Analysis of the Substrate-binding Domain of Chitinase and Enzyme Activity assistant Factor for Chitinase

• Main Authors: Chen, Yu-Chung, 陳昱仲
• Other Authors: Chang Ming-Chung
• Format: Others
• Language: zh-TW
• Published: 1996
• Online Access: http://ndltd.ncl.edu.tw/handle/51320943415467519257

碩士 === 國立成功大學 === 生物化學研究所 === 84 === Aeromonas hydrophila分泌一個由865個胺基酸組成，分子量約94 kDa的幾丁分解酵素，此酵素之蛋白質結構主要是由N端訊息序列，催化區 域以及含有兩個重覆序列區域的C端 (93個胺基酸) 所組成；而位於C端的 二個重覆序列 (CICII) 是具有51%相同性的35個胺基酸所構成。藉由蛋白 質序列相似性比對分析顯示，幾丁質分解酵素的兩重覆序列區域和 Bacillus sp. strain N-4的纖維水解酵素的C端區域有很高的相似性。　 　在結合作用的研究發現，C端截短的幾丁分解酵素及C端 (CICII) 融合 到glutathione S-transferase (GST) 的融合蛋白均顯示C端重覆序列能 調控幾丁分解酵素對膠狀幾丁質及纖維素的結合能力，且GST-CI (或GST- CII) 的融合蛋白本身即可和膠狀幾丁質進行結合作用。在上述的蛋白質 序列比對分析，發現有八個Trp是最具保存性的胺基酸，分別是Trp-773、 Trp-792、Trp-897、Trp-810、Trp-820、Trp-839、Trp-844以及Trp-856 ；將Trp-792以定點突變法將Trp (W)突變為Gly (G)，和野生株做幾丁質 結合能力的比較，發現不管是兩重覆序列或單一重覆序列結合區域的突變 株 (GST-CICIIW792G或GST-CIW792G)，其幾丁質結合能力分別比野生株 (GST-CICII或GST-CI) 約下降20-30%。 在幾丁分解酵素活性輔助因子 的研究中，發現在幾丁分解酵素活性突變株中，幾丁分解酵素結構基因上 游500 bp以及下游3 kb是影響酵素是否具有活性的重要因素。 Chitinase (ChiA) secreted by Aeromonas hydrophila is a 865-amino-acid (aa) polypeptide composed of an N-terminal signal peptide, a catalytic domain and a C-terminal chitin-binding domain containing two subdomains (CI and CII) which showed strong similarity (51% identity). By comparison with other glycosyl hydrolases, the C-terminal subdomains (CI or CII) showed significant similarity to C-terminal region of cellulase from Bacillus sp. strain N-4. In binding studies, intact ChiA binded strongly not only to colloidal chitin but also to cellulose, while the truncated ChiA derivatives lacking the C- terminal domain absorbed weakly to the colloidal chitin and cellulose. When CI-CII was fused to glutathione S-transferase (GST), it exhibited the binding property to colloidal chitin and cellulose, and furthermore, GST-CI (or GST-CII) also showed the signficant chitin-binding property. These observations indicated that C-terminal subdomains modulate the binding character of ChiA to colloidal chitin and cellulose. There are eight Trp residues (Trp-773, Trp-792, Trp-797, Trp-810, Trp-820, Trp-839, Trp-844 and Trp-856 in ChiA) conserved between the two C-termini of the A .hydrophila chitinase subdomains and the Bacillus cellulase. Among them, Trp-792 was chosen as the target for the amino acid substitution (Trp ?Gly) by site-directed mutagenesis. The results of binding experiments showed that the binding activites of the two mutants, GST-CICIIW792G and GST- CIW792G, were reduced to 70-80% when compared to those of GST- CICII and GST-CI, respectively. In the study of enzyme activity assistant factors for chitinase by using E. coli mutant, it was found that both the 0.5 kb upstream sequence and 3 kb downstream sequence may play an important role in determining the formation of the active form of chitinase.