Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J3/00—Working-up of proteins for foodstuffs
  • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
  • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
  • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
  • A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea

Definitions

• the present invention relates to a protease having a high activity at a low temperature and its utilization. ii) Description ofthe Related Art
• Psychrophilic bacteria have been known for a long time, and their existence can be cor-firmed extensively in low temperature circumstances. For example, psychrophilic bacteria can be isolated from soils, fishery products, milk products as well as artificial low temperature circumstances. Researches on psychrophilic bacteria have been conducted in accordance with food microbiological requirements, but they have principally been confined to those with respect to the phylogeny of microorganisms.
• Enzymes obtained from psychrophilic bacteria are expected to be cold-active enzymes having an optimal temperature in a low temperature range.
• the cold-active enzyme which acts efficiently at the low temperature is considered to be capable of being incorporated into, for example, a detergent which can be used even in water at the low temperature. It is also considered to be employed for a chemical reaction in the presence of an organic solvent which is volatile at ordinary temperature and for the quality improvement of foods at the low temperature at which the foods do not rot.
• the present inventors have now found that a protease can be isolated from the supernatant liquid of a culture medium of a Flavobacterium balustinum P104 strain and then purified, and that the isolated and purified protease has activity at a low temperature.
• the present invention is based on such knowledge.
• an object ofthe present invention is to provide a cold-active protease. Another object of the present invention is to provide a method for preparing the above-mentioned cold-active protease by the use of a Flavobacterium balustinum P104 strain.
• Still another object of the present invention is to provide a peptide comprising an amino acid sequence present at the N terminal ofthe cold-active protease.
• the protease according to the present invention has a part or all of the following physicochemical properties. - Specific activity and substrate specificity: The protease acts on casein, gelatin, hemoglobin and albumin to specifically decompose them in the order of casein, gelatin, hemoglobin and albumin.
• 8.0 - pH stability The protease is stable in the range of pH 6.5 to pH 10.0 at 30°C for
• the protease according to the present invention further has a part or all of the following physicochemical properties.
• the protease has about 50% or more of its maximum activity at 20°C.
• the active center ofthe enzyme is serine.
• the molecular weight of the protease is about 70 kDa as measured by SDS- PAGE.
• the protease according to the present invention consists of a protein containing a part or all of an amino acid sequence described in SEQ ID NO:l, or a protein containing a part or all of an amino acid sequence described in SEQ HD NO:l at its N terrninal.
• protease having about 50% or more of its maximum activity at 20°C.
• protease which consists of a protein containing a part or all of an amino acid sequence described in SEQ ID NO:l, and a protease which consists of a protein containing a part or all of an arnino acid sequence described in SEQ ID NO: 1 at its N terminal.
• a method for preparing the above-mentioned protease according to the present invention comprises the steps of culturing Flavobacterium balustinum P104 (FERM BP-5006) for producing the protease, and then collecting the protease from its culture medium.
• Fig. 1 is a drawing illustrating the results of the purification of an enzyme according to the present invention.
• Fig. 2 is a drawing illustrating a calibration curve for measuring the molecular weight ofthe enzyme according to the present invention.
• Fig. 3 is a drawing illustrating the effect of pH on an enzyme reaction of the enzyme according to the present invention.
• Each black circle represents the enzyme according to the present invention at pH 7
• each black triangle represents the enzyme according to the present invention at pH 10
• each white square represents Savinase at pH 7.
• Fig. 4 is a drawing illustrating the pH stability of the enzyme according to the present invention.
• Each black circle represents a measured value at 10°C
• each black triangle represents a measured value at 20°C
• each black square represents a measured value at 30°C
• each white circle represents a measured value at 40°C
• each white triangle represents a measured value at 50°C
• each white square represents a measured value at 60°C.
• Fig. 5 is a drawing illustrating the effect of temperature on the enzyme reaction of the enzyme according to the present invention.
• Fig. 6 is a drawing illustrating the temperature stability ofthe enzyme according to the present invention.
• the enzyme according to the present invention acts on casein, gelatin, hemoglobin and albumin to specifically decompose them.
• the substrate specificity of the enzvme decreases in the order of casein, gelatin, hemoglobin and albumin.
• the optimal pH of the enzyme according to the present invention is 8.0. Furthermore, the enzyme retains about 90% or more of a maximum activity in the range of pH 6.5 to pH 9.5.
• the enzvme according to the present invention is stable at 30°C for 1 hour in the range of pH 6.5 to pH 10.0. (4) Optimal temperature
• the optimal temperature ofthe enzyme according to the present invention is 40°C at pH 10 and pH 7. At a temperature of 30°C, the enzyme retains about 80% of the maximum activity at pH 10 and about 90% of the maximum activity at pH 7. At a temperature of 50°C, the enzyme retains about 10% ofthe maximum activity at pH 10 and about 80% ofthe maximum activity at pH 7.
