Patent Application
20140335553
1. Technical Field
The present invention relates to a novel L-amino acid oxidase, a method for measuring L-lysine using this novel L-amino acid oxidase, a kit employed in this method, and an enzyme sensor. More particularly, the present invention relates to a variant L-amino acid oxidase with high substrate specificity for L-lysine, a method for measuring L-lysine employing this variant enzyme, an L-lysine measurement kit, and an enzyme sensor.
2. Brief Description of the Related Art
L-lysine, one of the constituent amino acids of proteins, is an essential amino acid that cannot be produced within the body. The concentration of amino acids including L-lysine within the body is kept at homeostasis. However, congenital metabolic anomalies and internal disorders can cause great fluctuation in blood concentrations. The concentration of not just L-lysine, but of other amino acids within the body, affords a useful means of detecting disease. Thus, by measuring the blood concentration of one or many amino acids, it is possible to detect disease (International publication No. WO2006/129513 and Anal. Chem. 81: 307-314 (2009)).
In recent years, a large number of methods employing enzymes have become known as methods of quantifying amino acid levels. These methods employing enzymes are advantageous since they are less expensive and easier to implement than instrumental analysis methods. For example, enzymes such as dehydrogenase and oxidase are often employed. An example of a quantification method employing oxidase is the use of peroxidase to detect the hydrogen peroxide that is produced by subjecting amino acids to the action of oxidase (Japanese Un-examined patent publication No. Shou55-43409). This detection and quantification can be conducted with a method such as the colorimetric method, fluorescence method, or electrode method.
Methods employing enzymes are also known as methods of quantifying L-lysine. For example, in quantification with oxidase, L-lysine α-oxidase [EC 1.4.3.14] has been employed. L-lysine α-oxidase derived from Trichoderma viride affords a higher substrate specificity than other L-amino acid oxidases, and is commercially available. Thus, it has come to be employed in elements such as enzymatic sensors (Guerrieri et al., Sens. Actuators, B 126: 424-430 (2007); Endo et al. Anal. Bioanal. Chem. 391: 1255-1261 (2008); Anal. Bioanal. Chem. 406: 19-23 (2010)).
There are also reports that L-lysine monooxidase derived from Pseudomonas fluorescens exhibits an L-lysine oxygenase activity (Flashner et al., J. Biol. Chem. 249: 2579-2586 (1974); Flashner et al., J. Biol. Chem. 249: 2587-2592 (1974)). This enzyme employs L-lysine, L-threonine, and L-arginine as substrates.
However, the Trichoderma viride-derived L-lysine α-oxidase also exhibits oxidase activity on amino acids other than L-lysine. Thus, when employing L-lysine α-oxidase to quantify a sample containing multiple amino acids, such as blood plasma, excess evaluation tends to be involved.
Furthermore, it has recently been reported that L-lysine α-oxidase derived from the mucus of saltwater fish has greater substrate specificity than the above L-lysine α-oxidase (Endo et al., Anal. Bioanal. Chem. 391: 1255-1261 (2008)). However, this L-lysine α-oxidase is derived from the mucus of saltwater fish, and there are no reports of enzyme production by culturing. Accordingly, it is difficult to produce large quantities of this enzyme for use in quantifying L-lysine.
There have been no reports of using the Pseudomonas fluorescens-derived L-lysine monooxygenase to quantify L-lysine. Even when this enzyme is employed to quantify L-lysine, as set forth above, because it has oxygenase activity on L-ornithine and L-arginine in addition to L-lysine, it is impossible to strictly quantify L-lysine in samples containing multiple amino acids, such as blood plasma.
Methods of quantifying L-lysine based on oxidase, if successful, would be useful from the perspective of being less expensive and more convenient than instrumental analysis methods. However, as stated above, for samples containing multiple amino acids, such as blood plasma, the substrate specificity is low or there are problems with enzyme productivity with respect to the enzymes that are currently available. From this perspective, these enzymes are not practical.
Accordingly, it is an aspect of the present invention to provide an enzyme with high substrate specificity for L-lysine that enables specific quantification of L-lysine even in biological samples containing multiple amino acids, and to provide a method for enzymatically measuring L-lysine using this enzyme.
It is a further aspect of the present invention to provide a quantifying kit that can be used when implementing the above enzymatic measurement method.
It is a further aspect of the present invention to provide an enzyme sensor that can be used in the above enzymatic measurement method.
It is described that a variant L-amino acid oxidase, obtained by replacing the cysteine at position 254 in the amino acid sequence of L-amino acid oxidase derived from Pseudomonas sp. H-8-1-3 with a prescribed amino acid, exhibited high substrate specificity for L-Lysine.
It is a first aspect of the invention to provide a protein selected from the group consisting of:
(1) a protein comprising the amino acid sequence of SEQ ID NO: 2, but wherein the cysteine at position 254 has been replaced with an amino acid selected from the group consisting of methionine, phenyl alanine, tyrosine, tryptophan, alanine, glycine, valine, isoleucine, leucine, lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, and proline;
(2) a second protein comprising the amino acid sequence of the protein specified in (1) above, wherein one or a plurality of amino acids other than the amino acid at position 254 have been deleted, substituted, and/or added, wherein said second protein has an oxidase activity with a higher substrate specificity for L-lysine than the amino acid oxidase consisting of the amino acid sequence of SEQ. ID NO: 2; and
(3) a third protein comprising the amino acid sequence having at least 90% sequence identity with the amino acid sequence of the protein specified in (1) above, wherein the amino acid at position 254 of the third protein is selected from the group consisting of methionine, phenyl alanine, tyrosine, tryptophan, alanine, glycine, valine, isoleucine, leucine, lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, and proline, and wherein said third protein has oxidase activity with a higher substrate specificity for L-lysine than the amino acid oxidase consisting of the amino acid sequence of SEQ. ID NO: 2.
It is a further aspect of the invention to provide the protein as described above, wherein the 254th amino acid is selected from the group consisting of methionine, phenyl alanine, tyrosine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, and serine.
It is a further aspect of the invention to provide the protein as described above, wherein the 254th amino acid is isoleucine or tyrosine.
It is a further aspect of the invention to provide the protein as described above, wherein the amino acid at position 254 is other than tryptophan, glycine, lysine, arginine, histidine, threonine, asparagine, glutamine, or proline, and the oxidase activity of the second or third protein on L-arginine is 15% or less than the oxidase activity on L-lysine, and the oxidase activity of the second or third protein on L-ornithine is 80% or less than the oxidase activity on L-lysine.
It is a further aspect of the invention to provide a nucleic acid coding for the protein as described above.
It is a further aspect of the invention to provide a vector containing the nucleic acid as described above.
It is a further aspect of the invention to provide a transformant that has been transformed by the vector as described above.
It is a further aspect of the invention to provide a method for detecting or quantifying L-lysine, comprising:
(A) maintaining a specimen and the protein as described above in the presence of water and oxygen; and
(B) detecting or quantifying at least one reaction product produced in the reaction solution by the effect of the oxidase activity of the protein on L-lysine.
It is a further aspect of the invention to provide a method as described above, wherein the reaction product that is detected or quantified in step (B) is hydrogen peroxide.
It is a further aspect of the invention to provide a method as described above, wherein the hydrogen peroxide is detected or quantified using peroxidase.
It is a further aspect of the invention to provide a method as described above, wherein the reaction product that is detected or quantified in step (B) is ammonia.
It is a further aspect of the invention to provide a method as described above, wherein the ammonia is detected or quantified using an ammonia-detecting reagent.
It is a further aspect of the invention to provide a method as described above, wherein the reaction product that is detected or quantified in step (B) is a deamination product of L-lysine.
It is a further aspect of the invention to provide a kit for detecting or quantifying L-lysine, comprising the protein as described above.
It is a further aspect of the invention to provide a kit as described above, further comprising a kit component selected from the group consisting of a reaction buffer, a hydrogen peroxide-detecting reagent, an ammonia-detecting reagent, and an L-lysine deamination product-detecting agent.
It is a further aspect of the invention to provide a L-lysine-detecting or quantifying enzyme sensor comprising an electrode for detecting hydrogen peroxide, wherein the protein as described above is disposed on the surface or in the vicinity of the electrode for detecting hydrogen peroxide.
It is a further aspect of the invention to provide a sensor as described above, wherein the electrode for detecting hydrogen peroxide is an enzymatic hydrogen peroxide electrode or a diaphragm hydrogen peroxide electrode.
