JPS6151879B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel enzyme N-carbobenzoxyamino acid amide hydrolase, which differs in various properties from known aminoacylases, and a method for producing the same. Aminoacylase is an enzyme that acts on the acid amide bond of an N-acylamino acid in which the α-amino group of the amino acid is acylated, and catalyzes a reaction that produces an amino acid and a fatty acid corresponding to the acyl group. In addition, the enzyme called hiprate hydrolase or hiplicase is a type of aminoacylase, and it acts on hipric acid, a compound in which benzoic acid and glycine are dehydrated and condensed through an acid amide bond, and initiates the reaction that produces benzoic acid and glycine. It is a catalytic enzyme. Generally, these enzymes are N〓-acyl-DL-
When acting on a mixture of amino acids, N-acyl-L-amino acids can be selectively hydrolyzed. Based on this principle, acylase has been put to practical use in the production process of L-amino with high optical purity.
Although several aminoacylases produced by microorganisms have already been invented and put into practical use, it is important to provide new aminoacylases that are different from these known acylases in the production of amino acids, peptides, pharmaceutical raw materials, reagents, etc. This is one of the important issues in the field of chemistry, and it is also an important issue from the perspective of enzyme chemistry or analytical chemistry. It has been known that enzymes that hydrolyze N-acylamino acids, in which the α-amino group of L-amino acids is acylated, exist in mammalian tissues, molds, bacteria, actinomycetes, and various plant seeds. has been
In particular, aminoacylase derived from pig kidney cortex [J.Biol.
Chem., 194 , 455 (1952)] is well known. In microbial origin, Aspergillus or Rhizopus [Bull.Agr.Chem.
Soc.Jpn., 21 , 291, 296, 300, 304 (1957)], Corynebacterium genus [Special Publication No. 49-13989], Pseudomonas genus [Special Publication No. 56-43353], Lactobacillus (Lactobacillus) genus [J.Biol.Chem.,
235 , 3193 (1960)], and the genus Streptomyces [JP-A-53-59092]. These known aminoacylases have a degrading effect on various acylamino acids, but
It has no effect on compounds in which the α-amino group of an amino acid is protected with a carbobenzoxy group, ie, N-carbobenzoxy amino acids. Therefore, in view of the current situation, the present inventor conducted a wide range of studies on microbial cultures with the aim of searching for enzymes that act on N-carbobenzoxyamino acids.
We have discovered an activity that acts on N-carbobenxyamino acids in cultures of bacteria belonging to lactic acid bacteria. As a result of extensive research into this active factor, we discovered a novel enzyme that had never been reported before, and named it N-carbobenzoxyamino acid amide hydrolase. The present invention was completed based on this discovery. That is, the present invention has the general formula: [In the formula, R ₁ represents a carbobenzoxy group or benzoyl group and R ₂ represents a hydrogen, methyl group or hydroxymethyl group] to produce alanine, serine or glycine and benzyl alcohol or benzoic acid. The present invention relates to an enzyme N-carbobenzoxyamino acid amide hydrolase having the following properties and its production method: Substrate specificity: The α-amino group of glycine, L-alanine and L-serine is a carbobenzoxy group, a benzoyl group, or a nucleus. It acts on compounds protected with a carbobenzoxy group having a substituent or a benzyl group having a nuclear substituent and N〓-carbobenzoxyglycylglycine, but N〓-acetylglycine, N〓
-Does not act on acetyl-L-alanine. Optimum pH: 5-7 Working temperature: 37-60℃ Molecular weight: Approximately 235,000 Isoelectric point: 4.3-4.6 Activation: Activated with Co or Zn ions The enzyme of the present invention can be produced from a strain belonging to the genus Lactobacillus. I solved it. The bacterial strain may be any bacterial strain as long as it has the ability to produce N-carbobenzoxyamino acid amide hydrolase, or may be a variant or mutant of such a bacterial strain. A specific example of the above-mentioned strain belonging to the genus Lactobacillus is Lactobacillus casei. This strain is from the American Type Culture Collection.
