JPH06153945A - New protease and microorganism having new protease activity - Google Patents

Abstract

PURPOSE:To obtain an entirely new protease and a microorganism having the new protease activity. CONSTITUTION:A protease is obtained by replacing an amino acid residue located in the active central site or a substrate binding site of a serine protease derived from Lactococcus.lactis with another amino acid.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel serine protease, and more specifically to Lactococcus lactis.
Subspecies Lactis NCDO763 ( Lactoco
The present invention relates to a novel protease in which a part of the constituent amino acids of the serine protease derived from ccus lactis subsp. lactis NCDO763) is replaced with another amino acid.

[0002]

2. Description of the Related Art Conventionally, the protein degradation system of dairy lactic acid bacteria has played an important role in satisfying the complicated amino acid requirements and growing well in milk. Lactococcus lactis (hereinafter referred to as "L. lactis"), which is mainly used for cheese production, is a proteolytic cell wall-bound protease that is used not only for the growth of bacteria but also for the flavor and aging period of products. Has been attracting attention because it is thought to have an important effect.

L. Proteases produced by lactis include PI type that mainly decomposes β-casein and α
P that decomposes s1-casein and β-casein to the same degree
There are III types. A part of the degradation product of β-casein by PI type protease has been identified as a bitter peptide by its amino acid sequence.

The above-mentioned PI type protease and PIII type protease have very high homology in their DNA base sequences and amino acid sequences, and the amino acid sequences of their respective active center sites are the activities of Bacillus subtilis-derived protease, subtilisin. It is known that the homology with the central region is high.

[0005]

SUMMARY OF THE INVENTION In a lactis-derived serine protease, a novel protease having a novel enzyme specificity is obtained by substituting an amino acid residue located at the active center site or the substrate binding site with another amino acid, and the novel protease L. based on the produced gene information. It is intended to obtain a novel microorganism introduced into lactis and introduced with a novel protease activity.

More specifically, it is considered that lactic acid bacteria decompose proteins in the medium by the action of protease and utilize them as the amino acid source. Although various proteases derived from lactic acid bacteria have been isolated and research is progressing, it has been found that there is a large difference in the substrate specificity for casein despite the fact that the amino acid sequence homology is very high. There is. It is, for example, Lactococcus L. Lactis subspecies cremoris SK11 protease produced by SK11; 763 produced by Lactis subspecies Lactis NCDO763
This is remarkable when compared with protease.

The amino acid sequence homology between SK11 protease and 763 protease is as high as 97.5%, and there are only 49 differences in 1902 amino acid residues except for the SK11 repeating portion. However, SK11 protease cleaves α, β, κ-casein as a substrate, but 763 protease mainly cleaves β-casein, producing a bitter peptide. What is the difference in this substrate specificity? It was completely unknown if it was.

Therefore, in the present invention, L. By converting an amino acid residue of a lactis-derived serine protease into another amino acid, we tried to create a mutant enzyme with different specific activity, thermostability, optimum pH, or substrate specificity, and produced a completely new enzyme. The purpose is to obtain a microorganism that does.

[0009]

The novel protease according to the invention described in claim 1 comprises L. The amino acid residue located at the active center site or substrate binding site of lactis-derived serine protease is substituted with another amino acid.

Specifically, in the novel protease I216L according to the invention described in claim 2, L. In 763 serine protease derived from lactis, isoleucine at position 216 of the amino acid sequence of the 763 serine protease is replaced with leucine.

Furthermore, in the novel protease D353K or D353N according to the invention described in claim 3, L. In the 763 serine protease derived from lactis, the
The aspartic acid at position 353 of the amino acid sequence of 63 serine protease is substituted with lysine or asparagine.

The novel protease T409S according to the invention of claim 4 is L. 7 derived from lactis
In the 63 serine protease, threonine at the 409th position of the amino acid sequence of the 763 serine protease is replaced with serine.

Furthermore, in the novel protease T325K or T325D according to the invention described in claim 5, L. In the 763 serine protease derived from lactis, the
The threonine at position 325 of the amino acid sequence of 63 serine protease is replaced with lysine or aspartic acid.

