Classifications

• • 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
  • C12R2001/64—Xanthomonas

Definitions

• the present invention relates to a protease having keratinolytic activity under low temperatures and alkaline conditions, its use and microorganisms which produce the protease.
• Keratinases have conventionally been known to be proteases that degrade insoluble proteins such as keratins. However, all keratinases are also highly active in degrading water soluble proteins such as caseins; and, insofar as the present inventor know, there has been no report on proteases which act specifically on keratins.
• psychrophilic bacteria have long been known, and their presence in low temperature environments has been widely recognized. They are found in soil, fish and shell fish, and dairy products, as well as in environments artificially controlled at low temperatures. Research on psychrophilic bacteria has progressed in the field of food microbiology; however, it has been based on their phylogeny and is not related to their biological features or functions.
• Enzymes obtained from psychrophilic bacteria are expected to be low temperature enzymes, which are most active at cold temperatures. Such low temperature enzymes are expected to be useful as an additive in laundry detergents which would clean effectively in a cold water. Another expected use is in the food industry to improve the quality of food products which are difficult to process at high temperatures. Furthermore, the study of enzymes derived from psychrophilic bacteria will be significant in order to elucidate the physiological functions of psychrophilic bacteria and their mechanism to adapt under low temperatures and alkaline conditions.
• the present inventor isolated and purified a protease having a keratinolytic activity from the culture supernatant of Xanthomonas maltophiiia EK3 and found that this protease has enzyme activity under low temperatures and alkaline conditions.
• the present invention is based on this finding.
• Action and substrate specificity Degrades caseins and keratins derived from human and animal hair.
• the keratinolytic activity is higher than the casein degrading activity.
• pH stability 100% of the activity is retained at pH 10.0 and about 70% is retained at pH 6-9 when maintained at 30°C for 1 hour.
• a method for the production of the protease having the keratinolytic activity according to the present invention comprises culturing the microorganism that produces this protease and obtaining this protease from the culture medium.
• microorganism according to the present invention is Xanthomonas maltophiiia EK3, which produces the protease having the keratinolytic activity.
• Figure 1 shows the results of gel filtration chromatography carried out in Example 2.
• Figure 2 shows the effect of pH on the activity of the enzyme according to the present invention. ⁇ :MES, 0:MOPS, O:TAPS, ⁇ :CHES, • :CAPS.
• Figure 3 shows the pH stability of the enzyme according to the present invention. ⁇ Sodium citrate(pH5-7), 0:Tris-HCI(pH7-9), O:Glycine- NaOH(pH9-11).
• Figure 4 shows the effect of temperature on the activity of the enzyme according to the present invention.
• Figure 5 shows the temperature stability of the enzyme according to the present invention. :10°C, 0:20°C, O:30°C, ⁇ :50°C and •:60°C.
• Figure 6 shows the growth of Xanthomonas maltophiiia EK3 and the activity of the produced protease, at different culture temperatures.
• the protease having the keratinolytic activity according to the present invention can be produced using microorganisms. Any microorganism which belongs to genus Xanthomonas and produces the protease having the properties can be used for the production of the enzyme.
• a preferable embodiment of a microorganism capable of producing the protease according to the present invention is Xanthomonas maltophiiia EK3. This strain is a microorganism isolated by the present inventor from soil in an elephant house at a zoo, and is deposited with the accession number of FERM BP-5806 at the National Institute of Bioscience and Human-Technology of Industrial Science & Technology dated January 31 , 1997.
• the Xanthomonas maltophiiia EK3 according to the present invention has the following microbiological properties:
• the strain has mobility, and a spherical or spheroidal shape.
• the strain grows on an agar medium and a liquid medium, and shows white color.
• Proteases are secreted extracellularly from cells grown either at 20°C or 30°C.
• Aerobiosis/anaerobiosis The stain is considered to be facultatively anaerobic as judged from biochemical tests.
• Gram stain The strain is recognized to be a gram-negative bacteria according to Gram stain.
• Biochemical features Primary biochemical features of Xanthomonas maltophiiia EK3 are shown in Table 1.
• Oxidation-fermentation test Nitrate Tryptophane Glucose(covered with oil) Arginine Dihydrolase(covered with oil)
