WO1998006832A1

WO1998006832A1 - Protopectinase

Info

Publication number: WO1998006832A1
Application number: PCT/JP1997/002794
Authority: WO
Inventors: Takuo Sakai
Original assignee: Takuo Sakai
Priority date: 1996-08-12
Filing date: 1997-08-08
Publication date: 1998-02-19
Also published as: AU3785297A

Abstract

A novel protopectinase; a DNA encoding this protopectinase; an expression vector containing this DNA; host cells containing this expression vector; and a process for producing the above-mentioned protopectinase.

Description

Description Technical field

TECHNICAL FIELD The present invention relates to a novel broktocutinase and a method for producing the same. Background art

Prototinase is a general term for enzymes that release pectin from plant fiber. Brotobectinases are roughly classified into the following two types. One is the A-type (fermentation and industry: 37, 928-938, 1978), which is produced by yeast and has the activity of releasing pectin from plant fibers and limiting the degradation of free pectin. The other is produced by Bacillus genus bacteria and releases pectin from plant fiber, but has no activity to decompose free pectin. B-Eve (Agri Biol. Chem., 52 Agric. Biol. Chem., 53, 1213-1223, 1989; Agric. Biol. Chem., 54, 879-889, 1990; Eur. J. Biochem., 226, 285-291,

1994). In particular, the B-type has wide utility in the production of pectin (Japanese Patent Publication No. 6-8322), the refining of fibers (JP-A-6-220772), and the pulping of non-wood fibers (Japanese Patent Publication No. 57-39636). It has been shown.

Conventionally, a culture solution of a microorganism producing this enzyme has been used as a source of brotobectinase. However, for industrial applications, it is desirable to develop an inexpensive manufacturing method by improving productivity.

Accordingly, the invention of the present application described herein encodes a novel protease. To make use of this enzyme by isolating DNA and using genetic engineering techniques. Disclosure of the invention

According to the present invention, the following properties:

(1) it has a protopectin-degrading activity;

(2) molecular weight on SDS polyacrylamide electrophoresis gel is about 30,000;

(3) the optimum pH is 6.0;

(4) the optimum temperature is 60; and

(5) Inhibited by Hg and Mn,

Is provided.

In one embodiment, the proteinase has further arabinase activity.

In one embodiment, the protease is produced by Bacillus subtil is strain BS.

In one embodiment, the proteinase has the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence in which one or several amino acid residues are deleted, substituted, or inserted in the amino acid sequence. It has an activity equal to or higher than that of a protease having the amino acid sequence of brackets SEQ ID NO: 1. According to the present invention, a DNA encoding the protease is provided.

In one embodiment, the DNA has the nucleotide sequence of SEQ ID NO: 2, or has a nucleotide sequence in which one or several nucleotides are deleted, substituted or added in the nucleotide sequence, Activity equal to or better than that of the protectinase encoded by the nucleotide sequence of SEQ ID NO: 2 Is encoded.

According to the present invention, there is provided an expression vector containing the DNA.

According to the present invention, there is provided a host cell comprising the expression vector.

According to the present invention, the following properties are included in the genus Bacillus:

(1) Has protopectin degrading activity

(2) the molecular weight on an SDS polyacrylamide electrophoresis gel is about 30,000;

(3) the optimum pH is 6.0;

(4) the optimum temperature is 60; and

(5) subject to inhibition by Hg and min,

The present invention provides a method for producing protease, comprising a step of culturing a microorganism having the ability to produce protease.

In one embodiment, the proteinase has further arabinase activity.

In one embodiment, in the method, the microorganism is a Bacillus subtilis BS strain.

