| Cellulose is a polymer, which is composed of repeating glucose linked byβ-1,4-glucosidic bonds. It is the most abundant renewable natural biological resource, the bioenergy from cellulosic materials is important for the sustainable development of human beings. There are three types of cellulase:endoglucanases (EC 3.1.2.4), exoglucanases (EC 3.2.1.91), and P-glucosidases (EC 3.2.1.21), which are also abundant in nature. Cellulase can be produced by higher plant, insect, mollusc, bacteria, actinomycetes, fungi and so on. Besides, the bacteria in rumen of ruminant also can produce cellulase. With the growing shortage of energy, the research on exploiture and application of cellulase will develop in-depth. Now, the application of cellulase has been extended to many industries, including textile, medicine, food processing, daily chemical industry, wastewater treatment, as well as beverage additive and other fields.Many cellulase-producing bacteria reported usually come from land-based sources, our research focus on development and utilization of bacteria resources in sea. We have screened a cellulase-producing bacterium Martelella mediterranea from marine bacteria which are supplied by Third Institute of Oceanography State Oceanic Administration. This is the first time we have cloned the cellulase gene of this bacterium by construction of DNA genomic library. Then, the gene is expressed in E.coli, purified, and characterized. The major findings are as follows:1. From the bacteria supplied by Third Institute of Oceanography State Oceanic Administration, M. mediterranea labeled 1A02928 shows cellulase activity. It can produce hydrolysis circle in the cellulose plate by Congo red staining method, which confirms its cellulase activity. The 16S rDNA was obtained by PCR with genomic DNA of 1A02928 as the temple, and was sequenced by BGI (Shanghai). The results of sequence alignments show 1A02928 shared 100% identity with M. mediterranea strain 2PR511-4 and Martelella sp. AD-3.2. The genomic DNA from M. mediterranea was partially digested with Sau3AI and cloned into the BamHI site of a dephosphorized pUC18 plasmid to construct genomic library. The positive clone with CMC-hydrolyzing-activity was obtained which formed a clear halo following the Congo red staining method, named cel5D. The result of sequencing showed it include a 1,113 bp open reading frame (ORF) which had an overall G+C content of 62.98%. The ORF encoded a 370-residue polypeptide including a putative signal peptide of 41 residues, named Ce15D. The overall sequence of cel5D shared 51 identity with putative endoglucanases from Rhizobium etli CIAT 652, indicating cel5D is a novel gene.3. The coding sequence, excluding the predicted signal peptide, was amplified by PCR using two primers. The PCR product was ligated into the corresponding sites of the vector pGEX-6p-1. The recombinant plasmid pGEX-ce15D was then transformed into E.coli DL21 (DE3) and expressed. The GST-tagged Ce15D was purified through a GST-Bind purification kit. The purified Ce15D yielded a band at 36 KDa which was confirmed by SDS-PAGE. And concentration of the protein is 0.04314 mg/ml.4. Purified Ce15D was characterized and it showed the maximum enzyme activity occurred at 60℃, pH 5.0. It was stable at pH 3.0-11.0, belowing 60℃. The specific activity of purified Ce15D towards CMC-Na was 1.6 U/mg. The Km, Vmax and Kcat values, using CMC-Na as the substrate, were 28.4 mg/ml,455μmol/(min·mg) and 36.4 /s, respectively. When barley glucan was used as the substrate, the respective values were 8.8 mg/ml,87.7μmol/(min-mg) and 3.5/s. The metal ions and chemical reagents had no significant effect on enzyme activity, but SDS significantly inhibited enzyme activity. Ce15D produced mainly cellobiose, cellotriose and cellotetraose from cellopentaose and CMC-Na which were separated by thin-layer chromatography, indicating it is an endo-type glucanase.Through characterization of enzyme, Ce15D is a relatively stable and well-tolerated endoglucanase with potential applications, compared with other endoglucanase of glycoside hydrolase family 5.
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