Purification and Characterization of Creatinase by Recombinant Escherichia coli

基因重組大腸菌生產肌酸分解酵素之純化及特性分析

徐維富(Wei-Fu Hsu) ； 吳意珣(I-Son Ng) ； 蔡少偉(Shau-Wei Tsai) ； 張敏政(Ming-Cheung Chang)

《Journal of the Chinese Institute of Chemical Engineers》 34卷3期 (2003/05) Pp. 305-310

https://doi.org/10.6967/JCICE.200305.0305

摘要

本基因重組大腸桿菌含有來自Pseudomonas putida NTU-8的肌酸分解酵素基因，並在其前端插入一段來自於Aeromonas hydrophila幾丁質分解酵素的信號序列，故所生產之肌酸分解酵素可分泌至細胞間質。於LB培養液中以IPGT為誘導劑進行培養時，發現獲得最大菌體濃度及酵素產量之最佳誘導時間為1.75h。使用一系列分離步驟如離心、細胞打破、超過濾及FPLC自醱酵液進行酵素分離與純化，得到76.3%之酵素回收率以及提高2倍酵素比活性。純化酵素經SDS-PAGE及IEF電泳分析得到分子量為48.8kDa及等電點為4.52結果。不論粗醱素感純化酵素在37℃、pH為7.7附近均有最大活性，並受到Ni2+,Cu2+, Ag+ 和Hg2+等金屬離子抑制。粗醱素或純化酵素在37℃以下以及pH為5.5至9.4間均有較佳的穩定性。此外，肌酸分解酵素對肌酸的化學選擇性十分專一。

並列摘要

A recombinant Escherichia coli containing a gene from Pseudomonas putida NTU- 8 for producing creatinase and a signal peptide from Aeromonas hydrophila for transporting the enzyme into the periplasmic space was incubated in LB medium by employing IPTG as an inducer. The cell growth and creatinase production were optimized at the induction time of 1.75h by adding 100 mg/L of IPTG in a batch fermentor. By employing a series of separation steps such as centrifugation, cell breakage, ultrafiltration and FPLC purification, 76.3% of enzyme recovery with 2-fold enhancement of the specific creatinase activity from the broth was obtained. By using SDS-PAGE and IEF electrophoresis, the molecular weight of 48.8 kDa and pI of 4.52 for the enzyme was estimated. The maximum activity for the crude and purified creatinases was at 37℃ and pH around 7.7 Good enzyme thermal stability below 37℃ at pH 7.7 or at the pH between 5.5 and 9.4 at 37℃ was found. Inhibition or both enzymes by Ni2+,Cu2+, Ag+ and Hg2+ ions and good substrate specificity toward creatine were also reported.

並列關鍵字

Creatinase ； Recombinant E. coli ； Fermentation ； Purification