WO1996025489A1 - Protease active au froid et bacteries actives au froid - Google Patents

Protease active au froid et bacteries actives au froid Download PDF

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Publication number
WO1996025489A1
WO1996025489A1 PCT/US1996/002115 US9602115W WO9625489A1 WO 1996025489 A1 WO1996025489 A1 WO 1996025489A1 US 9602115 W US9602115 W US 9602115W WO 9625489 A1 WO9625489 A1 WO 9625489A1
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Prior art keywords
protease
psychrophilic
enzyme
temperature
activity
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PCT/US1996/002115
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English (en)
Inventor
Eiichi Tamiya
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The Procter & Gamble Company
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Filing date
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Application filed by The Procter & Gamble Company filed Critical The Procter & Gamble Company
Priority to MX9706272A priority Critical patent/MX9706272A/es
Priority to EP96906484A priority patent/EP0871719A1/fr
Priority to AU49848/96A priority patent/AU4984896A/en
Priority to BR9607452A priority patent/BR9607452A/pt
Publication of WO1996025489A1 publication Critical patent/WO1996025489A1/fr

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    • 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
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/20—Flavobacterium

Definitions

  • the present invention relates to a protease having a high activity at a low temperature range, its use and a psychrophilic bacterium producing the protease.
  • Psychrophilic bacteria have been known for a long time, and their existence can be confirmed extensively in low temperature circumstances. Psychrophilic bacteria can be isolated from various low temperature circumstances such as soil, fishery products, milk products as well as artificial low temperature circumstances. Studies on psychrophilic bacteria have been conducted from the food microbiological requirement but principally confined to those with respect to the phylogeny of microorganisms.
  • enzymes obtained from psychrophilic bacteria are expected to be the psychrophilic enzymes having an optimal temperature in a low temperature range.
  • the psychrophilic enzyme which works efficiently at low temperatures is considered capable of being incorporated into for example a detergent which can be used even in water at a low temperature. It is also considered to be employed for the chemical reaction in the presence of an organic solvent which is volatile at the room temperature and for improving the quality of foods at a temperature that the foods will not be rotten. Furthermore, the study on the enzyme derived from the psychrophilic bacteria is fairly interesting to reveal the physiological functions and adaptation mechanism to a low temperature of the psychrophilic bacteria.
  • an object of the present invention is to provide a novel psychrophilic protease.
  • Another object of the present invention is to provide a novel microorganism which produces the psychrophilic protease.
  • Further object of the present invention is to provide a process for preparing the psychrophilic protease with the novel microorganism.
  • the psychrophilic protease according to the present invention has the following physicochemical properties.
  • protease acts on casein and dimethylcasein to decompose them but does not act on ribonuclease.
  • the present protease also has the following physicochemical properties:
  • the protease acts optimally at pH 7.5;
  • the protease is stable at a pH in the range of 6.0 - 10.0 under the condition of storage at 20°C for 1 hour;
  • novel microorganism according to the present invention is Flavobacterium balustinum having the psychrophilic protease producing ability described above.
  • the process for preparing the psychrophilic protease according to the present invention comprises culturing Flavobacterium balustinum described above, and collecting the psychrophilic protease from the culture.
  • Figure 1 is a graph illustrating the result of Example 2, or shows the relationship between temperature and the activity of proteases derived from the strain P104 and the protease, Subtilysin Carlsberg.
  • Figure 2 is a graph illustrating the result of Example 4 (2), or shows the influence of initial pH on the activity and growth of the extracellular protease of Flavobacterium balustinum P104.
  • Figure 3 is a graph illustrating the result of Example 4 (3), or shows the influence of culturing temperatures on the activity and growth of the extracellular protease of Flavobacterium balustinum P104.
  • Figure 3 (A), (B) and (C) show the results at 10°C, 20°C, and 30°C, respectively.
  • Figure 4 is a graph illustrating the result of the elution by gel filtration in Example 5 (2) (b).
  • Figure 5 is a graph illustrating the result of the elution by chromatography in Example 5 (2) (c).
  • Figure 6 illustrates the result of SDS-PAGE for the measurement of molecular weight in Example 6.
