%) were moderate thermophiles and 12 bacterial isolates (21%) were extreme thermophiles, 46% of the isolates were optimally grown at 55°C, 33% were optimally grown at 60°C, 12% were optimally grown at 65°C and 9% were optimally grown at 70°C.
Thirty-one bacterial isolates with optimum temperature of ≥ 60°C were characterized genotypically by BOX-PCR for grouping the
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isolates. The results of BOX-PCR of the genomic DNA of the 31thermophilic bacterial isolates showed that the maximum number of bands was 8 distinct bands recovered from isolate PBG-6 while the lowest number of bands (2) was recovered from isolate PBG-16. Cluster analysis of BOX-PCR showed that the isolates were divided into three main clusters, cluster A included twenty isolates, cluster B, two isolates and cluster C, three isolates and 6 distinct separate branches.
The whole cell protein of the isolates with optimum temperature of ≥ 60°C were analyzed by SDS-PAGE. The results of the SDS-PAGE fingerprint of whole cell protein showed distinct bands with molecular weight ranging from 11 to 135 kDa. SDS-PAGE profile showed that the tested isolates had similar protein profile except for some unique bands. Cluster analysis of SDS-PAGE showed that the isolates were divided into three main clusters, cluster A, twenty-one isolates, cluster B included three isolates and cluster C, four isolates, 3 distinct separate branches.
Data analysis of the results of BOX-PCR fingerprints and whole cell protein profiling by SDS-PAGE showed 90% identity between the two methods used for grouping the isolates.
Six bacterial isolates which have the ability to grow at 37°C and high temperatures were selected for the comparison between the whole
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cell protein profile at 37°C and 60°C for the same isolate. The analysis of the result of the SDS-PAGE profile of the 6 bacterial isolates grown at 2 different temperatures of 37 and 60°C showed that different incubation temperatures had no impact on the protein profile where the protein profile was similar at both temperatures. So, the change of protein structure due to the high temperature couldn’t be detected by SDS-PAGE.
Ten representative bacterial isolates from the cluster of the BOX-
PCR fingerprints were selected for molecular identification by sequencing the 16S rRNA gene. Five isolates were identified as Geobacillus thermodenitrificans (PBG-1, PBG-4, PBG-5, PBG-10 and PBG-11) .Isolate PBG-2 was identified as Anoxybacillus flavithermus, PBG-3 was identified as Bacillus hisashii, PBG-6 was identified as Bacillus licheniformis, PBG-7 was identified as Bacillus kexueae and PBG-8 was identified as Bacillus thermoamylovorans.
All the isolates were screened for amylase, cellulase and protease enzymes at 4 different temperatures of 50, 55, 60 and 65°C. The results showed that 45.6% of the isolates could produce amylase, 84% could produce cellulase and 26% could produce protease. The result of the amylase activity showed that isolate PBG-2 showed maximum activity of 3.6517 IU/ml while isolate PBG-11 showed the lowest level of amylase activity of 1.2219 IU/ml among ten isolates