JPH0824577B2 - Peptidase and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel peptidase secreted by yeast, particularly Saccharomyces yeast, and a method for producing the same. INDUSTRIAL APPLICABILITY The peptidase of the present invention is useful as an enzyme that specifically cleaves the Leu C-terminal side of a peptide having an amino acid sequence of ... Leu-lys ... in its primary structure.

(Prior Art) In recent years, with the development of genetic engineering, it has become possible to microbially produce various higher animal hormones and the like, and it is possible to convert a prepro-peptide, which is a translation product of mRNA, into a mature peptide. Processing enzymes are attracting attention.

For example, in yeast, Kurjan J. et al. (Cell
30 933-943 (1982)) and Julius D. et al. (Cell 32 839-852 (1983)) processed a precursor polypeptide to obtain a yeast α-mate factor (hereinafter referred to as α-MF). A membrane-bound dipeptidyl aminopeptidase which gives rise to (abbreviation) has been reported. This enzyme
-Glu-Ala-cleaves the Ala carboxyl side of the repeat sequence. Also, Julius D. et al. Reported the structure of a membrane-bound endopeptidase and a gene encoding it as a processing enzyme of prepro α-MF (Cell 37 1075-
1089 (1984)). This enzyme cleaves the Lys-Arg bond. Furthermore, Matsuo et al., As an activating enzyme of yeast prohormone, intracellular enzyme Phorcesin Y-
1 is reported. (Nature (London) 309 (5968) 558
-60 (1984) and JP-A-60-217894). Its mechanism of action is to specifically cleave between Arg-Arg and Arg-Lys bonds. In addition to these, as yeast-produced peptidases, carboxyl peptidase Y, proteinase A, proteinase B, aminopeptidase, and carboxypeptidase S are known (H. Holzer, H. Betz and E. Ebner). (Ebner): Curr.Top, Cel
L. Regul., 9, 103-156 (1975)).

On the other hand, for α-MF, the following formula (I); It is a peptide having a molecular weight of 1684 having a primary structure, and is obtained by shaking culture of yeast (S. cerevisiae) α-type cell X-21801B in a Barkholder medium, and further, as the culture time becomes longer, the medium It is known that the α-MF activity in the medium rapidly decreases, and various peptides other than α-MF are formed in the medium. Tanaka et al.
As a result of analyzing various peptides formed by long-term culture, it was confirmed that α-MF was cleaved at the arrow portion of the above formula (I) (J. Biochem., 82 1689-1693 ( 197
7)), its decomposition activity is very weak (even if it is reacted for 24 hours, it decomposes 50% of a few μg / ml of α-MF production), and there are many side reactions. It has not been clarified yet. Thorner et al. Presume that the main body of this α-MF degrading activity is the hormone peptidase secreted outside the yeast cells (Sonar, J. 1981, Ph.
oremonal regulation of development in Saccharomyce
s cerevisiae. In the molecular biology of the yeast
Saccharomyces. I. Life cycle and inheritance (edited by J. Strathern et al.), P143. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. However, its body is not clear.

(Problems to be Solved by the Invention) The present invention clarifies the main body of the degradation activity of the Leu-Lys bond of α-MF, and provides a novel Leu-Lys bond-specific peptidase and a method for producing the same. To do.

(Means for Solving Problems) The present inventors immobilized yeast cells at a high density (1-2 g
Wet cells / 1g gel), when cultured, α-MF of the culture solution
Degradation activity is more than α-MF degradation activity in free cell culture medium,
It was found to be much higher, and the present invention was completed based on this finding.

Properties of enzyme The properties of the enzyme of the present invention are as follows.

(1) Action: Leucine-lysine (Leu-Ly) in the peptide
When the amino acid sequence s) exists, the peptide bond between them is specifically cleaved by hydrolysis.

(2) Substrate specificity: For example, the site shown by the arrow in the peptide shown below is cleaved. That is, in the molecule Only the C-terminal side of Leu of the bond is cleaved, and when Lys and Leu are bonded to other amino acids, hydrolysis is not performed.

(3) Molecular weight: The estimated molecular weights in the active form measured by molecular sieve chromatography using silica gel resin and Cefacrylic resin are about 100,000 and about 75, respectively.
It is about 55 kilodaltons and about 63 by SDS / PAGE.
It is composed of kilodalton subunits.

