Purification And Characterization Of α-L-rhamnosidase In Enterococcus Duerans

α-L-rhamnosidase is a kind of hydrolase , it can hydrolyze compounds of flavonoids nucleoside which are common compounds in daily diet.α-L-rhamnosidase is widely distributed in bacteria and fungi. It has a lot of potential applications in actual processes. Enterococcus durans was selected as fermentation strain. Enzyme activity, purification and enzymatic property ofα-L-rhamnosidase were studied in this paper.The main experimental results are as follows:(1) This work identified the determination method ofα-L-rhamnosidase from Enterococcus durans as following: in a 10 mL colorimetric tube accessed 2 mL 0.02 mol/L pH 5.0 citric acid-disodium hydrogen phosphate buffer solution, then add crude enzyme extract in it, keep in water bath at 45℃for 5 min, and then incorporated 0.2 mL the preheated substrate pNPR solution into them, after 14 min get out immediately and add 2 mL 1 mol/L Na2CO3 solution to terminate the reaction, then measure the absorbence at 400 nm when it was cooled to room temperature.(2) With a single factor and orthogonal experiment, the fermentation conditions ofα-L-rhamnosidase producing of Enterococcus durans were optimized to be the best basis for enzyme production medium nutrient as : saccharose 1.25%, soybean peptone 1.25%, K2HPO3 2 mmol/L, beef extract 0.3%, peptone 1%, NaCl 0.5%, Fe2(SO4)3 3 mmol/L; the correspondent fermentation conditions of Enterococcus duransα-L-rhamnosidase producing were as following: initial pH 7.0 of nutrient medium, fermentation temperature is 34℃, fermentation time is 4 days, rotation speed is 180 r/min, 250 mL flask with 50 mL liquid volume, inoculation size 3%.(3) Purification ofα-L-rhamnosidaseâ‘ It is determined that the concentration for the ammonium sulphate was between 50% and 80%.â‘¡In order to concentrate the precipitation after ammonium sulphate to 5 mL, dissolved it with 4℃distilled water, and then enclosed in dialysis membrane and sink it into 4℃distilled for 30 hours.â‘¢Purify theα-L-rhamnosidase by means of anion-exchange chromatography on DEAE-Sephadex 52: The balance buffer at pH 5.0 was used as eluent and the concentration range of sodium chloride was controlled between 0.2 and 0.6 mol/L. Below 0.2 mol/L or above 0.6 mol/L, there was almost no protein eluted out. The velocity eluate is 0.6 ml/min, and collect each tube every 15 min. The NO.12 tube displayed the highest activity among all tubes, and the first protein peak has no activity. When the sodium chloride concentration was between 0.3 mol/L and 0.5 mol/L, theα-L-rhamnosidase can be eluted completely.â‘£The unrefinedα-L-rhamnosidase was applied to the column of gel filtration chrmatography on Sephadex G-100. The velocity was 0.4 mL/min, and collect each tube every 15 min. Three protein peaks were eluted out, the third one is target peak.(4) The homogeneity of the enzyme was verified by using SDS-PAGE after salting out and Gel-filtration chromatography. The concentration of separate glue is 10% and the concentrate glue is 4%. The result showed that after the ammonium suphate fractional precipitation it contained some proteins with different molecular weights. However, after gel-filtration chromatography on Sephadex G-100, theα-L-rhamnosidase was purified completely and it showed homogeneity by SDS-PAGE. The molecular weight was about 90 K Da.(5) Enzymatic properties study indicate that this enzyme has high acid tolerance and thermal stability. The enzyme activity synergistically increased by the addition of metal cations such as K⁺ ,and Mg²⁺, meanwhile, Ca²⁺ and Fe²⁺ can significantly inhibit its activity. Some kinds of organic solvents can inhibit its activity as well, especially acetone. Kinetic studies showed that Michaelis constant Km is 4.58 mmol/L, Vmax is 16.13 U/L for the enzyme, Pnpr was used as substrate.