腊肠杆菌中几丁质酵素的选育与表现的研究
摘要：本研究由Bacillus cereus NCTU2的染色体DNA选殖得几丁质酵素(ChiNCTU2)之基因，并成功的接进载体pPCR上。经由定序，几丁质酵素(ChiNCTU2)之基因共有1083个核苷酸，相当於360个胺基酸。经与Genebank基因资料库上B. cereus ChiA比对，发现两段基因有27个核苷酸不一样，导致7个胺基酸的差异。经与先前纯化酵素之N端序列比较推测，其前27个胺基酸为讯息胜肽，剩余之333个胺基酸形成胞外酵素，分子量为36184 Da.
此酵素经胺基酸序列比对，在醣类水解酵素中，属於家族18,其蛋白质结构应为(α/β)8的结构，且仅具有单一催化区域(catalytic domain)，而不具备其他的辅助区域。
此基因被建构於pRSET A载体上并以大肠杆菌[BL21(DE3)]表达之。其中含讯息胜肽之基因，(p)chi/21,无法大量表现几丁质酵素，但去除讯息胜肽之基因，(m)chi/21,则可表现出蛋白质，可惜所得到之酵素为包涵体而不具活性。尝试利用透析的方式对包涵体进行蛋白质再摺叠，尚未找到可以使几丁质酵素恢复活性之条件。
另外，ChiNCTU2此刻正被建构於Bacillus megaterium之表现系统，本报告亦将涵盖此系统之先期研究成果。
Cloning and Expression of the Chitinase from Bacillus cereus NCTU2
Abstract：A chitin-degrading Bacillus strain, designated as NCTU2, was screened from soil and identified. An extra-cellular chitinase was purified to > 90% homogeneity from the culture filtrate. chitobiose is the predominant product through out the enzymatic hydrolysis of the colloidal chitin, indicating that the purified chitinase is an exo-chitinase. The first 15 N-terminal amino acid sequence of the enzyme is determined to be ANNLGSKLLVGYWHN, which is highly homologous to that of ChiA from Bacillus cereus. PCR cloning technique was employed for obtaining the corresponding gene from the Bacillus NCTU2. The gene sequence was determined to be 1080 bp encoding a polypeptide of 360 amino acids. By inspecting the N-terminal sequence of the purified enzyme and the amino acid sequence deduced from the gene, the signal peptide is identified as the first 27 amino acids of the enzyme. As compared with the gene of Chi36 and that of NCTU2, though both enzymes are similar in molecular size, 70 nucleotides resulting in 16 amino acids are different. Since the recombinant NCTU2 produced in E. coli was exhibited as an inclusion body, a Bacillus megaterium strain was attempted to be used as the expression host. The preliminary study on construction of the NCTU2 in this system was also included in this report.
目录
第一章 绪论
1-1 前言
1-2 几丁质(chitin)
1-3 几丁质酵素(chitinase)
1-3.1几丁质酵素(chitinase)的概述及其种类
1-3.2几丁质酵素在演化上的分类
1-3.3几丁质酵素的水解反应机制
1-3.4几丁质酵素的活性测定方法
1-4 几丁质酵素与几丁寡醣之应用
1-4.1几丁质酵素的应用
1-4.2 N-乙醯几丁质寡醣的性质与应用
1-5枯草杆菌属(Bacillus spp. )重组基因表现系统
1-5.1枯草杆菌属之简介
1-5.2以枯草杆菌属作为生产重组蛋白质的宿主细胞
1-5.3 Bacillus megaterium表现系统之简介
1- 6研究目的
第二章 实验方法与步骤
2-1 概述
2-1.1一般叙述
2-1.2药品、器材与仪器总览
2-1.3几丁质酵素活性之测定
2-2几丁质酵素的基因选殖及定序
2-2.1 B. cereus NCTU2染色体DNA的.纯化
2-2.2几丁质酵素基因的选殖
2-2.3几丁质酵素基因与pPCR载体的接合(ligation)
2-2.4基因片段之定序(cycle-sequencing
2-3 E. coli系统中几丁质酵素之表现
2-3.1 chi gene与表现型载体(expression vector)pRSET A的接合(ligation)
2-3.2质体DNA之转型
2-3.3重组几丁质酵素诱导表现之测试
2-3.4重组几丁质酵素之活性测试
2-3.5重组蛋白质在细胞内的分布情形
2-4重组几丁质酵素的再折叠(Refolding)
2-4.1概述--文献回顾
2-4.2重组几丁质酵素的再折叠
2-5 Bacillus megaterium系统中几丁质酵素之表现
2-5.1质体DNA 之建构
2-5.2 B. megaterium WH320原生质体(protoplasts)的制备
2-5.3质体DNA的转型
2-5.4 B. megaterium质体DNA的纯化
2-5.5蛋白质表现之测试
第三章 结果与讨论
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