According to the present invention, the following properties are included, including the step of culturing a host cell transformed with the expression vector:

(1) Has protopectin degrading activity

(2) molecular weight on SDS polyacrylamide electrophoresis gel is about 30,000; (3) optimal pH is 6.0;

(4) the optimum temperature is 60; and

(5) subject to inhibition by Hg and Mn,

And a method for producing a protease having the formula:

In one embodiment, the protease is further treated with arabinase activity. Has the property. BEST MODE FOR CARRYING OUT THE INVENTION

The term proproteininase (hereinafter abbreviated as PPase) refers to an enzyme having an activity of releasing water-soluble pectin from insoluble proto-pectin in plant tissue. PPases are classified into A-type, which has the activity of degrading free pectin, and B-type, which has no activity of degrading free pectin. The PPase activity can be measured by reacting an enzyme solution with protopectin as a substrate and colorimetrically quantifying the released pectin. The molecular weight of the enzyme can be determined from its mobility on an SDS polyacrylamide electrophoresis gel or its behavior on a gel filtration column. The optimal pH or temperature of an enzyme can be determined by measuring enzyme activity at various pHs or temperatures. Substances that affect the action of the enzyme can be specified by adding various substances to the reaction solution during the enzyme reaction.

The arabinase refers to an enzyme having an activity of releasing arabinose by acting on arabinan. The arabinase activity can be measured by reacting an enzyme solution with arabinan as a substrate and quantifying the released arabinose by the Nelson-Somogy method or the like. The molecular weight of the enzyme can be determined from its mobility on an SDS polyacrylamide electrophoresis gel or from its behavior on a gel filtration column. The optimal pH or temperature of an enzyme can be determined by measuring enzyme activity at various pHs or temperatures. Substances that affect the action of the enzyme can be identified by adding various substances to the reaction solution during the enzyme reaction.

PPase can be produced by culturing a microorganism that produces this enzyme or by culturing a host cell into which an expression vector containing DNA encoding this enzyme has been incorporated. Microorganisms that produce PPase include yeast, filamentous fungi, and bacteria. Bacillus bacteria are preferably used. More preferably, Bacillus subtilis BS strain is used. When the PPase is produced using a genetic recombination technique, any commonly used host cells can be used, but microorganisms such as Escherichia col i or Baci 1 lus subt i 1 is preferably used. Used.

Bacillus subtilis BS strain was previously called Bacillus subtilis IF03134 strain. This strain is Gram-positive, has spore-forming ability, is a bacillus, has low protease-producing ability, and requires phosphate for production of the active substance enzyme (ie, PPase). Has the characteristic that its production is suppressed by glucose. This microorganism was obtained on July 30, 1997, 1-3 1-3 Higashi, Tsukuba City, Ibaraki Prefecture, Japan (zip code: 305) by the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology, and the accession number FERM BP- Deposited under 6031.

Cloning of a gene encoding PPase can be carried out by preparing a library containing chromosomal DNA extracted from a microorganism that produces PPase and screening this library. Screening can be carried out by expressing the encoded protein and using the activity of the protein of interest or the reactivity with an antibody specific to this protein as an indicator. Alternatively, screening can be performed using a labeled probe designed based on the amino acid sequence of the purified protein. Preferably, labeled probes are prepared using fragments generated by the polymerase chain reaction (PCR) using primers designed based on the amino acid sequence of the purified protein. Cloning of the PPase gene can also be achieved by combining fragments obtained by PCR.

An expression vector that is compatible with the host cell used is selected. Generally, high copy number vectors with strong promoters are used. If the transcription efficiency of the promoter originally contained in the target gene is high, this can be used. Eye If expression of the target gene adversely affects the growth of the host cell, the use of an inducible promoter is preferred.

Cultivation of the microorganism is performed under conditions appropriate for the microorganism used. Either a commonly used solid medium or liquid medium can be used. The cultivation is performed under aerobic or anaerobic conditions, depending on the microorganism used. When culturing under aerobic conditions, aeration agitation or shaking culturing may be performed. When Bacillus genus bacteria are used, the culturing is preferably performed using a liquid medium under aerobic conditions. Although the medium components are not limited, a medium containing a carbon source and a nitrogen source that can be assimilated by the microorganism in a suitable combination and concentration for the growth of the microorganism and the production of the substance can be used. Preferably, a medium containing soybean powder having an effect of inducing the production of PPase and / or arabinase is used. The cultivation time is determined depending on the timing of the production of the substance to be produced and the properties of the microorganism used, but is generally preferably about 16 to 48 hours. Purification of the enzyme can be performed as follows. When the target enzyme is secreted out of the cells, the cells are removed from the culture by centrifugation or filtration to obtain a supernatant. If the target enzyme accumulates in the cells, collect the cells from the culture solution, lyse them by dissolving them with an enzyme, or crush them with ultrasonic waves, and then centrifuge to obtain a cell-free extract. obtain. From these solutions, conventional means for protein purification such as concentration by drying or ultrafiltration, precipitation by salting out or solvent precipitation, dialysis, gel filtration chromatography, or ion exchange chromatography can be used. By combining them, a purified enzyme can be obtained.

Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to these embodiments. (Example l: Purification of enzyme)

PPase activity was measured according to the method of Sakai et al. (Methods in Enzymology, 161, 335-350, 1988, edited by WA Wood and Τ · Kellogg, Academic Press) using sugar beet pulp protopectin as a substrate as follows. Add 10 z! Of the enzyme solution to 990 1 acetate buffer (100 ιιιΜ, pH 6.0) containing 10 mg of the substrate, incubate this at 37 ° C for 1 hour, and filter the reaction solution: filtrate 125 1 Mix water, 1251, water, 250 I ethanol containing 0.2% strength rubazole, and 3 ml of 35N sulfuric acid and incubate at 75 for 20 minutes. After cooling the reaction mixture on ice, measure the absorbance at 525 nm. The PPase activity was calculated from the relationship between the amount of galacturonic acid and the degree of color development, with the activity of producing pectin corresponding to 1 mol of galacturonic acid per minute as one unit.

The Bacillus subiilis BS strain, SP medium (1.5% soy flour (manufactured by Fuji Oil Co., _{_{Ltd.), 1.2¾ KH 2 P0 4,}} 2.8¾K 2 HP0 4, pH7.0) was inoculated into and cultured with shaking 37 for 22 hours. The culture was centrifuged to obtain a supernatant. The supernatant was concentrated on a rotary evaporator and dialyzed against 20 mM acetate buffer (PH6.0) at 4 overnight. An ammonium sulfate 0-60% saturated fraction was recovered from the dialyzed enzyme solution, and the precipitate was dissolved in 20 mM Tris-HCi (pH 7.5) and dialyzed against the same buffer at 4X: overnight. .

Ammonium sulfate is added to the dialyzed enzyme solution to a final concentration of 30, and the solution is applied to a Buty Toyopearl 650M column (manufactured by Tosoichi) equilibrated with 20 mM Tris-HCl (ρΗ7.5) containing 30% ammonium sulfate. Was. After thorough washing with the same buffer, elution was performed with a linear concentration gradient of ammonium sulfate 30 to 10% saturation, and fractions having PPase activity were collected and collected. Next, this fraction was applied to a Superdex 75 (Pharmacia Biotech) column equilibrated with 20 mM acetate buffer (pH 5.0) containing 100 mM NaCl. Elution with a 20 mM acetate buffer (ρΗ5.0) containing 100 mM NaCl, fractions with PPase activity Collected and collected. The obtained enzyme solution showed a single band on an SDS-polyacrylamide gel (hereinafter abbreviated as SDS-PAGE) gel, and this was used as a purified enzyme sample in the following procedure. A summary of the purification is shown in Table 1. Table 1 Purification of PPase

Procedure Total protein Total activity Specific activity Purity Yield

(mg) (unit) (unit / mg) (fold) (¾) Culture solution 47,880 277, 200 5.8 100 Ammonium sulfate precipitation 1,900 151,900 79.9 13.8 54.8

Butyl-Toyopearl 650M 40.0 2,620 65.5 11.3 0.95

Superdex 75 15.3 1,950 127 21.9 0.70

(Example 2: Characterization of enzyme)

2.1 Molecular weight

The molecular weight of PPase estimated from the mobility on a 10 SDS-PAGE gel was about 30,000. The molecular weight was estimated to be about 27,000 when this enzyme was applied to a Superdex 75 gel filtration column equilibrated with 20 mM acetate buffer (pH 7.0) containing 100 mM NaCl.

