KR20060092663A - Detergent and oxidant-stable haloalkalophilic protease and a gene coding it - Google Patents

Abstract

The present invention relates to a protease produced in Bacillus cloudi I-52 strain and a gene encoding the same, specifically, a protease derived from Bacillus cloudi I-52 strain that maintains activity even under extreme environmental conditions and It relates to a gene encoding. The protease of the present invention can be used not only as a component of a detergent for clothing or contact lenses, but also useful for processing food, feed, natural leather and the like and processing of organic waste.

Description

Detergent and oxidant-stable haloalkalophilic protease and a gene coding it} which is very stable to surfactants and oxidants

1 is a graph showing the enzyme activity of the protease of the present invention according to the pH change,

Figure 2 is a graph showing the enzyme activity of the protease of the present invention with temperature changes,

3 is a graph showing the relative activity of the protease of the present invention according to the salt concentration change,

4 is a graph showing the enzyme activity of the protease of the present invention according to the concentration of sodium dodecyl sulfate, an anionic surfactant,

5 is a graph showing the enzyme activity of the protease of the present invention according to the concentration of hydrogen peroxide solution,

6 is a graph showing the enzymatic activity of the protease of the present invention according to the concentration of sodium perborate,

7 is a graph showing the results of experiments to determine whether the protease of the present invention maintains enzyme stability when the protease of the present invention is placed with a commercial detergent.

Figure 8 is a graph showing the washing power by the protease produced by Bacillus Cluj in the presence of detergent component LAS (15%),

9 is a photograph showing the soybean meal decomposition ability by the protease produced by Bacillus Cluj,

Lane M: protein molecular weight marker,

Lane 1: before treatment, lane 2: 2 hours after treatment,

Lane 3: 4 hours after treatment, lane 4: 8 hours after treatment,

Lane 5: 16 hours after treatment

FIG. 10 is a graph showing the degradability of soybean trypsin inhibitor (SBTI) of soybean meal by protease produced by Bacillus clown,

11 is a schematic diagram showing the positions and amplified portions of primers used in the polymerase chain reaction using the chromosomal DNA of Bacillus cloud I-52 as a template;

12 is a gel electrophoresis picture showing the results of polymerase chain reaction with each primer combination,

Lane M: 100 bp ladder DNA molecular weight marker,

Lane 1: primer F2 / R1 combination lane 2: primer F2 / R2 combination

Lane 3: primer F2 / R3 combination lane 4: primer F3 / R2 combination

Lane 4: Primer F3 / R2 Combination Lane 6: Primer F3 / R3 Combination

FIG. 13 is a gel electrophoresis photograph showing the results of introducing a polymerase chain reaction product obtained from a primer F3 / R2 combination into a pGEM-Teay plasmid, and then cutting the DNA fragment with the restriction enzyme to confirm the DNA fragment inserted therein.

Lane M: lambda DNA molecular weight marker,

Lane 1: EcoRI treatment product of the inserted DNA fragment

14 is a schematic diagram of the plasmid for expression of mature protease (BCAP) (pT7-BCAP),

15 is a gel electrophoresis photograph showing the results of confirming the DNA fragment inserted by introducing a gene encoding a mature protease (BCAP) into the pT7-7 plasmid, and then cleaved with a restriction enzyme.

Lane M: lambda (λ) DNA molecular weight marker,

Lanes 1, 2 and 3: Nco I and Hind III treatment products of inserted DNA fragments, clones 1, 2 and 3

16 is a photograph showing the result of SDS-PAGE analysis of a mature protease induced by culturing by adding IPTG to a transformant expressing a mature protease,

Lane M: protein molecular weight marker,

Lane 1: before IPTG induction, lane 2: 1 hour after IPTG induction,

Lane 3: 2 hours after IPTG induction, lane 4: 3 hours after IPTG induction,

Arrow: expression induced mature protease band,

FIG. 17 is a schematic diagram of a Bacillus clausii genome insertion plasmid (pHPS-BCAP) comprising the gene set forth in SEQ ID NO: 1. FIG.

The present invention relates to a protease produced in Bacillus cloudi I-52 strain and a gene encoding the same, and more particularly, a protease derived from Bacillus cloudi I-52 strain that maintains activity even under extreme environmental conditions. And to a gene encoding the same.

Proteolytic enzymes are used in a variety of applications, and can be used not only for treating human diseases such as dissolution of blood clots, but also as a component of clothing detergents and contact lens cleaners, as well as milk protein modification, silk degumming, Various applications include improving feed, soaking leather, dehairing, recovering silver from X-rays, and regenerating ultrafiltration membranes used to concentrate protein solutions. The usefulness of enzymes in microbial products is well known and the industrial enzyme market is said to be over $ 1 billion. Proteolytic enzymes account for the largest portion of the industrial enzyme market (over 60%), and its marketability is increasing. Proteolytic enzymes have been used industrially for quite some time, with a wide range of uses, including pharmaceuticals, dairy, leather, food, feed, film making, waste disposal and detergents.