(4) Optimum pH and stable pH range: The optimum pH is about 4,
It is stable within a pH range of 3 to 4.5 after treatment at 30 ℃ for 24 hours.

(5) Optimum temperature range of action: Operates in the range of 20 ℃ -60 ℃,
The optimum temperature is 35 ℃ -42 ℃.

(6) Response to various inhibitors: It is not inhibited by phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DFP) 2 × 10 ⁻⁴ M which are serine protease inhibitors.

Monoiodoacetic acid, a thiol protease inhibitor, and P-chloromercury benzoic acid (PCM
Not hindered by B).

 It is not inhibited by the metal chelating agent EDTA.

Common trypsin inhibitors tosyl-L-lysine chloromethyl ketone (10 ^-3 M) and leupeptin (10 ^-4
Not inhibited in M).

Pepstatin (10
^-5 M) is not inhibited.

Method for producing enzyme Peptidase of the present invention, yeast of the genus Saccharomyces is immobilized at high density on an immobilizing carrier, and after culturing for 24 hours,
It can be obtained by purification from the culture solution.

The Saccharomyces yeast used in the production of the peptidase of the present invention is preferably an α-mating yeast, but it may be any yeast that produces and produces the peptidase of the present invention, for example, Saccharomyces cerevisiae (Saccharomyces ce
revisiae), specifically, for example, the X2180-1B strain (ATC), which is a representative strain of haploid Saccharomyces cerevisiae.
C26787).

The yeast is precultured using an appropriate medium, and the obtained bacterial cells are collected and immobilized in a carrier. Any medium can be used as long as it can grow the yeast and produce the peptidase. As the carbon source, for example, glucose, dextrin, sugar concentrate, etc. can be used, but the use of glucose is most preferable. As the nitrogen source, for example, proteolytic products such as peptone and casamino acid, meat extract, yeast extract, amino acids, ammonium salt and the like can be used, but amino acid and ammonium salt are preferably used. In addition, vitamins and nucleic acid-related compounds can be added for the purpose of promoting the growth of bacteria. In the pre-culture, a solid medium can be used, but in order to obtain a large amount of cells, it is preferable to carry out aeration stirring culture or shaking culture using a liquid medium. Conditions such as culturing temperature, culturing time, and liquidity of the medium are appropriately selected and adjusted so as to maximize the growth of bacterial cells, but usually, the culturing is carried out under aerobic conditions at 30 ° C for 24 to 30 hours Is preferred. The yeast cultivated by the above method is collected and immobilized by a conventional method.

As the immobilization carrier, it is sufficient if the cells immobilized at high density can grow, and κ (kappa) -carrageenan, natural polysaccharides such as sodium alginate, photocrosslinkable resin (ENT1000 to 6000), polyvinyl Although there are synthetic prepolymers such as alcohol resin, hydrophilic photo-crosslinkable resin
It is convenient to use ENT2000 to immobilize the cells at high density. The microbial cell concentration is 1 to 2 g wet wet microbial cells per 1 g carrier. It should be noted that if the cell concentration is low, the desired enzyme cannot be produced sufficiently. 200m per 1g carrier
The amount of enzyme produced when g of wet-cells was immobilized was 1/50 or less of that when the cells in the above range were immobilized.

Immobilization method may be according to a conventional method, for example, when using the hydrophilic photocrosslinkable resin, a polymerization accelerator, for example 2 mg of benzoin ethyl ether per 1 g of the photocrosslinkable prepolymer.
, Mix this with bacterial cells, and near UV (300-400nm)
By irradiating with, solidified bacterial cells can be prepared.

The yeast thus immobilized is cultured in the above-mentioned medium, preferably a Burkholder synthetic medium for 24 hours to produce the enzyme, and the enzyme is secreted out of the immobilized cells. Since the enzyme of the present invention is leaked out of the immobilized carrier, it can be obtained in the culture medium.