• the isolated bacteria are identified to be a strain of Xanthomonas maltophiiia (99.9%) as judged from the properties given above. The result were given by the use of Profile Index (API 20NE, NIHON Bio Merieux Biotech Co., Ltd.) Culture of microorganisms
• Bacterial cells of the strain to be used in the present invention may be cultured using either a liquid medium or solid medium. Generally, a culture with shaking or culture with stirring and aeration in a liquid medium is used.
• the medium for culturing the microorganism may be any medium in which the cells can grow and produce the protease.
• usable carbon sources are, for example, glucose, trehalose, fructose, maltose, sucrose, starch, and malto-oligosaccharides.
• Usable nitrogen sources are, for example, peptone, yeast extract, malt extract, meat extract, soybean powder, cotton seed powder, corn steep liquor, various amino acids and salts thereof, and nitrates.
• a synthetic medium or natural medium which additionally contains an appropriate amount of minerals and inorganic salts, such as magnesium phosphate, calcium, sodium, potassium, iron and manganese and, if necessary, other nutrients, can be used.
• Culture conditions such as pH of a medium and incubation temperature can be appropriately changed within a range in which the protease can be produced. However, when a culture with shaking or culture with stirring and aeration in a liquid medium is used, culturing at pH 10 and at 20°C is appropriate.
• protease usable in the present invention is present in the culture medium, on the cell wall and also inside the cell. Further, this protease can be used either in the form of crude enzyme secreted intracellularly or extracellularly, or extracted and purified enzyme, or directly as the cell form. Alternatively, these enzymes can be used in an immobilized form on a carrier. Extraction and purification of enzyme
• Extraction and purification of the protease of the present invention from the fluid cell culture can be carried out using known purification procedures alone or in combination.
• the protease of the present invention is primarily secreted extracellularly, i.e., into the culture medium; therefore the crude enzyme fraction can be easily obtained by removing cells, for example, by filtration or centrifugation.
• preferable purification methods include salting out method using ammonium sulfate or the like, precipitation method using organic solvents (e.g., methanol, ethanol and acetone), absorption method using raw starch, ultra filtration, gel filtration chromatography, ion exchange chromatography and various kinds of chromatography. Embodiments of preferable purification methods will be illustrated in Examples hereinafter.
• Properties of enzyme The protease according to the present invention has the following properties:
• the protease degrades caseins and keratins derived from human and animal hair in particular.
• the keratinolytic activity is higher than the casein degrading activity.
• the protease of this invention is specific to keratins.
• Optimum temperature is 50°C at pH 10.5. About 40% of the maximum activity is retained at 40°C and about 30% of the maximum activity is retained at 20°C and 30°C.
• the protease has a molecular weight of about 30 kDa as determined by gel chromatography.
• the protease according to the present invention has a keratinolytic activity under low temperature and alkaline conditions. Accordingly, degradation of keratins, which are insoluble proteins, can be carried out at low temperatures.
• the addition of the protease to a laundry detergent will provide a detergent which can degrade insoluble keratins.
• This detergent composition can be formulated according to a conventional method, except that the protease of the present invention is added. That is, it can be produced in combination with customary detergent compositions such as surfactants for cleaning purpose, bleaching agents and builders.
• Quantitative determinations of proteins hereinafter were carried out by a pigment binding method using BioRad Protein Assay (BioRad) unless otherwise mentioned. Further, detection of proteins in chromatography was carried out by measuring absorption at a wave length of ultraviolet range, i.e., at 280 nm.
• Protease activity was measured according to the following method:
• a sample enzyme solution (0.2 ml) was added to 0.3 ml of 50 mM glycine-NaOH buffer (pH 10.5) containing a 1% (w/v) substrate (e.g., keratins) solution and the mixture was maintained at 30°C for 60 minutes. Then, the enzyme reaction was stopped by adding 1 ml of a trichloroacetic acid solution (0.11 M trichloroacetic acid, 0.22 M sodium acetate and 0.33 M acetic acid).
• a trichloroacetic acid solution (0.11 M trichloroacetic acid, 0.22 M sodium acetate and 0.33 M acetic acid).
• reaction mixture was centrifuged (at 10,000 rpm, at room temperature for 10 minutes), 0.5 ml of a 2% sodium carbonate solution (0.001% copper sulfate) was added to 50 ⁇ l of the resultant supernatant and the mixture was allowed to stand for 30 minutes.