The present invention provides a novel variant L-amino acid oxidase with high substrate specificity for L-lysine. Using this variant L-amino acid oxidase, it is possible to conveniently and rapidly detect and quantify L-lysine with specificity and good precision even in samples containing numerous impurities such as other amino acids. In particular, the present invention is effective for biological samples such as blood plasma, blood serum, and urine. By inducing coupling with enzymes such as peroxidase, not only is it possible to quantify L-lysine by the fluorescence method and chromogenic method, but it is also possible to provide an electrode-type enzyme sensor.
a show the results of an alignment analysis of the amino acid sequence of the above wild-type enzyme, the amino acid sequence of the variant enzyme C254I, and the amino acid sequence of a known homologue gene. In
The present invention relates to proteins such as those described herein, including a protein having the amino acid sequence of SEQ ID NO: 2, but wherein the cysteine at position 254 is replaced with methionine, phenyl alanine, tyrosine, tryptophan, alanine, glycine, valine, isoleucine, leucine, lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, or proline, variants of such a protein, in which one or a plurality of amino acids other than the 254th amino acid, or a region containing the 254th amino acid, have been deleted, substituted, and/or added, wherein the variants can have at least 67% sequence identity. The variant proteins can have oxidase activity with higher substrate specificity for L-lysine than the amino acid oxidase consisting of the amino acid sequence denoted by SEQ. ID NO: 2.
The protein having the amino acid sequence denoted by SEQ. ID NO: 2 was obtained by separating for the first time a bacterial strain of genus Pseudomonas from the natural world, and cloning the gene coding for the L-amino acid oxidase produced by the microorganism belonging to the genus Pseudomonas. This point will be described in detail in the Examples.
The protein consisting of the amino acid sequence denoted by SEQ. ID NO: 2, as confirmed in the Examples, is a protein having L-amino acid oxidase activity. The L-amino acid oxidase activity of the protein is one that acts on L-lysine, L-ornithine, and L-arginine at pH of 7.0 (30° C.) in the presence of oxygen and water, producing hydrogen peroxide and ammonia. In the present description, the oxidase activity on L-lysine, L-ornithine, and L-arginine will be separately referred to as L-lysine oxidase activity, L-ornithine oxidase activity, and L-arginine oxidase activity. These oxidase activities can be measured using the measurement reagents and measurement methods described in the Examples.
The protein as described herein can be a variant L-amino acid oxidase that can be obtained by replacing the cysteine at position 254 in the amino acid sequence denoted by SEQ. ID NO: 2 with methionine, phenyl alanine, tyrosine, tryptophan, alanine, glycine, valine, isoleucine, leucine, lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, or proline. The protein obtained by replacing the 254th amino acid with these amino acids, as is indicated in the Examples, exhibits higher specificity of oxidase activity on L-lysine than the amino acid oxidase consisting of the amino acid sequence denoted by SEQ. ID NO: 2.
The protein having “oxidase activity with higher substrate specificity for L-lysine than the amino acid oxidase consisting of the amino acid sequence denoted by SEQ. ID NO: 2” can refer to a protein of relatively higher substrate specificity for L-lysine than the wild-type amino acid oxidase (“wild-type enzyme” hereinafter). Specifically, this means that compared to the wild-type enzyme, the ratio of L-arginine oxidase activity to L-lysine oxidase activity is lower, the ratio of the L-ornithine oxidase activity to L-lysine oxidase activity is lower, or both the ratio of L-arginine oxidase activity to L-lysine oxidase activity and the ratio of L-ornithine oxidase activity to L-lysine oxidase activity are lower. The protein having “oxidase activity with higher substrate specificity for L-lysine than the amino acid oxidase consisting of the amino acid sequence denoted by SEQ. ID NO: 2,” from the perspective of a higher substrate specificity for L-lysine can be a protein in which both the ratio of L-arginine oxidase activity to L-lysine oxidase activity, and the ratio of L-ornithine oxidase activity to L-lysine oxidase activity, are lower than in the above wild-type enzyme.
From the perspective of imparting “oxidase activity with higher substrate specificity for L-lysine” than the wild-type enzyme, the 254th amino acid in the protein as described herein can be methionine, phenyl alanine, tyrosine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, or serine. That is because when the 254th amino acid is replaced with one of these amino acids, as indicated in the Examples below, both the ratio of L-arginine oxidase activity to L-lysine oxidase activity and the ratio of L-ornithine oxidase activity to L-lysine oxidase activity are lower than those of the wild-type enzyme. In addition, from the perspective of obtaining a protein (amino acid oxidase) in which the substrate specificity of the oxidase activity to L-lysine is markedly enhanced, the 254th amino acid can be isoleucine or tyrosine.
Within the ranges as described herein, the protein of the present invention is not specifically limited so long as it has oxidase activity with high substrate specificity for L-lysine. When the L-lysine oxidase activity of the protein is defined as 100%, the L-arginine oxidase activity of the protein can be 15% or lower, 10% or lower, 5% or lower, 4% or lower, 3% or lower, 2% or lower, 1% or lower, 0.5% or lower, 0.01% or lower, or 0%. Additionally, the L-ornithine oxidase activity of the protein can be 80% or less, 60% or less, 50% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.01% or less, or 0%. In this connection, in the case of the wild-type enzyme serving as a reference, when the L-lysine oxidase activity at pH 7.0 and 30° C. is defined as 100%, the L-arginine oxidase activity can be about 20% and the L-ornithine oxidase activity is about 83%.
So long as the protein of the present invention exhibits the “oxidase activity of high substrate specificity for L-lysine” set forth above, the absolute value of the L-lysine oxidase activity is not specifically limited. For example, the L-lysine oxidase activity of the protein of the present invention can be at least equivalent to or greater than the L-lysine oxidase activity of the wild-type enzyme. Specifically, the protein of the present invention, at pH 7.0, can have an L-lysine oxidase activity of 0.6 U/mg or greater, or 1.0 U/mg or greater.
The variant L-amino acid oxidase as described herein does not exhibit activity toward L-tyrosine, L-alanine, L-cysteine, L-aspartic acid, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-methionine, L-asparagine, L-proline, L-glutamine, L-serine, L-threonine, L-valine, L-phenylalanine, and L-tryptophan, which are constituent amino acids of proteins other than L-lysine, L-ornithine, and L-arginine.
In the present invention, the variant protein can be a protein in which the amino acid at position 254 is replaced, and in addition, one or a plurality of amino acids in a region excluding the 254th amino acid are deleted, substituted, and/or added, wherein the protein exhibits oxidase activity with higher substrate specificity for L-lysine than the amino acid oxidase consisting of the amino acid sequence denoted by SEQ. ID NO: 2. Here, the expression of “one or a plurality” is not specifically limited so long as oxidase activity with high substrate specificity for L-lysine is present. Since the proportion of proteins having oxidase activity with high substrate specificity for L-lysine is high, the range of “one or a plurality” is, for example, 1 to 30, 1 to 20, 1 to 10, 1 to 7, 1 to 5, 1 to 3 or 1 to 2, and can be about 1.
Additionally, the variant protein can be a protein having an amino acid sequence which has at least 67% sequence identity with the amino acid sequence of the protein of SEQ ID NO: 2, and has oxidase activity with greater substrate specificity for L-lysine than the amino acid oxidase consisting of the amino acid sequence denoted by SEQ. ID NO: 2, wherein the amino acid corresponding to the 254th amino acid is methionine, phenyl alanine, tyrosine, tryptophan, alanine, glycine, valine, isoleucine, leucine, lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, or proline.
The expression “sequence identity” is not specifically limited so long as the enzyme has oxidase activity with high substrate specificity for L-lysine. The sequence identity of the amino acid sequences is not specifically limited to 67% or greater. It can be 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, or 74% or more. It can be 75% or more, 80% or more, 85% or more, or 90% or more. It is even more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more. The expression “sequence identity” can mean a degree of identity of amino acids in two or more amino acid sequences relative to each other. Accordingly, the higher the degree of identity of two given amino acid sequences, the higher the identity or similarity of the sequences. Whether or not two amino acid sequences have similarity can be analyzed by direct comparison of the sequences. Specifically, it can be analyzed using commercial sequence analysis software.
The term “mutation” can mean, specifically, the deletion or substitution of amino acids. That is, the amino acid corresponding to the 254th amino acid in the amino acid sequence of the protein of SEQ ID NO: 2, can be deleted or substituted with the specified amino acids. Accordingly, so long as the amino acid that is substituted into the protein of SEQ ID NO: 2 above is isoleucine, the amino acid corresponding to the 254th amino acid in the amino acid sequence will be isoleucine.
In the third protein as described above, the amino acid corresponding to the 254th amino acid in the amino acid sequence of the protein of SEQ ID NO: 2, which can serve as the reference for determining sequence identity can be checked by the above-described sequence identity or homology. Specifically, using commercial sequence analysis software, by analyzing the alignment of the amino acid sequences that are the targets of analysis against the amino acid sequence of SEQ. ID NO: 2 or the amino acid sequence of the protein of (1) above, it is possible to search for the amino acid corresponding to the 254th amino acid. Such methods of alignment analysis are widely known to persons having ordinary skill in the art.
