WO2006022113A1 - METHOD OF QUANTIFYING L-PHENYLALANINE BY USING IMMOBILIZED ENZYME CHIP HAVING His-Tag-FUSED PHENYLALANINE DEHYDROGENASE - Google Patents

Abstract

A phenylalanine dehydrogenase having an oligopeptide comprising 3 to 12 histidine residues fused at the N-terminus; a phenylalanine dehydrogenase having an oligopeptide comprising 3 to 12 histidine residues fused at the C-terminus; an immobilized enzyme chip having a plural number of wells on the substrate surface wherein the phenylalanine dehydrogenase as described above has been immobilized in these wells; an immobilized enzyme chip for analyzing L-phenyl alanine which has a plural number of wells on the substrate surface and in which a phenylalanine dehydrogenase having an oligopeptide comprising 6 to 9 histidine residues fused at the N-terminus has been immobilized in these wells; and a method of analyzing L-phenylalanine contained in a test sample which comprises incubating the test sample in the wells of the immobilized enzyme chip as described above together with a liquid reaction mixture containing resazurin, diaphorase and nicotinamide adenine dinucleotide (NAD) and detecting color development of the liquid reaction mixture.

Description

 Specification

 Method for quantification of L-phenylalanine by immobilized enzyme chip using His-Tag fusion phenylalanine dehydrogenase

 Technical field

 [0001] The present invention relates to a measurement method capable of quantifying L-phenylalanine contained in a biological sample in a simple and trace amount without requiring complicated pretreatment and a large amount of reagents. In particular, it is a test method that can be used for neonatal mass screening for early detection or periodic diagnosis of the patient for the disease of phenylketonuria, an inborn error of metabolism.

 Background art

 [0002] The global spread of newborn mass screening for the early detection of congenital metabolic disorders is the discovery of a cure for phenylketonuria (PKU) and the L- in dry filter paper blood. It begins with the development of a semi-quantitative method for phenylalanin (L-Phe). The most important reason why congenital anomaly is a disease subject to mass screening is because of its medical benefits, such as the expectation of normal development in children by early detection, and its economic efficiency. This is because it was revealed. Comparing the case of finding a child with disability lately, developing a detention facility and paying high medical expenses to care for the patient, and the case of growing up as a healthy adult by early detection and treatment, the latter This is much more economically efficient and improves the quality of life of the child. For this reason, prevention of mental retardation and developmental disorders due to congenital metabolic disorders is recognized as a public health issue in each country, and newborn mass screening is being developed with national and administrative support.

[0003] PKU, which represents inborn errors of metabolism, is a loss of the innate gene of L-Phe hydroxylase, which converts the essential amino acid L-Phe to tyrosine, or a decrease in its activity. It is a disease caused by the cause. Abnormal accumulation of L-Phe is confirmed in the body, and in addition to L-Phe, a large amount of ferrule birubic acid is excreted in the urine. Clinical symptoms include intellectual impairment such as mental retardation, neuropathy, and melanin deficiency. Treatment of this disease requires a diet that limits L-Phe intake and is preferred at least until adulthood. Or treatment for a lifetime. Since L-Phe is one of the essential amino acids for the human body, the intake is within the range of the maximum amount that does not cause brain damage and the minimum amount necessary for physical development. Must be maintained strictly. In addition, maternal PKU increases the amount of L-Phe in the mother's blood when a woman with PKU becomes pregnant, causing developmental disorders, intelligence impairment, microcephaly, and heart malformations in the fetus. However, pre-pregnancy can be prevented by controlling the blood L-Phe concentration.

 [0004] Here, the screening test method for diagnosis is a method for inhibiting bacterial growth using Bacillus subtilis and an antimetabolite inhibitor using dry filter paper blood that has been punctured in the heels of a newborn and soaked in blood collection filter paper as a specimen ( Bacterial inhibition assay (BIA) was developed by Guthrie et al. (Pediatrics, Vol. 32, p. 338 (1963), Non-Patent Document 1) and screening was started. The current multiple screening system is the BIA method! /, The Paigen method using E. coli and phage (Journal of Lab. Clin. Med., Vol. 99, p. 895 (1982), non-patent literature. Based on 2), the foundation was established in the 1960s. These test methods are very simple methods for determining the size of the bacterial growth circle after arranging a blood disk punched by a puncher on agar medium and culturing overnight, using dry filter paper blood as a specimen. This is a method that does not require special equipment, has excellent reagent costs, and enables multi-item testing of a large number of samples. However, since the final inspection result is determined visually, it is difficult to make the determination result objective and record it.

 [0005] As an improvement, imaging with a camera (Nippon Mass Screening Society Journal, Vol. 6, p. 23 (1996), Non-Patent Document 3) has been proposed. Quantifying the growth of bacteria with large fluctuations is extremely difficult, and the convenience is also impaired. On the other hand, high performance liquid chromatography (journal of Chromatography, Vol. 274, p. 318 (1983), Non-Patent Document 4, Journal of Japan Mass Screening Society, Vol. 5, p. 86 (1995), Non-Patent Document 5) The ability to apply automatic analyzers (Clinical Chemistry, Vol. 30, p. 287 (1984), Non-Patent Document 6) Target The disease is only an amino acid metabolism disorder or the use of an expensive measuring device, There are also questions about simplicity and speed.

[0006] In such circumstances, recently, enzymatic methods (Screening, Vol. 1, p. 63 (1992), Non-Patent Document 7, Medicine and Pharmacy, Vol. 31, p. 1237 (1994), non-patent document 8) or microplate fluorescence method (Clini cal Chemistry, Vol. 35, p. 1962 (1989), non-patent document 9) A kit was developed to perform the subsequent fluorescence reaction on a microplate and to quantify L-Phe in the specimen from the fluorescence intensity (Medicine and Pharmacy, Vol. 37, p. 1211 (1997), Non-Patent Document 10). In addition to the high sample throughput, this kit realizes quantitative analysis and recording, which is an objective determination of test results that was difficult with conventional methods. Furthermore, test results can be obtained in about 3 hours, and the speed is greatly improved. As described above, a mass specimen processing method in newborn mass screening is being established.

 [0007] However, in the kit used in the above enzymatic method, it was necessary to dispense a predetermined amount of a solution containing ferroalanine dehydrating enzyme together with other reagents into each well of the microplate. Inspection efficiency has been improved by using microplates. The above sorting work was still a human task.

 [0008] Therefore, an object of the present invention is to provide means capable of carrying out an enzymatic method without separating a solution containing ferulalanin dehydrogenase in each well of a microplate.

