Enzymeatic Characterization And Catalysis Of An ?-L-Rhamnosidase HFM-Rha78

?-L-rhamnosidase can specifically cleave the ?-rhamnose group from the terminal of natural products and effectively improve biological activity and bioavailability.We had obtained a novel ?-L-rhamnosidase HFM-Rha78 with independent intellectual property through conserved motif-based PCR and high-throughput sequencing from human fecal metagenome.The molecular mass of this enzyme is 85.5 k Da and the isoelectric point is 5.58.The ?-L-rhamnosidase HFM-Rha78 carries the N-terminal(His)6-tag by cloning into the vector p ET-28 a.This study systematically investitaged the enzymatic properties and biotransformation for flavonoid glycosides,and provided the basis for further application of this enzyme,which is of great significance and value for mining new ?-L-rhamnosidase resources.Bioinformatic analysis showed that the hydrophilic amino acids distribution of HFM-Rha78 is uniformed with no obvious hydrophobic region,and the grand average of hydropathicity is-0.425,appears holistic hydrophilicity.The N-terminal raw cleavage site score is 0.135,the signal peptide mean score is 0.205,and the combined cleavage site score is 0.185,all of the scores are at a low level,indicating that the enzyme is lack of signal peptide.The secondary structure of HFM-Rha78 mainly contains ?-helix and random coil,accounting for 33.43% and 41.47%,respectively.And the rests are ?-turn and extended strand,accounting for 6.8% and 18.31%,respectively.Both the general acid and the general base of the enzyme HFM-Rha78 are Glu,and the catalytic residues are highly conserved.The enzymatic properties of HFM-Rha78 were determined by UV-vis spectrophotometer using p-nitrophenol-?-L-rhamnopyranoside(p NPR)as the substrate.The results revealed that the optimal conditions were p H 6.5 and 50 oC.The enzyme possessed certain thermal stability,there's no effect on its enzyme activity during thermal incubation for 30 minutes at 46 oC.It could generally tolerate low concentrations of organic solvents,even 1-5% of isopropanol and 1% of ?-mercaptoethanol could significantly increase its enzymatic activity.Enzyme kinetics demonstrated that HFM-Rha78 had a weak affinity towards p NPR.The hydrolytic activity of HFM-Rha78 on the flavonoid glycosides rutin,naringin,and hesperidin was studied by high performance liquid chromatography(HPLC).The results showed that this enzyme could specifically biotransform three substrates into isoquercetin,prunin,and hesperetin-7-O-glucoside,respectively.The conversion rates from large to small are naringin,rutin,and hesperidin.The optimum temperature for the hydrolysis of rutin,naringin and hesperidin were 39 oC,45 oC,and 42 oC,respectively,and the optimum p H were 7.5,6.5 and 5.5-6.0.The optimum reaction time of naringin was the shortest,2 h,and those of rutin and hesperidin was 12 h.