As a detergent additive, protease is widely used for washing, contact lens cleaning, denture cleaning, and about 25% of industrial protease is sold for washing. Since the introduction of the bacterial protease for washing under the trade name BIO-40 in 1956, 13 billion tons of proteolytic enzymes have been produced for detergents. Detergent proteases should have a broad substrate specificity to remove food, blood or bodily fluids, an alkaline environment, surfactants and stability that will not lose enzyme activity at high temperatures. In the past, plant-derived proteases were mainly used, but nowadays, microorganisms, particularly those of the genus Bacillus, are most commonly used for protease production.

Proteolytic enzymes are classified into exoproteases, endoproteases, and serine proteases, aspartic proteases, and cysteine proteases, depending on the functional site of the active site, depending on the mode of action. Acidic proteases, depending on the location of production, may be classified as intracellular protease or extracellular protease. Alkaline proteases are mainly used in the detergent industry, mainly because they use microorganisms such as Bacillus licheniformis and fungi such as Aspergillus oryzae , but most of them are of the genus Bacillus. Produced using microorganisms.

When proteolytic enzymes are used in industrial applications, there is a need for highly stable proteolytic enzymes because the changes are severe compared to the internal environment of the living body, and the protease must be kept active in the more extreme external living environment. For example, protease used in detergents is extremely limited because most proteins decrease or disappear completely in the presence of a surfactant. When using detergents at high concentrations, the protease activity contained in them is expected. Difficult to do In addition, in the external environment, changes in conditions such as exposure to extreme pH, exposure to heavy metals, and degree of oxidation-reduction are much more severe than in vivo. All of these conditions generally strongly affect the activity of enzymes, and these conditions are appropriate. Outside the range, the enzyme rapidly loses its activity. Therefore, in order for protease to be used in industry in earnest, it is required to maintain activity despite severe and unstable physical and chemical conditions.

That is, in order for the protease to be used in industry, first, the activity of the protease should not be inhibited by the surfactant, and the activity of the protease should not be inhibited by the oxygen-based bleach. Must be alkaline protease to be maintained, must have high enzyme activity at low temperature (20 ~ 30 ℃) of the fourth, enzyme activity should not be inhibited by the heavy metal, and calcium ion should not be needed for enzyme activity of the sixth. do. When preparing enzymes in granule form for use as detergent additives, it is a basophilic protease that maintains the activity of enzymes even in the presence of high salt (more than 10% salt) because salt is used as the core substance. It is known to be of further value as an industrial enzyme.

The value of basophil protease can be found in applications especially for high salt foods. In Korea, the unique flavors of salted fish and jangjang, which are traditional high-salt foods, are made up of amino acids produced from proteins by the action of proteolytic enzymes during the ripening process. These fermented foods add more than 15% of salt for long-term distribution. It is necessary to maintain enzyme activity even at high concentrations of salinity.

In order to prepare a protease that satisfies the above conditions, US Pat. Nos. 534075, 6136553, and Republic of Korea Patent No. 10-0258740 apply specific mutations from existing proteases to provide better and higher stability. It is stated that proteolytic enzymes have been developed. In addition, US Pat. No. 6162635 and the like describe an attempt to find industrial strains that produce protease that satisfies the above conditions from natural systems such as soil. However, the mutant has a problem in that the activity of the enzyme is lowered in many cases, and so far, proteolytic enzymes satisfying all the above conditions have not been prepared. In particular, proteolytic enzymes that are stable to both oxygen-based bleaches and surfactants have been reported to be extremely limited.

Thus, the inventors of the Bacillus Cluj (Bacillus clausiiI-52, KCTC 10277BP,Republic of Korea Patent Application 10-2002-0037420, Registration No. 046582The present invention was completed by confirming that alkaline proteolytic enzymes derived from c) can maintain a very high activity even in the presence of a surfactant, an oxidizing agent, and a high concentration of salt, and thus can be used industrially.

An object of the present invention is to provide a protease which is stable to a surfactant, an oxidizing agent, and a high concentration of salt, which can be useful industrially.

In order to achieve the above object, the present invention provides a protease, characterized in that it comprises an amino acid sequence set forth in SEQ ID NO: 3.

The present invention also provides a gene encoding the protease.

The present invention also provides an expression vector comprising the gene.

The present invention also provides a transformant into which the expression vector is introduced.

In addition, the present invention provides an expression vector for Bacillus genome insertion comprising the gene.

The present invention also provides a transformant into which the expression vector is introduced.

The present invention also provides a detergent for clothing or contact lenses containing the protease.

In addition, the present invention provides a method for processing food, feed, textiles, natural leather using the protease.

In addition, the present invention provides a method for decomposing organic waste using the protease.

Hereinafter, the present invention will be described in detail.

The present invention provides proteolytic enzymes comprising the amino acid sequence set forth in SEQ ID NO: 3.