 [0009] More specifically, an object of the present invention is to provide a fixed chip that fixes a phenylalanine dehydrogenase to each well of a microplate, and to use a phenylalanine using the chip. It is to provide an analysis method.

 Furthermore, another object of the present invention is to provide phenylalanine dehydrogenase suitable for immobilization.

 Disclosure of the invention

 [0010] The present invention for achieving the above object is as follows.

 [1] Ferulalanine dehydrogenase fused with 3 to 12 oligopeptides with histidine strength at the N-terminus.

 [2] Ferulalanine dehydrogenase fused with 3 to 12 histidine-resistant oligopeptides at the C-terminus.

 [3] An immobilized enzyme chip having a plurality of wells on the surface of the substrate, in which the phenylalanine dehydrogenase described in [1] or [2] is immobilized.

[4] The immobilized enzyme chip according to [3], which is used for analysis of L-ferallanine. [5] The substrate surface has a plurality of wells, and in these wells, L-Folamine immobilized with phenylalanine dehydrogenase fused with 6-9 histidine oligopeptides at the N-terminus. Immobilized enzyme chip for analysis of nenylalanine.

 [6] The immobilized enzyme chip according to [5], wherein the ferulalanin dehydrogenase is immobilized in the well via Ni-chelate.

 [7] A reaction solution containing resazurin, diaphorase, and nicotinamide adenine dinucleotide (NAD) as a test sample in the well of the immobilized enzyme chip according to any one of [4] to [6] A method for analyzing L-fetalanin contained in a test sample, which comprises incubating together with the mixture and detecting color development of the reaction solution.

 [8] The method according to [7], wherein the color development of the reaction solution is detected using a DNA microarray scanner.

 [9] The method according to [7] or [8], wherein L-ferallanin contained in the test sample is quantified.

 [10] The method according to any one of [7] to [9], wherein the test sample is a blood sample and is used for diagnosis of phenylketonuria.

[0011] According to the present invention, L-phenylalanine contained in a biological sample can be used in a measurement method that can be easily and quantified in a trace amount without requiring complicated pretreatment and a large amount of reagents. A dehydrogenase and an immobilized enzyme chip on which the dehydrogenase is immobilized can be provided. Furthermore, according to the present invention, it is possible to provide an analytical method capable of quantifying L-phenylalanine contained in a biological sample in a simple and minute amount without requiring complicated pretreatment and a large amount of reagents. These enzymes, kits, and analytical methods are extremely useful for neonatal mass screening for early detection or periodic diagnosis of the patient in the disease of inborn metabolic disorder, fluorketuria.

 BEST MODE FOR CARRYING OUT THE INVENTION

[0012] [His-Tag fusion phenylalanine dehydrogenase]

The present invention relates to a pheralanine dehydrogenase in which 3 to 12 oligopeptides having histidine activity are fused to the N-terminus, and a pheralanine dehydrogenase in which 3 to 12 oligopeptides having histidine activity are fused to the C-terminus. . Hereafter, ferrolanine dehydrogenase fused with an oligopeptide that also has histidine power is referred to as His-Tag fusion phalalinine dehydrogenase. Sometimes called an enzyme.

 The His-Tag fusion phalalanin dehydrogenase of the present invention is an enzyme in which an oligopeptide (His-Tag) that also has histidine power is fused to the N-terminus or C-terminus of ferro-alanine dehydrogenase.

 [0013] The phalalanin dehydrogenase (EC. 1.4.1.20) in the present invention is an enzyme that specifically acidifies and deaminates the amino group of L-phenol. Various biogenic enzymes are known. As long as the enzyme catalyzes the same reaction, it is not particularly limited to the name of furanine dehydrogenase. In addition, this enzyme requires nicotinamide adenine dinucleotide (acidic type; NAD +) as a coenzyme. The acid-oxidation reaction by this enzyme is reversible, and the reductive amination reaction is carried out in the presence of ammonia ion and -cotinamide adenine dinucleotide (reduced form; NADH) in the neutral to weakly alkaline pH range. Use as a catalyst.

 [0014] An oligopeptide (His-Tag) composed of histidine is used when immobilizing ferruleanine dehydrogenase on a substrate surface via a metal chelate. The number of histidines in the His-Tag can be appropriately determined in consideration of the affinity with the metal chelate and the enzyme activity after immobilization. In the present invention, 3 to 12 histidines are considered in consideration of these points. An oligopeptide consisting of histidine is used as the His-Tag. However, the affinity with the metal chelate and the enzyme activity after immobilization change depending on the number of histidines in the His-Tag, so it can be changed appropriately according to the type of metal chelate used and the enzyme reaction system. .

 [0015] In addition, an oligopeptide (His-Tag) that also has histidine power is fused to the N-terminus or C-terminus of ferroalanine dehydrogenase. The ability to fuse the His-Tag to the end of the shift changes the affinity with the metal chelate and the activity of the enzyme after immobilization. Therefore, the use of phenylalanine dehydrogenase fused with His-Tag at any end can be appropriately changed according to the type of metal chelate used and the enzyme reaction system.

 [0016] [Immobilized enzyme chip]

The immobilized enzyme chip of the present invention has a plurality of wells on the substrate surface, and the His-Tag fusion ferallanine dehydrogenase of the present invention is immobilized in these wells. Multiple tags used to immobilize His-Tag fusion phenylalanine dehydrogenase Although there is no limitation in particular in the board | substrate which has L, the micro well array sheet | seat made from a polydimethylsiloxane demonstrated in the Example mentioned later can be mentioned, for example. However, it may be made of a material other than polydimethylsiloxane. For example, a printing type microwell array sheet using special ink (such as non-fluorescent or fluorine resin) may be used.

 [0017] There are no particular restrictions on the shape and dimensions of the microwells in the microwell array sheet, and the arrangement and number of microwells. For example, a special ink is printed directly on the surface of a slide glass in advance, and a microwell array sheet of a type that immobilizes the enzyme in the microwell formed there, or a microwell array sheet made of polydimethylsiloxane. There is a type that covers the surface of the slide glass after the enzyme immobilization.

 [0018] The immobilized enzyme chip of the present invention is used, for example, for analysis of L-phenylalanine.

 An analysis of L-felanin includes quantification of L-fetalanin in filter paper blood. In addition, it can be applied to the analysis of L-feralanine in food component analysis (L-feralanine).

 [0019] The His-Tag fusion phalalanin dehydrogenase to be immobilized can be appropriately determined according to the type of metal chelate used and the enzyme reaction system. If the His-Tag fusion phalalanin dehydrogenase is immobilized in the well via Ni-chelate, the His-Tag fusion phalalanin dehydrogenase will contain 6-9 histidine-resistant oligos. It is particularly preferable to use a ferulanine dehydrogenase in which a peptide is fused to the N-terminus when the immobilized enzyme chip is used for L-feralanine analysis.

