Effectt of Cell Membrane on D-Amino Acid Production by Agrobacterium radiobacter NRRL B11291

• Main Author: 李宗賢
• Other Authors: 李振綱
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/56102368942446779495

碩士 === 國立臺灣科技大學 === 化學工程技術研究所 === 86 === 　　D-p-hydroxyphenylglycine (D-pHPG) is a valuable synthon for the production of semisynthetic penicillins and cephalosporins such as ampicillin and amoxicillin. Several optically active amino acids including D-pHPG, are produced by using enzymatic route, starting from the corresponding racemic 5-substituted hydantoins. The latter can be cheaply synthesized from aldehydes, potassium cyanide and ammonium carbonate, and they easily undergo spontaneous racemization under a mild alkaline condition. Consequently, enzymatic cleavage and simultaneous racemization lead to high yields in production of the corresponding asymmetric N-carbamyl amino acids, which can be converted to free amino acids either by chemical methods or by a second enzymatic step catalyzed by an N-carbamy1-amino acid amidohydrolase. 　　A bacterium that stereospecifically produces D-pHPG from DL-5-p-hydroyphenylhydantoin (DL-pHPH) involves D-hydantoinase and N-carbamy1-amino acid amidohydrolase and identified as Agrobacterium radiobacter NRRL B11291.Their biosynthesis was found to be inducible by addition of 0.5 g/L DL-pHPH to the cultivation media. Optimun temperature and pH were respectively 40℃ and 8.0 when resting cells were used. In order to reuse the D-hydantoinase and amidohydrolase, a simple immobilization method was proposed. Chitosan solution [0.025%(w/v)] was added into cells suspension (pH 8.0, 0.1 M phosphate buffer) and the floculated cells were further cross-linked with glutaraldehyde. After five repeated batch reaction with high concentration substrate (4% w/v DL-pHPH), the D-pHPG conversion decreased from 73% to 25%. In this D-pHPG production process, the permeability of D-p-carbamoyl hydroxyphenylglycine for Agrobacterium radiobacter cell membrane is 0.0234 mL/min, much smaller than that of DL-pHPH and D-pHPG. This low permeability causes the D-pHPG production rate of using intact cell higher than that using toluene treatment cell.