Purification,Characterization And Structural Analysis Of A ?-L-rhamnosidasefrom Aspergillus Niger

?-L-rhamnosidase?Rha,EC 3.2.1.40?,which cleaved terminal?-L-rhamnose specifically from a large number of natural products,had many important application in food and pharmaceutical industries.The result from Genome sequence of Aspergillus niger CBS 513.88indicated the existence of five Rha genes of the fungus Aspergillus niger,of which four Rha genes had been identified.The purpose of this paper was to purify and characterized the Rha from the solid-state fermentation of Aspergillus niger from the aspectives of both enzymatic property and structure.The main results were shown in the following:?1?The Rha purified to homogeonity by the combined procedure consisting of ammonium sulphate precipitation,HiTrap Phenyl Fast Flow,HiTrapTM Bllue HP and Sephacyl^TM S 300HR gel chromatography.It was monomer with a mass weight at 164.48 kDa,through the analysis of SDS-PAGE and gel filtration chromatography.The specific activity and purification fold was79180.2 U/mg and 117.8.The results of glycosidase hydrolysis indicated the enzyme did not contain sugar group.?2?The optimum pH and optimum temperature of the Rha were 4.0-5.0 and 50-60°C,respectively.It was stable within pH3.0-8.0 and below 60°C.The Rha showed derhamnosylation activity for naringin,myricetrin,rutin,but non activity for p-nitrophenyl-?-L-rhamnoside.For the hydrolysis of naringin at pH5.0 and 50°C,the Rha showed the michaelis constant?K_m?of 137.25?g/mL?0.24?mol/mL?and maximum velocity(V_max)of 312.5 U/mL.?3?The MALDI-TOF/TOF analysis indicated that the Rha had a homology of 90%with the Rha from Aspergillus kawachii.The cDNA encoding the Rha was cloned from A.niger according to the identified sequence using both 3'and 5'Race method.The gene was observed with great similarity to that of a Rha from Aspergillus kawachii with the accessory number of AB374267.1and the hypothetical protein ANI₁₁896104?Aspergillus niger 513.88,XP₀01395635.2?,their homogenty reached 90%and 86%,respectively.The obtained cDNA was 2667 bp which was predicted to encode 888 amino acid residues.In addition,the homology,physicochemical properties,hydrophobicity and the primary structure was cpmpared to some other Rha,which indicate that the new purified Rha has great differences from other Rha from the nucleic acid sequence,structure,and enzymatic property paramaters.The study has verified an unknown Rha from Aspergillus niger which was demonstrated having novel properties,providing an valuable materials and reference to explore an practical Rha with specficial properties.