基因改造黏質沙雷氏桿菌幾丁質酵素之特性

Abstract

本研究由Serratia marcescens 的染色體DNA上選殖出幾丁質酵素ChiA之基因，並成功地接進載體pRSET A上。經由定序，得知此幾丁質酵素（ChiA_NCTU）之基因共有1692個核苷酸，轉譯後可得563個胺基酸，分子量相當於61033 Da。 轉殖後的JM 109宿主，經培養15小時後取胞內酵素，經硫酸銨鹽沈澱、 HIC管柱純化後，再經陰陽離子交換樹脂串聯管柱層析，可得90 %以上純度的ChiA_NCTU，經質譜儀鑑定為一成熟蛋白質（mature protein）。 純化所得的ChiA_NCTU其最適反應溫度、pH分別為55 ℃、7.0，酵素在pH 5.0 ~ 8.0穩定性良好。酵素水解膠狀幾丁質所得之產物為雙醣及少數單醣。經與幾丁寡醣反應後，證實為一exo-chitinase。本研究也對此基因進行改造，希望藉由基因改造在酵素中形成雙硫鍵，以期增加酵素的熱穩定性，並研究其對酵素水解最終產物的影響。 利用此轉殖系統可大量表現並得一高純度ChiA_NCTU，其具有可生產單一幾丁寡醣的特性，再透過萃取的方式以提高產物純度，已可達到大量生產具經濟價值N-乙醯幾丁二醣的目的。

A chitinase gene of Serratia marcescens (ChiA) was PCR cloned. The ChiA was further constructed into pRSETA vector and transformed into Escherichia coli JM109 cells for protein expression. DNA sequence analysis and comparison revealed that the region requires for protein expression involving an open reading frame of 1689 base pairs, correspondent to 563 amino acids with a N-terminal signal peptide of 23 residues. An intracellular chitinase was further purified to＞90% homogeneity by the hydrophobic interaction chromatography following by ion-exchange separation. Chitobiose is the predominant product through out the enzymatic hydrolysis of the colloidal chitin, indicating that the purified chitinase is an exo-chitinase. In addition, the gene was mutagenized to insert an extra disulfide bond between residues 441 and 521, 521 and 551. The activities and the end product (chitobiose) of these doubly mutated enzymes did not perturbed. With the application of the wild-type chitinase, a 10-gramed scale reaction was performed for chitobiose preparation.