The plasmid pHD-IL2 having the novel protease activity according to the invention of claim 6 is also provided.
16, pHD-TK325, pHD-TD325, pH
D-DK353, pHD-DN353 and pHD-TS
In 409, genetic information for producing the novel protease according to any one of claims 2 to 5 is incorporated.

Further, in the microorganism having novel protease activity according to the invention described in claim 7, the method according to claim 6
Which has any of the plasmids incorporating the gene information for producing the novel protease described in 1.

[0016]

In the present invention, L. A novel protease having a novel enzyme specificity can be obtained by substituting an amino acid residue located in the active center site or substrate binding site of a lactis-derived serine protease with another amino acid. Based on the gene information of the production of this novel protease, L. It is possible to obtain a novel microorganism introduced into lactis and introduced with a novel protease activity.

Specifically, the serine protease, which is the basis for introducing the mutation, is a lactic acid bacterium L. Lactis Subspecies
Lactis NCDO (National collection of dairy orga
nisms) 563 strain 55Kb plasmid (pLP763)
It is a serine protease whose production information is retained above. The 763 protease contributes to the serine proteinase family, and, like other serine proteases, Asp, His, and Ser residues form the active center.

In the present invention, the site for introducing the mutation was determined as follows. Converting all 43 amino acid residues cannot be said to be efficient and must be targeted among them. Therefore, BIOCES [E], a support system for protein engineering and pharmaceutical / agrochemical design (NEC Corporation)
Manufactured) was used. That is, subtilisin (B. subtilis), which is believed to have three-dimensional structural homology with 763 protease
Computer graphics (hereinafter C) of the three-dimensional structure of the serine protease secreted by Bacillus subtilis
The structure of 763 protease was visually captured in a three-dimensional image, and mutations were introduced at sites corresponding to the following conditions.

(1) Hydrophobic region at the center of protein molecule (Hydr
Select positions and amino acid residues that do not disrupt the ophobic core (2) N-terminal region (18) that has homology with the entire subtilisin
8th-679th) only. (3) Of the N-terminal region, the homology with subtilisin is particularly high, and only the portion considered to have a high similarity in three-dimensional structure is narrowed down. (4) Select residues that are predicted to be sterically close to the substrate binding site

As a candidate for mutation introduction satisfying these conditions, the 325th Thr (T) residue, the 353rd Asp (D) residue and the 409th Thr (T) residue are raised, Thr-325
Is Lys [T325K mutant] or Asp [T325D mutant], Asp-353 is SK11 type Asn (N) [D353N].
Mutant] and Thr-409 also turned into SK11 type Ser (S) [T
409S mutant], respectively. In addition, D that replaces Asp-353 with negative charge with Lys (K) with positive charge
A 353K mutant was also prepared at the same time.

Subtilisin, which is a serine protease, loses its activity when the Asp residue at the active center is converted to Asn.
Substitution with u has been shown to enhance activity.
Therefore, H in which the C-terminal of 763 protease is deleted
The 217th position which is considered to be the active center of D2 protease
The 216th Ile (I) residue next to the Asp (D) residue was replaced with Leu [I216L mutant], and at the same time, the predicted active center Asp (D) residue was replaced with Asn for comparison. We decided to make [D217N mutant].

That is, Lactococcus L. For the purpose of converting the substrate specificity of the protease 763 protease produced by lactis, a mutation was introduced in the region around the substrate binding site. Specifically, Ile-216 is Leu [I216L mutant], Asp-217 is Asn [D217N mutant], Thr-32.
5 is Lys [T325K mutant] or Asp [T325D
Mutant] and Asp-353 were replaced with Asn (N) [D353N mutant] or Lys with [D353K mutant], and Thr-409 with Ser (S) replaced with [T409S mutant]. FIG. 1 is an explanatory diagram showing a display of a site where a mutation has been introduced.