2.2 pH and temperature stability

After incubating PPase in 100 mM acetate buffer (pH 6.0) containing 50 g / ml pepsin serum albumin at a concentration of 5 units / ml at various pHs for 1 hour at 37, the procedure described in Example 1 was followed. When the enzyme activity was measured by the method, 80% or more remained at pH 6-9. P-Pase was also added to a 20 mM phosphate buffer (pH 6.0) containing 50 zg / ml of serum albumin. After incubation at various temperatures for 30 minutes at a concentration of about 10 ml / ml, the enzyme activity was measured.

2.3 Optimum pH and temperature

When the PPase activity was measured by the method described in Example 1 except that the reaction pH or reaction temperature was changed, PH6.0 and 60 showed the highest activity.

2.4 Effects of various ions

The PPase activity was measured at at pH 6. 0, 50 in a reaction solution of various substances in a concentration of 1 mM in Table 2, the activity was completely inhibited in the presence ¾C1 _2, Even MnCl ₂ It was inhibited by 7 5%.

Effect of ions on activity

Substance name Relative activity (X) None 100

AgN0 ₃ 60

BaCl ₂ 100

CaCI ₂ 95

CdCl ₂ 85

CoCl ₂ 105

CuS0 ₄ 90

FeCl ₂ 105

HgCl ₂ 0

KC1 100

MgCI ₂ 100

MnCl ₂ 25

NaCl 100

Ni Cl ₂ 95

ZnCl ₂ 90

NaF 95

NaN ₃ 105

NaN0 ₂ 80 p-Mercuric benzoate 110 EDTA 90 2.5 Substrate specificity

When PPase was allowed to act on various substrates, as shown in Table 3, the PPase activity on protopectin (derived from sugar beet) and L-arabinan (derived from sugar beet) and the arabinase activity on arabinogalactan (derived from soybean) were shown. did. The activity of arabinase was measured as follows: To a 100 mM acetate buffer (PH 7.0) 1901 containing 0.5% L-arabinane, add 10i1 of the enzyme solution, and incubate at 50 ^ for 10 minutes. Add 2001 Somogyi reagent, boil for 10 minutes, then cool in ice for 10 minutes; add 2001 Nelson's reagent, leave at room temperature for 20 minutes, absorbance at 660 runs Is measured. The arabinase activity was calculated from the relational expression between the amount of arabinose and the chromaticity, with the activity of releasing the amount of reducing sugar corresponding to 1 mol of arabinose per minute as one unit, and displayed. L-arabinane to be used as a substrate was prepared as follows: Sugar beet pulp was treated in 0.1 NaOH for lOOt for 1 hour; after treatment, the liquid obtained by filtration and centrifugation was mixed with 3 volumes of ethanol The resulting precipitate is collected by centrifugation; the precipitate is dissolved in water, and the insoluble matter is removed by centrifugation. Then, the precipitate is dissolved in 50 mM acetate buffer (pH 5.0) with the action of galactanase (100 units / ml). Dialyze at 37 for 24 hours; apply the dialyzed solution to a DEAE-Celluline ine AH column (Tosoichi) equilibrated with 50 mM acetate buffer (pH 5.0) to collect the non-adsorbed fraction. The solution is pooled, dialyzed against water, mixed with 3 volumes of ethanol, and the resulting precipitate is washed twice with ethanol and lyophilized.

It did not act on other substrates. Other activities were measured at pH 7.0 according to the method of FM Rombouts et al., "Carbohydrate Polymers 9 Vol. 9, pp. 25-47 (1988)". Table 3 PPase activity for various substrates

Activity Substrate (Origin) Activity Presence Brodactinase Brotopectin (sugar beet) Yes

L-arabinan (sugar beet) yes arabinase arabinogalactan (soybean) yes

Arabinogalactan (larch)

Galactanase /3-1.4-galactan (Dice ') No xylanase / 3-1,4-xylan (spelled wheat)

Cellulase CM-cellulose

Pectin lyase 髙 Methoxylated polygalacturonic acid (orange) None ぺ Pectate enzyme Polygalacturonic acid (Orange) None Polygalacturonic acid (orange)

i3-D-galactosidase 0 -ditrophenyl -iS-D-galactoside None

a -L-arabinofuranosidase P-nitrophenyl -α-L-arabinofuranoside None

a-L-Alappinoviranosidase p-Ditrophenyl-α-rearabinoviranoside None

From the above results, it was confirmed that the PPase of the present invention is a novel PPase different from the conventionally known PPase.

