Characterization of an Allylic/Benzyl Alcohol Dehydrogenase from Yokenella sp. Strain WZY002, an Organism Potentially Useful for the Synthesis of α,β-Unsaturated Alcohols from Allylic Aldehydes and Ketones

Abstract

A novel whole-cell biocatalyst with high allylic alcohol-oxidizing activities was screened and identified as Yokenella sp. WZY002, which chemoselectively reduced the CAO bond of allylic aldehydes/ketones to the corresponding ␣,␤-unsaturated alcohols at 30°C and pH 8.0. The strain also had the capacity of stereoselectively reducing aromatic ketones to (S)-enantioselective alcohols. The enzyme responsible for the predominant allylic/benzyl alcohol dehydrogenase activity was purified to homogeneity and designated YsADH (alcohol dehydrogenase from Yokenella sp.), which had a calculated subunit molecular mass of 36,411 Da. The gene encoding YsADH was subsequently expressed in Escherichia coli, and the purified recombinant YsADH protein was characterized. The enzyme strictly required NADP(H) as a coenzyme and was putatively zinc dependent. The optimal pH and temperature for crotonaldehyde reduction were pH 6.5 and 65°C, whereas those for crotyl alcohol oxidation were pH 8.0 and 55°C. The enzyme showed moderate thermostability, with a half-life of 6.2 h at 55°C. It was robust in the presence of organic solvents and retained 87.5% of the initial activity after 24 h of incubation with 20% (vol/vol) dimethyl sulfoxide. The enzyme preferentially catalyzed allylic/benzyl aldehydes as the substrate in the reduction of aldehydes/ketones and yielded the highest activity of 427 U mg ؊1 for benzaldehyde reduction, while the alcohol oxidation reaction demonstrated the maximum activity of 79.9 U mg ؊1 using crotyl alcohol as the substrate. Moreover, kinetic parameters of the enzyme showed lower K m values and higher catalytic efficiency for crotonaldehyde/benzaldehyde and NADPH than for crotyl alcohol/benzyl alcohol and NADP ؉ , suggesting the nature of being an aldehyde reductase.

Efficient Whole-Cell Oxidation of α,β-Unsaturated Alcohols to α,β-Unsaturated Aldehydes through Cascade Biocatalysis of Alcohol Dehydrogenase, NADPH Oxidase and Hemoglobin

“…The alcohol dehydrogenase from Yokenella sp. WZY002 (YsADH) was highly active in the oxidation of various α,β-unsaturated alcohols to the corresponding α,β-unsaturated aldehyde [7]. To be compatible with YsADH activity (e.g., temperature and pH optima), a thermostable NADPH oxidase from Thermococcus kodakaraensis (TkNOX) was chosen to catalyze the oxidation of NADPH, predominantly converting O 2 to H 2 O [29].…”

“…For example, citral (3,7-dimethyl-2,6-octadien-1-al) is in high demand for the production of ionones, vitamins A and E, as well as carotenoids [4]. α,β-unsaturated aldehydes can be obtained through the selective oxidation of the corresponding α,β-unsaturated alcohols via either chemical catalysis or biocatalysis [3,[5][6][7]. Traditional chemical oxidation methods have required the use of equimolar amounts of oxidizing reagents, which atom e ciency is relatively low.…”

“…Compared with isolated enzymes, the use of whole-cell catalysts simplify the procedure, reduces the cost and bene ts the enzyme stability. Despite great potentials, the number of characterized alcohol oxidases was very limited in contrast to a large array of alcohol dehydrogenases with various substrate speci cities [7,[13][14][15][16].…”

“…Our previous work reported that the NADP + -speci c YsADH, an α,β-unsaturated alcohol dehydrogenase from Yokenella sp. WZY002, demonstrated high activity and stability in the selectivie oxidation of crotonyl alcohol to crotyonyl aldehyde [7]. In this work, the selective oxidation of 3-methyl-2-buten-1-ol to 3-methyl-2-butenal was chosen as the model reaction, considering that both 3-methyl-2-buten-1-ol and 3methyl-2-butenal serve as important organic synthesis intermediates.…”

Lipid degradation during grain storage: markers, mechanisms and shelf-life extension treatments

“…Structural isomers have a different ordering of bonds and/or different bond connectivity from one another (McNaught & McNaught, 1997), as can be appreciated in Figure 2-3. On the other hand, ADH reduces unsaturated aldehydes and ketones to the corresponding unsaturated alcohols (Ying et al, 2014), stabilising the volatile compounds, since they will not react further.…”