Next, the activity of the mutant protease prepared as described above was measured. N-suc-A as a substrate for activity measurement
Using la-Ala-Pro-Phe-pNA (hereinafter abbreviated as PF substrate),
The absorbance at 410 nm was used as an index of activity. The protease activity of the culture supernatant of the strain producing 763 protease was 1
00, the activity of each mutant protease was expressed numerically.

The characteristics of each mutant protease specifically obtained are as follows. (1) Protease I216L The protease activity was 60, and the activity was decreased.

(2) Protease D353K The protease activity was 5, and the activity was decreased. However, when N-suc-Ala-Ala-Pro-Leu-pNA (hereinafter abbreviated as PL substrate) is used as a substrate, the activity is increased and the PL substrate shows about 1.8 times the activity of the PF substrate. The activity was stronger when used. The affinity of the 763 protease for the substrate of the D353K mutant protease was clearly different from that of the 763 protease because the activity of the 763 protease was stronger when the PF substrate was used. To confirm the change in affinity for the substrate, α-Mating Factor (Mating Fac
tor) was used as a substrate, the 763 protease and the D353K mutant protease showed a clearly different tendency of enzymatic reaction.

(3) Protease D323N The protease activity was 130, and the activity was increased.

(4) Protease T409S The protease activity was 70, which was decreased.

(5) Protease T325K The protease activity was 50, and the activity was decreased.

(6) Protease T325D The protease activity was 5, and the activity was decreased. Further, this mutant protease was characterized in that the progress of processing was extremely slow, and it took time until the activity was expressed.

The above-mentioned plasmid pHD-IL216 producing the protease I216L was used as a microorganism
No. 019 (June 23, 1992), the plasmid pHD-TK325 producing the protease T325K.
Is a plasmid p that produces the protease T325D, which is based on Microtechnology Research Institute No. 13022 (June 23, 1992).
HD-TD325 is based on Micro Engineering Research Institute No. 13018 (1992
June 23, 2013), the plasmid pHD-DK353 which produces protease D353K
No. 1 (June 23, 1992), Protease D353
The plasmid pHD-DN353 which produces N is the plasmid No. 13020 of Micro Incorporated (June 23, 1992), and the plasmid pHD-TS which produces protease T409S.
Reference numeral 409 is Microbiology Research Institute No. 13023 (23 June 1992)
The date) has been deposited.

[0031]

EXAMPLES The present invention will be described in detail below with reference to specific examples. I) Preparation of mutant protease FIG. 2 is an explanatory view showing the preparation process of pHD-IL216. The manufacturing process will be described below. In addition, * in the figure indicates a portion into which a mutation has been introduced.

1) Construction of plasmid pHD2 A lactic acid bacterium vector pHD2 was obtained in which the 763 protease gene lacking the C-terminal of 763 protease was incorporated into pGKV11.

2) Construction of plasmid pHD119 The obtained HpaI fragment of pHD2 was incorporated into the SmaI fragment site of plasmid pUC119 (Takara Shuzo Co., Ltd.) using E. coli as a host to obtain pHD119. This plasmid pH
Based on D119, a mutation was introduced in E. coli by the site-directed mutagenesis method.

3) Mutagenesis protease gene (plus
(Mido) site-Directed mutagenesis method is dU
Was performed by the Kunkel method using a single-stranded DNA containing DNA as a template. A Mutan-K kit manufactured by Takara Shuzo Co., Ltd. was used as a reagent. That is, specifically, the gene whose mutation is to be
This plasmid was inserted into C119, and this plasmid was introduced into Escherichia coli BW313 of dut ⁻ , ung ⁻ , and single-stranded DNA in which a part of thymine was replaced with uracil was extracted and used as a template. A mutation-introducing primer is annealed to this template DNA,
4 Complementary strands were synthesized using DNA polymerase and E. coli DNA ligase and reintroduced into E. coli as circular double-stranded DNA. Plasmids in which the desired mutation was introduced were selected from the obtained transformants and introduced into lactic acid bacteria to express the mutant protease.

By the above operation, a plasmid into which the D217N mutation in which Asp-217 had been replaced with Asn was introduced was transformed into pDN217.
(Alternatively, a plasmid introduced with the I216L mutation in which Ile-216 was replaced with Leu was designated as pIL216), and the AvaII fragment was excised from these plasmids and ligated to pHD2, and the plasmid pHD-DN217 ( Or pHD-IL
216) was created.

