Molecular Redesign For The Catalytic Activity Of ?-L-Rhamnosidase On Rutin

?-L-Rhamnosidase catalyzes the natural products containing ?-Lrhamnose at the end.This enzyme is widely distributed in nature,and has been reported in bacteria,fungi,yeast,animals and plants.It has a wide range of industrial applications and is biological technology uses important enzymes.The purpose of this study is to construct a high through-put screening method to obtain highly active ?-L-rhamnosidase and or muatnts,study the enzymatic properties of the mutants,and hydrolyze natural flavonoid glycoside rutin to obtain a rare flavonoid monosaccharide glycoside isoquercetin with various biological activities.The target gene Bt Rha78 A had been successfully amplified by searching CAZy database and using the whole genome of Bacteroides thetaiotaomicron VPI-5482 as the template,and cloned into the vector p ET-28a to characterize the enzymatic properties.The tertiary structure and amino acid sequence of Lrhamnosidase were compared,and the key residues catalyzed by Bt Rha78 A were determined by alanine scanning mutagenesis verification.Studies on its broad acid motif showed that some of the residues in the motif were noncatalytic functional residues.The flavonoids rutin,isoquercetin and quercetin were measured in acidic,methanol and basic conditions at 0 min and 250-500 nm absorption after 30 min incubation.Rutin had characteristic peaks at 400 nm and quercetin at 320 nm has characteristic peaks.Based on this characteristic,we prepared a method to convert rutin to quercetin directly on the basis of ?-L-rhamnosidase and ?-D-glucosidase.A dual wavelength high throughput screening method was established,and highly active ?-D-glucosidase(Tn Bgl1A-DM)was successfully amplified by POE-PCR,and the target protein was purified.Bt Rha78 A changes with the enzyme concentration(0-80 ?g/m L)and reaction time(0-120 min),and its corresponding absorption values at 320 nm and 400 nm change,which confirms the feasibility of this dual wavelength screening method.Nine ?-L-rhamnosidases in the laboratory were screened for activity.The relative activity of hydrolyzed rutin was obtained by ?-L-rhamnosidase whole cell combined with dual wavelength screening method,which laid the foundation for high throughput screening.Based on previous research in the laboratory and semi-rational design thinking,it was determined that a saturated mutation library of Bt Rha78A-V338 was constructed,and the 96-well plate was pre-screened,re-screened and final-screened to obtain the mutant V338 S with an activity increase of 20%.Alanine scanning mutation technology was used to mutate the residues of Bt Rha78 A generalized acid motif,using full plasmid PCR to mutate the residue of the sequence alignment of the generalized acid motif.Four highly active mutants V338 A,V338I,S340 A and G341 A were screened by dual wavelength screening method.The results were consistent with the initial screening by HPLC.Enzymatic properties and whole cell properties of biotransformation rutin were studied on mutants V338 A,V388I,V338 S,S340A and WT with higher catalytic activity to obtain enzyme reaction time curves.Five enzyme reactions took 180 min to plateau,the conversion rate of V338 S was 98% for 240 min and 86% for WT.Studies on enzyme kinetic constants show that each constant of V338 A and V388 S is better than WT,and the enzyme kinetic constants of V388 I and S340 A are worse than WT.The whole cell reaction time curve shows that the conversion rate of WT whole cell reaction is 73.9% at 4.5 h to plateau,while V338 S whole cell reaction is 100% at 4.5 h.The number of whole cell recycling showed that V338 S was stable for the first 4 times and WT was stable for the first 3 times.In order to obtain natural flavonoid glycoside rutin from cheap raw materials,microwave-assisted organic solvent extraction was used to investigate the effects of solid-liquid ratio and ethanol concentration on rutin extraction from tartary buckwheat leaves and tartary buckwheat seeds.The optimal extraction conditions of tartary buckwheat seeds were as follows: ethanol concentration was 55%,solid-liquid ratio was 1:20.The optimal extraction conditions of tartary buckwheat leaves were as follows: ethanol concentration was 65% and the ratio of solid to liquid was 1:40.