Production of Chitinase and N-Acetylchitooligosaccharides from Bacillus sp. DYU-Too 20 陳岳澤、吳淑姿
E-mail: 387188@mail.dyu.edu.tw
ABSTRACT
In this study, Bacillus sp. DYU-Too 20 was isolated from Hsinchu. The aim of this study was to investigate the effects of an optimal condition for production of N-acetylchitotetrasaccharide by Bacillus sp. DYU-Too 20. In addition, the chitinase produced by this strain was purified and characterized. The optimal condition of Bacillus sp. DYU-Too 20 to investigate the effects of carbon source on the production of N-acetyltetrasaccharide. When α-chitin was the sole carbon source, the major product was
N-acetyltetrasaccharide. Especially, the highest production of N-acetyltetrasaccharide (0.144 g/L) was obtained in a medium of 4%
α-chitin; NH4Cl seemed to be a better nitrogen source to produce N-acetyl- tetrasaccharide, and the production was 0.256 g/L in a medium containing 0.1 g/L NH4Cl; The highest yield of N-acetyltetrasaccharide (0.284 g/L) was obtained at 35℃. The crude enzyme was obtained from a culture of Bacillus sp. DYU-Too 20 in medium containing 4% α-chitin and 0.1 g/L NH4Cl at 35℃.
The purification procedures included precipitation by ammonium sulfate, dialysis, and anion exchange chromatograph
(DEAE-Sepharose CL-6B). From DEAE-Sepharose gel chromatographic diagram, one peak of Fraction 83-95 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-acetyl- glucosamine, N-acetylchitodisaccharide and N-acetyltetrasaccharide. . Through electrophoresis, the molecular weight of the chitinase was 26 kDa.
Keywords : chitinase、Bacillus sp. DYU-Too 20、N-acetyl-glucosamine、N-acetylchitodisaccharide、N-acetyltetrasaccharide
、Optimum condition
Table of Contents
封面內頁 簽名頁 中文摘要 iii 英文摘要 iv 誌謝 v 目錄 vii 圖目錄 xi 表目錄 xiv 1. 緒論 1 2. 文獻回顧 2 2.1 幾丁質 2 2.1.1 幾丁 質類之分子結構與性質 2 2.2 幾丁質相關衍生物與應用 4 2.2.1 免疫活性 4 2.2.2 幾丁質之應用 5 2.3 N-乙醯幾丁寡醣與幾丁 寡醣 6 2.4 幾丁質分解酵素 6 2.4.1 幾丁質分解酵素之來源 7 2.4.2 幾丁質分解酵素之應用 8 2.4.3近年幾丁質分解?之研究 9 3.
材料與方法 12 3.1 實驗架構 12 3.2 實驗藥品 12 3.3 實驗器材 14 3.4 試劑及培養基配製 15 3.4.1培養基組成 15 3.4.2膠態幾丁 質製備 15 3.4.3 McIlvaine buffer之配製 16 3.5實驗方法 16 3.5.1菌株篩選、保存與活化 16 3.5.2 菌株生長曲線之測定 19 3.5.3 酵素法製備N-乙醯幾丁寡醣 19 3.6 分析方法 19 3.6.1幾丁質分解酵素之活性測定 19 3.6.2蛋白質濃度測定 20 3.6.3幾丁質之 水解產物的處理與分析 20 3.7幾丁質分解酵素之純化與特性分析 21 3.7.1幾丁質分解酵素之純化 21 3.7.2 酵素分子量 22 3.7.2.1聚丙烯醯胺膠體電泳分析 22 4. 結果與討論 24 4.1 菌株於膠態幾丁質培養生長情形 24 4.2 篩選幾丁質酵素生產菌株 24 4.3 菌株粗發酵液之特性分析 28 4.3.1 菌株Ze1 28 4.3.2 菌株Ze2 28 4.3.3 菌株Ze3 31 4.3.4 菌株Ze4 33 4.3.5 菌株Ze5 33 4.3.6 篩選菌株之水產分析 35 4.4 菌株DYU-Too 20之基本特性分析 38 4.5 不同碳源濃度培養菌株 42 4.5.1 幾丁質?活性分析 42 4.5.2 還原醣與pH值變化 42 4.5.3 水解產物分析 46 4.6 α-幾丁質濃度 48 4.6.1 幾丁質?活性分析 48 4.6.2 還原醣與pH值變 化 48 4.6.3 水解產物分析 50 4.7 氮源 54 4.7.1 幾丁質?活性分析 54 4.7.2 還原醣量與pH值變化 54 4.7.3 水解產物分析 57 4.8 NH4Cl濃度 60 4.8.1 幾丁質?活性分析 60 4.8.2 還原醣與pH值變化 60 4.8.3 水解產物分析 64 4.9 溫度 64 4.9.1 幾丁質?活性分 析 66 4.9.2 還原醣量與pH值變化 66 4.9.3 水解產物分析 70 4.10 幾丁質?之分離純化 70 4.10.1 硫酸銨沉澱 70 4.10.2 離子交換 層析 72 4.10.3 膠體過濾層析 72 4.10.4 酵素分子量測定 75 5. 結論 79 5.1 結論 79 5.2 展望 80 參考文獻 81 附錄 88
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