Modified Leucine Dehydrogenase

Abstract

The present invention provides a means and method useful for measurement of a total branched-chain amino acid concentration. Specifically, the present invention provides a modified enzyme in which at least one amino acid residue is mutated so as to improve a property of a leucine dehydrogenase which is associated with the measurement of the total branched-chain amino acids, such as, for example, substrate specificities of leucine dehydrogenase for total branched-chain amino acids, activity of leucine dehydrogenase for any branched-chain amino acids, and thermal stability of leucine dehydrogenase; and a method of analyzing the total branched-chain amino acids, comprising measuring the total branched-chain amino acids contained in a test sample using the modified enzyme.

1 . A method of producing a derivative of a branched-chain amino acid, comprising producing the derivative from the branched-chain amino acid using a modified leucine dehydrogenase enzyme,
 wherein said modified leucine dehydrogenase enzyme comprises at least one amino acid mutation as compared to a non-modified leucine dehydrogenase enzyme, wherein said modified leucine dehydrogenase is improved in one or more properties selected from the group consisting of:   (a) substrate specificities for L-leucine, L-isoleucine and L-valine;   (b) activity for any branched-chain amino acid;   (c) thermal stability, and   (d) combinations thereof,   wherein said modified leucine dehydrogenase is selected from the group consisting of:   (A) a protein comprising an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, but having a substitution of isoleucine in the TGI motif with an amino acid selected from the group consisting of methionine, arginine, histidine, phenylalanine, leucine, lysine, cysteine, tyrosine, alanine, glycine, serine, asparagine, and tryptophan,   (B) a protein comprising an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, but having a substitution of isoleucine in the GVI motif with an amino acid selected from the group consisting of phenylalanine, histidine, asparagine, tyrosine, leucine, lysine, glutamine, arginine, aspartic acid, threonine, glutamic acid, serine, cysteine, alanine, glycine, valine, tryptophan, and methionine,   (C) a protein comprising an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, but having a substitution of isoleucine in the TGI motif with an amino acid selected from the group consisting of methionine, arginine, histidine, phenylalanine, leucine, lysine, cysteine, tyrosine, alanine, glycine, serine, asparagine, and tryptophan, and a substitution of isoleucine in the GVI motif with an amino acid selected from the group consisting of phenylalanine, histidine, asparagine, tyrosine, leucine, lysine, glutamine, arginine, aspartic acid, threonine, glutamic acid, serine, cysteine, alanine, glycine, valine, tryptophan, and methionine, and   (D) a protein as described in (A), (B), or (C) above, but also comprising one to ten additional mutations of amino acid residues.