The present inventors isolated Bacillus clausii strain I-52 from the western coastal tidal flat with extremely poor growth conditions and obtained protease having excellent stability even under extreme physical and chemical conditions. The inventors determined the nucleotide sequence ( SEQ ID NO: 1 ) and amino acid sequence ( SEQ ID NO: 2 and 3 ) of the protease.

The protease of the present invention is composed of 381 amino acids, the 32 amino acid residues of the N-terminal is a signal sequence, the 107th amino acid Alanine (Alanine, Ala) was confirmed to be the N-terminal amino acid of the active protein. Among them, the active protease moiety is composed of 275 amino acid residues as set forth in SEQ ID NO: 3, and Asp32, His64, and Ser221 residues, which are three activity-related amino acids common to serine protease It was confirmed that the protease is a type of a typical serine protease.

Proteolytic enzymes of the present invention are stable enzymes that maintain activity even under various physicochemical conditions such as high pH, surfactants, oxidants, organic solvents and heavy metals. The protease of the present invention exhibits proteolytic activity in a wide pH range of pH 7.0 to pH 12.0, in order to exhibit maximum activity, it is preferably pH 9.0 to pH 12.0, more preferably pH 10.0 to pH 11.0, Most preferred is pH 11.0 (see FIG. 1 ). Further, the protease of the present invention exhibits proteolytic activity at 10 to 70 ° C, and in order to exhibit maximum activity, it is preferably 40 to 65 ° C, more preferably 55 to 65 ° C, and 60 to 65 ° C. Most preferred (see FIG. 2 ). Proteolytic enzymes of the present invention was confirmed that the salt is a basophilic protease that not only shows the maximum activity of the enzyme when present in about 2 to 5%, but also maintains the enzyme activity even when present in a high concentration of 20% (see Figure 3 ). ). The proteolytic enzyme of the present invention maintains high proteolytic activity even in the presence of not only anionic surfactants but also strong anionic surfactants such as sodium dodecyl sulfate (SDS), especially when treated with 5% SDS for 3 days. It turned out to be a very stable enzyme that almost lost its activity (see FIG. 4 ) and almost lost its activity even in the presence of hydrogen peroxide and sodium perborate, known as a powerful oxidant (see FIGS . 5 and 6 ). From this series of enzymatic properties, it could be confirmed that the protease derived from Bacillus cluj of the present invention has various industrial applications.

The present invention also provides a gene encoding the protease.

Gene sequences encoding the protease of the present invention having the characteristics as described above were analyzed as follows. Specifically, the chromosomal DNA is isolated from the Bacillus cloud, and then the isolated DNA is used as a template to perform a polymerase chain reaction to obtain a DNA fragment of the protease of the present invention (see FIGS. 11 and 12 ). Was determined ( SEQ ID NO: 1 ).

The present invention also provides an expression vector comprising the gene.

In the present invention, a protease gene having an amino acid sequence as set forth in SEQ ID NO: 3 was introduced into an expression vector for transformation in order to produce the protease of the present invention, which can maintain activity even under extreme conditions, in an active form. The expression vector thus prepared was named “ pT7 -BCAP” (BCAP: Bacillus clausii Alkaline Protease) in the present invention (see FIG. 14 ).

The present invention also provides a transformant into which the expression vector is introduced.

The prepared expression vector may be transformed to express the protease of the active form by transduction into a suitable host cell. In the present invention, a transformant capable of producing the protease of the present invention in a stable form by introducing the transforming vector pT7-BCAP into Escherichia coli (see FIG. 15 ).

The present invention also provides a recombinant expression vector for Bacillus genome insertion comprising the gene.

A recombinant expression vector for integrating the DNA of SEQ ID NO: 1 containing the promoter portion encoding the protease of the present invention into the genome of the Bacillus cloud was constructed and named "pHPS-BCAP" (see FIG. 17 ). . The expression vector can be inserted into the chromosome of Bacillus cloud and used to increase the number of BCAP copies in the chromosome.

The present invention also provides a transformant into which the expression vector is introduced.

The expression vector pHPS-BCAP was transformed into E. coli JM109 and deposited on January 26, 2005 with the Korea Biotechnology Research Institute Gene Bank (Accession Number: KCTC 10772BP).

In addition, the present invention is a detergent for clothing or contact lenses comprising the protease, a method for processing food, feed, textiles, natural leather using the protease and organic waste using the protease Provide a way to disassemble.

The use of proteolytic enzymes in industrial applications requires a very stable proteolytic enzyme because the changes are more severe than the internal environment of the living body, and the protease must remain active in the more severe external environment. In other words, in the external environment, changes in conditions such as exposure to extreme pH, exposure to heavy metals, and degree of oxidation-reduction are much more severe than in vivo. All of these conditions generally strongly affect the activity of enzymes, and these conditions are appropriate. Outside the range, the enzyme rapidly loses its activity. Therefore, in order for protease to be used in industry in earnest, it is required to maintain activity despite severe and unstable physical and chemical conditions.

