Influence of Flavonoids on the Stability of an (S)-Hydroxynitrile Lyase from Hevea brasiliensis

Roland Geyer¹*, Theodor Kartnig², Herfried Griengl³ and Walter Steiner<sup>1

1</sup>Institute of Biotechnology, SFB Biocatalysis Technical University of Graz, Petersgasse 12, A-8010 Graz, Austria
²Institute of Pharmacognosy University of Graz, Universitätsplatz 4, A-8010 Graz, Austria
³Institute of Organic Chemistry, SFB Biocatalysis Technical University of Graz, Stremayrgasse 16, A-8010 Graz, Austria

Article history:
Received December 7, 2000
Accepted March 23, 2001

Key words:
hydroxynitrile lyase, Hevea brasiliensis, stabilization, flavonoids

Summary:
The (S)-hydroxynitrile lyase from leaves of the rubber tree Hevea brasiliensis (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 chemical background reaction. To reduce the chemical background reaction, lower pH values (below pH=5) are necessary, but the enzyme in this case is not stable enough. To stabilize the enzyme at these conditions the addition of distinct effectors might be helpful. The starting point of the stabilizing experiments was the extract of rubber tree leafs. Therefore the leafs were extracted with potassium citrate/ phosphate buffer and this extract then chromatographically separated into 5 fractions. The total extract and the fractions thereof were tested for their effect on the enzyme half-life. The extract improved the half-life up to 5 times and one chromatographic fraction increased the stability by 50 %. This fraction was further analyzed and it was found that flavonoids were present. In the next step some pure flavonoids 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 improve enzyme stability up to 50 %. In addition, some synthetic flavonoids (Venoruton^® and monohydroxyethylrutoside) were tested and they also prove this stabilizing effect. The increase of half-life was even higher, almost fivefold 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 hand. The impact of these effects cannot be presently differentiated.

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