US3905869A - Bacillopeptidase C, a new alkaline protease and its production by cultivating bacillus bacteria - Google Patents

Bacillopeptidase C, a new alkaline protease and its production by cultivating bacillus bacteria Download PDF

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US3905869A
US3905869A US392426A US39242673A US3905869A US 3905869 A US3905869 A US 3905869A US 392426 A US392426 A US 392426A US 39242673 A US39242673 A US 39242673A US 3905869 A US3905869 A US 3905869A
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bacillopeptidase
indicated
process according
enzyme
production medium
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Hidemasa Hidaka
Kenji Yoshida
Uichi Shibata
Yasukatsu Yuda
Tomizo Niwa
Hitoshi Goi
Shinji Miyado
Yujiro Yamada
Takemi Koeda
Kazuo Saito
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Meiji Seika Kaisha Ltd
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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
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/832—Bacillus

Definitions

  • Kenji Yoshida, Kawasaki Uichi Shibata, Tokyo; Yasukatsu Yuda, Yokohama; Tomizo Niwa, Kawasaki; Hitoshi Goi, Kawasaki; Shinji Miyado, Yokohama; Yujiro Yamada, Yokohama; Takemi Koeda, Yokohama; Kazuo Saito, Fujisawa, all of Japan Assignee: Meiji Seika Co., Ltd., Japan Filed: Aug. 29, 1973 Appl. No.: 392,426
  • ABSTRACT A novel protease named Bacillopeptidase C and hav ing strong anti-inflammatory activity is produced by Bacillus sp. No. 794 when this microorganism is cultivated.
  • the Bacillopeptidase C differs from other alkaline proteases in isoelectric point and behaviors to inhibitors.
  • This invention relates" to a novel and strong antiinflammatory alkaline protease named Bacillopeptidase C, and further relates to the fermentative production of this anti-inflammatory enzyme.
  • the present inventors now'satisfied with the functions and the productivity of the known alkaline proteases, have 'made extensive searches for proteaseproducing strains in the natural world and have confirmed that a strain of Bacillaceae produces a protease quite different in characteristics from the known alkaline proteases.
  • the present invention was accomplished on the basis of this observation.
  • the alkaline protease obtained in the present invention has physical and chemical'properties intrinsically different from those of the known alkaline proteases and displays exceedingly high anti-inflammatory and proteolytic activities. Having pronounced difference from the known alkaline proteases, the protease obtained according to the present invention has been characterized as a novel protease and thus nominated as Bacillopeptidase C. i
  • DFP diisopropylfluorophos-- phatc
  • the novel protease of the present invention Bacillopeptidase C
  • Bacillopeptidase C Bacillopeptidase C
  • the strain which produces the novel protease Bacillopeptidase C according to the present invention is the one newly isolated from the soil by the present inven tors and is deposited in the Fermentation Research Institute of the Agency of Industrial Science and 'Technology, Chiba, Japan, under PERM-P No. 1522, and in the American Type Culture Collection, Rockville, Md, U.S.A. under ATCC. No. 21964. This strain is characterized as follows:
  • Vegetative rods 0.7 to 0.9 by 2.5 to 3.5 microns. Motile by means of peritrichous flagella; Gram variable; spores, 1.3 to 1.6 microns spherical, terminal; optimum growth at pH 7 to 9; good growth at 28 to 40C; no growth at 50C; aerobic;
  • Colonies on meat extract agar Smooth surface with lustre, opaque, and light yellowish brown in color.
  • Glucose nutrient agar slants Growth equivalent to or better than nutrient agar slants;
  • Meat extractbroth containing NaCl Growth in 5 percent NaCl, no growth in 10 percent NaCl;
  • Meat extract broth Uniformly turbid, no bacterial film formed
  • Meat extract gelatine stab Strati form liquefaction at 22C within one week;
  • Glucose asparagine agar No growth
  • pH of glucose broth pH 7.8 8.0
  • the strain used in the present invention has been named as a new strain, Bacillus sp. No. 794, belonging to Bacillaceae.
  • the novel protease Bacillopeptidase C, is produced by cultivating in a medium a microorganism which be longs to Bacillaceae and is represented by Bacillus sp. No. 794.
  • the cultivation is performed, as a rule, in the usual manner adopted in the cultivation of general microorganisms, by either solid cultivation or liquid cultivation.
  • liquid cultivation is preferable.
  • Utilization cultivation medium are synthetic, semi-synthetic and natural media.
  • carbon source there are used glucose, sucrose, maltose, gluconic acid, starch, hydroly sates of starch and other carbohydrates, and as nitrogen source peptone, meat extract, corn steep liquor, Soybean meal, dry yeast, gluten, casein degradation product's, urea, ammonium sulfate and ammonium nitrate, singly or two or more in combination.
  • carbonate, sulfate or hydrochloride of magnesium, manganese or calcium and further sodium chloride or defoaming agent may be added in a suitable amount.
  • the preferred cultivation temperature is 25-40C.
  • the cultivation time depends upon the cultivation temperature, cultivation volume and cultivation system, but the production of the novel protease, Baciilopeptidasc C, is generally completed within 40 100 hours. Isolation of the produced protease from the medium can be carried out according to any of the conventional methods for the purification of enzymes.
  • the present enzyme is active at pH 6 l l on a milk casein substrate.
  • the optimum pH is 9 93, indicating that this enzyme is an alkaline protease.
  • One enzyme activity unit was defined as the enzyme quantity capable of liberating peptide to such an amount that is equivalent in the said optical density to l ,u.g of tyrosin in a reaction mixture at pH 9.0, 40C in 1 minute.
  • FIG. I The influence of pH value upon the activity of the present enzyme on a milk casein substrate is shown in FIG. I.
  • the optimum pH is 9.3, and the activity is reduced to half at pH 6.5 7, or 10.5 11.
  • FIG. 2 shows the influence of various pH values upon the stability of the enzyme, wherein the residual activity after treatment with M/ CAPS buffer solution (CAPS: Dotite reagent, cyclohexyl-aminopropanesulfonic acid) at a variety of pH values at 40C for 60 minutes was determined.
