Study on D-hydantoinase process for the preparation of D-amino acids

• Main Authors: Chia-Hsi Fan, 范加錫
• Other Authors: Cheng-Kang Lee
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/34525094367859184515

博士 === 國立臺灣科技大學 === 化學工程系 === 87 === Abstract Optically active D-amino acids are widely used as intermediates for the production of semisynthetic antibiotics, peptides, hormones, and pesticides. For example, D-phenylglycine and D-p-hydroxyphenylglycine are used as side chains for semisynthetic antibiotic ampicillin and amoxicillin. Generally, D-amino acids are produced by various biotransformation methods. The biotransformation method involving D-hydantoinase is most often employed because its substrate 5-substituted hydantoins are readily available. The D-hydantoinase catalyzes the hydrolysis of D-5-substituted hydantoins into N-carbamoyl-D-amino acids. On the other hand, the L-hydantoin will spontaneously racemize to D-hydantoin to be hydrolyzed. N-carbamoyl-D-amino acids can be further converted into D-amino acids by diazotization at very low pH or by enzyme D-amidohydrolase. In this thesis, the immobilized D-hydantoinase was found to be able to catalyze the cyclization of N-carbamoyl-D-amino acids into D-hydantoins at pH 5.5. By taking advantage of this feature, D-5-substituted hydantoins were prepared from one pot reaction by using D-hydantonase to hydrolyze the racemic substrate at pH 8.5 and adjusted the pH to 5.5 to initiate the D-5-substituted hydantoins synthesis. The prepared D-5-substituted hydantoins were employed to study the effect of pH and temperature on its spontaneous racemization rates. The racemization rate increased with pH and temperature. The racemization rate of D-5-p-hydroxyphenylhydantoin was slightly higher than that of D-5-phenylhydantoin. The racemization half-life of the two hydantoin at pH 8.5 and 40 oC were 16.3min and 18.7min, respectively. Based on the racemization rate constant of hydantoin and the reaction kinetic parameter Vm , Km of the immobilized D-hydantoinase, the D,L-p-hydroxy phenylhydantoin hydrolysis was simulated and found that the racemization will not limit the overall reaction. D-hydantoinase from adzuki bean and recombinant E. coli were extracted, purified and immobilized by a novel enzyme immobilization method. In which, the enzyme was adsorbed covalently or noncovalently onto the surface of PGL microparticles. The size of the immobilized enzyme particle was enlarged by entrapping the PGL microparticles into the calcium alginate gel. The immobilized D-hydantoinase from adzuki bean shows very good operational stability that no appreciable activity loss was detected after 5 repeated batch reaction at 40 oC with > 95% conversion. Filter separated reactor with pressure swing was developed to carry out the immobilized D-hydantoinase reaction with solid suspension of p-hydroxyphenylhydantoin. The immobilized enzyme and the solid substrate suspension was separated by the filter. The reactor can prevent the impurities from fouling on the immobilized enzyme particles and stirrer from breaking the immobilized enzyme particles. The performance of this reactor will approach that of stirred tank reactor as the pressure swing frequency increased. The hydrolysis of p-hydroxyphenylhydantoin with concentration as high as 15 % (w/v) can be carried out in this reactor without fouling the immobilized enzyme and prolonging the reaction time. The product D-N-carbamoyl-p-hydroxyphenylglycine (D-CpHPG) obtained from D-hydantoinase reaction was converted into D-p-hydroxyphenylglycine (D-pHPG) by recombinant E. coli cell. The cell contained the gene of D-amidohydrolase and was highly expressed. The cells were flocculated with chitosan and cross-linked by glutaraldehyde that made the cells recovery after repeated use much easier. However, the cross-linking made the substrate inhibition concentration increased from 100 mM to 150 mM. The cross-linked cell showed good operation stability for at least 10 one hour batch reactions that > 95% conversion was achieved.