Cloning, expression and enzymology properties of α-L-rhamnosidase gene from Enterococcus avium

Abstract:

[Background] A bacterium strain producing α-L-rhamnosidase was screened and identified as Enterococcus avium by molecular biological methods in the preliminary work. α-L-rhamnosidase can specifically cut terminal rhamnose from natural flavonoid compounds, which has great development prospect and application value in food production, pharmaceutical processing and chemical industry. [Objective] The α-L-rhamnosidase gene from E. avium was cloned and expressed, and the enzymatic properties of the recombinant protein were further studied. [Methods] Based on the putative α-L-rhamnosidase gene sequence in the genome of Enterococcus avium strain 352, specific primers were designed to amplify its coding sequence. Recombinant expression plasmid was constructed using pET-28a(+) as vector and the recombinant protein was expressed in Escherichia coli BL21(DE3) competent cells. The recombinant protein was purified by nickel affinity chromatography, and the enzymatic properties were determined using pNPR as a substrate. [Results] The molecular weight of the fusion protein EaRha1 is about 130 kDa. The optimal pH of EaRha1 is 7.0, the optimal temperature is 50℃, EaRha1 is stable at pH 5.0-8.0 and can maintain higher enzyme activity below 40℃. Metal ions can promote or inhibit EaRha1 in different degrees. Methanol has inhibitory effect on EaRha1, and the inhibitory effect increases with the increase of methanol concentration. The kinetic characteristic constants K_m and V_max of EaRha1 were 0.35 mmol/L and 4.2 μmol/(mg·min) (R²=0.999) respectively. The recombinant EaRha1 could catalyze the hydrolysis of neohesperidin, naringin and rutin. [Conclusion] In this study, the hydrolysis characteristics of the protein to flavonoids was determined by studying the enzymatic properties of recombinant protein EaRha1, which laid a theoretical foundation for the biotransformation of flavonoids.