Enzymatic Synthesis Of L-2-Aminobutyric Acid And The Immobilization Of Enzyme

Part I:First, three kinds of enzyme catalytic substrates, N-acetyl-DL-2-aminobutyric acid, DL-2-aminobutyric acid methyl ester hydrochloride and DL-2-aminobutanamide hydrochloride were synthesized and the structure were confirmed by spectroscopy characterization. Then the stereoselective hydrolysis of these substrates by L-aminoacylase was explored. And N-acetyl-DL-2-aminobutyric acid was proved to be the suitable substrate to generate optically active L-2-aminobutyric acid. Via single factor experiments, effect of experimental conditions on enzyme catalytic activity was studies as well, which are consist of substrate concentration, buffer pH, temperature, enzyme dosage, reaction time, etc. Results showed that the conversion rate was optimum when substrate concentration, enzyme dosage, pH, tris-HCl buffer solution’s volume and concentration, temperature, time was O.Olmol/L,100U,7.5,10mL,50mM,30℃and4h respectively. The conversion and e.e value of chiral L-2-aminobutyric acid prepared by enzyme split methodwere48.6%and98.9%, respectively.Part Ⅱ:This part focused on two kinds of immobilization methods of L-aminoacylase.1. L-aminoacylase was immobilized on the magnetic carrier which was prepared by combination of chitosan and magnetic iron oxide, through adding crosslinking agent glutaraldehyde (crosslinking method).2. L-aminoacylase and magnetic iron oxide were entrapped within calcium alginate (entrapment method), Comparing crosslinking method with entrapment method, the residue enzyme activity of later method was higher, keeping82.8%activity of free enzyme. Different entrapment conditions were optimized to obtain the best immobilization conditions. It was found that the thermostability of immobilized enzyme was higher than that of free enzyme.at high temperature. However, the pH stability of immobilized enzyme was similar with that of free enzyme. The immobilized enzyme can be used for five times with51.9%residue activity.