High-efficiency Expression,molecular Modification Of Aspergillus Niger α-L-rhamnosidase And Its Application In The Hydrolysis Of Rutin

α-L-Rhamnosidase（EC 3.2.1.40）is a glycoside hydrolase that can hydrolyze the terminal L-rhamnose residues of natural glycosides such as flavonoids,saponins,terpene glycosides,steviol alcohols,etc.In recent years,α-L-rhamnosidases have attracted more attention due to its important application value in the food industry,such as improving the quality of fruit juice,increasing the flavor of wine and preparing sweeteners,and in the pharmaceutical industry,such as preparing antibiotics and bioactive substances.Although severalα-L-rhamnosidases have been commercially produced,the price is expensive.At present,the expression level ofα-L-rhamnosidase is low,meanwhile,due to the large volume and low solubility of glycosides,the catalytic efficiency of the enzyme is affected,which seriously hinders its application in food and pharmaceutical fields.In this study,anα-L-rhamnosidase was screened,cloned and expressed,the expression level ofα-L-rhamnosidase was significantly improved;the catalytic efficiency ofα-L-rhamnosidase towards substrate rutin was improved by molecular modification based on the study;the conversion efficiency ofα-L-rhamnosidase for rutin was improved by studying the catalytic reaction medium.Finally,magnetic metal-organic framework（MOF）material was used to immobilizeα-L-rhamnosidase,and the reusability of the enzyme was significantly improved.The main research contents are as follows:（1）Screening,cloning and expression ofα-L-rhamnosidase.Anα-L-rhamnosidase-producing strain Aspergillus niger CCTCC M 2018240 was obtained from nature.Theα-L-rhamnosidase（An Rha）gene rha was obtained by cloning.Through phylogenetic tree analysis,An Rha was belonged to the GH 78 family.The 3D structure of An Rha was obtained through homology modeling,and it contains a（α/α）₆-barrel structure,which is typical in GH 78 family enzymes.Using p PIC9K as the expression plasmid,the codon-optimized gene optrha was highly expressed in Pichia pastoris GS115.The recombinant strain GS115/9K-optrha-98 had the highest enzymatic activity of 0.45 U/m L;enzymatic properties analysis showed that the optimum temperature and p H of An Rha were 65℃and p H 5.0,respectively,53.08%of the enzyme activity was remained after incubation at 65°C for 8 h,with t_1/2 of 9.67 h.This enzyme can hydrolyze the disaccharide unit in natural glycosides,which containedα-1,2 andα-1,6glycoside bonds.After 120.95 h of high-cell density fermentation in a 5-L fermenter,the enzyme activity was 17.08 U/m L,which was 37.96 times of that in shaking flasks.（2）Improving the catalytic efficiency of 53.08%based on the 3D structure:The 3D structure of An Rha was obtained through homology modeling,and the key amino acid residues in the active pocket were mutated,and 4 mutants L234A,L234C,F345S and L234A/S339V/F345S were obtained with more than 2-fold catalytic efficiency that of WT,and a mutant S339V with significantly improved thermal stability.The k_cat/K_m value of F345S was43.84 s^-1m M^-1,which was 2.5 times that of WT(17.51 s^-1m M^-1).The optimum temperature and p H of F345S were 65°C and 5.0,respectively,and the residual enzyme activity remained55.25%after incubation at 65°C for 8 h with a t_1/2of 9.69 h.The optimum temperature of L234A decreased to 60°C,and the optimum p H decreased to 4.0.After incubation at 65°C for10 min,the residual enzyme activity was 46.4%,and the t_1/2 was only 11.27 min.Structural analysis shows that the enlargement of active pocket entrance of F345S was the main reason for improving the catalytic efficiency;MD simulation showed that the fluctuation of loop near the active pocket entrance and catalytic residue of L234A was increased,which improved the catalytic efficiency of the enzyme,but also reduced the thermostability of the enzyme.F345S was then high-cell dentisty fermented in a 5-L fermentor for 190 h,the OD₆₀₀ was 682,and the enzyme activity reached 53.84 U/m L,which was 119.64 times that of WT in the shaking flask.（3）Conversion of rutin in DES（deep eutectic solvent）solution:24 kinds of DESs were synthesized,and An Rha had the highest conversion rate for rutin in the DES（Ch Cl-U）solution synthesized by choline chloride and urea.In 40%Ch Cl-U solution,130 g/L rutin was completely converted within 60 min by 6 U/m L of An Rha,however,in buffer solution,the conversion rate of 130 g/L rutin by 8 U/m L An Rha was only 67.18%.When conversion of 130g/L rutin for 150 min using 6 U/m L mutant F345S enzyme,the conversion rate reached95.3%.In 40%Ch Cl-U solution,the conversion efficiency of F345S was slightly higher than that of WT.L-rhamnose was extracted by a two-phase system formed by K₂HPO₄ and 40%Ch Cl-U solution,meanwhile,the DES was reused.The purity of L-rhamnose prepared was96.28%.The average conversion rate of rutin was 95.63%,and the average purity of the prepared isoquercetin was 94.22%after reutilization of CHCl-U solution for 5 times.（4）Magnetic MOF material was prepared to immobilizeα-L-rhamnosidase:Magnetic MOF material was synthesized and for immobilization of An Rha,the activity of immobilized enzyme Rha@MOF was 25.09 U/g.SEM,TEM,FTIR,XRD and MS characterization indicating that the magnetic MOF carrier was successfully synthesized.Kinetic parameters showed that the affinity of the enzyme was improved after immobilization,but the catalytic rate decreased.The conversion rate of 20 g/L rutin in buffer was 91.42%by 1.2 U/m L of Rha@MOF.The enzyme activity of the immobilized F345S enzyme was 32.3 U/g,and the optimum substrate concentration was 30 g/L when the dosage of the enzyme was 1.2 U/m L with the conversion rate of 94.9%.After reusability of F345S@MOF for 30 cycles,the conversion rate was 73.21%.