Structure And Characterization Of The Naringinase From Aspergillus Aculeatus Jmudb058for The Hydrolysis Of Naringin

Naringinase, containing both activities of α-L-rhamnosidase （EC3.2.1.40） andβ-D-glucosidase （EC3.2.1.21）, has many important applications in food and pharmaceuticalindustries. Reseachers has been studied the sources and fermentation process of naringinase, thestructure of naringinase hasn`t been revealed, and its characterization for the hydrolysis ofnaringin hasn`t been illustrated yet. The naringinase from Aspergillus aculeatus JMUdb058waspurified and inverstigated in the primary structure and the characteristics for the hydrolysis ofnaringin in the present thesis. The main results were listed below.（1） The HPCL method was applied to measure the activities of naringinase,α-L-rhamnosidase and β-D-glucosidase in concern to the hydyolysis of naringin. Theconcentrations of naringin, prunin and naringenin were determined on a Waters2695HPLCinstrument coupled with a2487UV detector and Symmetry C18reversed-phase column.Detection was performed at280nm with injection volume of20μL, the column temperature of35C and a flow rate of0.4mL/min. The mobile phase was composed of water （A）, methanol （B）and acetonitrile （C）. The gradient procedure were0-7min, A/B/C maintained at62/12/26;7-9min, A/B/C changed to15/35/50;9-15min A/B/C maintaind at15/35/50;15-17min, A/B/Cchanged to62/12/26;17-20min, A/B/C maintained at62/12/26. The method could detectnaringin, prunin and naringenin accurately, making the determination of naringinase,α-L-rhamnosidase and β-D-glucosidase accurately.（2） The naringinase purified by the procedure of ammonium sulphate precipitation,DEAE-Sepharose Fast Flow and SephacylTMS300HR gel chromatography had four submitsthrough SDS-PAGE analysis. The four subunits had MW at69kDa,84kDa,95kDa and100kDa, respectively. The smallest subunit was identified to be α-L-rhamnosidases, and the otherthree were all β-D-glucosidase.（3） It was found that the α-L-rhamnosidase of Aspergillus aculeatus JMUdb058had onecatalytical domain of the clan-L （alpha/alpha）（6）barrel with the possible active sites of Asp247,Asp252，Asp259and Glu523, and the β-D-glucosidase had one （alpha/beta）（8）barrel domain andone six-stranded beta sandwich domain with the possible essential catalytic residues of thenucleophile Asp280and the acid/base Glu509by means of homology analysis and modeling ofthree dimentional structure.（4） The optimum pHs of the naringinase and its α-L-rhamnosidase, β-D-glucosidase were all4.0and the optimum temperature were50°C,50-60°C and50°C respectively. They werestable within pH3-6and below50°C, and were strongly inhibited by mental ions of Ag⁺, Fe²⁺and Fe3+at1mM and10mM.（5） For the hydrolysis of naringin at pH4.0and50°C, the naringinase and itsα-L-rhamnosidase and β-D-glucosidase showed michaelis constant （Km） of0.11mM,0.23mMand0.53mM, respectively; maximum velocity （Vmax）of258.6U/mg,565.1U/mg and707.6U/mg, respectively; turnover constant （Kcat） of14034s-1×mM-1,14146s-1×mM-1and7733s-1×mM-1, respectively.In conclusion, the study has revealed the primary structure of naringinase from A. aculeatusis a tetramer consists of three β-D-glucosidase subunits and one α-L-rhamnosidase subunit. It hasdemonstrated the mechanism of acid and base catalysis clear, in addition, it also has illustratedthat the α-L-rhamnosidase was the main factor affecting the velocity of naringin hydrolysis.These results set up a foundation for further research of naringinase and its application in foodindustry.