Process for the Enzymatic Production of D-p-Hydroxyphenylglycine

• Main Authors: I-ming Lee, 李儀鳴
• Other Authors: Jan-Hsiung Huang
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/15064862753171861619

碩士 === 國立臺灣大學 === 農業化學研究所 === 89 === D-p-Hydroxyphenylglycine (D-p-HPG) is an industrial useful D-amino acid in the synthesis of antibiotics, peptide hormones, pyrethroids and pesticides. A fully enzymatic process employing D-hydantoinase (Dht) and N-carbamoylase (Nca) for the production of D-p-HPG in the same reaction system from DL-p-hydroxyphenylhydantoin (DL-p-HPH) was investigated, and we have also developed a HPLC system of detecting DL-p-HPH, NC-D-p-HPG and D-p-HPG. The conversion of DL-p-HPH to D-p-HPG was effected not only by the pH but also the variety of buffer. Moreover, enzyme activity will lose dramatically during the reaction. The optimal conditions were used 50mM Tris-HCl as buffer, 45oC as reaction temperature, besides pH was also maintained constantly at 7.5 by 3N HCl and NaOH. The results also indicated that additionally fresh supplementing enzyme at intervals of time can reach the highest production of D-p-HPG. In detail, both the enzyme activities were 3.2 U/mL at the beginning of reaction system, then fresh Dht (3.2 U/mL) and Nca (0.8 U/mL) were added at intervals of 1.5 h and 1 h, respectively. Based on the optimal condition in 5-liter jar fermentor (100 rpm, 0.5 vvm), 30 g/L DL-p-HPH can be converted into 25.78 g/L D-p-HPG in 8 h, at the same time, 50 g/L DL-p-HPH can be also converted into 40.72 g/L in 9 h. Their yields were approximately 98.8% and 93.6%. According to our result, we also found that the conversion activity of the enzyme was not effected when the product D-p-HPG reached its saturated concentration (about 200 mM), and new formed D-p-HPG were produced as crystal. Therefore, we used dialysis bag to separate crystal from insoluble DL-p-HPH and enzyme, and recovery of crystal was possible. Finally it was identified as D-p-HPG by Mass, IR and NMR.