Catalytic Properties And Functional Study Of ?-L-rhamnosidase From Bacillus Sp. GL1

Flavonoids compounds and their derivatives have various physiological functions.The mechanism of microbial enzymes has been extensively studied.?-L-Rhamnosidase can release the terminal rhamnose from a series of flavonoids compounds,and the corresponding products have higher biological activity than flavonoids.The?-L-rhamnosidase gene from Bacillus sp.GL1 was cloned and expressed in Escherichia coli Rosetta,purified to homogeneity,and characterized.In this study,the conversion of several flavonoids by recombinant enzymes was tested to provide an experimental basis for the application of?-L-rhamnosidase.Based on the results of domain analysis,a mutant that deleted the carbohydrate binding module?CBM?was constructed,and the effect of CBM on the function of?-L-rhamnosidase was discussed.The main experimental results were as follows.?1?RhaA,the approximately 98 kDa recombinant gene product,was expressed in E.coli Rosetta.The optimal pH and temperature were 40°C and pH 7.0,respectively.RhaA was stable at 4°C–40°C and pH 6.0–11.0.Moreover,0.1%glycerol,which was approximately 10%higher than the control group,exerted an optimal effect on RhaA.However,RhaA activity was inhibited by methanol,ethanol,propanol,butanol,Tween-80,Triton X-100,acetylacetone,succinic anhydride,chloramine-T,PMSF,and DTT.The activity of RhaA was inhibited by Mn²⁺and Ca²⁺divalent metal ions.Metal ions such as Na⁺,K⁺,Mg²⁺,Fe³⁺,Fe²⁺,Co²⁺,Zn²⁺,and especially Cu²⁺were potent inhibitors of RhaA.Almost half of RhaA activity was inhibited by 1 M NaCl,and 0–1.5 M KCl maintained over 60%of RhaA activity.The effect of K⁺on RhaA activity was lower than Na⁺.Furthermore,1 and 2.5 M L-rhamnose maintained RhaA activity at approximately 50%and 25%,respectively.?2?RhaA can hydrolyze naringin and rutin to prunin and isoquercetin,respectively.Under the optimal conditions,the optimal time for RhaA to transform naringin and rutin to prunin and isoquercetin were 36 and 24 h,respectively,which indicated that RhaA exhibited higher selectivity in cleaving the?-1,6 glycosdic bond between rhamnoside and glycoside than the?-1,2 glycosdic bond.Given the different mother ring structures between naringin and ginsenoside Re,RhaA cannot hydrolyze Re.?3?The activity and substrate specificity of the CBM-deleted mutant RhaA-CD was tested.The specific activity of RhaA-CD was 103.02 U/mg,which was lower than that of RhaA at 659.77 U/mg.RhaA-CD was active against pNP?Rha and showed weak degradation activity against pNP?Glu,pNP?Arab,pNP?Gal,pNP?Fuc,pNP?GluA,and pNP?Cel.RhaA-CD also cannot convert naringin and rutin,indicating that CBM may affect enzyme activity and substrate properties by influencing the interaction between enzyme and substrate.We further used the MBP of the pMAL-c2x vector to replace the CBM and determined whether the effect of the N-terminal sequence on RhaA was related to the sequence of CBM.Results showed that the MBP increased the specific activity of RhaA-CD to 345.75 U/mg,which was still lower than that of RhaA.The substrate specificity also changed significantly,indicating that the N-terminal sequence of RhaA considerably affected enzyme activity and substrate specificity,and this effect was related to the amino acid composition of the sequence.