• the enzyme according to the present invention can be considered to be a psychrophilic enzyme.
• the enzyme according to the present invention is scarcely inactivated at a temperature up to 30°C, but it is inactivated at 40°C as much as about 40% and completely inactivated at 50°C in about 10 minutes. Therefore, the enzyme according to the present invention can be considered to be a psychrophilic enzyme.
• the enzyme according to the present invention has about 50% or more of its maximum activity at 20°C.
• the protease activity of the enzyme according to the present invention is not inhibited by any of pepstatin, L-trans-epoxysuccinylleucylamido-4-guanidinobutane (E- 64), iodoacetamide and 1,10-phenanthroline, but it is notably inhibited by phenylmethanesulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA).
• PMSF phenylmethanesulfonyl fluoride
• EDTA ethylenediaminetetraacetic acid
• the enzyme according to the present invention has a molecular weight of about 70 kDa as measured by SDS-PAGE.
• amino acid sequence at the N terminal ofthe enzyme according to the present invention is described in SEQ ID NO: 1.
• the amino acid sequence at the N terminal of the enzyme according to the present invention With regard to the amino acid sequence at the N terminal of the enzyme according to the present invention, its homology with each amino acid sequence of known proteins was inspected by the use of a data bank "Entrez", and as a result, it was apparent that the amino acid sequence at the N terminal had no homology with any amino acid sequences of the known proteins.
• the protease according to the present invention may consists of a protein containing a part or all of an amino acid sequence described in SEQ ID NO:l or a protein containing a part or all of an amino acid sequence described in SEQ ID NO:l at its N terminal.
• protease consisting of a protein containing a part or all of an amino acid sequence described in SEQ ID NO:l
• protease consisting of a protein containing a part or all of an amino acid sequence described in SEQ ID NO: 1 at its N terminal.
• This protease may have such characteristics as described in the above-mentioned (1) to (8).
• the protein containing a part or all of an amino acid sequence described in SEQ LD NO:l includes a protein in which an optional amino acid sequence is added to an N terminal and/or a C terminal of a part or all of the amino acid sequence described in SEQ ID NO:l.
• a protease according to the present invention can be produced by the use of microorganisms.
• the production microorganisms belong to a Flavobactrium genus, and any microorganisms are usable, so far as they have an ability for producing the protease.
• a preferable example of the microorganisms having the ability for producing the protease according to the present invention is a Flavobacterium balustinum P104 strain. This strain is microorganisms separated from the intestine of salmon by the present inventors, and they are deposited in Agency of Industrial Science and Technology, Biotechnology Research Institute under Deposition No. FERM BP-5006 on Febmary 17, 1995.
• a culture medium may be liquid or solid, but a shake culture or an aeration spinner culture using a liquid culture medium is usually used.
• any medium is usable, so far as it can produce the protease.
• a carbon source there can be used, for example, glucose, trehalose, fructose, maltose, sucrose, starch and malt oligo-saccharide.
• a nitrogen source there can be used, for example, peptone, yeast extract, malt extract, meat extract, soybean powder, cotton seed powder, cone steep liquor, various amino acids and their salts, and nitrates.
• a synthetic medium or a natural medium which can be used in the present invention suitably contains the above ⁇ mentioned carbon source and nitrogen source, inorganic salts such as magnesium phosphate, calcium, sodium, potassium, iron and manganese as well as other nutrients, as needed.
• Culturing conditions such as the pH and the culture temperature of the culture medium can be suitably altered, so far as they permit the production of the protease, but in the case ofthe shake culture or the aeration spinner culture, it is preferred that the pH is about neutral and the culture temperature is from 10°C to 20°C.
• the protease ofthe present invention is present in cell walls of bacteria, cells ofthe bacteria and the supernatant of a culture medium, and it may be used in any form such as bacterial cells, a crude enzyme obtained from the bacterial cells or the supernatant of the culture medium, or an extracted and purified enzyme. Alternatively, the protease immobilized by a known method can also be used.
• protease ofthe present invention In order to collect and purify the protease ofthe present invention from the culture medium, known purification methods can be utilized singly or in combination thereof. Since the protease according to the present invention is mainly excreted extracellularly, namely into the culture medium, a crude enzyme solution can easily be obtained by removing the bacterial cells with the aid of filtration or centrifugation. This crude enzyme can be further purified by a known purification method.
• Examples of the known preferable purification method include a salting-out method using a salt such as ammonium sulfate, a precipitation method using an organic solvent (e.g., methanol, ethanol or acetone), an adsorption method using raw starch, an ultrafiltration method, and various chromatographical methods such as gel filtration chromatography and ion exchange chromatography.
• a salting-out method using a salt such as ammonium sulfate
• a precipitation method using an organic solvent e.g., methanol, ethanol or acetone
• an adsorption method using raw starch e.g., methanol, ethanol or acetone
• an adsorption method using raw starch e.g., methanol, ethanol or acetone
• an ultrafiltration method e.g., adsorption method using raw starch
