JPH03251175A - Alkali protease or modified aspergillus oryzae - Google Patents

Abstract

PURPOSE:To advantageously produce and obtain the subject protease having improved heat stability by having disulfide bondings in a molecule by transforming a host with a recombinant plasmid transduced with a gene coding a specific alkali protease of modified Aspergillus oryzae and then culturing. CONSTITUTION:At least, two amino acids in alkali protease of wild Aspergillus oryzae are respectively substituted with cystine to prepare a gene coding alkali protease of modified Aspergillus oryzae having disulfide bondings in a molecule. Next, said gene is transduced into a plasmid to prepare a recombinant plasmid. Then, a host is transformed by using said recombinant plasmid. Thus, the resultant transformant is cultured in a medium and above-mentioned alkali protease of modified Aspergillus oryzae is produced, collected by separation to afford the objective alkali protease of modified Aspergillus oryzae useful in a field of food, etc., having excellent heat resistance.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a mutant of alkaline protease derived from Aspergillus oryzae, which is useful as a proteolytic enzyme.

[Conventional technology] Conventionally, Aspergillus oryzae (
The structure of the alkaline protease gene derived from Spergillus oryzae is completely unknown, and the reality is that the gene has not even been isolated.

Alkaline protease is a hydrolytic enzyme that acts on proteins or partially hydrolyzed products thereof to decompose peptide bonds, and is widely used in medicines, food and drink products, and washing plants.

Therefore, as a result of various studies on the alkaline protease gene derived from Aspergillus oryzae, the present inventors succeeded in isolating and determining the structure of the alkaline protease gene and prepro-type alkaline protease gene derived from Aspergillus oryzae for the first time. , filed a patent application (Japanese Patent Application No. 63-51777 and
3-170018 patent application specification).

Now, in order to utilize useful enzymes such as alkaline protease as effective catalysts in vitro, temperature, pH, oxygen,
It is necessary to increase the stability of proteins against solvents, pressure, etc.

In particular, attempts to increase temperature: that is, thermal stability, are actively being carried out by so-called protein engineering using site-specific mutation technology of genes. For example, it has been reported that by substituting one or two amino acids in the wild-type subtilisin E enzyme with other amino acids, a mutant subtilisin E with improved thermostability compared to the wild type can be prepared ( JP-A No. 1-137972).

[Problems to be Solved by the Invention] Therefore, the first object of the present invention is to develop a mutant alkaline protease that is superior in properties such as thermostability compared to the wild-type Aspergillus oryzae alkaline protease, and a method for producing the enzyme. The object of the present invention is to provide a new method, and also to provide a gene for the mutant alkaline protease into which site-specific mutations have been introduced, a plasmid recombined with the gene, and a transformant containing the plasmid.

[Means to solve the problem]

As a result of intensive research, the present inventors have discovered a mutant form of Aspergillus oryzae that has improved thermostability (heat resistance) compared to the wild type by replacing two or more of the alkaline proteases of the wild type Aspergillus oryzae with cysteine. They discovered that alkaline protease can be prepared and completed the present invention.

For example, among the mutant-type Aspergillus oryzae alkaline proteases of the present invention, those in which the 169th and 200th amino acids from the N-terminus of the wild-type Aspergillus oryzae alkaline protease described in (1) above are each replaced with cysteine have a molecular weight of (1) 25,000;
~30,000 or so.

(2) Shows thermal stability (heat resistance).

Specifically, 90% or more of the enzyme activity remains after 11 hours at 50°C.

(3) The optimal temperature for enzyme activity is increased by +5°C or more compared to the wild type.

(4) The introduced cysteine forms a disulfide bond.

(5) Enzyme activity is similar to wild type.

It shows the properties such as

The method for preparing this mutant Aspergillus oryzae alkaline protease is carried out using genetic engineering techniques from the gene encoding the wild-type Aspergillus oryzae alkaline protease.

That is, the present invention has the following features (1) to (7).

