Engineering Promiscuous Alcohol Dehydrogenase Activity of a Reductive Aminase AspRedAm for Selective Reduction of Biobased Furans

Catalytic promiscuity is a promising starting point for improving the existing enzymes and even creating novel enzymes. In this work, site-directed mutagenesis was performed to improve promiscuous alcohol dehydrogenase activity of reductive aminase from Aspergillus oryzae (AspRedAm). AspRedAm showed the cofactor preference toward NADPH in reductive aminations, while it favored NADH in the reduction reactions. Some key amino acid residues such as N93, I118, M119, and D169 were identified for mutagenesis by molecular docking. Variant N93A showed the optimal pH and temperature of 8 and 30°C, respectively, in the reduction of 5-hydroxymethylfurfural (HMF). The thermostability was enhanced upon mutation of N93 to alanine. The catalytic efficiency of variant N93A (kcat/Km, 23.6 mM−1 s−1) was approximately 2-fold higher compared to that of the wild-type (WT) enzyme (13.1 mM−1 s−1). The improved catalytic efficiency of this variant may be attributed to the reduced steric hindrance that stems from the smaller side chain of alanine in the substrate-binding pocket. Both the WT enzyme and variant N93A had broad substrate specificity. Escherichia coli (E. coli) cells harboring plain vector enabled selective reduction of biobased furans to target alcohols, with the conversions of 35–95% and the selectivities of >93%. The introduction of variant N93A to E. coli resulted in improved substrate conversions (>98%) and selectivities (>99%).

Alcohol Dehydrogenase Activity Converts 3″-Hydroxy-geranylhydroquinone to an Aldehyde Intermediate for Shikonin and Benzoquinone Derivatives in Lithospermum erythrorhizon

Shikonin derivatives are red naphthoquinone pigments produced by several boraginaceous plants, such as Lithospermum erythrorhizon. These compounds are biosynthesized from p-hydroxybenzoic acid and geranyl diphosphate. The coupling reaction that yields m-geranyl-p-hydroxybenzoic acid has been actively characterized, but little is known about later biosynthetic reactions. Although 3″-hydroxy-geranylhydroquinone produced from geranylhydroquinone by CYP76B74 has been regarded as an intermediate of shikonin derivatives, the next intermediate has not yet been identified. This study describes a novel alcohol dehydrogenase activity in L. erythrorhizon cell cultures. This enzyme was shown to oxidize the 3″-alcoholic group of (Z)-3″-hydroxy-geranylhydroquinone to an aldehyde moiety concomitant with the isomerization at the C2′–C3′ double bond from the Z-form to the E-form. An enzyme oxidizing this substrate was not detected in other plant cell cultures, suggesting that this enzyme is specific to L. erythrorhizon. The reaction product, (E)-3″-oxo-geranylhydroquinone, was further converted to deoxyshikonofuran, another meroterpenoid metabolite produced in L. erythrorhizon cells. Although nonenzymatic cyclization occurred slowly, it was more efficient in the presence of crude enzymes of L. erythrorhizon cells. This activity was detected in both shikonin-producing and nonproducing cells, suggesting that the aldehyde intermediate at the biosynthetic branch point between naphthalene and benzo/hydroquinone ring formation likely constitutes a key common intermediate in the synthesis of shikonin and benzoquinone products, respectively.

Nutritional Quality of Red Dates (Zizyphus Jujube Mill.) in Response to Modified and Controlled Atmospheric Storage Conditions

Quality maintenance and ethanol metabolism of red date (Zizyphus jujube Mill.) fruits in response to modified atmosphere and controlled atmosphere (7% CO2, 3% O2 plus 90% N2) were investigated in the present study. Results showed that modified atmosphere and controlled atmosphere significantly maintained higher titratable and ascorbic acid contents during storage at 0°C for 32 days. In addition, ethanol accumulation and alcohol dehydrogenase activity indicated that ethanol metabolism in red dates was substantially inhibited by modified and controlled atmospheric storage conditions. Furthermore, the browning and polyphenoloxidase activity was also delayed by both atmospheric conditions compared with control. By evidence of sensory evaluation, results confirmed that both modified and controlled atmosphere packages contributed to the maintenance of better sweetness, sourness, firmness, juiciness and date flavor as well as overall preference after cold storage. Nonetheless, no significant difference on decay index of red dates was observed between changed atmospheres and untreated control after storage. Results from the present study are of importance to the red date industry on theoretical and practical aspects.