So long as the above protein of the present invention falls within the scope specified herein, the origin thereof is not specifically limited. For example, the protein of the present invention can be a recombinant protein fabricated with various genetic engineering techniques, a synthetic protein fabricated by chemical synthesis, or a protein produced by extracting or generating a protein that produces a variant after obtaining a variant capable of producing the variant enzyme of the present invention by subjecting a specific species of organism (such as a bacterium) having a genetic homologue to the L-amino acid oxidase consisting of the amino acid sequence denoted by SEQ. ID NO: 2 to a mutagen.
When producing the recombinant protein of the present invention by a genetic engineering technique, nucleic acid (DNA or RNA) coding for the above-described proteins can be prepared and combined into various expression vectors to express the protein of the present invention. In producing the nucleic acid coding for the protein of the present invention, amino acid substitutions, deletions, or addition or mutations can be achieved, for example, by the error-prone PCR method, DNA shuffling method, various site-specific mutagenic methods, or the like. By introducing the nucleic acid coding for the protein of the present invention that has been produced in this manner into a suitable expression system, it is possible to produce the protein of the present invention.
The expression system that can be used to produce the protein of the present invention is not specifically limited. However, by way of example, various expression vectors that are capable of expressing recombinant proteins in various species of organism (hosts) can be employed. Potential expression vectors that can be used include various expression vectors that are capable of expressing proteins in hosts such as microorganisms such as bacteria and fungi (for example, yeast), plants, insect cells, and mammalian cells. These can also be viral vectors (including phage vectors) and plasmid vectors. Alternatively, a cell-free protein expression system employing rabbit reticulocyte lysate, wheat germ lysate, E. coli lysate, or the like can be employed to fabricate the protein of the present invention.
When the protein of the present invention is being expressed in an expression system employing a host in the form of a specific species of organism, it can be prepared by a method comprising loading the nucleic acid coding for the protein of the present invention into a vector, using the vector to transform the host cell, culturing the transformed host cell to cause the protein coded for by the gene to accumulate in the culture solution, and collecting the accumulated protein.
The nucleic acid coding for the protein of the present invention, the vector containing the nucleic acid, and the transformant that is transformed by the vector are one embodiment of the present invention.
The method used to obtain the nucleic acid coding for the protein of the present invention is not specifically limited. For example, as described below in the Examples, the nucleic acid coding for the protein of the present invention can be obtained using a material in the form of nucleic acid coding for the L-amino acid oxidase gene (SEQ. ID NOS: 1 and 2) that has been isolated from Pseudomonas sp. H-8-1-3. Alternatively, nucleic acid coding for the protein of the present invention can be fabricated by isolating from various bacteria a gene with homology to the amino acid sequence (L-amino acid oxidase) described in SEQ. ID NO: 2 of the protein of the present invention, preparing nucleic acid coding for the gene, and conducting amino acid substitution corresponding to the 254th amino acid set forth above. Further, the nucleic acid coding for the protein of the present invention can be fabricated by any method known to persons having ordinary skill in the art, such as chemical synthesis, genetic engineering techniques, or mutagenesis based on information about the base pair sequence described in SEQ. ID NO: 1, the amino acid sequence described in SEQ. ID NO: 2, known base pair sequences having a certain identity to the base pair sequence described by SEQ. ID NO: 1, or known amino acid sequences having a certain identity to the amino acid sequence described in SEQ. ID NO: 2.
The amino acid sequence denoted by SEQ. ID NO: 2 is the amino acid sequence of L-amino acid oxidase (wild-type enzyme) derived from Pseudomonas sp. H-8-1-3 isolated by the present inventors from the natural world. However, a large number of genes exhibiting a certain homology with this L-amino acid oxidase are known. For example, genes derived from the bacteria shown in Table 1 are known.
Pseudomonas putida
Pseudomonas
entomophila
Pseudomonas
fluorescens Pf-5
Pseudomonas
fluorescens F113
Pseudomonas
fluorescens Pf-5
Pseudomonas
fluorescens WH6
Pseudomonas syringae
Pseudonocardia sp. P1
Gordonia amarae
Roseobacter
denitrificans Och 114
Mycobacterium
abscessus ATCC
The results of an alignment analysis of the amino acid sequence of wild-type enzyme, the amino acid sequence of the variant enzyme C254I and the amino acid sequence of known genes shown in Table 1 are provided in
Specifically, the nucleic acid coding for the protein of the present invention can be prepared using the method of contacting DNA having the base pair sequence given by SEQ. ID NO: 1 of the SEQUENCE LISTING, for example, with a reagent serving as a mutagen, the method of irradiation with ultraviolet radiation, genetic engineering methods, and the like. The site-specific mutagenic method, a type of genetic engineering method, can be a method permitting the introduction of a mutation at a specific position. Thus, it is useful to introduce a mutation at a specific site in a nucleic acid when producing a nucleic acid coding for the protein of the present invention.
For example, it is possible to prepare nucleic acid serving as a material for fabricating nucleic acid coding for the protein of the present invention by preparing suitable probes and primers based on information about the base pair sequence given in SEQ. ID NO: 1 or the amino acid sequence given in SEQ. ID NO: 2 of the SEQUENCE LISTING in the present Description, and using them to screen cDNA of bacteria including the strain Pseudomonas sp. H-3-1-3 or a genome library. The cDNA or genome library can be prepared by the conventional methods.
Material for preparing nucleic acid coding for the protein of the present invention can also be obtained by the PCR method. For example, a genome library, cDNA, or genomic DNA of bacteria including Pseudomonas sp. H-8-1-3 can be employed as template and PCR can be conducted using a pair of primers designed to amplify the base pair sequence given by SEQ. ID NO: 1. The PCR reaction conditions can be suitably set. An example of conditions is 30 cycles of a reaction process consisting of 30 seconds at 94° C. (denaturation), 30 seconds to 1 minute at 55° C. (annealing), and 2 minutes at 72° C. (elongation), followed by a 7 minute reaction at 72° C. The DNA fragment being amplified can be employed as the material for fabricating the nucleic acid coding for the protein of the present invention. Further, a vector obtained by cloning the amplified DNA fragment in a vector suited to amplification in a host such as E. coli can also be employed as the material for fabricating the nucleic acid coding for the protein of the present invention.
The nucleic acid coding for the protein of the present invention, or a vector containing this nucleic acid, can be fabricated by substituting base pairs by various mutagenic methods into the base sequence (codon) coding for the amino acid corresponding to the 254th amino acid in the material for fabricating the nucleic acid coding for the protein of the present invention that has been prepared as set forth above. The operations of preparing the above probe or primers, constructing a genome library, screening the genome library, and cloning the target gene are known to persons having ordinary skill in the art.
The nucleic acid coding for the protein of the present invention can be employed after having been inserted into a suitable vector. The type of vector that is employed in the present invention is not specifically limited. For example, an independently replicating vector (such as a plasmid) will do, as will a vector that recombines into the genome of a host cell in the course of being introduced into the host cell, and is replicated along with the chromosome into which it has recombined. The vector is desirably an expression vector. In an expression vector, elements (such as promoters) that are essential for transcription are functionally linked to the above nucleic acid. A promoter is a DNA sequence that exhibits transcription activity in a host cell, and can be suitably selected based on the type of host cell.
Examples of promoters that can function in bacterial cells are promoters of the Geobacillus stearothermophilus maltogenic amylase gene, Bacillus licheniformis alpha-amylase gene, Bacillus amyloliquefaciens BAN amylase gene, Bacillus subtilis alkaline protease gene, and Bacillus pumilus xylosidase gene; phage lambda PR or PL promoters, and lac, trp and tac promoters of E. coli.
Examples of promoters that function in mammalian cells are the SV40 promoter, MT-1 (metallothionein gene) promoter, or the adenovirus 2 major late promoter. Examples of promoters that function in insect cells are the polyhedrin promoter, P10 promoter, Autographa californica polyhedrosis basic protein promoter, Baculovirus immediate early gene 1 promoter, and the Baculovirus 39K delayed-early gene promoter. Examples of promoters that are capable of functioning in yeast host cells are promoters derived from yeast glycolytic genes, alcohol dehydrogenase gene promoters, TPI1 promoters, and ADH2-4-c promoters. Examples of promoters that function in filamentous fungal cells are ADH3 promoters and tpiA promoters.