(1) A mutant Aspergillus oryzae alkaline protease in which two or more amino acids in the wild-type Aspergillus oryzae alkaline protease are each replaced with cysteine to form a disulfide bond within the molecule.

(2) The mutant Aspergillus oryzae alkaline protease according to (1) above, wherein glycine at position 169 and valine at position 200 from the N-terminus of wild-type Aspergillus oryzae alkaline protease are each replaced with cysteine.

(3) A gene encoding the mutant Aspergillus oryzae alkaline protease described in (1) or (2) above.

(4) A recombinant plasmid into which the gene described in (3) above has been introduced.

(5) A transformant transformed with the recombinant plasmid described in (4) above.

(6) (a) Prepare the gene described in (3) above.

(b) Prepare a recombinant plasmid by introducing the gene described in (3) above into a plasmid.

(c) Transform a host using the recombinant plasmid of (b) to prepare a transformant. and (d)
The transformant of (c) is cultured to produce the mutant Aspergillus oryzae alkaline protease described in (1) or (2) above.

A method for producing mutant Aspergillus oryzae alkaline protease comprising the above steps (a) to (d).

The present invention will be explained in more detail below.

The mutant Aspergillus oryzae alkaline protease of the present invention has a disulfide bond formed within the molecule by substituting two amino acids with cysteine. Specifically, (1) the N of the wild-type Aspergillus oryzae alkaline protease is Glycine at position 169 and valine at position 200 from the end are each replaced with cysteine.

(2) Tyrosine at position 36 and arginine at position 125 from the N-terminus of wild-type Aspergillus oryzae alkaline protease are each replaced with cysteine.

(3) Serine at position 69 and glycine at position 101 from the N-terminus of wild-type Aspergillus oryzae alkaline protease are each replaced with cysteine.

(4) Isoleucine at position 180 and threonine at position 254 or valine at position 253 from the N-terminus of wild-type Aspergillus oryzae alkaline protease are each replaced with cysteine.

To do this, first, using site-specific mutagenesis, the base sequence of the mutation target site of the gene encoding wild-type Aspergillus oryzae alkaline protease is changed to the base sequence corresponding to cysteine, and the mutant form of Aspergillus oryzae alkaline protease is Prepare the protease gene.

In this site-specific mutation method, a mutant gene is synthesized using a single-stranded DNA of a plasmid into which the original gene DNA has been integrated as a template and a synthetic oligonucleotide containing the base sequence to be mutated as a primer. The giant Sambrook et al.

Molecular cloning, 2nd ed
ition, Colorado Spring Harbor
Laboratory, Co1d Spring H
arbor, New York (1989)].

In the present invention, although it is possible to anneal with the single-stranded DNA of the wild-type Aspergillus oryzae alkaline protease gene,
Oligonucleotides that differ in that the base sequence corresponding to the target site to be replaced encodes cysteine are chemically synthesized, and this synthetic oligonucleotide is used as a primer, and the wild-type Aspergillus oryzae alkaline protease gene is A mutant type of Aspergillus oryzae alkaline protease gene is synthesized using the single-stranded DNA of the integrated plasmid as a template.

Next, the gene encoding the mutant Aspergillus oryzae alkaline protease is inserted into an expression vector system and used as an expression host.
Construct a vector system.

Examples of the host used in the present invention include Escherichia coli, yeast, Bacillus subtilis, etc., but are not particularly limited thereto. As expression vectors, plasmid vectors containing replicons and control sequences derived from species convertible with these host cells are used, and the plasmid vectors generally have a replication site and are phenotypic in transformed cells. For example, E. coli, which has a marker sequence that allows the selection of
var et al., Gene, 2.95 (1
977)). pBR322 contains genes for ampicillin and tetracycline resistance and therefore provides an easy method for detecting transformed cells. The pBR322 plasmid, as well as other microbial plasmids, have a promoter that can be used by the microbial organism to express the fluorescent matter that it controls, or have a promoter sequence inserted therein. Promoters commonly used for constructing recombinant DNA include β-lactamase (penicillinase) and lactose promoter systems (Chang et al.
I, Nature, 275.615 (1978)
; Itakura et al, 5science,
198.1056 (1977); Goeddel
et al., Nature, 28L 544 (
1979)] or the tryptophan (trp) promoter system (Goeddel et al., Nucl.
, Ac1ds Res, 8, 4057 (19
79) ; European Patent Application Publication No. 0036776). Although these are the most commonly used, other microbial promoters have also been discovered and used.

