WO2014122698A1

WO2014122698A1 - Protease, and protease-containing cleaning agent and production method therefor

Info

Publication number: WO2014122698A1
Application number: PCT/JP2013/000724
Authority: WO
Inventors: 大助杉森
Original assignee: 国立大学法人福島大学
Priority date: 2013-02-08
Filing date: 2013-02-08
Publication date: 2014-08-14
Also published as: JPWO2014122698A1; JP6043371B2

Abstract

Provided is a protease exhibiting excellent protein breakdown performance, and excellent detergency, particularly with respect to protein-based stains caused by blood or the like. This protease is: a protease obtained by culturing Actinomadura sp. strain RD001933 (NBRC domestically isolated RD strain) of Actinobacteria; a protease obtained by culturing Actinomadura miaoliensis strain RD000920 (NBRC domestically isolated RD strain) of Actinobacteria; or a protease obtained by mixing and culturing the RD001933 strain and the RD000920 strain.

Description

Protease, protease-containing detergent and method for producing the same

The present invention relates to a novel protease (proteolytic enzyme), a protease-containing detergent containing the protease, and a method for producing the same.

Conventionally, as a cleaning agent for a medical device, a cleaning composition comprising a nonionic surfactant, a protease, a sequestering agent, benzotriazole, and the like is known (for example, see Patent Document 1). A cleaning composition containing a protease, a nonionic surfactant, and an enzyme stabilizer is also known (see, for example, Patent Document 2).

JP-A-5-279700 JP 2001-31999 A

Currently, it is said that alkaline detergents have the highest detergency, but nonetheless have not been able to completely clean medical equipment. The main components of dirt are cells and proteins consisting of blood and body fluids attached at the time of surgery, including infectious prions. In particular, steel tools such as scissors and tweezers are frequently reused among medical instruments, so it is essential to clean blood and proteinaceous dirt adhering to these steel accessories before the sterilization process. It has become. Further, if harmful substances such as infectious prions remain in these steel accessories, infection may spread to many surgical patients. Therefore, it is desired to develop a powerful cleaning agent that can completely remove such dirt.

The present invention has been made in view of the above points, and provides a protease, a protease-containing detergent, and a method for producing the same, which have a strong ability to degrade proteins, and particularly have a high detergency against protein stains such as blood. It is for the purpose.

The protease according to the present invention is a protease obtained by culturing actinomycetes sp. RD001933 strain (NBRC domestic isolate RD strain).

The protease according to the present invention is a protease obtained by culturing the actinomycete Actinomadura myaoliensis RD000920 strain (NBRC domestic isolate RD strain).

The protease according to the present invention is a protease obtained by co-culturing RD001933 strain belonging to the genus Actinodura sp. Of actinomycetes and RD000920 strain belonging to the genus Actinomadura miaoliensis of actinomycetes. .

It is preferable that the protease is a polypeptide described in the following (a1), (a2) or (a3).

(A1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2.

(A2) A polypeptide comprising an amino acid sequence in which one or more amino acid residues are substituted, inserted, deleted, and / or added in the amino acid sequence set forth in SEQ ID NO: 2.

(A3) A polypeptide having at least 65% homology with the amino acid sequence set forth in SEQ ID NO: 2.

The molecular weight of the protease by SDS-PAGE is preferably about 31,000 Da (31 kDa).

It is preferable that the polynucleotide encoding the protease includes the polynucleotide described in (b1), (b2) or (b3) below.

(B1) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 1.

(B2) A polynucleotide that hybridizes with a base sequence complementary to the base sequence described in SEQ ID NO: 1 under stringent conditions.

(B3) A polynucleotide having at least 70% sequence identity with the base sequence set forth in SEQ ID NO: 1.

The protease according to the present invention contains the protease.

The method for producing a protease-containing detergent according to the present invention is produced by culturing RD001933 strain belonging to the genus Actinomadura sp.

The method for producing a protease-containing detergent according to the present invention is produced by culturing RD000920 strain belonging to the genus Actinodura miaoliensis of actinomycetes.

The method for producing a protease-containing detergent according to the present invention comprises RD001933 strain belonging to the genus Actinodura sp. Of Actinomyces and RD000920 strain belonging to the genus Actinomadura miaoliensis of mixed actinomycetes. To manufacture.

The protease according to the present invention can be obtained from two types of highly safe microorganisms (strains belonging to the same), both of which have a strong ability to degrade proteins, and particularly have a high detergency against protein stains such as blood. In particular, these protease-containing detergents can wash denatured and fixed blood stains, which were difficult to wash with conventional alkaline detergents, at a low temperature and in a short time. In addition, a protease-containing detergent obtained by culturing each of two types of microorganisms has a high cleaning effect, but a protease-containing detergent obtained by mixing and culturing two types of microorganisms has a higher cleaning effect. In addition, these protease-containing detergents can be mass-produced easily and inexpensively, and can also be powdered or tableted in addition to being liquid, and can be stored for a long time.

Furthermore, the protease according to the present invention can be applied to various fields other than the detergent. For example, meat processing, squid peeling, and tanning.

It is an electrophoretic photograph showing the results of SDS-PAGE analysis of a purified fraction obtained from a culture solution of RD001933 strain belonging to the genus Actinomadura sp. It is an electrophoresis photograph showing the results of SDS-PAGE analysis of a purified fraction obtained from a culture solution of RD000920 strain belonging to the genus Actinomadura miaoliensis. It is a graph which shows the temperature dependence of the relative activity about the protease (purified enzyme) derived from RD001933 strain, and the protease (purified enzyme) derived from RD000920 strain. It is a graph which shows the temperature dependence of the specific activity about protease (purified enzyme) derived from RD001933 strain and protease (purified enzyme) derived from RD000920 strain. It is a graph which shows the pH dependence of the relative activity about the protease (purified enzyme) derived from RD001933 strain, and the protease (purified enzyme) derived from RD000920 strain. It is a graph which shows the pH dependence of the specific activity about the protease (purified enzyme) derived from RD001933 strain and the protease (purified enzyme) derived from RD000920 strain. It is a graph which shows the temperature dependence of the relative activity about the protease (crude enzyme) derived from RD001933 strain. It is a graph which shows the pH dependency of relative activity about protease (crude enzyme) derived from RD001933 strain. It is a graph showing the CaCl ₂ final concentration dependence of the relative activity for RD001933 strain derived protease (crude enzyme). It is a graph which shows the temperature dependence of relative activity about protease (crude enzyme) derived from RD000920 strain. It is a graph which shows the pH dependence of the relative activity about the protease (crude enzyme) derived from RD000920 strain. It is a graph showing the CaCl ₂ final concentration dependence of the relative activity for RD000920 strain derived protease (crude enzyme). It is a graph which shows the temperature dependence of the relative activity about the crude enzyme obtained from the mixed culture solution of RD001933 strain and RD000920 strain. It is a graph which shows the pH dependence of the relative activity about the crude enzyme obtained from the mixed culture solution of RD001933 strain and RD000920 strain. Is a graph showing the CaCl ₂ dependence of the relative activity for the crude enzyme obtained from a mixed culture of RD001933 strain and RD000920 strain. It is a graph which shows the daily change of the residual activity about the crude enzyme obtained from the mixed culture solution of RD001933 strain and RD000920 strain.

[Protease]
The protease according to the present invention is a protease obtained by culturing actinomycetes Actinomadura sp. RD001933 strain (NBRC domestic isolate RD strain). From the results of 16S rDNA nucleotide sequence analysis, it is presumed that the belonging classification group of strain RD001933 is Actinomadura sp. Closely related to Actinomadura miaoliensis.

The protease according to the present invention is a protease obtained by culturing the actinomycete Actinomadura myaoliensis RD000920 strain (NBRC domestic isolate RD strain). From the results of 16S rDNA nucleotide sequence analysis, it is presumed that the belonging classification group of strain RD000920 is Actinomadura miaoliensis.

The protease according to the present invention includes actinomycetes Actinomadura sp. RD001933 (NBRC domestic isolate RD strain) and actinomycetes Actinomadura miaoliensis RD000920 strain (NBRC domestic isolate). RD strain) is a protease obtained by mixed culture.

Proteases exhibit hydrolytic activity on proteins and are not limited to purified enzymes, but also include crude purified products and immobilized products. Purification of the protease can be performed, for example, using a culture solution of microorganisms, using a method such as ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography, or the like. As a result, enzymes having various degrees of purification (including enzymes purified to almost a single level) can be obtained.

The microorganism may be any strain such as a wild strain, a mutant strain (for example, induced by ultraviolet irradiation), or a recombinant derived by genetic engineering techniques such as cell fusion or genetic recombination. May be. A genetically engineered microorganism such as a recombinant is used, for example, using a technique described in Molecular Cloning A Laboratory Manual, 2nd edition (Sambrook, J. et al., Cold Spring Harbor Press, 1989). Created easily. The culture solution of microorganisms means both a culture solution containing microbial cells and a culture solution from which microbial cells have been removed by centrifugation or the like. Further, it may be a protease contained in the microbial cells.