In the above vectors, the nucleic acid coding for the protein of the present invention can be functionally bound to a suitable terminator as needed. The vector containing the nucleic acid coding for the protein of the present invention can also have elements such as polyadenylation signals (such as those derived from SV40 or the adenovirus 5E1b region) and transcription enhancing sequences (such as the SV40 enhancer). A recombinant vector containing the L-amino acid oxidase gene can also comprise a DNA sequence enabling replication of the vector within the host cell. One example is the SV40 replication origin (when the host cell is a mammalian cell).
The vector containing the nucleic acid coding for the protein of the present invention can also contain selection markers. Examples of selection markers are a gene the complement of which is lacking in the host cell, such as the dihydrofolate reductase (DHFR) or the Schizosaccharomyces pombe TPI gene, or a gene imparting resistance to a drug such as ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, or hygromycin. The methods of splicing the nucleic acid coding for the protein of the present invention, the promoters, and as needed, terminators and/or secretion signal sequences and inserting them into suitable vectors are widely known to persons having ordinary skill in the art.
The vector containing the nucleic acid coding for the protein of the present invention can be introduced into a suitable host to fabricate the transformant of the present invention. The host cell into which the vector of the present invention is introduced can be any cell in which the vector of the present invention can be replicated. When a transformant expressing the protein of the present invention is being fabricated, in addition to vector replication, it must be a cell that is capable of expressing the protein of the present invention. Examples of such host cells are bacteria, yeast, fungi, and higher eukaryotic cells.
Examples of bacterial cells are Gram-positive bacteria such as Bacillus and Streptococcus, and Gram-negative bacteria such as E. coli. The transformation of the bacteria can be done by the protoplast method, or using competent cells, which is a known method. Examples of mammalian cells are HEK293 cells, HeLa cells, COS cells, BHK cells, CHL cells, and CHO cells. Methods of transforming mammalian cells and expressing the DNA sequence that has been introduced into the cells are also known. For example, the electroporation method, calcium phosphate method, and lipofection method can be employed.
Examples of yeast cells are cells belonging to Saccharomyces and Schizosaccharomyces, such as Saccharomyces cerevisiae and Saccharomyces kluyveri. Examples of methods of introducing recombinant vectors into a yeast host are the electroporation method, spheroplast method, and lithium acetate method.
Examples of other fungal cells are cells belonging to the filamentous fungi such as Aspergillus, Neurospora, Fusarium, and Trichoderma. When employing a filamentous fungi as host cell, it is possible to conduct transformation by obtaining a recombinant host cell by recombining a DNA construct into the host chromosome. The recombination of the DNA construct into the host chromosome can be conducted by known methods, such as homologous recombination or heterogeneous recombination.
When employing an insect cell as a host, a vector into which a recombinant gene has been introduced and a Baculovirus can be jointly introduced into an insect cell to obtain a recombinant virus in the supernatant of an insect cell culture, and then infecting insect cells with the recombinant virus and inducing protein expression.
For example, the Autographa californica nuclear polyhedrosis virus, a virus that infects insects of the family Noctuidae, or the like can be employed.
Sf9 and Sf21, which are Spodoptera frugiperda of the Noctuidae, and HiFive (manufactured by Invitrogen), which are ovarian cells of Trichoplusia ni, can also be employed as insect cells.
For example, the calcium phosphate method or the lipofection method can be used to jointly introduce a recombinant gene vector and the above Baculovirus into an insect cell to prepare a recombinant virus.
The above transformant is cultured in a suitable nutrient medium under conditions permitting the replication of the vector of the present invention or under conductions permitting expression of the protein of the present invention. When expressing the protein of the present invention, the usual protein isolation and purifying methods can be used to isolate and purify the protein of the present invention from the culture product (containing the transformant and culture medium) of the transformant. For example, when the protein of the present invention is expressed in a dissolved state within the cell, following the end of culturing, the cells are recovered by centrifugation, suspended in an aqueous buffer, and crushed in an ultrasonic crusher to obtain a cell-free extract. The cell-free extract is then centrifuged. The protein of the present invention can be obtained as a purified preparation from the supernatant using just one or a combination of the usual protein isolating and purifying methods, such as the solvent extraction method, salting out with ammonium sulfate or the like, desalination method, precipitation with an organic solvent, anion-exchange chromatography employing a resin such as diethylaminoethyl (DEAE) Sepharose, cation-exchange chromatography employing a resin such as S-Sepharose FF (made by Pharmacia Co.), hydrophobic chromatography employing a resin such as butyl Sepharose or phenyl Sepharose, gel filtration with a molecular sieve, affinity chromatography, the chromatofocussing method, and electrophoresis such as isoelectric point electrophoresis.
<Method of Quantifying L-Lysine>
The method of quantifying L-lysine in the present invention can include the following steps:
(A) maintaining a specimen and the protein according to the present invention in the presence of water and oxygen; and
(B) detecting or quantifying at least one reaction product produced in the reaction solution by the effect of the oxidase activity of the protein on L-lysine.
In the method of the present invention, the biological sample that is employed as the specimen can be any sample potentially containing L-lysine. The biological sample is subjected to the action of the protein of the present invention, and some product thereof is quantified to measure the concentration of L-lysine in the biological sample, making it possible to suitably select biological samples. For example, when using a color-developing agent or a fluorescent agent to quantify the product, a colorless aqueous solution is desirable, with blood serum and blood plasma being examples.
Reaction scheme A below gives the oxidation reaction of L-lysine by the protein of the present invention.
The above protein employed in the method of the present invention catalyzes the reaction given by Scheme A above.
Step (A)
The quantity of protein of the present invention that is admixed in step (A) is suitably 10 mU/mL or greater (a quantity of enzyme exhibiting activity such that it consumes 1 μmol of lysine in one minute is denoted as 1 U). The quantity of water that is admixed can be suitably determined based on the concentration of lysine in the sample, but normally falls within a range of 5 to 95%, for example. There is not a specific upper limit to the quantity of protein of the present invention that is admixed. In practical terms, it can be 100 mU/mL or less, for example. However, there is no intent to limit the quantities of the protein of the present invention and water that are added to these ranges, and they can be suitably adjusted.
In addition to water and the protein of the present invention, a reaction buffer is also desirably incorporated. The pH of the reaction buffer is not specifically limited so long as the oxidase activity with high substrate specificity for L-lysine of the above protein of the present invention is ensured. It can be suitably adjusted taking into account the relation between the optimal pH for oxidase activity on L-lysine and the substrate specificity for L-lysine. For example, it can be set to within a range of from pH 1.0 to 14 by taking into account the properties of the various enzymes to ensure oxidase activity on L-lysine and high substrate specificity for L-lysine that permits the detection of L-lysine. When employing the variant enzyme C254I described in the Examples below, taking into account a range yielding an oxidase activity with high substrate specificity for L-lysine, the pH of the reaction buffer can be adjusted, for example, to within a range of pH 6.0 to 10.5, pH 6.5 to 10, pH 7 to 9, oe, pH 7.0 to 8.0.
The reaction solution obtained by the above mixing is maintained for a prescribed period in the presence of oxygen. In the L-lysine oxidation reaction based on the protein of the present invention, as shown in reaction Scheme A, 2-oxo-6-aminohexanoic acid, which is an L-lysine deamination product, ammonia (NH3), and hydrogen peroxide (H2O2) are obtained as products. By conducting the reaction in air, the oxygen is supplied as oxygen dissolved in the reaction solution. It is normally not necessary to forcefully supply an oxygen-containing gas such as air to the reaction solution to supply oxygen to the reaction solution. A trace amount of oxygen is required for the enzymatic reaction based on the protein of the present invention, which is adequately covered by dissolved oxygen. The maintenance time for the enzyme reaction will depend on the quantity of enzyme employed (quantity of protein of the present invention), for example, and can range from 10 minutes to an hour, for example. Additionally, so long as the catalytic reaction based on the protein of the present invention that is shown in reaction Scheme A can take place when L-lysine is present in the specimen, the maintenance temperature of the enzyme reaction is not specifically limited. It can be constant over the maintenance period, or can vary. The maintenance temperature will depend on the optimal temperature of the enzyme employed (the protein of the present invention) and the substrate specificity for L-lysine that is exhibited at that temperature, for example. A suitable reaction temperature can be selected from within a range of 10 to 60° C.; for example, a range of 20 to 55° C., or a range of 25 to 40° C., can be employed. However, there is no intent to limit the retention time and retention temperature to these ranges, and they can be suitably adjusted as needed.
Step (B)
In step (B), at least one of the reaction products that is present in the reaction solution due to the action of the protein of the present invention following maintenance in step (A) is detected or quantified.
