仙人掌桿菌幾丁質酶基因選殖及表現之研究

Molecular Cloning and Gene Expression of the Chitinase from Bacillus cereus BCRC 11026

林姿秀(Zih-Shiou Lin)

指導教授 ： 許輔

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.01696

摘要

葡萄糖胺與幾丁寡糖為幾丁質 (chitin) 之水解產物，在許多工業及生物技術方面佔有重要角色。葡萄糖胺與幾丁寡糖可由幾丁質酶分解幾丁質而獲得，因而本研究主要之目的為純化、轉殖並於大腸桿菌系統表現仙人掌桿菌屬 Bacillus cereus BCRC 11026 中之幾丁質酶。實驗上自仙人掌桿菌屬之 B. cereus BCRC 11026 萃取粗蛋白，經硫酸銨沉澱、快速蛋白質液相層析及活性測試等步驟，分離出具有幾丁質分解能力之幾丁質水解酶，經由 SDS-PAGE 分析結果顯示蛋白質分子量約為 61 kDa，後續進行 N 端胺基酸序列分析，發現其 N 端胺基酸序列為 SDIKY。之後以 B. cereus BCRC 11026 之全 DNA 為模板，以N 端胺基酸序列搜尋基因庫所得之同源基因序列為引子進行聚合酶連鎖反應，獲得 B. cereus BCRC 11026 之幾丁質酶基因 GD1A4，並構築成含有幾丁質酶基因的質體 pET32a-GD1A。將 pET32a-GD1A 轉殖入 Escherichia coli BL-21 表現後，進行 SDS-PAGE 與 western blotting 分析，顯示此重組蛋白分子量約為 38.5 kDa。分析發現此重組幾丁質水解酶之純化倍率為 48.78，回收率為 1.67 %，比活性為 12.536 U/mg，其最適反應溫度約為 60℃，最適反應 pH 值為 6.0，在 60 ℃以下熱安定。此幾丁質水解酶可利用於葡萄糖胺與幾丁寡糖之生產。

關鍵字

幾丁質 ； 幾丁質酶

並列摘要

N-acetyl-D-glucosamine (GlcNAc) and N-acetyl chitooligosaccharides, the products of chitin, are important to several industries and biotechnology sectors. They could be hydrolyzed from chitin by chitinase. The aim of this study was to purify and clone the gene encoding chitinase from Bacillus cereus BCRC 11026 and to perform recombinant expression in a prokaryotic E. coli system. Chitinase was purified from B. cereus BCRC 11026 through ammonium sulfate fractionation and Mono S ion-exchange chromatography. Its molecular mass was about 61 kDa, as determined by SDS-PAGE. The N-terminal amino acid sequence of the purified chitinase was further determined. Cloning was carried out using PCR technique, using the homogeneous gene sequences of the purified chitinase as the primers. The chitinase fragment (GD1A4) amplified from the genomic DNA of B. cereus BCRC 11026 was ligated with pET-32a (+) vector to generate an expression vector, pET32a-GD1A4. In order to achieve successful expression of the recombinant chitinase, the plasmid pET32a-GD1A4 was then transferred into E. coli BL-21 to produce the recombinant protein with a molecular weight of 38.5 kDa. Furthermore, the recombinant chitinase were purified and concentrated by 48.78 fold with a recovery of 1.67 % and a specific activity of 12.536 U/mg. The optimal pH and temperature for the recombinant chitinase wase 6.0 and 60 ℃, respectively, and the enzyme activity was relatively stable below 60 ℃. This chitinase could be utilized for the production of N-acetyl-D-glucosamine and N-acetyl chitooligosaccharides.

並列關鍵字

chitin ； chititnase

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