7.1 Materials:
Naringin, naringenin, rutin, quercetin, hesperidin, L-rhamnose were from Sigma- Aldrich Chemical Company, St.Louis,(USA). All other chemicals were either from Merck Ltd. (Mumbai, India) or from S.D. Fine Chem.Ltd., (Mumbai, India) and were used without further purifications.
7.2 Methods
7.21: The fungal strain, its growth and preparation of the enzyme solution
The fungal strain F. moniliforme MTCC-2015 was procured by MTCC Centre and Gene Bank Institution of Microbial Technology, (Chandigarh, India).
It was maintained on the agar slant of composition potato (50 g), sucrose (5 g), agar (5 g) and water (250 mL) as mentioned in in the Catalogue of Stains, 2000 published by the Institute of Microbial Technology, (Chandigarh, India) [16].
For secretion of the enzyme, the fungal strain was grown in the medium and using the method reported in the literature [17].
The cultures was grown in under stationary condition for 7 days in a liquid culture growth medium(20mL) kept in 100mL culture flasks which were incubated in a B.O.D. incubator at 25℃. After 7 days the mycelia mats were removed, cultures were pooled and filtered through filter paper to remove particles and the filtrate was used as enzyme solution for enzymatic transformations.
7.22 The assay of diglycosidase activity
The activity of the enzyme was determined using naringin as the substrate and monitoring the decrease of naringin concentration with time spectrophotometrically by Davi’s method [18]. The assay solution consisted of 2.5mL of 0.86mM naringin in 0.1M sodium phosphate buffer pH 4.0 maintained at 40℃ in a water thermostat and 0.5 mL of the enzyme extract (culture filtrate). Aliquots of 0.2 mL were withdrawn at regular intervals of 5 minutes which was added to 2.5 mL of 90% diethyl glycol followed by addition of 0.1 mL 4N NaOH solution. The samples were maintained at room temperature for 10 minutes and absorbance values were measured at 420nm. The absorbance values were converted to naringin concentration with the help of a calibration curve. The concentrations of naringin were plotted against time and decrease in naringin concentration in µMole/min was calculated. UV/Vis spectrophotomer Hitach(Japan) U-2900 was used. The enzyme unit is defined as the amount of enzyme which decreased naringin concentration at the rate of 1µmole/min in 0.1M sodium phosphate buffer pH 4.0 at 40℃.
The enzymatic transformations of rutin, naringin and hesperidin by the enzyme solution:
The enzymatic transformations of rutin, naringin and hesperidin were tested by suspending 10mg of these in 2 mL of 0.1M sodium phosphate buffer pH 4.0 in three test tubes kept at 40℃ in a water thermostat and adding 0.2mL of the enzyme solutions containing 18.0 × 10-3 IU/mL in each of the test tubes. The reaction solutions were kept in the thermostat for two hours and the reaction solution was analysed for the transformation by TLC using silica gel on glass plates. For the analysis of flavonols, the mobile phase was n-butanol: acetic acid: water in the ratio 40:11:29 (v/v/v). For the analysis of carbohydrates, the mobile phase was ethyl acetate: 2-propanol: water in the ratio 3:2:2 (v/v/v). The products were located by keeping the silica gel glass plates in iodine chamber.
The identities of the transformation products were confirmed by purifying the products by preparative TLC and analysing those by mass spectrometry using LC/MS dual channel method at the Sophisticated Analytical Instrument Facilities (SAIF), CDRI, Lucknow (India).
Determination of the yield of transformations of rutin to quercetin and rutinose:
In 100mg of rutin suspended in 20mL of 0.1M sodium phosphate buffer pH 4.0, 1mL of the enzyme solution containing 18.0×10-3 IU as prepared above was added, the reaction was allowed to occur at 40℃ in water bath for 24 hours, the precipitated quercetin was filtered through filter paper, dried and weighed. In the filtrate 0.05g of each of calcium hydroxide, cerite and charcoal were added, the solution was boiled for 20 minutes, filtered through filter paper and the filtrate was dried to achieve rutinose which was weighed for the calculation of the yield [8].