Details of their nucleotide sequences have also been published, allowing researchers to functionally introduce them into plasmid vectors (Siebenlist et al, Ce1
l.

Hou, 269 (1980)).

In the present invention, plasmid vectors containing these promoters or plasmid vectors commonly used for E. coli may be used as appropriate, and examples of the E. coli host include E. coli strains H8101, C600, and H3110. I can do it.

As the host yeast in the present invention, for example, Saccharomyces cerevisiae (Saccharon+yces ce
revisiae) NA37-11A strain, GRF18
strain, AH22 strain, etc. can be used.

In addition, 3-phosphoglycerate kinase (
PGK) promoter (Chang et al.,
Mol Ce11. Biol, Dan, 1812 (1
986) ) and other glycolytic enzymes [He5s et al.
,, J, ^dv, Enzyme, Reg,, 71
49 (1968); Holland et al.
Biochemistry, Jl.

4900 (1978)), enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPD
II) of enzymes such as hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate phosphate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, glucokinase. Examples include promoters, but among these,
The GAPDH promoter and PGK promoter are useful. When constructing a suitable expression vector, the transcription termination sequence present in these genes can also be inserted into the 3′ region of the gene to be expressed.
It is inserted at the side so that polyA conversion of mRNA and transcription termination occur. Promoters that have the advantage of being able to control transcription depending on growth conditions include promoters of alcohol dehydrogenase 2, isocytochrome C1 acid phosphatase, or degrading enzymes related to nitrogen metabolism, or enzymes related to the use of maltose or galactose. You can also use In the present invention, all plasmid vectors containing these yeast convertible promoters, replication origins, and transcription termination sequences can be used.

Furthermore, examples of Bacillus subtilis that serve as a host include, for example, No.
Natto (B, natto), Bacillus subtilis (
B, 5ubtilis) etc. are used. These Bacillus subtilis secretion expression vectors include plasmid pUBll
.

Plasmids can be used that have a plasmid replication origin that can be reproduced in Bacillus subtilis, and also have a promoter, signal peptide, and terminator, such as an α-amylase gene or alkaline protease (subtilisin) gene.

In addition, no matter which host is used, if the vector is integrated into the chromosome of the host cell, an origin of replication is not required in the vector, and the replication mechanism of the host cell chromosome can be utilized.

Transformation of host cells can be carried out using known methods, such as calcium chloride, protoplast polyethylene glycol fusion method,
This is done by electroporation method etc.

The obtained transformed strain is cultured in a medium known per se for host cells. Examples of the medium include L medium for E. coli and Bacillus subtilis, and YPD medium for yeast.

Culture is usually carried out at 15-43°C (preferably 30-37°C).
It is also possible to add aeration and stirring if necessary.

After culturing, the culture supernatant is collected, and the mutant Aspergillus oryzae alkaline protease is purified by a method known per se, such as affinity chromatography or fractionation.

(Effects of the Invention) The mutant Aspergillus oryzae alkaline protease of the present invention has the same enzymatic activity as the wild type, and has significantly improved thermostability (heat resistance). The mutant Aspergillus oryzae alkaline protease of the invention is expected to be useful in the fields of food, drinking water, reagents, and pharmaceuticals.

[Example] Examples will be given to explain the present invention in more detail, but
The present invention is not limited to these in any way.

