JP2892171B2 - Polypeptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a polypeptide having the amino acid sequence of aminoacylase I and its DNA sequence.

[0002]

BACKGROUND OF THE INVENTION Aminoacylase I [N-acylamino ac
id aminohydrolase, (EC 3.5.1.14.)]
Is an enzyme that hydrolyzes N-acyl amino acids into organic acids and amino acids. Widely found in animal tissues, molds and bacteria,
In mammals, it has a particularly high activity on the kidney. This enzyme has been known for more than a century and its enzymatic properties are well known. For example, the enzyme has high substrate specificity, and uses only L-amino acids as substrates. Therefore, it is used on a laboratory or industrial scale to optically resolve L-amino acids from chemically synthesized racemic N-acyl amino acids [John, New York City].
Wiley & Sons, 1961, Greenstein J. M. (Greenstein, JM) and Winitz, M., Amino Acid Chemistry (Che
mistry of the Amino Acids), Volume 2, 1753-
1816]. Structurally, the molecular weight is 46,000 ± 200.
0 Da (sodium lauryl sulfate-polyacrylamide electrophoresis), 98,000 ± 5000 Da (sedimentation equilibrium method), and are presumed to exist in the form of a dimer composed of two identical subunits [Heese D (Heese, D., et al.). ),
Lofrel H. G. FIG. (Loeffler, HG) and ROHM K. H. (Roehm, KH), Biological Chemistry Hop-Seyler.
), Vol. 369, pp. 559-566 (1988)].
By the way, in small cell lung cancer and renal cancer,
Chromosomal deletions have been found at high frequency in p14-p23 of the chromosome short arm. It is presumed that this enzyme is encoded in this portion, and it has been confirmed that the activity of this enzyme is actually lost in some types of small cell lung cancer cell lines [Miller Y. E. FIG. (Miller, YE),
Khao B. (Kao, B.) and Gazda A. (Gazder,
A. ), American Journal of Human
Genetics (Amer. J. Hum. Genet.), Vol. 41,
A32 (1987)]. Thus, aminoacylase I is an extremely effective means for optical resolution of racemized amino acids. Furthermore, D encoding human aminoacylase I
The NA sequence is considered to be useful as a marker for small cell lung cancer or renal cancer in genetic diagnosis or treatment of the cancer.

[0003]

SUMMARY OF THE INVENTION Aminoacylase I
Is widely distributed in animal tissues and purified especially for porcine kidney enzymes [Henseling J. et al. (Henseling, J.) and ROHM K. H. , Biosimica and Biophysica
Acta (Biochim. Biophys. Acta), Vol. 959, pp. 370-377 (1988)] and commercially available.
However, its gene structure and amino acid sequence are still unknown. Also, no industrially advantageous method for producing aminoacylase I is disclosed. An object of the present invention is to clarify the gene structure and amino acid sequence of aminoacylase I, and to provide a nucleotide sequence necessary for genetic diagnosis or treatment of small cell lung cancer or kidney cancer and a polypeptide having aminoacylase I activity. It is in.

[0004]

SUMMARY OF THE INVENTION In summary of the present invention, a first invention of the present invention relates to a polypeptide represented by the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, The present invention relates to a nucleic acid encoding a polypeptide having an amino acid sequence having at least one of deletion, substitution, insertion, and addition of one or more amino acid residues in the sequence, the polypeptide having aminoacylase I activity. Further, the second invention of the present invention relates to a polypeptide represented by the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, or deletion, substitution, and insertion of one to several amino acid residues in the sequence. A polypeptide having an amino acid sequence having at least one addition, and having aminoacylase I activity.

Hereinafter, the present invention will be described specifically. Known methods are used for cloning the cDNA encoding aminoacylase I. For example, from a kidney where aminoacylase I is particularly prevalent, RN containing poly (A)
A is extracted and purified with a cellulose carrier or the like to which oligo (dT) is bound. Using this as a template, cDNA is synthesized by the action of reverse transcriptase, and the Okayama-Berg method or Gubler-Hofman method (Gubler-Hofman method) is used.
fmann method) and the like, connecting to a plasmid or phage vector, introducing into a host, and preparing a cDNA library. Such libraries are also commercially available and can be purchased, for example, from Clonetech. In order to screen a cDNA clone encoding the desired aminoacylase I from a cDNA library, first, the partial amino acid sequence of aminoacylase I must be determined, and a deduced synthetic DNA probe must be prepared therefrom. In order to determine the partial amino acid sequence, first, purified aminoacylase I is hydrolyzed by acting a highly specific protein hydrolase, and the peptide is separated and purified using reverse phase HPLC. It is effective to determine this by performing amino acid sequence analysis by the Edman degradation method. There are two methods for designing a synthetic DNA probe from this partial amino acid sequence. One is to synthesize all possible combinations of sequences. The other is a method of synthesizing and using a long DNA using a codon frequently used, which has been investigated so far. In addition, when the nucleotide sequence of a cDNA encoding a certain aminoacylase I has been determined, in order to screen a cDNA clone encoding aminoacylase I of another source, the sequence has already been revealed. It is effective to use a cDNA fragment of aminoacylase I as a probe. The DNA probe may be chemically synthesized, or may be cut out of a vector containing cDNA with a restriction enzyme and purified. As a means for screening the library with a DNA probe, first, the library is amplified on a plate, the grown colonies or plaques are transferred to a nitrocellulose or nylon filter, and the DNA is fixed to the filter by denaturation treatment. This filter is incubated in a solution containing a DNA probe previously labeled with ³² P or the like to form a hybrid between the DNA on the filter and the probe DNA (hereinafter, this operation is referred to as hybridization). The incubation temperature is set using the Tm (melting temperature) of the probe to be used as a guide. After hybridization, non-specific adsorption is washed away, and clones that have hybridized with the probe are identified by autoradiography. This operation is performed again to isolate the clone, and the next analysis is performed. When the recombinant is Escherichia coli, a small amount of culture is performed in a test tube or the like, the plasmid is extracted by a conventional method, a cleavage reaction is performed with a restriction enzyme, and subjected to agarose or acrylamide gel electrophoresis. Examine the generation of. Further, the electrophoresis pattern is transferred to a nitrocellulose or nylon membrane, and subjected to hybridization according to the above-described method to examine whether or not the inserted fragment forms a hybrid with the DNA probe. Finally, the base sequence of the inserted fragment is determined by a known method. When the recombinant is a phage, the clone is analyzed in basically the same steps. The cloned phage is infected to host Escherichia coli cultured in advance, and phage DNA is prepared from the lysate. Phage DNA
The specific preparation method is described, for example, in page 100 of Seismic Chemistry Laboratory Course 1 “Genetic Research Method II”, page 100 (published by Tokyo Chemical Dojin). The phage DNA is cleaved with a restriction enzyme and subjected to gel electrophoresis to confirm the inserted fragment, and further examined to hybridize with the probe DNA. Finally, the clone is confirmed by determining the nucleotide sequence. The determined base sequence is used for amino acid sequence analysis of aminoacylase I peptide fragment, C-terminal analysis,
The gene structure and amino acid sequence can be known by comparing with amino acid composition analysis, molecular weight and the like. In addition, a polypeptide having aminoacylase activity can be obtained by connecting the structural gene of the obtained cDNA to an appropriate host cell, for example, an expression vector so that it can be expressed in yeast, introducing into the host cell, and culturing it. Can be produced. Confirmation of expression can be performed by measuring the activity by a conventional aminoacylase I activity measuring method. For example, a cell extract of a recombinant yeast is added to a system for measuring the activity of aminoacylase I containing 2-acetamidoacrylic acid, L-alanine dehydrogenase, and β-nicotinamide adenine dinucleotide disodium salt (NADH). At this time, after 2-acetamidoacrylic acid is hydrolyzed by aminoacylase I, it is deaminated to produce pyruvic acid and ammonia. Aminoacylase I activity can be measured by measuring the decrease in NADH consumed when L-alanine dehydrogenase synthesizes alanine with a spectrophotometer. For purification of the aminoacylase I polypeptide from the culture of the transformant, a usual chromatography technique is used. That is, for example, the cultured cells are disrupted to obtain a supernatant. This is fractionated by salting out using ammonium sulfate, and then the desired polypeptide can be obtained by chromatography such as hydrophobicity, ion exchange, and gel filtration. From the above, the nucleotide sequence and primary structure of aminoacylase I have been elucidated by the present invention, and a nucleotide sequence for gene diagnosis of small cell lung cancer or renal cancer, and a method for producing aminoacylase I by genetic engineering are provided. It became possible.

