Abstract:

Objective: To design bio-specific affinity chromatography for alliinase purification. Methods: Sepharose CL-4B coupled with competitive alliinase inhibitor, S-alkyl-L-cysteine, was developed as the affinity chromatography gel, which was activated by 1,4-butanediol diglycidyl ether in an alkaline condition. Crude enzyme was obtained through cryogenic comminution and precipitation with PEG-5000. Further purification of alliinase was conducted on prepared affinity chromatography column and its purity was examined by SDS-PAGE. Results: Alliinase was successfully purified through activated Sepharose CL-4B column coupled with S-alkyl-L-cysteine. A single band with 52 kD was shown in SDS-PAGE. A total amount of 0.1296 mg of alliinase with high purity (the specific activity was as high as 9.493 U/mg protein) was obtained from 1 g of garlic. Compared with the supernatant of comminuted garlic, its activity recovery rate was 68.42%, which exhibited 22.17-fold enhancement in purity. The optimal reaction temperature and pH for alliinase were 35 and 6.32, respectively. Km and Vmax were 9.845 mmol/L and 24.93 mol/mg min. Conclusion: S-alkyl-L-cysteine as the binding ligand can be applied to affinity chromatography for alliinase purification.

Key words: alliinase, isolation and purification, affinity chromatography, S-alkyl-L-cysteine