US3622459A - Method of preparing a proteolytic enzyme by fermentation - Google Patents

Method of preparing a proteolytic enzyme by fermentation Download PDF

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Publication number
US3622459A
US3622459A US6277A US3622459DA US3622459A US 3622459 A US3622459 A US 3622459A US 6277 A US6277 A US 6277A US 3622459D A US3622459D A US 3622459DA US 3622459 A US3622459 A US 3622459A
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US
United States
Prior art keywords
protease
strains
growth
chelating agents
bacillus subtilis
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Expired - Lifetime
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US6277A
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English (en)
Inventor
Kojo Mitsugi
Yoshiteru Hirose
Masami Hoshino
Sadanobu Tobe
Kohei Hashimoto
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Ajinomoto Co Inc
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Ajinomoto Co Inc
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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
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • 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
    • Y10S435/814—Enzyme separation or purification
    • Y10S435/816—Enzyme separation or purification by solubility
    • 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
    • Y10S435/839—Bacillus subtilis

Definitions

  • This invention relates to a method of preparing proteolytic enzymes by fermentation, and particularly to the production of proteolytic enzymes suitable for laundry use.
  • Laundry products containing alkaline proteases are finding rapidly increasing acceptance. It is known that proteolytic enzymes are produced by certain fungi and bacteria, but the enzymes obtained heretofore do not, satisfactorily meet the requirements for use in laundry products. More specifically, they fail to maintain their protease activity under one or more of the following conditions normally encountered in launder- 1. an alkaline medium, particularly at pH 9 to l 1;
  • elevated temperature typically 40 to 60 C.
  • proteolytic enzymes which meet the requirements of laundry service can be obtained in high yields from cultures of newly isolated strains of bacterium belonging to the species Bacillus subtilis.
  • the several strains were identified as Bacillus subtilis by comparison of their characteristics with data in Bergeys Manual of Determinative Bacteriology," 7th ed.
  • the several strains differ slightly from each other in their resistance to chelating agents and in the protease activity of the enzymes produced, but are very similar in their taxonomical characteristics as evidenced by comparison of the following data on two strains, A.I3205 and AJ-3208.
  • strains of Bacillus subtilis may or may not produce alkaline protease
  • strains of this invention hereinafter identified as A.l-3205, AJ-3206, AJ-3207 and AJ-3208, which were isolated from soil samples, not only produce enzymes having protease activity, but are also capable of growing on media containing as much as 8 percent sodium tripolyphosphate (TPP) or 3 percent sodium nitrilotriacetate (NTA), whereas most known strains of Bacillus subu'lis cannot grow on media containing 3 percent TPP or 1 percent NTA, and are similarly sensitive to 0.2 percent tetrasodium ethylenediaminetetraacetate (EDTA).
  • TPP sodium tripolyphosphate
  • NTA sodium nitrilotriacetate
  • Stains of Bacillus subtilis incapable of growth on media including such chelating agents include ATCC 605i, and [AM 1026, H45, H07, 1193,1214, 1108,1033 and 1076.
  • the strains ofthe invention are selected among other strains of Bacillus subtilis by conventional screening methods based on their resistance to chelating agents and their ability of producing alkaline protease.
  • table I lists the turbidity values of cultures as a measure of microbial growth 24, 48 and 72 hours after inoculation.
  • Bacillus subtilis ATCC 6051, AJ-3208 and AJ-3205 were cultured under uniform conditions in media containing 0.5 g./dl. soluble starch, 1.0 g./dl. meat extract, 1.0 g./dl. polypeptone, 0.25 g./dl. NaCl, and the amounts of sodium tripolyphosphate listed in the table.
  • Ten ml. batches of the several media were inoculated with the micro-organisms and cultured at 31.5" C. with shaking.
  • the micro-organisms of the invention when grown on suitable nutrient media, not in themselves unusual, produce alkaline protease which is stable at pH 9 to l l and at temperatures of 40 to 60 C., and is not deactivated by chelating agents.
  • the nutrient media must include sources of assimilable carbon and nitrogen and small amounts of inorganic salts. Suitable carbon sources are starch, soluble starch, dextrin and acid-treated starch, and are preferably present in the medium in amounts of 5 to g./dl.
  • Suitable nitrogen sources include soybean flake, soybean flour, soybean cake extract, milk casein, whey, polypetone, meat extract, peptides and amino acids.
  • the cultivation is preferably performed at pH 5.5 to 7.5 and between 25 and 37 C., preferably between 31 and 34 C. for 24 to 60 hours under aerobic conditions.
  • the protease formed may be recovered from the broth by conventional methods.
  • the bacterial cells may first be removed by filtration or centrifuging, and the protease precipitated from the aqueous phase by salting out with ammonium sulfate or sodium sulfate, or by means of organic solvents miscible with water. The precipitate is most conveniently recovered by centrifuging, and is then dried.
  • the enzyme powders so prepared from cultures of strains of the invention compare favorably in their temperature stability in the presence of chelating agents with a proteolytic enzyme commercially available under the name Maxatase" (Koninklijke Nederlandische Gisten Spiritusfabriek N.V., Delft, Netherlands) and widely used in laundry detergents.
  • Maxatase a proteolytic enzyme commercially available under the name Maxatase