 [0020] The immobilized enzyme chip of the present invention is a metal chelate for immobilizing a His-Tag fusion ferulalanin dehydrogenase on the bottom of each well of a substrate having a plurality of wells, for example, Ni-chelate. And apply a solution containing His-Tag fusion phalaranin dehydrogenase dropwise on it, or immerse the entire substrate in a solution containing His-Tag fusion phalaranine dehydrogenase. be able to.

In the immobilized enzyme chip of the present invention, the amount of His-Tag fusion ferulalanine dehydrogenase immobilized was determined by adding 500 mM imidazole and 0.5 M NaCl to the enzyme previously immobilized on a slide glass. Dissociate with mM Tris-HCl buffer, pH 8.0, and determine the activity and protein content in the recovered dissociation solution to determine the activity per unit area (U / cm ² ) or specific activity (U / mg / cm ² ). The amount of enzyme immobilized is also affected by the density of the amino group on the surface of the slide glass or the binding capacity of N- (5-amino-1-carboxypentyl) iminodiacetic acid (AB-NTA).

The amount of immobilized His-Tag fusion phalalanin dehydrogenase in the immobilized enzyme chip of the present invention is expressed in terms of activity (U / cm ² ) or specific activity (U / mg / cm ² ), for example, Capability to be in the range of 0.01 U / mg / cm ^{2 to} 1000 U / mg / cm ^{2 For} preparing fixed-enzyme chips, for preparing His-Tag fusion ferulalanine dehydrogenase and fixed-glass slide glass In addition to reducing the cost involved, the viewpoint power of maintaining quantitative accuracy is also preferable.

 [0022] [Method for Analyzing L-Phenenolealanine]

 The present invention includes incubating a test sample with a reaction solution containing resazurin, diaphorase, and nicotinamide adenine dinucleotide (NAD) in the well of the immobilized enzyme chip of the present invention, and detecting color development of the reaction solution. Thus, a method for analyzing L-ferallanin contained in a test sample is included. This enzymatic reaction itself is a method known as the enzymatic method described above. In the present invention, except for using a His-Tag fusion phalalanin dehydrogenase as the phenylalanine dehydrogenase, the conventional method known as an enzyme method is known as an enzyme method except for the method for analyzing L-phenylalanine. The method conditions can be used as they are.

 [0023] In the usual enzymatic method, the eluate (0. 0) is used to extract L-phenylalanine from filter paper blood.

 1 M glycine 'KCl' KOH buffer, pH 9.6) force S used. However, in the present invention, the extraction of L-phenylalanine using 50 mM Tris-HCl buffer, pH 8.9, containing 40 μΜ resazurin instead of the eluate is due to the reduction of complicated dispensing operations. The viewpoint power of improving the accuracy of quantification, preventing contamination, shortening the extraction process, and reducing the cost of the reagent used is also preferable. However, it is also possible to extract L-phenylalanine with an eluate used in a normal enzyme method, and use 50 mM Tris-HCl buffer containing 40 M resazurin, pH 8.9 as a buffer for enzyme reaction.

[0024] More specifically, a blood sample that may contain L-feralanin is obtained from a well of a microwell array chip to which a first enzyme (His-Tag fusion ferralanin dehydrogenase protein) is immobilized. In the second product, a substrate reductant (resorufin) and a coenzyme oxidant (NAD +) are produced from a coenzyme reductant (NADH) and a substrate oxidant (resazurin) of the second enzyme (diaphorase). Incubation in a reaction system containing an enzyme (diaphorase) and a substrate oxidized form (resazurin) of the second enzyme (diaphorase) produces a substrate reduced form (resorufin) of the second enzyme, and the resulting substrate Detect fluorescence of reductant (resorufin). This detection can be performed using an optical detection method, for example, using a DNA microarray scanner. If a calibration curve between the fluorescence emission intensity and L-ferranin is prepared in advance, L-ferranin can be quantified, and the method of the present invention is usually used for quantification of L-phenylalanine. Used for.

[0025] Thus, when the test sample is a blood sample, by quantifying L-ferrananin in the blood sample, the result can be used for diagnosis of phenylketonuria.

 Example

 [0026] 1. Method for measuring furanine dehydrogenase activity

The ferulanine dehydrogenase activity was measured using a double beam spectrophotometer (Model 11-3210, manufactured by Hitachi, Ltd.) according to the method of Asano et al. (Eur J Biochem. 168 (1): 153-9 (1987)). It was measured with a PMMA cuvette (BRAND) with an optical path length of 1 cm. The composition of the reaction solution was as follows. 1 M glycine-KC to KOH buffer solution, 0.1 ml of pH 10.4, 0.1 ml of 25 mM NAD + (produced by Oriental Yeast Co., Ltd.), 0.1 M L-ferallanin (Nippon Rigaku Pharmaceutical Co., Ltd.) (Made by a company) 0.1 ml of an aqueous solution and an appropriate amount of enzyme solution were added to make a total volume of 1.0 ml. The molecular extinction coefficient (ε) of coenzyme NAD + was 6,220 L'mole— ^ cm— ¹ . One unit (U) of this enzyme activity was defined as the amount of enzyme that produces NADH of L mole per minute: Specific activity (U / mg) was defined as enzyme activity (U) per 1 mg of protein.

 [0027] In addition, as a simple enzyme activity measurement method, a total amount of 200 μ1 was used in the above reaction solution composition, a 96-well UV plate (manufactured by Grainer) was used, and a fluorescence 'absorption' luminescence microplate reader (JEOS) was used. , Tecan 'Japan Co., Ltd.) increases the absorbance at 340 °! The enzyme activity was determined from

 [0028] The protein concentration was quantified using a protein assay kit manufactured by Bio-Rad. Bovine serum albumin was used as a standard protein.

