Preparation Of ?-L-rhamnosidase And Its Application In The Modification Of Flavonoids

As a glycoside hydrolase,?-L-rhamnosida(EC 3.2.1.40)can hydrolyze the glycosidic bonds in flavonoids containing rhamnose,thus increasing the water-solubility and physiological activity of these compounds.Therefore,?-L-rhamnosida has important application value in the fields of medicine,food and cosmetics.Aspergillus Niger is a kind of commonly used strains producing?-L-rhamnosidase,but most wild Aspergillus Niger produces?-L-rhamnosidase at the same time as?-glucosidase,which not only increases the difficulty of enzyme isolation and purification,but also interferes with the application of enzyme research.Through genetic engineering technology,the genetic engineering strain producing only?-L-rhamnosidase could be obtained.Through previous experiments,we constructed and screened several engineered strains of Pichia pastoris containing?-L-rhamnosidase encoding protein gene.In this paper,an engineered strain N12 with high yield of?-L-rhamnosidase was further screened,and its enzyme-producing conditions,isolation and purification methods,enzymological properties,application and immobilization were studied.The main research contents and results are as follows:1.Eleven engineered Pichia pastoris strains containing?-L-rhamnosidase encoding genes obtained in the previous study were induced to express?-L-rhamnosidase under the same fermentation conditions,and the strain N12 with the highest expression level was screened out for subsequent study.In order to optimize the fermentation conditions of recombinant Pichia pastoris,the effects of different inoculation amount and fermentation time on enzyme activity were investigated.The results showed that when the initial OD value of fermentation broth was 1 and the induction fermentation time was 7 days,the enzyme activity was the highest,which was 3250 U/m L.2.Recombinant?-L-rhamnosidase N12-rha was isolated and purified,and its molecular weight was determined by SDS-PAGE.The results showed that the molecular weight of the enzyme purified by nickel column was about 97k D.The size of N12-rha after digestion with N-glycosidase F was about 70 k D,which was consistent with the molecular weight of protein calculated by computer simulation.Through the ultrafiltration concentration method,the crude enzyme solution can be concentrated50-100 times,and the concentrated enzyme solution can be freeze-dried into high-activity enzyme powder,which is convenient for transportation and storage.3.The optimal reaction temperature of N12-rha is 50?,and the optimal reaction p H is 4.8;Under 60?,can maintain a stable enzyme activity,with a certain heat resistance,at p H value of 3-6,can maintain a stable enzyme activity,with a certain acid resistance.N12-rha can hydrolyze naringin,rutin,hesperidin and neohesperidin,but can not hydrolyze myricin and icariin.Using different concentrations of rutin solutions as substrates,the Vmax and Km of the enzyme were calculated as 1.316m M/L·min and6.765mmol/L.Mg²⁺,Fe³⁺,Fe²⁺and Zn²⁺could be increased by adding 1 m M,10 m M,50 m M,100 m M and 150 m M.The concentration of Ba²⁺at 1 m M,10 m M,50 m M,100 m M and 150 m M inhibited the enzyme activity to some extent.EDTA and?-ME had little effect on the enzyme activity,while high concentration of DTT had some effect on the enzyme activity,while SDS could inhibit the enzyme activity.4.N12-rha was used to hydrolyze new hesperidin,hesperidin,rutin and naringin,respectively,to explore the hydrolysis capacity and hydrolysis yield of the enzyme for flavonoid compounds containing different glycosidic bonds.The results showed that the hydrolyzing ability of N12-rha was higher than that of Hesperidin under the same conditions.According to the calculation,the yield of new hesperidin and hesperidin hydrolyzed into hesperidin 7-O-glucoside was 94.5 and 93.9%,respectively.N12-rha hydrolyzed rutin more than naringin.The yields of rutin and naringin hydrolyzed into isoquercetin and pulunin were 93.6%and 91.2%,respectively.5.The immobilization of N12-rha was studied by using the embedding method,and the optimal immobilization material ratio was determined to be 3%chitosan solution and 8%polyvinyl alcohol solution;The optimal concentration of glutaraldehyde was 2%,the optimal activation time was 2h,and the optimal cross-linking time was 24h.The optimal reaction temperature of immobilized enzyme was60?,the stable temperature was 30-80?,the optimal reaction p H was 5,and the stable p H was 3-8.Compared with the free enzyme,the acid and alkali resistance and thermal stability of the immobilized enzyme were improved to some extent.In addition,the immobilized enzyme can maintain a relatively high enzyme activity after multiple hydrolysis reactions,and can be used repeatedly.