Isolation, Purification And Characterization Of S-Stereoselective Amidase From Paracoccous Sp. W10173

Amidase, which catalyzes the hydrolysis of amides to the corresponding acids andammonia, is a promising synthesis tool for chiral amides and related derivatives. The（S）-amidase produced by Paracoccous sp. W10173, which was screened by our lab, cantransform （R,S）-piperazine-2-carboxamide to yield S-piperazine-2-carboxylic acidstereoselectively. Thus it can be used in the producting of important drug intermediates ofpiperazine-2-carboxylic acid single enantiomers.Firstly, the study on isolation and purification methods of the （S）-amidase of W10173were carried out. The ultrasonic treatment, salting out, and ion exchange chromatographyconditions of （S）-amidase from W10173were explored and established preliminary. Theoptimal ultrasonic treatment conditions were determined at the output power of400W,radition time of3s （with a5s interval）, treat for240cycles altogether. The best range ofammonium sulfate saturation for salting-out was20%-60%and the salting-out should berepeated three times. The optimal pH of potassium phosphate buffer for DEAE-52ionexchange chromatography was determined at pH7.0, the velocity of flow was250mL/cm2/hr,and the eluent follow were c（NaCl）0.25mol/L100mL, c（NaCl）0.275mol/L60mL, c（NaCl）0.30mol/L60mL, c（NaCl）0.325mol/L60mL, c（NaCl）0.35mol/L60mL and c（NaCl）0.375mol/L60mL. Finally, the （S）-amidase purification fold was428.3times, the specific activity fold was10.2times. The （S）-amidase was a monomer of37.6kDa by SDS-PAGE.Secondly, the characterization of the （S）-amidase was explored to set up the optimalreaction conditions of the free enzyme, and the effects of pH, temperatue, metal ions,EDTA-Na₂and urea were determined. The pH optimum was9.0and the temperature optimumwas37℃by detected the activity of （S）-amidase in different pHand various temperatures. Itwas also found that the amidese was sensitive to the environmental pH. The pH in theenvironment above10.5or below9.0caused a sharp decrease in the （S）-amidase activity. Theactivities of （S）-amidase were significantly differences to different metal ions. The enzymewas inactivated distinctly in the presence of Ca²⁺, Zn²⁺, Ag⁺, Pb²⁺, Na⁺at1mmol/L or10mmol/L, and completely inactivated even at the presence of1mmol/L of Ca²⁺, Zn²⁺, Ag⁺. Ascontrast, this （S）-amidase could be activitied by Co²⁺, Fe3⁺, Mn²⁺, Mg²⁺at1mmol/L or10mmol/L. The activity of （S）-amidase in the presence of10mmol/L Mn²⁺was about2.9timesto the control. No inhibition was observed in the presence of the urea. Inhibition was observed in the presence of the chelating agent EDTA-Na₂, suggesting that the metal ion wasparticipated in the catalytic process of the （S）-amidase.