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
• • 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
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
• • 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
  • C12R2001/07—Bacillus
  • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass 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
  • Y10S435/839—Bacillus subtilis

Definitions

• proteolytic enzymes obtained by fermenting strains of Bacillus subtilis are widely used in industry, especially in the detergent industry.
• the invention relates to a new mutant strain of Bacillus subtilis which has the property of being substantially asporogenic whilst showing proteolytic activity similar to that of the native strain from which it is derived.
• the invention also relates to a process for mutating a native strain of Bacillus subtilis and isolating the asporogenic strain according to the invention.
• the mutant strain obtained by this process has the same identification characteristics as the original native strain except that it; does not sporulate. Its identification which is thus already possible by comparison with the well-known and defined native strain is facilitated by the fact that the mutant strain has been deposited in the collection of the Pasteur Institute in Paris and given the identification number 71-l00l.
• mutant strain according to the invention is no different from the native strain already used in the production of protease, i.e., an extracellular enzyme used in detergents, apart from its asporogenic character, this mutant can readily be subjected to known processesfor commercially growing the native strain with a view to obtaining enzymes suitable for use in household detergents.
• the enzymes thus obtained by applying a conventional process to the new mutant strain according to the invention also have the advantage of not giving off the unpleasant odour attending the use of detergents containing enzymes obtained from spore-forming strains. Finally, the absence of spores avoids any subsequent microbial growth.
• the process comprises (A) subjecting the spores of the native strain to a mutagenic treatment, (B) spreading the spores subjected to the mutagenic treatment over an agar-containing nutrientbroth accommodated in Petri dishes to obtain after incubation spore-containing colonies and (C) isolating colonies of the asporogenic strain by comparison between the homologous Petri dishes of one series emanating from operation (B) and having been subjected to treatment with chloroform vapours followed by replication and incubation, and a series having undergone the same operations except for exposure to the chloroform vapours, and finally (D) selecting the mutant strain satisfying the required sporulation conditions from the mutant strains isolated showing proteolytic activity.
• the mutagenic treatment (A) of the spores of the native strain of Bacillus subtilis comprises initially heating a suspension of spores for 10 minutes to C., followed by germination in Difco nutrient broth in the presence of chloramphenicol ug/ml), the culture being stirred for 2 hours at 39C., and finally by the actual mutagenic treatment with N-methyl-N'-nitro-N-nitrosoguanidine (50 tog/ml), the culture being stirred for 1 hour at 37C.
• the culture medium used for germination has the following composition:
• Isolation (C) which includes a treatment with chloroform vapours applied to certain colonies comprises the following operations:
• the sp colonies are absent from series lIl but present in series Il because the cells are killed by the chloroform vapours. Samples of the sp colonies are taken from series II and then isolated again. After the second isolation, the strain retained for their sp character are tested for their proteolytic activity.
• the medium used is'preferably agar-containing Difco nutrient broth with casein added.
• a Difco medium of the following composition is used:
• strain y proteolytic sporulation 4 activity sp/ml strain y proteolytic sporulation 4 activity sp/ml.
• the cells are in the form of more or less long chains (4 to 10 cells). After growth, the chains are shorter (2 to 4 cells) and the cells are less long.
• the spores formed are oval (0.8 1.4/1.1 11.).
• Mutant strain Identical colonies except that, after 3 to 4 days at 37C., the colonies become translucent (destruction of the cells). Yellow pigment is again excreted.
• the gradient of each straight line is characteristic of an enzyme.
• the gradients k log e are the same for the enzyme emanating from the mutant and for the enzyme emanating from the native strain.
• the velocity constant of the denaturation reaction is:
• k (70C) 0.1 min are not inhibited by o-phenantroline hydrochloride b.
• the degree of inhibition caused by diisopropyl fluorophosphate IO M) is'of the order of 95 percent for the mutant and the native strain. Accordingly, the principal enzyme would be seryl-protease in both cases.
• mutant strain according to the invention is an asporogenie mutant of the native strain of Bacillus subtilis which does not have any effect either upon production or upon the properties of the extracellular protease, i.e., the enzyme produced by fermentation from the strain, except in regard to the formation of spores.
• the enzyme produced by fermentation of the Bacillus subtilis strain is present in solution in the aqueous phase of the culture broth.
• the following procedure can be adopted for producing the enzymes according to the invention on a commercial scale:
• a fermentation process is carried out in a variablesized fermenter aerated in depth, agitated and equipped with a heat-regulating system.
• the culture medium contains organic nitrogen compounds such as distillery residues, maize maceration liquor, soya flour, casein, mineral elements etc.
• This culture medium is inoculated with an asporogenic Bacillus subtilis culture (strain according to the invention as deposited) obtained in agitated flasks or from an inoculum fermenter.
• the aeration required amounts for example to between 100 and 500 m" per hour for a fermenter with a useful capacity of m.
• Fermentation is carried out at 37C.
• the fermentation temperature can be in a range from 30 to 38C.
• the pH-value is adjusted to 7.0 at the beginning of fermentation.
• the culture medium After having been inoculated, the culture medium is sterilised with live steam for 30 minutes at 121C.
• the air injected at the bottom of the fermenter is sterilised by passage through a cylinder filled with glass wool.
• the liquid phase is separated from the bacteria and solid particles by fermentation, generally in a filter press comprising :1 Nylon cloth.
• a filtration additive is added to the fermentation juice to be filtered.
• the filtrate is precipitated with a solvent such as ethanol, butanol, or even with ammonium sulphate.
• the precipitate obtained is centrifuged in a centrifuge, for example of the Sherpless type and filtered in a filter press.
• the wet cake obtained is introduced in trays into an oven in which it is dried in vacuo at a temperature not exceeding 60C.
• the powder obtained does not contain any spores. It can be used in detergents made up in accordance with the usual formulations and-does not produce any of the allergic reactions observed with enzymes obtained from sporular strains.
• a process for selectingasporogenic bacteria from the genus Bacillus subtilis which comprises:
• Process for preparing protease without spore which comprises culturing in a known manner for the wild Bacillus subtilis an asporogenie bacteria selected by the process of claim 1.
• asporogenic bacteria is a Bacillus subtilis of the type identified by No. 71-1-00] deposited in Institut Pasteur de Paris.

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• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Genetics & Genomics (AREA)
• Organic Chemistry (AREA)
• Wood Science & Technology (AREA)
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• Bioinformatics & Cheminformatics (AREA)
• Biotechnology (AREA)
• Biomedical Technology (AREA)
• General Engineering & Computer Science (AREA)
• Microbiology (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Molecular Biology (AREA)
• Medicinal Chemistry (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Physics & Mathematics (AREA)
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• Oil, Petroleum & Natural Gas (AREA)
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• Tropical Medicine & Parasitology (AREA)
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• Enzymes And Modification Thereof (AREA)

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Citations (2)

• 1971
  • 1971-07-28 FR FR7127671A patent/FR2147470A5/fr not_active Expired
• 1972
  • 1972-07-25 US US00275069A patent/US3833474A/en not_active Expired - Lifetime
  • 1972-07-27 GB GB3514972A patent/GB1396964A/en not_active Expired
  • 1972-07-27 BE BE786826A patent/BE786826A/xx unknown
  • 1972-07-27 IT IT9612/72A patent/IT968106B/it active
  • 1972-07-27 DE DE2236926A patent/DE2236926A1/de active Pending
  • 1972-07-28 NL NL7210486A patent/NL7210486A/xx unknown
  • 1972-07-28 JP JP47075829A patent/JPS4835078A/ja active Pending

Patent Citations (2)

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