Lactobacillus casei
It is ATCC7469. In the present invention, the medium used for producing N-carbobenzoxyamino acid amide hydrolase using N-carbobenzoxyamino acid amide hydrolase-producing bacteria may be a medium used for the cultivation of ordinary lactic acid bacteria. It will be done. That is,
Any material may be used as long as it contains a carbon source, a nitrogen source, an inorganic salt, and, if necessary, micronutrients that can be assimilated by the N-carbobenzoxyamino acid amide hydrolase producing bacteria. Examples of carbon sources include carbohydrates such as glucose, fructose, lactose, sucrose, dextrin, starch hydrolyzate, malt extract, and blackstrap molasses, organic acids such as citric acid, succinic acid, fumaric acid, and acetic acid, and mannitol and glycerin. Alcohols are used. The nitrogen source for the medium may be any nitrogen compound that can be assimilated or one containing it, such as polypeptone, meat extract, hydrolysates of proteins such as soybean, various amino acids, ammonium salts, nitrates, and the like. In addition, as inorganic salts, for example, inorganic salts such as manganese, phosphoric acid, potassium, and magnesium may be used as appropriate, or as organic micronutrients, amino acids, vitamins, purine bases, peptones containing these, yeast extracts, etc. may be used as appropriate. I can do it. The bacteria are cultured by static culture. Stirring submerged culture is suitable for industrial production such as mass culture, but culture can also be carried out under aerobic conditions such as aerated stirring culture and permeation culture. The culture temperature is 30 to 40°C, preferably around 37°C, and the medium pH is 8 to 5, preferably
It is around 7.0. The culture time varies depending on the culture format, but is usually 14 to 17 hours. The N-carbobenzoxyamino acid amide hydrolase of the present invention exists in the culture solution and within the bacterial cells of N-carbobenzoxyamino acid amide hydrolase-producing bacteria, but most of it exists within the bacterial cells. By prolonging the culture time, autolysis can be induced and N-carbobenzoxyamino acid amide hydrolase can be released into the culture solution. In order to extract and purify the N-carbobenzoxyamino acid amide hydrolase of the present invention from a culture, conventional enzyme protein extraction and purification methods can be applied. For example, after collecting bacterial cells from a culture using an appropriate operation such as centrifugation, the bacterial cells are crushed mechanically with an appropriate abrasion agent such as glass beads, or by ultrasonic irradiation. The bacterial cells can be disrupted using a French press, a lytic enzyme such as lysozyme, or an osmotic shock, or the bacterial cells can be dissolved in water, physiological saline, or a buffer solution. After suspending the enzyme and extracting it by standing or permeating in the presence of toluene, the solution is subjected to an appropriate operation such as centrifugation to remove insoluble materials, and the crude enzyme solution is obtained as it is. Ordinary protein concentration methods, such as freeze-drying a crude enzyme solution,
Alternatively, a method of fractional precipitation using an organic solvent such as ethanol, acetone, or isopropanol, or a method of performing a dialysis operation using an ultrafiltration membrane, hollow fiber membrane, collodion membrane, etc. after salting out using salts such as ammonium sulfate. A crude enzyme powder can be obtained by appropriately selecting and implementing the following methods. In order to separate the purified enzyme from the above-mentioned crude enzyme solution or crude enzyme powder, an appropriate combination of ion exchange, gel filtration, adsorption, electrophoresis, affinity chromatography, etc. is carried out. For example, ion exchange chromatography using an ion exchanger such as diethylaminoethyl-cephadex, adsorption chromatography using an adsorbent such as aminohexyl-cephalose or hydroxyapatite, or hydrophilic carriers such as sephadex or cephalose or cephacryl. Gel filtration method used, electrophoresis method using polyacrylamide gel or carrier ampholite, affinity chromatography method using a hydrophilic carrier to which an appropriate ligand compound is chemically bonded, molecular weight method using molecular sieve membrane or hollow fiber membrane, etc. The purified enzyme can be obtained by appropriately selecting a fractionation method and performing a combination of these methods. A typical method for producing the novel enzyme of the present invention and its collection method is to culture a strain of the genus Lactobacillus having the ability to produce N-carbobenzoxyamino acid amidohydrolase, such as Lactobacillus casei, collect the cultured strains, and adjust the pH to neutral. By disrupting in a buffer solution of
