الفهرس 
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Abstract
The aim of the present study is to isolate bacteria that produce industrially important enzymes with the possibility to isolate DNA fragments that code for these enzymes. To fulfill this aim, seventy two bacterial isolates were isolated, purified and examined for their ability to produce extracellular enzymes (protease and amylase enzymes). Twenty one of them had the ability to produce protease while eleven of them plus other five isolates have the ability to produce amylase enzyme. Most of the isolates have the ability to produce both enzymes (protease and amylase) as extracellular enzymes. The bacterial isolate (SHG10) was selected as a high protease and amylase producer.
The extracellular protease production was monitored during the growth of SHG10 isolate on sporulation medium (1XSG). Expression of the alkaline protease gene stared early stationary phase of bacterial growth. Considerable levels of this enzyme were noticed during this phase. The present finding is in a good agreement of other findings concerning the onset of alkaline protease gene expression of other members belonging to the genus Bacillus. Similarly, the production of α- amylase enzyme of the bacterial isolate SHG10 was monitored during the bacterial growth. Data showed that expression of the α- amylase gene started at log phase of growth and considerable level of this enzyme was produced.
Identification of the most promising protease and amylase-producing bacterial isolate was carried out based on Microscopic examination and several biochemical tests using Bergey’s Manual of Systemic Bacteriology. The bacterial isolate under study (SHG10) was a spore-forming Gram positive bacterium and it belongs to the family Bacillaceae and it is a member of the genus Bacillus. Further identification was carried out utilizing 16S rRNA (DNA) technique. Data were submitted to the GenBank sequence database with a given accession number JN853580. The bacterial isolate was identified as Bacillus licheniformis.
Isolation of protease and amylase genes was carried out through designing of specific primers (oligonucleotides) that have the ability to bind precisely to protease or amylase genes of the whole B. licheniformis SHG10 chromosome. B. licheniformis alkaline protease and α-amylase gene sequences which submitted in the GenBank (NCBI), were used as a reference to design the new primers. The alkaline protease gene as PCR product (1350 bp) was first sequenced using the automated DNA sequencer and analyzed using Basic Local Alignment Search Tool programme (BLASTN 2.2.26). Results indicated that, the sequenced protease gene (1298 b.p) was so close to B. licheniformis strain RPk keratinase gene by identity percent (99%) and query coverage percent (100%). The nucleotide sequence (1298 bp) was deposited in the GenBank database and given the accession number JN853581. A possible two putative (-10) promoters TATAAT and TATAAT, a possible ribosomal binding site AGGAGGAG , then ATG (Methionine) and an open reading frame were found. Moreover, the alanine (coded by GCG) as start codon of the mature alkaline protease enzyme and the translational stop codon (TAA) were also found in this sequence. This finding is in a very good agreement with that reported earlier. Based on result for nucleotides translated to the amino acids, using http://web.expasy.org/translate web site, The pre-protease gene belongs to B. licheniformis SHG10 contain 379 amino acids while the mature alkaline protease has 274 amino acids.
Similarly, the isolation of α-amylase gene (depending on some α-amylase genes of various B. licheniformis strains) was carried out using specific primers. The alpha amylase gene as PCR product (1920 bp) was first sequenced using the automated DNA sequencer and analyzed using Basic Local Alignment Search Tool programme (BLASTN 2.2.26). The sequence was compared to other alpha amylase genes of B. lichenifirmis that have been sequenced and submitted to the GenBank database so far. Results indicated that, the DNA fragment sequence was similar to B. licheniformis strain 3TB2 alpha-amylase (bla) gene by maximum identity percent (99%) and the query coverage percent was (100%).The nucleotide sequence (1845 bp) was deposited in the GenBank database and given the accession number JN853583. On the other hand, a putative promoter with (-10) TACAAT and (-35) TTGTTA, a possible ribosomal binding site, were found in the sequence GAAAGG, an ATG (methionine) and an open reading frame are found in the α-amylase DNA fragment sequence. Moreover, GCA code as the alanine amino acid is the start codon of the mature amylase gene and TAG code as the translational stop codon were also present. Results are in agreement with those reported earlier. Based on result for nucleotides translated to the amino acids, using http://web.expasy.org/translate web site, the pre-amylase gene belongs to B. licheniformis SHG10 contain 512 amino acids, while the mature α-amylase has 483 amino acids .