Park Da Yeon, Chi Won-Jae, Park Jae-Seon, Chang Yong-Keun, Hong Soon-Kwang
Division of Bioscience and Bioinformatics, Myongji University, San 38-2, Namdong, Yongin, Gyeonggi-do, 449-728, South Korea.
Appl Biochem Biotechnol. 2015 Jan;175(2):733-47. doi: 10.1007/s12010-014-1294-3. Epub 2014 Oct 24.
An agarase gene (agaH71) was identified from Pseudoalteromonas hodoensis, an agar utilizing marine bacterium. The nucleotide sequence revealed that AgaH71 had significant homology to glycosyl hydrolase (GH) 16 agarases. agaH71 encodes a primary translation product (32.7 kDa) of 290 amino acids, including a 21-amino-acid signal peptide. The entire AgaH71 was expressed in a fused protein with glutathione-S-transferase (GST) at its N-terminal (GST-AgaH71) in Escherichia coli. Purified GST-AgaH71 had an apparent molecular weight of 59 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which was consistent with the calculated molecular weight (58.7 kDa). Agarase activity of the purified protein was confirmed by zymogram assay. GST-AgaH71 could hydrolyze p-nitrophenyl-β-D-galactopyranoside, but not p-nitrophenyl-α-D-galactopyranoside. The optimum pH and temperature for GST-AgaH71 were 6.0 and 45 °C, respectively. GST-AgaH71 retained more than 95 and 90 % of its initial activity at 40 and 45 °C after heat treatment for 60 min, respectively. The K m and V max for agarose were 28.33 mg/ml and 88.25 U/mg, respectively. GST-AgaH71 did not require metal ions for its activity, but severe inhibition by divalent metal ions was observed. Thin-layer chromatography (TLC) analysis, mass spectrometry, and nuclear magnetic resonance (NMR) spectrometry of the GST-AgaH71 hydrolysis products revealed that GST-AgaH71 is an endo-type β-agarase that hydrolyzes agarose into predominantly neoagarotetraose and small proportions of neoagarobiose and neoagarohexaose.
从嗜琼假交替单胞菌（一种利用琼脂的海洋细菌）中鉴定出一种琼脂酶基因（agaH71）。核苷酸序列显示，AgaH71与糖基水解酶（GH）16琼脂酶具有显著同源性。agaH71编码一个由290个氨基酸组成的初级翻译产物（32.7 kDa），包括一个21个氨基酸的信号肽。完整的AgaH71在大肠杆菌中以N端与谷胱甘肽 - S - 转移酶（GST）融合的蛋白形式表达（GST - AgaH71）。纯化后的GST - AgaH71在十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS - PAGE）上的表观分子量为59 kDa，与计算分子量（58.7 kDa）一致。通过酶谱分析证实了纯化蛋白的琼脂酶活性。GST - AgaH71能够水解对硝基苯基 - β - D - 吡喃半乳糖苷，但不能水解对硝基苯基 - α - D - 吡喃半乳糖苷。GST - AgaH71的最适pH和温度分别为6.0和45℃。热处理60分钟后，GST - AgaH71在40℃和45℃下分别保留了超过95%和90%的初始活性。琼脂糖的K m和V max分别为28.33 mg/ml和88.25 U/mg。GST - AgaH71的活性不需要金属离子，但观察到二价金属离子对其有严重抑制作用。对GST - AgaH71水解产物的薄层色谱（TLC）分析、质谱分析和核磁共振（NMR）光谱分析表明，GST - AgaH71是一种内切型β - 琼脂酶，可将琼脂糖主要水解为新琼脂四糖，并少量水解为新琼脂二糖和新琼脂六糖。