  • Figure 7 is a calibration curve for the measurement of molecular weight in Example 6.
  • Figure 8 is a calibration curve of gel filtration for the measurement of molecular weight in Example 6.
  • Figure 9 illustrates the result of isoelectric focusing in Example 7.
  • Figure 10 is a calibration curve of isoelectric focusing in Example 7.
  • Figure 11 is a graph illustrating the result of Example 8, or shows the influence of pH on the enzyme reaction of the enzyme of the present invention.
  • Figure 12 is a graph illustrating the result of Example 9, or shows the stability of the enzyme of the present invention to pH.
  • Figure 13 is a graph illustrating the result of Example 10, or shows the influence of temperature on the enzyme reaction of the enzyme of the present invention.
  • Figure 14 is a graph illustrating the result of Example 11, or shows the stability of the enzyme of the present invention to temperature.
  • Figure 15 is a graph illustrating the result of Example 13, or a graph illustrating the influence of the protein modifier SDS on the enzyme of the present invention.
  • Figure 16 is a graph illustrating the result of
  • Example 13 or a graph illustrating the influence of the protein modifier urea on the enzyme of the present invention.
  • Figure 17 is Lineweaver-Burk plot of the enzyme of the present invention examined in Example 16.
  • Figure 17 (A) and (B) show the change in 1/v value in the range of 0 to 2.0, and in the range of 0 to 0.2, respectively.
  • Novel protease producing bacterium Novel protease producing bacterium
  • novel protease according to the present invention is produced by microorganisms which belongs to Flavobacterium genus and have the ability to produce a protease having the properties described above.
  • a specific example of the microorganisms having the ability to produce a protease according to the present invention preferably includes Flavobacterium balustinum P104.
  • This strain is a microorganism isolated from the internal organs of salmon and has been deposited in National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology as the deposit number of FERM BP-5006 on February 17, 1995.
  • Flavobacterium balustinum P104 The bacteriological properties of Flavobacterium balustinum P104 according to the present invention are listed in the following.
  • the strain is in the form of short bacillus having a size of 0.4 - 0.5 X 1.7 - 1.9 ⁇ m.
  • the strain grew on an agar medium and produced a yellow pigment.
  • the strain grew at a temperature in the range of 10 - 30°C, and the optimal temperature for growth was around 20°C.
  • Flavobacterium balustinum P104 had the main biochemical properties shown in Table 1 below.
  • Flavobacterium indolgenes from these properties it was judged suitable to be classified into Flavobacterium balustinum by the comparison of the base sequence of DNA coding for 16S ribosomal RNA as is below described in
  • Example 3 with the base sequence in a known microorganism.
  • the culture medium may be either liquid or solid, but shaking culture or aeration culture with a liquid culture medium is generally used.
  • the culture medium for culturing the microorganism may be any one which is suitable for growth and can produce protease.
  • the carbon source include glucose, trehalose, fructose, maltose, sucrose, starch, and malt oligo-saccharides.
  • the nitrogen source include yeast extract, malt extract, beef extract, soybean powder, cotton seed powder, corn steep liquor, various amino acids and their salts, and nitrates. It is also possible to use synthetic media or natural media which contain properly inorganic salts such as magnesium, calcium, sodium potassium, iron and maganese phosphate, and the other nutrients according to necessities.
  • the protease of the present invention is produced in the cell wall of the bacterial cell, within the cell, and in the supernatant of the culture solution, and may be in either form of the bacterial cell, a crude enzyme obtained from the bacterial cell or the supernatant of the culture solution, or of an extracted and purified enzyme. It is also possible to be in the form of protease immobilized on a substrate by the well-known method.
  • the well-known methods can be used alone or in combination thereof.
  • the protease according to the present invention is mainly excreted extracellularly, namely into a culture solution, so that the bacterial cell can be easily removed for example by filtration or centrifugation to obtain a crude enzyme solution.
  • the crude enzyme solution can be further purified by a known method.
  • the method includes preferably the salting-out method with a salt such as ammonium sulfat; the precipitation method with an organic solvent such as methanol, ethanol or acetone; the adsorption method with raw starch; the ultrafiltration method; and a variety of chromatographical methods such as gel filtration chromatography or ion exchange chromatography. Specific examples of the preferred methods are described in Examples below.
