Biodiesel can replace petroleum diesel as it is produced from animal fats and vegetable oils, and it produces about 10 % (w/w) glycerol, which is a promising new industrial microbial carbon, as a major by-product. One of the most potential applications of glycerol is its biotransformation to high value chemicals such as 1,3-propanediol (1,3-PD), dihydroxyacetone (DHA), succinic acid, etc., through microbial fermentation. Glycerol dehydratase, 1,3-propanediol dehydrogenase (1,3-propanediol-oxydoreductase), and glycerol dehydrogenase, which were encoded, respectively, by dhaB, dhaT, and dhaD and with DHA kinase are encompassed by the dha regulon, are the three key enzymes in glycerol bioconversion into 1,3-PD and DHA, and these are discussed in this review article. The summary of the main research direction of these three key enzyme and methods of glycerol bioconversion into 1,3-PD and DHA indicates their potential application in future enzymatic research and industrial production, especially in biodiesel industry.

Serine hydroxymethyltransferase (SHMT) is the key enzyme in L-serine enzymatic production, suggesting the importance of obtaining a SHMT with high activity. Here, a novel SHMT gene, glyA, was obtained through degenerate oligonucleotide-primed PCR and encoded a novel SHMT with 54.3% similarity to the known SHMT from Escherichia coli. The obtained protein AnSHMT showed the optimal activity at 40°C and pH 7.5, and was more stable in weakly alkali conditions (pH 6.5-8.5) than Hyphomicrobium methylovorum’s SHMT (pH 6.0-7.5), In order to improve the catalytic efficiency of the wild type, the site-directed mutagenesis based on sequences alignment and bioinformatics prediction, was used and the catalytic efficiency of the mutant I249L was found to be 2.78-fold higher than that of the wild-type, with the replacement of isoleucine by leucine at the 249 position. This research provides useful information about the interesting site, and the application of DOP-PCR in cloning a novel glyA gene.

With the advantages of biocatalytic method, enzymes have been excavated for the synthesis of chiral amino acids by the reductive amination of ketones, offering a promising way of producing pharmaceutical intermediates. In this work, a robust phenylalanine dehydrogenase (PheDH) with wide substrate spectrum and high catalytic efficiency was constructed through rational design and active-site-targeted, site-specific mutagenesis by using the parent enzyme from Bacillus halodurans. Active sites with bonding substrate and amino acid residues surrounding the substrate binding pocket, 49L-50G-51G, 74M,77K, 122G-123T-124D-125M, 275N, 305L and 308V of the PheDH, were identified. Noticeably, the new mutant PheDH (E113D-N276L) showed approximately 6.06-fold increment of kcat/Km in the oxidative deamination and more than 1.58-fold in the reductive amination compared to that of the wide type. Meanwhile, the PheDHs exhibit high capacity of accepting benzylic and aliphatic ketone substrates. The broad specificity, high catalytic efficiency and selectivity, along with excellent thermal stability, render these broad-spectrum enzymes ideal targets for further development with potential diagnostic reagent and pharmaceutical compounds applications.

Ancestral sequence reconstruction (ASR) is an effective semirational strategy for enzyme engineering that integrates evolutionary information with bioinformatic analysis. Here, ASR was combined with structure-guided mutagenesis using FuncLib and experimental validation to improve the catalytic performance and stability of the reductive aminase from Asp ergillus oryzae ( Asp RedAm). Phylogenetic analysis of Asp RedAm and its homologs enabled the reconstruction of ancestral variants, including Anc43, Anc74, and Anc75, and guided the identification of key substrate-binding pockets and catalytically relevant residues. Targeted mutagenesis was then performed to generate improved variants. Among the resulting enzymes, Anc74_7 and Anc75_20 exhibited markedly enhanced catalytic activity and thermostability relative to Asp RedAm. In particular, their specific activities increased by up to 1.6- and 2.2-fold, respectively. Molecular docking together with kinetic analysis suggested that these improvements arose from active-site reconfiguration and an optimized substrate-binding microenvironment. Collectively, these results demonstrate the utility of integrating ASR with structure-guided engineering for the development of high-performance biocatalysts and provide insight into enzyme functional evolution and rational design.