In producing a protease-containing detergent, it is preferable to use at least one of the RD001933 strain-derived protease and the RD000920 strain-derived protease described below as the enzyme.

The protease preferably contains the polypeptide described in the following (a1), (a2) or (a3).

(A3) A polypeptide having at least 65%, preferably at least 70%, more preferably at least 80% homology with the amino acid sequence set forth in SEQ ID NO: 2.

In addition, the polynucleotide encoding the protease preferably includes the polynucleotide described in (b1), (b2) or (b3) below.

(B1) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 1.

(B3) A polynucleotide having at least 70%, preferably 75%, more preferably 80% sequence identity with the base sequence set forth in SEQ ID NO: 1.

The polynucleotide having the base sequence described in SEQ ID NO: 1 can encode the polypeptide (amino acid sequence) described in SEQ ID NO: 2.

The enzyme activity of the protease can be confirmed by the following method. First, 50 μL of 3% azocasein (final concentration 0.6%) is mixed with 198 μL of Tris-HCl buffer (pH 7.5, final concentration 80 mM). And 2 microliters of samples which confirm enzyme activity are added, and it is made to react at 65 degreeC for 5 minutes or 10 minutes. After the enzyme reaction, 50 μL of 20% trichloroacetic acid is added to stop the reaction. After stopping the reaction, the reaction solution is centrifuged, and the absorbance of the supernatant is measured at 340 nm. An enzyme amount of 1 U (unit) is an amount that generates 1 μmol of an azo dye equivalent per minute.

Protease can act at 20 to 80 ° C., for example. The optimum temperature can be within this range. Preferably it is in the range of 60-90 ° C, more preferably in the range of 70-80 ° C, and even more preferably about 75 ° C.

Protease can act at a pH of 4.0 to 9.5, for example. The optimum pH can be within this range. The pH is preferably around 7.5, but exhibits an activity of 50% or more at pH 6.0 to 8.8.

Protease may vary slightly depending on electrophoresis conditions and the like, but preferably has a molecular weight of about 31,000 Da (31 kDa) by SDS-PAGE. For example, in a natural enzyme derived from the RD001933 strain belonging to the genus Actinodura sp. Of Actinomyces and the RD000920 strain belonging to the genus Actinomadura miaoliensis of Actinomyces, the molecular weight by SDS-PAGE is about 31, 000 Da.

One embodiment of the protease consists of the amino acid sequence set forth in SEQ ID NO: 2. The protease preferably has an amino acid sequence from position 111 to position 386 of SEQ ID NO: 2 (hereinafter also referred to as “amino acid sequence described in SEQ ID NO: 2”). The amino acid sequence from position 1 to position 26 of SEQ ID NO: 2 is a signal sequence, and the amino acid sequence from position 27 to position 110 of SEQ ID NO: 2 is a pro sequence.

Protease is an amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted and / or added to the amino acid sequence described in SEQ ID NO: 2. It may be an enzyme. For example, site-directed mutagenesis (Nucleic Acid Ａ Res., 1982, 10, pp. 6487; Methods in Enzymol., 1983, 100, pp. 448; Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Laboratories, Cold Spring Harbor, NY, 1989; PCR: APPROCICAL Approach, IRL Press, 1991, pp. 200), etc., by introducing substitutions, deletions, insertions and / or addition mutations as appropriate. The structure of (protein) can be modified. The number of amino acid residues that can be substituted, deleted, inserted and / or added is usually 50 or less, such as 30 or less, or 20 or less, preferably 16 or less, more preferably 5 or less, and even more preferably 0 to 3. is there. Proteases include not only enzymes with artificially mutated amino acid residues but also enzymes with mutated amino acid residues in nature.

An enzyme having an amino acid sequence having homology to the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 2 is also included in the protease. The protease preferably has an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 65%, preferably at least 70%, more preferably at least 80% homology with the amino acid sequence within SEQ ID NO: 2. It has a protein.

Protein homology (homology) search, for example, databases such as SWISS-PROT, PIR, DAD and other protein amino acid sequences, DNA databases such as DDBJ, EMBL, Gene-Bank, etc., programs such as BLAST and FASTA Can be done through the Internet. The protein activity can be confirmed using the procedure described above.

Protease sources are RD001933 strain (accession number: NITE P-1467) belonging to the genus Actinodura sp. Of Actinomycetes and RD000920 strain (trusted) belonging to the genus Actinodura miaoliensis of actinomycetes. Number: NITE P-1468). These have a polynucleotide represented by the base sequence described in SEQ ID NO: 1 in DNA.

For example, RD001933 strain (accession number: NITE P-1467) belonging to the genus Actinodura sp. Of actinomycetes, and RD000920 strain (accession number: NITE P) belonging to the genus Actinomadura miaoliensis of actinomycetes In all cases, 1468), the protease is secreted outside the cells by liquid culture in an appropriate nutrient medium, so that the culture supernatant is treated with freeze-drying, salting-out, organic solvent, etc., or this treatment What fixed the thing etc. can be manufactured as an enzyme formulation.

More specifically, first, the above-mentioned bacteria are cultured in a suitable medium, for example, a medium containing a suitable carbon source, nitrogen source, and inorganic salts to secrete protease. At this time, the RD001933 strain belonging to the genus Actinodura sp. Of actinomycetes may be cultivated alone, or the RD000920 strain belonging to the genus Actinomadura miaoliensis is cultivated alone. These may be mixed and cultured.

Here, as the carbon source, starch and starch hydrolysate, sugars such as glucose and sucrose, alcohols such as glycerol and organic acids (for example, acetic acid and citric acid) or salts thereof (for example, sodium salt), etc. Can be mentioned. The concentration of the carbon source is, for example, in the range of 1 to 20% (w / v), preferably 1 to 10% (w / v).

Examples of the nitrogen source include organic nitrogen sources such as yeast extract, peptone, meat extract, corn steep liquor, and soybean powder, and inorganic nitrogen compounds such as ammonium sulfate, ammonium nitrate, and urea. The concentration of the nitrogen source is, for example, in the range of 1 to 20% (w / v), preferably 1 to 10% (w / v).

Examples of inorganic salts include sodium chloride, monopotassium phosphate, magnesium sulfate, manganese chloride, calcium chloride, and ferrous sulfate.

The culture temperature is preferably a temperature at which the protease is stable and the cultured microorganism can sufficiently grow, and is preferably 25 to 50 ° C., for example. The culture time is preferably a time during which protease is sufficiently produced, and is preferably about 1 to 7 days, for example. Culturing can be performed preferably under aerobic conditions, for example, with aeration stirring or shaking.

Polypeptides contained in proteases are fractionated by protein solubility (precipitation with organic solvents, salting out with ammonium sulfate, etc.); cation exchange, anion exchange, gel filtration, hydrophobic chromatography; chelates, dyes, antibodies, etc. It can be purified by an appropriate combination of methods such as affinity chromatography using. For example, after recovering the culture supernatant of the microorganism, it can be purified by ammonium sulfate precipitation, further anion exchange chromatography, hydrophobic chromatography and / or cation exchange chromatography. Thereby, it can be purified to almost a single band in polyacrylamide gel electrophoresis (SDS-PAGE). That is, the polypeptide constituting the protease can be estimated as a monomer by HPLC analysis and gel filtration chromatography analysis.

[Microorganisms]
Proteases can be produced by microorganisms. As for the protease, a polypeptide can be artificially generated from the amino acid sequence information shown in SEQ ID NO: 2 and the base sequence information shown in SEQ ID NO: 1, and obtained by artificial synthesis for the production of a protease-containing detergent. Enzymes (polypeptides) may be used. However, when an enzyme is produced by a microorganism, the above enzyme can be easily obtained.

As the microorganism, RD001933 strain belonging to the genus Actinodura sp. Of actinomycetes and RD000920 strain belonging to the genus Actinomadura miaoliensis of actinomycetes can be used. You may use the introduced microorganism. That is, the microorganism is a polynucleotide having the base sequence described in SEQ ID NO: 1, a polynucleotide that hybridizes under stringent conditions with a base sequence complementary to the base sequence described in SEQ ID NO: 1, A microorganism into which at least one polynucleotide selected from polynucleotides having at least 70%, preferably at least 75%, more preferably at least 80% sequence identity with the described base sequence has been introduced. The microorganism into which the above polynucleotide is introduced can be obtained by using a vector or preparing a transformant. For example, by introducing the above-described polynucleotide into a vector and introducing the vector into a host such as Escherichia coli, a transformant having the ability to produce a protease can be produced.