  • the present enzyme decomposed well macromolecular proteins such as casein or dimethylcasein or denaturated proteins. It also decomposed gelatin which is the denaturated protein of collagen in a proportion of about 50% as compared with the case of casein. It acted however little on the other natural proteins, and it did not act at all particularly on ribonuclease.
  • subtilisin may have no substrate specificity and act on almost of proteins. On the contrary, the enzyme of the present invention acts only on macromolecular proteins or on denaturated proteins into which the enzyme gets comparatively easily.
  • the protease which is distinct from the enzyme of the present invention, also decomposes natural globular proteins such as hemoglobin and bovine serum albumin in a proportion of about 40% as compared with the case of dimethylcasein.
  • the enzyme of the present invention is likely to have a substrate specificity higher than the well-known enzymes.
  • the enzyme of the present invention acts at a temperature of about 40'C .
  • the enzyme of the present invention thus is the so-called psychrophilic enzyme which exhibits efficiently catalytic action at a low temperature.
  • the enzyme is thus a neutral protease, which will not work in an extremely acidic or alkaline range. Furthermore, it will be inactivated during storage in an extremely acidic or alkaline range.
  • the enzyme of the present invention has a molecular weight of about 38 kDa as measured by SDS-PAGE and gel filtration methods.
  • the enzyme of the present invention has an isoelectric point of about 4.5 as measured by isoelectric focusing.
  • the protease activity of the enzyme is not inhibited by phenylmethyl-sulfonyl fluoride or iodoacetamide, but inhibited noticeably by ethylenediaminetetraacetic acid, 2,2-bipyridyl, citric acid or oxalic acid.
  • the protease activity is thus found to depend on a metal ion, so that it is suggested that the enzyme of the present invention is a metal protease. It is also considered from the inhibition of the protease activity by either of citric acid and oxalic acid that the activity depends on calcium.
  • the activity of the enzyme is inhibited noticeably by metal ions such as Ag + , Cu 2+ , Zn 2+ ,
  • the enzyme of the present invention shows the Michaelis-Menten type reaction rate to the concentration of a substrate such as casein.
  • the Km value decreases and the Vmax value increases along with the increase of temperature.
  • the Kcat value of the enzyme exhibits a remarkably high value in the range from 10 to 40°C.
  • An enzyme is generally tends to be inhibited at an excessive amount of substrates.
  • the Kcat value increases when the system approaches the optimal working temperature in the case of the enzyme of the present invention.
  • the enzyme is therefore advantageous in the point that appreciable inhibition will not be observed by decomposed products. It is also believed from the Lineweaver-Burk plotting that inhibition due to temperature is of a mixed form, that is, the influence of temperature is of non-competitive inhibition or uncompetitive inhibition and the influence of decomposed products is of competitive inhibition.
  • the psychrophilic protease according to the present invention has an optimal temperature at a low temperature range.
  • a detergent which can be used even in water at a low temperature is prepared by incorporating the protease of the present invention into a detergent composition.
  • This detergent composition can be prepared according to the conventional method except that the psychrophilic protease of the present invention is incorporated. Briefly, it can be prepared by combining the protease of the present invention with an ordinary detergent component such as a surface active agent for detergent, a bleach or a mul.
  • the enzyme reaction of the psychrophilic protease according to the present invention can be carried out at a low temperature.
  • the reaction system involves an organic solvent which is volatile at a room temperature
  • the reaction can be conducted at a low temperature where the organic solvent will not be volatilized.
  • protease according to the present invention is provided, advance in the study of the physiological mechanism of psychrophilic bacteria and their application mechanism at a low temperature is expected.
  • proteins were quantitatively determined by the protein staining method, Bio-Rad protein assay, and the protein fractions of the eluate in the chromatographical procedure were determined by the absorption in the ultraviolet range at 280 nm unless otherwise specified.
  • a 0.05 ml portion of a sample enzyme solution was added to 0.3 ml of a 0.067M phosphate buffer containing 1% (W/V) azocasein (pH 7.0), and the mixture was kept at 30°C for 30 minutes. The reaction was then terminated with a 6% trichloroacetic acid solution. After 15 minutes, the reaction mixture was centrifuged at 14,000 rpm at room temperature for 5 minutes. The absorbancy of the supernatant at 340 nm was measured. The enzyme activity was defined on the basis of ACU (azocasein digestion unit) which means the increase of absorbancy of 0.001 per minute at 340 nm.