The enzyme is purified from the obtained culture solution by appropriately combining commonly used methods. For example, the culture solution is centrifuged or filtered to remove insoluble matter, the solution is concentrated by ultrafiltration, and then dialyzed against water or a buffer solution. Next, fractionation by column chromatography is performed using an adsorption resin composed of DEAE-cellulose or DEAE-Sephadex, such as DEAE-Sephacel,
Collect the fractions eluting at 0.1-0.6 M, preferably 0.2 M sodium chloride. The peptidase of the present invention can be obtained by subjecting this section to molecular sieve chromatography and collecting the sections of high molecular weight (about 100,000 or about 750,000).
This eluate may be desalted if necessary and used as it is, or may be dried by a conventional method such as vacuum drying or freeze drying.

Measurement of Enzymatic Activity The activity of the peptidase of the present invention was determined by synthesizing α-MF (Tr
p-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pr
o-Met-Tyr) is used as a substrate for enzymatic reaction, and -Leu-Lys
-Trp-His-Trp-Leu-Gl that accompanies cleavage between bonds
It is determined by measuring the amount of n-Leu peptide (referred to as 1-6 peptide). For example, 1.0 nmol (1.6 μg)
10 μl of the enzyme solution to be tested was added to 0.4 ml of 0.05 M acetate buffer (pH 4.0) containing the chemically synthesized α-MF as a substrate.
The reaction was carried out at ℃ for 24 hours, and the decomposition product (Trp
-His-Trp-Leu-Gln-Leu) is quantified by high performance liquid chromatography (hereinafter abbreviated as HPLC).

 HPLC can be performed, for example, under the following conditions.

Column: μ Bonder Pack C ₁₈ (Japan Waters) Eluent: Solution A = 50 mM phosphate buffer containing 5% acetonitrile
pH2.0 Solution B = 60% acetonitrile-containing 50 mM phosphate buffer pH2.
0 Elution method: Elute from solution A 100% to solution B 100% at a flow rate of 1.5 ml / min by a linear gradient method for 30 minutes.

Detection: 280 nm and 214 nm.

Example 1. Production and purification of enzyme and estimation of molecular weight (A) Yeast S. cerevisiae X2180-1B was cultivated with shaking in a Barkholder medium at 30 ° C for 28 hours, centrifuged at 3000 rpm for 10 minutes, and wet. The cells were obtained. 1.5 g of these cells were mixed with 1 g of hydrophilic photo-crosslinkable prepolymer ENT2000 (manufactured by Kansai Paint Co., Ltd.) and 2 mg of benzoin ethyl ether, a polymerization accelerator, and polymerized by irradiating with near-ultraviolet rays (300 to 400 nm), and solid The microbial cells were created. The immobilized cells were cultured in a Bark holder medium at 30 ° C for 24 hours. The α-MF degrading activity of this culture was 1.5 μg, α-MF / 1 ml / 24 hours. 400 ml of this culture solution was aseptically passed through a 2.2 μm Millipore filter and further concentrated using an ultraperforated membrane PU-10 (manufactured by Amicon). Overspeed is about 25ml / 3 with a 43mm filter
It was performed at 0 minutes for about 50 ml / 30 minutes with a 60 mm filter.

The impermeable section was further dialyzed against water using cellophane tubing. The dialyzed solution was centrifuged at 10,000 rpm for 10 minutes and then freeze-dried to obtain a white powdery crude product. still,
The enzyme activity of this powder was stable for at least 3 months, and the enzyme activity in an aqueous solution was also stable.

The powder obtained above was mixed with 10 mM Tris-hydrochloric acid buffer (pH 8.
2) Dissolved in 4 ml, 3.5 ml of which was subjected to column chromatography using DEAE-Sephacel (Pharmacia) (0.
5 × 2.0 cm) and 0.2, 0.4, 0.6, 0.8, 1.0M Na at each concentration
Elution was performed stepwise with 10 mM Tris-hydrochloric acid buffer (pH 8.2) containing Cl (flow rate 1 ml / 15 minutes). The results are shown in Fig. 1. The 0.2 M NaCl elution fraction shown in FIG. 1 was collected as the peptidase active portion.

