Cloning and characterisation of a novel neoagarotetraose-forming-β-agarase, AgWH50A from Agarivorans gilvus WH0801
Cloning and characterisation of a novel neoagarotetraose-forming-β-agarase, AgWH50A from Agarivorans gilvus WH0801
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DOI：
10.1016/j.carres.2014.02.019
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发表时间：
2014-03-31
影响因子：
3.1
通讯作者：
Wei, Dongzhi
中科院分区：
文献类型：
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作者：
Liu, Nan;Mao, Xiangzhao;Wei, Dongzhi
利用简并和巢式PCR技术从Agarivorans gilvus WH 0801中克隆到一种新的β-琼胶酶AgWH 50 A。它由942个氨基酸（105 kDa）组成，包括21个氨基酸的信号肽。AgWH 50 A与Agarivoranssp.QM38的AgaD 02具有最高的氨基酸同源性（53%）。重组琼胶酶基因在大肠杆菌中表达，亲和层析纯化。在pH 6.0和30 ℃下观察到最大酶活性（K-m 5.97 mg/mL和V-max 0.781 U/mg）。利用基质辅助激光解吸/电离飞行时间质谱、傅立叶变换核磁共振谱和薄层色谱分析了AgWH 50 A的水解产物，并得出结论：AgWH 50 A是一种能将琼脂糖裂解为新琼脂四糖的β-琼脂糖酶。该酶在新琼胶四糖的工业化生产中具有潜在的应用价值，为今后的研究提供了一种新的琼脂糖水解模型。(C)2014爱思唯尔有限公司版权所有。
AgWH50A, a novel beta-agarase, was cloned from Agarivorans gilvus WH0801 by degenerate and nested PCR. It consists of 942 amino acids (105 kDa), including a 21-amino acid signal peptide. AgWH50A shares the highest amino acid sequence homology with AgaD02 from Agarivorans sp. QM38 (53%). The recombinant agarase gene was expressed in Escherichia coli and purified by affinity chromatography. Maximum enzymatic activity (K-m 5.97 mg/mL and V-max 0.781 U/mg) was observed at pH 6.0 and 30 degrees C. Using matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry, Fourier transform-nuclear magnetic resonance spectrometry and thin-layer chromatography, we analysed the hydrolysis products and concluded that AgWH50A is a neoagarotetraose-forming beta-agarase, which can cleave agarose into neoagarotetraose. This novel agarase has potential applications in the industrial production of neoagarotetraose and provides a new agarose hydrolysis model for future research. (C) 2014 Elsevier Ltd. All rights reserved.