[0006]

EXAMPLES Next, examples of the present invention will be described, but these do not limit the present invention.

Example 1 Porcine aminoacylase IcDN
A cloning (1-1) Screening from cDNA library <Identification and isolation of positive clone> Porcine kidney poly (A) RNA was obtained from Clonetech (USA) (code No. 6683). This poly (A) RNA 2μ
g, cDNA was synthesized with an oligo (dT) primer using a cDNA synthesis kit (code number RPN.1256) manufactured by Amersham. Next, using a cDNA cloning system λgt10 (code No. PRN.1257) also manufactured by Amersham, in a cell-free system, cDN
The product obtained by incorporating A into the EcoRI site of λgt10 was packaged in lambda phage to prepare a cDNA library. However, the EcoRI linker represented by SEQ ID NO: 3 in the Sequence Listing was manufactured by Takara Shuzo Co., Ltd., and Gigapack Gold (Stratagene, USA) was used for packaging. Using the C600hfl strain as a host bacterium,
Approximately 1600 per 8 pieces of 4 cm x 10 cm square petri dishes
0 plaques were formed. That is, 0.1 ml of a phage solution was mixed with 0.2 ml of a culture obtained by culturing C600hfl at 37 ° C. overnight in L medium containing 4 mg / ml maltose.
It was kept at 7 ° C. for 15 minutes. To this was added 8 ml of soft agar (agarose was added to L medium to a final concentration of 0.6%, treated in an autoclave, and kept at 50 ° C.)
After spreading on an L-plate and solidifying, the mixture was kept at 37 ° C. for about 10 hours to form phage plaques (hereinafter, this operation is abbreviated as plating). Next, 2 from this plate
One piece of hybridization filter was prepared.
That is, a nylon film made by Amersham (trade name: Highbond (Hybond-N)) was brought into contact with the plate surface for 30 seconds,
This was placed on a filter paper dipped in a solution of 0.5 M NaOH and 1.5 M NaCl for 5 minutes (denatured),
(Tris) -HCl buffer (pH 7.0), 1.5 M Na
After 5 minutes (neutralization) treatment on a filter paper immersed in a Cl solution, 2 × SSC [17.53 g of NaCl and 8.82 g of sodium citrate dissolved in 1 liter of water]
And dried on filter paper (this process is abbreviated as filter treatment). The second filter treatment was performed with the contact time between the plate and the nylon membrane being 2 minutes. U
This filter was irradiated with a V lamp for 5 minutes to immobilize DNA. As a hybridization probe, a mixture of 17 bases of synthetic DNA (represented by SEQ ID NO: 4 in the sequence listing) estimated from the partial amino acid sequence of porcine kidney aminoacylase I (Sigma) was used. The partial amino acid sequence consists of purified porcine kidney aminoacylase I
Was digested with HPLC after digestion of the protease Achromobacter protease I, and determined by a gas phase peptide sequencer. SEQ ID NO: 5 in the sequence listing is the amino acid sequence. 50 ng of this synthetic DNA was labeled with ³² P using a Takara Shuzo Mega Label Kit (code No. 6070), and probe D having a specific activity of 8.6 × 10 ⁸ cpm / μg was used.
NA was obtained. Using the total amount of this probe and the filter prepared above, 6 × SSC, 1% SDS 100 μg / ml herring sperm DNA, 5 × Denhardt's [bovine serum albumin, polyvinylpyrrolidone, and ficoll 0.1% each. ) At 45 ° C overnight. The filters were then washed for 10 minutes in 6 × SSC at room temperature. Further, after washing twice in 2 × SSC at 40 ° C. for 10 minutes, the filter was transferred onto a filter paper to remove excess water. This was adhered to Whatman 3MM filter paper, and an intensifying screen was applied thereto to perform autoradiography at -70 ° C overnight.
As a result, a total of 51 positive signals were obtained. Plaques at positions corresponding to these signals were placed on agar plates with 0.
2ml of SM solution [NaCl 5.8g, MgSO ₄ · 7
H ₂ O 2g, 1M Tris -HCl buffer (pH 7.
5) 50 ml of 2% gelatin was dissolved in water to make the total volume 1 liter]. Ten of these phage solutions were appropriately selected and subjected to the following operation. These phage solutions were diluted and plated (about 300 plaques / φ9 cm round petri dish) and subjected to the same operation as described above (hereinafter abbreviated as secondary screening). As a result, a single plaque could be isolated from all clones. These clones were named λpkAmA 1-10. (1-2) Nucleotide Sequence Analysis <Preparation of λDNA> Liquid culture using E. coli L87 strain as a host phage which could be cloned
II, page 100, Tokyo Chemical Doujinshi). Thus, about 40 μg of λDN was prepared from 40 ml of the culture solution.
A was obtained. <Identification of Insertion Fragment and Extraction and Purification>
μg was incubated at 37 ° C. for 1.5 hours with 120 units of EcoRI in 100 μl of 1 × EcoRI buffer (composition described in the catalog of reagents for Takara Shuzo / Genetic Engineering), and the final concentration was 5%.
RNase A was added to a concentration of 0 μg / ml, and the mixture was kept warm for 10 minutes. 5 μl was taken out of the reaction solution and electrophoresed on a 1.0% agarose gel to determine the size of the inserted fragment. As a result, it was found that a fragment of 0.6 to 1.4 kb was inserted. Among these, λpkAmA10 having the longest insert fragment of 1.4 kb was selected and subjected to the following operation. The total amount of the remaining reaction solution previously digested with EcoRI was 1.0%
Electrophoresis was performed on an agarose gel. 1 μg /
After staining with 10 ml of ethidium bromide solution for 10 minutes,
The portion containing the insert fragment of interest was cut out from the gel under ultraviolet irradiation. This was extracted and purified using an EASYTRAP kit manufactured by Takara Shuzo Co., Ltd. to obtain about 200 ng of a 1.4 kb DNA fragment. <Identification of restriction enzyme site of inserted fragment and determination of base sequence> λ
50 ng of pkAmA10 insert and M13mp18 (Takara Shuzo)
Was ligated using a ligation kit (Takara Shuzo Code No. 6021). Escherichia coli JM109 was transformed using a part of this ligation reaction solution, and X-Gal
(5-Bromo-4-chloro-3-indolyl-β-D-
Galactoside) and IPTG (isopropyl-β-D-thiogalactoside) were mixed with 0.2 ml of a culture solution obtained by culturing JM109 in L medium overnight, and the mixture was spread on an L-plate. The plate was incubated overnight at 37 ° C., and a recombinant in which the fragment was inserted was obtained by selecting a formed white plaque. The obtained white plaque was used for E. coli JM1.
09 was used as a host cell for liquid culture, and RF DNA was prepared by an alkaline lysis method [Reference: 1982, published by Cold Spring Harbor Laboratory, T.C. T. Maniastis et al., Molecular Cloning, A Laboratory Manual (Molecula)
rCloning, A Laboratory Manual), No. 368
page〕. A part of this RF DNA was digested with EcoRI and 1%
It was confirmed by agarose gel electrophoresis that the 1.4 kb fragment was inserted. Next, the orientation of the inserted fragment and the presence of a restriction enzyme site were examined. A part of the obtained RF DNA was digested with several kinds of restriction enzymes and analyzed by 1% agarose gel electrophoresis. As a result, the restriction enzymes KpnI, S
It was found that one or two sites such as acI, StuI, PstI, HindIII, and HincII were present. Also, M1
In the opposite direction to the 3mp18 vector DNA,
It was also found that a clone in which the 1.4 kb fragment was inserted was obtained. MpkAmA 10-
2. The other was named MpkAmA 10-5. Next, to determine the entire base sequence of the 1.4 kb insert,
MpkAmA 10-2, MpkAmA 10-
From No. 5, derivatives in which DNAs of various lengths were deleted were prepared. First, MpkAmA 10-2 is converted to KpnI, StuI and Sma
I, PstI, HindIII, MpkAmA 10-5 to HindII
The DNA was digested with I, PstI, SacI, and KpnI, cut at restriction enzyme sites in the multiple cloning site and sites in the inserted fragment to delete DNAs of various sizes. Next, these vector DNAs were self-ligated and then introduced into E. coli JM109 to obtain a transformant. These derivative clones, MpkAmA 10-2, Mpk
AmA 10-5 was cultured and single-stranded DNA (s
sDNA) was prepared and subjected to DNA sequencing by the dideoxy method. As a result, the base sequence of the inserted fragment of λpkAmA10 was determined. The result is shown in FIG. That is, FIG. 1 is a diagram showing a restriction enzyme map of porcine aminoacylase I cDNA, a portion whose base sequence has been determined, and its orientation. From the results of the cDNA base sequence analysis, the entire base sequence and amino acid sequence of the porcine aminoacylase I constituent gene of SEQ ID NO: 6 in the sequence listing were determined. That is, SEQ ID NO: 6 shows the nucleotide sequence of one example of aminoacylase I and the corresponding amino acid sequence obtained by cDNA nucleotide sequence analysis.