The protease is an alkaline environment, a surfactant, and stability at a high temperature does not lose the activity of the enzyme and has a broad substrate specificity that can remove food, blood or body components, so the protease comprises SDS, octyl It may be included as a component of the detergent together with known synthetic surfactants or natural surfactants such as glucoside, octylglucoside, Brij-35, Triton series and Tween series. In addition, since it maintains its activity in a wide range of pH, redox environment, it can be widely used in processing processes such as food, feed, textiles, natural leather, etc., and can be used for waste decomposition because it maintains high activity even in the presence of heavy metals.

Detergents containing the protease may be prepared according to a general method of manufacturing a detergent for clothing or contact lenses, and there is no particular limitation on the processing method or the degradation method using the protease. All methods performed by adding the protease of the present invention to known methods are included in the scope of the present invention.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Base Sequence and Amino Acid Sequence Analysis of Protease

<1-1> Genomic DNA Isolation

Bacillus clausii was inoculated into 3 ml Trypitic soy broth (TSB) and shaken at 250 rpm at 37 ° C for 500 ml of Erlenmeyer flasks containing 100 ml of TSB medium. Inoculation was incubated for 24 hours. The culture solution was centrifuged at 5,000 × g and lysozyme and proteinase K were reacted at room temperature for 18 hours, and the same volume of phenol solution was added at 20 ° C. The mixture was mixed well for 1 minute and centrifuged at 13,000 × g for 20 minutes to recover the supernatant. After repeating the same process, add the same volume of chloroform and isoamyl alcohol mixture (24: 1) to the supernatant, mix well at room temperature for 20 minutes, and centrifuge at 13,000 × g for 20 minutes. Recovered. Add the same volume of chloroform and mix well at room temperature for 20 minutes, then centrifuge at 13,000 × g for 20 minutes to recover the supernatant, add 2 volumes of ethanol, and slowly mix at room temperature until a DNA precipitate is formed. gave. The sterile Pasteur pipette recovers genomic DNA from the solution, and the recovered DNA is washed with 70% ethanol to remove remaining salts, dried on a sterile bench, and then in 1 ml TE buffer (10 mM Tris-). HCl, pH 8.2 / 1 mM EDTA). The obtained DNA was confirmed by performing 1% agarose electrophoresis.

Example 1-2 Sequence Analysis

Forward primers F2 described in SEQ ID NOs: 4, 5 and reverse primers R1, R2, R3 described in SEQ ID NOs: 6, 7, 8, respectively, were synthesized (see FIG. 11 ).

Bacillus cloud genomic DNA isolated in <1-1> was used as a template, and the above primers were combined in various ways to perform a polymerase chain reaction. After 30 minutes of per-denaturation at 94 ° C using Taq DNA polymerase, PCR was carried out for 30 cycles under conditions of 1 minute at 94 ° C, 1 minute at 60 ° C, and 1 minute at 72 ° C. , PCR products were obtained according to each primer combination (see FIG. 12 ). Among them, PCR products obtained by the combination of primers F3 / R2 were cloned into the expression vector pGEM T easy (Promega, USA) (see FIG. 13 ) . As a result, the total DNA size was 2280 bp (SEQ ID NO: 1), TCTACT (872-877) was -35 sequence and TACAAT (897-902) was -10 sequence. It was confirmed that the whole gene of the Bacillus cloudage-derived protease including the part.

<1-3> amino acid sequence analysis

As a result of analyzing the nucleotide sequence ( SEQ ID NO: 1 ) of the entire DNA obtained in <1-2> by Blast, the DNA was 1143 bp of open reading frame encoding 381 amino acids of SEQ ID NO: 2. 32 amino acid residues of the N-terminal were identified by the signal sequence, 107th amino acid Alanine (Ala), as the N-terminal amino acid of the active protein. Among them, the active protease moiety is composed of 275 amino acid residues as set forth in SEQ ID NO: 3, and Asp32, His64 and Ser221 residues, which are three activity-related amino acids common to serine protease It was confirmed that the protease is a type of a typical serine protease. The active protein portion described in SEQ ID NO: 3 was named BCAP in the sense of Bacillus clausii Alkaline Protease.

Example 2 Construction and Transformation of Active Protease Expression Vector

In order to prepare an expression vector comprising the gene of the Bacillus cloudage-derived active protease analyzed in the present invention, pGEM T easy-BCAP is used as a template, and the polymerases are prepared using primers described in SEQ ID NOs: 9 and 10. Chain reactions were performed. Amplify by 30 cycles of PCR using Taq DNA polymerase for 2 minutes per-denaturation at 94 ° C, 1 minute at 94 ° C, 1 minute at 60 ° C, and 1 minute at 72 ° C. DNA fragments of about 870 bp comprising a 825 bp DNA sequence encoding 275 amino acid residues as set forth in SEQ ID NO: 3 were subjected to electrophoresis and isolated with a DNA elution kit and the DNA fragment and pT7-7 plasmid ( Roche Applied Sscience) was double digested with Nco I and Hind III and conjugated to name the vector pT7-BCAP and transformed E. coli BL21 (DE3). Specifically, 0.1 to 0.5 ug of the expression vector pT7-BCAP was added to 100 ul of the transformed cell E. coli BL21 (DE3), and left on ice for 30 minutes, and then subjected to heat shock at 42 ° C. for 1.5 minutes. After incubation for 1 hour at 37 ° C by adding ml of LB medium, smeared on LB agar plate medium to which ampicillin (50 ug / ml), IPTG and X-gal were added and incubated for 16 hours at 37 ° C A convert was obtained. The transformed E. coli was inoculated and cultured in LB liquid medium to which ampicillin was added, and a portion of the culture was taken to extract a plasmid according to the alkaline lysis method, and then digested with restriction enzymes at the cloned site to confirm that the gene was introduced. ( See FIG. 15 ).

