Lu Chengyu, Chen Ziwei, Ravikumar Yuvaraj, Zhang Guoyan, Tang Xinrui, Zhang Yufei, Zhao Mei, Sun Wenjing, Qi Xianghui
School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, China.
School of Life Sciences, Guangzhou University, 230 Wai Huan Xi Road, Guangzhou 510006, China.
Foods. 2024 May 31;13(11):1727. doi: 10.3390/foods13111727.
L-Arabinose isomerase (L-AI) has been commonly used as an efficient biocatalyst to produce D-tagatose via the isomerization of D-galactose. However, it remains a significant challenge to efficiently synthesize D-tagatose using the native (wild type) L-AI at an industrial scale. Hence, it is extremely urgent to redesign L-AI to improve its catalytic efficiency towards D-galactose, and herein a structure-based molecular modification of CY6 L-AI (LpAI) was performed. Among the engineered LpAI, both F118M and F279I mutants showed an increased D-galactose isomerization activity. Particularly, the specific activity of double mutant F118M/F279I towards D-galactose was increased by 210.1% compared to that of the wild type LpAI (WT). Besides the catalytic activity, the substrate preference of F118M/F279I was also largely changed from L-arabinose to D-galactose. In the enzymatic production of D-tagatose, the yield and conversion ratio of F118M/F279I were increased by 81.2% and 79.6%, respectively, compared to that of WT. Furthermore, the D-tagatose production of whole cells expressing F118M/F279I displayed about 2-fold higher than that of WT cell. These results revealed that the designed site-directed mutagenesis is useful for improving the catalytic efficiency of LpAI towards D-galactose.
L-阿拉伯糖异构酶（L-AI）通常被用作一种高效的生物催化剂，通过D-半乳糖的异构化反应来生产D-塔格糖。然而，在工业规模上使用天然（野生型）L-AI高效合成D-塔格糖仍然是一项重大挑战。因此，迫切需要对L-AI进行重新设计，以提高其对D-半乳糖的催化效率，在此进行了基于结构的CY6 L-AI（LpAI）分子修饰。在工程改造的LpAI中，F118M和F279I突变体均表现出D-半乳糖异构化活性增强。特别是，双突变体F118M/F279I对D-半乳糖的比活性相比野生型LpAI（WT）提高了210.1%。除了催化活性外，F118M/F279I的底物偏好也从L-阿拉伯糖大幅转变为D-半乳糖。在酶法生产D-塔格糖中，与WT相比，F118M/F279I的产率和转化率分别提高了81.2%和79.6%。此外，表达F118M/F279I的全细胞产生的D-塔格糖比WT细胞高出约2倍。这些结果表明，设计的定点诱变对于提高LpAI对D-半乳糖的催化效率是有用的。
