Abstract: The present invention aims to provide a mutant L-pipecolic acid hydroxylase for highly productive and highly selective production of cis-5-hydroxy-L-pipecolic acid by hydroxylation of L-pipecolic acid, and to provide a novel method for industrially producing cis-5-hydroxy-L-pipecolic acid from L-pipecolic acid with high productivity at low cost. The present invention provides a mutant L-pipecolic acid hydroxylase having an amino acid sequence that a particular amino acid mutation(s) is/are introduced in the amino acid sequence of SEQ ID NO:2.

Abstract: A method for producing an active-form mutant enzyme, by specifying a protein of which a native form exhibits an enzyme activity but which has 10% or less enzyme activity of the native form when a gene of the protein is expressed to provide an inactive-form enzyme; determining a sequence conservation of amino acid residues in an amino acid sequence of the inactive-form enzyme and specifying amino acid residue(s) for which sequence conservation in the inactive-form enzyme is lower than sequence conservation of other amino acid(s) of the same residue; preparing a gene having a base sequence that codes for the amino acid sequence of the inactive-form enzyme in which at least one said specified amino acid residue is substituted by another amino acid with a higher sequence conservation; and expressing the gene to obtain an enzyme that exhibits an enzyme activity of the native form protein.

Abstract: Provided are a method for obtaining an HNL gene and HNL derived from a millipede other than Chamberlinius hualienensis, and preparing a practically useable amount of HNL; and a method for producing optically active cyanohydrin using this HNL. A method for producing a millipede-derived HNL gene. A method that includes the selection of a gene having a base sequence that encodes a conserved amino acid sequence TAX1DIX2G (SEQ ID NO: 15) or VPNGDKIH (SEQ ID NO: 16) of millipede-derived HNL from genes present in an organism belonging to the Diplopoda. A protein having an amino acid sequence of any of (1)-(3) and having HNL activity. (1) An amino acid sequence listed in any of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 83, 85, 87, or 89; (2) an amino acid sequence having amino acids deleted, substituted, and/or added in an amino acid sequence of (1); or (3) an amino acid sequence having 90% or greater identity to an amino acid sequence of (1).

Abstract: As a method for producing a 3-hydroxyisobutyric acid ester using a biocatalyst, a method for producing a 3-hydroxyisobutyric acid ester, including a step of allowing an alcohol or phenol to act on 3-hydroxyisobutyryl-CoA in the presence of alcohol acyltransferase to produce a 3-hydroxyisobutyric acid ester is provided.

Abstract: A method for producing an active-form mutant enzyme, by specifying a protein of which a native form exhibits an enzyme activity but which has 10% or less enzyme activity of the native form when a gene of the protein is expressed to provide an inactive-form enzyme; determining a sequence conservation of amino acid residues in an amino acid sequence of the inactive-form enzyme and specifying amino acid residue(s) for which sequence conservation in the inactive-form enzyme is lower than sequence conservation of other amino acid(s) of the same residue; preparing a gene having a base sequence that codes for the amino acid sequence of the inactive-form enzyme in which at least one said specified amino acid residue is substituted by another amino acid with a higher sequence conservation; and expressing the gene to obtain an enzyme that exhibits an enzyme activity of the native form protein.

Abstract: A method for expressing, as a soluble protein or an active-form mutant enzyme, an enzyme that cannot be expressed as a soluble protein or an active-form enzyme in a heterologous expression system or that is obtained in a minute amount even when an active-form enzyme is expressed, the method including a technique for selecting an effective mutation site and a mutated amino acid. A new active-form mutant enzyme is also disclosed. The method involves: specifying an insoluble protein or an inactive-form enzyme; specifying a hydrophilic amino acid in a hydrophobic domain and/or a hydrophobic amino acid in a hydrophilic domain of an ?-helix structure portion of the insoluble protein or the inactive-form enzyme and preparing a gene that codes for an amino acid sequence in which a substitution is made to the hydrophilic amino acid in the hydrophobic domain and/or the hydrophobic amino acid in the hydrophilic domain.

Abstract: Provided is a method for producing a methacrylic acid ester using a biocatalyst, said method comprising a step of reacting an alcohol or a phenol with methacrylyl-CoA in the presence of an alcohol acyltransferase originated from a plant selected from the group consisting of a plant belonging to the genus Osmanthus, a plant belonging to the genus Vitis, a plant belonging to the genus Citrus, a plant belonging to the genus Durio, a plant belonging to the genus Magnolia and a plant belonging to the genus Chamaemelum to thereby synthesize the methacrylic acid ester.

