Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Gachibowli, Hyderabad 500046, Telangana, India.
Department of Botany, Indira Gandhi National Tribal University, Amarkantak 484887, Madhya Pradesh, India.
Int J Biol Macromol. 2018 Oct 1;117:62-71. doi: 10.1016/j.ijbiomac.2018.05.129. Epub 2018 May 22.
Chitin and its derivatives are used for a variety of applications. Flavobacterium johnsoniae UW101 is an aerobic Gram-negative bacterium. Genome analysis of F. johnsoniae UW101 revealed the presence of 10 glycoside hydrolases (GHs) that may degrade or modify chitin. The gene encoding chitinase B (FjchiB), which encodes a single catalytic GH18 domain has been cloned and heterologously expressed in Escherichia coli. FjChiB was optimally active in 50 mM sodium citrate buffer (pH 6.0) at 40 °C. FjChiB was salt-tolerant and catalytically versatile, with substrate specificity towards 75% DDA (degree of de-acetylation) chitosan, followed by colloidal chitin. Chitotetraose (DP4) was the shortest of the oligomeric substrates used by FjChiB. The K and V values of FjChiB for colloidal chitin were 49.38 mg/ml and 11.2 nanokat mg, respectively. The overall catalytic efficiency (k/K) of FjChiB was 1.40 × 10 mg ml s. FjChiB exhibited transglycosylation (TG) with chitopentaose (DP5) and chitohexaose (DP6) substrates. The TG by FjChiB was fine-tuned by introducing a tryptophan (G106W) and asparagine (D148N) in the highly conserved catalytic groove and catalytic center, respectively. Hydrolytic products profile and homology modelling indicated that FjChiB is an endochitinase that holds promise for the conversion of chitin into useful products through both TG and/or hydrolysis.
几丁质及其衍生物被广泛应用于各种领域。黄杆菌 UW101 是一种需氧革兰氏阴性菌。对 UW101 基因组的分析表明，它含有 10 种糖苷水解酶（GHs），这些酶可能会降解或修饰几丁质。几丁质酶 B（FjchiB）的编码基因已被克隆并在大肠杆菌中异源表达，该基因编码一个单一的催化 GH18 结构域。FjChiB 在 50 mM 柠檬酸钠缓冲液（pH 6.0）中 40°C 时具有最佳活性。FjChiB 具有耐盐性和多功能的催化特性，对 75%脱乙酰度（DDA）壳聚糖的底物特异性最强，其次是胶体几丁质。FjChiB 作用的寡聚底物中最短的是四糖（DP4）。FjChiB 对胶体几丁质的 K 和 V 值分别为 49.38 mg/ml 和 11.2 纳克·分钟·毫克。FjChiB 的总体催化效率（k/K）为 1.40×10 mg ml s。FjChiB 对五糖（DP5）和六糖（DP6）底物表现出转糖苷作用（TG）。通过在高度保守的催化沟和催化中心分别引入色氨酸（G106W）和天冬酰胺（D148N），FjChiB 的 TG 活性得到了精细调控。水解产物图谱和同源建模表明 FjChiB 是一种内切几丁质酶，有望通过 TG 和/或水解作用将几丁质转化为有用的产物。