• various chromatographical methods such as gel filtration chromatography and ion exchange chromatography.
• the psychrophilic protease according to the present invention has an optimal temperature in a low temperature range.
• the psychrophilic protease of the present invention permits the decomposition reaction of a protein to be carried out in low temperature environments.
• a detergent utilizable even in low temperature water can be prepared by adding the protease according to the present invention to a detergent composition for clothes.
• This detergent composition can be prepared in accordance with a conventional method except that the psychrophilic protease according to the present invention is added. That is to say, the detergent can be formed by blending the protease of the present invention with ordinary detergent components such as a surface active agent for the detergent, a bleach, a builder and the like.
• the psychrophilic protease according to the present invention enables the reaction to proceed at a low temperature. Therefore, even if an organic solvent which is volatile at ordinary temperature is present in the reaction system, the reaction can be carried out at a low temperature at which the organic solvent component is not volatilized. Moreover, when it is attempted to improve the quality of a food by the use of the protease according to the present invention, the reaction proceeds advantageously at a low temperature, whereby the food can be effectively prevented from rotting.
• the protease according to the present invention since the protease according to the present invention is provided, it can be expected to advance the elucidation ofthe physiological mechanism of psychrophilic bacteria and their application mechanism to a low temperature.
• Protein having amino acid sequence at N terminal we provide a peptide consisting of a part or all of an amino acid sequence described in SEQ LD NO:l, a protein comprising a part or all of an amino acid sequence described in SEQ ED NO:l, and a protein comprising a part or all of an amino acid sequence described in SEQ ID NO: 1 at its N te ⁇ riinal.
• This protein may have a protease activity.
• This peptide or protein consists of a part or all of an amino acid sequence present at the N terminal ofthe enzyme according to the present invention, or comprises a part or all ofthe amino acid sequence (preferably at the N terminal). Therefore, the above ⁇ mentioned peptide or protein is useful as an antigen in forming an antibody to the enzyme according to the present invention.
• the quantitative analysis of a protein was carried out by the protein staining method using Bio-Rad protein assay (Bio-Rad Co., Ltd.).
• the detection of a protein by chromatography was carried out by measuring the absorption of an ultraviolet portion at 280 nm.
• the activity of a protease was measured by the following method (a) or (b).
• reaction solution was centrifuged (10,000 rpm, room temperature, 10 minutes), and 500 ⁇ l of a 0.5 M sodium carbonate solution and 100 ⁇ l of a phenol solution twice diluted with distilled water were added to 100 ⁇ l of the resultant supernatant liquid. After the solution was allowed to stand at room temperature for 1 hour, the absorbency ofthe solution at 660 nm was measured.
• Example 1 Purification of protease derived from a Flavobacterium balustinum P 104 strain (1) Culture of a bacterial strain
• a bacterial strain was inoculated into 150 ml of the undermentioned culture medium (which was separately poured into six 100-ml Erlenmeyer flasks), and rotary shaking culture was then carried out at 10°C for 48 hours at 140 ⁇ m by the use of a triple shaker NR-80 (Tietec Co., Ltd.).
• 150 ml ofthe pre-culture medium was inoculated into 3 liters of the undermentioned culture medium, and rotary culture was then carried out at 10°C for 75 hours at 140 m by the use of a laboratory fermenter LS-5 (Oriental Yeast Co., Ltd.).
• the culture medium and the like were sterilized with high-pressure vapor for 15 minutes under 1.2 kgf/cm 2 (gauge pressure) (121 C C) by an autoclave.
• the culture medium obtained in the above-mentioned (1) was clarified by centrifugation (7,200xg, 4°C, 30 minutes).
• the resultant supernatant liquid was subjected to ion exchange chromatography as a crude enzvme solution.
• As a column there was used an LNdEX 100 column (Parmacia Biotec Co., Ltd.) filled with 2 liters of a DEAE Sephalose fast flow anion exchanger (Parmacia Biotec Co., Ltd.).
• a 20 mM tris buffer solution (pH 7.0) was introduced into the above-mentioned column at a linear velocity of 150 cm/hr to equilibrate the column five times or more (10 liters) as much as a gel volume.
• the fractions obtained above were treated by a new model stirring type cell 8400 (Amicon Co., Ltd.) to which a Diaflo Membrane PM30 (which can fractionate a substance having a molecular weight of 30,000 or more) (Amicon Co., Ltd.) was set, whereby proteins having a molecular weight of 30,000 or more were concentrated.
• Ammonium sulfate was added to the thus concentrated solution under ice cooling so that the solution might be saturated as much as 50% with ammonium sulfate. After slow stirring at 0°C for 4 hours, the solution was sedimented by centrifugation (27,000xg, 4°C, 20 minutes) to obtain a 0 to 50% saturated fraction. The amount of added ammonium sulfate was an amount necessary to achieve a saturated concentration at 25°C.
• a Bis- tris buffer solution (pH 7) was caused to flow through the HiLoad 16/60 Superdex 200 prep grade column at a linear velocity of about 60 cm/hr to equilibrate the column, the amount ofthe Bis-tris buffer solution being three times or more (400 ml) as much as the gel volume. Afterward, 5 ml of the sample enzyme solution which had been subjected to the salting out with ammonium sulfate was introduced into the column by the use of a Superloop. Then, elution was carried out at a linear velocity of 60 cm hr by the use of the Bis-tris buffer solution (pH 7) as an eluent to collect fractions every 5 ml.