Next, the molecular weight of the enzyme is TS, which is a silica gel resin.
It was measured by molecular sieve chromatography (0.75 × 60 cm) using K-Gel G3000SW (manufactured by Toyo Soda Co., Ltd.). That is, 0.2M NaCl purified by DEAE-Sephacel obtained above
Elute 0.1 ml of the eluted fraction with 0.1 M potassium phosphate buffer (0.2 M
Elution was performed with M NaCl (pH 7.0) (flow rate 0.7 ml / min, 280
nm0.10OD). As a result, a peak of the enzyme activity of the present invention was obtained at a retention time of 21.21 minutes, and the molecular weight of the active form was estimated to be about 100,000 by comparison with the elution pattern (retention time) of a standard protein of known molecular weight. (Fig. 2).

(B) A peptidase concentrate obtained by the same culture method and concentration method as described in Example 1- (A) above was used as Example 1-
Apply to DEAE-Sephacel column as in (A), 0.2M NaC
The experiment of collecting the fractions eluted at 1 was repeated 5 times.

Next, the molecular weight of the fraction thus obtained was estimated by molecular sieve chromatography (1 × 45 cm) using Sephacryl S500 (Pharmacia), which is a Cefacrylic resin. That is, 0.1 m of the active fraction obtained above
l was eluted with 0.1 M acetate buffer solution (pH 6.0) (flow rate 0.15 ml
/ Min). As a result, an activity peak of the enzyme was obtained at a retention volume of 30 ml, and the molecular weight of the active form of the enzyme was estimated to be about 750,000 by comparing the elution patterns of proteins of known molecular weight (Fig. 3). ).

The difference from the molecular weight estimated in Example 1- (A) is largely due to the difference in the measuring method (molecular sieving column), and the purified enzymes are essentially the same. It seems that the molecular weight was small due to the adsorptivity of the silica gel column.

Next, the subunit structure of this enzyme was analyzed using SDS polyacrylamide gel electrophoresis (SDS-PAGE). As a sample, a fraction obtained by fractionating with the above Sephacryl S-500 column was dialyzed and desalted, and then freeze-dried. Freeze-dried preparation containing SDS 2.3% 60 mM
SDS-PAGE by 13.6 polyacrylamide gel (16 × 18cm × 0.75mm) after dissolving in Tris-HCl buffer solution (pH6.8)
Was performed (current value 40 mA, electrophoresis for 3 hours), and the resulting protein band was stained with Coomassie Brilliant Blue R250. As a result, the enzyme was judged to be a protein composed of subunits of about 55 kilodaltons and about 63 kilodaltons by comparison with electrophoresis of proteins of known molecular weight (Fig. 4).

Example 2. Properties of enzyme (1) Optimal temperature of action 0.2 M NaCl elution fraction 10 μm purified by DEAE-Sephacel
1.5 μg of chemically synthesized α-MF as a substrate
g as a substrate in 0.05M acetate buffer (pH 4.0) at 20 ℃ to 50 ℃
The rate of action was measured at each temperature of ° C. As a result, the optimum temperature was 35 to 42 ° C (Fig. 5).

(2) Optimum action pH 0.2 μM NaCl elution fraction 10μ purified by DEAE-Sephacel
About 1 liter, chemically synthesized α-MF 1.5 μg as a substrate, 30 ℃
The action rates were measured in 0.05M acetate buffer and phosphate buffer at pH 2 to 8 respectively. As a result, pH 3 to 4.5 was optimal (Fig. 6).

(3) Substrate specificity of the enzyme The substrate specificity of the enzyme was determined by using DEAE-Sephacel chromatography 0.2M elution section and using 0.05M acetate buffer (pH 4.0) at 30 ° C, 24
Analysis was carried out by reacting for a period of time and examining the rate of substrate decomposition by HPLC. As a substrate 1. Having a Leu-Lys bond in the center of the molecule 2. Having a Lys-X or X-Lys bond inside the molecule 3. Leu-X or X-Leu bond inside the molecule The peptides shown in Table 1 were used as those having a bond. (X is 2.
In the case of, an amino acid other than Leu is shown, and in the case of 3, an amino acid other than Lys is shown. ) The results are shown in Table 2. According to this result, only the peptide having a Leu-Lys bond in the molecule and contained in the above 1. was decomposed, and it was shown that the enzyme of the present invention is specific to the Leu-Lys bond.

