THE ENZYMATIC RACEMIZATION OF D-ASPARTIC ACID AND THE ON-LINE COUPLING OF A SOLID-PHASE-EXTRATION COLUMN WITH A LIGAND-EXCHANGE HPLC CHROMATOGRAPHY

• Main Authors: Shouh Chwan Wu, 吳碩傳
• Other Authors: Cheanyeh Cheng
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/27254488981956350275

碩士 === 中原大學 === 化學系 === 88 === D-aspartic acid can be enzymatically biotransformed with D-amino acid oxidase and aminotransferase clonezyme to L-aspartic acid. The reaction temperature of this biotransformation was maintained around 90℃ and the reaction period was 3 days. The analysis of D-aspartic acid and L-aspartic acid in the coupled system with the ligand-exchange chromatography was subjected to matrix interference. Therefore, column switching technique and on-line coupling of a solid-phase-extraction (SPE) column to the ligand-exchange analytical column were used to solve the interference. The pretreatment of reaction samples with the solid-phase-extraction column was actually a combination of size- exclusion chromatography and an ion-pair chromatography with the use of an ion-pair reagent sodium 1-octanesulphonate. Part of the first eluted peak from solid-phase-extraction column containing the D- and L- aspartic acid was switched through the ligand-exchange column and analyzed qualitatively and quantitatively. The method of quantitative analysis was performed by the standard addition method. Overall, the separation and analysis of D-aspartic acid and L-aspartic acid in the enzymic solution was successful with the developed on-line LC-LC column coupling system and the column switching technique. D- and L-alanine and some other unknowns were also produced by the coupled reaction system of the D-amino acid oxidase and the aminotransferase clonezyme by the analysis of liqund-exchange chromatography. In order to understand the mechanism of the coupled reaction system and to know further about the individual effect of the two enzymes on the racemization of D-aspartic acid, the one-pot coupled system was modified to become a sequential two—step reaction. That is, firstly, by reacting the D-aspartic acid with D-amino acid oxidase alone then adding the aminotransferase clonezyme into the solution. The racemization of D-aspartic acid can be performed by using D-amino acid oxidase only. This reaction has been reacted at three temperatures 30℃﹑60℃﹑and 90℃ for a reaction period of 3 days. L-aspartic acid was found only in the solution with the reaction temperature 90℃ by using the crown-ether chromatography. Also the racemization can be performed with either D- or L-aspartic acid in the solution and without using any enzyme at 90℃. From the above studies, many byproducts such as DL-malic acid, D- and L-alanine, oxalacetic acid, and an oligomer (1,2,4,6-tetracaroxyl- 1,3,5-hexatriene) produced in the one-pot coupled enzyme reaction were thus identified separately. The production of these byproducts indicated that there also exists a reaction pathway with malic acid as an intermediate for the racemization besides the enzyme coupled reaction system. Although the racemization of D-aspartic acid can be performed at high temperature without the help of enzyme, the use of enzyme will be much helpful for the racemization from the results of quantitative analysis.