[0029] 2. Construction of His-Tag fusion ferulanine dehydrogenase plasmid

Preparation of N-terminal His-Tag fusion ferulalanine dehydrogenase fragment by PCR I went there. Bacillus badius IAM11059-derived ferulalanin dehydrogenase gene (p dh; Asano Y. et al., Eur J Biochem. 168 (1). 153-9. (1987), Yamada A. et al "Biosci B iotechnol Biochem. 59 (10). Plasmid DNA (pBBPDHl) incorporating 1994-5. (1995)) was used as the vertical DNA, including His-Tag sequences (3, 6, 9, 12), respectively. synthetic O Rikonukure old Tet (Sensufufima ^{1 ~~; fBBn3n: 5- gctcatatgcatcatcatgcgatgagcttagtagaa aaaaca} -3, fBBn6h: 5 -gctcatatgcatcatcatcatcatcatgcgatgagcttagtagaaaaaaca-3, f BBn9n: 5 -gctcatatgcatcatcatcatcatcatcatcatcatgcgatgagcttagtagaaaaaaca-3, fBBnl 2h: 5'-gctcatatgcatcatcatcatcatcatcatcatcatcatcatcatgcgatgagcttagtagaaaaaaca-3 'id and anti Sense primers rBBnh2, 5'-taatctcgaggattagttgcgaatatccca-3 '(manufactured by Hokkaido System Science Co., Ltd.) were prepared, and PCR was carried out using the above-described DNA and synthetic oligonucleotides. 10 ng of cocoon-type DNA (pBBPDHl), 100 pmol / μ 1 of the above synthetic oligonucleotides 1 μ 1 each, 10χ Εχ-Taq buffer (Takara Shuzo Co., Ltd.) 5 μ 1 , 2.5 mM dNTP mixture (Takara Shuzo Co., Ltd.) 5 μ1, and Takara Εχ-Taq DNA Polymerase (Takara Shuzo Co., Ltd.) 0.5 μ1 were added to make the total volume 50 μ1. The conditions were 30 seconds at 94 ° C, 30 seconds at 55 ° C, and 2 minutes at 72 ° C for 30 minutes.The PCR reaction was performed by PTC-200 (MJ Research Japan Co., Ltd.). ).

 [0030] The PCR reaction solution was run on a 1.5% agarose gel, and the target amplification product (about 1.2 kb) obtained was excised and amplified using a gel extraction kit (Ge 抽出 M ™ Gel Extraction System, manufactured by VIOGENE). The product was extracted and purified. Collect the total volume of the extract 29 1, 10x H buffer (Takara Shuzo Co., Ltd.) 3.5 μ1, Ndel (New England BioLabs) 0.8 μ1, Xhol (MBI Fermentas) 0.8 μ1 The restriction enzyme treatment was performed at both ends by reacting at 37 ° C for 3 hours. Each of the obtained restriction enzyme-treated fragments was used as an N-terminal His-Tag fusion phenylalanine dehydrogenase insert.

[0031] A vector plasmid for inserting an N-terminal His-Tag fusion ferulalanin dehydrogenase fragment was prepared as follows. For the expression of the fusion protein, pRSET-B vector DNA (manufactured by Invitrogen) having a T7 promoter was used. 10 μg of pRSET-Β vector I DNA, 10x H buffer (Takara Shuzo) 2 μ 1, Ndel (NewEngland BioLabs) 1 μl, 1 μl of Xhol (manufactured by MBI Fermentas) and sterilized water were added to make a total volume of 20 μ1, and the reaction was carried out at 37 ° C. for 3 hours, followed by restriction enzyme treatment.

 [0032] The restriction enzyme-treated vector was subjected to dephosphorylation treatment as follows. Restriction-treated vector DNA 20 μ1, 10x SAP buffer (Boehringer Mannheim) 5 1, Shrimp-derived alkaline phosphatase (Boehringer Mannheim) 2 1 and sterilized water are added to make a total volume of 50 μ1, 37 The mixture was allowed to react at 1 ° C for 1 hour, and further 1 µl of alkaline phosphatase was added and reacted at 50 ° C for 30 minutes. Thereafter, phenol 'chloroform extraction and ethanol precipitation were performed to purify the vector DNA. The insert was ligated downstream of the T7 promoter of pRSE T-B vector DNA.

 [0033] The ligation reaction was performed as follows. Dephosphorylation vector DNA 1 μ1, insert DN A 51, Τ4 DNA ligase (New England BioLabs) 1 μ1, 10x Reaction buffer 2 μ1 and sterile water 11 1 are added to make a total volume of 16 A plasmid for the expression of the Ν-terminal His-Tag fusion furanalanine dehydrogenase was constructed by reacting at CC. The expression plasmid constructed above was transformed into E. coli (E. coli JM109 (Novagen)) by the heat shock method. Luria-Bertani agar medium (l% Bacto tryptone (Difco), 0.5% Bacto yeast (Difco)), l% containing 50 μg / ml ampicillin-strength light tester Co., Ltd. Apply to NaCl (Pure Chemical Co., Ltd.) and 1.5% agar (Nacalai Testa Co., Ltd.), pH 7.5), and incubate at 37 ° C for 10 hours. The cells were cultured at 37 ° C. for 10 hours, showed phenylalanine dehydrogenase activity, and colonies containing the desired insert DNA in the extracted plasmid were selected. Respective plasmids extracted from the respective transformants (E. coli JM109 / pBBPDHNH3, pBBPDHNH6, pBBPDHNH9, pBBPDHNH12) obtained above were used as expression host E. coli (E. coli BL21 (DE3), manufactured by Invitrogen). Transformed. The procedure is described below.

[0034] The transformant (E. coli JM109 / pBBPDHNH3, pBBPDHNH6, pBBPDHNH9, pBBPDH NH12) was cultured in 3 ml LB test tube medium containing 50 g / ml ampicillin at 37 ° C for 12 hours, Extracted by alkaline 'mini' prep method, 100 µ1 (dissolved in TE buffer. 5 mg / ml ribonuclease (Sigma's Aldrich) solution 51 was added to the extracted plasmid solution. Boil and react for 3 hours at 37 ° C. Then, the ethanol-precipitated plasmid DNA was dissolved in 100 μl of TE buffer and subjected to a polyethylene glycol precipitation treatment. That is, a 20% (w / v) polyethylene glycol 6000 (PEG 6000: manufactured by Nacalai Tester Co., Ltd.) solution containing an equal amount of 2 M NaCl was added to the extracted plasmid 100 1 at 4 ° C. After standing for 1 hour, the supernatant was removed by centrifugation (15,000 rpm, 20 minutes, 4 ° C; himac CF15D, Hitachi, Ltd.), and the precipitate was dissolved with 30 1 TE buffer. . Each of the obtained purified plasmids was transformed into T7 expression system E. coli (E. coli B L2KDE3) by the heat shock method. After applying the transformant to LB agar medium and incubating at 37 ° C for 10 hours, the resulting colonies were filled with 300 μ1 LB medium (containing 50 μg / ml ampicillin) in 1 ml. The cells were transferred to a volumetric deep well plate and cultured at 37 ° C for 12 hours with shaking. The obtained culture solution was centrifuged (1,890 X g, 15 minutes, 4 ° C; himac CR20, manufactured by Hitachi, Ltd.) to precipitate the cells, and lysozyme (5 mg / ml lysozyme (derived from egg white; biochemical) Kogyo Co., Ltd.) and 0.1 M phosphate buffer solution containing 5 mM MgCl, pH 7.5)

 2

After treatment at 37 ° C for 30 minutes, the enzyme solution was extracted by freeze-thawing. Ferulanine dehydrogenase activity was measured with a microplate reader (Genius, manufactured by Tecan), and highly active colonies were selected. The obtained colonies were used as N-terminal His-Tag fusion furan-lauranin dehydrogenase transformants (E. coli BL21 (DE3) / pBBPDHNH3, pBB PDHNH6, pBBPDHNH9, pBBPDHNH12), respectively.