Preparation of 1LfLL template DNA and mutation primer
Based on the knowledge of Aqualysin I, a serine protease secreted by sYT-1 to the outside of the bacterial cell, an attempt was made to introduce a disulfide bond into Aspergillus oryzae alkaline protease to increase its thermal stability. That is, Aqualysin I is an alkaline serine protease consisting of 281 amino acids and a molecular weight of approximately 28,000, and is a thermostable enzyme with an optimum temperature around 80°C (Matsuzaga, H.).

et al., Eur, J., Biochem, 17L 44
1-447 (1988)), the results of comparing the amino acid sequences of Aqualysin I and Aspergillus oryzae alkaline protease revealed that Aqualysin I is highly homologous to Aspergillus oryzae alkaline protease (44% identity), and that Aqualysin I has heat resistance within the molecule. Disulfide bonds, which are thought to be one of the causes of heat resistance, exist in two locations, and if a cysteine residue is introduced at a position corresponding to Aspergillus oryzae alkaline protease, which does not have a cysteine residue, and a disulfide bond is introduced, heat resistance can be achieved. It was thought that it could be modified into Aspergillus oryzae alkaline protease with improved properties.

To form a disulfide bond, it is necessary to substitute cysteine for amino acid residues at even positions in Aspergillus oryzae alkaline protease. Therefore, as shown below, we decided to substitute cysteine for the amino acid residues at the two positions homologous to the disulfide bonds of Aqualysin I, and named these mutants SS-1 and 5s-n. .

SS-r: 5et-69/Gly-101SS-I
I: Gly-169/Val-200 As a method for introducing the above amino acid substitution, site-directed mutagenesis using synthetic oligonucleotides was used. Introducing a mutation using this method requires a -terminal DNA template and a mutation primer.

The alkaline protease cDNA cloned from Aspergillus oryzae ATCC20386 strain was transferred to the plasmid vector pUc119 (Vieira,
J. et al., Methodin Enzymology,
153.3-11 (1987)) and named pOAPlo (Patent Application No. 1987).
-170018). The base sequence and amino acid sequence of alkaline protease cDNA are shown in FIG. pUc11
9 is interg of M13 phage that infects E. coli.
Because the enic region (IG) is inserted, the plasmid becomes a single-stranded DNA upon infection with M13 helper phage, and is released outside the E. coli cells while being encapsulated within the phage particle. Therefore, pOAP
lo to Escherichia coli JM109 strain [Messing+ J, .
Gene+ 33103-119 (1985))
After introducing D, one-end strand D of poAPlo was added according to the attached protocol of Zenshuzo 0) pUC119.
NA was prepared and used as a template for site-directed mutagenesis.

The following four types of oligonucleotides were used as mutation primers using an Applied Biosystems DNA synthesizer 3.
After synthesis using Type 81A, it was purified using Oligonucleotide Purification Cartridge J manufactured by the same company.

IA: 9 1 B = 01 1rA: 169^sn Se
r Asp Ala Cys Gln Thr
Ser Pr.

8ACTCT GAT GCA TGCC multiplication table
ACCAGCCCTSph I** I[B: 200 Asn Phe Gly Lys Cys
Val Asp Val Phe AlaAAA
CTTT GGCAAG TGCGTCGACGT
CTTCGCT*Umbrella 5all indicates substituted bases. As a result of base substitution, the restriction enzyme site is shortened or shortened. The numbers indicate amino acid numbers from the N-terminus of mature alkaline protease.

Evidence I: Production of mutant alkaline protease cDNA For the production of mutant alkaline protease cDNA,
The "Oligonucleotide-Buillefted In Vitro Mutagenesis System" manufactured by JAM Co., Ltd. was used. In addition, the operation was in accordance with the manual attached to the system.

(1) Site-directed mutagenesis 6 μg (approximately 4 pmol) of the single-stranded DNA of pOAPlo obtained in Example 1 11. cz l ) and a mutant primer phosphorylated at the 5' end (40-60 ng each, approximately 4 p
After annealing two types of mutant primers at once using the combinations of ■I A + I B and ■ I [A + IrB, mol), complementary strand extension and ligation in the presence of dCTPαS were carried out according to the manual, and unnecessary single-stranded DNA was removed. removal, nifking with NciJ, exonuclease treatment, and complementary strand extension. The final DN
Escherichia coli TGI strain (attached to the kit) was transformed using 175 amounts of A.

