Abstract
Recently, an in vitro enzymatic cascade was constructed to transform glycerol into the high-value platform chemical pyruvate. However, the low activity of dihydroxy acid dehydratase from Sulfolobus solfataricus (SsDHAD) limited the efficiency. In this study, the enzymatic reduction of pyruvate catalyzed by d-lactate dehydrogenase from Pseudomonas aeruginosa PAO1 was used to assay the activities of dihydroxy acid dehydratases. Dihydroxy acid dehydratase from Paralcaligenes ureilyticus (PuDHT) was identified as the most efficient candidate for glycerate dehydration. After the optimization of the catalytic temperature for the enzymatic cascade, comprising alditol oxidase from Streptomyces coelicolor A3, PuDHT, and catalase from Aspergillus niger, 20.50 ± 0.27 mM of glycerol was consumed in 4 h to produce 18.95 ± 0.97 mM of pyruvate with a productivity 12.15-fold higher than the previous report using SsDHAD. The enzymatic cascade was further coupled with the pyruvate decarboxylase from Zymomonas mobile for the production of another platform compound, acetoin. Acetoin at a concentration of 8.52 ± 0.12 mM was produced from 21.62 ± 0.19 mM of glycerol with a productivity of 1.42 ± 0.02 mM h−1.
Highlights
World production of glycerol, the main by-product of biodiesel and bioethanol generation, is higher than the existing market demand for its industrial applications [1,2,3]
The low efficiency of the dehydration of glycerate to pyruvate catalyzed by SsDHAD restricted the application of this enzymatic cascade
The results indicate that ScALDO and PuDHT are affected differently by heat
Summary
The main by-product of biodiesel and bioethanol generation, is higher than the existing market demand for its industrial applications [1,2,3]. Biological conversion is generally considered to be the preferred method of glycerol utilization due to its advantages of high selectivity, modest reaction condition, and low pollution risk [8,9]. Various valuable compounds such as 1,3-dihydroxyacetone [10,11], 1,3-propanediol [12], and succinate [13] have been produced by microbial fermentation using glycerol as a carbon source. The low efficiency of the dehydration of glycerate to pyruvate catalyzed by SsDHAD restricted the application of this enzymatic cascade. (d) High-performance liquid chromatography (HPLC) analysis of the glycerate dehydration catalyzed by SsDHAD, CcXylD, or PuDHT. PuDHT was selected as the optimal dehydratase for successive experiments
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