3. Construction of C-terminal His-Tag fusion phenylalanine dehydrogenase plasmid

A C-terminal His-Tag fusion ferulalanine dehydrogenase fragment was prepared by PCR reaction as follows. Plasmid DNA (pBBPDHl) incorporating a bacillus badius IAM11059-derived ferulalanin dehydrogenase gene was used as the vertical DNA. Synthetic oligonucleotides (sense primers; fflBnchl, 5-aaggatccgatgagcttagtagaaaaaaca-ύ and "> Nonsense Fuma ¹ ~~; rBBc ^ h, 5 each containing His-Tag sequences (3, 6, 9, 12) - c gtaatctcgagtcagtggtggtggttgcgaatatcccattt-, ri3Bc6h, 5- cgtaatctcgagtcagtggtggtg gtggtggtggttgcgaatatcccattt-3 ', rBBc9h, 5'- cgtaatctcgagtcagtggtggtggtggtggtggtgg tggtggttgcgaatatcccattt-3', rBBcl2h, 5'- cgtaatctcgagtcagtggtggtggtggtggtggtggtggt ggtggtggtggttgcgaatatcccattt-3 ':. were produced in Hokkaido system Science Co., Ltd.) wherein PCR was performed using vertical DNA and synthetic oligonucleotides. The composition was as follows. 1 μl each of 10 ng vertical DNA (pBBPDHl), 100 pmol / 1 of the above synthetic oligonucleotide, 5 μl of 10x Ex-Taq buffer (Takara Shuzo), 2.5 mM dNTP mixture (Takara Shuzo) 5 μ 1 and Takara Εχ-Taq DNA Polymerase (Takara Shuzo Co., Ltd.) 0.5 1 were added to make the total amount 50 1. PCR reaction conditions were 30 cycles of 94 ° C for 30 seconds, 55 ° C for 30 seconds and 72 ° C for 2 minutes. PCR reaction was performed using PTC-200 (manufactured by MJ Research Japan Ltd.).

 [0036] The PCR reaction solution was run on a 1.5% agarose gel, and the target amplification product (about 1.2 kb) obtained was excised and amplified using a gel extraction kit (Ge 卜 M ™ Gel Extraction System, manufactured by VIOGENE). The product was extracted and purified. Add 29 1 and 10x K buffer (Takara Shuzo Co., Ltd.) 3.5 μ1, BamHI (TOYOBO) 0.8 μ1, Xhol (MBI Fermentas) 0.8 μ1 to make the total volume 35 / zl. Then, the reaction was carried out at 37 ° C for 3 hours to perform restriction enzyme treatment at both ends. Each of the obtained restriction enzyme-treated fragments was used as a C-terminal His-Tag fusion phenylalanine dehydrogenase insert.

 [0037] A vector plasmid for inserting a C-terminal His-Tag-fused phenylalanine dehydrogenase fragment was prepared as follows. For the expression of the fusion protein, pET21 (+) vector DNA (manufactured by Invitrogen) having a T7 promoter was used. 5 μg of ρΕΤ21 (+) vector D ΝΑ ゝ 10x K buffer (Takara Shuzo Co., Ltd.) 2 μ1, BamHI (TOYOBO) 1 μ1, Xhol (M BI Fermentas) 1 μ1 1 and sterilized water were added to make the total volume 20 μl and reacted at 37 ° C for 3 hours, followed by restriction enzyme treatment.

 [0038] The restriction enzyme-treated vector was subjected to dephosphorylation treatment as follows. Restriction-treated vector DNA 20 μ1, 10x SAP buffer (Boehringer Mannheim) 5 1, Shrimp-derived alkaline phosphatase (Boehringer Mannheim) 2 1 and sterilized water are added to make a total volume of 50 μ1, 37 The mixture was allowed to react at 1 ° C for 1 hour, and further 1 µl of alkaline phosphatase was added and reacted at 50 ° C for 30 minutes. Thereafter, phenol 'chloroform extraction and ethanol precipitation were performed to purify the vector DNA. The insert was ligated downstream of the T7 promoter of pET2 1 (+) vector DNA.

[0039] The ligation reaction was performed as follows. Dephosphorylation vector DNA 1 μ1, insert DN A 51, Τ4 DNA ligase (New England BioLabs) 1 μ1, 10x Reaction buffer 2 μ1 Then, sterilized water 111 was added to make a total volume of 1, and the mixture was allowed to react at 16 ° C to construct a plasmid for C-terminal His-Tag fusion furanine dehydrogenase expression. The expression plasmid constructed above was transformed into E. coli (E. coli JM109 (Novagen)) by the heat shock method. Luria-Bertani agar medium (l% Bacto tryptone (Difco), 0.5% Bacto yeast (Difco)), l% containing 50 μg / ml ampicillin-strength light tester Co., Ltd. Apply to NaCl (Pure Chemical Co., Ltd.) and 1.5% agar (Nacalai Testa Co., Ltd.), pH 7.5), and incubate at 37 ° C for 10 hours. The cells were cultured at 37 ° C. for 10 hours, showed phenylalanine dehydrogenase activity, and colonies containing the desired insert DNA in the extracted plasmid were selected. Respective plasmids extracted from the respective transformants (E. coli JM109 / pBBPDHCH3, pBBPDHCH6, pBBPDHCH9, pBBPDHCH12) obtained above were used as expression host E. coli (E. coli BL21 (DE3), manufactured by Invitrogen). Transformed. The procedure is described below.

