Influence of flavonoids on the stability of an (S)-hydroxynitrile lyase from Hevea brasiliensis

The (S)-hydroxynitrile lyase from leaves of the rubber tree Hevea brasilien sis (HbHnl) (EC 4.1.2.39) catalyzes the industrially interesting formation of (S)-cyanohydrins from aldehydes or ketones and HCN. The overall yield of the (S)-cyanohydrin is reduced by the non-stereoselective base catalyzed c hemical background reaction. To reduce the chemical background reaction, lo wer pH values (below pH=5) are necessary, but the enzyme in this case is no t stable enough. To stabilize the enzyme at these conditions the addition o f distinct effectors might be helpful. The starting point of the stabilizin g experiments was the extract of rubber tree leafs. Therefore the leafs wer e extracted with potassium citrate/phosphate buffer and this extract then c hromatographically separated into 5 fractions. The total extract and the fr actions thereof were tested for their effect on the enzyme half-life. The e xtract improved the half-life up to 5 times and one chromatographic fractio n increased the stability by 50 %. This fraction was further analyzed and i t was found that flavonoids were present. In the next step some pure flavon oids were tested for their effect and it was found that low concentrations of rutin (5-20 ng/mL) and hyperoside (1.5-6 ng/mL) were sufficient to impro ve enzyme stability up to 50 %. In addition, some synthetic flavonoids (Ven oruton (R) and monohydroxyethylrutoside) were tested and they also prove th is stabilizing effect. The increase of half-life was even higher, almost fi vefold in optimal conditions. The increase in the stability is assigned to a chelating effect on the one hand and a structural effect on the other han d. The impact of these effects cannot be presently differentiated.