  • the table lists residual protease activity in percent of the initial activity.
  • enzyme powder of 60,000 protease units per gram was dissolved in 0.1 M H 80 Kc1-Na,co, buffer solution (pH 9.5) in a concentration of 0.06 percent. Solutions of each enzyme powder were further mixed with TPP in two concentrations, NTA, EDTA and the four solutions of each enzyme powder were kept under the following conditions:
  • Test A 2% TPP, 60 minutes at 40 C.
  • Test B 4% TPP, 30 minutes at 55 C.
  • Test C l% NTA, 60 minutes at 45 C.
  • Test D 2% EDTA, 60 minutes at 45 C.
  • Protease activity was determined by Anson's method in which milk casein is hydrolyzed in the above buffer at 37 C. for 10 minutes, trichloroacetic acid soluble tyrosine is dyed with Folin's reagent, and the amount of released dye is determined.
  • One unit is defined as the protease activity which releases 1 pg. tyrosine per minute.
  • EXAMPLE l A culture medium containing l g./dl. meat extract, 1 percent polypeptone, 0.5 g./dl. soybean flake extract and 5 g./dl. soluble starch was adjusted to pH 7.0, and 50 ml. batches of the medium were sterilized in 500 ml. shaking flasks at C. for 20 minutes.
  • Each medium was inoculated with Bacillus sublilis Al-3205 (NRRL B-3699) which had been cultured on a bouillon slant at 3 l C. for 24 hours.
  • the flask was kept at 315 C. for 48 hours with shaking.
  • the broth thereafter contained 2650 units/ml. protease (about 5.3 g./l. protease).
  • a product suitable for laundry use and containing 60,000 protease units per gram was prepared by uniformly mixing the enzyme powder with 16.5 g. anhydrous sodium sulfate.
  • Bacillus subtilis AJ-3206 was cultured on a medium containing, per deciliter, l g. soybean cake extract, 1.5 g. casein, 7 g. soluble starch, 0.05 g. KH PO 0.02 g. MgSO -7H O, and 0.2 g. CaCl at pH 7.0 as described in example 1.
  • the cultured broth was found to contain 3960 protease units per milliliter (about 8 g./l. protease) after 48 hours.
  • EXAMPLE 4 Bacillus subtilis AJ-3208 (NRRL 13-3700) was cultured for 48 hours as described in example 2, and protease was produced at a rate of 4050 units per milliliter (about 8. l g./l. When 900 ml. of the broth were worked as described in example l, 29 g. of a crude enzyme powder containing 112,000 protease units per gram were recovered.
  • suitable artificial mutants can be derived from strains of Bacillus subtilis which are themselves incapable of producing alkaline protease resistant to chelating agents. Diethyl sulfate, nitrosoguanidine, or nitrogen mustard are suitable chemical mutation inducing agents, and X ray irradiation is similarly effective.
  • the mutants produced are screened according to their ability of growing on a culture medium containing 4 g./dl. sodium tripolyphosphate or corresponding amounts of the other chelating agents mentioned above, and of producing extracellular alkaline protease when grown on culture media which are conventional in themselves.
  • the following example illustrates the method of obtaining alkaline protease from the mutant strain, and the properties of the protease.
  • EXAMPLE 5 An aqueous culture medium containing, per deciliter, l g. deoiled soybean extract, 1.5 g. casein, 0.5 g. amino acid mixture, 8 g. soluble starch, 0.1 g. KH PO and 0.02 g. MgSO 7 H O was adjusted to pH 7.0, and 50 ml. batches of the medium were sterilized in 500 ml. shaking flasks. Each batch was then inoculated with Bacillus subtilis D-4628 (NRRL B-375l and the cultures were kept at 315 C. for 48 hours with shaking. The culture broth so obtained was found to contain 4,050 protease units per ml. (about 8.2 g./l. enzyme protein).
  • the enzyme powder was subjected to comparison tests with the alkaline protease produced by the parent strain AJ-3 168 and with Maxatase as described with reference to table 2. The results are listed below in table 4 which also indicates the optimum pH for ea'ch of the enzymes and its stable range as determined in the absence of chelating agents.
  • Test G the remaining protease activity was determined in percent of initial activity after 20 minutes in the presence of 2 percent TPP at 50 C.
  • Test H residual protease activity was measured after 40 minutes in 2 percent TPP at 50 C.
  • Specimen cultures of all strains of Bacillus subtilis referred to hereinabove by NRRL numbers are available without our permission from the ARS Culture Collection, Northern Utilization Research and Development Division of the United States Department of Agriculture in Peoria, Illinois.
  • a method of producing alkaline protease which comprises:
  • micro-organism is of one of the strains AJ3205 (NRRL B-3699), AJ-3208 (NRRL B-3700), and D-4628 (NRRL B375l

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
US6277A 1969-05-02 1970-01-27 Method of preparing a proteolytic enzyme by fermentation Expired - Lifetime US3622459A (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP44034235A JPS507157B1 (de) 1969-05-02 1969-05-02

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US (1) US3622459A (de)
JP (1) JPS507157B1 (de)
DE (1) DE2005232A1 (de)
FR (1) FR2041793A1 (de)
GB (1) GB1287212A (de)
NL (1) NL6919594A (de)

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Publication number Priority date Publication date Assignee Title
DK386586D0 (da) * 1986-08-14 1986-08-14 Novo Industri As Protease, fremgangsmaade til fremstilling deraf og anvendelse deraf

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3451935A (en) * 1966-04-25 1969-06-24 Procter & Gamble Granular enzyme-containing laundry composition

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3451935A (en) * 1966-04-25 1969-06-24 Procter & Gamble Granular enzyme-containing laundry composition

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JPS507157B1 (de) 1975-03-22
NL6919594A (de) 1970-11-04
GB1287212A (en) 1972-08-31
FR2041793A1 (de) 1971-02-05
DE2005232A1 (de) 1970-11-12

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