The transformed transformants (E. coli JM109 / pBBPDHCH3, pBBPDHCH6, pBBPDHCH9, pBBPDHCH12) were cultured in 3 ml of LB test tube medium containing 50 g / ml ampicillin at 37 ° C for 12 hours, and the plasmid was alkaline 'Extracted by prep method and dissolved in 100 µ \ <TE buffer. A 5 mg / ml ribonuclease (Sigma's Aldrich) solution 51 was added to the extracted plasmid solution and reacted at 37 ° C for 3 hours. Phenolic black mouth form extraction was performed, and the ethanol-precipitated plasmid DNA was dissolved in 100 μl of TE buffer, followed by polyethylene glycol precipitation. That is, a 20% (w / v) polyethylene glycol 6000 (PEG 6000: manufactured by Nacalai Tester Co., Ltd.) solution containing an equal amount of 2 M NaCl was added to the extracted plasmid 100 1 at 4 ° C. After standing for 1 hour, the supernatant was removed by centrifugation (15,000 rpm, 20 minutes, 4 ° C; himac CF15D, Hitachi, Ltd.), and the precipitate was dissolved with 30 1 TE buffer. . Each of the obtained purified plasmids was transformed into T7 expression system E. coli (E. coli B L2KDE3) by the heat shock method. After applying the transformant to LB agar medium and incubating at 37 ° C for 10 hours, the resulting colonies were filled with 300 μ1 LB medium (containing 50 μg / ml ampicillin) in 1 ml. The cells were transferred to a volumetric deep well plate and cultured at 37 ° C for 12 hours with shaking. The obtained culture broth was centrifuged (1,890 Xg, 15 minutes, 4 ° C; himac CR20, manufactured by Hitachi, Ltd.) to precipitate the cells, and lysozyme (5 mg / ml lysozyme (egg white derived; raw) Chemical Industry Co., Ltd.) and 0.1 M phosphate buffer solution containing 5 mM MgCl, pH 7.5)

 2

 After treatment at 37 ° C for 30 minutes, the enzyme solution was extracted by freeze-thawing. Ferulanine dehydrogenase activity was measured with a microplate reader (Genius, manufactured by Tecan), and highly active colonies were selected. The obtained colonies were used as C-terminal His-Tag fusion ferulanine dehydrogenase transformants (E. coli BL21 (DE3) / pBBPDHCH3, pBB PDHCH6, pBBPDHCH9, pBBPDHCH12), respectively.

 [0041] 4. Expression and purification of N-terminal His-Tag fusion ferulanine dehydrogenase

 Expression of the N-terminal and C-terminal His-Tag fusion phenylalanine dehydrogenases was achieved by inoculating transformant colonies into 3 ml of LB tube medium containing 50 μg / ml ampicillin at 37 ° C. After 10 hours of incubation, transfer to a 2 L Erlenmeyer flask containing 500 ml of LB medium (containing 50 g / ml ampicillin and 0.5 mM isopropyl-β-D-galactoside (IPTG; manufactured by Nacalai Testa)) The cells were cultured at 30 ° C for 18 hours (C-terminal His-Tag fusion ferulalanine dehydrogenase was expressed at 30 ° C for 12 hours). Each culture solution obtained was centrifuged (6,760 X g, 10 minutes, 4 ° C; himac CR20, manufactured by Hitachi, Ltd.), and the precipitated cells were washed with physiological saline (0.85% (w / v ) Concentration). 20 mM Tris containing 5 times the volume of binding buffer (0.5 M NaCl and 2 mM 2-mercaptoethanol (manufactured by Junsei Kagaku Co., Ltd.) with respect to the wet weight of each washing cell obtained. Suspended in hydrochloric acid buffer (pH 8.0) and cooled to 4 ° C with a constant temperature circulating water device (TATEC COOLNIT BATH EL-15, manufactured by Tytec Co., Ltd.) 201M 19 kH z (manufactured by Kubota Corporation), and centrifuge (28,400 X g, 20 minutes, 4 ° C; himac CR20, Hitachi, Ltd.) supernatant (cell-free extract) Got.

 [0042] Purification of His-Tag fusion ferulalanine dehydrogenase by affinity chromatography was performed as follows. 0.1 M NiSO 6Η 0 (Wako Pure Chemical Industries, Ltd.)

 4 2

The above-mentioned cell-free solution was applied to a chelating 'Sepharose FF resin column (5 ml; manufactured by Amersham Fanolemacia Co., Ltd.), which was half the volume of the resin and equilibrated with the binding buffer. The extract was added. After thoroughly washing with a washing buffer (20 mM Tris-HCl buffer, pH 8.0 containing 0.5 M NaCl, 2 mM 2-mercaptoethanol and 5 mM imidazole), an elution buffer (0.5 M NaCl, 2 mM 2-mercaptoethanol and 500 mM The adsorbed protein was eluted with 20 mM Tris-HCl buffer (pH 8.0) containing midazole (manufactured by Junsei Co., Ltd.). The resulting active fraction is concentrated with a centrifugal concentrator (Centrebrep YM-10, Amicon) and 50 mM potassium phosphate buffer with a desalting column (Ampure ™ SA, Amersham Japan). The solution was replaced with pH 7.0. Eluted protein fractions are combined and concentrated to about 2 m in a centrifugal concentrator, and previously adjusted to 50 mM potassium phosphate buffer, pH 7.0 using the AKTA FPLC system (Amersham Falmacia Biotech). Equilibrated Mono Q (HR 5/5 column (Pharmacia Biotech)) was added. After 10 column volume washes with the same buffer, the NaCl concentration in the buffer was changed from 0 to 1 M. Elution was performed using a linear concentration gradient method with a volume of column volume, and the active fractions were collected and used as purified enzymes.

 [0043] 5. Production of polydimethylsiloxane microwell sheet

 The microwell array sheet was prepared using polydimethylsiloxane (PDM S; manufactured by DOW CORNING), which is a silicone-based resin. In preparing the microwell array sheet, first, a well of a microwell was prepared using a thick film resist HNANO ™ XP SU-850 (manufactured by Micro Chem). Production of a microwell saddle using a thick film resist (manufactured by HMicroChem) was performed according to the following procedure. A substrate (38 mm × 26 mm) obtained by cutting acetone (made by Wako Pure Chemical Industries, Ltd.) in advance and cutting a commercially available slide glass subjected to ultrasonic cleaning with ultrapure water was used as the substrate. Place the above-treated slide glass on a spinner single turntable (KYOWARIKEN model K-359S-1, manufactured by Kyowa Riken Co., Ltd.), apply resist adhesive resin 0 mmnicoat (manufactured by MicroChem), rotate at a rotation speed of 2,500 rpm Rotated under conditions of 15 seconds to spread the resist adhesive grease. A few drops of negative thick film resist NANO ™ XP SU-8 50 (manufactured by MicroChem) is dropped on the slide glass coated with the adhesive resin, and the spinner is rotated at a rotation speed of 2,500 rpm and a rotation time of 15 seconds. The thick film resist was spread by rotating one turntable. This was placed in a 100 ° C oven and soft baked for 30 minutes. The above operation was repeated 6 times to form a thick film resist layer having a height of 0.6 mm.