Example 2 Human aminoacylase IcDN
A cloning (2-1) Screening from cDNA library Human liver poly (A) RNA was obtained from Clonetech. From 2 μg of this poly (A) RNA, a human liver-λgt10-cDNA library was prepared in the same manner as in Example 1, and a filter was prepared. As a probe DNA for hybridization, an NcoI-StuI fragment (about 530 bp) in the λpkAmA10 insert fragment was used. That is, MpkAm containing porcine aminoacylase I cDNA
A10-2 DNA was digested with NcoI and StuI and subjected to agarose gel electrophoresis. After the electrophoresis, the gel was stained with a 1 μg / ml ethidium bromide solution for 10 minutes, and a portion containing the target DNA fragment was cut out from the gel under ultraviolet irradiation. This was extracted and purified using an easy trap kit. The resulting fragment, using a random primer DNA labeling kit (Takara Shuzo code No. 6045) ³²
It was labeled with P and used for hybridization. Hybridization was performed under the same conditions as in Example 1.
However, the hybridization temperature is 65 ° C.
The washing was finally performed twice in 0.2 × SSC at 55 ° C. for 30 minutes. As a result, three positive signals were obtained. These plaques were subjected to a secondary screening and isolated. This clone was named λhAmA1-3. (2-2) Nucleotide sequence analysis [lambda] DNA was prepared from the above three clones, digested with EcoRI, and the size of the inserted DNA fragment was examined. As a result 1.
It was found that a DNA fragment of 1 to 1.4 kb was inserted. The insert was excised with EcoRI from λhAmA3 having the longest insert of 1.4 kb and incorporated into M13mp18. The resulting clones in both directions were isolated from MhAmA3-
2. It was named MhAmA3-3. Next, in order to determine the entire base sequence of the 1.4 kb insert, M
DNA of various sizes from hAmA3-2 and MhAmA3-3
Derivatives were deleted. First, each RFDNA is converted to H
The DNA was digested with incII and PstI, cut at the restriction enzyme site in the multiple cloning site and the site in the inserted fragment, and DNAs of various sizes were deleted. Next, these vector DNAs were self-ligated and then introduced into Escherichia coli JM109 to obtain transformants. Also Takara
(TaKaRa) Using the Kilo Sequencing Delation Kit (Takara Shuzo), for MhAmA3-2 and MhAmA3-3, insert fragments were degraded from the annealing position side of the sequencing primer, and DNAs of various sizes were deleted. A derivative was prepared. Suitable ones of these derivatives,
MhAmA3-2 and MhAmA3-3 were cultured to prepare ssDNA, and DNA sequencing was performed by the dideoxy method. As a result, the base sequence of the inserted fragment of λhAmA3 was determined. The result is shown in FIG. That is, FIG. 2 is a diagram showing a restriction enzyme map of human aminoacylase I cDNA, a portion where a base sequence is determined, and a direction thereof. This cDNA
From the results of the nucleotide sequence analysis, the entire nucleotide sequence and amino acid sequence of the human aminoacylase I component gene were determined. The result is shown in SEQ ID NO: 7 in the sequence listing. That is, SEQ ID NO: 7 is aminoacylase I obtained by cDNA base sequence analysis.
1 shows the base sequence of Example 1 and the corresponding amino acid sequence.

Example 3 Construction of Plasmid Expressing Aminoacylase I Polypeptide A portion encoding aminoacylase I in the λpkAmA10 or λhAmA3 insert was cut out by the following procedure.
Expression plasmids (pYAmA101 and pYhAmA101, respectively) integrated into pJM124 which is an E. coli-yeast shuttle vector having a yeast alcohol dehydrogenase promoter (Reference: The EMBO Journal, Vol. 8, p. 2067 (1989)) Named). (3-1) Construction of porcine aminoacylase I-like polypeptide expression plasmid pYAmA101 MpkAmA 10-2 RFDNA having EcoRI insert of λpkAmA10 incorporated into M13mp18 was converted to restriction enzyme NcoI.
Approximately 1230 bp DNA digested and released with Eco47III
The fragments were separated and purified. And DNA branding
Kit (Blunting Kit) (Takara Shuzo code number 602)
Using 5), the NcoI overhang was blunted. this,
Plasmid pJM124 was digested with BamHI to remove the BamHI insert, which was then blunt-ended and integrated. The resulting plasmid was named pYAmA101. That is, the plasmid pYAmA101 encodes a polypeptide of 407 amino acid residues of porcine aminoacylase I downstream of the yeast alcohol dehydrogenase promoter. This plasmid pYAmA101 was transformed into yeast BJ2168 [MAT
a, ura3-52, leu2, trp1, prc-4
07, prb1-1122, pep4-3] or DKD-5DH (MATa, leu2-3, leu2
-112, trp1, his3] with an alkali metal treatment method [Literature: Journal of Bacteriology (Journ
al of Bacteriology) Volume 153, Page 163 (198
3)]. The yeast BJ2168 into which pYAmA101 was introduced was transformed into Saccharomyces cerevisiae BJ216.
8 / pYAmA101 and deposited with the Institute of Microbial Industry and Technology, National Institute of Advanced Industrial Science and Technology.
MP-12002)]. (3-2) Construction of Human Aminoacylase I-like Polypeptide Expression Plasmid pYhAmA101 MhAmA3-3 RFDNA having EcoRI inserted fragment of λhAmA3 incorporated into M13mp18 was subjected to restriction enzymes SacI and EcoR.
The DNA fragment of about 1360 bp released after digestion with I was separated and purified. Then, the protruding end was blunted using a DNA branding kit (Takara Shuzo Code No. 6025). This was incorporated into a plasmid pJM124 which had been digested with BamHI to remove the BamHI insert and blunt-ended. The resulting plasmid was named pYhAmA101. That is, plasmid pYhAmA101 encodes a polypeptide of 408 amino acid residues of human aminoacylase I downstream of the yeast alcohol dehydrogenase promoter. This plasmid pYhAmA101 was transformed into yeast BJ
2168 [MATa, ura3-5, leu2, trp
1, prc-407, prb1-1122, pep4-
3] or DKD-5DH (MATa, leu2
-3, leu2-112, trp1, his3] by an alkali metal treatment method [Literature: Journal of Bacteriology, vol. 153, p. 163 (1983)]. Yeast BJ2168 into which pYhAmA101 was introduced
To Saccharomycescerevisiae BJ2168 / pYhAmA1
01 and deposited with the Institute of Microbial Industry and Technology, National Institute of Advanced Industrial Science and Technology [Microtechnical Laboratory No. 12003 (FERM P-12)
003)].

