Background
Wadi El Natrun microorganisms have been considered as a new resource for natural products due to its extreme condition of salinity and alkalinity. Therefore, this study was devoted to generate metagemic library from soils collected from such an extreme environment in order to clone a novel cellulase for physique industrial applications.
Results
Total soil-DNA was successfully extracted, and then digested by different restriction enzymes. Purified fragments ranged ~ 200–6500 bp were ligated and were cloned into plasmid cloning vector (pUC19) by using Escherichia coli DH5α (E. coli) host cells. A constructed metagenomic library composed of 270 clones was screened on carboxymethylcellulose (CMC) agar plate where the active clones had been characterized by the formation of the yellowish halo zone. Thereafter, clone 1 was selected as the most active as being based on cellulase activity quantification (19 μ/ml). Plasmid related to clone 1 encoded cellSNSY gene of approximately 1.5 kb was subjected to molecular characterization; the obtained partial sequence of 861 bps encoded 287 amino acids showing 76% similarity to the endoglucanase gene of Bacillus amyloliquefaciens. The recombinant cellSNSY was expressed under lacz promoter at 1 mM of isopropyl β-d-1-thiogalactopyranoside (IPTG), giving 21 μ/ml cellulase after ~ 27 h. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and an activity staining of the recombinant cellSNSY which revealed an active band with a molecular mass ~ 59 kDa appeared in the induced sample. The maximum enzyme activity of crude cellSNSY was observed at 45 °C and for a pH of 8.5. Interestingly, the enzyme activity was slightly inhibited by ethylenediamine tetraacetic acid (EDTA) and methanol. It showed high resistance to the tested heavy metals and the surfactant which ordered Zn> (SDS,Fe)>Mn>Cu.
Conclusions
This study established an easy and a skillful way to clone/express a new found cellulase gene(s) under lacZ promoter. The isolated recombinant cellSNSY showed 76% similarity to endoglucanase gene, and the enzyme showed tolerance to the mostly tested agents including heavy metals, surfactant, solvents, and EDTA. Additionally, the studied recombinant showed a high stability up to 55 °C and for alkaline pH 8.5. These features make it an ample and viable for many applications.
Of 24 fungi belonging to more than five genera isolated from tubers of rotten Helianthus tuberosus, 11-inulinolytic active isolates were able to develop halo zones around their fungal colonies, indicating inulinase activity. Alternaria, Aspergillus, Fusarium, Pencillium and Trichoderma were the most common inulinolytic genera, representing more than 90 % of the total positive inulinolytic fungi. Aspergillus tamarii and Pencillium citrinum quantitatively recorded better growth (5.5 and 4.7 mg ml(-1)) and inulinase production (21.53 and 20.15 U ml(-1)) in submerged culture. The enzyme preparation showed also invertase activity. Aspergillus tamarii, as the most potent producer of inulinase, was identified using the Inter Transcribed Spacer marker. The sequence comparisons showed that our molecularly identified strain (GU295949) is related more closely to A. tamarii strains of the gene bank. Statistical screening using the fractional factorial Plackett-Burman design with 12 run was applied for screening ten variables, the low levels of pH (4.8), inoculum size (10(3) spore g(-1)), NH(4)NO(3) (1.0 mg g(-1)) and MgSO(4) (0.12 mg g(-1)), were the most significant variables on A. tamarii inulinase production. The high inulinase/invertase ratio (1.841-4.293) classified the enzyme preparation as inulinases, which can be used efficiently in production of fructose syrup from tubers of H. tuberosus.
Three kinds of processed meat (Luncheon, Burger , Minced meat) were examined for the presence of Listeria species. Collected samples purchased from local supermarkets were from three different Egyptian companies. All the bacterial isolates were cultured on specific media after pre-enrichment and enrichment cultures. The colonial morphology depends strongly up on the media used and the incubation conditions provided. (Oxford Listeria Agar Base) was used as specific medium for Listeria. The bacterial distribution of Listeria was, 2.22% in Beef burger, 13.6%. in luncheon and there was no bacterial growth in minced meat. In this study; gram staining., morphological characterization, molecular identification using 16S rDNA partial sequencing and specific gene Iap gene of Listeria were carried out.
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