Purification and characterization of naringinase from a newly isolated strain of Aspergillus niger 1344 for the transformation of flavonoids

Abstract

An extracellular naringinase (an enzyme complex consisting of a-L-rhamnosidase and b-D-glucosidase activity, EC 3.2.1.40) that hydrolyses naringin (a trihydroxy flavonoid) for the production of rhamnose and glucose was purified from the culture filtrate of Aspergillus niger 1344. The enzyme was purified 38-fold by ammonium sulphate precipitation, ion exchange and gel filtration chromatography with an overall recovery of 19% with a specific activity of 867 units per mg of protein. The molecular mass of the purified enzyme was estimated to be about 168 kDa by gel filtration chromatography on a Sephadex G-200 column and the molecular mass of the subunits was estimated to be 85 kDa by sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 4.0 and temperature of 50°C, respectively. The naringinase was stable at 37°C for 72 h, whereas at 40°C the enzyme showed 50% inactivation after 96 h of incubation. Hg 2+ , SDS, p-chloromercuribenzoate, Cu 2+ and Mn 2+ completely inhibited the enzyme activity at a concentration of 2.5-10 mM, whereas, Ca 2+ , Co 2+ and Mg 2+ showed very little inactivation even at high concentrations (10-100 mM). The enzyme activity was strongly inhibited by rhamnose, the end product of naringin hydrolysis. The enzyme activity was accelerated by Mg 2+ and remained stable for one year after storage at )20°C. The purified enzyme preparation successfully hydrolysed naringin and rutin, but not hesperidin.

Kinetic Properties of Extracellular Thermophilic Naringinase Produced by Rhizophus Stolonifer Isolated from Palmyrah Fruit Pulp

“…Palmyrah Fruit Pulp at 37 o C for 72 hours which showed its optimum activity at 50 o C and at pH 4.0 (Puri and Kalra, 2005). The stability was determined at low temperature (37 o C) but in the present study, stability was determined at 60 o C. The enzyme activity is influenced by different factors such as temperature, pH, substrate concentration, enzyme concentration, inhibitors, metal ions and incubation period.…”

“…When Rhizophus stolonifer strain was grown in the solid state fermentation system using paddy husk as media, naringinase production was higher (269.84 U/gram of dry substrate) in seven days (Karuppaija et al, 2016b). The activity of the crude naringinase obtained from this strain was maximum at 65 o C. Activity of naringinase from Aspergillus flavus and Aspergillus niger were optimum at 50 o C (Radhakrishnan andSampath, 2013 andPuri andKalra, 2005). Another study indicates that the activity of crude naringinase from Aspergillus niger and Penicillium decumbens were optimum at 55 o C (Norouzian et al, 2000 andThammawat et al, 2008).…”

Evaluation and characterization of new α-L-rhamnosidase-producing yeast strains

“…The purified enzyme having the optimum pH in the neutral pH range is more suitable for the enhancement of wine aroma (Yadav et al, 2012). The α-L-rhamnosidases reported from Curvularia lunata (Feng et al, 2007), Aspergillus niger (Puri and Kalra, 2005) and Aspergillus kawachii (Koseki et al, 2008) have same optimum pH i.e., 4. Yanai and Sato (2000) reported that the enzyme from yeast Pichia angusta showed an optimum activity at pH 6.…”

“…The optimum temperature obtained was similar to the α-L-rhamnosidases from Bacillus sp. GL1 (Hashimoto et al, 1999), Lactobacillus plantarum NCC 245 (Avila et al, 2009), Aspergillus flavus (Scaroni et al, 2002), Curvularia lunata (Feng et al, 2007), Aspergillus niger (Puri and Kalra, 2005) and Aspergillus kawachii (Koseki et al, 2008). Enzymes from Pichia angusta (Yanai and Sato, 2000), Aspergillus kawachii, Penicillium aureatiogriseum and Trichoderma longibrachiatu (Scaroni et al, 2002) were optimally active at 40°C and 60°C.…”

“…α-L-rhamnosidases catalyze the hydrolysis of a wide spectrum of natural glycosides containing terminal L-rhamnose, enhancement of aroma such as the debittering of grapefruit juices by the hydrolysis of naringin and derived beverages (Manzanares et al, 2003;Puri and Kalra, 2005), the elimination of hesperidin crystals from orange (Terada et al, 1995) and a large number of natural products which include naringin, rutin, quercitrin, diosgene and ter-penyl glycosides (Feng et al, 2005).…”