A procedure for producing a transformant and construction of a recombinant vector suitable for the host can be performed according to techniques commonly used in the fields of molecular biology, biotechnology, and genetic engineering (for example, Sambrook et al. , Molecular Cloning: Laboratory Manual 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). In particular, for actinomycetes, it can be performed with reference to “PRACTICAL STREPTOMYCES GENETICS (Kieser et al., John Inns Foundation, 2000)”.

In order to express a polynucleotide encoding the above enzyme in a microorganism, this DNA is first introduced into a plasmid vector or a phage vector that is stably present in the microorganism, and the genetic information is transcribed and translated. Therefore, it is preferable to incorporate a promoter corresponding to a unit for controlling transcription / translation 5 'upstream of the DNA strand. Further, it is preferable to incorporate a terminator, which is a unit for controlling transcription / translation, downstream of the 3 ′ side of the DNA strand. More preferably, both the promoter and terminator are incorporated at each site. As the promoter and terminator, promoters and terminators known to function in microorganisms used as hosts are used. Regarding the vectors, promoters, and terminators that can be used in these various microorganisms, “Basic Course of Microbiology 8, Genetic Engineering, Kyoritsu Shuppan”, especially regarding actinomycetes, “PRACTICAL STREPTOMYCES GENETICS (Kieser et al., John Inns Foundation, 2000) It is possible to use this method. Moreover, it can be efficiently secreted and produced extracellularly by using a signal sequence as necessary.

The host to be transformed is not particularly limited as long as it is an organism that can be transformed with a vector containing a polynucleotide encoding the enzyme and express the enzyme activity. For example, bacteria, actinomycetes, Bacillus subtilis, Escherichia coli, yeast, mold and the like can be mentioned. More specifically, for example, genus Escherichia, genus Bacillus, genus Pseudomonas, genus Serratia, genus Brevibacterium, genus Corynebacterium, genus Corynebacterium Bacteria for which host vector systems such as Streptococcus and Lactobacillus have been developed; Actinomycetes for which host vector systems such as Rhodococcus and Streptomyces have been developed; Saccharomyces (Saccharomyces) Saccharomyces), Kluyveromyces, Shizosa A genus of the genus Schizosaccharomyces, the genus Zygosaccharomyces, the genus Yarrowia, the genus Trichosporon, the genus Rhodosporidium, the genus Rhodaspodium, and the genus C And yeasts that have been developed for host vector systems such as the genus Neurospora, the genus Aspergillus, the genus Cephalosporum, the genus Trichoderma, and the like. Escherichia coli is preferred for ease of gene recombination, and actinomycetes are preferred for ease of gene expression.

In addition to microorganisms, various host / vector systems have been developed in plants and animals. For example, insects such as moths (Nature 315, 592-594 (1985)), rapeseed, corn, potatoes, and other plants in large quantities. Systems for expressing heterologous proteins have been developed, and these may be used.

The obtained transformant can be used for enzyme production as described above. Specifically, the transformant is liquid-cultured in an appropriate nutrient medium, the expressed polypeptide is secreted outside the cell, and the culture supernatant is lyophilized, salted out, treated with an organic solvent, etc. Can be manufactured.

Although the culture conditions can vary depending on the host cells, the culture can be performed under normal conditions. For example, when an actinomycete such as Streptomyces is used as a host, a tryptic soy medium containing thiostrepton (for example, Becton Dickinson) can be used. The enzyme produced by the transformant can be further purified as described above.

[Protease-containing detergent]
The protease-containing detergent according to the present invention comprises RD001933 strain belonging to the genus Actinodura sp. Of Actinomycetes and RD000920 strain belonging to the genus Actinomadura miaoliensis, respectively, individually or mixedly. Then, it can be obtained as a crude enzyme by salting out (ammonium sulfate precipitation, acetone precipitation, ethanol precipitation, etc.). That is, depending on the purpose, a single culture solution or a mixed culture solution is filtered through a filter paper or a filter to obtain a protease-containing detergent as a crude enzyme solution, or centrifuged to obtain a protease-containing detergent as a supernatant (culture supernatant). You can also. Filtration is performed to remove impurities that cause turbidity. If the turbidity is not worrisome, filtration is unnecessary, and a sufficiently transparent protease-containing detergent can be obtained by centrifugation. If necessary, it may be further purified and obtained as a purified enzyme.

The protease obtained as described above can be obtained from two types of highly safe microorganisms (strains belonging to the same), both of which have a strong ability to degrade proteins, particularly high washing against protein stains such as blood. Have power. In particular, protease-containing detergents containing these proteases can wash denatured and adhered blood stains, which were difficult to wash with conventional alkaline detergents, at a low temperature and in a short time. In addition, the protease-containing detergent obtained by culturing each of the two types of microorganisms alone has a strong proteolytic ability and a high cleaning effect. Strong and high cleaning effect. In addition, these protease-containing detergents can be mass-produced easily and inexpensively, and can also be powdered or tableted in addition to being liquid, and can be stored for a long time.

Furthermore, the above protease can be applied to various fields other than detergents. For example, meat processing, squid peeling, and tanning.

[Purification of protease from RD001933 strain]
The RD001933 strain belonging to the genus Actinodura sp. Of actinomycetes is cultured, and the RD001933 strain is cultured on the culture supernatant using ammonium sulfate fractionation, hydrophobic chromatography, cation exchange chromatography, and anion exchange chromatography. The derived protease was purified. Details are shown below.

(A) Culture of microorganism RD001933 strain (accession number: NITE P-1467) belonging to the genus Actinodura sp.

First, 490 mL of ISP2 medium (yeast extract 0.6%, malt extract 1.4%, glucose 0.6%) was prepared, and 70 mL each was dispensed into a 500 mL Erlenmeyer flask. One spring coil was put in this, and steam sterilization was performed at 121 ° C. for 20 minutes. Furthermore, 30 mL of 2.5% skimmed milk that was sterilized separately was added to a final concentration of 0.75%.

Then, 50 μL of glycerol stock cells were inoculated into a φ18 test tube (18 × 180 mm) containing 5 mL of ISP2 medium, and cultured with shaking at 45 ° C. until good growth was obtained. 1 mL of this culture solution was inoculated into 100 mL of the previously sterilized medium, and cultured with shaking at 45 ° C. for about 96 hours. The supernatant was recovered from this culture using a centrifuge.

(B) Acetone fractionation (acetone precipitation)
Acetone was added to the culture supernatant collected in the above (a) so as to be 60% (v / v) or more, and the resulting precipitate was collected by centrifugation (10,000 rpm, 10 minutes, 4 ° C.). This precipitate was dissolved in 15 mL of 20 mM Tris-HCl buffer (pH 8.0) containing ammonium sulfate having a final concentration of 1 M to obtain a crude enzyme solution.

(C) Toyopearl Phenyl-650M column chromatography The Toyopearl Phenyl-650M column (inner diameter) obtained by previously equilibrating the crude enzyme solution obtained in (b) above with 20 mM Tris-HCl buffer (pH 8.0) containing 1 M ammonium sulfate. 26 mm, height 38 mm, manufactured by Tosoh Corporation). After washing the column with the same buffer, the protein was eluted with a linear gradient of ammonium sulfate (from 1 M to 0 M).

(D) HiTrap Q HP column chromatography The active fractions obtained in (c) above were collected and desalted by dialysis using 20 mM Tris-HCl buffer (pH 9.0). The column was applied to a HiTrap Q HP (5 mL) column (manufactured by GE Healthcare Biosciences) previously equilibrated with 20 mM Tris-HCl buffer (pH 9.0), washed with the same buffer, and then sodium chloride ( The protein was eluted with a linear gradient from 0M to 1M.

(E) HiTrap SP HP column chromatography The active fractions obtained in (d) above were collected and desalted by dialysis using 20 mM MES-sodium hydroxide buffer (pH 5.5). This was applied to a HiTrap SP (1 mL) column (manufactured by GE Healthcare Biosciences) previously equilibrated with 20 mM MES-sodium hydroxide buffer (pH 5.5), and the column was washed with the same buffer. The protein was eluted with a linear gradient of sodium chloride (0M to 1M).

As described above, a purified enzyme was obtained from the RD001933 strain belonging to the genus Actinomadura sp. The single purified enzyme was confirmed by the following (f) SDS-PAGE analysis.

(F) SDS-PAGE
The active fractions eluted in (e) above were collected and analyzed for molecular weight by SDS-PAGE (12% (w / v) polyacrylamide gel). FIG. 1 is an electrophoretogram showing the results of SDS-PAGE analysis of the eluted fraction. The left lane in FIG. 1 is a protein molecular weight marker (M), and the right lane in FIG. 1 shows a band of the purified enzyme (protease from RD001933 strain) obtained in (e). As shown in FIG. 1, a single band was observed in the active fraction, and the molecular weight of the purified enzyme (polypeptide) was about 31,000. The unit of molecular weight is Da (Dalton). Therefore, in the kDa notation, the molecular weight of this enzyme is about 31 kDa.

