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Screening, Identification And Enzymatic Properties Studies Of Chitinase From Submarine Sediment

Posted on:2015-08-10Degree:MasterType:Thesis
Country:ChinaCandidate:R X ZhangFull Text:PDF
GTID:2180330482471945Subject:Biochemistry and Molecular Biology
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Among the natural polysaccharides, chitin is the second most abundant next to cellulose. Chitin can be degraded specifically by chitinase and its degradation products have a great application value in biology, energy, medicine, agriculture, etc. The ocean is a rich source of microorganisms, and screening chitinase producers from marine sediments is therefore important for the development of chitinase with high stability and activity.In this study, sediments from Beibu Gulf of South China Sea were chosen as our samples. Using the clear zone method, we obtained 21 strains of chitinase producers. We then screened out a strain with high chitinase activity by DNS method. After PCR amplification of its 16S rDNA, we identified that this strain belonged to the Bacillus cereus family and named it as Bacillus cereus B04.Our preliminary study showed that the optimum time for collecting the enzyme was the fifth day, and the optimum initial pH is 9.0, and the optimum quantum is 50/250ml, and the optimum substrate concentration is 2g/l. We also showed that B. cereus B04 used inorganic nitrogen more efficiently than organic nitrogen.In the isolation procedure of chitinase, we found there were 6 protein containing chitin binding domain. We designed a reasonable purification protocol:(NH4)2SO4 precipitation----hydrophobic chromatography----ion exchange chromatography----gel filtration. After the purification, we isolated a 40kDa chitinase with a purity greater than 80%. The specific activity of the final purified chitinase was 2.12 U mg-1, the activity recovery rate was 15.27% and the purification factor was 21.20. After sequencing the N-terminal amino acid and construction of a phylogenetic tree, we identified this chitinase as Chi36 and named it as Chi36B04. We also determined that the optimum pH, reaction temperature and reaction time and substrate concentration for Chi36B04 was pH4.0,60℃,80min and 1.5%, respectively.To facilitate the industrial production of Chi36B04, we used molecular biology technologies to amplified the gene sequence encoding Chi36B04 protein. We also construct the Chi36B04/pET-28a(+) recombinant plasmid and expressed the soluble protein in E. coli. After eluted from Ni+ affinity column, purity of the recombinant protein reached 90% with high activity and stability.In summary, we established a repertoire of screening, identification, and research of the chitinase from marine sediments, and laid the foundation for the future large-scale screening of new chitinase.
Keywords/Search Tags:Beibu Gulf, chitinase, Bacillus cereus, enzymatic property, soluble expression
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