As the host, five species (55 Kb, 15 Kb, 8.5 Kb, 3 Kb, 1.5 K, which are retained by Lactococcus lactis NCDO505 strain, are retained.
Lactococcus lactis MQ954, which was prophage-free by removing the plasmid of b), was used.

By the same operation, pHD-TK
325, pHD-TD325, pHD-DK353, p
HD-DN353, pHD-TS409 was obtained. In addition, the primer DNA corresponding to the target mutation introduction is from ABI
It was synthesized using DNA synthesizer 380B. The synthesis method was the phosphorous acid solid phase method. The reaction was performed on a 0.5 μmol scale to obtain a DNA oligomer of about 2 to 5 OD. 0.00 of the DNA oligomers synthesized in this way
1 OD was used as a primer for the production of the mutated protease described below.

The design of the mutation-introducing primer DNA is carried out by selecting the codon corresponding to the amino acid residue to be mutated.
Of these, we chose codons that are commonly used in lactic acid bacteria. The length of the primer was 17 to 27 bases and was designed so that mutations could be introduced efficiently. The sequences of the primers used for mutagenesis are as shown below.

I216L mutation primer; TCT CGG TTC TTG ACA GT
(17mer) D217N mutation primer; CGG TTA TTA ACA GTG GC
(17mer) D353N mutation primer; TGT CCT TAG GTT CGA ATT C
AG GCA A (25mer) D353K mutation primer; TGT CCT TAG GTT CGA AAT C
AG GCA ACC (27mer) T409S Mutation primer; AAT GGT GGG ATC TCC CGG G
AC ATC AC (26mer) T325K mutation primer; ACA CTT CTG CTA AAA CCG G
GT CA (23mer) T325D mutation primer; ACA CTT CTG CTG AAA CCG G
GT CA (23mer)

II) Production of Mutant Protease and Its Characteristics Each mutant protease-introduced plasmid prepared in the above step was transformed into strain MQ954, and this lactic acid bacterium was grown to a steady state. Then, the supernatant liquid from which the bacterial cells were removed was collected and filtered through a cellulose acetate filter, and 50 μl of this filtrate was taken to confirm the characteristics of each mutant protease.

1) Proteases I216L and D217
N Mutant protease producing plasmid pHD-IL obtained
216 and pHD-DN217 were used to detect the protease activity and anti-7 activity of proteases I216L and D217N.
The reaction with 63 protease antiserum was confirmed.

First, pHD-IL217 and pHD-IL incorporating the D217N and I216L mutant genes were incorporated.
The culture supernatant of 216 was subjected to pHD2 using SDS-PAGE.
We compared and examined. As a result, in D217N, a thick band was observed on the high molecular weight side as compared with pHD2, and in I216L, a migration pattern almost the same as that of pHD2 was obtained.

Therefore, an antigen-antibody reaction was tried using an anti-763 protease antiserum to determine whether the bands at D217N and I216L were derived from protease. As a result, the band on the high molecular weight side was judged to be derived from protease because the band cross-reacted with the antiserum. In D217N, 3 out of 8 clones were targeted, and in I216L, 1 out of 8 clones was targeted.

Next, the activity of protease was measured.
No activity was detected for D217N. I21
Regarding 6L, when HD2 protease was used as a control, the measured value of about half thereof was shown. By this mutation introduction, D217N was inactivated and I216L also decreased in activity. This suggests that Asp-217 is strongly involved in the expression of activity.

Sample 5 was used to measure the protease activity.
2x reaction solution (0.1 M acetate buffer; pH 5.
4,10 mM calcium chloride, 1 mM Suc-Ala-Ala-Pr
50 μl of o-Phe-pNA) was added, the mixture was reacted at 37 ° C. for 12 hours, and the activity was measured by absorption of visible light at 410 nm. In addition, pNA substrate (Suc-Ala-Ala-Pro-Phe-pNA)
Was purchased from Peptide Institute Inc.