  • ACU azocasein digestion unit
  • Isolation of a novel bacterial strain was conducted on an agar plate medium.
  • An isolated sample of internal organs of salmons was suspended in aqueous physiological saline, and the supernatant was used as a stock solution.
  • a 10 2 dilution was prepared from the stock solution.
  • a 0.2 ml portion of each of the stock solution and the 10 dilution was sprayed on an agar plate medium for screening (3 g/liter of polypeptone, 10 g/liter of yeast extract, 10 g/liter of sodium casein, 0.2 g/liter of MgSO 4 ⁇ 7H 2 O, 2.0 g/liter of agar, on a 9 cm Petri dish), and cultured at 10°C for 3 days. Colonies grown well among the colonies which had been grown on the agar plate were selected and subcultured as well as inoculated on an agar plate for stock.
  • the activity of an exoenzyme was assayed on an agar plate medium.
  • the bacterial strain isolated was inoculated on an agar plate medium for screening as described above by streaking and cultured at 10°C for 3 days.
  • a 10% trichloroacetic acid solution was then sprayed on the agar plate medium on which the bacteria were grown to assay the protease producing bacterium by the presence of clear plaques.
  • the isolated bacterium from the stock medium was inoculated on 25 ml of a pre-culture medium (10 g/liter of polypeptone, 10 g/liter of endoextract, 0.2 g/liter of MgSO 4 ⁇ 7H 2 O, pH 7.0, in a 100 ml Erlenmeyer flask) and rotary-shake cultured at 10°C at 150 rpm for 48 hours in TAITECNR-80 for stabilizing the growth activity of the bacterium.
  • a pre-culture medium (10 g/liter of polypeptone, 10 g/liter of endoextract, 0.2 g/liter of MgSO 4 ⁇ 7H 2 O, pH 7.0, in a 100 ml Erlenmeyer flask
  • rotary-shake cultured at 10°C at 150 rpm for 48 hours in TAITECNR-80 for stabilizing the growth activity of the bacterium.
  • the bacterial strain isolated was stored in an agar plate medium for storage at 10°C, and subcultured after 2 weeks - 1 month.
  • the culture solution obtained in the preceding step (2) was clarified by centrifugation (17,000 X g, 4°C, 15 minutes). The supernatant was used as a crude enzyme solution.
  • the protease activity was measured by the decomposition of azocasein.
  • a 0.05 ml portion of the crude enzyme solution was added to 0.3 ml of a 0.067M phosphate solution containing 1% (W/V) azocasein (pH 7.0), and the mixture was kept at 20°C for 30 minutes.
  • the reaction was then terminated with a 6% trichloroacetic acid solution, and after 15 minutes the reaction mixture was centrifuged at 14,000 rpm at room temperature for 5 minutes.
  • the absorbancy of the supernatant at 340 nm was measured with a spectrophotometer (Beckman DU640).
  • the enzyme activity was defined on the basis of ACU (azocasein digestion unit) which means the increase of absorbancy of 0.001 per minute at 340 nm.
  • the bacterial strain P104 having a protease activity was isolated.
  • the bacterial strain had a protease activity shown in the following table.
  • the growth rate of the strain was obtained by comparing with the growth of the divided strain Cytophaga xantha IFO 14972.
  • the optimal temperature was 40°C for the exoprotease of the strain P104 and 60°C or more for Subtilisin Carlsberg, respectively.
  • the exoprotease of the strain P104 retained the protease activity at 40% or more of the activity at the optimal temperature at a temperature of about 20°C.
  • Subtilisin Carlsberg retained only about 10% of the protease activity at an optimal temperature.
  • Example 2 The culture solution obtained in Example 2 was sampled in a 1.5 ml microtube, and the bacteria was collected by centrifugation. Genomic DNA was extracted to amplify the base sequence of DNA coding for 16S ribosomal
  • RNA by PCR polymerase chain reaction
  • the base sequence was then determined using the Sanger method, compared with the data base of GenBank for identification.
  • the primers used are listed below, and lF-Link and 5R-Link were used as PCR.
  • Results of comparison with the data base of GenBank are shown in the following tables.
  • Query represents 16S ribosomal RNA gene derived from the strain PI04
  • Subject represents Flavobacterium balustinum 16S ribosomal RNA (FVBRR16SH).
  • FVBRR16SH Flavobacterium balustinum 16S ribosomal RNA
  • the culture solution obtained in Example 2 was diluted with physiological aqueous saline to ensure that 0 - 5 cells were contained in a bacterial counter cell.
  • the cells were countered with an optical microscope.
  • the turbidities of the culture dilutions were measured spectroscopically at 660 nm to obtain the correlation between the cells and turbidity.
  • the relationship between the turbidity and the bacterial cell concentration of Flavobacterium balustinum P104 were represented by the following equation:
  • the bacterial strain was cultured at various initial pHs of the protease producing culture medium in the range from 5 to 9 in order to examine the influence of the initial pH on exoprotease activity and growth of it.