Table 1 shows the yield, yield and the like in the purification of protease from RD001933 strain.

In addition, the enzyme activity of the protease derived from RD001933 strain was measured as follows. First, the reaction liquid shown in Table 2 was allowed to react at 65 ° C. and pH 7.5 for 5 minutes, and then 50 μL of 20% trichloroacetic acid was added to stop the reaction. Thereafter, the reaction solution was centrifuged, and the absorbance at 340 nm of the supernatant was measured. The amount of enzyme 1U (unit) was set to an amount capable of producing 1 μmol of an azo dye corresponding to 1 minute.

[Properties of protease derived from RD001933 strain]
The enzymatic properties of the RD001933-derived protease (purified enzyme) were examined.

(1) Action temperature The reaction liquid shown in Table 2 was allowed to react at each temperature and pH 7.5 for 5 minutes, and then 50 μL of 20% trichloroacetic acid was added to stop the reaction. Thereafter, the reaction solution was centrifuged, and the absorbance at 340 nm of the supernatant was measured to determine the enzyme activity.

FIG. 3 is a graph showing enzyme activities at various reaction temperatures as relative activities based on the activity (100%) when the reaction temperature is 75 ° C. As shown in the graph of FIG. 3, the RD001933-derived protease (purified enzyme) exhibits activity at 50 to 90 ° C., and the optimum temperature for the reaction is in the range of 60 to 80 ° C., preferably 70 It was about ~ 75 ° C. FIG. 4 is a graph showing specific activities at various reaction temperatures.

(2) Working pH
The reaction liquid shown in Table 3 was allowed to react at each pH and 75 ° C. for 5 minutes, and then the reaction was stopped by adding 50 μL of 20% trichloroacetic acid. Thereafter, the reaction solution was centrifuged, and the absorbance at 340 nm of the supernatant was measured to determine the enzyme activity.

The buffer solution used is as follows.

MES-sodium hydroxide buffer: pH 5.5, pH 6
Bis-Tris buffer: pH 6, pH 6.5, pH 7.2
Tris-HCl buffer: pH 7.2, pH 8, pH 8.8
Glycine-NaOH buffer: pH 9, pH 9.5

FIG. 5 is a graph showing enzyme activities at various reaction pHs as relative activities based on the enzyme activity when the reaction pH is 7.2 (100%). As can be seen from the graph of FIG. 5, the RD001933-derived protease (purified enzyme) exhibits activity in a wide range of pH 5.5 to 9.0, and the optimum pH of the reaction is around 7.2 (for example, pH 6 0.0 to 8.8). FIG. 6 is a graph showing specific activities at various reaction pHs.

(3) Effect of added reagent First, the enzyme solution to which the added reagent was added was incubated at room temperature for 30 minutes, and then the reaction solution shown in Table 4 (excluding the added reagent and enzyme solution) was added to 75 ° C, pH 7. 5 and allowed to react for 5 minutes. Thereafter, 50 μL of 20% trichloroacetic acid was added to stop the reaction. And the enzyme activity was calculated | required by centrifuging a reaction liquid and measuring the light absorbency of 340 nm of a supernatant liquid.

The additive reagents used were EDTA, PMSF (phenylmethylsulfonyl fluoride) (final concentration 5 mM), ZnCl ₂ , MnCl ₂ , CaCl ₂ , CuCl ₂ , CoCl ₂ , MgCl ₂ (final concentration 1 mM).

Table 5 shows the enzyme activity when the additive reagent is added as a relative activity based on the activity (100%) when the additive reagent is not added. RD001933 strain-derived protease (purified enzyme) showed an activity of about 60% in the presence of Zn ²⁺ , Mn ²⁺ , Ca ²⁺ , Cu ²⁺ , Co ²⁺ and Mg ²⁺ . Further, in the presence of EDTA and PMSF, the activity was greatly reduced and activity inhibition was observed.

[Analysis of N-terminal amino acid sequence of protease (purified enzyme) derived from RD001933 strain]
The purified enzyme was electroblotted after SDS-PAGE, and the target enzyme was transferred to a PVDF (polyvinylidene fluoride) membrane. This was analyzed for the N-terminal amino acid sequence by a protein sequencer. From the analysis, it was confirmed that the N-terminal amino acid sequence of this purified enzyme was as shown in SEQ ID NO: 3.

[Analysis of internal amino acid sequence of protease (purified enzyme) derived from RD001933 strain]
For the purified enzyme, after SDS-PAGE, the target enzyme was excised and digested in gel using trypsin. The obtained peptide sample was analyzed for internal amino acid sequence using a mass spectrometer (nanoLC-MS / MS). Thereby, it was confirmed that the internal amino acid sequences are those shown in SEQ ID NOs: 4, 5, 6, and 7.

Here, SEQ ID NO: 4 is the sequence shown from position 277 of SEQ ID NO: 2. SEQ ID NO: 5 is the sequence shown from position 331 of SEQ ID NO: 2. SEQ ID NO: 6 is the sequence shown from position 348 of SEQ ID NO: 2. SEQ ID NO: 7 is the sequence shown from position 364 of SEQ ID NO: 2. In addition, since de novo amino acid sequence analysis by nanoLC-MS / MS cannot distinguish Leu and Ile, there may be portions where these amino acids do not partially match.

[Separation of chromosomal DNA of strain RD001933]
The RD001933 strain was cultured at 45 ° C. for 3 days using 50 mL of ISP2 medium (yeast extract 0.6%, malt extract 1.4%, glucose 0.6%) and collected.

Next, this microbial cell was suspended in 2.5 mL of a solution consisting of 75 mM NaCl, 25 mM EDTA, 20 mM Tris-HCl buffer (pH 7.5) and 1 mg / mL lysozyme, and treated at 37 ° C. for 1 hour. To this, 900 μL of 10% (w / v) SDS and 3.75 mg of proteinase K were added and treated at 55 ° C. for 2 hours. To this solution, 2 mL of 5 M NaCl and 5 mL of chloroform were added and stirred, and 5 mL of the aqueous phase was collected by centrifugation.

3 mL of isopropanol was added to and mixed with this aqueous phase to recover the DNA fraction, which was rinsed with 1 mL of 70% ethanol and then centrifuged. This precipitate was collected, dried under reduced pressure, and dissolved in 500 μL of a solution composed of 10 mM Tris-HCl buffer (pH 8.0) and 1 mM EDTA, and left at 55 ° C. overnight. RNaseA was added to this so that it might become 0.1 mg / mL, and after processing for 20 minutes at 37 ° C., 500 μL of 13% PEG solution containing 0.8 M NaCl was added and stirred, and after standing at 4 ° C. for 1 hour, The precipitate was collected by centrifugation. This precipitate was dissolved in 500 μL of 10 mM Tris-HCl buffer (pH 緩衝 7.5) containing 1 mM EDTA. 500 μL of a phenol / chloroform mixed solution was added thereto and stirred, and 500 μL of an aqueous phase was collected by centrifugation.

To this aqueous phase, 50 μL of 3M sodium acetate buffer (pH 5.2) and 1 mL of 100% ethanol were added and mixed to recover the chromosomal DNA of RD001933 strain.

The DNA was rinsed with 70% (v / v) ethanol and then centrifuged to collect the precipitate. The collected precipitate was dried under reduced pressure, and then dissolved in 200 μL of a solution composed of 10 mM Tris-HCl buffer (pH 8.0) and 1 mM EDTA.

[Cloning of core region of protease gene from RD001933 strain]
Primers were designed based on the N-terminal and internal sequences highly conserved in the Actinomadura-derived protease.

In preparing the primer, the above N-terminal sequence (SEQ ID NO: 3) and the above two internal amino acid sequences (SEQ ID NO: 4 and SEQ ID NO: 5) were picked up. In designing the primer, an appropriate sequence in the base sequence encoding these amino acid sequences was used.

The N-terminal amino acid sequence information was “Sense primer” and the internal amino acid sequence information was “Antisense primer”. For degenerate codons, a codon with high codon usage in the genus Actinomadura was selected and a primer was designed. Also, mixed base primers were used for those with the same codon usage frequency. Also, Leu and Ile, which are difficult to be separated by mass spectrometry, were excluded. Thus, degenerate oligonucleotide primer S1 (SEQ ID NO: 8) for PCR was designed as a sense primer (Sense プライマー primer). In addition, as two types of antisense primers (Antisense primer), “primer A1-1” (SEQ ID NO: 9) designed from SEQ ID NO: 4 and “primer A1-2” (SEQ ID NO: 10) designed from SEQ ID NO: 5 And designed. Here, s in the sequence represents c or g, and w represents a or t.