The reaction with the anti-763 protease antiserum was carried out by fractionating the culture supernatant by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotting on a nitrocellulose filter, which was supplied by Promega. Protoblot Western Blot A
The P system (Proto Blot Westerm Blot AP System) was used.

From the above results, it is suggested that Asp-217 is the active center of 763 protease, which is one of serine proteases, as expected from comparison of the amino acid sequences with other serine proteases. It was In addition, from the result of the analysis of the DNA base sequence, it was presumed that the first expressed 763 protease was a precursor substance before. After that, it is considered to be cleaved at the time of membrane permeation to become a precursor substance, and further cleaved at the time of becoming a mature form in which the activity is expressed. A band was detected on the high molecular weight side only for D217N that does not express activity, suggesting that the cleavage of the precursor substance requires the protease activity of 763 protease.

2) Protease D353K In the test using the PF substrate and the PL substrate, which are synthetic substrates, the present protease was found to have reversed substrate specificity. Also, β
-The cleavage activity for casein was inactivated. But,
There was no change in the viable cell count in the skim milk medium at steady state. Therefore, as a result of investigating the tendency of the substrate specificity using the culture supernatant using the synthetic peptide as a substrate, an enzymatic reaction which is clearly different from the wild type when α-Mating Factor was used as the substrate. Showed a tendency.

Furthermore, in order to understand the changes in the substrate specificity of the mutant protease, L. et al. Lactis MQ954 p
Using the cells transformed with the HD2, D217N, and D353K plasmids, a synthetic peptide substrate; Bs
It was reacted with each substrate of ite2 (synthetic peptide), model peptide (manufactured by Serva), and α-mate factor (manufactured by Peptide Institute, Inc.).

The characteristics of each substrate are as follows. (1) B-site2; β-casein partial sequence (10 residues) 763 has one of the positions at which the protease specifically cleaves in the center. Val-Pro-Tyr-Pro-Gln-Arg-Asp-Met-Pro-
It has the structure of Ile-NH ₂ . (2) Model peptide; a peptide (5 residues) designed to investigate the specificity of various proteases. Leu-Met-Ar
It has the structure of g-Phe-Ala. (3) α-mate factor; 13 residues, 763
One of the arbitrarily selected peptide hormones on the market to investigate the specificity of proteases.

HPLC is 60 manufactured by Waters.
It is performed by the OE system and also Maxi of Waters
The analysis was performed by the ma825 system. The column is also Waterus μBondasphere 5μm C18-100A 3.9m
m × 15 cm was used.

The enzyme solution was prepared by using M17 medium (polypept
one 5.0g, phytone pepton 5.0g, yeast extract 2.5g,
beef extract 5.0g, ascorbic acid 5.0g, β-glycero
phosphate 19.0g ； H ₂ O 900ml / After sterilization 1M MgSO ₄ 1ml,
1 ml of cells pre-cultured with 5% glucose 100 ml) was added to 50 ml of CAYE-GP medium (yeast extract 2.5 g, sugar 5.0).
g, NaCl 1.5g, CaCl ₂ 0.55g; H ₂ O 890ml / 20% after sterilization
casamino acid 25ml, 5mg / ml Tryptophane 10ml, 1M Mg
SO ₄ 1ml, 1M β-glycerophosphate 75ml)
The culture was allowed to stand overnight at 0 ° C. Culture medium at 5000 xg for 10
After cooling and centrifuging for minutes, the supernatant is dialyzed at 4 ° C (water, 10 mM
2 times each 2 liters in order of ammonium carbonate aqueous solution),
Lyophilized. The dialysis dry powder was dissolved in 2 ml of 50% glycerol aqueous solution and used in the subsequent experiments.

The reaction with the synthetic peptide substrate was carried out using 30 μl of water.
10 × Assay buffer (0.5M AcONH ₄ (pH6.0), 10mM
(AcO) ₂ Ca) 20 μl, peptide substrate solution (10 mM)
After adding 10 μl, add 40 μl of enzyme solution to 30
The reaction was carried out at ° C. 20 μl of reaction solution was taken out at a predetermined time (10, 30, 60 minutes, 3 hours, overnight), 180 μl
Was added to 1% aqueous TFA solution to stop the reaction, and HPLC
Was analyzed.