• the sample enzyme solution of the protease activity fraction eluted by the gel filtration was introduced into the column at a linear velocity of 90 cm/hr by the use of the Superloop.
• elution was carried out at a linear velocity of 90 cm/hr with 150 ml of the Bis-tris buffer solution (pH 7) containing IM NaCl in accordance with a linear ion strength increasing gradient (0 to 1 M) to collect fractions every 5 ml.
• the results ofthe above-mentioned purification are shown in Table 1. 10
• the purity and the molecular weight of the purified enz me according to the present invention were measured by the use of sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
• a 10% polyacrylamide gel having a thickness of 1 mm was used as a support. Electrophoresis was carried out by applying 20 mA of an electric current to the gel until Bromophenol Blue (BPB) reached a lower end. The gel plate was stained with an aqueous 30% methanol- 10% acetic acid solution containing 0.02% of Coomassie Brilliant Blue R250 for 1 hour, and then decolored with a decolorant (a solution of 30% of methanol and 10% of acetic acid) overnight.
• BBPB Bromophenol Blue
• Example 4 (Effect of pH on enzyme reaction) Azocasein was decomposed with the enzyme of the present invention at various pH values.
• Buffer solutions for a reaction solution had each a concentration of 100 mM and they were a sodium acetate-acetic acid buffer solution (pH 4.0 - 5.5), MES (2- mo ⁇ holinoethanesulfonic acid monohydrate) buffer solution (pH 5.5 - 6.5), MOPS (3- mo ⁇ holinopropanesulfonic acid) buffer solution (pH 6.5 - 8.0), TAPS (N- tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) buffer solution (pH 8.0 - 9.0), CHES (N-cy ohexyl-2-aminoethanesulfonic acid) buffer solution (pH 9.0 - 10.0), CAPS (N-cyclohexyl-3-aminopropanesulfonic acid) buffer solution (pH 10.0 - 11.0) and g
• Example 5 pH stability of enzyme according to the present invention
• the enzyme according to the present invention was maintained in each of the above ⁇ mentioned buffer solutions at 30°C for 1 hour, and the remaining protease activity was then inspected. The results are shown in Fig. 4. It was found that the enzyme according to the present invention was stable over the range of pH 6.5 to pH 10.0 at 30°C for 1 hour, but the same was inactivated at pH 4.0 and pH 11.0 under the same conditions.
• Example 6 (Effect of temperature on enzyme reaction) Azocasein was decomposed with the enzyme according to the present invention at various temperatures in a 67 mM phosphoric acid buffer solution (pH 7) and a 100 mM glycine-sodium chloride (pH 10).
• reaction temperatures were changed from 5°C to 70°C. Furthermore, also with regard to a commercially available enzyme, Savinase (Novonordisc Co., Ltd.), an influence of a temperature at pH 7 was measured. The results are shown in Fig. 5.
• the optimal temperature of the enzyme according to the present invention at pH 10 and pH 7 were 40°C. At a temperature of 30°C, the enzyme of the present invention could maintain about 80% of the activity at pH 10 and about 90% of the activity at pH 7. At a temperature of 50°C, the enzyme ofthe present invention could maintain about 10% of the activity at pH 10 and about 80% of the activity at pH 7.
• the optimal temperature of commercially available Savinase was 60°C, which was much higher as compared with that ofthe enzyme according to the present invention.
• Example 7 (Temperature stability of enzyme according to the present invention)
• the enzyme according to the present invention was maintained at 10 to 60°C for 1 hour.
• the variation of its activity with time is shown in Fig. 6.
• the enzyme according to the present invention was scarcely inactivated at 10°C, 20°C and 30°C for 1 hour. However, it was inactivated at 40°C to about 60% ofthe protease activity, and afterward, it was gradually inactivated. At 50°C and 60°C, the enzyme was rapidly inactivated, and it was completely inactivated in 10 minutes.
• Example 8 Effect of inhibitor on enzyme according to the present invention
• pepstatin acting on aspartic protease
• iodoacetamide acting on cysteine protease
• PMSF phenylmethanesulfonyl fluoride
• EDTA ettiylenediaminetetraacetic acid
• protease activity of the enzyme according to the present invention was not inhibited by pepstatin, L-tr-ms-epoxysuccinylleuc ⁇ lamido-4-guanidinobutane (E-64), iodoacetamide and 1,10-phenanthroline, but it was notably inhibited by phenylmethanesulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA). From these results, it is implied that the enzyme according to the present invention is serine protease in which an active center is serine.
• PMSF phenylmethanesulfonyl fluoride
• EDTA ethylenediaminetetraacetic acid
• Example 9 Substrate specificity of protease The proteolytic activities ofthe protease to casein, hemoglobin, albumin and gelatin as substrate proteins were measured by a phenol reagent method. The results are shown in Table 3.
• the enzyme according to the present invention specifically acted on casein at a low temperature, and the substrate specificity of the enzyme decreased in the order of gelatin, hemoglobin and albumin.
• Example 10 (Determination of amino acid sequence at N terminal)
• 30 residues of the amino acid sequence at its N terminal were determined.
• the results are shown in SEQ LD NO: 1.
• the amino acid sequence at the N terminal ofthe enzyme according to the present invention was determined, and its homology with known amino acid sequences was inspected by the use of a data bank "Entrez". As a result, it was apparent that there was no homology. Accordingly, since the enzyme according to the present invention has no homology with any amino acid sequences of known proteins, it has been confirmed that the enzyme according to the present invention possesses the novel amino acid sequence at the N terminal.
• Sequence type Amino acid Strandedness: Single strand Topology: Linear Molecule type: Peptide Fragment type: N terminal Original source: Organism: Flavobacterium balustinum

