Hong J, Tamaki H, Akiba S, Yamamoto K, Kumagai H
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
J Biosci Bioeng. 2001;92(5):434-41. doi: 10.1263/jbb.92.434.
A gene encoding an endo-beta-1,4-glucanase, which is highly resistant to high temperature, protease and surfactant treatment, was isolated from Aspergillus niger IFO31125 and designated as eng1. The deduced amino acid sequence encoded by eng1 showed high homology with the sequence of a not-well-characterized cellulase encoded by eglB which has not yet been shown to be a stable enzyme. To confirm the sequence of the gene encoding the highly stable endo-beta-1,4-glucanase, the cloned gene was expressed in the yeast Saccharomyces cerevisiae, in which no cellulase activity was found, and the gene product was purified and subjected to enzymatic characterization. The enzyme retained 56% of the initial activity after 1 h of incubation at 80 degrees C and was stable in the range of pH 3.0-10.0. The optimal temperature for enzyme activity was 70 degrees C and the optimal pH was 6.0. The enzyme was highly protease-resistant and retained more than 80% of the initial activity after protease treatment for 3 d at 40 degrees C. The enzyme was also resistant to various surfactants. From these results, eng1 was confirmed to encode a very stable endo-beta-1,4-glucanase.
从黑曲霉IFO31125中分离出一个编码内切β-1,4-葡聚糖酶的基因，该酶对高温、蛋白酶和表面活性剂处理具有高度抗性，被命名为eng1。eng1编码的推导氨基酸序列与eglB编码的一种尚未充分表征的纤维素酶序列具有高度同源性，而eglB尚未被证明是一种稳定的酶。为了确认编码高度稳定的内切β-1,4-葡聚糖酶的基因序列，将克隆的基因在未发现纤维素酶活性的酿酒酵母中表达，对基因产物进行纯化并进行酶学特性分析。该酶在80℃孵育1小时后保留了56%的初始活性，在pH 3.0 - 10.0范围内稳定。酶活性的最佳温度为70℃，最佳pH为6.0。该酶对蛋白酶具有高度抗性，在40℃用蛋白酶处理3天后保留了超过80%的初始活性。该酶对各种表面活性剂也具有抗性。从这些结果可以确认，eng1编码一种非常稳定的内切β-1,4-葡聚糖酶。