When the product that is used in detection or quantification is hydrogen peroxide, for example, a known method such as a peroxidase reaction can be used for measurement, making it possible to detect or quantify the hydrogen peroxide. When employing a peroxidase reaction for measurement, it suffices for the peroxidase employed to be an enzyme that can be used to detect or quantify hydrogen peroxide. An example is peroxidase derived from horseradish. It is also possible to use a color-developing agent that is capable of serving as a substrate for the peroxidase employed. When employing peroxidase derived from horseradish, an example is 4-aminoantipyrine: phenol. The reaction for detecting or quantifying hydrogen peroxide employing peroxidase derived from horseradish is as indicated below.
2H2O2+4-aminoantipyrine+phenol->quinoimine dye+4H2O Chem. 2:
Color-developing agents and fluorescence agents such as 4-aminoantipyrine can be suitably selected based on the type of peroxidase employed.
The hydrogen peroxide that is the product of the L-amino acid oxidase reaction based on the protein of the present invention can be detected with a current detection sensor employing a hydrogen peroxide electrode. An example of a hydrogen peroxide electrode is a sensor employing an electrode in the form of a membrane upon which peroxidase and bovine serum albumin have been immobilized with glutaraldehyde, with ferrocene incorporated into a carbon paste.
When the product that is used for detection or quantification is ammonia, detection can be conducted with an ammonia-detecting agent. An example of an ammonia-detecting agent is the indophenol method in which phenol and hypochlorous acid are combined. Specifically, the sample is mixed with a phenol nitroprusside solution and a perchloric acid solution, developing color. Absorbance at 635 nm is measured, permitting the detection or quantification of ammonia.
When the product used for detection or quantification is 2-oxy-6-aminohexanoic acid, which is a deamination product of L-lysine, the 2-oxo-6-aminohexanoic acid reacts with 3-methyl-2-benzothiazolone hydrazine hydrochloride, making it possible to quantify the 2-oxo-6-aminohexaonic acid by spectrally quantifying hydrazone derivatives.
<The L-Lysine Quantification Kit>
The present invention further includes a kit for detecting or quantifying L-lysine, containing the above protein of the present invention.
The kit of the present invention can contain a reaction buffer, hydrogen peroxide-detecting reagent, ammonia-detecting agent, and at least one agent for detecting 2-oxy-6-aminohexanoic acid, which is an L-lysine deamination product.
The reaction buffer is used to maintain a pH suited to the enzymatic reaction by the protein of the present invention in the reaction solution. The pH of the reaction buffer solution is not specifically limited so long as the oxidase activity with high substrate specificity for L-lysine of the protein of the present invention is ensured. It can be suitably adjusted taking into account the relation between the optimal pH of oxidase activity on L-lysine and substrate specificity for L-lysine. For example, a range ensuring the oxidase activity on L-lysine and high substrate specificity permitting the detection of L-lysine can be set within the range of pH 1.0 to 14 taking into account the properties of the various enzymes. When variant enzyme C245I described in the Examples below is used, taking into account a range yielding oxidase activity with high substrate specificity for L-lysine, the pH of the reaction buffer can be adjusted to, for example, a range of pH 6.0 to 10.5, pH 6.5 to 10, pH 7 to 9, andor, pH 7.0 to 8.0.
A hydrogen peroxide-detecting reagent is employed when detecting hydrogen peroxide by color development or fluorescence, for example. Examples of hydrogen peroxide-detecting reagents are combinations of peroxidase and a color-developing agent that is capable of using it as a substrate. A specific example is a combination of horseradish peroxidase and 2-aminoantipyrine phenol.
An example of an ammonia-detecting agent is the indophenol method based on a combination of a phenol and hypochlorous acid.
As a reagent for detecting 2-oxo-6-aminohexanoic acid, which is a deamination product of L-lysine, for example, 2-oxo-6-aminohexanoic acid and 3-methyl-2-benzothiazolone hydrazine hydrochloride can be reacted and the method of spectrally quantifying the hydrazone derivatives can be used.
<The Enzyme Sensor>
The present invention includes an L-lysine-detecting or quantifying enzyme sensor that includes an electrode for detecting hydrogen peroxide, wherein the protein of the present invention is disposed on the surface or in the vicinity of the electrode for detecting hydrogen peroxide.
The detection electrode is an electrode that detects hydrogen peroxide. The hydrogen peroxide-detecting electrode can be an enzymatic hydrogen peroxide electrode or a diaphragm-type hydrogen peroxide electrode. The protein of the present invention reacts with L-lysine to produce hydrogen peroxide. Thus, the hydrogen peroxide can be detected by a hydrogen peroxide-detecting electrode. By way of example, the enzymatic hydrogen peroxide electrode can be in the form of a sensor employing an electrode in the form of a membrane upon which peroxidase and bovine serum albumin have been immobilized with glutaraldehyde, with ferrocene incorporated into a carbon paste. The diaphragm hydrogen peroxide electrode is a type of electrode in which an electrode reacting with hydrogen peroxide is separated by a membrane.
The protein of the present invention is desirably disposed on the surface of the detection electrode or in the vicinity of the detection electrode. When disposed on the surface of the detection electrode, it can be immobilized, or not immobilized, on the surface of the detection electrode. Immobilization on the surface of the detection-use electrode is advantageous in that the sensor of the present invention can be repeatedly used.
The present invention will be specifically described based on the following non-limiting Examples.
In the Examples, the active measurement reagents and L-lysine measuring reagent composition are prepared by the following composition. The measurement conditions were as set forth below. The activity of the L-amino acid oxidase was measured as set forth below.
(1) Preparation of L-Amino Acid Oxidase Activity Measuring Reagent
(2) Method Measuring the Activity of L-Amino Acid Oxidase
The activity of L-amino acid oxidase was determined by the colorimetric method using the color-developing solutions of Table 2 from the quantity of hydrogen peroxide produced in the oxidation of L-amino acid. When measuring activity with microplates, to 100 μL of color-developing solution were added 100 μL of 100 mM solutions of the amino acids shown in Table 3 and 50 μL of enzyme solution. After dispensing on ice, reactions were conducted for 0, 0.5, 1, 1.5, 2, 3, 4, and 5 hours at 30° C. A microplate reader was then used to measure the absorbance at 550 nm. For the substrates with L-amino acid oxidase activity, the 20 types of amino acid (100 mM solutions) shown in Table 3 were employed. For blanks, 100 mM potassium phosphate buffer solution (pH 7.0) was added instead of the substrate.
In measurement with an absorption spectrometer with 1 cm quartz cells in enzyme purification, a 1 mL total quantity of reaction solution was employed. Based on the change in absorbance obtained, the L-lysine oxidase enzymatic activity was calculated with the calculation equation given below. A quantity of enzyme that yielded one micromol of substrate per minute under the above conditions was defined as 1 U. Based on the change in absorbance obtained, the enzymatic activity of the L-amino acid oxidase was calculated from the calculation equation given below.
(3) Equation for Calculating L-Amino Acid Oxidase Activity
Activity level (U/mL)={ΔOD/min(ΔODtest−ΔODblank)×3.1 (mL)×dilution rate}/{13×1.0 (cm)×0.1 (mL)}
3.1 (mL): Total quantity of solution
13: Millimolar extinction coefficient
1.0 cm: Length of optical path of cell
0.1 (mL): Amount of enzyme sample solution
(4) Enzyme Purification of Pseudomonas Sp. H-8-1-3-Derived L-Amino Acid Oxidase
Method of Culturing Pseudomonas Sp. H-8-1-3
Pseudomonas sp. H-8-1-3 was precultured by being inoculated onto 5 mL of TGY medium (0.5% polypeptone, 0.5% yeast extract, 0.1% KH2PO4, 0.1% D-glucose, pH 7.0) and cultured for 12 hours at 30° C. and 200 rpm. Subsequently, it was inoculated onto a two-liter Sakaguchi flask containing 500 mL of TGY medium and cultured for 48 hours at 30° C. and 96 rpm. Following culturing, the product was centrifuged for 20 minutes at 5,000×g, yielding a bacterial mass.
(ii) Preparation of Cell-Free Extract
Strain H-8-1-3 was precultured (200 rpm, 30° C., 12 hours) in 5 mL of TGY medium. The precultured solution was inoculated onto 500 mL of TGY medium and cultured with shaking (96 rpm) for 2 days at 30° C. using two-liter Sakaguchi flasks (total 20 L). The bacteria were collected with a large centrifuge (5,000 rpm, 10 minutes, 4° C.), washed with physiological saline (0.9% NaCl), after which the bacterial mass of 5 liters of medium was suspended in 100 mL of 20 mM phosphate buffer solution (pH 7.0) (KPB). A 100 mL quantity of the bacterial solution was ultrasonically processed for 15 minutes and centrifuged (8,000 rpm, 20 minutes, 4° C.). The supernatant obtained was employed as the cell-free extract.