(5) Responses to Various Inhibitors Using 0.05 μm of α-MF chemically synthesized using the purified enzyme described in Example 2- (4) as a substrate, 0.05 M acetate buffer (p
The effect of various inhibitors on the enzyme activity was investigated in H4.0). Table 3 shows the inhibitors used, their concentrations, and the presence or absence of inhibition. As a result, the peptidase of the present invention was not inhibited by any of the inhibitors used. The results are shown in Table 3.
Shown in

[Brief description of drawings]

FIG. 1 shows DEAE-Seph of a crude purified solution containing the enzyme of the present invention.
It is a graph which shows the elution progress in the column chromatography using acel. 2 and 3 show TSK-Gel G of the enzyme of the present invention.
It is a graph which shows the result of molecular weight measurement in the high performance liquid chromatography which used 3000SW and Sephacryl S500 molecular sieve. Figure 4 shows the results of SDS-PAGE of this enzyme. FIG. 5 is a graph showing the temperature characteristics of the enzyme of the present invention. FIG. 6 is a graph showing pH characteristics of the enzyme of the present invention.

Claims (7)

[Claims] 1. The following properties: 0.1-0.6 M NaCl adsorbed on DEAE cellulose or DEAE dextran that specifically cleaves the C-terminal side of Leu of the amino acid sequence Leu-Lys ... existing in the peptide chain. The molecular weight estimated by molecular sieving chromatography using silica gel resin is about 100,000, and the molecular weight estimated by molecular sieving chromatography using Cefacrylic resin is about 750,000. Composed of subunits of about 55 kilodaltons and about 63 kilodaltons, optimum pH of action is 3 to 4.5, optimum temperature of action is 35 ° C to 42 ° C, 2 × 10 ^-4 molar concentration of monoiodoacetic acid, Phenylmethyl sulfonyl fluoride (PMSF), P-chloro-mercury benzoic acid (PCMB), diisopropyl fluorophosphate (DFP), 1 × 10 ^-3 molar toshi L-L-lysine chloromethyl ketone (TLCK), EDTA,
A peptidase secreted from yeast, which is characterized in that the enzyme activity is not inhibited by leupeptin at a concentration of 1 × 10 ⁻⁴ molar and pepstatin at a concentration of 1 × 10 ⁻⁵ . 2. A yeast α-factor, Trp-His-Tr.
p-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Ty
Acting on r, Trp-His-Trp-Leu-Gln-Leu and Lys
A peptidase according to claim 1, which provides -Pro-Gly-Gln-Pro-Met-Tyr. 3. The peptidase according to claim 1, wherein the yeast is Saccharomyces yeast. 4. Saccharomyces yeasts are immobilized on an immobilizing carrier at a high density and cultivated, and the peptidase is recovered from the culture supernatant. The following properties are present in peptide chains: Leu-Lys ... The specific molecular weight estimated by molecular sieving chromatography using silica gel-based resin, which is adsorbed to DEAE cellulose or DEAE dextran and eluted with 0.1-0.6 M NaCl, which specifically cleaves the C-terminal side of Leu in the amino acid sequence 100,000, and the estimated molecular weight by molecular sieve chromatography using Cefacrylic resin is about 750,000. By SDS / PAGE, it is composed of subunits of about 55 and 63 kilodaltons. is 3-4.5, is 35 ° C. through 42 ° C. effect optimum temperature, 2 × 10 ^-4 molar concentration of monoiodoacetic acid, phenylalanine methyl sulfonyl fluoride (PMSF), P- Lolo - Mercury benzoic acid (PCMB), diisopropylfluorophosphate phosphine benzoate (DFP), 1 × 10 ^-3 molar concentration of tosyl -L- lysine chloromethyl ketone (TLCK), EDTA,
1. A method for producing a peptidase secreted from yeast, which comprises not inhibiting enzyme activity with leupeptin at a concentration of 1 × 10 ⁻⁴ mol and pepstatin at a concentration of 1 × 10 ⁻⁵ . 5. The method according to claim 4, wherein the immobilized carrier is a hydrophilic photocrosslinkable resin. 6. The method according to claim 4, wherein yeast is immobilized at a density of 1 to 2 g per 1 g of the immobilized carrier. 7. The method according to claim 4, wherein the culture supernatant is purified using a DEAE cellulose or dextran carrier and a molecular sieve carrier.