Next, PCR was performed using the above primers. The composition of the reaction solution for PCR is as follows.

Sterile water was added to 100 ng of the template chromosomal DNA obtained in the above [Separation of chromosomal DNA of RD001933 strain], 2 × MightAmp Buffer 25 μL, each primer 300 nM, and 0.5 unit of HighAmp DNA Polymerase so that the total amount was 50 μL.

PCR reaction conditions are as follows.

Step 1: 98 ° C., 2 minutes;
Step 2: 98 ° C., 10 seconds;
Step 3: 80 ° C., 15 seconds;
Step 4: 68 ° C., 1 minute;
Repeat steps 2 to 4 for 30 cycles;
Step 5: 68 ° C., 2 minutes.

A specific amplification product of about 700 bp was obtained by PCR using the above primers.

The PCR reaction solution was subjected to agarose gel electrophoresis, and the target band portion of about 700 bp was cut out and bound to pMD20-T Vector using Mighty TA-cloning Kit (TaKaRa) to transform Escherichia coli. The transformed strain is cultured in an LB medium (tryptone 1%, yeast extract 0.5%, sodium chloride 0.5%, pH 7.5) containing ampicillin 50 μg / mL, Miniprep method (miniprep method) or High Pure Plasmid. A plasmid for DNA sequencing was extracted and purified using Isolation Kit (Roche).

Subsequently, the base sequence of the inserted fragment was determined by an automatic sequencer using T7 primer and SP6 primer derived from a vector (pMD20-T Vector). This base sequence (692 bp) is shown in SEQ ID NO: 11.

[Cloning upstream and downstream of core region of protease gene from RD001933 strain]
In order to clarify the sequence of the peripheral region of the gene sequence determined in [Cloning of core region of protease gene derived from RD001933 strain] above, the chromosomal DNA obtained above was completely digested with PstI or SmaI, and Ligation high Ver. The digested fragment was self-cyclized using 2 (Toyobo). Using this as a template, PCR was performed using two inverse PCR primers (SEQ ID NO: 12 and SEQ ID NO: 13) prepared based on the partial gene sequence of the protease derived from the RD001933 strain. The DNA fragment on the downstream side was amplified.

The PCR reaction solution composition is as follows.

Sterile water was added to 25 units of template DNA 25 ng, 2 × MightAmp Buffer 25 μL, each primer 300 nM, and LightAmp DNA Polymerase 0.5 unit so that the total amount was 50 μL.

PCR reaction conditions are as follows.

Step 1: 98 ° C., 2 minutes;
Step 2: 98 ° C., 10 seconds;
Step 3: 62 ° C., 15 seconds;
Step 4: 68 ° C., 3 minutes;
Repeat steps 2 to 4 for 25 cycles;
Step 5: 68 ° C., 3 minutes.

A specific amplification product of about 3000 bp was obtained by PCR using the above primers.

The PCR reaction solution was subjected to agarose gel electrophoresis, and the target band portion of about 3000 bp was cut out and bound to pMD20-T Vector using Mighty TA-cloning Kit (TaKaRa) to transform Escherichia coli. The transformed strain is cultured in an LB medium (tryptone 1%, yeast extract 0.5%, sodium chloride 0.5%, pH 7.5) containing ampicillin 50 μg / mL, Miniprep method (miniprep method) or High Pure Plasmid. A plasmid for DNA sequencing was extracted and purified using Isolation Kit (Roche).

Subsequently, the base sequence of the inserted fragment was determined by an automatic sequencer using SP6 primer and M13M4 primer derived from a vector (pMD20-T Vector). By this sequence, the base sequence (330 bp) shown in SEQ ID NO: 14 was obtained as the base sequence on the N-terminal side (upstream side). This sequence is a signal sequence (78 bp) and a pro sequence (252 bp) from the N-terminal side. Moreover, the base sequence (139 bp) shown in SEQ ID NO: 15 was obtained as the base sequence on the C-terminal side (downstream side).

[Determination of nucleotide sequence of protease gene derived from RD001933 strain]
Based on the base sequence determined above, the base sequence (1161 bp) of the region containing the protease gene derived from the RD001933 strain was determined (SEQ ID NO: 1). SEQ ID NO: 2 is an amino acid sequence corresponding to the codon of this sequence (SEQ ID NO: 1).

From the results of sequence analysis, it was revealed that the gene encoding protease derived from RD001933 strain consists of 1161 bp nucleotides and encodes 386 amino acids.

The N-terminal and internal amino acid sequences of the RD001933 strain-derived protease (purified enzyme) determined above were present in the above deduced amino acid sequence and were almost completely identical.

[Protease from RD001933 strain (crude enzyme)]
RD001933 strain belonging to the genus Actinodura sp. Of actinomycetes was cultured, and a crude enzyme solution was obtained from the culture supernatant by three kinds of precipitation methods. Details are shown below.

First, 700 mL of ISP2 medium (yeast extract 0.6%, malt extract 1.4%, glucose 0.6%) was prepared, and 70 mL was dispensed into a 500 mL Erlenmeyer flask. One spring coil was put in this, and steam sterilization was performed at 121 ° C. for 20 minutes. Furthermore, 30 mL of 2.5% skimmed milk that was sterilized separately was added to a final concentration of 0.75%.

(B1) Acetone precipitation Acetone at −20 ° C. was added to the culture supernatant collected in (a) so as to be 40%, 50%, 60%, 70%, and 80% (v / v). The precipitate produced in 1 was recovered by centrifugation (10,000 rpm, 10 minutes, 4 ° C.). This precipitate was dissolved in 30 mL of 20 mM Tris-HCl buffer (pH 9.0) to obtain a crude enzyme solution. Table 6 shows enzyme recovery.

(B2) Ethanol precipitation At −20 ° C. ethanol was added to the culture supernatant collected in (a) above so that it would be 40%, 50%, 60%, 70%, and 80% (v / v). The precipitate produced in 1 was recovered by centrifugation (10,000 rpm, 10 minutes, 4 ° C.). This precipitate was dissolved in 30 mL of 20 mM Tris-HCl buffer (pH 8.0) to obtain a crude enzyme solution. Table 6 shows enzyme recovery.

(B3) Ammonium sulfate precipitation To the culture supernatant collected in (a) above, ammonium sulfate powder was added so that the saturation amount was 40%, 50%, 60%, 70%, 80%, and the precipitates generated at each concentration were added. It was recovered by centrifugation (10,000 rpm, 10 minutes, 4 ° C.). This precipitate was dissolved in 30 mL of 20 mM Tris-HCl buffer (pH 9.0) to obtain a crude enzyme solution. Table 6 shows enzyme recovery.

Thus, when the enzyme is concentrated and recovered, three types of precipitation methods can be used as appropriate.

Next, the enzymatic properties of the RD001933-derived protease (crude enzyme solution) were examined.

(1) Working temperature After reacting the reaction solution shown in Table 2 (enzyme solution obtained by 80% saturated ammonium sulfate precipitation) at each temperature and pH 7.5 for 10 minutes, 50 μL of 20% trichloroacetic acid was reacted. In addition, the reaction was stopped. Thereafter, the reaction solution was centrifuged, and the absorbance at 340 nm of the supernatant was measured to determine the enzyme activity.

FIG. 7 is a graph showing enzyme activities at various reaction temperatures as relative activities based on the activity (100%) when the reaction temperature is 80 ° C. As shown in the graph of FIG. 7, the RD001933 strain-derived protease (crude enzyme solution) exhibits activity at 50 to 95 ° C., and the optimal temperature for the reaction is in the range of 70 to 85 ° C., preferably It was around 75 to 85 ° C.

(2) Working pH
The reaction solution shown in Table 3 (enzyme solution obtained by 80% saturated ammonium sulfate precipitation) was allowed to react for 10 minutes at each pH and 65 ° C., and then the reaction was stopped by adding 50 μL of 20% trichloroacetic acid. It was. Thereafter, the reaction solution was centrifuged, and the absorbance at 340 nm of the supernatant was measured to determine the enzyme activity.

The buffer solution used is as follows.

Acetic acid-Na acetate buffer: pH 5
Bis-Tris buffer: pH 6
Tris-HCl buffer: pH 7.2, pH 8
Glycine-NaOH buffer: pH 9
FIG. 8 is a graph showing enzyme activities at various reaction pHs as relative activities based on the enzyme activity when the reaction pH is 8.0 (100%). As can be seen from the graph of FIG. 8, the RD001933 strain-derived protease (crude enzyme solution) exhibits activity in a wide range of pH 5.0 to 9.0, and the optimum pH of the reaction is around 7.5 (for example, pH 7.0-8.0).

