Cloning and Expression of the Chitinase from Bacillus cereus NCTU2
Dr. Yaw-Kuen Li
|關鍵字:||仙人掌桿菌;幾丁質;幾丁質酵素;選殖;表現;包涵體;Bacillus cereus;chitin;chitinase;cloning;expression;inclusion bodies;Bacillus megaterium|
|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，並成功的接進載體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個胺基酸為訊息胜肽，剩餘之333個胺基酸形成胞外酵素，分子量為36184 Da。
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.
|Appears in Collections:||Thesis|