State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, People's Republic of China.
Mar Biotechnol (NY). 2010 Feb;12(1):62-9. doi: 10.1007/s10126-009-9200-7. Epub 2009 May 30.
A novel endo-type beta-agarase gene, agaA, was cloned from a newly isolated marine bacterium, Agarivorans sp. LQ48. It encodes a protein of 457 amino acids with a calculated molecular mass of 51.2 kDa. The deduced protein contains a typical N-terminal signal peptide of 25 amino acid residues, followed by a catalytic module, which is homologous to that of glycoside hydrolase family 16. A sequence similar to a carbohydrate-binding module is found in the C-terminal region of the enzyme. The overall amino acid sequence shares a highest identity of 73% with the sequence of beta-agarase AgaB from Pseudoalteromonas sp. strain CY24. The mature agarase was highly expressed extracellularly in Escherichia coli. At pH 7.0 and 40 degrees C, the purified recombinant AgaA had a high specific activity of 349.3 micromol min(-1) mg(-1), a K(m) of 3.9 mg ml(-1), and a V(max) of 909.1 micromol min(-1) mg(-1) for agarose. The recombinant enzyme hydrolyzed the beta-1,4-glycosidic linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the main products. Enzyme activity analysis revealed that the optimal temperature and pH of the recombinant AgaA were 40 degrees C and 7.0, respectively. Notably, AgaA still retained more than 95% activity after incubation at pH 3.0-11.0 for 1 h, a characteristic much different from other agarases reported. It is the first agarase identified to have so wide a pH range stability. This favorable property could make AgaA to be attractive to the food, cosmetic, and medical industrial applications.
从一株新分离的海洋细菌 Agarivorans sp. LQ48 中克隆到一个新型内切β-琼脂酶基因 agaA。它编码一个 457 个氨基酸的蛋白质，计算分子量为 51.2 kDa。推断的蛋白质含有一个典型的 25 个氨基酸残基的 N 端信号肽，其后是一个与糖苷水解酶家族 16 同源的催化模块。在酶的 C 端区域发现了一个与碳水化合物结合模块相似的序列。该酶的整个氨基酸序列与来自 Pseudoalteromonas sp. strain CY24 的β-琼脂酶 AgaB 的序列具有最高 73%的同源性。成熟的琼脂酶在大肠杆菌中高度表达于细胞外。在 pH 7.0 和 40°C 时，纯化的重组 AgaA 具有 349.3 μmol min(-1) mg(-1)的高比活性、3.9 mg ml(-1)的 K(m)和 909.1 μmol min(-1) mg(-1)的 V(max)琼脂糖。重组酶水解琼脂糖的β-1,4-糖苷键，产生 neoagarotetraose 和 neoagarohexaose 作为主要产物。酶活性分析表明，重组 AgaA 的最适温度和 pH 分别为 40°C 和 7.0。值得注意的是，AgaA 在 pH 3.0-11.0 下孵育 1 h 后仍保留超过 95%的活性，这一特性与其他报道的琼脂酶有很大不同。它是第一个被鉴定为具有如此宽 pH 范围稳定性的琼脂酶。这种有利的特性可能使 AgaA 在食品、化妆品和医疗工业应用中具有吸引力。