Example 3 Induction of Recombinant Protein Expression

In <Example 2> E. coli BL21 (DE3) transformed with the expression vector pT7-BCAP was inoculated in LB medium containing ampicillin (50 ug / ul) and incubated overnight at 37 ℃ was used as a seed. 1 ml of the seed was inoculated into 100 ml of LB medium (500 ml Erlenmeyer flask) containing ampicillin, and then cultured at 600 nm until the absorbance reached 0.6 to 0.8. To induce the synthesis of protein, IPTG was added to 1 mM, and then 1 ml of the culture was taken every hour. The cells were suspended in 20 ul of SDS-PAGE sample treatment solution (0.05 M Tris-HCl, pH 6.8, 0.1 M DTT, 2% SDS, 10% glycerol and 0.1% bromophenol blue) and heated at 100 ° C. for 5 minutes. After destruction, the cells were electrophoresed by Lamley et al. (Laemmli, UK, 1970, Nature, 227, 680-685). After the electrophoresis gel was stained with Coomassie brilliant blue R-250 solution (Coomassie brilliant blue R-250) to confirm the protein produced in E. coli transformed with pT7-BCAP.

As a result, it was confirmed that the mature protease of the present invention is expressed over time by ITPG induction (see FIG. 16 ).

Example 4 Preparation of Plasmid for Protease Gene Insertion Including Promoter

The pGEM-Teay plasmid and the Bacillus genome insertion plasmid pHPS9 (ACTC), each of which was cloned from the DNA sequence of SEQ ID NO: 1 including the promoter of Bacillus cluj derived protease analyzed in the present invention, were respectively treated with EcoRI and electrophoresed. And then separated by DNA elution kit. After conjugation, E. coli JM109 was transformed as in <Example 3> . Marker used chloroamphenicol instead of ampicillin. After extracting the plasmid from the transformant and cutting it with the restriction enzyme of the cloned site to confirm that the DNA fragment of SEQ ID NO: 1 was introduced, the transformed vector into which the DNA fragment of SEQ ID NO: 1 was introduced was referred to as "pHPS-BCAP". Named (see FIG. 17 ).

Example 5 Activity Determination of Protease

In order to analyze the activity of proteolytic enzymes, cell culture supernatants were collected and measured for activity. Specifically, 0.1 ml of enzyme solution diluted 100 to 1000 times was added to 0.5 ml of substrate solution in which Hammerstan casein was dissolved in 0.1 M glycine-NaOH buffer (pH 11.0) to a final concentration of 0.5%. After the reaction, the reaction was stopped at 60 ° C. for 10 minutes and 0.5 ml of 10% Trichlor acetic acid (TCA) was added to stop the reaction. The solution was left on ice for 10 minutes to precipitate unreacted protein, centrifuged to obtain supernatant, and the amount of tyrosine produced at 275 nm was measured. At this time, one unit of protease was defined as the amount of enzyme required to produce 1 microgram (ug) of tyrosine in 1 minute.

Example 6 Analysis of Optimal Conditions for Protease Activity

<6-1> Optimal pH of protease activity

The present inventors wanted to find out the optimal pH at which the protease produced in <Example 2> can exhibit maximum activity. Protease activity was measured using 0.1 M sodium phosphate buffer at pH 7.0-7.5, 0.1 M tris-hydrochloride buffer at pH 8.0-9.5, and 0.1 M glycine-NaOH buffer at pH 10.0-pH 12.0. It was.

As a result, the protease was found to have high activity in the pH 8.0 ~ 12.0 range and the optimum pH is 11.0 (see Figure 1 ).

<6-2> Optimum Temperature of Protease Activity

The inventors have found the optimum temperature at which protease shows activity. Specifically, casein (0.5%) was prepared using 0.1 M glycine-NaOH buffer solution (pH 11.0), and enzyme activity was measured at a temperature of 10 to 70 ° C.

As a result, it was found that the optimum temperature of the protease activity was 60 ~ 65 ℃ (see Figure 2 ).