  • CAPS buffer solution CAS: Dotite reagent, cyclohexyl-aminopropanesulfonic acid
  • FIG. 3 of the drawings shows the effect of temperature on the enzyme activity.
  • the optimum temperature for the activity of the present enzyme is around 45C, and more than 80% of activity is detectable in the range from 35C to C.
  • the enzyme is quite stable at below 40C and displays 50% remaining activity at C.
  • the enzyme is substantially inactivated at a pH value of below 6 and above l2, and completely inactivated through l-hour treatment at 40C at pH 4 or 12. It is also considerably unstable at pH 7 and 60C, and com- 4s pletcly inactivated at pH 7 and C, and likewise at pH 9.5 and C.
  • the enzyme of the present invention is clearly a novel one; it is in one way a serine enzyme having serine at its active site as alkaline proteases do but in the other way obviously influenced by EDTA, indicating the important roles of bivalent metals in the enzyme action.
  • T enzyme and A enzyme were used as representatives of neutral protease and alkaline protease, respectively.
  • T enzyme Thermolysin (Daiwa Kasei Kabushiki Kaisha), neutral protease, crystal A enzyme: Alkaline protease ll (Seikagaku Kogyo),
  • novel protease Bacillopeptidase C
  • Bacillopeptidase C is useful not only as an anti'inflammatory agent but also as a digestive, softener for leathers and edible meats, detergent, etc., by virtue of its high proteolitic activity.
  • Each of four Donryu rats (weighed 130-150 g) was given intraperitoneally 0.5 mg per rat of each enzyme sample solution, and after 1 hour, 0.05 ml of an 1% carrageenin solution was given subcutaneously to the sole of one hind limb of each rat. After 3 hours, the extent of edema formed was measured by a slide caliper to assess the effect of each enzyme.
  • Bacillopeptidase C has the above-mentioned properties that distinctly differ from the known proteases. Unlike the known proteases which are completely inactivated by DFP but not by ElDTA, the enzyme of the present invention is completely affected by both DP? and EDTA. In contrast to the known alkaline proteases having an isoelectric point of pH 8 or higher, the enzyme of the present invention has an isoelectric point of pH 6.0 and exibits far better anti-inflammatory activity.
  • the Bacillopeptidase C is low in toxicity. Acute toxicity of the Bacillopeptidase C was tested by oral administration.
  • the BacillopeptidaseC of this invention is, therefore, used as an effective anti-inflammatory agent.
  • 2.5 10 mg of the Bacillopeptidase C may be formulated into tablets or capsule with starch and/or lactose, and orally administered 2 to 3 of it for 3 to 4 times a day.
  • EXAMPLE 1 Twenty liters of a medium containing 1% of starch, 2% of soybean oil cake, 1% of wheat bran, 0.5% of potassium phosphate, 0.5% of calcium carbonate, and 0.05% of magnesium sulfate was charged into a jar fermenter of 30 liter volume and sterilized at 120C for 20 minutes under pressure. The medium was then inoculated with 200 ml of a seed culture of the Bacillus No. 794 (identified as ATCC No. 21964 or PERM-P No. 1522), which had been cultivated separately in a neutrient broth. Cultivation was performed with stirring at 300 rpm at 30C for 3 days under 100% aeration.
  • the cultures were subjected to a centrifugal separation to remove the bacterium and there was obtained liters of the filtrate.
  • the filtrate was then concentrated to 3 liters under reduced pressure and then its pH value was adjusted to 8.0 by addition of 27 g of calcium acetate.
  • the precipitate formed was removed by filtration and the filtrate was saturated with ammonium sulfate up to 0.6 saturation and subjected to centrifugal separation to recover the precipitate formed. 80% of the activity at the completion of the fermentation was thus recovered.
  • the precipitate was again dissolved in 300 ml of a solution containing M/500 of calcium acetate in M/200 of Tris hydrochloric acid buffer solution of pH 7.5 and the solution was then dialyzed against said buffer solution.
  • the resulting enzyme solution was adsorbed on a column of 3 liters of DEAE Sephadex A50 equilibrated with said buffer solution and washed with about 10 liters of similar buffer solution, and then with the same buffer solution containing 0.1 NaCl whereupon the enzyme of the invention was eluted. In 3 liters of the eluate, about 70% activity was recovered. Re-chromatography of the eluate under the same condition and concentration to its 1/10 volume gave 0.8 g of crystal of the Bacillopeptidase C.
  • EXAMPLE 2 Two liters of the culture filtrate obtained as described in Example 1 was subjected without being concentrated to a salting-out operation with ammonium sulfate, then to dialysis and finally to chromatography using DEAE-Sephadex. The eluate obtained by a concentration gradient elution method was concentrated to its 1 /8 volume to obtain 60 mg of crystal of the Bacillopeptidase C.
  • FIG. 1 is a graph showing the active pH range of Bacillopeptidase C
  • FIG. 2 a graph showing the stable pH range thereof
  • FIG. 3 a graph showing the active temperature, range thereof
  • FIG. 4 a graph showing the stable temperature range
  • FIG. 5 a graph showing the isolation condition when using a 8 ml column of DEAS Sephadex A-50.
  • Bacillopeptidase C which is an effective anti-inflammatory agent, having the following properties:
  • the production medium contains at least one member selected from the group consisting of glucose, sucrose, maltose, gluconic acid, starch, hydrolysates of starch and other carbohydrates.
  • the production medium contains at least one member selected from the group consisting of peptonc, meat extract, corn steep liquor, soybean meal, dry yeast, gluten, casein degradation products, urea, ammonium sulfate and ammonium nitrate.
  • the production medium contains a. a salt of magnesium, manganese or calcium consisting of a carbonate, sulfate or hydrochloride;
  • Bacillopeptidase C which is an effective anti-inflammatory agent, and which is produced by a process of (a) cultivating Bacillus sp. No. 794 (ATCC 21964 or FERM-P No. 1522) in a production medium until the said enzyme is substantially accumulated in the medium, and (b) recovering the resulting Bacillopeptidase C from the medium and which has the following properties:

