Rapid Evaluation Of The Selectivity Of α-L-rhamnosidase To Citrus Flavonoid Glycoside Substrates And Molecular Modification

α-L-Rhamnosidase is a glycoside hydrolase that can specifically catalyzeα-L-rhamnose at the end of natural glycosides such as rutin,hesperidin,naringin,and ginsenoside.As an important class of biocatalysts,they widely exist in nature and have high application value in industries such as food,medicine,beauty,and chemical engineering.However,at present,there is a lack of efficient and specific evaluation methods for the catalytic activity ofα-L-rhamnosidase,and the lack ofα-L-rhamnosidase with good stability and high catalytic activity.It is urgent to develop efficient library building strategies to obtainα-L-rhamnosidase with excellent properties quickly,so as to promote its development and application in industrial production.In this study,firstly,four citrus flavonoid diglycosides,hesperidin,neohesperidin,naringin,narirutin,and their corresponding aglycones hesperetin and naringenin,were scanned at a wavelength of 250 nm to 550 nm under acidic,alkaline,and methanol conditions.The results showed that the spectra of citrus flavonoid diglycosides and their corresponding aglycones were different under alkaline conditions,that is,the aglycones had characteristic peaks at 320 nm under alkaline conditions.Therefore,α-L-rhamnosidase can be used to removeα-L-rhamnosidase from citrus flavonoid diglycosides and convert it to flavonoid glucoside,andβ-D-glucosidase can catalyze flavonoid glucoside to produce corresponding aglycones.The absorption value at 320 nm was determined by ultraviolet-visible spectrophotometry to quantify aglycans,so as to evaluate the activity ofα-L-rhamnosidase to catalyze citrus flavonoid diglycosides.Based on the above studies,a series of intestinal bacteria fromα-L-rhamnosidase HFM-RhaA,HFM-RhaC,HFM-Rha78,BtRha78A,BtRha78D combined with thermophilic and highly activeβ-D-glucosidase Tn Bgl1A-DM were selected in this study.Ultraviolet-visible spectrophotometry was used to rapidly evaluate its substrate selectivity to four citrus flavonoids diglycosides.The results showed that HFM-RhaA,HFM-Rha78 and BtRha78D showed high catalytic activity on neohesperidin and naringin（preferentially hydrolyzedα-1,2 glucoside bond）,and BtRha78A could specifically hydrolyzed narirutin and hesperidin（α-1,6 glucoside bond）,but had no hydrolyzed activity on neohesperidin and naringin.HFM-RhaC can hydrolyze all the four citrus flavonoid diglycosides,among which the catalytic ability of hesperidin and narirutin is stronger（preferring to hydrolyzeα-1,6 glucoside bonds）.Subsequently,the catalytic activities of the above fiveα-L-rhamnosidases on four citrus flavonoid diglycosides were detected by high performance liquid chromatography with fixed enzyme concentration and reaction time.The chromatographic peak graph shows that the results are consistent with the above,confirming the feasibility of ultraviolet-visible spectrophotometry.Secondly,a novel semi-rational molecular modification strategy-six-codons combinatorial mutagenesis（SCCM）was used to modifyα-L-rhamnosidase BtRha78A derived from Bacteroides polymorphoides to improve its catalytic activity against hesperidin.Based on the analysis of the tertiary structure and homologous sequence alignment of BtRha78A,three mutant target motifs at the entrance of the substrate binding pocket were identified.A library of six-codons combinatorial mutants was constructed by whole plasmid PCR,initial screening by ultraviolet-visible spectrophotometry and final screening by high performance liquid chromatography.Four excellent mutants with higher hydrolytic capacity than the wild type were obtained by sequencing,which were TM1-6-F5,TM1-6-H6,TM1-7-G1 and TM1-8-F9.The relationship between the biological conversion rate and reaction time of wild type and mutants hesperidin was determined by high performance liquid chromatography.The mutant TM1-6-F5 had the highest catalytic activity of hesperidin,and the conversion rate reached 50.67%when the reaction time was 20 min,while the wild type had only 53.97%when the reaction time was 60 min.Finally,the enzyme kinetic constants,thermal stability and substrate selectivity of the wild type and mutants were measured.The results showed that the kinetic constants of the mutants were higher than those of the wild type.The catalytic efficiency of TM1-6-F5 was the highest,and K_cat/K_Mwas 1.4 times that of the wild type.The thermal stability of the four mutants decreased compared with that of the wild type,and TM1-6-F5 and TM1-8-F9 showed better thermal stability at 37℃,with more than95%enzyme activity after being held for 240 min.In addition,the determination of substrate selectivity showed that,compared with the wild type,the catalytic activity of the four mutants on the target substrates hesperidin,rutin and pNPR had varying degrees of change.