Example 4 Expression of aminoacylase I polypeptide in yeast Saccharomyces cerevisiae BJ2 obtained in Example 3
168 / pYAmA101 (FERM P-12002) or Saccharomyces cerevisiae BJ2168 / pYhA
20 μg / ml of mA101 (FERM P-12003)
L-tryptophan, 30 μg / ml L-leucine,
5 ml of SD medium containing 0.6 μg / ml L-histidine and 20 μg / ml uracil [0.67% bactoeast nitrogen-based w / o amino acid (Difco <Difco>), 2% dextrose) was inoculated into 30 ml. The cells were cultured at C for 2 nights and collected. The supernatant was discarded, and the cells were washed with 2 ml of water, centrifuged, and then suspended in 400 μl of 0.2 M sodium phosphate buffer (pH 7.2). Equal volumes of glass beads (0.35 to 0.5 mm in diameter) were added thereto, and the cells were disrupted by vigorous stirring under ice cooling. After centrifugation to remove the glass beads and cell debris, the supernatant was recovered and used as a yeast extract. The aminoacylase I activity of the extract thus obtained was measured by the acetamidoacrylic acid method [Reference:
Analytical Biochemistry
ochemistry), 165, 142 (198
7)]. This will be described in detail below. First, the reaction buffer (40
mM phosphoric acid, 40 mM acetic acid, 40 mM boric acid, 0.08 mM cobalt chloride, 20 mM ammonium sulfate, and the pH was adjusted to 7.6 with sodium hydroxide). Next, a substrate solution (1 mmol of 2-acetamidoacrylic acid was added to 1.2 ml of 1
Dissolved in an aqueous solution of N sodium hydroxide and added with 0.8 ml of a reaction buffer), an NADH solution (a solution of NADH dissolved to 3.2 mM in the reaction buffer), 30
A U / ml L-alanine dehydrogenase solution was prepared. Reaction buffer 700 μl, substrate solution 100 μl, N
After mixing 50 μl of the ADH solution and 50 μl of the L-alanine dehydrogenase solution and preheating at 37 ° C. for about 5 minutes,
The extract was added in an amount of 00 μl, and kept at 37 ° C. for 30 minutes. At this time, aminoacylase I activity was measured by measuring the decrease in absorption at a wavelength of 340 nm by NADH using a spectrophotometer. The quantification of the enzyme activity was performed in comparison with the measurement of porcine kidney aminoacylase I (Sigma) having a known enzyme titer in the same manner. As a result of the aminoacylase I activity measurement, 100 μl of each extract prepared above was used.
Had about 1 unit of aminoacylase I activity. In addition, yeast BJ2168 which does not carry a plasmid is used.
Was treated in the same manner, but almost no aminoacylase activity was observed (0.03 units / 100 μl extract or less).

[0011]

From the above results, the present invention has revealed the amino acid sequence of porcine and human aminoacylase I and the DNA sequence thereof, and provided a method for producing aminoacylase I polypeptide by genetic engineering. In addition, by providing a target for examining the presence or expression of aminoacylase I in vivo, it is possible to prepare probes and primers based on this DNA sequence, and to prepare antibodies based on the amino acid sequence. Became possible.