<6-3> Optimal Salinity of Protease Activity

Inoculate 2% of the seedlings incubated for 24 hours in a culture medium (2% soybean meal, 1% wheat flour, 2% starch syrup, 0.5% sodium citrate, 0.5% potassium phosphate, magnesium sulfate, 0.5 mM, 0.6% sodium carbonate), Shake incubation for 48 hours at 37 ℃. After centrifugation of the strain, the culture supernatant was recovered, and the concentration of salt was added to the culture supernatant to 0-20%, and the enzyme activity was measured in the same manner as in <Example 5> . As a result, the enzyme activity increased when the salt was added at a concentration of 0.1 to 10%, it was preferable to add 0.5 ~, it was more preferable to add the salt at a concentration of 1%.

As a result, the Bacillus Cluj-derived protease did not inhibit the enzyme activity even in the presence of a high concentration of salt (10% or more), and in particular, at least 75% of the enzyme activity was maintained even in the presence of 20% salt. It can be seen that the degrading enzyme is a halophilic proteolytic enzyme, thereby confirming the possibility of use for business, in particular, detergent additives and food processing (see FIG. 3 ).

Example 7 Stability Analysis of Protease

<7-1> Stability of Protease against Surfactant

The present inventors examined the activity of the enzyme by treating with an anionic surfactant in order to determine whether the protease produced in Example 6 is stable to the surfactant. Specifically, the activity of protease was measured when SDS was treated with 1%, 5% or 10% as a surfactant.

As a result, it was confirmed that the protease of the present invention was a very stable enzyme for SDS that hardly loses its activity even after treatment with 5% SDS for 3 days (see FIG. 4 ).

<7-2> Stability of Protease against Oxidizer

In order to find out whether the protease is stable to the oxidizing agent, the present inventors measured the activity of the enzyme remaining at each time after treatment with an oxidizing agent such as hydrogen peroxide and sodium perborate. Specifically, as an oxidant, protease activity was measured when 1%, 5%, or 10% of hydrogen peroxide solution, which is known as a powerful oxidant, was measured, and proteolysis when sodium perborate was treated with 1% or 2.5%. The activity of the enzyme was measured.

As a result, it was confirmed that the protease of the present invention is an enzyme that is very stable to an oxidant which almost loses its activity even after treatment with 10% hydrogen peroxide solution and 2.5% sodium perborate for 3 days (see FIGS . 5 and 6 ). ).

Example 8 Stability of Protease to Commercial Detergent

In order to find out whether the protease is stable to an oxidizing agent, the present inventors measured the stability of a commercial detergent commercially available in Korea.

As a result, the proteolytic enzyme of the present invention was found to be a very stable enzyme that does not lose enzymatic activity even after being left at room temperature for 4 weeks after treatment with commercially available commercial detergent at 2.5%. See FIG. 7 ).

Example 9 Detergency Test of Protease

The present inventors were subjected to the washing power test using the protease produced in Example 6 in order to determine whether the Bacillus cluj derived protease is suitable for use as a detergent additive. The used contaminated cloth was tested for washing power using an international standard cotton test cloth (EMPA 116) contaminated with blood and milk and an international standard mixed fiber test cloth (EMPA 117) contaminated with blood and milk.

As a result, the proteolytic enzyme of the present invention was found to show a higher washing power than the control in the condition that the commercial surfactant LAS (linear alkylbenzene sulfonates) 15% is present it was confirmed that it can be used as a detergent additive (see Figure 8 ).

<Example 10> Soybean meal digestion test of protease

<10-1> soybean meal decomposition test

The inventors performed soybean meal digestion test using the protease produced in Example 6 to determine whether the Bacillus cluj derived protease is suitable for use as a feed additive. Soybean meal was added to distilled water to 10%, and the enzyme was added to 0.5% of the total soybean meal volume at room temperature, followed by stirring. The degree of soybean meal degradation was measured by SDS-PAGE.

As a result, it was confirmed that the protease of the present invention efficiently degrades soybean meal within 2 hours (see FIG. 9 ).

<10-2> Trypsin Inhibitor Degradation Test in Soybean Meal

The inventors performed soybean meal digestion test using the protease produced in Example 6 to determine whether the Bacillus cluj derived protease is suitable for use as a feed additive. Soybean meal has a soybean trypsin inhibitor (SBTI), which is known as an anti-nutritive factor, and is known to reduce protein digestibility when soybean meal is the main protein source. Therefore, we investigated whether it can be used as feed additive by inactivating soybean meal trypsin inhibitor by treatment with Bacillus cloudage-derived protease.

As a result, the protease of the present invention inactivated about 80% of soybean meal trypsin inhibitors within 2 hours, and almost completely inactivated trypsin inhibitors after 16 hours of treatment (see FIG. 10 ).

The protease of the present invention is active in heavy metals, organic solvents, low temperatures and extreme reducing environments, high pH, high salt concentration, anionic surfactants such as SDS, strong oxidizing agents such as hydrogen peroxide, commercially available It is a very stable enzyme even in the presence of a commercial detergent. Therefore, the protease may be usefully used for various industrial purposes, such as processing of clothing or contact lens detergents, processing of feed, food, textiles, natural leather and the like, and processing organic wastes. The property enables the production of amino acids in a short time when fermenting high-salt foods such as traditional soy sauce and salted fish, so that the enzyme can be used to shorten the ripening time of the food.