Abstract: Provided are a method for obtaining an HNL gene and HNL derived from a millipede other than Chamberlinius hualienensis, and preparing a practically useable amount of HNL; and a method for producing optically active cyanohydrin using this HNL. A method for producing a millipede-derived HNL gene. A method that includes the selection of a gene having a base sequence that encodes a conserved amino acid sequence TAX1DIX2G (SEQ ID NO: 15) or VPNGDKIH (SEQ ID NO: 16) of millipede-derived HNL from genes present in an organism belonging to the Diplopoda. A protein having an amino acid sequence of any of (1)-(3) and having HNL activity. (1) An amino acid sequence listed in any of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 83, 85, 87, or 89; (2) an amino acid sequence having amino acids deleted, substituted, and/or added in an amino acid sequence of (1); or (3) an amino acid sequence having 90% or greater identity to an amino acid sequence of (1).

Abstract: A method for expressing, as a soluble protein or an active-form mutant enzyme, an enzyme that cannot be expressed as a soluble protein or an active-form enzyme in a heterologous expression system or that is obtained in a minute amount even when an active-form enzyme is expressed, the method including a technique for selecting an effective mutation site and a mutated amino acid. A new active-form mutant enzyme is also disclosed. The method involves: specifying an insoluble protein or an inactive-form enzyme; specifying a hydrophilic amino acid in a hydrophobic domain and/or a hydrophobic amino acid in a hydrophilic domain of an ?-helix structure portion of the insoluble protein or the inactive-form enzyme and preparing a gene that codes for an amino acid sequence in which a substitution is made to the hydrophilic amino acid in the hydrophobic domain and/or the hydrophobic amino acid in the hydrophilic domain.

Abstract: The present invention provides a process for industrial production of chiral-1,1-difluoro-2-propanol. More specifically, a microorganism having the activity to cause asymmetric reduction of 1,1-difluoroacetone or an enzyme having the same activity is allowed to act on 1,1-difluoroacetone, whereby chiral-1,1-difluoro-2-propanol can be produced with high optical purity and in good yield. The process for production of the present invention is easy for industrial implementation.

Abstract: Disclosed is a method for quantifying L-tryptophan involving a step for mixing a specimen, L-tryptophan oxidase, and water, a step for allowing the obtained reaction solution to stand a predetermined period of time in the presence of oxygen, and a step for measuring the reaction product resulting from action of enzymes present in the reaction solution after allowing to stand. The L-tryptophan oxidase has a given amino acid sequence and has oxidase activity that generates hydrogen peroxide and ammonia by acting on the L-tryptophan in the presence of oxygen and water. The oxidase activity of the L-tryptophan oxidase on the L-phenylalanine is in the range of 0-3% of the oxidase activity thereof on the L-tryptophan, and the L-tryptophan oxidase does not have oxidase activity on protein-constituting amino acids other than L-tryptophan and L-phenylalanine. Also disclosed are a kit used to quantify the L-tryptophan containing L-tryptophan oxidase, and an enzyme sensor using the L-tryptophan oxidase.

Abstract: There is provided a method for quantifying a subject substance, of which typical examples are amino acids. The method of the present invention comprises the following steps: the step of allowing an enzyme that can generate pyrophosphate by using adenosine triphosphate (ATP) as a substrate with converting the subject substance to act on the subject substance to generate pyrophosphate; the step of allowing pyruvate pyrophosphate dikinase (PPDK) to act on the generated pyrophosphate in the presence of adenosine monophosphate (AMP) and phosphoenolpyruvate (PEP) to generate ATP, phosphoric acid, and pyruvate; and the step of quantifying the generated pyruvate, and amount of the subject substance is determined on the basis of the obtained amount of pyruvate. According to the present invention, an amino acid in a biological sample containing a lot of various kinds of contaminants such as inorganic phosphoric acid and urea can be conveniently and quickly quantified without being influenced by the contaminants.

Abstract: Disclosed is a method for quantifying L-tryptophan involving a step for mixing a specimen, L-tryptophan oxidase, and water, a step for allowing the obtained reaction solution to stand a predetermined period of time in the presence of oxygen, and a step for measuring the reaction product resulting from action of enzymes present in the reaction solution after allowing to stand. Also disclosed are a kit used to quantify the L-tryptophan containing L-tryptophan oxidase, and an enzyme sensor using the L-tryptophan oxidase. This method, kit and enzyme sensor use an L-tryptophan-specific enzyme, so are capable of quantifying L-tryptophan even in the presence of other amino acids.