(3) Effect of added reagent The reaction solution shown in Table 4 (enzyme solution obtained by 80% ethanol precipitation) was allowed to react at 65 ° C. and pH 7.5 for 10 minutes. Thereafter, 50 μL of 20% trichloroacetic acid was added to stop the reaction. And the enzyme activity was calculated | required by centrifuging a reaction liquid and measuring the light absorbency of 340 nm of a supernatant liquid.

The additive reagents used are CaCl ₂ , NaCl, KCl, MgCl ₂ , MnCl ₂ , FeCl ₂ , FeCl ₃ , CuCl ₂ , ZnCl ₂ , CoCl ₂ , EDTA.

Table 7 shows the enzyme activity when the additive reagent is added as a relative activity based on the activity when the additive reagent is not added (100%).

(4) Dependence on CaCl ₂ concentration After reacting the reaction solution shown in Table 8 (enzyme solution obtained by 80% ethanol precipitation) at 65 ° C. for 10 minutes, 50 μL of 20% trichloroacetic acid was added. The reaction was stopped. Thereafter, the reaction solution was centrifuged, and the absorbance at 340 nm of the supernatant was measured to determine the enzyme activity.

9, the enzymatic activity at various CaCl ₂ final concentration is a graph showing the activity when CaCl ₂ final concentration is 0.5mM as a relative activity to the reference (100%).

[Purification of protease from RD000920 strain]
The RD000920 strain belonging to the genus Actinomadura miaoliensis of actinomycetes is cultured, and the culture supernatant is derived from the RD000920 strain using ammonium sulfate fractionation, hydrophobic chromatography, cation exchange chromatography, and anion exchange chromatography. Protease was purified. Details are shown below.

(A) Culture of microorganisms RD000920 strain (accession number: NITE P-1468) belonging to the genus Actinomadura miaoliensis of actinomycetes was used as the cells.

First, 560 mL of ISP2 medium (yeast extract 0.6%, malt extract 1.4%, glucose 0.6%) was prepared, and 70 mL was dispensed into a 500 mL Erlenmeyer flask. One spring coil was put in this, and steam sterilization was performed at 121 ° C. for 20 minutes. Furthermore, 30 mL of 2.5% skimmed milk that was sterilized separately was added to a final concentration of 0.75%.

(B) Acetone fractionation (acetone precipitation)
-20 ° C. acetone is added to the culture supernatant collected in (a) above 60% (v / v) and the resulting precipitate is centrifuged (10,000 rpm, 10 minutes, 4 ° C.). It was collected. This precipitate was dissolved in 15 mL of 20 mM Tris-HCl buffer (pH 8.0) containing ammonium sulfate having a final concentration of 1 M to obtain a crude enzyme solution.

(C) Toyopearl Phenyl-650M column chromatography The Toyopearl Phenyl-650M column (equilibrated in advance with 20 mM Tris-HCl buffer (pH 8.0) containing 1 M ammonium sulfate was added to the crude enzyme solution obtained in (b) above. The inner diameter was 26 mm, the height was 38 mm, and manufactured by Tosoh Corporation. After washing the column with the same buffer, the protein was eluted with a linear gradient of ammonium sulfate (from 1 M to 0 M).

(D) HiTrap Q HP column chromatography The active fractions obtained in (c) above were collected and desalted by dialysis using 20 mM Tris-HCl buffer (pH 9.0). To this, 20 mM Tris-HCl buffer (pH 9.0) was added. The column was applied to a HiTrap Q HP (5 mL) column (manufactured by GE Healthcare Biosciences) previously equilibrated with 20 mM Tris-HCl buffer (pH 9.0), washed with the same buffer, and then sodium chloride ( The protein was eluted with a linear gradient from 0M to 1M.

(E) HiTrap SP HP column chromatography The active fractions obtained in (d) above were collected and desalted by dialysis using 20 mM MES-sodium hydroxide buffer (pH 5.5). To this, 20 mM MES-sodium hydroxide buffer (pH 5.5) was added. This was applied to a HiTrap SP (1 mL) column (manufactured by GE Healthcare Biosciences) previously equilibrated with 20 mM MES-sodium hydroxide buffer (pH 5.5), and the column was washed with the same buffer. The protein was eluted with a linear gradient of sodium chloride (0M to 1M).

As described above, a purified enzyme was obtained from the RD000920 strain belonging to the genus Actinomadura miaoliensis. The single purified enzyme was confirmed by the following (f) SDS-PAGE analysis.

(F) SDS-PAGE
The active fractions eluted in (e) above were collected and analyzed for molecular weight by SDS-PAGE (15% (w / v) polyacrylamide gel). FIG. 2 is an electrophoretogram showing the results of SDS-PAGE analysis of the eluted fraction. The left lane in FIG. 2 is the protein molecular weight marker (M), and the right lane in FIG. 2 shows the band of the purified enzyme (protease from RD000920 strain) obtained in (e). As shown in FIG. 2, a single band was observed in the active fraction, and the molecular weight of the purified enzyme (polypeptide) was about 31,000. The unit of molecular weight is Da (Dalton). Therefore, in the kDa notation, the molecular weight of this enzyme is about 31 kDa.

Table 9 shows the yield, yield and the like in the purification of protease from RD000920 strain.

The enzyme activity of the RD000920 strain-derived protease was measured in the same manner as the enzyme activity of the RD001933 strain-derived protease.

[Properties of protease derived from RD000920 strain]
The enzymatic properties of protease (purified enzyme) derived from RD000920 strain were examined.

(1) Working temperature After reacting the reaction liquid shown in Table 2 at each temperature and pH 7.5 for 10 minutes to react, 50 μL of 20% trichloroacetic acid was added to stop the reaction. Thereafter, the reaction solution was centrifuged, and the absorbance at 340 nm of the supernatant was measured to determine the enzyme activity.

FIG. 3 is a graph showing enzyme activities at various reaction temperatures as relative activities based on the activity (100%) when the reaction temperature is 75 ° C. As shown in the graph of FIG. 3, the RD000920 strain-derived protease (purified enzyme) exhibits activity at 50 to 90 ° C., and the optimum temperature for the reaction is in the range of 60 to 80 ° C., preferably 70 It was about ~ 75 ° C. FIG. 4 is a graph showing specific activities at various reaction temperatures.

(2) Working pH
The reaction liquid shown in Table 3 was stirred and reacted at each pH and 65 ° C. for 10 minutes, and then 50 μL of 20% trichloroacetic acid was added to stop the reaction. Thereafter, the reaction solution was centrifuged, and the absorbance at 340 nm of the supernatant was measured to determine the enzyme activity.

The buffer solution used is as follows.

MES-sodium hydroxide buffer: pH 5.5, pH 6
Bis-Tris buffer: pH 6, pH 6.5, pH 7.2
Tris-HCl buffer: pH 7.2, pH 8, pH 8.8
Glycine-NaOH buffer: pH 9, pH 9.5
FIG. 5 is a graph showing enzyme activities at various reaction pHs as relative activities based on the enzyme activity when the reaction pH is 7.2 (100%). As can be seen from the graph of FIG. 5, the RD000920 strain-derived protease (purified enzyme) exhibits activity in a wide range of pH 5.5 to 9.0, and the optimum pH of the reaction is around 7.2 (for example, pH 6 0.0 to 8.8). FIG. 6 is a graph showing specific activities at various reaction pHs.

Table 10 shows the enzyme activity when the additive reagent is added as a relative activity based on the activity (100%) when the additive reagent is not added. RD000920 strain-derived protease (purified enzyme) showed an activity of about 60% in the presence of Zn ²⁺ , Mn ²⁺ , Ca ²⁺ , Cu ²⁺ , Co ²⁺ and Mg ²⁺ . Further, in the presence of EDTA and PMSF, the activity was greatly reduced and activity inhibition was observed.

[Analysis of amino acid sequence of protease (purified enzyme) derived from RD000920 strain]
Analysis of the amino acid sequence of the RD000920 strain-derived protease (purified enzyme) was performed in the same manner as in the case of the RD001933 strain-derived protease (purified enzyme). As a result, the amino acid sequence of the RD000920 strain-derived protease (purified enzyme) was almost 100% identical to the amino acid sequence of the RD001933 strain-derived protease (purified enzyme), so it is considered that both enzymes consist of the same amino acid sequence. Moreover, since the base sequence of the protease gene derived from the RD000920 strain was almost 100% identical to the base sequence of the protease gene derived from the RD001933 strain, both genes are considered to have the same base sequence.

However, as shown in FIGS. 3 to 6, both enzymes showed slight differences in enzymological properties such as action temperature and action pH.

[RD000920 strain-derived protease (crude enzyme)]
The RD000920 strain belonging to the genus Actinodura miaoliensis of actinomycetes was cultured, and a crude enzyme solution was obtained from the culture supernatant by three kinds of precipitation methods. Details are shown below.