<110> INHA UNIVERSITY <120> Detergent and oxidant-stable haloalkalophilic protease and a gene          coding this protease <130> 4P-12-36 <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 2280 <212> DNA <213> 1 <400> 1 aacgttgaca ttcggcacac tccttttcat ttatatcgta accgaagaac gttcaaaaaa 60 ccaaatcatc aagccgccat tttcacttcg ccggcacatt gagagaataa tggacaaatc 120 cggtatcctc ttcatagccg ttttgctcat acaagcttct tgccttccgg ttgtggtgct 180 cagtctgaag cgttaaacat tttgccccgt tttcccctgc ataatccttt gcggcagaaa 240 gcagccggcc gccggctccc tttgtacgcg catgaggaac gacaaataag tcatttaata 300 tgtagatcct tttcattgac acagaagaaa acgttggata gagctgggta aagcctatga 360 attctccatt ttcttctgct atcaaaataa cagactcgtg attttccaaa cgagctttca 420 aaaaagcctc cgccccttgc aaatcggatg cctgtctata aaattcacga tattggttaa 480 acaacggcgc aatggcggcc gcatctgatg tctttgcttg gcgaatgttc atcttatttc 540 ttcctccctc tcaataattt ttttattcta tcccttttct gtaaagttta tttttcagaa 600 tacttttatc atcatgcttt gaaaaaatat cacgataata tccattgttc tcacggaagc 660 atatacaggt catttgaacg aattttttcg acaggaattt gcagggactc aggagcattt 720 aacctaaaaa agcatgacat ttcagcataa tgaacattta ctcatgtcta ttttcgttct 780 tttctgtatg aaaatagtta tttcgagtct ctacggaaat agcgagagat gatataccta 840 aatagagata aaatcatctc aaaaaaatgg gtctactaaa atattattcc atctattaca 900 ataaattcac agaatagtct tttaagtaag tctactctga atttttttaa aaggagaggg 960 taaagagtga gaagcaaaaa attgtggatc agcttgttgt ttgcgttaac gttaatcttt 1020 acgatggcgt tcagcaacac gtctgcgcag gctgccggaa aaagcagtac agaaaagaaa 1080 tacattgtcg gatttaagca gacaatgagt gccatgagtt ccgccaagaa aaaggatgtt 1140 atttctgaaa aaggcggaaa ggttcaaaag caatttaagt atgttaacgc ggccgcagca 1200 acattggatg aaaaagctgt aaaagaattg gaaaaagatc cgagcgttgc atatgtggaa 1260 gaagatcata ttgcacatga atatgcgcaa tctgttcctt atggcatttc tcaaattaaa 1320 gcgccggctc ttcactctca aggctacaca ggctctaacg taaaagtagc tgttattgac 1380 agcggaattg actcttctca tcctgactta aacgtcagag gcggagcaag tttcgtacct 1440 tctgaaacaa acccatacca ggacggcagt tctcacggta cgcatgtagc cggtacgatt 1500 gccgctctta ataactcaat cggtgttctg ggcgtagcgc caagcgcatc attatatgca 1560 gtaaaagtgc ttgattcaac aggaagcggc caatatagct ggattattaa cggcattgag 1620 tgggccattt ccaacaatat ggatgttatc aacatgagcc ttggcggacc tactggttct 1680 acagcgctga aaacagtcgt tgacaaagcc gtttccagcg gtatcgtcgt tgctgccgct 1740 gccggaaacg aaggttcgtc cggaagctca agcacagtcg gctaccctgc aaaatatcct 1800 tctactattg cggtaggtgc ggtaaacagc agcaaccaaa gagcttcatt ctcaagcgca 1860 ggttctgagc ttgatgtgat ggctcctggc gtatccatcc aaagcacact tcctggaggc 1920 acttacggtg cttacaacgg cacgtccatg gcgactcctc acgttgccgg agcagcagcg 1980 ttaattcttt ctaagcaccc gacttggaca aacgcgcaag tccgtgatcg tttagaaagc 2040 actgcaacat atcttggaaa ctctttctac tatggaaaag ggttaatcaa cgtacaagca 2100 gctgcacaat aatagtaaaa agaagcaggt tcctccatac ctgcttcttt ttatttgtca 2160 gcatcctgat gttccggcgc attctcttct ttttccgcat gttgaatccg ttccatgatc 2220 gacggatggc tgcctctgaa aatcttcaca agcaccggag gatcaacctg gctcagcccc 2280                                                                         2280 <210> 2 <211> 381 <212> PRT <213> 2 <400> 2 Met Arg Ser Lys Lys Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu   1 5 10 15 Ile Phe Thr Met Ala Leu Ser Asn Met Ser Ala Gln Ala Ala Gly Lys              20 25 30 Ser Ser Thr Glu Lys Lys Tyr Ile Val Gly Phe Lys Gln Thr Met Ser          35 40 45 Ala Met Ser Ser Ala Lys Lys Lys Asp Val Ile Ser Glu Lys Gly Gly      50 55 60 Lys Val Gln Lys Gln Phe Lys Tyr Val Asn Ala Ala Ala Ala Thr Leu  65 70 75 80 Asp Glu Lys Ala Val Lys Glu Leu Lys Lys Asp Pro Ser Val Ala Tyr                  85 90 95 Val Glu Glu Asp His Ile Ala His Glu Tyr Ala Gln Ser Val Pro Tyr             100 105 110 Gly Ile Ser Gln Ile Lys Ala Pro Ala Leu His Ser Gln Gly Tyr Thr         115 120 125 Gly Ser Asn Val Lys Val Ala Val Ile Asp Ser Gly Ile Asp Ser Ser     130 135 140 His Pro Asp Leu Asn Val Arg Gly Gly Ala Ser Phe Val Pro Ser Glu 145 150 155 160 Thr Asn Pro Tyr Gln Asp Gly Ser Ser His Gly Thr His Val Ala Gly                 165 170 175 Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu Gly Val Ala