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Cellulose is a glucose to β-1, 4 - glycosidic bond polysaccharide polymerization, it is the the nature most abundant renewable biological resources, generating bio-energy on sustainable human development is of great significance. Cellulase be divided into three categories: endo-glucanase (EC 3.1.2.4), exo glucanase (EC 3.2.1.91) and β-glucosidase enzymes (EC 3.2.1.21), they are in the nature distribution is extremely broad. Higher plants, insects, mollusks, bacteria, actinomycetes, fungi are capable of producing cellulase. In addition, there cellulase production in the rumen of ruminant and pig intestine bacteria. As energy increasingly scarce, people cellulase more and more extensive and in-depth. Cellulase application is now extended to many industries, including textile, pharmaceutical, food processing, household chemicals, waste water treatment, as well as drinks add various fields. Many have reported microbial cellulase production usually comes from the land, our research is dedicated to the development and utilization of the resources of marine bacteria. Marine bacteria from Xiamen marine San screening get the one producing cellulase bacteria Martelella mediterranea. By constructing a genomic library, the first cloned cellulase genes from the bacteria, and expressed in E. coli and purified protein and enzymatic properties of the protein. The main findings are as follows: 1, from the Xiamen marine three marine bacteria, M. mediterranea number 1A02928 cellulase activity. Cellulose plates, with the Congo Red staining detected that the strain can produce hydrolysis circle proved having cellulase activity. 1A02928 total DNA as a template, PCR amplification of 16S rDNA sent Genomics (Shanghai) sequencing. Sequence alignment results show 1A02928 and M. mediterranea strain2PR511-4 highest homology is 100%. PUC18 vector construct the genomic library of M. mediterranea, Congo red staining through a cellulose plate screening of recombinant clones obtained one positive clone, named ce15D. Sequence analysis showed that the clones contain a complete ORF, a total length of 1,113 bp, 62.98% GC content, encoding 370 amino acids. ORF only consists of a catalytic domain (belong to the glycoside hydrolase family 5). The protein named the ce15D translation obtained Ce15D. Protein level, Ce15D and the Rhizobium etli CIAT 652 amino acid homology of up to 51% the instructions ce15D is a new gene. 3, the design of primers, PCR amplified ce15D gene, and cloned into the expression vector pGEX-6P-1, inducible expression in E. coli DL21 (DE3), obtained recombinant protein the pGEX-Ce15D by affinity chromatography column purification remove the GST tag. SDS-PAGE analysis after purification the protein, Ce15D produce a clear bands at 36 KDa. The protein concentration of 0.04314 mg / ml. 4, the enzymatic properties of purified Ce15D. Enzyme at 60 ℃ and the highest enzyme activity at pH 5.0; pH 3.0-11.0,60 ℃ more stable enzyme activity. Cellulose as substrate, enzyme activity was 1.6 U / mg, Km = 28.4 mg / ml, Vmax = 455μmol / (min · mg), and Kcat = 36.4 / s. Barley glucan substrate Km = 8.8 mg / ml, Vmax = 87.7μmol, / (min · mg) kcat = 3.5 / s. Apart from SDS strongly inhibited Ce15D activity, metal ions and chemical reagents obvious effect on enzyme activity. The tlc results for the product after the CMC-Na, hydrolyzes cellobiose, cellotriose and fiber tetrasaccharide to proved Ce15D belong endoglucanase. Enzymatic properties of this enzyme, as well as with other within glycoside hydrolase family 5 endoglucanase Ce15D a broader pH tolerance, free from heavy metal ion suppression, has potential applications endo-β-1, 4 - glucanase.
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