[0044] Except for the well area printed by a laser printer (EPSON LP-9500C, manufactured by Epson Corporation) on the prepared thick film resist-coated substrate, the exposure apparatus (KYOWARIKEN K_307PS, manufactured by Kyowa Riken) and exposed for 90 seconds Let The exposed glass substrate was placed in an oven at 100 ° C. and heated for 10 minutes for PEB treatment. After that, the glass substrate was immersed in a thick film resist developer SU-8 developer (manufactured by MicroChem) to remove the thick film resist resin in the unexposed area (black area of the photomask). By the treatment, a saddle shape having a cylindrical thick film resist pattern having a height of 0.6 mm was obtained.

 [0045] A PDMS microwell array sheet was prepared by the following procedure. Mix SYLGARD 184 base (manufactured by DOW CORNING) and SYLGARD 184 curing agent (manufactured by DOW CORNING) at a ratio of 10: 1 each of which is based on silicone resin (polydimethylsiloxane: PDMS), 15-20 Deaerated for a minute with an aspirator. A silicon rubber sheet (thickness 0.5 mm) and a saddle were stacked on a metal plate, and one drop of PDMS was dropped on it. One drop of PDMS was also dropped on the OHP sheet, and the PDMS on the saddle and the PDMS on the OHP sheet were stacked together so that no air bubbles could enter. A silicone rubber sheet, a slide glass (S-1111, manufactured by Matsunami Glass Co., Ltd.), a silicone rubber sheet, and a metal plate were superposed on the bonded saddle mold and the OHP sheet. Then, it fixed with the clamp, and put into 60 degreeC oven (Iuchi DRYING OVEN D0-300, product made by Inouchi), and heat-processed for 50 minutes. After returning to room temperature, the clamp was removed, and it was immersed in 99.5% ethanol (manufactured by Wako Pure Chemical Industries, Ltd.) and peeled off so as not to break the PDMS sheet formed on the bowl. The prepared PDMS microwell array sheet had a diameter of 1 mm and a depth of 0.6 mm. Further, a sheet of PDMS lattice (1.6 mm × 1.6 mm) covering the periphery of each well was prepared according to the same procedure as described above, overlaid with the PDMS microwell sheet, squeezed, and bonded to prepare a microwell sheet. The prepared PDMS microwell array sheet with a lattice was coated on the surface of a His-Tag fusion-feralanine dehydrin enzyme-immobilized slide glass to obtain a microarray array enzyme-immobilized chip for L-phenylalanine quantification.

 [0046] 6. Preparation of slide glass for His-Tag fusion enzyme protein immobilization

The preparation of a Ni ²⁺ complexed slide glass for His-Tag fusion phenylalanine dehydrogenase protein immobilization is shown below. Coated slide glass for DNA microarray TYPE2 high density amino group introduction type (26 mm x 76 mm: manufactured by Matsunami Glass Co., Ltd.) was used as the slide glass substrate. The slide glass was immersed in a 12.5% (v / v) dartalaldehyde (manufactured by Junsei Chemical Co., Ltd.) solution for 1 hour, and activated by reacting an amino group with an aldehyde. Activate The glass slide was immersed in a petri dish containing 2 mM N- (5-amino-1-carboxypentyl) iminodiacetic acid (AB-NTA; manufactured by Dojin Chemical Research Co., Ltd.) and stirred for 1 hour. After that, it was immersed in ultrapure water to remove unreacted AB-NTA. In order to protect the unreacted activated functional group, the slide glass was immersed in an aqueous solution of 50 mM L-Lysine (manufactured by Nippon Ryugaku Pharmaceutical Co., Ltd.) for 1 hour and washed with ultrapure water. Then, slide glass 1% (w / v) NiSO 6Η Η (

4 2 Wako Pure Chemical Industries, Ltd.) It was immersed in an aqueous solution for 1 hour to complex Ni ²⁺ metal. Thereafter, the slide glass was thoroughly washed with ultrapure water and used as a slide glass for His-Tag fusion enzyme protein fixation.

[0047] Immobilization of the His-Tag fusion phenylalanine dehydrogenase protein was performed according to the following procedure. Immerse the Ni ²⁺ metal complexed glass slide in a petri dish containing the enzyme solution diluted with binding buffer (20 mM Tris-HCl buffer, pH 8.0 containing 0.5 M NaCl and 2 mM 2-mercaptoethanol), The mixture was gently stirred at 4 ° C for 1 hour. Thereafter, in order to remove unreacted His-Tag fusion enzyme, the slide glass was washed with the same buffer. The prepared His-Tag fusion phenylalanine dehydrogenase protein-immobilized slide glass was stored at 4 ° C until just before use.

 [0048] 7. Quantification of L-feuraranin in filter paper blood

 As a standard substance, a standard filter paper blood impregnated with L-feralanin at a prescribed concentration (manufactured by Safhiro Imnodiagnostic Laboratories) was used. The standard filter paper blood force was extracted by the following procedure. Each spot on the filter paper blood was punched into a disk with a diameter of 3 mm and placed in each well of a 96-well microplate (manufactured by Corning). For each well, add 10 μL of fixed liquid (acetone: ethanol: ultra pure water = 7: 7: 2 (v / v)) and soak it in the filter paper, and maintain a 37 ° C thermostat (ADVANTEC INCUBAT OR Cto 410, manufactured by Advantech Co., Ltd.) for 1 hour. Then, add 100 L of buffer solution (50 M Tris-HCl buffer, pH 8.9) containing 40 M resazurin (manufactured by Wako Pure Chemical Industries, Ltd.) to each well and leave at room temperature for 1 hour. Extraction was performed.

[0049] For the measurement, each extract was covered with a PDMS microwell array sheet on the surface of a His-Tag-fused phenylalanine dehydrogenase-immobilized slide glass. NAD + aqueous solution 15 L and diaphorase solution (0.1 M phosphate The solution was adjusted to 0.3 mg / mL with aqueous buffer solution, pH 7.5, and 10 L of the mixed solution was dispensed by 0.2 μL each using Pipetteman P-2 (Gilson). The surface of the PDMS sheet was covered with a cover glass (Matsunami Glass Co., Ltd.) to prevent the reaction solution from evaporating. The slide Dallas was placed in a thermostat at 25 ° C and allowed to react for 1 hour, and then the developed fluorescence image was captured with a DNA microarray scanner (CRBIO II, manufactured by Hitachi Software Co., Ltd.) (fluorescence wavelength) 585 nm; excitation wavelength 532 nm). The obtained fluorescence image data, analysis software ^{(DNASI S (R) Array ver} . 2.6.0.4, Hitachi Software Co., Ltd.) was analyzed by.

