Dept. of Biotechnology & Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, Felix-Hausdorff-Strasse 4, 17487 Greifswald, Germany.
Appl Microbiol Biotechnol. 2012 Dec;96(5):1243-52. doi: 10.1007/s00253-012-3885-7.
Glycerol as a by-product of biodiesel production is an attractive precursor for producing d-glyceric acid. Here, we demonstrate the successful production of d-glyceric acid based on glycerol via glyceraldehyde in a two-step enzyme reaction with the FAD-dependent alditol oxidase from Streptomyces coelicolor A3(2). The hydrogen peroxide generated in the reaction can be used in detergent, food, and paper industry. In order to apply the alditol oxidase in industry, the enzyme was subjected to protein engineering. Different strategies were used to enhance the substrate specificity towards glycerol. Initial attempts based on rational protein design in the active site region were found unsuccessful to increase activity. However, through directed evolution, an alditol oxidase double mutant (V125M/A244T) with 1.5-fold improved activity for glycerol was found by screening 8,000 clones. Further improvement of activity was achieved by combinatorial experiments, which led to a quadruple mutant (V125M/A244T/V133M/G399R) with 2.4-fold higher specific activity towards glycerol compared to the wild-type enzyme. Through studying the effects of mutations created, we were able to understand the importance of certain amino acids in the structure of alditol oxidase, not only for conferring enzymatic structural stability but also with respect to their influence on oxidative activity.
甘油作为生物柴油生产的副产品，是生产 d-甘油酸的有吸引力的前体。在这里，我们通过两步酶反应，以链霉菌 A3(2)中的 FAD 依赖性糖醇醛氧化酶为基础，成功地从甘油生产 d-甘油酸。反应中产生的过氧化氢可用于洗涤剂、食品和造纸工业。为了将醛糖氧化酶应用于工业，对该酶进行了蛋白质工程改造。使用了不同的策略来增强对甘油的底物特异性。最初在活性位点区域进行基于理性的蛋白质设计的尝试未能提高活性。然而，通过定向进化，通过筛选 8000 个克隆，发现了一种对甘油的活性提高了 1.5 倍的醛糖氧化酶双突变体(V125M/A244T)。通过组合实验进一步提高了活性，得到了一个四重突变体(V125M/A244T/V133M/G399R)，与野生型酶相比，对甘油的比活性提高了 2.4 倍。通过研究所产生的突变的影响，我们能够了解某些氨基酸在醛糖氧化酶结构中的重要性，这些氨基酸不仅对赋予酶结构稳定性很重要，而且对氧化活性也有影响。
