Liu Nan, Yang Meng, Mao Xiangzhao, Mu Bozhong, Wei Dongzhi
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, People's Republic of China.
College of Food Science and Engineering, Ocean University of China, Qingdao, People's Republic of China.
Biotechnol Appl Biochem. 2016 Mar-Apr;63(2):230-7. doi: 10.1002/bab.1363. Epub 2015 May 24.
A new α-neoagarobiose hydrolase (NABH) called AgaWH117 was cloned from Agarivorans gilvus WH0801. The gene encoding this hydrolase consists of 1,086 bp and encodes a protein containing 361 amino acids. This new NABH showed 74% amino acid sequence identity with other known NABHs. The molecular mass of the recombinant AgaWH117 was estimated to be 41 kDa. Purified AgaWH117 showed endolytic activity during neoagarobiose degradation, yielding 3,6-anhydro-l-galactose (l-AHG) and d-galactose as products. It showed a maximum activity at a temperature of 30 °C and a pH of 6.0 and was stable at temperatures below 30 °C. Its Km and Vmax values were 2.094 mg/mL and 6.982 U/mg, respectively. The cloning strategy used and AgaWH117 isolated in this study will provide information on the saccharification process of marine biomass. This study provides a method to produce l-AHG from agarose by using AgaWH117 without an acid and describes its one-step purification by using Bio-Gel P2 chromatography.
从琼胶嗜盐菌WH0801中克隆出一种名为AgaWH117的新型α-新琼脂二糖水解酶（NABH）。编码这种水解酶的基因由1086个碱基对组成，编码一种含有361个氨基酸的蛋白质。这种新型NABH与其他已知的NABH氨基酸序列同一性为74%。重组AgaWH117的分子量估计为41 kDa。纯化后的AgaWH117在新琼脂二糖降解过程中表现出内切活性，产生3,6-脱水-L-半乳糖（L-AHG）和D-半乳糖作为产物。它在30°C温度和pH 6.0时表现出最大活性，在30°C以下温度稳定。其Km和Vmax值分别为2.094 mg/mL和6.982 U/mg。本研究中使用的克隆策略和分离出的AgaWH117将为海洋生物质糖化过程提供信息。本研究提供了一种不使用酸通过AgaWH117从琼脂糖生产L-AHG的方法，并描述了使用Bio-Gel P2色谱法对其进行一步纯化的方法。