2.6 Amino acid sequence

The amino acid sequence of the purified PPase was analyzed for the N-terminus of the mature protein and the N-terminus of the peptide generated by treatment with V8 protease. The N-terminal sequence is Xaa-Phe-Trp-Gly-Al-Ser-Asn-Glu-Leu-Leu-His-Asp-Xaa-Thr-Met-Ile- (Lys) -GIu-Gly-Ser- Ser- (Trp) -Tyr-Ala-Leu-Gly-Thr-Xaa-Leu-Asn (where Xaa is Amino acid residues that could not be analyzed are shown, and amino acids in parentheses indicate uncertain amino acid residues.) Gly-Ser-Ser-Trp-Tyr-Ala-Leu-Gly-Thr-Gly, Gly-Leu-Val-I include the amino acid sequence obtained as a result of analysis of the peptide generated by V8 protease digestion. le-A rg-Ser, Leu-Thr-Phe-Asp-Lys-Asp-Gly-Asn-Pro and the like.

(Example 3: Gene cloning)

3.1 PCR

Genomic DM of Bacillus subtilis BS strain was prepared using CTAB according to Molecular Cloning 3rd Edition (1993). This was digested with the restriction enzymes HincHII and PsU, and the ends were ligated with a HindlU cassette (product number 3870, manufactured by Takara Shuzo) and a Pstl cassette (product number 387, manufactured by Takara Shuzo) to form a type II PCR. Based on the amino acid sequence obtained in Example 2, two kinds of reverse primers 1-1 (SEQ ID NO: 3) and II-1 (SEQ ID NO: 4) were designed, and were designed using a DNA synthesizer. The above cassette-bound genomic DNA was converted to type I using Primer 1-1 and 11-1 and Ex Taq DNA polymerase for 94 minutes at 30 seconds, 60 ° C for 2 minutes, and 72 minutes for 2 minutes. PCR was performed under the conditions of 30 cycles, and further primer 1-2 (SEQ ID NO: 5) was designed and synthesized based on the DNA sequence of the resulting fragment. Using 1 (primer for common sequence of HindlU cassette and Pstl cassette, product number 3875, manufactured by Takara Shuzo) and 1-1 or II-1 primer, 94 30 seconds, 6 (30 cycles of TC2 min. After performing the first PCR in step 2, the product generated in this PCR reaction is Chromatography (Hindlll cassette Bok and Pstl cassette primers against the consensus sequence Bok, No. 3876, Takara Shuzo) using a 1-2 primers second round of P

CR was performed under the conditions of 30 cycles of 94 30 seconds, 60 minutes 2 minutes, and 72 minutes 2 minutes. When the DNA sequence of the generated fragment was determined, the portion not used for primer design was determined. Since this fragment encoded the sequence obtained by the above amino acid sequence analysis, this fragment was determined to be a fragment encoding the desired PPase, and was used below. 3.2 Analysis of clones

The DNA sequence of the obtained fragment was determined, and a part thereof is shown in SEQ ID NO: 2. This DNA sequence had an open reading frame that could code for a protein. Since the N-terminal sequence obtained by amino acid sequence analysis of the purified protein was found in the amino acid sequence deduced from the DNA sequence, it was confirmed that this clone encodes the target PPase. When a homologous gene was searched using GenBank database, the sequence of endo-1,5-arabinase of Aspergillus niger (Flipphi, MJA et al., ApI. Microbiol. Biotechnol. 40, 318-326) , 1993) showed only low homology (23% or less), and it was confirmed from the amino acid sequence that the PPase obtained here was new, and was identified as protokinase C. Named.

