New nucleic acid sequence encoding Alcaligenes faecalis nitrilase polypeptide useful for converting racemic nitriles to chiral carboxylic acids

摘要

An isolated nucleic acid sequence (I) encoding an Alcaligenes faecalis nitrilase polypeptide is new. An isolated nucleic acid sequence (I) encoding a polypeptide with nitrilase activity is selected from: (1) a defined genomic DNA sequence (seq1) of 1071 base pairs (bp) given in the specification; (2) nucleic acid sequences derived from seq1 as a result of the degeneracy of the genetic code; and (3) derivatives of seq1 that code for a polypeptide with a defined sequence of 356 amino acids and have at least 95% homology at the amino acid level without essentially reducing the enzymatic activity of the polypeptide (sic). Independent claims are also included for: (1) an amino acid sequence encoded by (I); (2) a nucleic acid construct (II) comprising (I) linked to one or more regulatory sequences; (3) a vector containing (I) or (II); (4) a microorganism containing (I) or (II); (5) a process for preparing chiral carboxylic acids of formula (III), comprising converting a racemic nitrile of formula (IV) in the presence of an amino acid sequence as in (1) or a growing, resting or digested microorganism as in (4), where the nitrile conversion is at least 25 mmoles/hour per mg protein or 25 mmoles/hour per g dry weight. R1-R3 = H, optionally substituted 1-10C alkyl, 2-10C alkenyl, optionally substituted aryl, heteroaryl, OR4 or NR4R5, provided that R1-R3 are all different; R4 = H, optionally substituted 1-10C alkyl, 2-10C alkenyl, 2-11C alkanoyl, 3-11C alkenoyl, aryl, aroyl, heteroaryl or heteroaroyl; and R5 = H, optionally substituted 1-10C alkyl, 2-10C alkenyl, aryl or heteroaryl.