(iii) Processing with Protamine Sulfate to Remove Nucleic Acid
To the cell-free extract was added 0.5% of protamine sulfate sodium. After stirring the mixture for 30 minutes, it was centrifuged in a large centrifuge (3,000 rpm, 10 minutes, 4° C.) and the supernatant was recovered.
(iv) Ammonium Sulfate Fractionation
While stirring the cell-free extract, from which the nucleic acid had been removed, in ice with a stirrer, ammonium sulfate powder was added little by little to 30% saturation. After stirring for 30 minutes, the mixture was centrifuged (8,000 rpm, 10 minutes, 4° C.). While stirring the supernatant on ice, ammonium sulfate powder was added to 60% saturation. After stirring for 30 minutes, the mixture was centrifuged. Similarly, ammonium sulfate powder was added to 90% saturation and the mixture was centrifuged. The precipitates obtained from the various fractions (0 to 30% fraction, 30 to 60% fraction, and 60 to 90% fraction) were suspended in 10 mL of 20 mM KPB (pH 7.0) and dialyzed overnight in 5 L of the same buffer (×3 times).
(v) Anion-Exchange Column Chromatography (DEAE-Toyopearl)
A column was packed with 15 mL of DEAE-Toyopearl resin equilibrated with 20 mM KPB and the dialyzed 30 to 60% fraction of enzyme solution was adsorbed. The column was washed with 100 mL of 20 mM KPB, after which 200 mL of 20 mM KPB and 200 mL of 20 mM KPB containing 500 mM NaCl were used to gradually raise the NaCl concentration above gradient, causing the enzyme to elute. A fraction collector was used to collect each fraction in a 50 mL test tube. The fractions in which activity was found were collected and dialyzed overnight with the same buffer.
(vi) Hydroxyapatite Column Chromatography (GIGA-PITE)
A column was packed with 5 mL of GIGA-PITE resin equilibrated with 20 mM phosphate buffer and the enzyme solution was adsorbed. After washing the column with 50 mL of 20 mM KPB, 50 mL of 20 mM KPB and 200 mL of 500 mM KPB were employed to gradually raise the KPB concentration above gradient, causing the enzyme to elute. Non-adsorbing fractions in which activity was found were dialyzed overnight with 5 L of the same buffer (×3 times).
(vii) Strong Ion Exchange Chromatography (MonoQ HR 10/100)
Medium pressure high performance liquid chromatography (FPLC, column: 20 mM KPB-equilibrated MonoQ HR 10/100 column) was employed. A 200 μL quantity of enzyme solution concentrated by ultrafiltration (Centricon tube) was introduced into the sample loop, and the enzyme was eluted with an FPLC gradient system using the two solvents of 20 mM KPB and 20 mM KPB containing 0.5 mM NaCl. Those fractions (0.5 mL each) in which activity was found were collected and dialyzed overnight. Following dialysis, a Centricon was used to concentrate the enzyme solutions to 200 μL.
(viii) Gel Filtration Chromatography (Superdex 200 10/30)
Medium pressure high performance liquid chromatography (FPLC, column: Superdex 200 10/30 column equilibrated with 20 mM KPB containing 150 mM NaCl) was employed. A 200 μL quantity of enzyme solution was introduced into the sample loop, and the enzyme was eluted with an FPLC system using a solvent in the form of 20 mM KPB containing 150 mM NaCl. Those fractions in which activity was found were collected and dialyzed overnight.
Table 4 shows the quantities of protein and the enzyme activity in each purification step.
(5) Determination of Molecular Weight of H-8-1-3-Derived L-Amino Acid Oxidase by SDS Polyacrylamide Gel Electrophoresis
The electrophoretic gel employed consisted of a concentrated gel comprised of 0.5 mL of 36% polyacrylamide, 3.5 mL of 0.179M tris-HCl (pH 6.8), 0.5 mL of 1% SDS, 125 μL of 10% TEMED, and 375 μL of 2% APS layered over a gel comprised of 5.25 mL of 36% acrylamide, 8.25 mL of 0.68 M tris-HCl buffer (pH 8.8), 1.58 mL of 1% SDS, 187 μL of 10% TEMED, and 562.5 μL of 2% APS. A 10 μL quantity of purified enzyme sample mixed with an equal quantity of buffer (200 μL glycresol, 40 μL 1 M tris-HCl (pH 8.0), 360 μL water, 200 μL 2-mercaptoethanol, and 200 μL of 10% SDS) was subjected to electrophoresis at 30 mA in running buffer (tris 3.0 g, glycine 14.1 g, and SDS 10 g). Subsequently, it was stained for one hour with protein staining solution (CBB 2.5 g, methanol 500 mL, acetic acid 50 mL, and water 450 mL) and decolored with a destaining solution (methanol:acetic acid:water=3:1:6) until the bands became distinct.
Molecular weight markers (Bio-Rad) were employed in the form of phosphorylase (97,400), bovine serum albumin (66,267), aldolase (42,200), carbonic anhydrase (30,000), and soybean trypsin inhibitor (20,000).
(6) Determining the N-Terminal Amino Acid Sequence of Pseudomonas Sp. H-8-1-3-Derived L-Amino Acid Oxidase
Eight residues from the N-terminal of the purified Pseudomonas sp. H-8-1-3-derived L-amino acid oxidase were determined by the Edman degradation procedure.
(7) Cloning of the Pseudomonas Sp. H-8-1-3-Derived L-Amino Acid Oxidase Gene
(i) Extraction of Chromosomal DNA from Pseudomonas Sp. H-8-1-3
Pseudomonas sp. H-8-1-3 was inoculated onto 3 mL of TGY medium and cultured for 12 hours at 30° C. and 200 rpm. The cells were collected by centrifuging (15,000 rpm, 5 minutes, 4° C.) the bacterial mass from 1 mL of culture solution. After washing the cells with 1 mL of STE buffer (0.58 g of NaCl, 1 mL of 1 M tris-HCl (pH 8.0), and 200 μL of 0.5 M EDTA (pH 8.0) to which water was added to make 100 mL), the cells were suspended in the same buffer. They were heated for 15 minutes at 68° C. and centrifuged (15,000 rpm, 5 minutes, 4° C.). The supernatant was removed. The cells were suspended in 300 μL of lysozyme-RNase (lysozyme 5 mg and 10 mL of 10 mg/mL RNase per solution; dissolved in 1 mL of a solution of 0.9 g of glucose, 2.5 mL of 1 M tris-HCl (pH 8.0), 2 mL of 0.5 M EDTA (pH 8.0) to which ultrapure water was added to make 100 mL). Following incubation for 30 minutes at 37° C., 6 μL of proteinase K solution was added (10 mg/L proteinase K per mL of solution), the mixture was gently stirred, and the mixture was incubated for 10 minutes at 37° C. A 3 mg quantity of N-lauroylsarcosine was added. The mixture was gently stirred and then incubated for 3 hours at 37° C., and a phenol-chloroform treatment was gently conducted twice. To 300 μL of the supernatant were added 10 μL of 5 M NaCl solution and 600 μL of ethanol, the mixture was stirred, and centrifugation was conducted (15,000 rpm, 10 minutes, 4° C.). After washing with 70% ethanol, the product was air dried and dissolved in 100 μL of TE buffer, yielding the targeted chromosomal DNA.
(ii) Amplification of Pseudomonas Sp. H-8-1-3-Derived L-Amino Acid Oxidase Gene by PCR
The composition of the PCR reaction solution was 35 μL of water, 5 μL of 10×Ex Taq buffer, 5 μL of 2 mM dNTP, 1 μL of 100 pmol primer 1 (5′-ATGAACAANAANAACCGCCACCCSGCCGAC-3′) (SEQ. ID NO: 17), 1 μL of 100 pmol primer 2 (5′-TCARTCYGCCAGGGCGATYGGSCCGATYTC-3′) (SEQ. ID NO: 18), 2 μL of template DNA, and 1 μL of Ex Taq. The PCR reaction conditions were (i) 5 minutes at 98° C., (ii) 10 seconds at 96° C., (iii) 5 seconds at 50° C., (iv) 4 minutes at 60° C., and 31 cycles up to (ii). The amplified gene was confirmed by agarose gel electrophoresis. The amplified gene was extracted with a Gel-M gel extraction kit from Viogene (USA).
