PDF(Pubmed)
Abstract：
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 Lactobacillus plantarum 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.
摘要：
L-阿拉伯糖异构酶(L-Al)通常用作通过D-半乳糖异构化产生D-塔格糖的有效生物催化剂。然而,使用天然(野生型)L-AI以工业规模有效合成D-塔格糖仍然是一个重大挑战。因此，迫切需要重新设计L-AI以提高其对D-半乳糖的催化效率，并且在本文中对植物乳杆菌CY6L-AI(LpAI)进行基于结构的分子修饰。在工程LpAI中，F118M和F279I突变体均显示增加的D-半乳糖异构化活性。特别是,与野生型LpAI（WT）相比，双突变体F118M/F279I对D-半乳糖的比活性增加了210.1％。除了催化活性，F118M/F279I的底物偏好也从L-阿拉伯糖变为D-半乳糖。在D-塔格糖的酶生产中，F118M/F279I的收率和转化率分别提高了81.2%和79.6%,分别,与WT相比。此外，表达F118M/F279I的全细胞的D-塔格糖产量比WT细胞高约2倍。这些结果表明，设计的定点诱变可用于提高LpAI对D-半乳糖的催化效率。