The enzyme extract is obtained. This extract may be purified by the following method if necessary. For example, the above-mentioned extract is subjected to liquid chromatography using a column filled with dextrin or a cellulose derivative having anion exchange ability, and the active fraction thereof is collected. Further, the concentrated solution may be collected by ultrafiltration. If necessary, the ultrafiltration concentrate may be subjected to adsorption column chromatography, and the active fraction thereof may be fractionated and concentrated by an appropriate method, for example, using a suitable organic solvent. If further production is required, this may be fractionated by one or more gel filtration chromatographies and the active fraction may be collected. Although the above manufacturing method is the most preferred method, the method of the present invention is not limited thereto, and suitable changes may be made, such as substituting some of the steps with other methods or omitting some of the steps. Good too. process alternatives,
Additions and deletions may be appropriately selected depending on the culture conditions, type, by-products, required purity, etc. of the strain. The novel N-carbobenzoxyamino acid amide hydrolase obtained by the above method has the characteristics described below and is clearly distinguishable from known aminoacylases collected from microbial or mammalian tissues or plant seeds. be done. That is, these known enzymes act on N-acylamino acids in which the α-amino group of the amino acid is acylated, and catalyze the reaction that produces fatty acids and amino acids corresponding to the acyl group, but the α-amino acid of the amino acid It has no effect on N-carbobenzoxy amino acids whose amino group is carbobenzoxylated. On the contrary,
The N-carbobenzoxyamino acid amide hydrolase of the present invention exhibits an extremely high decomposition effect on N-carbobenzoxyamino acids, and also shows no decomposition effect on N-acyl amino acids, which are suitable substrates for aminoacylases in general. do not have. Based on the above findings, the enzyme of the present invention can be clearly distinguished from any known aminoacylases and is recognized as a novel enzyme. The physicochemical properties of the enzyme N-carbobenzoxyamino acid amide hydrolase of the present invention will be described below. (1) Action: Acts on N-carbobenzoxyglycine,
Benzyl alcohol and glycine are produced as reaction products. N〓-carbobenzoxy-L
-Acts on alanine and produces benzyl alcohol and alanine as reaction products. N〓-
It acts on carbobenzoxy-L-serine and produces benzyl alcohol and serine as reaction products. Compounds in which the α-amino group of glycine or alanine is benzoylated, that is, N-benzoylglycine or N-benzoyl-L-alanine, also exhibit a decomposition effect, and the products of the decomposition reaction are benzoic acid and glycine or alanine. generate. (2) Substrate specificity The α-amino group of glycine, L-alanine, and L-serine is protected with a carbobenzoxy group, a benzoyl group, a carbobenzoxy group with a nuclear substituent, or a benzoyl group with a nuclear substituent. It acts on compounds that are It has no effect on compounds in which the α-amino group of glycine or L-alanine is acetylated, that is, N-acetylglycine and N-acetyl-L-alanine, and this enzyme exhibits an aminoacylase-like action. do not have. When N-carbobenzoxy-D-alanine or N-benzoyl-D-alanine is used as a substrate, unlike in the case of the L-form of the compound, this enzyme does not act at all, and its degrading action is L
specific for compounds containing amino acids. Compounds in which the α-amino group of glycine is protected with a group other than the above, such as N-formylglycine, N-phenylacetylglycine, N-
p-Nitrophenylacetylglycine, N〓-
It has no effect on 2,4-dinitrophenylglycine, N-triphenylglycine, N-phthalylglycine, N-tolylsulfonylglycine, or N-t-butoxycarbonylglycine, and has no effect on L-alanine. Even in the case of , it has no effect on compounds protected with similar groups. A compound in which the carboxyl group of N-carbobenzoxyglycine is protected with a residue other than an amino acid residue, such as N-carbobenzoxyglycine,
