Ruiz-Sanchez Alejandro, Cruz-Camarillo Ramon, Salcedo-Hernandez Ruben, Ibarra Jorge E, Barboza-Corona Jose Eleazar
Instituto de Ciencias, Agrícolas, Universidad de Guanajuato, Guanajuato, Mexico.
Mol Biotechnol. 2005 Oct;31(2):103-11. doi: 10.1385/MB:31:2:103.
An endochitinase gene from the Serratia marcescens Nima strain (chiA Nima) was cloned, sequenced, and expressed in Escherichia coli DH5alphaF', and the recombinant protein (ChiA Nima) was purified by hydrophobic interaction chromatography. chiA Nima contains an open reading frame (ORF) that encodes an endochitinase with a deduced molecular weight and an isoelectric point of 61 kDa and 6.84, respectively. A sequence at the 5'-end was identified as a signal peptide, recognized by Gram-negative bacteria transport mechanism. Comparison of ChiA Nima with other chitinases revealed a modular structure formed by the catalytic domain and a putative chitin-binding domain. The purified chitinase was able to hydrolyze both trimeric and tetrameric fluorogenic substrates, but not a chitobiose analog substrate. ChiA Nima showed high enzymatic activity within a broad pH range (pH 4.0-10.0), with a peak activity at pH 5.5. The optimal temperature for enzymatic activity was detected at 55 degrees C.
克隆、测序了粘质沙雷氏菌尼玛菌株的一种内切几丁质酶基因（chiA Nima），并在大肠杆菌DH5alphaF'中进行表达，通过疏水相互作用色谱法纯化了重组蛋白（ChiA Nima）。chiA Nima包含一个开放阅读框（ORF），其编码的内切几丁质酶推导分子量和等电点分别为61 kDa和6.84。5'-端的一个序列被鉴定为信号肽，可被革兰氏阴性菌转运机制识别。将ChiA Nima与其他几丁质酶进行比较，发现其由催化结构域和一个假定的几丁质结合结构域形成模块化结构。纯化的几丁质酶能够水解三聚体和四聚体荧光底物，但不能水解壳二糖类似物底物。ChiA Nima在较宽的pH范围内（pH 4.0 - 10.0）显示出高酶活性，在pH 5.5时活性最高。检测到酶活性的最佳温度为55℃。
