綠竹筍幾丁質酶之純化與生化性質研究

Purification and characterization of chitinase from shoots of Green Bamboo Bambusa oldhamii

林宜蓁(I-Tsen Lin)

指導教授 ： 宋賢一

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.01952

摘要

藉由含有 4-MU (GlcNAc)3 之SDS-PAGE 及幾丁三糖酶活性染色，和含有乙二醇幾丁質之 SDS-PAGE 及幾丁質酶活性染色與活性測定，可知綠竹筍外殼中有多種幾丁質酶異構酶存在。將綠竹筍外殼經過 20 – 80% 硫酸銨飽和濃度沉澱、陰離子交換管柱、Con A 親和層析法後得到一種幾丁三糖酶，將其命名為 Chi A。綠竹筍外殼經 60 – 80% 硫酸銨飽和濃度沉澱及陰離子交換管柱，再經 Con A 親和層析法或疏水性作用管柱層析後，得到兩種幾丁質酶，將其命名為 Chi B 及 Chi C。幾丁三糖酶 Chi A 與兩種幾丁質酶 Chi B、Chi C 分子量分別為 30、30、29 kD。Chi A 之最適 pH 值為 3。以陰離子交換層析估計 Chi A 之等電點約在 7 – 9 之間，Chi B 及 Chi C 等電點在 7 以下。本研究顯示以二次元電泳進行幾丁質酶活性染色為可行之方法，故以二次元電泳及活性染色預測 Chi C 之等電點在 4.7。以 4-MU (GlcNAc)3 為基質，幾丁三糖酶 Chi A 之 Km 值為 2.5 μM。以4-MU (GlcNAc)2 為基質，Chi A 之 Km 值為 16 μM，推論 Chi A 對 4-MU (GlcNAc)3 之親和力較 4-MU (GlcNAc)2 大。Chi A 水解乙二醇幾丁質產物分析為 (GlcNAc)3，Chi B 產物為 (GlcNAc)13 至 (GlcNAc)15，Chi C 則得到 (GlcNAc)4 和 (GlcNAc)23 之產物。幾丁質酶 Chi C 之基質特異性顯示對乙二醇幾丁質及乙二醇幾丁聚糖具有較大活性，CM-chitin、CM-chitosan 及膠態幾丁質則次之。以 LC-MS-MS 進行胺基酸定序，Chi B 所含 LMGSALNTKR 序列和阿拉伯芥醣苷水解酶第十八家族中之第三類酸性幾丁質酶相同。

關鍵字

幾丁質 ； 純化 ； 幾丁質酶幾丁三糖酶

並列摘要

chitotriosidase activity assay, SDS-PAGE and enzyme activity staining was assayed by 4-methylimbelliferyl-β-D-N,N’,N’’-triacetylchitotrioside [4-MU (GlcNAc)3]. Chitinase activity assay, SDS-PAGE and enzyme activity staining was assayed by ethylene glycol chitin (EGC) embedded as substrate. Several chitinase isoenzyme were presented in the shell of bamboo shoots. One chitotriosidase, designated as Chi A was purified by 20 – 80% ammonium sulfate precipitation, DEAE-Sephacel and Con A Sepharose column chromatography. Two chitinase, designated as Chi B and Chi C, were isolated by 60 – 80% ammonium sulfate precipitation, DEAE-Sephacel, Con A Sepharose and Phenyl Sepharose 6 column chromatography. By SDS-PAGE and enzyme activity staining, the molecular mass of Chi A, Chi B and Chi C were 30, 30 and 29 kD, respectively. The Optimal temperature of Chi A was 70℃. Thermal stability of Chi A was 30 – 50℃. The Optimal pH of Chi A was 3. pH stability of Chi A was 3 – 5. The isoelectric point estimated by ion exchange chromatography was 7 – 9 (Chi A) and below 7 (Chi B and Chi C). This thesis established the feasibility of 2D electrophoresis and chitinase activity staining. The 2D electrophoresis and chitinase activity staining showed the isoelectric point of Chi C was 4.7. For hydrolysis of 4-MU (GlcNAc)3 ,Km of Chi A was 2.5 μM. For hydrolysis of 4-MU (GlcNAc)2 ,Km of Chi A was 16 μM. The substrate hydrolyzed of EGC by Chi A was (GlcNAc)3. The substrate hydrolyzed of EGC by Chi B were (GlcNAc)13 - (GlcNAc)15, but (GlcNAc)4 and (GlcNAc)23 by Chi C. Substrate stability showed the activity of Chi C to ethylene glycol chitin and ethylene glycol chitosan were higher than CM-chitin、CM-chitosan and colloidal chitin. Using LC-MS-MS for amino sequencing indicated Chi B contains the sequence of LMGSALNTKR which was found in arabidopsis (Arabidopsis thaliana) class III acidic chitinase.

並列關鍵字

purification ； 4mu ； chitin ； chitinase ； chitotriosidase

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