It has almost no effect on methyl esters, N-carbobenzoxyglycinamide, etc. Furthermore, when a peptide in which glycine is located at the amino terminal position and a compound in which the α-amino group of the glycine residue in the peptide is protected with a carbobenzoxy group is used as a substrate, this enzyme
-Carbobenzoxyglycylglycine and cleavage of the peptide bond between glycine residues was observed, but other compounds such as N〓-carbobenzoxyglycyl-L-leucine, N〓-carbobenzoxyglycyl Cyl-L-phenylalanine, N〓-carbobenzoxyglycyl-L-proline, N〓-carbobenzoxyglycylglycylglycine, N〓-carbobenzoxyglycylglycyl-L-phenylalanine or N〓
-It has no effect on carbobenzoxyglycylglycyl-L-serine, etc. A peptide in which glycine is located at the amino terminal position, and a compound in which the α-amino group of the glycine residue in the peptide is protected with a benzoyl group, such as N
It has almost no effect on benzoylglycylglycine or N-benzoylglycyl-L-histidyl-L-leucine. It has no effect on dipeptides containing glycine at the carboxyl terminal position, such as L-alanyl-glycine, L-tyrosylglycine, L-phenylalanyl-glycine or glycylglycine. It also has no effect on dipeptides in which glycine is located at the amino terminal position, such as glycyl-L-leucine or glycyl-L-phenylalanine. It does not act at all on compounds in which the α-amino group of a natural amino acid other than glycine, L-alanine, or L-serine is protected with a carbobenzoxy group, benzoyl group, or acetyl group. Table 1 shows the activity of this enzyme against various substrates. In addition, the relative activity in Table 1 is based on the degrading activity for N-carbobenzoxyglycine 100
It is shown as an activity ratio when

【table】

[Table] (3) Titer measurement method 0.025M CoCl ₂ 0.02ml and enzyme solution 0.05ml 0.05M
Add phosphate buffer (PH6.0) to make a total volume of 0.45 ml, add 0.05 ml of 0.01 M N-carbobenzoxyglycine, react at 50°C for 10 to 30 minutes, and then add 0.5 ml of distilled water. Add to make a total volume of 1 ml. Using this reaction mixture, the glycine released as a result of the enzymatic reaction was measured using the ninhydrin colorimetric method [EWYemm & E.C. Cocking., Analyst., 80 ,
209 (1955)]. The method for measuring enzyme activity is as shown below. In addition, the enzyme activity to produce 1 mM of glycine in 1 hour in the above reaction system was defined as 1 unit.
Further, the specific activity was expressed in enzyme units per mg of enzyme protein of the enzyme activity measured by the above method.
The protein was quantified by measuring absorbance in ultraviolet light. (4) Range of optimal PH and temperature for action As shown in Figure 1, the range of optimal PH for action is 5 to 7, and the optimal PH for action is around 6.0. As shown in Figure 2, the range of suitable temperature for action is 37
~60°C, and the optimum temperature for action is around 50°C. (5) Stable PH and stable temperature range The stable PH range is between 6 and 7, and as shown in Figure 3, the activity residual rate after 16 hours at PH6.5 and 35°C is 70%. The stable temperature range is up to 45℃, and as shown in Figure 4, the activity residual rate is 100% after 1 hour at PH6.2 and 40℃, and 70% after 1 hour at 50℃. It is almost completely inactivated in 1 hour at ℃. (6) Effect of various metal ions on enzyme activity Various metal ions were added to the enzyme solution so that the final concentration was as shown in Table 2, and after reacting at 50°C for 10 minutes, N-carbobenzo Table 2 shows the results of measuring enzyme activity using xyglycine as a substrate. Note that the relative activities in Table 2 are expressed as activity ratios when the decomposition activity in the presence of 1 mM CoCl ₂ is taken as 100.

【table】

[Table] (7) Effect of metal chelate compounds and SH on enzyme activity
Effects of reagents or protein modification reagents Various metal chelate compounds, SH reagents, organic mercury compounds, or protein modification reagents were added to enzyme solutions (1 ml) at final concentrations shown in Table 3.
Table ₃ shows the results of measuring the residual enzyme activity using N-carbobenzoxyglycine as a substrate after reacting at 50° C. for 10 minutes. The relative activities in Table 3 are expressed as activity ratios, with the enzyme activity measured without the addition of the above-mentioned drugs being taken as 100.