[Sequence list] SEQ ID NO: 1 Sequence length: 407 Sequence type: Number of amino acid chains: Single chain Topology: Linear Sequence type: Peptide Sequence features: 1-407 Porcine aminoacylase I Sequence: Met Ala Ser Lys Gly Arg Glu Gly Glu His Pro Ser Val Thr Leu 1 5 10 15 Phe Arg Gln Tyr Leu Arg Ile Arg Thr Val Gln Pro Glu Pro Asp 20 25 30 Tyr Gly Ala Ala Val Ala Phe Leu Glu Glu Arg Ala Arg Gln Leu 35 40 45 Gly Leu Gly Cys Gln Lys Val Glu Val Val Pro Gly His Val Val 50 55 60 Thr Val Leu Thr Trp Pro Gly Thr Asn Pro Thr Leu Ser Ser Ile 65 70 75 Leu Leu Asn Ser His Thr Asp Val Val Pro Val Phe Lys Glu His 80 85 90 Trp Ser His Asp Pro Phe Glu Gly Phe Lys Asp Ala Asp Gly Tyr 95 100 105 Ile Tyr Gly Arg Gly Ala Gln Asp Met Lys Cys Val Ser Ile Gln 110 115 120 Tyr Leu Glu Ala Val Arg Arg Leu Lys Val Glu Gly His His Phe 125 130 135 Pro Arg Thr Ile His Met Thr Phe Val Pro Asp Glu Glu Val Gly 140 145 150 Gly His Gln Gly Met Glu Leu Phe Val Lys Arg Pro Glu Phe Gln 155 160 165 Ala Leu Arg Ala Gly Phe Ala Leu Asp Glu Gly Leu Ala Ser Pro 170 175 180 Thr Asp Ala Phe Thr Val Phe Tyr Ser Glu Arg Ser Pro Trp Trp 185 190 195 Leu Arg Val Thr Ser Thr Gly Lys Pro Gly His Gly Ser Arg Phe 200 205 210 Ile Glu Asp Thr Ala Ala Glu Lys Leu His Lys Val Ile Asn Ser 215 220 225 Ile Leu Ala Phe Arg Glu Lys Glu Lys Gln Arg Leu Gln Ser Asn 230 235 240 Gln Leu Lys Pro Gly Ala Val Thr Ser Val Asn Leu Thr Met Leu 245 250 255 Glu Gly Gly Val Ala Tyr Asn Val Val Pro Ala Thr Met Ser Ala 260 265 270 Cys Phe Asp Phe Arg Val Ala Pro Asp Val Asp Leu Lys Ala Phe 275 280 285 Glu Glu Gln Leu Gln Ser Trp Cys Gln Ala Ala Gly Glu Gly Val 290 295 300 Thr Phe Glu Phe Val Gln Lys Trp Met Glu Thr Gln Val Thr Ser 305 310 315 Thr Asp Asp Ser Asp Pro Trp Trp Ala Ala Phe Ser Gly Val Phe 320 325 330 Lys Asp Met Lys Leu Ala Leu Glu Leu Glu Ile Cys Pro Ala Ser 335 340 345 Thr Asp Ala Arg Tyr Ile Arg Ala Ala Gly Val Pro Ala Leu Gly 350 355 360 Phe Ser Pro Met Asn His Thr Pro Val Leu Leu His Asp His Asp 365 370 375 Glu Arg Leu His Glu Ala Val Phe Leu Arg Gly Val Asp Ile Tyr 380 385 390 Thr Gln Leu Leu Ser Ala Leu Ala Ser Val Pro Ala Leu Pro Ser 395 400 405 Glu Ser SEQ ID NO: 2 Sequence length: 2 408 Sequence type: amino acid Number of chains: 1 chain Topology: linear Sequence type: peptide Sequence characteristics: 1-408 Human aminoacylase I Sequence: Met Thr Ser Lys Gly Pro Glu Glu Glu His Pro Ser Val Thr Leu 1 5 10 15 Phe Arg Gln Tyr Leu Arg Ile Arg Thr Val Gln Pro Lys Pro Asp 20 25 30 Tyr Gly Ala Ala Val Ala Phe Phe Glu Glu Thr Ala Arg Gln Leu 35 40 45 Gly Leu Gly Cys Gln Lys Val Glu Val Ala Pro Gly Tyr Val Val 50 55 60 Thr Val Leu Thr Trp Pro Gly Thr Asn Pro Thr Leu Ser Ser Ile 65 70 75 Leu Leu Asn Ser His Thr Asp Val Val Pro Val Phe Lys Glu His 80 85 90 Trp Ser His Asp Pro Phe Glu Ala Phe Lys Asp Ser Glu Gly Tyr 95 100 105 Ile Tyr Ala Arg Gly Ala Gln Asp Met Lys Cys Val Ser Ile Gln 110 115 120 Tyr Leu Glu Ala Val Arg Arg Leu Lys Val Glu Gly His Arg Phe 1 25 130 135 Pro Arg Thr Ile His Met Thr Phe Val Pro Asp Glu Glu Val Gly 140 145 150 Gly His Gln Gly Met Glu Leu Phe Val Gln Arg Pro Glu Phe His 155 160 165 Ala Leu Arg Ala Gly Phe Ala Leu Asp Glu Gly Ile Ala Asn Pro 170 175 180 Thr Asp Ala Phe Thr Val Phe Tyr Ser Glu Arg Ser Pro Trp Trp 185 190 195 Val Arg Val Thr Ser Thr Gly Arg Pro Gly His Ala Ser Arg Phe 200 205 210 Met Glu Asp Thr Ala Ala Glu Lys Leu His Lys Val Val Asn Ser 215 220 225 Ile Leu Ala Phe Arg Glu Lys Glu Trp Gln Arg Leu Gln Ser Asn 230 235 240 Pro His Leu Lys Glu Gly Ser Val Thr Ser Val Asn Leu Thr Lys 245 250 255 