• 50 ⁇ l of a phenol reagent solution was added after diluting in two times with distilled water. After being allowed to stand at room temperature for 1 hour, the mixture was subjected to the measurement of absorption at 660 nm.
• ISHIKAWA Prefectural Zoo was suspended in a physiological saline, and the supernatant was used as a stock solution. Furthermore, a 10 dilute solution of this stock solution was prepared therefrom. The stock solution and the 10 dilute solution were each sprayed on an agar plate culture medium for screening (25 g/liter of keratin, 1.0 g/liter of K2HSO4, 0.1 g/liter of NH4NO3, 0.1 g/liter of MgSO4, 0.1 g/liter of NaCI, 0.1 g/liter of CaCL2, 0.1 g/liter of FeCl2, 0.1 g/liter of EDTA and 15 g/liter of agar), and then cultured at 5, 10, and 20°C for 7 days. Among colonies which had grown on the agar medium, well grown colonies were selected, subcultured, and then inoculated into a retention culture medium.
• the isolated microorganisms were inoculated into the above-mentioned agar medium for screening, and then cultured at 20°C for
• the culture medium and the like were sterilized with high-pressure
• the culture medium obtained in Example 1 was centrifuged
• the crude enzyme solution was introduced into the column at a linear velocity of 100 cm/hr. Elution was carried out at a linear velocity of 100 cm/hr by the use of 3 liters of each of 20 mM tris- HCI buffer solutions (pH 9.0) containing 0.2 M, 0.4 M and 0.6 M NaCI, respectively, and only portions in which a protein was detected by a UV meter were fractionated.
• Ammonium sulfate was added to the thus obtained fraction under ice cooling so that the fraction might be saturated as much as 80% with ammonium sulfate. After the solution was slowly stirred at 4°C overnight in a cold chamber, centhfugation (18,000 g, 4°C, 30 minutes) was carried out to precipitate an enzyme, thereby obtaining a saturated fraction. The amount of ammonium sulfate to be added was an amount necessary to achieve a saturated concentration at 25°C.
• Degrading reaction of azocasein (1%) was carried out at various pH ranges and at 30°C for 1 hour using the enzyme purified in Example 2.
• Compositions of buffer solutions used for the reaction were 100 mM each of MES (2-morpholinoethanesulfonic acid hydrate) buffer (pH 5.5-6.5), MOPS (3-morpholinopropanesulfonic acid) buffer (pH 6.5-8.0), TAPS (N- tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) buffer (pH 8.0-9.0), CHES (N-cyclohexyl-2-aminoethanesulfonic acid) buffer (pH 9.0-10.0), CAPS (N-cyclohexyl-3-aminopropanesulfonic acid) buffer (pH 10.0-11.0), and glycine-NaCI-NaOH buffer (pH 11.0-13.0). Results are shown in Figure 2.
• Example 2 The enzyme purified in Example 2 was maintained in buffer solutions, i.e., 20 mM each of sodium citrate buffers (pH 5-7), tris-HCI buffer solutions (pH 7-9) and glycine-NaOH (pH 9-11), at 30°C for 1 hour, after which remaining protease activity against azocasein (1%) was measured.
• buffer solutions i.e., 20 mM each of sodium citrate buffers (pH 5-7), tris-HCI buffer solutions (pH 7-9) and glycine-NaOH (pH 9-11), at 30°C for 1 hour, after which remaining protease activity against azocasein (1%) was measured.
• Azocasein (1%) degrading reaction was carried out using the enzyme purified in Example 2 in a 50 mM glycine-NaOH buffer solution (pH 10.5) at various temperatures for 1 hour. The reaction temperatures ranged from 10 °C to 70°C. Results are shown in Figure 4.
• Optimum temperature for the reaction of the enzyme according to the present invention was 50°C at pH 10.5.
• the activity obtained was more than 30% at 30°C and about 40% at 40 °C, referring the activity obtained at 50°C as 100%.
• Example 6 Temperature stability of enzyme
• Example 2 The enzyme purified in Example 2 was maintained at various temperatures between 10°C and 60°C for 1 hour. The change of the resulting activity against azocasein (1%) with the lapse of time is shown in Figure 5. In the Figure, :10°C, 0: 20°C, O:30°C, ⁇ :50°C and •:60°C.
• Casein, hemoglobin, and albumin were used as water soluble substrate proteins. Keratins (derived from human and animal hair), collagen, and elastin were used as water insoluble or slightly water soluble substrate proteins. Activity to degrade these substrate proteins was determined using the phenol reagent.
• Keratin (derived from human hair)*2 190
• reaction temperature 30°C; reaction pH 10.5; reaction time: 60 minutes. All of the substrate concentrations were 1%.
• the enzyme according to the invention specifically degrades keratins.
• the activity on keratin derived from animal hair was 2.75 times higher than that on casein.

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• 1997
  • 1997-03-13 JP JP9059491A patent/JPH10248564A/ja not_active Withdrawn
• 1998
  • 1998-03-12 WO PCT/IB1998/000318 patent/WO1998040473A1/en active Application Filing
  • 1998-03-12 AU AU61105/98A patent/AU6110598A/en not_active Abandoned
  • 1998-03-13 AR ARP980101139A patent/AR011983A1/es unknown

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