(iii) Sequencing Pseudomonas Sp. H-8-1-3-Derived L-Amino Acid Oxidase Gene
To sequence both strands of the gene, a sequencing reaction was conducted using primer 1, primer 2, primer 3 (5′-AGCACGGTAATCGATCTGGA-3′) (SEQ. ID NO: 19), and primer 4 (5′-CATCGAGTGCCAGTTGCACG-3′) (SEQ. ID NO: 20). The composition of the reaction solution was 1.6 μL of each primer, 1.6 μL of template DNA, 1 μL of BigDye premixed solution, 1.6 μL of 5× BigDye sequencing buffer, and 2.8 μL of sterilized water. A total quantity of 10 μL was employed. The PCR reaction conditions were (i) 2 minutes at 96° C., (ii) 10 seconds at 96° C., (iii) 5 seconds at 50° C., (iv) 4 minutes at 60° C., (v) 25 cycles of (ii) to (iv), and (vi) 5 minutes at 72° C. To the PCR product were added 1 μL of 3 M sodium acetate (pH 5.2), 1 μL of 0.125 M EDTA, and 25 μL of ethanol. The mixture was left standing for 15 minutes at room temperature and then centrifuged (15,000 rpm, 8 minutes, 4° C.) to induce precipitation. The supernatant was discarded, 10 μL of Hi Di Formamide was added, and the mixture was heated for 5 minutes at 100° C. The mixture was then rapidly cooled with ice water and the base sequence thereof was read with an ABI PRISM 310 Genetic Analyzer. The sequence data obtained were analyzed with Genetyx and the base sequences of fragments amplified with the various primers were linked.
(iv) Transformation of E. coli (E. coli JM 109) with the Pseudomonas Sp. H-8-1-3-Derived L-Amino Acid Oxidase Gene
The composition of the ligation reaction was 5 μL of PCR product, 1 μL of pT7 Blue T-Vecter (Novagen), and 6 μL of ligation mix (Takara). The reaction was conducted for 30 minutes at 16° C. To 100 μL of competent cells of E. coli (E. coli JM 109) was added 12 μL of the ligation reaction solution and transformation was conducted by the heat shock method. Several colonies growing in LB medium (1.0% of polypeptone, 0.5% of yeast extract, and 1.0% of NaCl) containing 80 μg/mL of ampicillin were selected, extracted with plasmid, and subjected to 0.7% agarose electrophoresis to confirm the presence of the insert.
(8) Expression in E. coli (E. coli BL21) of Pseudomonas Sp. H-8-1-3-Derived L-Amino Acid Oxidase Gene
(i) Amplification of Pseudomonas Sp. H-8-1-3-Derived L-Amino Acid Oxidase Gene
PCR was conducted using the plasmids obtained in the above cloning as template DNA. The composition of the PCR reaction solution was 35 μL of water, 5 μL of 10×Ex Taq buffer, 5 μL of 2 mM dNTP, 1 μL of 100 pmol/μL primer 5 (5′-TATAATCATATGAACAAGAACAACCGCCA-3′) (SEQ. ID NO: 21), 1 μL of 100 pmol/μL primer 6 (5′-TATTACTCGAGTCAGTCCGCCAGGGCGATTG-3′) (SEQ. ID NO: 22), 100 ng of template DNA, and 5 units of Ex Taq. Primers 5 and 6 were provided with NdeI and XhoI restriction enzyme sites, respectively. The PCR reaction conditions were (i) 5 minutes at 98° C., (ii) 10 seconds at 96° C., (iii) 5 seconds at 50° C., (iv) 4 minutes at 60° C., and 31 cycles up to (ii).
(ii) Recombination of Pseudomonas Sp. H-8-1-3-Derived L-Amino Acid Oxidase Gene into pET15b Vector and Transformation of E. coli (E. coli BL21)
To 5 μL of the PCR product obtained in the PCR reaction were added 1 μL of NdeI and 1 μL of XhoI and the mixture was incubated for 1 hour at 37° C. to conduct restriction enzyme treatment. The ligation reaction was conducted with 5 μL of DNA, 1 μL of pET15B (which had been subjected to the same restriction enzyme treatment as the amplified gene), and 6 μL of ligation mix, which were incubated for 30 minutes at 16° C. The entire quantity of the ligation reaction solution obtained was introduced into E. coli (E. coli BL21) by the heat shock method. An expression plasmid was constructed so that the L-amino acid oxidase produced by the recombinant E. coli would be produced as a fused protein to which 6× histidine tags were added to the N-terminal side.
(iii) Expression of Pseudomonas Sp. H-8-1-3-Derived L-Amino Acid Oxidase Gene and Purification of Fused Enzyme with 6 Histidine Tags Using Ni-Sepharose
The recombinant E. coli (BL21) was inoculated onto 4 liters of LB medium (1.0% of polypeptone, 0.5% of yeast extract, 1.0% of NaCl, pH 7.0) containing 80 μg/mL of ampicillin and cultured for 12 hours at 37° C. Subsequently, 0.5 mM IPTG was added. The mixture was then cultured for 12 hours at 30° C. and L-amino acid oxidase was induced. The cells were collected using a large centrifuge (5,000 rpm, 10 minutes, 4° C.), washed with physiological saline (0.9% NaCl), and suspended in 100 mL of 20 mM phosphate buffer (pH 7.0) (KPB). A 100 mL quantity of the bacterial cell solution was ultrasonically treated for 15 minutes and centrifuged (8,000 rpm, 20 minutes, 4° C.). The supernatant obtained was adopted as a cell-free extract. The cell-free extract was adsorbed on a Ni-Sepharose column substituted with 20 mM KPB and the column was washed with 20 mM of KPB and then, with 20 mM of KPB containing 500 mM imidazole, causing the enzyme solution to elute.
(9) Measurement of the Activity of Pseudomonas Sp. H-8-1-3-Derived L-Amino Acid Oxidase
The enzymatic activity of the purified enzyme preparation was measured with measurement reagents containing the single amino acids indicated in Table 3. The enzyme preparation was found to have the following properties:
(a) It employed L-lysine, L-arginine, and L-ornithine as a substrate (Table 5). The L-amino acid oxidase did not exhibit activity for other amino acids constituting proteins (L-tyrosine, L-alanine, L-cysteine, L-aspartic acid, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-methionine, L-asparagine, L-proline, L-glutamine, L-serine, L-threonine, L-valine, L-phenylalanine, and L-tryptophan).
(b) At pH 6.5 and below, it only acted on L-lysine (
(10) Quantification of L-Lysine with Pseudomonas Sp. H-8-1-3-Derived L-Amino Acid Oxidase
The above L-lysine-detecting reagent composition was prepared using the L-amino acid oxidase preparation purified in (8) (iii) above in which histidine tags had been added to the N-terminal side. L-lysine was measured using this reagent composition. Specimens in the form of 1 to 6 mM L-lysine aqueous solutions were prepared.
The results revealed that when L-lysine aqueous solutions were employed as specimens, reactivity was observed. The L-lysine concentration and measurement data indicated a good, positive correlation (see
(11) the Introduction of Mutations into the Pseudomonas Sp. H-8-1-3-Derived L-Amino Acid Oxidase Gene
Variant L-amino acid oxidase having oxidase activity highly substrate specific to L-lysine was fabricated by conducting saturation mutation of the cysteine at position 254 in the amino acid sequence of the above L-amino acid oxidase.
The expression plasmids into which the L-amino acid oxidase genes had been incorporated, constructed in (8) (ii) above, were employed as templates in mutagenic PCR. In the PCR reaction, a QuikChange® Multi Site-Directed Mugagenesis Kit (Stratagene) was employed. The composition of the PCR reaction solution was 16.5 μL of sterile water (MilliQ), 2.5 μL of 10×QuikChange Lightening Multireaction buffer, 1.0 μL of 100 ng/μL template DNA, 1.0 μL of 100 ng/μL primer 7 (5′-GTGGTGAT-GACCAATNNSGACGACCACCAACAC-3′) (SEQ. ID NO: 23), 1.0 μL of 100 ng/μL primer 8 (5′-GCGGTGCTGACGACCNNSCAGAGTTGGCTGCTG-3′) (SEQ. ID NO: 24), 1.0 μL of 100 ng/μL primer 9 (5′-AAGCCAGGGGTGATCNNSCTGTCCTACGCGTGG-3′) (SEQ. ID NO: 25), 1.0 μL of dNTP mix, and 1.0 μL of QuikChange Lightning Multi enzyme blend. The PCR reaction conditions were (i) 2 minutes at 95° C., (ii) 20 seconds at 95° C., (iii) 30 seconds at 55° C., (iv) 4 minutes at 65° C., and (v) (ii) to (iv) for 5 minutes at 65° C. To the PCR product was added 1.0 μL of DpnI and the mixture was left standing for 5 minutes at 37° C. (sample 1). Similarly, 1.0 μL of 100 ng/μL primer 10 (5′-CATGTGCCAGAGCGTNNSGCGCACTGGCCCGAA-3′) (SEQ. ID NO: 26) and 1.0 μL of 100 ng/μL primer 11 (5′-ACCACCCAGATCGACNNSGAAGAGTCGTTGTTC-3′) (SEQ. ID NO: 27) were employed to conduct the above processing (sample 2).
