Cui Fangyuan, Dong Sujie, Shi Xiaochong, Zhao Xia, Zhang Xiao-Hua
College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China.
School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China.
Mar Drugs. 2014 May 12;12(5):2731-47. doi: 10.3390/md12052731.
Genome sequencing of Catenovulum agarivorans YM01T reveals 15 open-reading frames (ORFs) encoding various agarases. In this study, extracellular proteins of YM01T were precipitated by ammonium sulfate and separated by one-dimensional gel electrophoresis. The results of in-gel agarase activity assay and mass spectrometry analysis revealed that the protein, YM01-3, was an agarase with the most evident agarolytic activity. Agarase YM01-3, encoded by the YM01-3 gene, consisted of 420 amino acids with a calculated molecular mass of 46.9 kDa and contained a glycoside hydrolase family 16 β-agarase module followed by a RICIN superfamily in the C-terminal region. The YM01-3 gene was cloned and expressed in Escherichia coli. The recombinant agarase, YM01-3, showed optimum activity at pH 6.0 and 60 °C and had a K(m) of 3.78 mg mL⁻¹ for agarose and a Vmax of 1.14 × 10⁴ U mg⁻¹. YM01-3 hydrolyzed the β-1,4-glycosidic linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the main products. Notably, YM01-3 was stable below 50 °C and retained 13% activity after incubation at 80 °C for 1 h, characteristics much different from other agarases. The present study highlights a thermostable agarase with great potential application value in industrial production.
食琼脂链菌YM01T的基因组测序揭示了15个编码各种琼脂酶的开放阅读框（ORF）。在本研究中，YM01T的细胞外蛋白通过硫酸铵沉淀，并通过一维凝胶电泳进行分离。凝胶内琼脂酶活性测定和质谱分析结果表明，蛋白YM01-3是一种琼脂olytic活性最明显的琼脂酶。由YM01-3基因编码的琼脂酶YM01-3由420个氨基酸组成，计算分子量为46.9 kDa，在C末端区域包含一个糖苷水解酶家族16β-琼脂酶模块，随后是一个蓖麻毒素超家族。YM01-3基因被克隆并在大肠杆菌中表达。重组琼脂酶YM01-3在pH 6.0和60°C时表现出最佳活性，对琼脂糖的K(m)为3.78 mg mL⁻¹，Vmax为1.14×10⁴ U mg⁻¹。YM01-3水解琼脂糖的β-1,4-糖苷键，产生新琼脂四糖和新琼脂六糖作为主要产物。值得注意的是，YM01-3在50°C以下稳定，在80°C孵育1小时后仍保留13%的活性，这些特性与其他琼脂酶有很大不同。本研究突出了一种在工业生产中具有巨大潜在应用价值的耐热琼脂酶。