  • the proliferation was significantly lowered and the protease activity was also lowered.
  • insignificant difference was observed in either the proliferation or the protease activity in an acidic pH range.
  • the bacterial strain proliferated best and the protease activity was maintained at the highest level in a neutral pH range.
  • Flavobacterium balustinum P104 was cultured at a various temperature in the range from 10 to 30°C to examine the fluctuation of the exoprotease activity and the proliferation with the passage of the culture.
  • the culture solution obtained in the preceding step (1) was clarified by centrifugation (17,000 X g at 4°C for 15 minutes). The supernatant was used as a crude enzyme solution. Ammonium sulfate was added to the crude enzyme solution to ensure that the solution contained ammonium sulfate at 50% of the saturated concentration. After slow stirring for 1 hour, the solution was sedimented by centrifugation (17,000 X g at 4°C for 15 minutes) to give a 0 - 50% saturation fraction. The added amount of ammonium sulfate in the saturated concentration at 25°C was used as the amount ammonium sulfate added.
  • the HiLoad 16/60 Superdex 200 prep grade column was equilibrated by flowing a 20 mM Bis-Tris buffer (pH 6.0) at a linear rate of about 60 cm/hour in a proportion of at least 3 (400 ml) to the gel volume.
  • a 5 ml portion of the sample enzyme solution which had been subjected to salting out with ammonium sulfate was loaded on the column with a Superloop.
  • the column was eluted with 20 mM Bis-Tris buffer (pH 6.0) as an eluent at a linear rate of about 60 cm/hour to collect 5 ml fractions.
  • Ion exchange chromatography was next carried out with a Q Sepharose HP column.
  • the column was equilibrated by flowing a 20 mM Bis-Tris buffer (pH 6.0) at a linear rate of about 35 cm/hour in a proportion of at least 5 (25 ml) to the gel volume.
  • a 10% polyacrylamide gel having a thickness of 1 mm was used. Electrophoresis was carried out by applying 20 mA of an electric current to the gel until Bromophenol Blue (BPB) reached the lowermost terminal. The gel plate was stained with an aqueous mixture of 30% methanol and 10% acetic acid having 0.02% Coomassie Brilliant Blue R250 dissolved therein for 1 hour and then decolored with a decolorant -(30% methanol and 10% acetic acid) overnight.
  • BPB Bromophenol Blue
  • the molecular weight of the protease was determined by SDS-PAGE with phosphorylase, albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor, and ⁇ -lactalbumin as the markers.
  • the molecular weight was determined by the gel filtration method with Hiprep 16/60 Sephacryl S-100 HR.
  • the column was equilibrated by flowing a 50 mM phosphate buffer having 0.15 M NaCl added thereto (pH 7.0) at a linear rate of about 30 cm/hour in a proportion of at least 3 (400 ml) to the gel volume.
  • a 1 ml portion of the sample enzyme solution was loaded on the column, and eluted with the same buffer as above at a linear rate of about 30 cm/hour to collect 2 ml fractions.
  • the excluded volume was determined for Blue Dextran 2000 with albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), ribonuclease A (13.7 kDa) as the standard proteins.
  • Isoelectric focusing was carried out with a Phast system ( Farmacia-Biotec Co.). IEF3-9 gel was employed, and the sample was loaded at the point on the gel at a distance of 3 cm from the anode. Electrophoresis was carried out under the condition of 2,000 V, 2.5 mA at 15°C and 410 Vh. The gel plate was stained by fixing with a 20% TCA solution at 20°C for 5 minutes, and washed with a rinsing and decoloring solution (30% methanol and 10% acetic acid) for 2 minutes. The plate was finally rinsed and decolored with a solution having 0.02% Coomassie Brilliant Blue R250 dissolved therein at 50°C for 10 minutes. A Broad pI Calibration kit (Farmacia-Biotec Co.) was used as a pI marker.
  • Azocasein was decomposed with the enzyme at various pHs.
  • Buffer solutions in the reaction mixture had a concentration of 67 mM and comprised an acetate buffer (pH
  • the relative activity of the enzyme at pH 7.5 as the optimal pH was maintained at a level of about 80% in the pH range from 6.0 to 10.0.
  • the enzyme was thus found to act over a considerably wide range centering around neutral pH.
  • the enzyme did not work quite satisfactory in the ranges of pH 5.5 or less or 10.5 or more, and it was inactivated at pH 12 to lost the protease activity.
  • the enzyme was examined in buffer having various pHs with an Econo-Pac (Biorad Co.).
  • Buffer solutions used had a concentration of 67 mM and comprised an acetate buffer (pH 4 - 5), KH 2 PO 4 -Na 2 HPO 4 (pH 6 - 8), glycine-NaOH (pH 9 - 10), and Na 2 HPO 4 -NaOH (pH 11 - 12), respectively.
  • Azocasein was decomposed with the enzyme at various pHs at a reaction temperature in the range from 0 to 70°C. Similar reaction was conducted for commercially available enzymes such as Subtilisin Carlsberg, V8 protease which was the protease derived from Staphylococcus aurcub V8, Sabinase (Novonordisc) and Alkalase (Novonordisc).
