Purification And Characterization Of Naringinase From Aspergillus Niger DB056

Naringinase, which can specifically hydrolyze naringin into naringenin, is an enzymecomplex consisting of α-L-rhamnosidase （EC3.2.1.40） and β-D-glucosidase （EC3.2.1.21）. Itplays an important role in the processing of citrus fruit, food aroma, and preparation of rhamnoseand so on.In the earlier research, we have screened a high-yield, safe naringinase-producing strain--Aspergillus niger DB056and developed its optimal fermetation process for naringinaseproduction. This paper mainly studied on purification and characterization of naringinase fromAspergillus niger DB056. The research mainly included establishment of the method fordetecting activity of naringinase, purification and characterization of naringinase,α-L-rhamnosidase and β-D-glucosidase and so on. The main results were as follows:（1）The HPLC conditions for determination of naringinase activity with Waters1525machineand Symmetry C18column were a flow of0.4ml/min, the detection wavelength of280nm andthe temperature of the column of35℃; the mobile phase was composed of62%of ultra-purewater and38%of organic phase which included30%methanol and70%acetonitrile. Thecalibration curve was of good linearity in the range of50-300μg/mL for naringin （R2=0.9964）and for naringenin （R2=0.9999） respectively. The recoveries of naringin were96.85%-101.65%while the recoveries of naringenin were88.83-100.66%. The method detection limits andquantitative detection limits were0.00109μg/mL and0.00363μg/mL for naringin, and0.0046μg/mL and0.01534μg/mL for naringenin. The recoveries were100-102.57%forα-L-rhamnosidase,100-108.30%for β-D-glucosidase and100-116.43%for naringinase.（2）The naringinase, α-L-rhamnosidase and β-D-glucosidase were purified steply byammonium sulphate precipitation, ion exchange and gel filtration chromatography with specificactivities of1716.41,8256.14and662.07units per milligram of protein and were purified by17.52,77.68and68.35fold, respectively. The α-L-rhamnosidase and β-D-glucosidase had amolecular mass of90kDa and95kDa, respectively.（3）Naringinase, α-L-rhamnosidase and β-D-glucosidase had an optimum pH of4.0-5.0,5and4.5-5.0and temperature of50℃、50-55℃and55℃receptively, they were stable in pH4.5-5,3.5-7.5and5.0-6.0and had good stability in low temperature.（4）Ag⁺、 Hg²⁺strongly inhibited activity of naringinase, α-L-rhamnosidase and theβ-D-glucosidase; Fe²⁺, Fe³⁺, Al³⁺, Cu²⁺inhibited the α-L-rhamnosidase and the β-D-glucosidase; Zn²⁺, Mn²⁺, Ca²⁺, Na⁺and Mg²⁺at a concentration of1.0mM inhibited β-D-glucosidase, andFe²⁺, Al³⁺at concentration of5.0mM inhibited naringinase; Ca²⁺, K⁺, Al³⁺, Mn²⁺, Cu²⁺at aconcentration of0.1mM could improve the activity of naringinase; Na⁺and Ba²⁺could improvethe activity of α-L-rhamnosidase; K⁺, Ba²⁺, Ca²⁺, Na⁺and Mg²⁺at a concentration of5.0mMcould improve the activity of β-D-glucosidase.（5）Rutin, naringin and p-nitrophenol-α-rhamnoside were hydrolyzed by α-L-rhamnosidasewith michaelis constant （Km）157.24μg/mL and Vmax588.24μg/（mL min）, usingp-nitrophenol-α-rhamnoside as a substrate; Aescuin, arbutin, p-nitrophenol-β-glucoside,myricitrin and pruin were hydrolyzed by β-D-glucosaminidase with michaelis constant （Km）550.33μg/mL and Vmax1670μg/（mL min） using p-nitrophenol-β-glucoside as a substrate.In this paper, we had set up the method for determination of naringinase by HPLC, purifiednaringinase, α-L-rhamnosidase and β-D-glucosidase from Aspergillus niger DB056broth, andclarified their characterization. These studies set a foundation for the production and applicationof naringinase.