(B1) Acetone precipitation Acetone at −20 ° C. was added to the culture supernatant collected in (a) so as to be 40%, 50%, 60%, 70%, and 80% (v / v). The precipitate produced in 1 was recovered by centrifugation (10,000 rpm, 10 minutes, 4 ° C.). This precipitate was dissolved in 30 mL of 20 mM Tris-HCl buffer (pH 8.0) to obtain a crude enzyme solution. Table 11 shows enzyme recovery.

(B2) Ethanol precipitation At −20 ° C. ethanol was added to the culture supernatant collected in (a) above so that it would be 40%, 50%, 60%, 70%, and 80% (v / v). The precipitate produced in 1 was recovered by centrifugation (10,000 rpm, 10 minutes, 4 ° C.). This precipitate was dissolved in 30 mL of 20 mM Tris-HCl buffer (pH 8.0) to obtain a crude enzyme solution. Table 11 shows enzyme recovery.

(B3) Ammonium sulfate precipitation To the culture supernatant collected in (a) above, ammonium sulfate powder was added so that the saturation amount was 40%, 50%, 60%, 70%, 80%, and the precipitates generated at each concentration were added. It was recovered by centrifugation (10,000 rpm, 10 minutes, 4 ° C.). This precipitate was dissolved in 30 mL of 20 mM Tris-HCl buffer (pH 8.0) to obtain a crude enzyme solution. Table 11 shows enzyme recovery.

Next, the enzymatic properties of the RD000920 strain-derived protease (crude enzyme solution) were examined.

FIG. 10 is a graph showing enzyme activities at various reaction temperatures as relative activities based on the activity (100%) when the reaction temperature is 70 ° C. As shown in the graph of FIG. 10, the RD000920 strain-derived protease (crude enzyme solution) exhibits activity at 50 to 85 ° C., and the optimal temperature for the reaction is in the range of 65 to 80 ° C., preferably It was around 70 to 80 ° C.

The buffer solution used is as follows.

Acetic acid-Na acetate buffer: pH 5
Bis-Tris buffer: pH 6
Tris-HCl buffer: pH 7.2, pH 8
Glycine-NaOH buffer: pH 9
FIG. 11 is a graph showing enzyme activities at various reaction pHs as relative activities based on the enzyme activity when the reaction pH is 8.0 (100%). As can be seen from the graph of FIG. 11, the RD000920 strain-derived protease (crude enzyme solution) exhibits activity in a wide range of pH 5.0 to 9.0, and the optimum pH of the reaction is around 7.5 (for example, pH 7.0-8.0).

Table 12 shows the enzyme activity when the additive reagent is added as a relative activity based on the activity (100%) when the additive reagent is not added.

12, the enzyme activity at various CaCl ₂ final concentration is a graph showing the activity when CaCl ₂ final concentration is 0.5mM as a relative activity to the reference (100%).

[Protease (crude enzyme) derived from RD001933 and RD000920]
The RD001933 strain belonging to the genus Actinodura sp. Of Actinomycetes and the RD000920 strain belonging to the genus Actinomadura miaoliensis of Actinomycetes are mixed and cultured, and three kinds of precipitation methods are used to coarsely prepare the strain. An enzyme solution was obtained. Details are shown below.

(A) Culture of microorganisms As cells, RD001933 strain (accession number: NITE P-1467) belonging to the genus Actinodura sp. Of actinomycetes and RD000920 belonging to the genus Actinodura miaoliensis of actinomycetes The strain (Accession Number: NITE P-1468) was used.

First, 4.2 L of ISP2 medium (yeast extract 0.6%, malt extract 1.4%, glucose 0.6%) was prepared and placed in a 10 L desktop culture device (manufactured by Maruhishi Bioengineer), 121 ° C. For 20 minutes. Furthermore, 1.8 L of 2.5% skimmed milk that was sterilized separately was added to a final concentration of 0.75%.

Then, 500 μL of two glycerol stock cells were inoculated into 500 mL Erlenmeyer flasks containing 50 mL of ISP2 medium, and cultured with shaking at 45 ° C. until good growth was obtained. 30 mL of each of these two types of culture solutions was inoculated into 6 L of the previously sterilized medium and cultured at 45 ° C., 500 rpm, 1 vvm for 1 to 7 days, preferably 1 to 3 days. The ratio of the two types of culture solutions at the start of the culture is preferably 1: 1, but there is no special provision. The supernatant was recovered from this culture using a centrifuge.

(B1) Acetone precipitation Acetone at −20 ° C. was added to the culture supernatant collected in (a) so as to be 40%, 50%, 60%, 70%, and 80% (v / v). The precipitate produced in 1 was recovered by centrifugation (10,000 rpm, 10 minutes, 4 ° C.). This precipitate was dissolved in 50 mL of 20 mM Tris-HCl buffer (pH 8.0) to obtain a crude enzyme solution. Table 13 shows enzyme recovery.

(B2) Ethanol precipitation Ethanol was added to the culture supernatant collected in the above (a) so as to be 40%, 50%, 60%, 70%, 80% (v / v), and it was generated at each concentration. The precipitate was collected by centrifugation (10,000 rpm, 10 minutes, 4 ° C.). This precipitate was dissolved in 50 mL of 20 mM Tris-HCl buffer (pH 8.0) to obtain a crude enzyme solution. Table 13 shows enzyme recovery.

(B3) Ammonium sulfate precipitation To the culture supernatant collected in (a) above, ammonium sulfate powder was added so that the saturation amount was 40%, 50%, 60%, 70%, 80%, and the precipitates generated at each concentration were added. It was recovered by centrifugation (10,000 rpm, 10 minutes, 4 ° C.). This precipitate was dissolved in 50 mL of 20 mM Tris-HCl buffer (pH 8.0) to obtain a crude enzyme solution. Table 13 shows enzyme recovery.

Next, the enzymatic properties of proteases (crude enzymes) derived from strains RD001933 and RD000920 were examined.

FIG. 13 is a graph showing enzyme activities at various reaction temperatures as relative activities based on the activity (100%) when the reaction temperature is 70 ° C. As shown in the graph of FIG. 13, RD001933 strain and RD000920 strain-derived protease (crude enzyme) exhibit activity at 55 to 85 ° C., and the optimal temperature for the reaction is in the range of 65 to 80 ° C. Preferably, it was around 70 ° C.

The buffer solution used is as follows.

Acetic acid-Na acetate buffer: pH 5
Bis-Tris buffer: pH 6
Tris-HCl buffer: pH 7.2, pH 8
Glycine-NaOH buffer: pH 9
FIG. 14 is a graph showing enzyme activities at various reaction pHs as relative activities based on the enzyme activity when the reaction pH is 8.0 (100%). As can be seen from the graph of FIG. 14, the proteases (crude enzymes) derived from strains RD001933 and RD000920 exhibit activity in a wide range of pH 5.0 to 9.0, and the optimum pH of the reaction is around 7.5. (For example, pH 7.0 to 8.0).

Table 14 shows the enzyme activity when the additive reagent is added as the relative activity based on the activity (100%) when the additive reagent is not added.

Figure 15 is an enzyme activity at various CaCl ₂ final concentration is a graph showing the activity when CaCl ₂ final concentration is 0.5mM as a relative activity to the reference (100%).

(5) Storage stability Ammonium sulfate was added to the culture supernatant collected in (a) so as to be 60% saturation, and the resulting precipitate was collected by centrifugation (10,000 rpm, 10 minutes, 4 ° C.). . This precipitate was dissolved with 20 mM Tris-HCl buffer (pH 8.0) so as to be concentrated 90-fold or 18-fold to obtain a crude enzyme solution. The crude enzyme solution concentrated 90 times was allowed to stand at 4 ° C., room temperature (average 26.0 ° C.) and 40 ° C., and the crude enzyme solution concentrated 18 times was lyophilized in vacuo to obtain a lyophilized product at 4 ° C. It left still at 40 degreeC. After a predetermined number of days, the enzyme activity of each crude enzyme was measured as follows. First, the reaction solution shown in Table 2 (the crude enzyme solution was diluted 200-fold with tap water, and the freeze-dried product was dissolved in tap water to 1 mg / mL) and left at 65 ° C. for 10 minutes. After the reaction, 50 μL of 20% trichloroacetic acid was added to stop the reaction. Thereafter, the reaction solution was centrifuged, and the absorbance at 340 nm of the supernatant was measured. The activity on the first day was taken as the standard (100%).

FIG. 16 is a graph showing changes over time in residual activity for each crude enzyme. It can be seen from FIG. 16 that the activity is hardly lowered even when the freeze-dried product is stored at 40 ° C.