3.3 Expression

A 1.05 kb Pstl-EcoRV fragment containing the entire open reading frame was inserted into a multicopy vector pUC19 for E. coli to construct a plasmid pUC-Ppc, which was used to transform E. coli DH5a strain. . The resulting ampicillin-resistant transformant was cultured for 22 hours at 37T in the production medium used in Example 1, and an activity of 15 units / ml was detected in the culture. The fragment was confirmed to encode PPase. Industrial applicability

By cloning the gene encoding the protease of the present invention, the enzyme could be highly expressed using gene recombination technology. The protein vectorase of the present invention is useful for fiber refining, pectin production, pulp production, and the like. By improving expression efficiency and producing at low cost, this enzyme can be used industrially. can do.

Sequence listing

SEQ ID NO: 1

Sequence length: 291

Sequence type: amino acid

Topology: linear

Sequence type: protein

Array

Ala Phe Trp Gly Ala Ser Asn Glu Leu Leu His Asp Pro Thr Met lie 1 5 10 15 Lys Glu Gly Ser Ser Trp Tyr Ala Leu Gly Thr Gly Leu Asn Glu Glu

20 25 30

Arg Gly Leu Arg Val Leu Lys Ser Ser Asp Ala Lys Asn Trp Thr Val

35 40 45

Gin Lys Ser lie Phe Ser Thr Pro Leu Ser Trp Trp Ser Asn Tyr Val 50 55 60

Pro Asn Tyr Glu Lys Asn Gin Trp Ala Pro Asplie Gin Tyr Tyr Asn 65 70 75 80

Gly Lys Tyr Trp Leu Tyr Tyr Ser Val Ser Ser Phe Gly Asn Asn Thr

85 90 95 Ser Ala He Gly Leu Ala Ser Ser Thr Ser lie Ser Ser Gly Asn Trp

100 105 110

Lys Asp Glu Gly Leu Val lie Arg Ser Thr Ser Ser Asn Asn Tyr Asn

115 120 125

Ala lie Asp Pro Glu Leu Thr Phe Asp Lys Asp Gly Asn Pro Trp Leu 130 135 140

Ala Phe Gly Ser Phe Trp Ser Gly He Lys Leu Thr Lys Leu Asp Lys 145 150 155 160

Ser Thr Met Lys Pro Thr Gly Ser Leu Tyr Ser He Ala Ala Arg Pro

165 170 175

Asn Asn Asn Gly Ala Leu Glu Ala Pro Thr Leu Thr Tyr Gin Asn Gly

180 185 190

Tyr Tyr Tyr Leu Met Val Ser Phe Asp Lys Cys Cys Asn Gly Val Asn

195 200 205

Ser Thr Tyr Lys lie Ala Tyr Gly Arg Ser Lys Ser He Thr Gly Pro 210 215 220

Tyr Leu Asp Lys Ser Gly Lys Ser Met Leu Asp Gly Gly Gly Thr lie 225 230 235 240

Leu Asp Ser Gly Asn As Gin Trp Lys Gly Pro Gly Gly Gin Asp He

245 250 255

Val Asn Gly Asn lie Leu Val Arg His Ala Tyr Asp Ala Asn Asp Asn

260 265 270

Gly Thr Pro Lys Leu Leu lie Asn Asp Leu Asn Trp Ser Ser Gly Trp

275 280 285

Pro Ser Tyr

290 SEQ ID NO: 2

Sequence Length: 876 Sequence type: nucleic acid

Number of chains: double strand

Topology: linear

Sequence type: genomi c DNA

Origin

Organism name: Bac i 1 lus subt i l i s

Stock Name: BS

Array

GCATTTTGGG GTGCATCTAA CGAGCTGCTT CACGACCCGA CTATGATCAA AGAGGGGAGC 60 TCATGGTATG CGTTAGGAAC AGGGCTTAAT GAAGAACGGG GACTGCGGGT TTTGAAGTCT 120 TCGGATGCTA AAAACTGGAC CGTCCAAAAA TCTATTTTCA GCACACCGCT ATCGTGGTGG 180

TCCAATTATG TGCCGAATTA CGAGAAAAAC CAGTGGGCGC CGGATATCCA ATACTATAAC 240

GGAAAGTACT GGCTGTATTA TTCAGTTTCC TCTTTTGGAA ACAATACATC TGCCATCGGA 300

CTGGCATCCT CAACGAGCAT CAGTTCGGGG AACTGGAAAG ACGAAGGCTT GGTCATCCGT 360 TCGACAAGCT CCAATAATTA TAACGCGATT GATCCGGAGT TGACATTTGA CAAGGATGGC 420