  • the enzyme had an optimal temperature at 40°C and was rapidly inactivated at an enzyme reaction temperature over the optimal temperature. It was also found that all of the commercially available proteases had an optimal temperature of 50°C or more and that the Kcat value of the present enzyme was higher than any of those of the comparative proteases in the range from 10 to 40°C.
  • the present enzyme was maintained at a temperature in the range from 20 to 50°C.
  • the variation of the activity with the passage of time is shown in Figure 14, in which ⁇ represents the variation at 20°C, O at 30°C, O at 40°C, and ⁇ at 50°C.
  • the enzyme was scarcely inactivated at 20°C or 30°C, but it is gradually inactivated at 40°C to about 60% of the protease activity at 20°C or 30°C after 1 hour and rapidly inactivated at 50°C to completeness in about 15 minutes.
  • Example 12 The present enzyme is considered to be unstable to heat, since the above temperatures are lower than the optimal temperature of the comparative proteases employed in the preceding experiment.
  • Example 12 The present enzyme is considered to be unstable to heat, since the above temperatures are lower than the optimal temperature of the comparative proteases employed in the preceding experiment.
  • Phenylmethylsulfonyl fluoride (PMSF) acting on serine protease, iodoacetamide (IAA) acting on cysteine protease, ethylenediaminetetraacetic acid (EDTA) acting on metal protease, o-phenanthroline, 2,2'-bipyridyl, and a citrate and an oxalate specifically acting on calcium were employed as the inhibitors. After various concentrations of the inhibitors were added to the enzyme reaction system, it was maintained at 20°C for 1 hour to examine the survived protease activity.
  • protease activity of the enzyme was not inhibited by PMSF or IAA, but noticeably inhibited by EDTA, 2,2'-bipyridyl, a citrate or an oxalate. It was found from these observations that the protease activity is metal ion dependent, and thus the present enzyme is a metal protease. It is also considered from the inhibition by a citrate or an oxalate that the protease activity depends on calcium.
  • Example 13 Example 13:
  • SDS and urea were used as the protein denaturing agents. After various concentrations of the protein denaturing agents were added, the enzyme reaction system was maintained at 20°C for 1 hour to examine the remaining protease activity.
  • the protease was inhibited by SDS even in quite a low concentration and completely inactivated to completeness with 0.25% of SDS.
  • the protease was not inhibited by urea in a concentration up to 2 M, but it was inhibited to about 40% by 3 M urea and completely inactivated by 4 M urea.
  • MgSO 4 were employed as the metal sources. After the metal salt was added to ensure that the final concentration was
  • the present enzyme was extensively inhibited by Ag + , Cu 2+ , Zn 2+ , Co 2+ , and Fe 2+ . Above all, when inhibited with Ag + , only 10% of the protease activity was survived.
  • the present enzyme was not inhibited by Mg 2+ or Ca 2+ at all.
  • Casein (Hammarsten), dimethylcasein, gelatine, hemoglobin, bovine serum albumin, and ribonuclease were employed as the substrate proteins to measure the proteolytic activity by the modified Anson method. Azocasein and azoalbumin were used as the azoprotein modifying proteins.
  • the enzyme of the present invention decomposes well high molecular proteins and denaturated proteins such as casein and dimethylcasein, and also decomposed gelatine as the collagen denaturated protein in about 50% on the basis of casein.
  • the enzyme scarcely acted on the other natural proteins, and it did not act particularly on ribonuclease.
  • the Lineweaver-Burk plots at various temperatures from 5 to 40°C were obtained with solutions containing 0.05 - 1% of casein as the substrate.
  • the plots are shown in Figure 17, in which the upper graph (A) illustrates the change in 1/v value in the range of 0 to 2.0, and the lower graph (B) illustrates the change in 1/v value in the range of 0 to 0.2.
  • ⁇ represents the plots at 5°C,0 at 10°C, O at 20°C, ⁇ at 30°C, and ⁇ at 40°C
  • the kinetic constant of the enzyme reaction was obtained from the Lineweaver-Burk plots as shown in the table below.
  • the enzyme exhibited a reaction rate of the Michaelis-Mentne type for the concentration of casein.
  • the Km value decreased and the Vmax value increased with the increase of temperature.
  • the enzyme activity was inhibited in a high concentration of casein at 5°C and 10°C. in general, the enzyme tends to be inhibited in an excessive concentration of a substrate.
  • the enzyme did not inhibited in 1% of casein at an increased reaction temperature. This is considered due to the increase of the Kcat value by the approach to the optimal temperature and little inhibition by the decomposed product of casein.
  • the inhibition mode by temperature is of a mixed type. That is, it is considered that the influence of temperature is a non-competitive or uncompetitive inhibition, and the influence of the decomposed product of casein is a competitive inhibition.

