Study On The Immobilization Of S-Stereoselective Amidase By Magnetic Chitosan Microspheres

The non-protein amino acid piperazine-2-carboxylic acid is an important chiral drugintermediates. Its various single enantiomer derivatives, type R and type S, are characterizedby different pharmacological activities. The (S)-stereoselective amidase produced byParacoccous sp.W10173, which was screened by our group, can stereoselectively hydrolyze(S)-piperazine-2-carboxamide to generate the single enantiomer of the crucial pharmaceuticalintermediate (S)-piperazine-2-carboxylic acid. Compared with free enzyme, immobilizedenzyme possesses a series of advantages, such as easy to recover, high stability, continuousand controllable operation, and simple technology. Therefore, it is of great significance toresearch on the immobilization of (S)-stereoselective amidase for its application inindustrialized production.Firstly, crude enzyme was prepared by ultrasonic disruption and hierarchical salting outof biomass of Paracoccous sp. W10173. The conditions for ultrasonic disruption are asfollows:400W of power,3S of operating time,6S of intermittent time, and240times ofcirculation; the optimal ammonium sulfate saturation for fractional salting out is between20%and50%, which should be repeated for three times.Magnetic chitosan microspheres (MCNPs) were prepared by using Fe3O4magneticparticles, generated by chemical co-precipitation, as magneticnucleus, liquid paraffin asdispersion medium, Span-80as emulsifier, and glutaraldehyde as cross-linking agent withreverse phased suspension crosslinking method. The result indicates that magnetic chitosanmicrosphere is spherical. Fe3O4magnetic particle is wrapped in it, forming the core-shellstructure. By using MCNPs as carriers, this research prepared the immobilized(S)-stereoselective amidase through the adsorption-crosslinking method, and the effects of thefactors impacted on immobilization effects, such as enzyme given amount, adsorption time,crosslinking time, concentration of glutaraldehyde, etc., were also explored. As a result, theoptimum immobilization conditions for (S)-amidase are as follows:10mL of0.95mg/mL crude enzyme solution per50mg fluid carrier adsorbed for2h, then crosslinked for4.5h at1%of glutaraldehyde.The research results of immobilized S-stereoselective amidase enzyme properties showthat the optimal temperature for immobilized (S)-amidase enzyme is32℃, the optimal pH is11, which shifts2units to alkaline compared with that of the free enzyme. At the same time, itis also found that the immobilized enzyme has good thermal stability. It can still retain nearly88%of activity under60℃treatment for4hours. The activity of the obtained immobilizedenzyme S-stereoselective amidase is2.3U/mg carrier and the recovery rate of enzyme activityis36.9%. Immobilized enzyme activity remains more than71%after10consecutive batchesof repetitive use and it has good operation stability. Compared with the free enzyme, thestorage stability of (S)-amidase has been improved after its immobilization.