Department of Food Science and Biotechnology, Sejong University, Seoul, Republic of Korea.
Carbohydrate Bioproduct Research Center, Sejong University, Seoul, Republic of Korea.
PLoS One. 2018 Apr 23;13(4):e0196099. doi: 10.1371/journal.pone.0196099. eCollection 2018.
d-Tagatose has gained substantial interest due to its potential functionalities as a sucrose substitute. In this study, the gene araA, encoding l-arabinose isomerase (l-AI) from Clostridium hylemonae (DSM 15053), was cloned and expressed in Escherichia coli BL21 (DE3). This gene consists of 1,506 nucleotides and encodes a protein of 501 amino acid residues with a calculated molecular mass of 56,554 Da. Since l-AI was expressed as an intracellular inclusion body, this enzyme was solubilized with guanidine hydrochloride, refolded, and activated with a descending concentration gradient of urea. The purified enzyme exhibited the greatest activity at 50°C, pH 7-7.5, and required 1 mM of Mg2+ as a cofactor. Notably, the catalytic efficiency (3.69 mM-1sec-1) of l-AI from C. hylemonae on galactose was significantly greater than that of other previously reported enzymes. The bioconversion yield of d-tagatose using the C. hylemonae l-arabinose isomerase at 60°C reached approximately 46% from 10 mM of d-galactose after 2 h. From these results, it is suggested that the l-arabinose isomerase from C. hylemonae could be utilized as a potential enzyme for d-tagatose production due to its high conversion yield at an industrially competitive temperature.
d-塔格糖因其作为蔗糖替代品的潜在功能而引起了广泛关注。在本研究中，从 Clostridium hylemonae (DSM 15053) 中克隆并表达了编码 l-阿拉伯糖异构酶 (l-AI) 的 araA 基因。该基因由 1506 个核苷酸组成，编码 501 个氨基酸残基的蛋白质，计算分子量为 56554 Da。由于 l-AI 作为细胞内包涵体表达，因此该酶用盐酸胍溶解，复性并用尿素浓度下降的梯度激活。纯化的酶在 50°C、pH7-7.5 时表现出最大活性，需要 1mM 的 Mg2+作为辅助因子。值得注意的是，C. hylemonae 的 l-AI 对半乳糖的催化效率 (3.69mM-1sec-1) 明显大于其他先前报道的酶。在 60°C 下使用 C. hylemonae l-阿拉伯糖异构酶进行 d-塔格糖的生物转化，在 2 小时后，从 10mM 的 d-半乳糖中达到约 46%的 d-塔格糖产率。从这些结果可以看出，由于在具有工业竞争力的温度下具有较高的转化率，C. hylemonae 的 l-阿拉伯糖异构酶可用作生产 d-塔格糖的潜在酶。