[0050] [Result]

 1. Construction and expression of various His-Tag fusion phenylalanine dehydrogenase recombinants

 To fuse 3, 6, 9, and 12 His to the N-terminus (Fig. 1) or C-terminus (Fig. 2) of ferulalanin dehydrogenase from Bacillus badius IAM11059, Various plasmids were constructed by PCR using ligonucleotides. Table 1 shows the specific activities of the obtained cell-free extracts of each recombinant. When His was fused to the N-terminal side, a decrease in specific activity was observed in 6 and 9. On the other hand, when His was fused to the C-terminal side, the highest specific activity was obtained when six His were fused.

[0051] [Table 1]

i 伥 i £ ΌΟ sro 86 inch 0l N ..

SR ^ 960Ό £ 200 inch Γ05ΌU.

2. Purification of various His-Tag fusion ferulalanine dehydrogenase recombinants by Ni facility

The cell-free extract prepared from each recombinant was used as a Ni ²⁺ affinity chromatographer. Φ 瓛 * 一 ¾ 【CHis600545n,

Binding rate of His-Tag fusion enzyme to Ni ²⁺ affinity

His array 3x His 6x His 9x His 12x His

C-terminal His-Tag (Q /.) 6 2

N-terminal His-Tag (%)

* When the total enzyme activity detected in all fractions (non-adsorbed 'washing' elution) is 100%

 It was expressed as a relative value.

^ tLn ~ The above adsorption rate (binding ability) was obtained. With the C-terminal His-Tag fusion enzyme, an adsorption rate of 80% or more was observed with two Hisl. It became clear that the other His-Tag fusion enzymes hardly adsorbed (bound). Therefore, 6 and 9 for the N-terminal His-Tag fusion enzyme and 12 for the C-terminal His-Tag fusion enzyme are appropriate for the preparation of His-Tag fusion ferulalanine dehydrogenase immobilized microarray chips. It was thought that. In addition, the results showed that the binding ability was examined in the same way even when using Co ²⁺ in place of Ni ²⁺ . However, when Co ²⁺ was used, any His-Tag fusion enzyme showed a significant decrease in enzyme activity and was not used in subsequent experiments. Therefore, using BD Biocoat Ni-chelate assay plate manufactured by BD Bios ciences, we examined the enzyme activity after fixing the cell-free extract of each His-Tag fusion enzyme by changing the dilution ratio of the enzyme. The results are shown in Fig. 3. As a result, although the C-terminal His-Tag fusion enzyme (Fig. 3B) was found to have activity in 9 His, it had only a very low activity compared to the N-terminal His-Tag fusion enzyme result (Fig. 3A). However, there was a problem that there was a problem in maintaining the activity after fixing. Therefore, in the subsequent experiments, the immobilization of the N-terminal His-Tag fusion enzyme was performed.

[0055] 3. Examination of conditions using BD biocoat Ni ²⁺ chelate assembly plate

 N-terminal His-Tag fusion ferulalanine dehydrogenase His6 and 9 cell-free extracts were fixed on BD Biocoat Ni2 + chelate plate and contained in standard filter paper blood L- The results of the quantitative determination of phenylalanin are shown in FIG. As a result, the quantitativeness of both enzymes was confirmed up to a concentration of 12.8 mg / dl. We also examined the conditions for the amount of NAD and diaphorase added (Figure 5). As a result, the amounts of NAD and diaphorase added during the enzyme reaction were 25 mM and 0.2 mg / ml, respectively.

 [0056] 4. Quantification of L-Phe using a microarray chip with immobilized His-Tag fusion ferulanin dehydrogenase

N-terminal His-Tag fusion ferulalanine dehydrogenases His6 (Fig. 6A) and 9 (Fig. 6B) were immobilized on a microwell array chip and extracted from standard filter paper blood. Figure 6 shows the results of quantification of lulanin. As a result, when the reaction solution was 0.21, it was possible to quantify L-felananin in the filter paper blood to a concentration of 12.8 mg / dl in 1 hour of reaction. did it.

 Industrial applicability

、て利用可能な検査方法に利用可能である。 [0057] The immobilized enzyme chip of the present invention can be used for neonatal mass screening for early detection in patients with ferruketoneuria, which is an inborn error of metabolism, or for the determination of the patient.

It can be used for inspection methods that can be used.

 Brief Description of Drawings

 [0058] FIG. 1 is an explanatory diagram of a method for constructing a plasmid for fusing His to the N-terminal side of ferulalanin dehydrogenase.

 [Fig. 2] Explanatory diagram of how to construct a plasmid to fuse His to the C-terminal side of ferulalanin dehydrogenase.

 [Fig. 3] Results of examining enzyme activity after fixation of cell-free extract of His-Tag fusion enzyme at different enzyme dilutions.

 [Fig. 4] Quantitative results of L-fetalanin.

 [Fig. 5] Results of examination of conditions for the amount of NAD and diaphorase added.

 [Fig. 6] Quantitative results of L-fetalanin.

Claims

The scope of the claims  [1] Ferulalanin dehydrogenase fused with 3 to 12 oligopeptides with histidine strength at the N-terminus.  [2] Ferulalanin dehydrogenase fused with 3 to 12 oligopeptides with histidine power at the C-terminus.  [3] An immobilized enzyme chip having a plurality of tools on the surface of the substrate, wherein the phenylalanine dehydrogenase according to claim 1 or 2 is immobilized in these wells.  [4] The immobilized enzyme enzyme chip according to claim 3, which is used for analysis of L-ferallanin.  [5] L-ferrule having a plurality of wells on the surface of the substrate, and immobilizing phenylalanin dehydrogenase fused with 6-9 histidine oligopeptides at the N-terminus. Immobilized enzyme chip for analysis of lulanin. [6] The immobilized enzyme chip according to claim 5, wherein the phenylalanine dehydrogenase is immobilized in the well via Ni-chelate. [7] In the well of the immobilized enzyme chip according to any one of claims 4 to 6, the test sample is incubated together with a reaction solution containing resazurin, diaphorase, and nicotinamide adenine dinucleotide (NAD). A method for analyzing L-fetalanin contained in a test sample, which comprises detecting color development of the reaction solution. [8] The method according to claim 7, wherein the color development of the reaction solution is detected using a DNA microarray scanner.  [9] The method according to claim 7 or 8, wherein the L-phenylalanine contained in the test sample is quantified. [10] The method according to any one of [1] to [9] above, wherein the test sample is a blood sample and is used for diagnosis of phenylketonuria.