Purification and characterization of chitinase produced by indeqineous Aeromonas sp.
許桓銘、
E-mail: 321378@mail.dyu.edu.tw
ABSTRACT
In this study, 11 strains were screened from soil all over Taiwan to produce chitinases. Among them, strains DN15 and DN23 produced higher level of reducing sugars. According to the DNA sequences of these two strains identified by the NCBI (National Center for Biotechnology Information), Strains DN15 and DN23 were then named as Aeromonas hydrophila DYU-Too15 and Aeromonas punctata DYU-Too16. The α-chitin content and nitrogen species in the CB (chitin broth) media were altered to search for a better culture condition to produce N-acetyloligosaccharides. In a CB medium, when the content of α-chitin was altered, there was no effect on the variety of N-acetyloligosaccharides produced by A. hydrophila DYU-Too15 or A. punctata DYU-Too16, but the content of chitooligosaccharides increased with the increase of the α-chitin concentration. If the medium contained 4%
α-chitin, the highest amount of N-acetylglucosamine (about 3.65 g/L) was produced by A. hydrophila DYU-Too15 at 96 h. When the medium contained 5% α-chitin, N-acetylchitotriose was the major product and reached about 1.22 g/L at 96 h. There was no effect on the variety of N-acetyloligosaccharides produced by A. hydrophila DYU-Too15 in a CB medium with various nitrogen sources (yeast extract, peptone, tryptone, yeast extract + peptone, NH4Cl). The major product was N-acetylglucosamine. For A.
punctata DYU-Too16 in a CB medium with yeast extract and peptone as the nitrogen sources, the major products included N-acetylglucosamine and N-acetylchitotriose. However, the only major product was N-acetylglucosamine if each of the other four nitrogen sources was used as the nitrogen source in the CB medium. In order to obtain N-acetylchitooligosaccharides, the newly screened two strains and the early obtained strain, Aeromonas hydrophila DYU-Too14, in our laboratory were examined. A.
hydrophila DYU-Too14 can produce N-acetylchitopentaose and N-acetylchitohexaose in a CB medium by using NH4Cl as a nitrogen source. Since N-acetylchitopentaose and N-acetylchitohexaose can enhance the immune system, inhibit tumor cell growth and possess other physiological activities, and their values are much higher than N-acetylglucosamine and N-acetylchitotriose produced by the other two strains. Thereafter, A. hydrophila DYU-Too14 was used as the target strain for chitinase purification. To separate the chitinase produced by A. hydrophila DYU-Too14, this strain was cultivated in a CB medium containing 4% α-chitin as the carbon source and 0.7 g/L NH4Cl as the nitrogen source. The supernatant of the culture containing crude enzyme was first precipitated by ammonium sulfate, and then the precipitate was further purified through dialysis, anion gel (DEAE-Sepharose) chromatography. From DEAE-Sepharose gel chromatographic diagram, two peaks of Fractions 90-93 and 94-98 possessed chitinase activity. Hence, the above chitinase was used to hydrolyze colloidal chitin solution, the hydrolysates were separated through
centrifuge and lyophilization, and its composition was analyzed by HPLC. The hydrolysates contained N-acetylchitopentaose and N-acetylchitohexaose. Through electrophoresis, the molecular weight of the chitinase was identified to be 25 kDa.
Keywords : chitinase、Aeromonas hydrophila DYU-Too14、N-acetylglucosamine、N-acetylchitotriose、N-acetylchitopentaose
、N-acetylchitohexaose
Table of Contents
封面內頁 簽名頁 授權書 iii 中文摘要 iv 英文摘要 vi 致謝 vii 目錄 viii 圖目錄 xii 表目錄 xv 1. 緒論 1 2. 文獻回顧 2 2.1 幾丁質 2 2.2 N-乙醯幾丁寡醣相關衍生物與應用 2 2.2.1抗菌活性 2 2.2.2免疫活性 4 2.2.3 基因輸送載體 6 2.2.4 藥物輸送載體 7 2.3 N-乙醯幾丁寡醣的製備 9 2.3.1 化學法 9 2.3.2 酵素法 10 2.4 N-乙醯幾丁寡醣的分離與純化 11 2.4.1 膠體過濾層析法 11 2.4.2 離子交換層析法 12 3.材料方法 13 3.1 實驗藥品 13 3.2 實驗器材 14 3.3實驗試劑 15 3.3.1 培養基組成 15 3.3.2 膠態幾丁質之 製備 17 3.3.3 McIlvaine buffer之配製 17 3.3.4 呈色劑之配置 17 3.4 實驗方法 17 3.4.1菌株篩選、保存及活化 19 3.4.2幾丁質分 解?′〝吨尷R 19 3.4.3還原醣含量之測定 21 3.4.4蛋白質濃度測定 21 3.4.5幾丁質水解產物之HPLC分析 21 3.4.6分離純化幾 丁質? 22 3.4.7聚丙烯醯胺膠體電泳分析 24 4.結果與討論 27 4.1菌株於膠態幾丁質培養基生長情形 27 4.2分解幾丁質菌株之 篩選 27 4.2.1 菌株Aeromonas hydrophila DYU-Too15 32 4.2.2 菌株Aeromonas punctata DYU-Too16 32 4.2.3 菌株Aeromonas sp. DYU-Too14之特性 32 4.3菌株培養於不同含量α-幾丁質之CB培養基 35 4.3.1 菌株A. hydrophila DYU-Too15 35 4.3.1.1 幾 丁質?′〝吨坐尷R 35 4.3.1.2還原醣量與pH值變化 35 4.3.1.3幾丁質水解產物分析 37 4.3.2 菌株A. punctata DYU-Too16 37 4.3.2.1幾丁質?′〝吨坐尷R 42 4.3.2.2還原醣量與pH值變化 42 4.3.2.3幾丁質水解產物分析 42 4.4不同氮源培養菌株 46 4.4.1 菌株A. hydrophila DYU-Too15 46 4.4.1.1 幾丁質?′〝吨坐尷R 46 4.4.1.2 還原醣量與pH值變化 51 4.4.1.3 幾丁質水解產物分 析 51 4.4.2 菌株A. punctata DYU-Too16 55 4.4.2.1幾丁質?′〝吨坐尷R 55 4.4.2.2 還原醣量與pH值變化 55 4.4.2.3 幾丁質水
解產物分析 58 4.5 幾丁質分解?﹞坐擢鰳瞻 58 4.5.1 硫酸銨沉澱 61 4.5.2 離子交換層析 61 4.5.3 膠體過濾層析 66 5.結論 72 5.1 結論 72 5.2 展望 73 參考文獻 75 附錄 82
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