Production and immobilization of D-aminoacylase of Alcaligenes faecalis DA1 for optical resolution of N-acyl-DL-amino acids.
The production of D-aminoacylase by Alcaligenes faecalis DA1 was induced 5- to 50-fold by N-acetyl-D-amino acids. This strain produced about 443 units of D-aminoacylase and 52 units of L-aminoacylase per gram of cells (wet weight) when cultivated in a medium containing 1% N-acetyl-DL-leucine as the carbon source. The D-aminoacylase was partially purified by Fractogel DEAE 650 column chromatography and then immobilized on another Fractogel DEAE 650 column. The catalytic activity of the immobilized D-aminoacylase was 2,650 units per milliliter of gel. The Km values for the free and the immobilized enzymes were found to be 1.00 and 0.22 mM, respectively, using N-acetyl-D-methionine as a substrate. The optimal reaction pH and temperature for both soluble and immobilized enzyme were around 8.0 and 45 degrees C, respectively. The free enzyme was stable in the pH range from 5.0 to 11.0, whereas the immobilized enzyme tended to detach from the gel at pH values higher than 9.0. Both forms of enzyme were stable up to 40 degrees C. When used for the optical resolution of N-acetyl-DL-methionine, the immobilized enzyme maintained 90% initial activity after 17 days of continuous operation at 45 degrees C. The process of purification and immobilization of D-aminoacylase described in this report is very effective and easy to scale up.
Tsai
Y C
YC
Institute of Biochemistry, National Yang-Ming Medical College, Taipei, Taiwan, Republic of China.
Lin
C S
CS
Tseng
T H
TH
Lee
H
H
Wang
Y J
YJ
D000421:Alcaligenes / Q000187:drug effects / Q000378:metabolism*; D000581:Amidohydrolases / Q000096:biosynthesis / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism* / Q000494:pharmacology; D004790:Enzyme Induction; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006358:Hot Temperature; D007536:Isomerism; D007700:Kinetics; D013816:Thermodynamics
D000596:Amino Acids; D004800:Enzymes, Immobilized; D000581:Amidohydrolases; C019672:aminoacylase I