Li Ren Kuan, Hu Ya Juan, Ng Tzi Bun, Guo Bing Qi, Zhou Zi He, Zhao Jing, Ye Xiu Yun
The Key Laboratory of Marine Enzyme Engineering of Fujian Province, Fuzhou University, PR China; National Engineering Laboratory for High-efficient Enzyme Expression, PR China.
The Key Laboratory of Marine Enzyme Engineering of Fujian Province, Fuzhou University, PR China.
Int J Biol Macromol. 2020 May 1;158:1125-1134. doi: 10.1016/j.ijbiomac.2020.04.242.
Chitinases play an important role in the process of chitin bioavailability. In this study, we cloned a new chitinase gene and characterized its recombinant protein. The new 1251 bp gene of chitinase (ChiT-7) was cloned from the metagenome of the mangrove tidal flat soil in the city of Zhangzhou in Fujian Province (China) by genome walking. The gene encoded a mature protein with 381 amino acids, which manifested certain sequence similarity (59% identity) to characterized GH18 chitinases. The mature protein of ChiT-7 was successfully expressed in E. coli BL21 (DE3). After purification, the specific activity of the recombinant enzyme was 0.63 U/mg at the optimal pH of 6.0 and the optimal temperature of 45 °C. The rChiT-7 was active over a wide pH range, and the residual enzyme activity reached 80% or higher at 30 °C-50 °C. rChiT-7 hydrolyzed colloidal chitin with (GlcNAc) and GlcNAc as the main final products. Structural analysis of ChiT-7 indicated that ChiT-7 could be a processive chitinase. rChiT-7 manifested characteristics analogous to those of fungi and actinomycetes and exhibited sequence homology.
几丁质酶在几丁质生物利用过程中发挥着重要作用。在本研究中，我们克隆了一个新的几丁质酶基因并对其重组蛋白进行了表征。通过基因组步移技术，从中国福建省漳州市红树林潮滩土壤宏基因组中克隆到了一个新的1251 bp的几丁质酶基因（ChiT - 7）。该基因编码一个含有381个氨基酸的成熟蛋白，与已表征的GH18几丁质酶表现出一定的序列相似性（同一性为59%）。ChiT - 7的成熟蛋白在大肠杆菌BL21(DE3)中成功表达。纯化后，重组酶在最适pH 6.0和最适温度45℃下的比活性为0.63 U/mg。rChiT - 7在较宽的pH范围内具有活性，在30℃至50℃时残余酶活性达到80%或更高。rChiT - 7以（GlcNAc）和GlcNAc作为主要终产物水解胶体几丁质。ChiT - 7的结构分析表明ChiT - 7可能是一种连续作用的几丁质酶。rChiT - 7表现出与真菌和放线菌类似的特性并呈现出序列同源性。