[Table] (8) Molecular weight Molecular weight was determined by gel filtration chromatography using Sepharose 6B (manufactured by Pharmacia Fine Chemicals) equilibrated with 0.01M phosphate buffer (PH6.8) containing 0.1M NaCl. It was measured. Standard proteins include thyroglobulin,
Catalase and altolase were used. As a result, the molecular weight of this enzyme was found to be 235,000. (9) Isoelectric point Isoelectric point was measured by polyacrylamide gel isoelectric point separation method. As a result, it was found that the isoelectric point of this enzyme was in the range of 4.3 to 4.6. (10) Disk electrophoresis Disc electrophoresis was performed using polyacrylamide as a carrier and a Tris-barbital buffer solution with a gel concentration of 7.0% and a pH of 8.0. After running a current of 3 mA per column and running at 4°C for 1 hour, Kumasi Brilliant Blue R-
Stained with 250. As a result, the specific electrophoretic distance of this enzyme with respect to the marker (bromophenol blue) was Rm _BPB = 0.51. The novel enzyme N-carbobenzoxyamino acid amide hydrolase of the present invention has the above properties. EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereto. Example 1 5 parts of casamino acid (manufactured by Difco) per 1 part of water
g, Bactotryptone (manufactured by Difco) 5g, L
- Cysteine 0.2g, L-aspartic acid 0.1g,
DL-alanine 0.5g, DL-tryptophan 0.2
g, glucose 20g, sodium acetate 10g, sodium succinate 20g, KH ₂ PO ₄ 0.5g, K ₂ HPO ₄ 0.5
g, _FeSO4・_7H2O10mg , _MnSO4・_H2O10mg ,
MgSO ₄ 7H ₂ O 10mg, NaCl 10mg, Thiamine 1
mg, calcium pantothenate 1 mg, folic acid 0.25 mg,
A culture volume containing 0.5 mg of p-aminobenzoic acid, 5 μg of biotin, 5 mg of adenine sulfate, 5 mg of guanine hydrochloride, 5 mg of uracil, and 1 ml of Tween 80 was adjusted to pH 6.8.
After adjusting to 100 ml Erlenmeyer flasks, each
The mixture was dispensed into 100ml portions and sterilized at 120°C for 10 minutes. This sterilized medium contains Lactobacillus casei ATCC7469.
Three loopfuls of each were inoculated from the puncture medium of the strain,
Static culture was performed at 37°C for 24 hours. 200 ml of this preculture solution was transferred to a 30 volume stainless steel jar fermenter containing medium 20 having exactly the same composition as the medium used for preculture, and main culture was carried out at 37° C. with stirring at 45 revolutions per minute. The main culture was terminated after 16 hours, the culture solution was centrifuged, and the bacterial cells were collected. The culture was carried out 5 times in the same manner, and bacterial cells weighing 1 kg (wet weight) were obtained from a total of 100 culture solutions. The bacterial cells thus obtained were suspended in approximately 2000 ml of 0.01M phosphate buffer (PH6.8), and Dynomill (Willy) containing approximately 1.2 kg of 0.1 mm diameter glass beads was suspended.
(manufactured by A-Bakoohen Co., Ltd.), and the bacterial cells were destroyed while cooling with water at 0°C. This disrupted solution was centrifuged (0° C., 12,000 rpm, 30 minutes) to obtain 2,750 ml of a viscous pale yellow cell extract. Example 2 Solid ammonium sulfate was added to 2750 ml of the crude enzyme solution obtained in Example 1.
Add 662g little by little while stirring under ice cooling.
Set to 40% saturation. After leaving this item in the ice room for 30 minutes,
The resulting precipitate was removed by centrifugation, and 564 g of solid ammonium sulfate was added little by little to the supernatant with stirring under ice cooling to achieve 70% saturation. This product was left in an ice chamber overnight, and the resulting precipitate was collected by centrifugation. The obtained ammonium sulfate precipitate was dissolved in 0.01M phosphate buffer (PH6.8) and dialyzed against the same buffer for 16 hours to obtain 800 ml of dialysis solution. The N-carbobenzoxyglycine degrading activity of this enzyme solution is
The protein concentration was 9.35 units/ml and 13 mg/ml. Example 3 800 ml of the enzyme solution obtained in Example 2 was placed in a 3 m
7.0 cm of DEAE-Sephadex A-50 (manufactured by Pharmacia Fine Chemicals) equilibrated with 0.01M phosphate buffer (PH6.8) containing M _NaN3
Pour into a x86.0cm column, wash thoroughly with the same buffer, perform linear gradient elution from 0 to 1M NaCl using the same buffer, and collect 800ml of N-carbobenzoxyamino acid amide hydrolase active fraction.
This was subjected to ultrafiltration using a membrane concentrator (PM-10, manufactured by Amicon, USA) to obtain 145 ml of enzyme solution. The activity of this enzyme solution was 51 units/ml. Example 4 145 ml of the enzyme solution obtained in Example 3 was added in advance.
AH− equilibrated with 0.01M phosphate buffer (PH6.8)
Pour into a 3.0 cm x 58.0 cm column of Sepharose 4B (manufactured by Pharmacia Fine Chemicals), wash with 2.5 cm of the same buffer, and perform linear gradient elution from 0 to 1 M NaCl using the same buffer. - 116 ml of a carbobenzoxyamide hydrolase active fraction was obtained, which was concentrated using polyethylene glycol to obtain 29 ml of an enzyme solution. The activity of this enzyme solution was 208 units/ml. Example 5 29ml of the enzyme solution obtained in Example 4 was diluted with 3mM in advance.
