从马塞利亚提蒙纳中纯化和表征两种细胞外内切几丁质酶。

Purification and characterization of two extracellular endochitinases from Massilia timonae.

机构信息

Department of Pharmaceutical Biotechnology, Biotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, PO Box 14155-6451, Tehran 14174, Iran.

出版信息

Carbohydr Res. 2010 Feb 11;345(3):402-7. doi: 10.1016/j.carres.2009.11.015. Epub 2009 Nov 13.

DOI:10.1016/j.carres.2009.11.015

PMID:19962135

Abstract

Two extracellular chitinases (designated as Chi-56 and Chi-64) produced by Massilia timonae were purified by ion-exchange chromatography, ammonium sulfate precipitation, and gel-filtration chromatography. The molecular mass of Chi-56 was 56 kDa as determined by both SDS-PAGE and gel-filtration chromatography. On the other hand, Chi-64 showed a molecular mass of 64 kDa by SDS-PAGE and 28 kDa by gel-filtration chromatography suggesting that its properties may be different from those of Chi-56. The optimum temperature, optimum pH, pI, K(m), and V(max) of Chi-56 were 55 degrees C, pH 5.0, pH 8.5, 1.1 mg mL(-1), and 0.59 micromol microg(-1)h(-1), respectively. For Chi-64, these values were 60 degrees C, pH 5.0, pH 8.5, 1.3 mg mL(-1), and 1.36 micromol microg(-1)h(-1), respectively. Both enzymes were stimulated by Mn(2+) and inhibited by Hg(2+), and neither showed exochitinase activity. The N-terminal sequences of Chi-56 and Chi-64 were determined to be Q-T-P-T-Y-T-A-T-L and Q-A-D-F-P-A-P-A-E, respectively.

摘要

两株来自麦氏不动杆菌的胞外几丁质酶（分别命名为 Chi-56 和 Chi-64）经离子交换层析、硫酸铵沉淀和凝胶过滤层析纯化。SDS-PAGE 和凝胶过滤层析均显示 Chi-56 的分子量为 56 kDa。而 Chi-64 在 SDS-PAGE 中的分子量为 64 kDa，在凝胶过滤层析中的分子量为 28 kDa，这表明其性质可能与 Chi-56 不同。Chi-56 的最适温度、最适 pH 值、等电点、K(m)和 V(max)分别为 55°C、pH 5.0、pH 8.5、1.1 mg mL(-1)和 0.59 μmol μg(-1)h(-1)。对于 Chi-64，这些值分别为 60°C、pH 5.0、pH 8.5、1.3 mg mL(-1)和 1.36 μmol μg(-1)h(-1)。两种酶均受 Mn(2+)的刺激和 Hg(2+)的抑制，且均无外切几丁质酶活性。Chi-56 和 Chi-64 的 N 端序列分别为 Q-T-P-T-Y-T-A-T-L 和 Q-A-D-F-P-A-P-A-E。