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US392426A 1972-09-02 1973-08-29 Bacillopeptidase C, a new alkaline protease and its production by cultivating bacillus bacteria Expired - Lifetime US3905869A (en)

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4480037A (en) * 1982-02-08 1984-10-30 Showa Denko Kabushiki Kaisha Alkaline protease and preparation method thereof
WO1992017579A1 (en) * 1991-03-29 1992-10-15 Genencor International, Inc. Alkaline protease 3733, its production and use in cleaning contact lens
WO1992017576A1 (en) * 1991-04-03 1992-10-15 Novo Nordisk A/S Novel proteases
EP0277216B1 (en) * 1986-08-14 1993-08-11 Novo Nordisk A/S Alkaline protease derived from bacilles production and use thereof
US5308761A (en) * 1992-09-11 1994-05-03 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Process for acetylating seaweed alginate with pseudomonas syringae subsp. phaseoliocola
US5346822A (en) * 1992-06-04 1994-09-13 Solvay Enzymes Gmbh & Co. Kg Alkaline proteases from Bacillus pumilus
US5358865A (en) * 1990-10-12 1994-10-25 Novo Nordisk A/S Alkaline protease from Bacillus J 20
US5362414A (en) * 1991-04-03 1994-11-08 Novo Nordisk A/S Proteases
WO1996006636A1 (en) * 1994-08-26 1996-03-07 Wilson Trafton Crandall Topical anti-inflammatory composition and method
US5981255A (en) * 1995-11-02 1999-11-09 Novo Nordisk A/S Alkaline protease, process for the production thereof, use thereof, and microorganism producing the same
WO2001012795A1 (en) * 1999-08-12 2001-02-22 National Enzyme Company COMPOSITION AND METHOD FOR TREATING DISEASE BY INCREASING ACTIVATED α2 MACROGLOBULIN IN THE BLOOD AND EXTRAVASCULAR TISSUE
US6333057B1 (en) 1995-07-03 2001-12-25 Wilson T. Crandall Composition and method for topical treatment of androgenic alopecia
US20080081035A1 (en) * 2006-10-03 2008-04-03 National Enzyme Company Therapeutic protease compositions
US7998476B2 (en) 2006-11-22 2011-08-16 Standard Biologics, Inc. Method of treatment using Aspergillus oryzae protease