The analysis of enzyme digestion by HPLC was carried out by injecting 100 μl of the above-mentioned stop solution into the HPLC and adding 0.1% TFA.
Elution was performed with a linear gradient of a 10% to 45% acetonitrile aqueous solution containing the above, and the absorbance was detected at 220 nm.

The result is 1 when B-site 2 is used.
After 6 hours, it was not digested at all by any enzyme solution. This is because the enzyme concentration in this experiment is low,
Even in the produced wild-type protease, this peptide substrate was β
-Explained because it is less likely to be cleaved than casein.

When the model peptide was used as a substrate, almost all the enzymes were digested in the same manner. Since the digestive rate of this peptide was originally not so fast as B-site2, it was presumed that the observed digestive reaction was due to other peptidases existing as contaminants.

By the way, each protease D217N, D353K, using α-mate factor as a substrate,
Comparing the action of HD2, protease D353
It was confirmed that K suggests that it has a different substrate specificity from HD2 protease.

3) Protease D353N Obtained mutant protease producing plasmid pHD-DN
353 using the protease D353N as described above.
The protease activity was confirmed to be 130, and the activity was increased.

4) Protease T409S Obtained mutant protease producing plasmid pHD-TS
409 using the protease T409S as described above.
The protease activity was confirmed to be 70, and the activity was decreased.

5) Protease T325K Obtained mutant protease producing plasmid pHD-TK
325 with the protease T325K as described above.
The protease activity was confirmed to be 50, and the activity was decreased.

6) Protease T325D Obtained mutant protease producing plasmid pHD-TD
325 with the protease T325D as described above.
The protease activity was confirmed to be 5, and the activity was decreased. It was also confirmed that this mutant protease has an extremely slow progress of processing and has a characteristic that it takes time until the activity is expressed.

III) Comparison of Properties of Mutant Proteases D353K protease hardly decomposes not only α- and κ-caseins but also β-casein, and on the other hand, unlike inactive mutant (D217N) It has been confirmed that it is useful for the growth of the host in the milk medium. From this, it was inferred that the substrate specificity of D353K protease changed from the wild type, so that it digested other proteins derived from milk without degrading casein to supply amino acids to the host. The protease activity of the mutant protein prepared there was measured, and it was examined whether or not the substrate specificity was changed.

For the preparation of the mutant protein, lactic acid bacteria carrying the mutant gene were grown to a steady state, and after collecting the bacteria, the supernatant was collected and filtered with a cellulose acetate filter, and 50 μl was used for activity measurement. In addition, in order to confirm the production of protein, trichloroacetic acid (TC
A) was added to collect the protein as a precipitate, and SDS-
The production of protein and its molecular weight were confirmed by polyacrylamide gel electrophoresis.

As the activity measurement conditions, the following substrates were used, and 50 μl of buffer A (100 mM sodium aceta) was used.
50 μl of the above supernatant was added to te buffer (pH 5.4), 10 mM CaCl ₂ , 1 mM substrate) and the reaction was carried out at 30 ° C.
The absorption at 10 nm was measured and used as an index of activity. Synthetic peptide; N-suc-Ala-Ala-Pro-Phe-pNA (PF subs
trate) N-suc-Ala-Ala-Pro-Leu-pNA (PL substrate) protein; α-casein β-casein κ-casein

1) Each of mutated protein to PF substrate
Protease Activity of Species Variants FIG. 3 is a diagram showing the measurement results of the protease activity of various mutants with respect to the PF substrate of various mutant proteins. Further, the following Table 1 summarizes the activity of various mutant proteins on the PF substrate and the processing mode. In the figure, the activity is defined with the activity of the wild strain as 100 for convenience, and the activity of each mutant protein is shown by a numerical value. This measurement does not use purified protein, nor does it require specific activity, so the significance of the value is not very important, but a rough judgment such as the presence or absence of activity is made. It is effective when