0.01M _phosphate buffer (PH
6.8) and 5.0 cm of Cephadex G-150 (manufactured by Pharmacia Fine Chemicals).
Pour into a x 88.0 cm column and add 22.5
Elution was performed at a flow rate of ml/hr, and the N-carbobenzyloxyamino acid amide hydrolase active fraction
295 ml was obtained, which was concentrated using polyethylene glycol to obtain 34 ml of enzyme solution. The activity of this enzyme solution was 233 units/ml. Example 6 34 ml of the enzyme solution obtained in Example 5 was mixed with 0.01% mercaptoethanol and 3mM _NaN3 .
DEAE equilibrated with 0.01M phosphate buffer (PH6.8)
- Pour into a 5.0cm x 70.0cm column of Cephadex A-50 (manufactured by Pharmacia Fine Chemicals), wash the column thoroughly with the same buffer, and then elute with a linear gradient of 0 to 1M NaCl using the same buffer. 117 ml of N-carbobenzoxyamino acid amide hydrolase active fraction was obtained, which was concentrated with polyethylene glycol to obtain 22.5 ml of enzyme solution. The activity of this enzyme solution is 373 units/ml
It was hot. Example 7 22.5 ml of the enzyme solution obtained in Example 6 was added in advance to a concentration of 0.01
0.01M phosphate buffer ( _PH
6.8) of Cephacryl S-300 (manufactured by Pharmacia Fine Chemicals), 2.6 cm x
Pour into a 91.0 cm column and use the same buffer at 12.5 ml/h.
Elution was performed at a flow rate of r to obtain 60 ml of N-carbobenzoxyamino acid amidohydrolase active fraction, which was concentrated with polyethylene glycol.
14 ml of enzyme solution was obtained. The activity of this enzyme solution was 431 units/ml. Example 8 14 ml of the enzyme solution obtained in Example 7 was placed in a 2.6 cm x 91.0 cm Cefacryl S-300 (manufactured by Pharmacia Fine Chemicals) equilibrated in advance with a buffer solution under exactly the same conditions as in Example 7. Pour into the column and elute with the same buffer at a flow rate of 9.5 ml/hr.
75 ml of a carbobenzoxyamino acid amide hydrolase active fraction was obtained and concentrated with polyethylene glycol to obtain 25 ml of an enzyme solution. The activity of this purified enzyme solution is 362 units/ml, which is 995 units per mg of protein.
It showed the activity of the unit. Table 4 shows the total activity, specific activity, and yield in the above purification process.

[Table] Reference example 1 Dissolve N-benzoylglycine in 0.05M phosphate buffer (PH6.8) to prepare a 10 μmol/ml solution, and add 1 ml of the enzyme solution obtained by the above purification method to 20 ml of the solution.
After reacting at 37° C. for 1 hour, the reaction solution was extracted three times with 20 ml of ether. After drying this ether layer with anhydrous sodium sulfate,
When the ether was distilled off, white crystals were precipitated. As a result of examining this crystal using IR, MS, UV, and TLC,
The white crystals obtained from the ether layer were benzoic acid. In addition, after concentrating the aqueous layer after ether extraction, silica gel 60 Pre-Coated PLC plate
(manufactured by Merck & Co.), solvent, MeOH:
After developing with H ₂ O 60:40, the silica gel in the glycine developed area was scraped off and extracted with water in a beaker. After removing the silica gel by filtration, the aqueous solution was concentrated and the precipitated white crystals were recrystallized from a mixture of MeOH and water. As a result of IR, MP, and TLC elemental analysis, this crystal was found to be glycine. When N-benzoylalanine was treated in exactly the same manner, benzoic acid was detected in the ether layer and alanine was detected in the aqueous layer. Reference Example 2 Dissolve N-carbobenzoxyglycine in 0.05M phosphate buffer (PH6.8) to prepare a 10 μmol/ml solution, and add 1 ml of the enzyme solution obtained by the above purification method and M/ml to 10 ml of the solution. Add 0.1 ml of 10 CoCl ₂ aqueous solution,
After reacting at 37°C for 1 hour, the reaction solution was diluted with ether.
