الفهرس 
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Abstract
Interest in cellulolytic systems has increased in recent years with the need for novel cellulolytic enzymes to serve as biocatalysts for efficient conversion of cellulose to bioethanol. Cellulose is a complex carbohydrate biopolymer structured as numerous linear fibrils intertwined to form crystalline structures. For many years, cellulose degrading bacteria have been isolated and characterized for obtaining more effective cellulases from variety of sources such as soil, decayed plant materials, hot springs, organic matters, feces of ruminants and composts Researchers keep on working to isolate microorganisms with higher cellulase activity.
The present study aimed to isolate and screen bacteria, which can produce more efficient cellulase enzymes from soil. In addition to the production, purification and characterization of cellulose degrading bacteria.
Obtained results could be summarized as follows:
6.1. Isolation of cellulolytic bacteria
Eighty four bacterial strains were isolated from rhizospheric soil from “Kafr Wahb, Monofeya government, Egypt ”. Out of theses, twenty-four isolates formed clear zones around colonies when grown on nutrient agar medium amended with CMC as carbon source.
6.2. Screening of the most potent cellulolytic isolates
Further screening of the twenty-four bacterial isolates for having a relatively wider clear zone around the colony resulted in the selection of three prospective isolates, namely, Z7, Z9, and Z63 with diameter of hydrolyzed areas of 1.8, 2 and 1.6 cm respectively.
6.3. Scanning Electron Micrograph (SEM) for the selected isolates
The most potent isolated bacteria were scanned by electron microscope (JSM-5500 LV JEOL “JAPAN”) and appeared rod- shaped.
6.4. Morphological, physiological and biochemical characteristics of the three chosen bacterial isolates
The three chosen cellulolytic bacterial isolates were characterized morphologically, physiologically and biochemically.
6.5. Molecular identification of the most potent bacterial isolates.
PCR product of 16S rRNA for the three isolates, Z7, Z9 and Z63 was about 1.5 kb. The obtained sequence of strains Z7, Z9 and Z63 was assembled, analyzed and submitted in the GenBank under accession numbers (KT693283), (KT693282) and (KT693284) respectively. Multiple alignment of 16S rRNA genes and Phylogenetic tree of Bacillus cereus strain Z7, Bacillus licheniformis strain Z9 and Klebsiella oxytoca strain Z63 with the closest bacterial strains were constructed.
6.6. PCR product of cel9z gene from B. licheniformis strain Z9
PCR amplification was performed, giving a good band at about 1.5 kb on agarose gel. The sequence of the obtained cel9z gene assembled, analyzed and deposited in the GenBank database under accession number (MK814929). The entire open reading frame (ORFs) of cel9z containing 1453 bases encodes a protein with 484 amino acids and a molecular mass (54.4 KDa).
6.7. Cloning and subcloning of cel9z cellulase gene
The cellulase gene was successfully inserted into the pSC-A-amp/kan vector. The isolated fragments were ligated into pET-22b (+) expression vector. The isolated plasmids were digested and the products were analyzed on 0.8% agarose gel. The inserts were confirmed by comparing PCR products amplified from the clones with products amplified using genomic DNA from B. licheniformis strain Z9. Restriction enzyme map of enzyme cel9z was constructed. The protein sequences of cel9z cellulase was subjected to conserved domain analysis and hydropathy plots of cel9z of B. licheniformis strain Z9. The deduced amino acids of the open reading frame of cel9z were aligned to NBCI GenBank. Also, phylogenetic tree was constructed for cel9z using the neighbor-joining method.
6.8. Enzyme Purification
Ammonium sulphate precipitation gave purification fold of about 1.96 with 51.1% yield, while purification fold of about 2.1 and 3.07% yield was achieved for Gel exclusion chromatography (Sephadex G-100).
6.9. SDS-PAGE and Zymogram analysis
Cel9z of B. licheniformis strain Z9 showed a single band on SDS-PAGE with a molecular weight of approximately 54.4 kDa. The enzymatic activity of purified cel9z was confirmed by CMC zymographic analysis. It was shown as yellow halo against a red background.
6.10. characterization of (cel9z) cellulase protein
Glucose and protein standard curve were constructed. The optimal enzyme activity was observed at pH 7.4 and 30°C. The enzyme was strongly inhibited by Mg2+, EDTA and Na+, whereas strongly activated by Fe3+, Cu2+, Ca2+ and SDS.