[0066]

[Table 1]

I would like to touch on processing for a moment. Previously, we reported on a mutant protein in which a mutation was introduced at the active center to inactivate the activity, but the mutant protein has a slower mobility in electrophoresis and a shift to a higher molecular weight side than the wild type strain. It was revealed.
The N-terminal amino acid sequence of the mutant protein was determined in order to investigate what is responsible for this delay in mobility.
It was found that the fragments to be cleaved remained attached during the process of (uration). From these experiments, it was suggested that the activity expression of protease and the cleavage of pro sequence are closely related, and it is considered that the protease having lost the activity cannot cleave its own pro sequence.

As described above, among the above three kinds of mutant proteins, D353N was more active than the wild type strain, and
409S resulted in weak activity, but in any case, even if a mutation was introduced around the substrate binding site, it was possible to successfully create a mutant protein that retained the activity.

Next, paying attention to the D353K mutant, the activity using the PF substrate was only 1/20 as compared with the wild type strain, and was greatly reduced. However, it was confirmed from the result of electrophoresis that the processing was completely performed. As described above, it is considered that the protease whose activity has disappeared is not processed, and thus D353K
It was suggested that the cleavage activity of the mutant towards the PF substrate (AAPF) was significantly reduced, but the activity of cleaving the pro sequence was not significantly reduced. That is, the activity of the D353K mutant may be as high as that of the wild strain.

2) PF and P of wild strain and D353K mutant
Difference in activity for L substrate FIG. 4 is a diagram showing the difference in activity for the PF and PL substrates between the wild strain and the D353K mutant. In order to clarify that the substrate specificity of the D353K mutant is different from that of the wild type strain, various synthetic substrates were screened,
A PL substrate (AAPL) having a structure similar to that of the PF substrate was selected as a candidate. The activity of the D353K mutant was increased by about 1.8 times by using the PL substrate as compared with the case of using the PF substrate, but was decreased by 0.6 times in the wild strain. From the above results, it was revealed that the affinity for the PF and PL substrates was clearly different between the D353K mutant and the wild strain.

3) Difference in activity against casein Although the above results were obtained for synthetic substrates, the activity against actual substrate casein was examined next. The culture supernatant was treated by the method described above, a part thereof was taken, and an attempt was made to cleave α, β, κ-casein.

As a result, the wild type strain decomposed β-casein, but the D353K mutein did not cleave any casein. In conclusion, by converting the Asp residue at position 353 to a Lys residue, a mutant protease that retains protease activity but does not cleave β-casein could be constructed.

4) Effect of mutant protease on host growth
Effect (1) As mentioned above, the production of a mutant protein with an altered affinity for a substrate as compared to the wild strain was described. Then, this mutant protein actually cleaves the protein in the medium, It was verified whether or not the growth of bacteria was promoted. In addition, it was also measured to what extent the peptide, which is a degradation product, was released into the medium by cleaving the protein in the medium with a protease.

First, a skim milk medium (10% skim milk,
After culturing the strains retaining the above four kinds of plasmids with 75 mM β-glycerophosphate, 1% glucose) for 24 hours, the viable cell count was measured. The results are shown in Table 2 below.

[0075]

[Table 2]

Bacteria not having a protease gene (pGK
V11) and a bacterium having a protease gene whose activity has disappeared (pHD-DN217) have a concentration of 3 to 4 × 10 ⁸ / ml, whereas a bacterium having a 763 protease gene (pHD2) or a mutant protease gene (pHD-DK)
In 353), the viable cell count of 3 to 4 × 10 ⁹ / ml, which is about 10 times, was observed. From this result, as described in the previous section, D353
Although the activity of K mutant protein against β-casein disappeared, it was revealed that the protein in the skim milk medium was cleaved to supply the amino acid source, and the growth of the bacteria was promoted to the same extent as the wild strain.

5) Effect of mutant protease on host growth
Effect (2) Next, the OPA reagent (50 mM Na ₂ B ₄ O ₇ , 1% SDS, 0.2% (v
/ v) β-mercaptoethanol, 0.08% (w / v) OPA (o-phathaldia
ldehyde)). The results are shown in Table 3 below.