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Abstract

Nouvelle protéase psychrophile et micro-organisme ayant la capacité de produire ladite protéase. La protéase agit sur la caséine et la diméthylcaséine et la décompose, sans agir sur la ribonucléase, et présente une température optimale d'environ 40 °C. Dans des conditions de stockage à un pH 7 pendant 1 heure, elle est rarement inactivée à une température allant jusqu'à 30 °C et perd environ 40 % de son activité à 40 °C. A 50 °C, la protéase est rapidement inactivée et totalement inactivée en approximativement 15 minutes. Flavobacterium balustinum qui est capable de produire ladite protéase est également décrit.
PCT/US1996/002115 1995-02-17 1996-02-16 Protease active au froid et bacteries actives au froid WO1996025489A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
MX9706272A MX9706272A (es) 1995-02-17 1996-02-16 Proteasa psicrofilica y bacterias psicrofilicas.
EP96906484A EP0871719A1 (fr) 1995-02-17 1996-02-16 Protease active au froid et bacteries actives au froid
AU49848/96A AU4984896A (en) 1995-02-17 1996-02-16 Psychrophilic protease and psychrophilic bacteria
BR9607452A BR9607452A (pt) 1995-02-17 1996-02-16 Bactérias psicrofílicas e protease psicrofílica

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP7/29904 1995-02-17
JP2990495 1995-02-17

Publications (1)

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WO1996025489A1 true WO1996025489A1 (fr) 1996-08-22