Pro             180 185 190 Ser Ala Ser Leu Tyr Ala Val Lys Val Leu Asp Ser Thr Gly Ser Gly         195 200 205 Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu Trp Ala Ile Ser Asn Asn     210 215 220 Met Asp Val Ile Asn Met Ser Leu Gly Gly Pro Thr Gly Ser Thr Ala 225 230 235 240 Leu Lys Thr Val Val Asp Lys Ala Val Ser Ser Gly Ile Val Val Ala                 245 250 255 Ala Ala Ala Gly Asn Glu Gly Ser Ser Gly Ser Thr Ser Thr Val Gly             260 265 270 Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala Val Gly Ala Val Asn Ser         275 280 285 Ser Asn Gln Arg Ala Ser Phe Ser Ser Ala Gly Ser Glu Leu Asp Val     290 295 300 Met Ala Pro Gly Val Ser Ile Gln Ser Thr Leu Pro Gly Gly Thr Tyr 305 310 315 320 Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala                 325 330 335 Ala Ala Leu Ile Leu Ser Lys His Pro Thr Trp Thr Asn Ala Gln Val             340 345 350 Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr Leu Gly Asn Ser Phe Tyr         355 360 365 Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala Ala Ala Gln     370 375 380 <210> 3 <211> 275 <212> PRT <213> 3 <400> 3 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala Pro Ala Leu   1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp              20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val Arg Gly Gly Ala          35 40 45 Ser Phe Val Pro Ser Glu Thr Asn Pro Tyr Gln Asp Gly Ser Ser His      50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly  65 70 75 80 Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu                  85 90 95 Asp Ser Thr Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu             100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly         115 120 125 Pro Thr Gly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser     130 135 140 Ser Gly Ile Val Val Ala Ala Ala Ala Gly Asn Glu Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala                 165 170 175 Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Ala             180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr         195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr     210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr                 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala             260 265 270 Ala Ala Gln         275 <210> 4 <211> 24 <212> DNA <213> primer F2 <400> 4 gaagcacgtt tacatcggtc tggc 24 <210> 5 <211> 24 <212> DNA <213> Primer F3 <400> 5 ggggctgagc caggttgatc ctcc 24 <210> 6 <211> 23 <212> DNA <213> R1 <400> 6 gcatctgatg tctttgcttg gcg 23 <210> 7 <211> 23 <212> DNA <213> R2 <400> 7 aacgttgaca ttcggcacac tcc 23 <210> 8 <211> 24 <212> DNA <213> Primer R3 <400> 8 tggcaaaggc accagccagt cacc 24 <210> 9 <211> 30 <212> DNA <213> BCAP-F1 <400> 9 ggggcatatg atatgcgcaa tctgttcctt 30 <210> 10 <211> 32 <212> DNA <213> BCAP-R1 <400> 10 ccccaagctt ttattgtgca gctgcttgta cg 32  

Claims (13)

A proteolytic enzyme comprising an amino acid sequence set forth in SEQ ID NO: 3. The protease according to claim 1, wherein the degrading enzyme has an amino acid sequence set forth in SEQ ID NO: 2. Gene encoding the protease of claim 1. 4. The gene of claim 3, wherein the gene is set forth in SEQ ID NO: 1. An expression vector comprising the gene of claim 3. The expression vector of claim 5, wherein the expression vector is pT7-BCAP. A transformant into which the expression vector of claim 5 is introduced. An expression vector for Bacillus genome insertion comprising the gene of claim 3. 9. The expression vector of claim 8, wherein the expression vector is pHPS-BCAP. The transformant JM109 / pHPS-BCAP (accession number: KCTC 10772BP) into which the expression vector of claim 8 was introduced. A detergent for clothing or contact lenses comprising the protease of claim 1. A method for processing food, feed, textiles, and natural leather using the protease of claim 1. Method for decomposing organic waste using the protease of claim 1.

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