α-L-rhamnosidase from Penicillium tardum and Its Application for Biotransformation of Citrus Rhamnosides

Enzymatic deramnosylation of flavonoids is a convenient tool for improving the quality of citrus juices. α-L-rhamnosidase with a specific activity of 33.1 units/mg was isolated and characterized from the culture liquid of Penicillium tardum. The molecular weight of the enzyme was 95 kDa according to the data of gel filtration on Sepharose 6B and gel electrophoresis in SDS-PAGE. The pH optimum of the enzyme activity was 5.0, and the thermo optimum was 60 °C. Enzyme showed high stability in the temperature range of 45-50 and at 60-70 °C. It retained 80 to 50% of the initial activity for 90 min. The half-life of α-L-rhamnosidase at 70 °C increased twofold in the presence of 20-40% glycerol and 2.3-fold in the presence of 4 M sorbitol. The enzyme was completely inhibited in the presence of 10^-3 M Ag⁺ and Cd²⁺ and approximately by 90% in the presence of Fe²⁺, Fe³⁺, and Al³⁺ ions. More than 60%, the enzyme activity was inhibited by Hg²⁺, Co²⁺, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide. Activating effect of Ca²⁺ ions was also noted. K_m and V_max for the hydrolysis of p-nitrophenyl-α-L-rhamnopyranoside and naringin were 0.7 mM and 38.3 µM/min/mg and 1.34 mM and 43.7 µM/min/mg, respectively. Penicillium tardum α-L-rhamnosidase hydrolyzed naringin, neohesperidin, hesperidin, rutin, and narirutin at high rate, which allowed us to consider it as an effective tool for transformation of bioflavonoids in food industry.