臘腸桿菌中幾丁質(zhì)酵素的選育與表現(xiàn)的研究

時(shí)間：2020-08-26 08:16:15 生物科學(xué)畢業(yè)論文我要投稿

　　摘要：本研究由Bacillus cereus NCTU2的染色體DNA選殖得幾丁質(zhì)酵素(ChiNCTU2)之基因，并成功的接進(jìn)載體pPCR上。經(jīng)由定序，幾丁質(zhì)酵素(ChiNCTU2)之基因共有1083個(gè)核苷酸，相當(dāng)於360個(gè)胺基酸。經(jīng)與Genebank基因資料庫上B. cereus ChiA比對(duì)，發(fā)現(xiàn)兩段基因有27個(gè)核苷酸不一樣，導(dǎo)致7個(gè)胺基酸的差異。經(jīng)與先前純化酵素之N端序列比較推測(cè)，其前27個(gè)胺基酸為訊息勝肽，剩余之333個(gè)胺基酸形成胞外酵素，分子量為36184 Da.

臘腸桿菌中幾丁質(zhì)酵素的選育與表現(xiàn)的研究

　　此酵素經(jīng)胺基酸序列比對(duì)，在醣類水解酵素中，屬於家族18,其蛋白質(zhì)結(jié)構(gòu)應(yīng)為(α/β)8的結(jié)構(gòu)，且僅具有單一催化區(qū)域(catalytic domain)，而不具備其他的輔助區(qū)域。

　　此基因被建構(gòu)於pRSET A載體上并以大腸桿菌[BL21(DE3)]表達(dá)之。其中含訊息勝肽之基因，(p)chi/21,無法大量表現(xiàn)幾丁質(zhì)酵素，但去除訊息勝肽之基因，(m)chi/21,則可表現(xiàn)出蛋白質(zhì)，可惜所得到之酵素為包涵體而不具活性。嘗試?yán)猛肝龅姆绞綄?duì)包涵體進(jìn)行蛋白質(zhì)再摺疊，尚未找到可以使幾丁質(zhì)酵素恢復(fù)活性之條件。

　　另外，ChiNCTU2此刻正被建構(gòu)於Bacillus megaterium之表現(xiàn)系統(tǒng)，本報(bào)告亦將涵蓋此系統(tǒng)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.

　　第一章緒論

　　1-1 前言

　　1-2 幾丁質(zhì)(chitin)

　　1-3 幾丁質(zhì)酵素(chitinase)

　　1-3.1幾丁質(zhì)酵素(chitinase)的概述及其種類

　　1-3.2幾丁質(zhì)酵素在演化上的分類

　　1-3.3幾丁質(zhì)酵素的水解反應(yīng)機(jī)制

　　1-3.4幾丁質(zhì)酵素的活性測(cè)定方法

　　1-4 幾丁質(zhì)酵素與幾丁寡醣之應(yīng)用

　　1-4.1幾丁質(zhì)酵素的應(yīng)用

　　1-4.2 N-乙醯幾丁質(zhì)寡醣的性質(zhì)與應(yīng)用

　　1-5枯草桿菌屬(Bacillus spp. )重組基因表現(xiàn)系統(tǒng)