Leu Glu Gly Gly Val Ala Tyr Asn Val Ile Pro Ala Thr Met Ser 260 265 270 Ala Ser Phe Asp Phe Arg Val Ala Pro Asp Val Asp Phe Lys Ala 275 280 285 Phe Glu Glu Glu Gln Leu Gln Ser Trp Cys Gln Ala Ala Gly Glu Gly 290 295 300 Val Thr Leu Glu Phe Ala Gln Lys Trp Met His Pro Gln Val Thr 305 310 315 Pro Thr Asp Asp Ser Asn Pro Trp Trp Ala Ala Phe Ser Arg Val 320 325 330 Cys Lys Asp Met Asn Leu Thr Leu Glu Pro Glu Ile Met P ro Ala 335 340 345 Ala Thr Asp Asn Arg Tyr Ile Arg Ala Val Gly Val Pro Ala Leu 350 355 360 Gly Phe Ser Pro Met Asn Arg Thr Pro Val Leu Leu His Asp His 365 370 375 Asp Glu Arg Leu His Glu Ala Val Phe Leu Arg Gly Val Asp Ile 380 385 390 Tyr Thr Arg Leu Leu Pro Ala Leu Ala Ser Val Pro Ala Leu Pro 395 400 405 Ser Asp Ser SEQ ID NO: 3 Sequence length: 10 Sequence type: Nucleic acid Number of strands: 2 Main strand Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Sequence characteristics: 1-10 linker Sequence: CCGAATTCGG 10 SEQ ID NO: 4 Sequence length: 17 Sequence type: Nucleic acid Number of strands: 1 Main strand Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical sequence: NO Antisense: NO Sequence characteristics: 1-17 probe sequence: TGGATGGARA CNCARGT 17 SEQ ID NO: 5 Sequence length: 6 Sequence type: amino acid Number of chains: 1 chain Topology: linear Sequence type: peptide Fragment type: middle fragment Fragment (porcine aminoacylase I) SEQ ID NO: 6 Sequence length: 1221 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA hypothetical sequence: NO Antisense: NO Sequence characteristics: 1- 1221 CDS (porcine aminoacylase I) sequence: ATG GCC AGC AAG GGT CGC GAG GGC GAG CAC CCA TCC GTG ACG CTC 45 Met Ala Ser Lys Gly Arg Glu Gly Glu His Pro Ser Val Thr Leu 15 10 15 TTC CGT CAG TAC CTG CGC ATC CGC ACC GTC CAG CCT GAG CCC GAC 90 Phe Arg Gln Tyr Leu Arg Ile Arg Thr Val Gln Pro Glu Pro Asp 20 25 30 TAC GGG GCT GCT GTG GCC TTC CTT GAG GAG AGA GCC CGC CAG CTG 135 Tyr Gly Ala Ala Val Ala Phe Leu Glu Glu Arg Ala Arg Gln Leu 35 40 45 GGC CTG GGC TGT CAG AAA GTG GAG GTG GTA CCT GGC CAT GTG GTG 180 Gly Leu Gly Cys Gln Lys Val Glu Val Val Gly His Val Val 50 55 60 ACC GTG CTG ACC TGG CCG GGC ACC AAC CCC ACA CTC TCC TCC ATC 225 Thr Val Leu Thr Trp Pro Gly Thr Asn Pro Thr Leu Ser Ser Ile 65 70 75 TTG CTC AAC TCC CAC ACA GAT GTG GTG CCT GTC TTC AAG GAG CAT 270 Leu Leu Asn Ser His Thr Asp Val Val Pro Val Phe Lys Glu His 80 85 90 TGG AGT CAT GAC CCC TTT GAG GGC TTC AAG GAT GCA GAT GGC TAC 315 Trp Ser His Asp Pro Phe Glu Gly Phe Lys Asp Ala Asp Gly Tyr 95 100 105 ATC TAT GGC AGG GGC GCC CAG GAC ATG AAG TGC GTC AGC ATC CAG 360 Ile Tyr Gly Arg Gly Ala Gln Asp Met Lys Cys Val Ser Ile Gln 110 115 120 TAC CTG GAG GCT GTG AGG AGG CTG AAG GTT GAG GGC CAC CAT TTC 405 Tyr Leu Glu Ala Val Arg Arg Leu Lys Val Glu Gly His His Phe 125 130 135 CCC AGA ACC ATC CAC ATG ACC TTT GTG CCA GAT GAG GAG GTT GGA 450 Pro Arg Thr Ile His Met Thr Phe Val Pro Asp Glu Glu Val Gly 140 145 150 GGT CAC CAA GGC ATG GAG CTC TTT GTG AAG CGG CCC GAG TTC CAG 495 Gly His Gln Gly Met Glu Leu Phe Val Lys Arg Pro Glu Phe Gln 155 160 165 GCC CTG AGG GCT GGC TTT GCC CTG GAT GAA GGC CTA GCC AGC CCC 540 Ala Leu Arg Ala Gly Phe Ala Leu Asp Glu Gly Leu Ala Ser Pro 170 175 180 ACT GAC GCC TTC ACT GTC TTT TAC AGT GAG CGG AGC CCC TGG TGG 585 Thr Asp Ala Phe Thr Val Phe Tyr Ser Glu Arg Ser Pro Trp Trp 185 190 195 CTA AGG GTC ACG AGC ACT GGG AAG CCA GGG CAT GGC TCG CGC TTC 630 Leu Arg Val Thr Ser Thr Gly Lys Pro Gly His Gly Ser Arg Phe 200 205 210 ATT GAG GAC ACA GCA GCA GAG AAG CTG CAC AAG GTC ATC AAC TCT 675 Ile Glu