AACCCGTGGC TTGCATTCGG CTCGTTTTGG AGCGGAATTA AGCTGACAAA GCTTGATAAA 480

AGTACGATGA AGCCTACAGG CTCGCTCTAT TCGATTGCAG CCAGGCCGAA TAATAACGGG 540

GCGCTGGAAG CTCCTACTCT TACGTATCAA AATGGCTATT ACTATTTAAT GGTTTCATTT 600

GATAAATGTT GTAACGGGGT AAACAGTACG TACAAAATTG CTTATGGAAG ATCTAAAAGC 660 ATTACAGGGC CTTATCTTGA TAAAAGCGGG AAAAGCATGC TTGATGGCGG GGGCACCATT 720

TTGGATTCCG GCAACGACCA ATGGAAAGGC CCTGGCGGTC AGGATATTGT AAACGGAAAC 780

ATTCTTGTTC GTCATGCCTA TGACGCCAAT GACAACGGCA CTCCGAAGCT TCTCATCAAT 840

GATTTGAATT GGAGTTCGGG CTGGCCGTCC TATTAA 876 SEQ ID NO: 3

Array length: 30

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: other nucleic acid synthetic DNA

Array

GGGAGCTCAT GGTATGCGTT AGGAACAGGG 30 SEQ ID NO: 4

Array length: 31

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: other nucleic acid synthetic DNA

Array

GCCATCGGAC TGGCATCCTC AACGAGCATC A 31 SEQ ID NO: 5

Array length: 31

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: other nucleic acid synthetic DNA

^ιε 319ID3D0W3 mm

6LZO / L6dSH10d 890/86 OAV

Claims

Claim 1-The following properties:

(1) Has protopectin degrading activity

(2) molecular weight on SDS polyacrylamide electrophoresis gel is about 30,000;

(3) the optimum pH is 6.0;

(4) the optimum temperature is 60; and

(5) Inhibited by Hg and "Mn,

A prototinase having 2. The protease according to claim 1, which further has arabinase activity.

3. The proteinase according to claim 1, which is produced by Bacillus subtilis BS strain. 4. a proteinase having the amino acid sequence of SEQ ID NO: 1, or having the amino acid sequence in which one or several amino acids are deleted, substituted or added, and having the amino acid sequence of SEQ ID NO: 1 2. The proteinase according to claim 1, which has an activity equal to or higher than that of the prototinase. 5. A DNA encoding the prototinase according to any one of claims 1 to 4.

6. It has the nucleotide sequence of SEQ ID NO: 2, or has a nucleotide sequence in which one or several nucleotides have been deleted, substituted or added in the nucleotide sequence, and has a nucleotide sequence of SEQ ID NO: 2 Prototyped The DNA according to claim 5, which encodes a protease having an activity equal to or higher than that of cutinase.

7. An expression vector comprising the DNA according to claim 5 or 6.

8. A host cell comprising the expression vector according to claim 7.

9. For the genus Bacillus, the following properties:

(1) It has protobectin degradation activity

(3) the optimum pH is 6.0;

(4) the optimum temperature is 60; and

(5) Inhibited by Hg and ¾1,

A method for producing a protease, comprising a step of culturing a microorganism having the ability to produce a protease having the following characteristics.

10. The production method according to claim 9, wherein the proteinase has further arabinase activity.

1 1. The production method according to claim 9, wherein the microorganism is Bacillus subtil is BS strain.

12. A step of culturing a host cell transformed with the expression vector according to claim 7, comprising the following properties:

(1) Has protopectin degrading activity (2) the molecular weight on an SDS polyacrylamide electrophoresis gel is about 30,000;

(3) the optimum pH is 6.0;

(4) the optimum temperature is 61T; and

(5) subject to inhibition by Hg and min,

A method for producing a protease, comprising:

13. The production method according to claim 12, wherein the proteinase has further arabinase activity.