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Zoology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Wood Science & Technology (AREA)
• Organic Chemistry (AREA)
• Biochemistry (AREA)
• Genetics & Genomics (AREA)
• General Engineering & Computer Science (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Medicinal Chemistry (AREA)
• Biotechnology (AREA)
• Molecular Biology (AREA)
• General Health & Medical Sciences (AREA)
• Biomedical Technology (AREA)
• Microbiology (AREA)
• General Chemical & Material Sciences (AREA)
• Nutrition Science (AREA)
• Food Science & Technology (AREA)
• Polymers & Plastics (AREA)
• Enzymes And Modification Thereof (AREA)
• Detergent Compositions (AREA)
• Peptides Or Proteins (AREA)

Applications Claiming Priority (3)

Publications (2)

Family

ID=11798951

Family Applications (1)

Country Status (7)

Families Citing this family (3)

Citations (2)

• 1996
  • 1996-01-26 JP JP8012207A patent/JPH09201195A/ja not_active Withdrawn
• 1997
  • 1997-01-24 BR BR9707203-6A patent/BR9707203A/pt not_active IP Right Cessation
  • 1997-01-24 EP EP97903957A patent/EP0876500A4/de not_active Withdrawn
  • 1997-01-24 CN CN 97193195 patent/CN1214084A/zh active Pending
  • 1997-01-24 WO PCT/US1997/001148 patent/WO1997027313A1/en not_active Application Discontinuation
  • 1997-01-24 AR ARP970100288A patent/AR005541A1/es unknown
  • 1997-01-24 CA CA002243598A patent/CA2243598C/en not_active Expired - Fee Related

Patent Citations (2)

Non-Patent Citations (5)

Also Published As

Similar Documents

Legal Events