Three extractions were performed using 20 ml. After drying this ether layer over anhydrous sodium sulfate, the solvent was distilled off, and the remaining liquid was examined by IR, UV, TLC, and MS. As a result, what was extracted into the ether was benzyl alcohol. In addition, the aqueous layer after ether extraction was adsorbed to Amberlite IR-120 (manufactured by Rohm and Haas) adjusted to pH 2.0 using the batch method, and then
Elution was performed using % _NH4OH . The crystals precipitated by concentrating this were examined by TLC, MP, and IR elemental analysis, and the crystals obtained from the aqueous layer were found to be glycine. When N-carbobenzoxyalanine was treated in exactly the same manner, benzyl alcohol was detected in the ether layer and alanine was detected in the aqueous layer.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the optimum pH of N-carbobenzoxyamino acid amidohydrolase. Second
The figure shows the action temperature of N-carbobenzoxyamino acid amidohydrolase. FIG. 3 is a diagram showing the PH stability of N-carbobenzoxyamino acid amidohydrolase at 35°C. FIG. 4 is a diagram showing the thermostability of N-carbobenzoxyamino acid amidohydrolase.

Claims (1)

[Claims] 1. General formula: [In the formula, R ₁ represents a carbobenzoxy group or benzoyl group and R ₂ represents a hydrogen, methyl group or hydroxymethyl group] to produce alanine, serine or glycine and benzyl alcohol or benzoic acid. The enzyme N-carbobenzoxyamino acid amide hydrolase has the following properties: Substrate specificity: α-amino groups of glycine, L-alanine, and L-serine have carbobenzoxy groups, benzoyl groups, and nuclear substituents. It acts on compounds protected with a carbobenzoxy group or a benzyl group having a nuclear substituent and N〓-carbobenzoxyglycylglycine, but N〓-acetylglycine, N〓
-Does not act on acetyl-L-alanine. Optimum pH: 5-7 Working temperature: 37-60°C Molecular weight: Approx. 235000 Isoelectric point: 4.3-4.6 Activation: Activated with Co or Zn ions 2 Benzoxyamino acid amide hydrolase. 3. The N-carbobenzoxyamino acid amide hydrolase according to item 1, wherein DISC electrophoresis shows Rm=0.51. 4 Belongs to the genus Lactobacillus, general formula: [In the formula, R ₁ represents a carbobenzoxy group or benzoyl group and R ₂ represents a hydrogen, methyl group or hydroxymethyl group] to produce alanine, serine or glycine and benzyl alcohol or benzoic acid. A strain capable of producing an enzyme N-carbobenzoxyamino acid amide hydrolase having the following characteristics is cultured in a medium, and a novel N-carbobenzoxyamino acid amide hydrolase is collected from the culture. -Production method of carbobenzoxy amino acid amide hydrolase: Substrate specificity: α-amino groups of glycine, L-alanine and L-serine are carbobenzoxy groups, benzoyl groups, carbobenzoxy groups having a nuclear substituent, or nuclear It acts on compounds protected with benzyl groups having substituents and N〓-carbobenzoxyglycylglycine, but N〓-acetylglycine, N〓
-Does not act on acetyl-L-alanine. Optimum pH: 5-7 Working temperature: 37-60°C Molecular weight: Approximately 235000 Isoelectric point: 4.3-4.6 Activation: Activated with Co or Zn ions 5 The production method according to item 4, wherein the bacterial strain is Lactobacillus casei . 6. Collect the bacterial strain from the culture solution, disrupt it in a buffer with a neutral pH, salt out the disrupted extract, dissolve the precipitate in a buffer with a neutral pH, and dialyze it with the same buffer. The manufacturing method according to item 4, which is carried out by. 7. The production method according to item 4, wherein the collection is performed by treating the dialysate according to item 6 with anion exchange liquid chromatography, ultrafiltering the active fraction, and collecting the concentrated solution. 8. The production method according to item 4, wherein the collection is performed by subjecting the ultrafiltration concentrate according to item 7 to adsorption column chromatography and optionally concentrating the active fraction. 9. The production method according to item 4, wherein the collection is performed by subjecting the active fraction described in item 8 or its concentrated solution to gel filtration chromatography, and optionally concentrating the active fraction. 10. The production method according to item 9, in which the collected active fraction or its concentrate according to item 9 is further subjected to gel filtration chromatography.