DEGLYCOSYLATION OF FLAVONOIDS BY RHAMNOSIDASE BREVUNDIMONAS SP. CI19 AND THEIR IMMOBILIZATION BY CLEAS

A. E. ALVARENGA; M. I. ISLA; CASTRO, G.R.

Simposio; ESBES-IFIBIOP 2014. 10th European Symposium on Biochemical Engineering Sciences and 6th International Forum on Industrial Bioprocesses; 2014

The production of monoglycosylated flavonoids by rhamnosidases (EC 3.2.1.40) is an interesting application in the field of enzymatic biocatalysis for the removal of rhamnose. Pure flavonoid glycosides and those present in sweet orange (Citrus sinensis), grapefruit (Citrus paradise), lemon (Citrus limon) and sour orange (Citrus aurantium) juices were structurally modified by enzymatic de-glycosylation to obtain functional derivatives with higher antioxidant activity. The enzymatic reactions were carried out using partially purified extract from psychrotolerant Brevundimonas sp. Ci19. The enzymatic activity of Brevundimonas sp. Ci19 cell-free extracts were able to hydrolyze α(1→2) bonds of naringin and neohesperidine, and α(1→6) bond of rutine, but not of hesperidin. The scavenging activity was analyzed by ABTS in enzyme treated samples and controls after 1-hour incubation. The antioxidant activity of treated juices was higher than that of the untreated juices, confirming the enhancement of antioxidant activity made by the enzymatic reaction. The results presented herein are a new strategy to produce functional beverages by the use of α-L-rhamnosidases for deglycosylation of flavonoid glycosides. α-L-Rhamnosidase from Brevundimonas sp. Ci19 was immobilized as a cross-linked enzyme aggregate (CLEA) via precipitation with acetone followed by glutaraldehyde cross-linking. Sixty percent acetone solution and 2.0 % glutaraldehyde were used at pH 7.0 for 1-h cross-linking reaction. The rhamnosidase activity recovered in CLEAs starting from crude extracts o pure was approximately 80 % in both cases, suggesting non-purification steps are required for enzyme extended use. No significant differences in optimum pH and temperature values of the enzyme were recorded after immobilization. The rham-CLEA recycled 4 times showed about 80 % rhamnosidase activity; meanwhile, in the fifth and six recycled time, the enzyme activity was reduced to about 40 and 20 %, respectively.