Abstract: The objective of the present invention is to provide a (R)-hydroxynitrile lyase which is more stable than a hydroxynitrile lyase derived from a plant, a gene which encodes the hydroxynitrile lyase and by which heterologous expression is possible, and a method for producing the hydroxynitrile lyase. The (R)-hydroxynitrile lyase according to the present invention is characterized in having the specific amino acid sequence such as the amino acid sequence of SEQ ID NO: 3.

Abstract: Provided is a method for producing a methacrylic acid ester using a biocatalyst, said method comprising a step of reacting an alcohol or a phenol with methacrylyl-CoA in the presence of an alcohol acyltransferase originated from a plant selected from the group consisting of a plant belonging to the genus Osmanthus, a plant belonging to the genus Vitis, a plant belonging to the genus Citrus, a plant belonging to the genus Durio, a plant belonging to the genus Magnolia and a plant belonging to the genus Chamaemelum to thereby synthesize the methacrylic acid ester.

Abstract: Methods are provided measuring L-lysine using a variant enzyme, an L-lysine measurement kit, and an enzyme sensor. Variant L-amino acid oxidase having a predetermined amino acid mutation, and having oxidase activity that is highly substrate-specific for L-lysine; a method for measuring L-lysine using this variant enzyme; an L-lysine measurement kit; and an enzyme sensor are also provided.

Abstract: Disclosed is a method for producing (S)-1,1,1-trifluoro-2-propanol with high optical purity and high yield by having at least one kind of microorganism, which is selected from the group consisting of Hansenula polymorpha, Pichia anomala, Candida parapsilosis, Candida mycoderma, Pichia naganishii, Candida saitoana, Cryptococcus curvatus, Saturnospora dispora, Saccharomyces bayanus and Pichia membranaefaciens, act on 1,1,1-trifluoroacetone. Since microorganisms found in nature are made to act in a natural state, the problems to be raised when a transformant or the like is used can be avoided in this method. Consequently, the method can be easily put in industrial practice.

Abstract: There is provided a method for quantifying a subject substance, of which typical examples are amino acids. The method of the present invention comprises the following steps: the step of allowing an enzyme that can generate pyrophosphate by using adenosine triphosphate (ATP) as a substrate with converting the subject substance to act on the subject substance to generate pyrophosphate; the step of allowing pyruvate pyrophosphate dikinase (PPDK) to act on the generated pyrophosphate in the presence of adenosine monophosphate (AMP) and phosphoenolpyruvate (PEP) to generate ATP, phosphoric acid, and pyruvate; and the step of quantifying the generated pyruvate, and amount of the subject substance is determined on the basis of the obtained amount of pyruvate. According to the present invention, an amino acid in a biological sample containing a lot of various kinds of contaminants such as inorganic phosphoric acid and urea can be conveniently and quickly quantified without being influenced by the contaminants.

Abstract: Methods are provided measuring L-lysine using a variant enzyme, an L-lysine measurement kit, and an enzyme sensor. Variant L-amino acid oxidase having a predetermined amino acid mutation, and having oxidase activity that is highly substrate-specific for L-lysine; a method for measuring L-lysine using this variant enzyme; an L-lysine measurement kit; and an enzyme sensor are also provided.

Abstract: A method for analyzing L-threonine contained in an specimen, which includes the steps of mixing a sample containing the specimen with an L-threonine dehydrogenase derived from Cupriavidus necator and a coenzyme NAD+ and analyzing the amount of NADH or 2-amino-3-oxobutyric acid after a predetermined period; an L-threonine dehydrogenase derived from Cupriavidus necator, which is a novel L-threonine dehydrogenase (TDH; EC 1.1.1.103) and can be utilized in the above-mentioned analysis method; a method for preparing a gene or the like to be used in the preparation of the enzyme, or a method for preparing the enzyme; an L-threonine analysis kit which includes (A) the L-threonine dehydrogenase and (B) a coenzyme NAD+; an enzyme preparation for use in the analysis of L-threonine, which includes the L-threonine dehydrogenase contained in a buffer solution; and an enzyme sensor utilizing the L-threonine dehydrogenase.