摘要：本研究由Bacillus cereus NCTU2的染色體DNA選殖得幾丁質酵素(ChiNCTU2)之基因，併成功的接進載體pPCR上。經由定序，幾丁質酵素(ChiNCTU2)之基因共有1083個核苷酸，相當於360個胺基酸。經與Genebank基因資料庫上B. cereus ChiA比對，發現兩段基因有27個核苷酸不一樣，導致7個胺基酸的差異。經與先前純化酵素之N端序列比較推測，其前27個胺基酸爲訊息胜肽，剩餘之333個胺基酸形成胞外酵素，分子量爲36184 Da.
此酵素經胺基酸序列比對，在醣類水解酵素中，屬於家族18,其蛋白質結構應爲(α/β)8的結構，且僅具有單一催化區域(catalytic domain)，而不具備其他的輔助區域。
此基因被建構於pRSET A載體上並以大腸桿菌[BL21(DE3)]表達之。其中含訊息胜肽之基因，(p)chi/21,無法大量表現幾丁質酵素，但去除訊息胜肽之基因，(m)chi/21,則可表現出蛋白質，可惜所得到之酵素爲包涵體而不具活性。嘗試利用透析的方式對包涵體進行蛋白質再摺疊，尚未找到可以使幾丁質酵素恢復活性之條件。
另外，ChiNCTU2此刻正被建構於Bacillus megaterium之表現系統，本報告亦將涵蓋此係統之先期研究成果。
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.
目錄
第一章 緒論
1-1 前言
1-2 幾丁質(chitin)
1-3 幾丁質酵素(chitinase)
1-3.1幾丁質酵素(chitinase)的概述及其種類
1-3.2幾丁質酵素在演化上的分類
1-3.3幾丁質酵素的水解反應機制
1-3.4幾丁質酵素的活性測定方法
1-4 幾丁質酵素與幾丁寡醣之應用
1-4.1幾丁質酵素的應用
1-4.2 N-乙醯幾丁質寡醣的性質與應用
1-5枯草桿菌屬(Bacillus spp. )重組基因表現系統
1-5.1枯草桿菌屬之簡介
1-5.2以枯草桿菌屬作爲生產重組蛋白質的宿主細胞
1-5.3 Bacillus megaterium表現系統之簡介
1- 6研究目的
第二章 實驗方法與步驟
2-1 概述
2-1.1一般敘述
2-1.2藥品、器材與儀器總覽
2-1.3幾丁質酵素活性之測定
2-2幾丁質酵素的基因選殖及定序
2-2.1 B. cereus NCTU2染色體DNA的.純化
2-2.2幾丁質酵素基因的選殖
2-2.3幾丁質酵素基因與pPCR載體的接合(ligation)
2-2.4基因片段之定序(cycle-sequencing
2-3 E. coli系統中幾丁質酵素之表現
2-3.1 chi gene與表現型載體(expression vector)pRSET A的接合(ligation)
2-3.2質體DNA之轉型
2-3.3重組幾丁質酵素誘導表現之測試
2-3.4重組幾丁質酵素之活性測試
2-3.5重組蛋白質在細胞內的分佈情形
2-4重組幾丁質酵素的再摺疊(Refolding)
2-4.1概述--文獻回顧
2-4.2重組幾丁質酵素的再摺疊
2-5 Bacillus megaterium系統中幾丁質酵素之表現
2-5.1質體DNA 之建構
2-5.2 B. megaterium WH320原生質體(protoplasts)的製備
2-5.3質體DNA的轉型
2-5.4 B. megaterium質體DNA的純化
2-5.5蛋白質表現之測試
第三章 結果與討論