(2) Screening for mutants From the transformants obtained in (1), the method of Sakaki [Yoshiyuki Sakaki,
Hector DNA, Kodansha Scientific (1986)
)) Plasmids were prepared and screened for the presence or absence of a restriction enzyme recognition site designed on the mutant primer shown in Example 1. For clones showing a planned restriction enzyme digestion pattern, use Sakaki's method [described above].
After denaturing the plasmid with alkaline according to the method, the base sequence of the mutation site was determined by the dideoxy method, and it was confirmed that the designed mutation had been introduced. Plasmi F' (SS
-1: 5er69=Cys, Gly101→Cy
s) to pAPO61, a plasmid (ss-n: G1y16
9-+Cys, Va1200 →Cys) to pAPO6
It was named 2.

IL Example J - Construction of an alkaline protease secretory expression vector (1) Construction of a secretory expression vector using the ADFII promoter A method for constructing an alkaline protease secretory expression vector using the promoter and terminator of the alcohol dehydrogenase 1 gene (ADHI). Shown in Figure 2.

First, the EcoR (Al at the site) I (l promoter (indicated as P in the figure) and terminator (indicated as T in the figure)
pOA to the EcoRI site of AAR6 (managed by the Washington Research Foundation), which is connected to
1.5kb of Plo (Patent Application No. 170018/1983)
EcoRI fragment (wild type alkaline protease c
DNA (indicated as ALP in the figure) is ligated to form pOP^103.
It was named. Since this plasmid was predicted to be unstable in yeast because the replication origin is AR5I, the replication origin was changed to 2 μm DNA. That is, 2
AAH5 (obtained from Washington Research Foundation), which has an origin of replication derived from μta DNA, and p
The Ban+HI fragment of OAPlo3 was ligated and pOAPl
Got o7. This plasmid was used as a selectable marker.
Because EU2 is used, the size of the plasmid is 14.
It is large at 2kb. Therefore, in order to downsize the plasmid, the selection marker was changed from LEU2 to TRPI. That is, the Pstl fragment of pOAPlo7 (ADH
pOAPloB was created by ligating the Pstl fragment of MA56 (obtained from Washington Research Foundation) having TRPI as a selection marker and TRPI as a selection marker.

(2) ZygosaccharoIlyces r
Construction of a secretory expression vector using the GAPDH promoter derived from Zygosae oxii
Zyg.

The promoter of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Saccharomyces rouxii is
G of ccharomyces cerevisiae)
APDI (Imura et al.) has already shown that it is highly expressed like the promoter.

To et al., Agric, Biol, Chem, 53
．． 813-819 (1989)). Furthermore, the GAPDFI gene is known to be one of the most highly expressed yeast genes. (Holland, J.P.

et al., Biochemistry, 17.4900-
4907 (197B)).

Therefore, based on pQAP108 prepared in (1), Z,
A high-expression plasmid using the Ruxi GAPDH promoter was constructed. The construction method is shown in Figure 3.

First, Z, puc1 of the ruxi GAPDH promoter
Subcloning into 9 was performed. That is, Eco containing the GAPDH promoter of plasmid pTI43 (kindly provided by Professor Toyo, Hiroshima University) in which 4 bases upstream of the translation initiation codon (ATG) of the GAPDH promoter were deleted.
The RI-Pst I fragment was converted into pUc19 with EcoRI-
p[lG43 was obtained by inserting into the PstI site.

Furthermore, in preparation for adding the GAPDH terminator of Z and Ruxi, pUBG43 was prepared by converting the Ssp I site of pUG43 to a Bam) II site.

