2021年臘腸桿菌中幾丁質酵素的選育與表現(xiàn)的研究

1、臘腸桿菌中幾丁質酵素的選育與表現(xiàn)的研究 摘要：本研究由Bacillus _reus NCTU2的染色體DNA選殖得幾丁質酵素(ChiNCTU2)之基因，并成功的接進載體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. _reus ChiA比對，發(fā)現(xiàn)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個胺基酸為訊息勝肽，剩余之333個胺基酸形成胞外酵素，分子量為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,其蛋白質結構應為(/)8的結

2、構，且僅具有單一催化區(qū)域(catalytic do _in)，而不具備其他的輔助區(qū)域。 此基因被建構於pRSET A載體上并以大腸桿菌BL21(DE3)表達之。其中含訊息勝肽之基因，(p)chi/21,無法大量表現(xiàn)幾丁質酵素，但去除訊息勝肽之基因，(m)chi/21,則可表現(xiàn)出蛋白質，可惜所得到之酵素為包涵體而不具活性。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(xiàn)系統(tǒng)，本報告亦將涵蓋此系統(tǒng)之先期研究成果。 Cloning and Expression of the Chit

3、inase from Bacillus _reus NCTU2 Abstract：A chitin-degrading Bacillus strain, designated as NCTU2, was screened from soil and identified. An extra- _llular chitinase was purified to 90% homogeneity from the culture filtrate. chitobiose is the predominant product through out the enzy _tic hydrolysis o

4、f the colloidal chitin, indicating that the purified chitinase is an exo-chitinase. The first 15 N-terminal amino acid sequen _ of the enzyme is determined to be ANNLGSKLLVGYWHN, which is highly homologous to that of ChiA from Bacillus _reus. PCR cloning technique was employed for obtaining the corr

5、esponding gene from the Bacillus NCTU2. The gene sequen _ was determined to be 1080 bp encoding a polypeptide of 360 amino acids. By inspecting the N-terminal sequen _ of the purified enzyme and the amino acid sequen _ dedu _d from the gene, the signal peptide is identified as the first 27 amino aci

6、ds of the enzyme. As pared 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. Sin _ the rebinant NCTU2 produ _d in E. coli was exhibited as an inclusion body, a Bacillus megaterium strain was attempte

7、d 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

8、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(xiàn)系統(tǒng) 1-5.1枯草桿菌屬之簡介 1-5.2以枯草桿菌屬作為生產重組蛋白質的宿主細胞 1-5.3 Bacillus megaterium表現(xiàn)系統(tǒng)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. _reus 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(tǒng)中幾丁質酵素之表現(xiàn) 2-3.1 chi gene與表現(xiàn)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(xiàn)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布情形 2-4重組幾丁質酵素的再折疊(Refolding) 2-4.1概述-文獻回顧 2-4.2重組幾丁質酵素的再折疊 2-5 Bacillus megaterium系統(tǒng)中幾丁質酵素