R-stereoselective amidase from Rhodococcus erythropolis No. 7 acting on 4-chloro-3-hydroxybutyramide.
Ethyl (S)-4-chloro-3-hydroxybutyrate is an intermediate for the synthesis of Atorvastatin, a chiral drug used for hypercholesterolemia. A Rhodococcus erythropolis strain (No. 7) able to convert 4-chloro-3-hydroxybutyronitrile into 4-chloro-3-hydroxybutyric acid has recently been isolated from soil. This activity has been regarded as having been caused by the successive actions of the nitrile hydratase and amidase. In this instance, the corresponding amidase gene was cloned from the R. erythropolis strain and expressed in Escherichia coli cells. A soluble active form of amidase enzyme was obtained at 18 degrees . The Ni column-purified recombinant amidase was found to have a specific activity of 3.89 U/mg toward the substrate isobutyramide. The amidase was found to exhibit a higher degree of activity when used with midchain substrates than with short-chain ones. Put differently, amongst the various amides tested, isobutyramide and butyramide were found to be hydrolyzed the most rapidly. In addition to amidase activity, the enzyme was found to exhibit acyltransferase activity when hydroxyl amine was present. This dual activity has also been observed in other enzymes belonging to the same amidase group (E.C. 3.5.1.4). Moreover, the purified enzyme was proven to be able to enantioselectively hydrolyze 4-chloro-3-hydroxybutyramide into the corresponding acid. The e.e. value was measured to be 52% when the conversion yield was 57%. Although this e.e. value is low for direct commercial use, molecular evolution could eventually result in this amidase being used as a biocatalyst for the production of ethyl (S)-4-chloro-3-hydroxybutyrate.
Park
Ha-Ju
HJ
Division of Biotechnology, The Catholic University of Korea, Bucheon 420-743, Korea.
Uhm
Ki-Nam
KN
Kim
Hyung-Kwoun
HK
D000577:Amides / Q000378:metabolism; D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006836:Hydro-Lyases / Q000378:metabolism; D006868:Hydrolysis; D006885:Hydroxybutyrates / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D012240:Rhodococcus / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D012988:Soil Microbiology; D013379:Substrate Specificity
C511345:4-chloro-3-hydroxybutyric acid; D000577:Amides; D006885:Hydroxybutyrates; C006866:butyramide; D000581:Amidohydrolases; C019665:amidase; D006836:Hydro-Lyases; C048831:nitrile hydratase