Discovery And Characterization Of Novel Aldolases

Aldol reactions constitute a powerful methodology for carbon-carbon bond formation in synthetic organic chemistry. The aldolase, that catalyses thealdol reaction in nature, usually has anexcellent control over the regiochemistry and accept a wide variety of acceptor moleculesand made itself a most useful tool in organic synthesis. This study aimed to use genome mining to discover new aldolases and explore their application potential in the biocatalytic synthesis of pharmaceutical intermediates.By the means of genome mining, a N-acetylneuraminate lyase gene ?shnal? from Staphylococcus hominis was cloned into pET-28a and expressed in Escherichia coli BL21 ?DE3? host cells. The recombinant enzyme was purified and characterized. It is a homotetrameric enzyme with the optimum pH at 8.0 for the cleavage direction and the optimum pH and temperature were 7.5 and 45?for the synthetic direction. The activity of ShNAL is stable when incubated at 45? for two hours but decreased rapidly over 50?. ShNAL showed high stability in a wide range pH from 5.0 to 10.0 with the residual activity being>70% when the enzyme was incubated 24 h in different buffers at 4?. Its Km towards N-acetylneuraminic acid, pyruvate and ManNAc were ?4.0±0.2? mmol/L, ?35.1±3.2? mmol/L and?131.7±12.1? mmol/L, respectively. The kcat/Km value of Neu5Ac, ManNAc, and Pyr for ShNAL were 1.9 L/mmol-s,0.08 L/mmol-s and 0.08 L/mmol-s, respectively.The conversion and yield of condensation reaction catalyzed by ShNAL reached 91.5% and 89.4%, respectively. By Coupling N-acetyl-glucosamine-2-epimerase and N-acetyl-D-neuraminic acid lyase, the conversion from N-acetylglucosamine to N-acetyl neuraminic acid reached about 30%.Similarly, a N-acetylneuraminic acid lyase gene ?BmNAL GenBank Accession No. ZP01092134.1? from Blastopirellula marina, and two threonine aldolase genes from Deinococcus deserti ?DdTA? and Hoeflea phototrophica ?HpTA? ?Accession No. YP₀02785560.1 and ZP02165963.1? were selected and expressed in E. coli. The target proteins were purified and showed the desired enzyme activity.