| Summary: | 碩士 === 國立高雄海洋科技大學 === 海洋生物技術研究所 === 97 === A chitin-degrading bacterium was isolated from the cast shell of fresh shrimp (Neocaridina denticulate). The 16S rDNA sequence of the strain showed the 99% similarity to that of Chitinimonas taiwanensis, Therefore, we name the strain as Chitinibacter sp.. The chitinase-specific primers was designed according to the highly conserved region of family 18 chitinase, which was used to amplify a partial sequence of chitinase from Chitinibacter sp. with PCR. The full length chitinase gene was cloned from the genomic library of Chitinibacter sp. employing biotin-labeled specific DNA probe. The inserted DNA fragment, 2930 bp, possesses a complete open reading frame (1419 bp), which encodes a 52 kDa protein. Amino acid sequence analysis shows that the enzyme was a family 18 chitinase and just possesses a catalytic domain. A soluble chitinase was successfully over-expressed using the T7 promoter and E. coli BL21(DE3) host cell. The biochemical analysis show that the optimum reaction temperature and pH value are 45 oC and 6.0, respectively. Metal ions, such zinc, cupper and mercuric ion, significantly inhibit the enzyme activity. SDS also shows the inhibition on the enzyme activity. The dominant extracellular chitinase is capable of being purified with single column methyl hydrophobic interaction chromatography. The molecular mass of purified chitinase is 90 kDa and the purity is 90% above.
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