Asymmetric synthesis of d-glyceric acid by an alditol oxidase and directed evolution for enhanced oxidative activity towards glycerol

Abstract

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.

Production of alkenes and novel secondary products by P450 OleTJE using novel H2O2‐generating fusion protein systems

“…Glycerol ( S. coelicolor AldO enzyme reported a k cat = 17 s À1 and a k cat /K m = 1.2 9 10 4 M À1 s À1 for sorbitol, and values of k cat = 13 s À1 and k cat / K m = 4.1 9 10 4 M À1 s À1 for xylitol at pH 7.5 [17]; and a k cat /K m = 2.1 9 10 4 M À1 s À1 for glycerol at pH 9.0 [26]. Given the similar extents of oxidation of these different substrates by the OleT JE -HRV3C-AldO fusion enzyme (Table 2), it is likely that the kinetics of this fusion enzyme are similar to those reported previously.…”

“…This suggested that OleT JE can convert these hydroxylated fatty acids to further products (Figure 6). 0.5 ml reactions were set up with 5 µM OleT JE -HRV3C-AldO, 1% glycerol and 200 µM of either 2-OH or 3-OH myristic acid, or using 1 µM OleT JE /500 µM H 2 (27). Given the similar extents of oxidation of these different substrates by the OleT JE -HRV3C-AldO fusion enzyme ( Table 2), it is likely that the kinetics of this fusion enzyme are similar to those reported previously.…”

Structure-Based Engineering of Phanerochaete chrysosporium Alcohol Oxidase for Enhanced Oxidative Power toward Glycerol

“…The best alditol oxidase mutant for oxidation of glycerol was found to display a k cat value of only 0.06 s –1 . 44 …”

An artificial enzymatic reaction cascade for a cell-free bio-system based on glycerol

“…Glycerol is first oxidized with oxygen to glyceric acid via glyceraldehyde by the FAD-dependent alditol oxidase (Aldo)-catalyzed two-step oxidation reaction. [31][32][33] The substrate tolerance of the dihydroxy acid dehydratase (DHAD) in Sulfolobus solfataricus was reported recently. 16,34 Glyceric acid 10 produced from glycerol can be transformed into pyruvic acid and water without oxygen by the DHAD in S. solfataricus.…”