[Cleaning effect confirmation test]
(1) Dropping test As a protease-containing detergent, RD001933-derived protease (purified enzyme), RD000920 strain-derived protease (purified enzyme), RD001933 strain and RD000920 strain-derived protease (crude enzyme) were each contaminated with simulated blood. The solution was dropped on a piece (NITI-ON, washing evaluation indicator “TOSI-Gold”), and the pseudo blood was wiped off every predetermined time for washing. This washing was performed at 65 ° C. by adjusting the amount of each enzyme to 5 μL (concentration: about 5 U / mL). The cleaning result of the test piece was determined visually by the following criteria. The results are shown in Table 15.

“◎”: completely cleaned “○”: almost cleaned “△”: very little residue (pseudo-blood stain) “×”: residue (pseudo-blood stain) remains State

(2) Flask test As detergents, RD001933 strain and RD000920 strain-derived protease (crude enzyme), commercially available enzyme nattokinase (Wako Pure Chemical Industries, Ltd. “147-08801”), commercially available streptokinase (Wako Pure Chemical Industries, Ltd.) "593-20581"), commercially available enzyme urokinase (Mitsubishi Tanabe Pharma Corporation "873954"), commercially available clothing detergent (Kao Corporation "Attack Neo"), commercially available enzyme thermolysin (Sigma-Aldrich "P1512-1G") Using.

30 mL of each of the above cleaning agents was put in a 200 mL flask, and further a test piece (NITI-ON, cleaning evaluation indicator “TOSI-Gold”) was added and immersed. And the inside of said flask was stirred with the stirring bar on the conditions of temperature and time shown in Table 17. Then, after taking out said test piece and lightly washing with water, the washing | cleaning result was determined on the same basis as the above. The results are shown in Table 16.

As is clear from Table 16, it was confirmed that proteases (crude enzymes) derived from RD001933 and RD000920 strains had the highest cleaning effect, and then the commercially available enzyme thermolysin had the highest cleaning effect.

(3) Washer test 1
The cleaning effect of thermolysin (Sigma-Aldrich “P1512-1G”), which is considered to have the highest cleaning effect among commercially available cleaning agents, and protease (crude enzyme) derived from RD001933 and RD000920 strains is as follows. Confirmed.

A test piece (NITI-ON, cleaning evaluation indicator “TOSI-Gold”) was set in an instrument decontamination cleaner “WD3060” manufactured by smeg (Italy). Furthermore, protease (crude enzyme) or thermolysin (Sigma-Aldrich “P1512-1G”) derived from RD001933 and RD000920 strains was set as a protease-containing detergent, and the temperature X (° C.) shown in Table 17 in the following steps 1 to 5 was set. And the test piece was washed under the condition of time Y (min). The cleaning result of the test piece was determined visually by the same standard as described above. The results are shown in Table 17.

Step 1: pre-washing with tap water, 3 minutes;
Step 2: Washing, temperature X (° C.), time Y (min);
Step 3: First rinse, 40 ° C., 1 minute;
Step 4: Rinse second time, tap water, 1 minute;
Process 5: Sterilization, 90 degreeC, 5 minutes.

From Table 17, the RD001933 strain and the RD000920 strain-derived protease (crude enzyme) are considered to have a higher cleaning effect because they show the same cleaning effect with a smaller amount of enzyme than the commercially available enzyme thermolysin.

(4) Cleaner test 2
The relationship between Ca ²⁺ concentration and detergency assuming hard water in Europe and the like was examined as follows.

A test piece (NITI-ON, cleaning evaluation indicator “TOSI-Gold”) was set in an instrument decontamination cleaner “WD3060” manufactured by smeg (Italy). Further, RD001933 strain and RD000920 strain-derived protease (crude enzyme) were set as protease-containing detergents, and the test pieces were washed in the same steps 1 to 5 as described above. The results are shown in Table 18. The cleaning result of the test piece was determined visually by the same standard as described above.

As is apparent from Table 18, proteases (crude enzymes) derived from RD001933 and RD000920 strains are hard water (European water, etc.) having a high Ca ²⁺ concentration compared to soft water having a low Ca ²⁺ concentration (for example, tap water in Japan). ) It was confirmed that the lower one further improved the cleaning power.

(5) Cleaning device test 3
The cleaning effect of the alkaline detergents currently widely used was confirmed as follows.

A test piece (NITI-ON, cleaning evaluation indicator “TOSI-Gold”) was set in an instrument decontamination cleaner “WD3060” manufactured by smeg (Italy). Furthermore, set the existing detergent A (alkaline detergent, Borer Chemie's “deconex® 28ALKAONE-X”) and existing detergent B (multienzyme detergent, Borer Chemie ’s “deconex® POWER ZYME”) The test pieces were washed in the same steps 1 to 5 as described above. The results are shown in Table 19. The cleaning result of the test piece was determined visually by the same standard as described above.

Comparing the washing results of proteases (crude enzymes) derived from RD001933 and RD000920 shown in Table 17 and Table 18 with the washing results of existing alkaline detergents shown in Table 19, proteases from RD001933 and RD000920 (crude enzymes) ) Was confirmed to have a higher cleaning effect than existing alkaline cleaners.

[Actinomadura sp. RD001933 strain (Accession number: NITE P-1467)]
Accession Number: NITE P-1467
Name of depositary institution: National Institute for Product Evaluation Technology Patent Microorganism Depositary (NPMD)
Name of depositary institution: Japan 2-5-8 Kazusa Kamashitsu, Kisarazu City, Chiba Prefecture 292-0818, Japan
Date of deposit: November 22, 2012.
[Actinomadura miaoliensis RD000920 strain (Accession number: NITE P-1468)]
Accession Number: NITE P-1468
Name of depositary institution: National Institute for Product Evaluation Technology Patent Microorganism Depositary (NPMD)
Name of depositary institution: Japan 2-5-8 Kazusa Kamashitsu, Kisarazu City, Chiba Prefecture 292-0818, Japan
Date of deposit: November 22, 2012.

SEQ ID NO: 1: Protease expression gene SEQ ID NO: 2: Protease (polypeptide)
SEQ ID NO: 3: N-terminal sequence of protease SEQ ID NO: 4: Internal sequence of protease SEQ ID NO: 5: Internal sequence of protease SEQ ID NO: 6: Internal sequence of protease SEQ ID NO: 7: Internal sequence of protease SEQ ID NO: 8: Primer S1
SEQ ID NO: 9: Primer A1-1
SEQ ID NO: 10: Primer A1-2
Sequence number 11: Protease expression gene fragment (analysis result)
SEQ ID NO: 12: primer (inverse PCR)
SEQ ID NO: 13: primer (inverse PCR)
SEQ ID NO: 14: Protease-expressed gene fragment (analysis result, 5 ′ end sequence)
SEQ ID NO: 15: Protease expression gene fragment (analysis result, 3 ′ end sequence)

Claims

Protease obtained by culturing Actinomycetes sp. RD001933 strain (NBRC domestic isolate RD strain).
Protease obtained by cultivating actinomycete Myaoriensis RD000920 strain (NBRC domestic isolate RD strain).
Actinomycetes Actinomadura sp. RD001933 (NBRC domestic isolate RD) and Actinomycetes Actinomadura miaoliensis RD000920 (NBRC domestic isolate RD) are mixed and cultured. Protease obtained.
The protease according to any one of claims 1 to 3, wherein the protease comprises a polypeptide according to the following (a1), (a2), or (a3).
(A1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2.
(A2) A polypeptide comprising an amino acid sequence in which one or more amino acid residues are substituted, inserted, deleted, and / or added in the amino acid sequence set forth in SEQ ID NO: 2.
(A3) A polypeptide having at least 65% homology with the amino acid sequence set forth in SEQ ID NO: 2.
The protease according to any one of claims 1 to 4, wherein the protease has a molecular weight of about 31,000 Da (31 kDa) by SDS-PAGE.
The protease according to any one of claims 1 to 5, wherein the polynucleotide encoding the protease comprises the polynucleotide according to the following (b1), (b2) or (b3).
(B1) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 1.
(B2) A polynucleotide that hybridizes with a base sequence complementary to the base sequence described in SEQ ID NO: 1 under stringent conditions.
(B3) A polynucleotide having at least 70% sequence identity to the base sequence set forth in SEQ ID NO: 1.
A protease-containing detergent containing the protease according to any one of claims 1 to 6.
A method for producing a protease-containing detergent comprising culturing and producing actinomycetes, Actinomadura sp. RD001933 strain (NBRC domestic isolate RD strain).
A method for producing a protease-containing detergent, which is produced by culturing and producing actinomycetes, Actinomadura miaoliensis RD000920 strain (NBRC domestic isolate RD strain).
Actinomycetes Actinomadura sp. RD001933 (NBRC domestic isolate RD) and Actinomycetes Actinomadura miaoliensis RD000920 (NBRC domestic isolate RD) are mixed and cultured. A method for producing a protease-containing detergent.