Escherichia coli JM109 was transformed using each of samples 1 and 2 and cultured for 12 hours at 37° C. in 5 mL of LB liquid medium to which 500 μg of ampicillin had been added. Once the culturing had ended, the various samples were centrifuged (13,000 rpm, 4° C., 5 minutes) to collect the bacteria. The collected bacterial mass was subjected to plasmid extraction by the alkali mini prep method. The alkali mini prep method was conducted according to the following procedure. (1) E. coli culture solution containing plasmid DNA was moved to a 1.5 mL tube and centrifuged. The bacterial mass was recovered as pellets. (2) A 100 μL quantity of solution I (25 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.9% glucose) was added to the bacterial pellets to prepare a suspension. (3) A 200 μL quantity of solution II (0.2 M NaOH, 1% SDS) was added, the bacterial mass was dissolved, and the protein and nucleic acid were denatured. (4) A 150 μL quantity of solution III (3 M potassium acetate and 11.5% acetic acid) was added, the solution was neutralized, and the SDS was removed. These operations rendered the chromosomal DNA and protein in the form of an aggregated precipitate. However, the plasmid DNA remained in the dissolved portion. (5) The aggregated precipitate was removed from the solution by centrifugation, and the solution containing the plasmid DNA was recovered. (6) The solution containing the plasmid DNA that was obtained was subjected to phenol chloroform extraction to remove the protein. (7) A quantity of ethanol double that of the plasmid solution was added and the mixture was placed for 5 minutes on dry ice. (8) The plasmid DNA was precipitated by a centrifugal operation. (9) The precipitate of plasmid DNA obtained was dissolved in TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) to obtain a sample.
The product obtained by PCR1 was denoted as plasmid1 and the product obtained by PCR2 was denoted as plasmid2 among the plasmids obtained. These were used to transform E. coli BL21(DE3) by the heat shock method. The procedure of the heat shock method was as follows. To a 50 μL quantity of E. coli BL21(DE3) was added 5 μL quantity of each of the plasmids and the mixtures were left standing on ice for 60 minutes. Subsequently, heat shock was conducted for 90 seconds at 42 degrees, after which 1 mL of LB liquid medium was added and the bacteria were incubated for 1 hour at 37 degrees. The culture was spread on plates containing ampicillin and cultured for 16 hours at 37 degrees.
To each well of a 96-deep-well plate was dispensed a 300 μL quantity of LB liquid medium containing 100 μL/mL of ampicillin. A colony picker was then employed to inoculate the colonies obtained into the medium of each well. Following inoculation, a shaker was used to conduct culturing for 12 hours at 37 degrees and 700 rpm. Subsequently 20 μL of 7.5 mM IPTG was added and shake culturing was again conducted for 12 hours at 30 degrees. Following culturing, centrifugation (2,500 rpm, 4° C., 20 minutes) was used to collect the bacteria. To each mass of collected bacteria was added 200 μL of 10 mM KPB (pH 7.0) to form a suspension. The bacteria were transferred to a 96-well round-bottomed plate and the bacterial mass was crushed in an eight-section sonicator (output 3, 30 seconds). Following centrifugation under identical conditions, the supernatant obtained was collected as a cell-free extract. A 50 μL quantity of this cell-free extract was employed to measure relative activity with L-lysine as substrate by the above active measurement method.
The transformants were screened for those exhibiting marked activity. As a result, 175 colonies were obtained from the 4,000 colonies obtained in PCR1 and 50 colonies were obtained from the 500 colonies obtained in PCR2.
The supernatant of the colonies obtained was used to again measure the activity with p-chloromercuribenzoate (PCMB). The SH group in the protein reacted with the metal ion, forming a mercaptide. Although this reaction was reversible, equilibrium is generally highly skewed by mercaptides. Hg2+ is highly hydrophilic with respect to SH groups. For enzymes having oxidase activity or monooxygenase activity, it is widely known among persons having ordinary skill in the art that the addition of PCMB reagent skews oxidase activity.
Based on the above, screening was conducted to find enzymes exhibiting no difference in activity when PCMB was added to the cell-free extract and when it was not. As a result, four colonies were obtained among the 175 colonies from PCR1 and four colonies were obtained among the 50 colonies from PCR2. A Big Dye Terminator v3.1 Cycle Sequencing Kit (made by Applied Biosystems) and a DNA Sequencer 310 Genetic Analyzer (also made by Applied Biosystems) were used to sequence the plasmids contained in the colonies obtained. As a result, it was found that the TGC base sequence of numbers 760 to 762 in the coding region had mutated to ATC, and that this mutation had replaced the cysteine at position 254 in the amino acid sequence with isoleucine. This variant enzyme was named variant enzyme C254I.
The variant enzymes obtained as the results of this screening were purified by the method described in (8) (iii) above. As shown in
To examine the effect of pH on variant enzyme C254I during the reaction, the pH of the reagent composition was varied and the oxidase activity at various pH levels was measured for substrates in the form of L-lysine, L-ornithine, and L-arginine.
As shown in
The oxidase activity of variant enzyme C254I was measured for substrates in the form of amino acids other than L-lysine, L-arginine, and L-ornithine. The activity measuring reagents and activity measuring method were as indicated in (1) and (2) above. The reaction conditions were pH 7.0 and 30° C. The results are given in
As shown in
Further, the above L-lysine measuring reagent composition was prepared using the purified wild-type enzyme prepared in (8) (iii) above and variant enzyme C254I. L-lysine was measured with this reagent composition. As specimens, 1 to 10 mM L-lysine aqueous solutions were prepared.
As a result, reactivity was observed when L-lysine aqueous solutions were employed as specimens. The L-lysine concentration and measurement data exhibited a good, positive correlation (see
Further, in the variant obtained by the above saturation mutation, even when the cysteine at position 254 had been replaced with an amino acid other than isoleucine, oxidase activity was measured for L-lysine, L-arginine, and L-ornithine. The activity measuring reagents and activity measurement method were identical to those indicated in (1) and (2) above. Reaction conditions of pH 7.0 and 30° C. were employed. The results are given in Tables 6 and 7 and
As shown in Table 6 and
L-amino acid oxidase activity was measured for substrates in the form of the constituent amino acids of proteins in the form of L-tyrosine, L-alanine, L-cysteine, L-aspartic acid, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-methionine, L-asparagine, L-proline, L-glutamine, L-serine, L-threonine, L-valine, L-phenylalanine, and L-tryptophan when the cysteine at position 254 was substituted with an amino acid other than isoleucine in the variants obtained by the above saturation mutation. As a result, no L-amino acid oxidase activity was detected in any of the variant enzymes when these amino acids were employed as substrates. Additionally, no L-amino acid oxidase activity was detected in any of the variant enzymes even when D-lysine, D-arginine, and D-ornithine were employed as substrates.
(12) Measurement of Blood Plasma Samples
Human blood plasma samples to which L-lysine, L-ornithine, or L-arginine had been added were used to measure oxidase activity in the above variant enzyme C254I. Amicon Ultra 0.5 (UFC501096, Millipore) was used as the human serum and a deproteinized solution was prepared by centrifugation for 30 minutes at 16,100×g and 4° C. To the prepared human plasma were added L-lysine, L-ornithine, or L-arginine to final concentrations of 0, 5, 10, 15, 20, 25, 30, 35, 40, and 45 μM to prepare plasma samples. The measurement reagent composition and measurement method were the same as those indicated in (1) and (2) above. The reaction conditions were pH 7.0 and 30° C. The results are given in
As shown in
Based on the above test examples, the use of the variant enzyme of the present invention permitted the accurate detection or quantification of L-lysine even in clinical samples containing amino acids other than L-lysine.
While the invention has been described in detail with reference to exemplary embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. The foregoing description of the preferred embodiments of the invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and modifications and variations are possible in light of the above teachings or may be acquired from practice of the invention. The embodiments were chosen and described in order to explain the principles of the invention and its practical application to enable one skilled in the art to utilize the invention in various embodiments as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto, and their equivalents. The entirety of each of the aforementioned documents is incorporated by reference herein.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2012-016165 | Jan 2012 | JP | national |
This application is a Continuation of, and claims priority under 35 U.S.C. §120 to, International Application No. PCT/JP2013/051894, filed Jan. 29, 2013, and claims priority therethrough under 35 U.S.C. §119 to Japanese Patent Application No. 2012-16165, filed on Jan. 30, 2012, the entireties of which are incorporated herein. Also, the Sequence Listing electronically filed herewith is hereby incorporated by reference (File name: 2014-07-29T_US-517_Seq_List; File size: 86 KB; Date recorded: Jul. 29, 2014.)
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP2013/051894 | Jan 2013 | US |
| Child | 14445597 | US |