Next, a GAPDH terminator was added to pU13G43. That is, pUBG43 was mixed with EcoRI and Ba
Digested with IIIHT and added EcoRI-BamH of pGAP4-Zr (from Hiroshima University, professor of fruit juice).
pUGT43 into which the I fragment (including 700 bp downstream of the GAPDH stop codon) was inserted, and the GAPDH promoter and terminator were linked via the EcoRI site.
I got it. Next, a 1.5 kb EcoRI site containing the alkaline protease cDNA of pOAPlo (Japanese Patent Application No. 170018/1983) was inserted into the EcoRI site of pUGT43.
The fragment was inserted to obtain pGAT43. Subsequently, pGAT
GAPDH promoter/alkaline protease cDNA, -GAPDH terminator, possessed by 43
An origin of replication in yeast and a selection marker were added to the expression unit consisting of. That is, pOAPlo8 prepared in (1)
pGAT43 between the BamHT-5ph I sites of
By inserting the BamHI-5ph r fragment of I) OAP106B was obtained.

Construction of mutant alkaline protease expression vector Using pAPO61 and pAPO62 obtained in Example 2 and the alkaline protease secretion expression vector ρ0AP106B obtained in Example 3, mutant alkaline protease was produced in yeast according to the steps shown in FIG. A vector for secretory expression was constructed. That is, pAPO61 and ρ
By digesting APO62 with Bgl II and Afl II, the region encoding the mature alkaline protease in the mutant alkaline protease cDNA was converted to 860b.
It is obtained as a DNA fragment of p. On the other hand, the vector pO^P106B for secretory expression of yeast alkaline protease constructed in Example 3 was similarly transformed into Bglll and Afl.
After digestion with
I got a piece of it. Next, by ligating these two types of DNA fragments, a vector for expressing the mutant alkaline protease in yeast was obtained. pAPO
The vector incorporating the 61 fragment was called pAPO66, pA
The vector incorporating the PO62 fragment was named pAPO67.

! Secretory production of mutant alkaline protease by yeast Two types of mutant alkaline protease expression vectors pAPO66 and pAPO67 obtained in Example 4 and Example 3
The wild-type alkaline protease expression vector p obtained in
Using OAP106B, the method of Hinnen et al.
nenAo et al., Proc, Natl, Acad, Si
c, USA, 75.1929-1933 (1978)
), Saccaromyces cerevisiae (Sacc
haromyces cerevisiae) NA3
7-11A was transformed using the release of tryptophan requirement as an indicator, and S. cerevisiae NA37-11A/pAP was transformed.
O66 and S, Cereviche NA37-11^/pAP0
67, S9 Celebishe NA37-11^/po^P10
Three transformed yeast strains of 6B were obtained.

Next, the three types of transformants obtained were transferred to SD medium [142
Win Yeast Nitrogen Ba5e wl
o Am1n.

Ac1ds (manufactured by Difco) 6.7g, glucose 20g.

L-leucine 30■, L-histidine hydrochloride 20■]5
Inoculate a 300 d Erlenmeyer flask containing 0 d and heat at 30°C.
The cells were cultured with shaking for 72 hours. 25 d of this culture solution was mixed into YPD medium [20 g of Bacto-Peptone (manufactured by Difco), Yeast extract (manufactured by Difco), Log of yeast extract (manufactured by Difco),
The cells were transferred to two 3i Erlenmeyer flasks containing 500 d of glucose (20 g) and cultured with shaking at 30°C for 72 hours. After completion of the culture, the culture supernatant was collected by centrifugation at 3.50 rpm for 20 minutes, and this was used for purification of mutant alkaline protease.

! Purification of mutant alkaline protease Purification of mutant alkaline protease was performed by anion exchange column chromatography, hydrophobic interaction column chromatography, and gel filtration column chromatography. The pH of the yeast culture supernatant is about 7.5 and 10-15++ U,
After filtration with a 0.4 μm filter, unadsorbed fractions were collected by equilibration with water and chromatography on an Ω-5 epharose column. This unadsorbed fraction contains approximately 80%
of alkaline protease activity was recovered. Ammonium sulfate was added to this unadsorbed fraction to a final concentration of 2M, and the pH was adjusted to 6.0.
After adjusting Millipore Filter H
Filtration was performed at V 0.45 μn+ to remove insoluble matter.

