| Summary: | 博士 === 大葉大學 === 生物產業科技學系 === 95 === The biological function of protease, chitinase and hydrolysates were investigated in this study. In the first part, the protease of M. purpureus BCRC31499 was produced under the optimized culture condition. In the first step, the protease was precipitated and dialyzed by using ammonium sulfate. The further purification and separation procedures of the protease were processed by the use of DEAE Sepharose CL-6B ionic exchange chromatography. Purification was 27-fold with the crude enzyme solution. The overall activity yield of the purified protease was 6%, with specific protease activities of 10 U/mg. The final amount of the protease obtained was 1.6 mg. The protease had a molecular weight of 40 kDa and a pI of 7.9. The optimal pH, optimum temperature, pH stability of the protease were pH 7-9, 40℃, pH 5-9, and 40℃, respectively. In addition to protease activity, amino acids and peptides from the hydrolysis of the SCSP proteins by proteases also exhibited activity of enhancing vegetable growth. The protease would be used to produce biofertilizer in the future.
In the second part, we investigated the optimized hydrolysis condition for chitinous materials (water-soluble chitosan, chitin and colloidal chitin). The chitinous materials were hydrolyzed by B. amyloliquefaciens V656 crude enzyme solution. The optimized hydrolysis conditions were that 1% of water-soluble chitosan or 1% of chitin or 3% of colloidal chitin with 20% of crude enzyme solution, pH 5, at 40℃. The composition of the hydrolysates were analyzed by HPLC. It was found that the optimum temperature and reaction time for production of (GlcNAc)6 were 40℃ and 12 hours. Longer reaction time lead to the generation of (GlcNAc)n with lower DP’s.
In the third part, we investigated the antitumor actions of the hydrolysates produce by B. amyloliquefaciens V656 and M. purpureus BCRC31499 crude enzymes on the growth of colon carcinoma cell line, CT26. However, we found that the hydrolysates of crude enzyme solution produce by M. purpureus BCRC31499 had no significant effect on the growth of CT26 cell. But colon carcinoma cell was challenged with the hydrolysates of chitinous materials by B. amyloliquefaciens V656 crude enzyme solution for 1,2,3 days. The cell growth has been measured by MTT assay. The change of cell cycle distribution and induction of apoptosis caused by the hydrolysates of chitinous materials were examined by flow cytometry. Results indicated that when cells were treated with 500 g/mL of the hydrolysates, cell proliferation rate was significantly inhibited. The hydrolysates-treated cells indicated a block of S-phase and an elevated level of DNA fragmentation. Additionally, sub-G1 fraction (apoptotic cells) increased with increasing concentrations of the hydrolysates as analyzed by flow cytometry, using agarose gel electrophoresis to analyze the hydrolysates-treated cells, a similar result was found as that of flow cytometry. For the hydrolysates-induced apoptosis, loss of mitochondrial membrane potential was noted. These results suggested that the hydrolysates of chitinous materials by B. amyloliquefaciens V656 crude enzyme solution inhibited the growth of colon carcinoma cell line, CT26 through an accumulation of cell cycle at S-phase and an induction of apoptosis. We expect that these results will provide a new strategy for therapy of colon carcinoma in human beings.
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