| Summary: | 碩士 === 中華醫事科技大學 === 生物醫學研究所 === 101 === N-acetyl glucosamine (NAG) is the final hydrolysis product of a natural polysaccharide, chitin. It has been found to have many biological functions. Previously, Chen et al’s study had found the bacteria, Chitinibacter tainanensis, can degrade chitin particles into soluble reducing sugar, NAG. However, they don’t further investigate the extracellular chitinase. In this study, we recorded 0 ~ 20 hours growth curve of Chitinibacter tainanensis in LB medium (containing 1 mg / mL β-chitin), and found that Chitinibacter tainanensis bacterial reached a growth peak at 14 hours. Comparing the chitinase activity between bacteria pellet and culture supernatant by chitin degradation assay, we found that only the latter has enzymatic activity, which was suggested as an extracellular chitinase. Furthermore, with or without chitin induction, Chitinibacter tainanensis can secrete chitinase, suggested that chitinase may be a non-inductive enzyme. After the culture supernatant was first precipitated with 30% ammonium sulfate to filtrate contaminating proteins, and then with 75% ammonium sulfate precipitation, this can be achieved initial purification and concentration. In this study, we purified target chitinase by hydrophobic, ion-exchange and gel filtration chromatography. SDS-PAGE and zymogram assay had been performed to analyze chitinase activity as well as their molecular weight. Our results showed that they display chitinase activity around molecular weight of 33, 66.2 and 120 kDa after hydrophobic chromatography. In the Native-PAGE experiment, we found that the distribution of chitinases purified from hydrophobic chromatography shift to the larger molecular weight, which indicated that Chitinibacter tainanensis extracellular chitinase may be a group of CDFs. In addition, we have purified the chitinase with the molecular weight of 33 kDa by Superdex 75 chromatography. In the chitinase plate assay, we compare the chitinase activity before and after hydrophobic chromatography. The results showed that the purified chitinase from hydrophobic chromatography range significantly greater than the non-purified supernatant crude protein. In the future, we will establish the quantitative methods of extracellular chitinase analysis. Cloning of extracellular chitinase genes is the future work in this study as the basis of the future production of recombinant enzyme.
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