Asp Thr Ala Ala Glu Lys Leu His Lys Val Ile Asn Ser 215 220 225 ATC CTG GCT TTT CGG GAG AAG GAG AAG CAG AGG CTG CAG TCA AAC 720 Ile Leu Ala Phe Arg Glu Lys Glu Lys Gln Arg Leu Gln Ser Asn 230 235 240 CAG CTG AAG CCG GGG GCT GTG ACC TCC GTG AAC CTG ACT ATG CTA 765 Gln Leu Lys Pro Gly Ala Val Thr Ser Val Asn Leu Thr Met Leu 245 250 255 GAG GGT GGC GTG GCC TAT AAC GTC GTT CCT GCC ACC ATG AGT GCC 810 Glu Gly Gly Val Ala Tyr Asn Val Val Pro Ala Thr Met Ser Ala 260 265 270 TGC TTT GAC TTC CGC GTA GCA CCG GAT GTG GAC CTG AAG GCT TTC 855 Cys Phe Asp Phe Arg Val Ala Pro Asp Val Asp Leu Lys Ala Phe 275 280 285 GAG GAG CAG CTG CAG AGT TGG TGC CAG GCA GCT GGC GAG GGG GTC 900 Glu Glu Gln Leu Gln Ser Trp Cys Gln Ala Ala Gly Glu Gly Val 290 295 300 ACC TTT GAG TTT GTT CAG AAG TGG ATG GAG ACG CAA GTG ACA TCT 945 Thr Phe Glu Phe Val Gln Lys Trp Met Glu Thr Gln Val Thr Ser 305 310 315 ACT GAT GAC TCA GAC CCC TGG TGG GCA GCA TTT AGT GGG GTC TTC 990 Thr Asp Asp Ser Asp Pro Trp Trp Ala Ala Phe Ser Gly Val Phe 320 325 330 AAG GAT ATG AAG CTT GCC CTA GAG CTA GAG ATC TGC CCT GCT TCC 1035 Lys Asp Met Lys Leu Ala Leu Glu Leu Glu Ile Cys Pro Ala Ser 335 340 345 ACT GAC GCC CGC TAT ATT CGT GCG GCG GGG GTT CCG GCT CTG GGC 1080 Thr Asp Ala Arg Tyr Ile Arg Ala Ala Gly Val Pro Ala Leu Gly 350 355 360 TTC TCA CCC ATG AAC CAC ACA CCG GTG CTG CTC CAT GAC CAT GAT 1125 Phe Ser Pro Met Asn His Thr Pro Val Leu Leu His Asp His Asp 365 370 375 GAG CGG CTG CAT GAG GCC GTG TTT CTC CGT GGG GTT GAC ATA TAC 1170 Glu Arg Leu His Glu Ala Val Phe Leu Arg Gly Val Asp Ile Tyr 380 385 390 ACT CAG CTG CTG TCT GCC TTG GCC AGC GTG CCC GCA CTG CCC AGT 1215 Thr Gln Leu Leu Ser Ala Leu Ala Ser Val Pro Ala Leu Pro Ser 395 400 405 GAA AGC 1221 Glu Ser SEQ ID NO: 7 Sequence length: 1224 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Hypothetical sequence: NO Antisense: NO Sequence characteristics: 1-1224 CDS (human aminoacylase I) sequence : ATG ACC AGC AAG GGT CCC GAG GAG GAG CAC CCA TCG GTG ACG CTC 45 Met Thr Ser Lys Gly Pro Glu Glu Glu His Pro Ser Val Thr Leu 1 5 10 15 TTC CGC CAG TAC CTG CGT ATC CGC ACT GTC CAG CCC AAG CCT GAC 90 Phe Arg Gln Tyr Leu Arg Ile Arg Thr Val Gln Pro Lys Pro Asp 20 25 30 TAT GGA GCT GCT GTG GCT TTC TTT GAG GAG ACA GCC CGC CAG CTG 135 Tyr Gly Ala Ala Val Ala Phe Phe Glu Glu Thr Ala Arg Gln Leu 35 40 45 GGC CTG GGC TGT CAG AAA GTA GAG GTG GCA CCT GGC TAT GTG GTG 180 Gly Leu Gly Cys Gln Lys Val Glu Val Ala Pro Gly Tyr Val Val 50 55 60 ACC GTG TTG ACC TGG CCA GGC ACC AAC CCT ACA CTC TCC TCC ATC 225 Thr Val Leu Thr Trp Pro Gly Thr Asn Pro Thr Leu Ser Ser Ile 65 70 75 TTG CTC AAC TCC CAC ACG GAT GTG GTG CCT GTC TTC AAG GAA CAT 270 Leu Leu Asn Ser His Thr Asp Val Val Pro Val Phe Lys Glu His 80 85 90 TGG AGT CAC GAC CCC TTT GAG GCC TTC AAG GAT TCT GAG GGC TAC 315 Trp Ser His Asp Pro Phe Glu Ala Phe Lys Asp Ser Glu Gly Tyr 95 100 105 ATC TAT GCC AGG GGT GCC CAG GAC ATG AAG TGC GTC AGC ATC CAG 360 Ile Tyr Ala Arg Gly Ala Gln Asp Met Lys Cys Val Ser Ile Gln 110 115 120 TAC CTG GAA GCT GTG AGG AGG CTG AAG GTG GAG GGC CAC CGG TTC 405 Tyr Leu Glu Ala Val Arg Arg Leu Lys Val Glu Gly His Arg Phe 125 130 135 CCC AGA ACC ATC CAC ATG ACC TTT GTG CCT GAT GAG GAG GTT GGG 450 Pro Arg Thr Ile His Met Thr Phe Val Pro Asp Glu Glu Val Gly 140 145 150 GGT CAC CAA GGC ATG GAG CTG TTC GTG CAG CGG CCT GAG TTC CAC 495 Gly His Gln Gly Met Glu Leu Phe Val Gln Arg Pro Glu Phe His 155 160 165 GCC CTG AGG GCA GGC TTT GCC CTG GAT GAG GGC ATA GCC AAT CCC 540 Ala Leu Arg Ala Gly Phe Ala Leu Asp Glu Gly Ile Ala Asn Pro 170 175 180 ACT