This filtrate was diluted with 2.0M (NH,) zsoa-o, os%
Equilibrated with NaNz-10mM acetate buffer (pH 6) and subjected to Butyl Toyopeal 630M column chromatography, 2M→OM (NH,)
Elution was performed using a zsO4 linear gradient, and alkaline protease activity fractions were collected. Enzyme activity recovery rate is approximately 8
It was 0% to 90%. This alkaline protease activity fraction was pretreated with IQOmM NaC]-1mMεDTA.
-0,05% NaN3-50111M acetate buffer pH 6
The mixture was equilibrated and subjected to 5uperose 12 column chromatography, and the alkaline protease activity fractions were collected and used as a final sample. This specimen is single hand SO3 electrophoretically and 95% reversed phase chromatographically.
% purity.

Lohry method (Lohry+ 0.H, et al., J, Bio
l,Chem,.

193, 265 (1951)), assuming that the molecular weight of alkaline protease is 29,000 daltons, SS-1 is 34.9μ and ss-n is 30.
The concentration was 5 μm. Wild type and S purified using this method
The specific activities of S-I and SS-■ were comparable when azocasein was used as a substrate. The substrate solution is 1% azocasein 1 mM EDTA-50 oiM sodium phosphate buffer pH 7. The reaction temperature is 30°C.

Confirmation of disulfide bond formation in W-purified mutant alkaline protease was performed using the DTNB method and a method combining DTT and DTNB methods (Butterwort).
h, P, H.

Wetal Arch, Biochem, Bioph
ys, 118, 716 (1967)) with modifications. In other words, free SH groups are quantified under non-reducing conditions, and furthermore, free SH groups are quantified under reducing conditions. A method for quantifying free SH groups under non-reducing conditions is described below. Equilibrate the PD-10 column with 0.1 M) Lys buffer pH 8.0. Apply 2.5 samples of the sample and elute with 3 and 5 samples of the same buffer. PD-1
Add 8M guanidine hydrochloride in an amount equal to 0 column eluate (
Guanidine final concentration 4M). The absorbance of this solution was measured at 280 nm, and the molecular weight of alkaline protease was determined to be 2900 nm.
0 Dalton and Er=,=7.5tA280(Naka
dai, T, et al, Agr, Biol, chem
, 37.2685 (1973)). This solution 400 (10mM DTNB-50 in tl)
Add 800 uffi of (mM) Lys buffer pH 8.0, stir instantly, and measure the absorbance at 412 nm. Blank is 0.1M) Perform the same operation as above using Liss buffer pH 8.0. To quantify the SH group, subtract the blank value and divide that value by 13600 to determine the molar concentration of the SH group. The number of SH groups per protein molecule is determined from the protein concentration.

The above measurement results show that per molecule of alkaline protease,
The number of SH groups in both wild type and SS-1,5S-II was 0.

A method for quantifying free SH groups under reducing conditions is described below.

Guanidine hydrochloride is added to the protein solution to give a final concentration of 4M, and 1710 volumes of 100mM DTT solution is added to this solution, stirred and left to stand at room temperature for 30 minutes or more. PD-10 column
Equilibrate with n+M EDTA-10mM acetic acid pH 4.

Specimen 2. Od is applied, washed with the same buffer for 0.5 d, and then eluted with the same buffer for 3.0 d. Add an equal volume of 8M guanidine hydrochloride to the same eluate (final guanidine concentration: 4
M). The protein concentration is determined by measuring the absorbance of this solution at 280 nm. Add 10mM DTNB to 400uf of this solution.
-50mM) Squirrel buffer pH 8,0 800μ! In addition, while stirring instantly, the absorbance at 412 nm is measured. Flank contains 1mM EDT instead of protein solution.
The same operation is performed using A-10mM acetate buffer pH 4.0. To quantify SH groups, subtract the blank value and divide that value by 13,600 to determine the molar concentration of SH groups. protein 1
The number of SH groups per molecule is determined from the protein concentration.