[0078]

[Table 3]

As is clear from this result, the bacteria having no protease activity (pGKV11, pHD-DN21
Bacteria that have protease activity compared to 7) (pHD-DK
353, pHD2), it is found that a large amount of peptide is released in the medium. Although the amount of free peptide is higher in pHD2, which is considered to be due to cleavage of casein, if the amount of peptide is such that pHD-DK353 is released, it can help the growth of bacteria.

As described above, among the three types of mutants, D
Although the 353K mutant has been mainly described, it has an activity in which the affinity for the substrate (affinity for β-casein and affinity for PF and PL substrates) is changed as compared with the wild strain due to a point mutation near the substrate binding site. A mutant could be created. However, it is doubtful whether it has the activity as strong as the wild strain.

Mutant proteases such as the aforementioned D353K mutants are enzymes that have not been found in nature and are novel substances. Like the method used this time, 76
It was possible to create a new protein by comparing 3 protease and SK11 protease and focusing on the site where the substitution occurred. Since the 353rd residue may be a hotspot that regulates the substrate specificity, this residue may be further converted into various amino acid residues, and not only point mutation but also deletion, duplication, reverse A novel enzyme can be constructed by a method such as various mutations of position and chimera gene.

[0082]

As described above, the present invention can be applied to the L. A novel protease having a novel enzyme specificity can be obtained by substituting an amino acid residue located in the active center site or substrate binding site of a lactis-derived serine protease with another amino acid. Based on the gene information of the production of this novel protease, L. There is an effect that a novel microorganism introduced into lactis and having a novel protease activity can be obtained.

[Brief description of drawings]

FIG. 1 is an explanatory diagram showing a display of a site where a mutation has been introduced.

FIG. 2 is an explanatory diagram showing a production process of pHD-IL216.

FIG. 3 is a diagram showing the measurement results of the protease activity of various mutants against PF substrates of various mutant proteins.

FIG. 4 is a diagram showing the difference in the activity of the wild strain and the D353K mutant on PF and PL substrates.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C12R 1:01) (C12N 1/21 C12R 1:01) (72) Inventor Mayumi Kiwaki Tokyo Port Higashi-Shimbashi, 1-119, Yakult Honsha Co., Ltd. (72) Inventor Hirashima Showa, Minato-ku, Tokyo Higashi-Shimbashi, 1-1-19 Yakult Honsha (72) Inventor, Hiroshi Kaneko 5-7-1, Shiba Ward, NEC Corporation (72) Inventor Toshikazu Takada 5-7-1, Shiba, Minato-ku, Tokyo NEC Corporation

Claims (7)

[Claims] 1. A Lactococcus ( Lactococcus)
A novel protease characterized in that an amino acid residue located at the active center site or substrate binding site of a lactis- derived serine protease is substituted with another amino acid. 2. Lactococcus Lactococcus
lactis ) -derived 763 serine protease, wherein isoleucine at position 216 of the amino acid sequence of the 763 serine protease is replaced with leucine. 3. Lactococcus Lactococcus
lactis ) -derived 763 serine protease, wherein the aspartic acid at position 353 of the amino acid sequence of the 763 serine protease is substituted with lysine or asparagine.
Or D353N. 4. Lactococcus
lactis ) -derived 763 serine protease, wherein the threonine at the 409th position of the amino acid sequence of the 763 serine protease is substituted with serine, a novel protease T409S. 5. Lactococcus
lactis ) -derived 763 serine protease, wherein a threonine at position 325 of the amino acid sequence of the 763 serine protease is substituted with lysine or aspartic acid, T325K or T325D. 6. A plasmid pHD-IL216, pHD-TK325, pH in which gene information for producing the novel protease according to any one of claims 2 to 5 is incorporated.
D-TD325, pHD-DK353, pHD-DN3
53 and pHD-TS409. 7. A microorganism having any of the plasmids into which the gene information for producing the novel protease according to claim 6 is incorporated.