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Country Link
EP (1) EP0871719A1 (fr)
AU (1) AU4984896A (fr)
MX (1) MX9706272A (fr)
WO (1) WO1996025489A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0876500A1 (fr) * 1996-01-26 1998-11-11 The Procter & Gamble Company Protease cp70 active a froid
WO1999025848A1 (fr) * 1997-11-14 1999-05-27 The Procter & Gamble Company Polynucleotide codant une protease active froide cp70
WO2000005352A1 (fr) * 1998-07-24 2000-02-03 The Procter & Gamble Company Pseudomonas sp. gk-15 et leur protease
DE102007033104A1 (de) 2007-07-13 2009-01-15 Henkel Ag & Co. Kgaa Mittel enthaltend Proteasen aus Stenotrophomonas maltophilia
DE102007032111A1 (de) 2007-07-09 2009-01-15 Henkel Ag & Co. Kgaa Neue Proteasen und Wasch- und Reinigungsmittel enthaltend diese Proteasen
DE102007036756A1 (de) 2007-08-03 2009-02-05 Henkel Ag & Co. Kgaa Neue Proteasen und Wasch- und Reinigungsmittel, enthaltend diese neuen Proteasen
WO2013024143A1 (fr) 2011-08-18 2013-02-21 Unilever Plc Système enzymatique

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
ANNALS AGRICULTURAL SCIENCE, 1991, Volume 36, Number 2, ISMAIL et al., "Production of Proteases by Some Dairy Psychrotrophic Bacteria", pages 525-534. *
CULTURED DAIRY PRODUCTS JOURNAL, November 1991, Volume 26, Number 4, MAGDOUB et al., "Utilization of Psychrotrophic Bacterial Protease for Acceleration of Flavor Development in Ras Cheese Slurry", pages 24-28. *
FEMS MICROBIOLOGY LETTERS, 1991, Volume 79, MARGESIN et al., "Characterization of a Metalloprotease from Psychrophilic Xanthomonas Maltophila", pages 257-261. *
INDUSTRIES ALIMENTAIRES ET AGRICOLES, May 1986, DOUSSET et al., "Action des Proteases des Bacteries Psychrotrophes du Lait Sur la Qualite des Produits Laitiers", pages 325-333. *
JOURNAL OF APPLIED BACTERIOLOGY, 1985, Volume 59, MILLIERE et al., "An Inventory of Peptide Hydrolases and Arylamidases in Flavobacterium II b", pages 459-468. *
JOURNAL OF DAIRY SCIENCE, 1991, Volume 74, Suppl. 1, MAGDOUB et al., "Flavor Development Acceleration in Ras Cheese Slurry Using Bacterial Protease", page 127, Abstract D139. *
JOURNAL OF GENERAL APPLIED MICROBIOLOGY, 1992, Volume 38, MARGESIN et al., "A Comparison of Extracellular Proteases from Three Psychrotrophic Strains of Pseudomonas Fluorescens", pages 209-225. *
MILCHWISSENSCHAFT, October 1986, Volume 41, Number 10, JOOSTE et al., "The Significance of Flavobacteria as Proteolytic Psychrotrophs in Milk", pages 618-621. *
REVISTA ASOCIACION ARGENTINA DE MICROBIOLOGIA, 1976, Volume 8, Number 1, JUAN et al., "Produccion de Proteasa Extracellular Por Una Bacteria Criofila de Agua Dulce", pages 8-13. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0876500A1 (fr) * 1996-01-26 1998-11-11 The Procter & Gamble Company Protease cp70 active a froid
EP0876500A4 (fr) * 1996-01-26 2002-11-27 Procter & Gamble Protease cp70 active a froid
WO1999025848A1 (fr) * 1997-11-14 1999-05-27 The Procter & Gamble Company Polynucleotide codant une protease active froide cp70
WO2000005352A1 (fr) * 1998-07-24 2000-02-03 The Procter & Gamble Company Pseudomonas sp. gk-15 et leur protease
DE102007032111A1 (de) 2007-07-09 2009-01-15 Henkel Ag & Co. Kgaa Neue Proteasen und Wasch- und Reinigungsmittel enthaltend diese Proteasen
DE102007033104A1 (de) 2007-07-13 2009-01-15 Henkel Ag & Co. Kgaa Mittel enthaltend Proteasen aus Stenotrophomonas maltophilia
DE102007036756A1 (de) 2007-08-03 2009-02-05 Henkel Ag & Co. Kgaa Neue Proteasen und Wasch- und Reinigungsmittel, enthaltend diese neuen Proteasen
WO2013024143A1 (fr) 2011-08-18 2013-02-21 Unilever Plc Système enzymatique

Also Published As

Publication number Publication date
MX9706272A (es) 1997-11-29
AU4984896A (en) 1996-09-04
EP0871719A1 (fr) 1998-10-21

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