The above measurement results show that per molecule of alkaline protease,
There were 0 Sr1 groups in the wild type, and 2 SH groups in 55-1 and ss-n.

From the above results, it is concluded that one set of intramolecular disulfide bonds is formed in the SSN type and the ss-n type.

"IL Example" - Comparison of thermostability and optimal temperature for enzyme activity expression (i) Measurement of thermostability The final specimen obtained in Example 6 was incubated at 50°C for 1 nc each time.
After ubation, the activity was measured at 30°C. Solution composition for 1 incubation at 50°C is 100mM NaCl-1mM EDTA-0.05%
NaN3-50mM acetate buffer pH6. 30°C
The substrate solution for activity measurement was 1% aso'casein-1.
mMEDT^-50mM sodium phosphate buffer pH7
It is. The results are shown in Figure 5. Wild type and SS-I type alkaline protease were incubated at 50°C, 6
A rapid decrease in residual activity was observed within 0 minutes, but s
Almost no decrease in activity of sn-type alkaline protease was observed. In order to find a decrease in the residual activity of the ss-n type, incubate at 50°C for 10 hours or more under the same conditions.
The half-life of heat inactivation at 50°C, which required ncubation, was 20 minutes for the wild type, and 1 for the SS-1 type.
The half-life of the ss-n type was 330 minutes, and the half-life of the ss-n type was 16.5 times that of the wild type.

(11) Measurement of optimal temperature for expression of enzymatic activity Measurement of optimal temperature for expression of enzymatic activity for wild type and mutant alkaline protease SS and ss-n type was carried out using 1% azocasein as a substrate, 2 mM EDTA-50 mM phosphate. carried out in a sodium acid buffer solution. The results are shown in Figure 6. Figure 6 is 30
The enzyme activity at each temperature is shown as a relative value, with the activity of each mutant enzyme at °C being 1. The optimum temperature for the wild type was 51°C, and the optimum temperature for the SS-1 type was 48°C, which was 3°C lower than the wild type. The optimal temperature of the ss-n type was 56°C, which was 5°C higher than that of the wild type. The enzyme activity of 5s-n type at 56℃ is 30℃
The enzyme activity was three times that of

[Brief explanation of drawings]

FIG. 1 shows the base sequence and amino acid sequence of Aspergillus oryzae alkaline protease cDNA. FIG. 2 shows the steps for constructing an alkaline protease secretion expression vector using the ADHI promoter. FIG. 3 shows the steps for constructing an alkaline protease secretion expression vector using the GAPDH promoter of Z and Ruki. FIG. 4 shows the steps for constructing a mutant alkaline protease secretion expression vector. Figure 5 shows 1 of wild type and mutant alkaline proteases.
The relationship between ncubation time and residual activity is shown. Figure 6 shows 1 of wild type and mutant alkaline proteases.
Relationship between ncubation temperature and residual activity (optimum temperature)
shows. Figure 4

Claims (1)

[Scope of Claims] 1. A mutant Aspergillus oryzae alkaline protease in which two or more amino acids in the wild-type Aspergillus oryzae alkaline protease are each replaced with cysteine to form a disulfide bond within the molecule. 2. 1 from the N-terminus of wild-type Aspergillus oryzae alkaline protease
The mutant Aspergillus oryzae alkaline protease according to claim 1, wherein glycine at position 69 and valine at position 200 are each replaced with cysteine. 3. A gene encoding the mutant Aspergillus oryzae alkaline protease according to claim 1 or 2. 4. A recombinant plasmid into which the gene according to claim 3 has been introduced. 5. A transformant transformed with the recombinant plasmid according to claim 4. 6. (a) Preparing the gene according to claim 3. (b) A recombinant plasmid is prepared by introducing the gene according to claim 3 into a plasmid. (c) Transform a host using the recombinant plasmid of (b) to prepare a transformant. and (d) culturing the transformant of (c) to produce the mutant Aspergillus oryzae alkaline protease according to claim 1 or 2. A method for producing mutant Aspergillus oryzae alkaline protease comprising the above steps (a) to (d).