GAT GCC TTC ACT GTC TTT TAT AGT GAG CGG AGT CCC TGG TGG 585 Thr Asp Ala Phe Thr Val Phe Tyr Ser Glu Arg Ser Pro Trp Trp 185 190 195 GTG CGG GTT ACC AGC ACT GGG AGG CCA GGC CAT GCC TCA CGC TTC 630 Val Arg Val Thr Ser Thr Gly Arg Pro Gly His Ala Ser Arg Phe 200 205 210 ATG GAG GAC ACA GCA GCA GAG AAG CTG CAC AAG GTT GTA AAC TCC 675 Met Glu Asp Thr Ala Ala Glu Lys Leu His Lys Val Val Asn Ser 215 220 225 ATC CTG GCA TTC CGG GAG AAG GAA TGG CAG AGG CTG CAG TCA AAC 720 Ile Leu Ala Phe Arg Glu Lys Glu Trp Gln Arg Leu Gln Ser Asn 230 235 240 CCC CAC CTG AAA GAG GGG TCC GTG ACC TCC GTG AAC CTG ACT AAG 765 Pro His Leu Lys Glu Gly Ser Val Thr Ser Val Asn Leu Thr Lys 245 250 255 CTA GAG GGT GGC GTG GCC TAT AAC GTG ATA CCT GCC ACC ATG AGC 810 Leu Glu Gly Gly Val Ala Tyr Asn Val Ile Pro Ala Thr Met Ser 260 265 270 GCC AGC TTT GAC TTC CGT GTG GCA CCG GAT GTG GAC TTC AAG GCT 855 Ala Ser Phe Asp Phe Arg Val Ala Pro Asp Val Asp Phe Lys Ala 275 280 285 TTT GAG GAG CAG CTG CAG AGC TGG TGC CAG GCA GCT GGC GAG GGG 900 Phe Glu Glu Gln Leu Gln Ser Trp Cys Gln Ala Ala Gly Glu Gly 290 295 300 GTC ACC CTA GAG TTT GCT CAG AAG TGG ATG CAC CCC CAA GTG ACA 945 Val Thr Leu Glu Phe Ala Gln Lys Trp Met His Pro Gln Val Thr 305 310 315 CCT ACT GAT GAC TCA AAC CCT TGG TGG GCA GCT TTT AGC CGG GTC 990 Pro Thr Asp Asp Ser Asn Pro Trp Trp Ala Ala Phe Ser Arg Val 320 325 330 TGC AAG GAT ATG AAC CTC ACT CTG GAG CCT GAG ATC ATG CCT GCT 1035 Cys Lys Asp Met Asn Leu Thr Leu Glu Pro Glu Ile Met Pro Ala 335 340 345 345 GCC ACT GAC AAC CGC TAT ATC CGC GCG GTG GGG GTC CCA GCT CTA 1080 Ala Thr Asp Asn Arg Tyr Ile Arg Ala Val Gly Val Pro Ala Leu 350 355 360 GGC TTC TCA CCC ATG AAC CGC ACA CCT GTG CTG CTG CAC GAC CAC 1125 Gly Phe Ser Pro Met Asn Arg Thr Pro Val Leu Leu His Asp His 365 370 375 GAT GAA CGG CTG CAT GAG GCT GTG TTC CTC CGT GGG GTG GAC ATA 1170 Asp Glu Arg Leu His Glu Ala Val Phe Leu Arg Gly Val Asp Ile 380 385 390 TAT ACA CGC CTG CTG CCT GCC CTT GCC AGT GTG CCT GCC CTG CCC 1215 Tyr Thr Arg Leu Leu Pro Ala Leu Ala Ser Val Pro Ala Leu Pro 395 400 405 AGT GAC AGC 1224 Ser Asp Ser

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram showing a restriction enzyme map of porcine aminoacylase I cDNA, and a portion subjected to DNA sequencing and its orientation.

FIG. 2 is a diagram showing a restriction enzyme map of human aminoacylase I cDNA, and a portion subjected to DNA sequencing and its orientation.

Continuation of front page (56) References Biochim. Biophys. Acta 959 p. 370-377 (1988) (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 15/00 C12N 9/80

Claims (4)

(57) [Claims] 1. A polypeptide represented by the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing, or at least one of deletion, substitution, insertion, and addition of one or more amino acid residues in the sequence. A nucleic acid which encodes a polypeptide having an aminoacylase I activity. 2. SEQ ID NO: 6 or SEQ ID NO: 7 in the sequence listing
The nucleic acid according to claim 1, comprising a nucleic acid represented by a base sequence represented by: 3. A nucleic acid according to claim 1 or 2, comprising a nucleic acid hybridizable under stringent conditions,
And a nucleic acid encoding a polypeptide having aminoacylase I activity. 4. A polypeptide represented by the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, or one to
A polypeptide having an amino acid sequence having at least one of deletion, substitution, insertion, and addition of several amino acid residues, wherein the polypeptide has aminoacylase I activity.