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5148489A (en) * 1974-10-25 1976-04-26 Ajinomoto Kk Biseibutsu nyoru tanpakushitsuno seizoho
GB1519148A (en) * 1974-11-19 1978-07-26 Gist Brocades Nv Compositions of matter
JPS58189122A (ja) * 1982-04-30 1983-11-04 Kaken Pharmaceut Co Ltd 高脂血症予防治療剤

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3576719A (en) * 1968-04-23 1971-04-27 Squibb & Sons Inc Alkaline proteinase
US3684658A (en) * 1969-04-10 1972-08-15 Astra Laekemedel Ab Enzymes and process for their preparation
US3813319A (en) * 1971-03-23 1974-05-28 Sir Soc Italiana Resine Spa Process for the manufacture of proteases

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BE626101A (lm) *
GB1133579A (en) * 1966-03-09 1968-11-13 Shionogi & Co Process for preparing proteases and enzymatic composition prepared thereby
FR1585121A (lm) * 1968-07-24 1970-01-09
FR2129947A1 (en) * 1971-03-23 1972-11-03 Amano Pharma Co Ltd Anti-inflammatory protease prepn - from aspergillus

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3576719A (en) * 1968-04-23 1971-04-27 Squibb & Sons Inc Alkaline proteinase
US3684658A (en) * 1969-04-10 1972-08-15 Astra Laekemedel Ab Enzymes and process for their preparation
US3813319A (en) * 1971-03-23 1974-05-28 Sir Soc Italiana Resine Spa Process for the manufacture of proteases

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4480037A (en) * 1982-02-08 1984-10-30 Showa Denko Kabushiki Kaisha Alkaline protease and preparation method thereof
EP0277216B1 (en) * 1986-08-14 1993-08-11 Novo Nordisk A/S Alkaline protease derived from bacilles production and use thereof
US5358865A (en) * 1990-10-12 1994-10-25 Novo Nordisk A/S Alkaline protease from Bacillus J 20
WO1992017579A1 (en) * 1991-03-29 1992-10-15 Genencor International, Inc. Alkaline protease 3733, its production and use in cleaning contact lens
WO1992017576A1 (en) * 1991-04-03 1992-10-15 Novo Nordisk A/S Novel proteases
US5650315A (en) * 1991-04-03 1997-07-22 Novo Nordisk A/S Alkaline proteases obtainable from Bacillus sp. JA16-38A
US5362414A (en) * 1991-04-03 1994-11-08 Novo Nordisk A/S Proteases
US5346822A (en) * 1992-06-04 1994-09-13 Solvay Enzymes Gmbh & Co. Kg Alkaline proteases from Bacillus pumilus
US5444160A (en) * 1992-09-11 1995-08-22 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Acetylated alginates, and method for producing acetylated alginates
US5308761A (en) * 1992-09-11 1994-05-03 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Process for acetylating seaweed alginate with pseudomonas syringae subsp. phaseoliocola
WO1996006636A1 (en) * 1994-08-26 1996-03-07 Wilson Trafton Crandall Topical anti-inflammatory composition and method
US5560910A (en) * 1994-08-26 1996-10-01 Crandall; Wilson T. Topical anti-inflammatory composition and method
US6333057B1 (en) 1995-07-03 2001-12-25 Wilson T. Crandall Composition and method for topical treatment of androgenic alopecia
US5981255A (en) * 1995-11-02 1999-11-09 Novo Nordisk A/S Alkaline protease, process for the production thereof, use thereof, and microorganism producing the same
US6413512B1 (en) * 1998-02-13 2002-07-02 National Enzyme Company Composition and method for treating disease by increasing activated α2 macroglobulin in the blood and extravascular tissue
WO2001012795A1 (en) * 1999-08-12 2001-02-22 National Enzyme Company COMPOSITION AND METHOD FOR TREATING DISEASE BY INCREASING ACTIVATED α2 MACROGLOBULIN IN THE BLOOD AND EXTRAVASCULAR TISSUE
US20080081035A1 (en) * 2006-10-03 2008-04-03 National Enzyme Company Therapeutic protease compositions
US7998476B2 (en) 2006-11-22 2011-08-16 Standard Biologics, Inc. Method of treatment using Aspergillus oryzae protease

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DE2343963A1 (de) 1974-04-11
CA990672A (en) 1976-06-08
DE2343963B2 (de) 1980-03-20
JPS4942883A (lm) 1974-04-22
FR2197569B1 (lm) 1978-07-28
GB1451074A (en) 1976-09-29
DE2343963C3 (de) 1980-11-13
FR2197569A1 (lm) 1974-03-29
JPS5039151B2 (lm) 1975-12-15

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