Purification and characterization of naringinase from a newly isolated strain of Aspergillus niger 1344 for the transformation of flavonoids Munish Puri* and Sukirti Kalra Fermentation and Protein Biotechnology Laboratory, Department of Biotechnology, Punjabi University, Patiala 14 7002, India *Author for correspondence: Tel.: +91-175-2282461-65 ext. 6281, Fax: +91-175-2283073, E-mail: mpuri@pbi.ac.in Keywords: Aspergillus niger, debittering, naringin, naringinase, purification, transformation Summary 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-chloromercuribenzo- ate, 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. Introduction The presence of bitterness has been a major limitation in the commercial acceptance of citrus juices. Citrus tissues possess naringin, a flavonoid responsible for the bitter taste (Hasegawa & Maier 1993). Different approaches have been used to avoid naringin bitterness or to eliminate this flavonoid from citrus juices but few have achieved significant success (Johnson & Chandler 1988; Kimball 1991). Naringinase (EC 3.2.1.40), an enzyme detected in different species of microorganisms, is widely used to remove rhamnose and glucose from naringin and other glycosides to obtain the aglycone (Puri et al. 1996). Naringin is 4,5,7-trihydroxyflavonone 7-rhamnogluco- side and on hydrolysis with naringinase yields rhamnose and naringenin (4,5,7-trihydroxyflavonone), a non-bitter derivative, which cannot be reconverted to naringin (Chandler & Nicol 1975; Habelt & Pittner 1983). Although naringinases are not common enzymes, several industrial applications have been reported (for details see review, Puri & Banerjee 2000). The use of naringinase for removing naringin, the main bitter component of several citrus juices, is a common industrial practice (Thomas et al. 1958; Ono et al. 1978; Habelt & Pittner 1983). The deglycosylation of the novel glyco- peptide antibiotic, chloropolysporin from Faenia inter- jecta, was achieved successfully by the rhamnosidase activity of naringinase (Sankyo 1988). Also, the hydro- lysis of hesperidin by a-L -rhamnosidase to release L -rhamnose and hesperetin glucoside, an important precursor in sweetener production has been described (Manzanares et al. 1997). The enzyme is used to produce L -rhamnose which is a chiral intermediate in organic synthesis and it is used as a pharmaceutical and plant protective agent (Daniels et al. 1990). The production and characterization of an Aspergillus terus a-L -rham- nosidase activity of naringinase in combination with b-D -glucosidase is considered suitable for aroma enhancement in wine making (Caldini et al. 1994). Among the naringinase-producing fungi, the enzyme preparation from Penicillium decumbens (Young et al. 1989) is commercially available. Naringinase obtained from P. decumbens had been used for immobilization studies and for the transformation of flavonoids (Man- jon et al. 1985; Romero et al. 1985; Puri et al. 2001). On using commercial naringinase, incomplete hydro- lysis of naringin was observed (Puri et al. 1996, 2001). In World Journal of Microbiology & Biotechnology (2005) 21:753–758 Ó Springer 2005 DOI 10.1007/s11274-004-5488-7