Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
  • C12P7/6409—Fatty acids
  • C12P7/6418—Fatty acids by hydrolysis of fatty acid esters
• • 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)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
  • C12N9/20—Triglyceride splitting, e.g. by means of lipase
• • 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
  • Y10S435/882—Staphylococcus

Definitions

• the present invention relates to a method for producing a novel lipase. For more information, see the staphylococ
• the present invention relates to a method for producing a novel lipase for producing free fatty acids and glycerin from triglycerides by a microorganism belonging to (S taphycoccus cus pitis). Background technology
• FIG. 1 shows the relative activity of lipase of the present invention to various substrates assuming that the activity when using olive oil as a substrate is 100%.
• Fig. 2 shows the activity when using triolein as a substrate.
• % Shows the relative activity of the lipase of the present invention to various substrates.
• FIG. 3 shows the effect of pH on the activity of this lipase.
• -Sin- is the activity measured using 0.1 M acetate buffer.
• - ⁇ -indicates activity measured using 0.1 M phosphate buffer and --mouth-indicates activity measured using 0.1 M glycine-NaOH buffer. Show.
• FIG. 4 shows the stability of the rivase of the present invention to PH.
• the symbols in the figure have the same meanings as in FIG.
• FIG. 5 shows the effect of temperature on the activity of the ribose of the present invention.
• FIG. 6 shows the stability of the rivase of the present invention with respect to temperature.
• FIG. 7 is a thin-layer chromatogram comparing the lipase of the present invention with the lipase of the prior art with respect to the reaction product.
• FIG. 8 is a thin-layer chromatogram showing the activity of the lipase of the present invention for various oleic acid glycerides.
• FIGS. 9 and 10 are thin-layer chromatograms showing products when trioin is degraded by various enzyme solutions (different strains) of the present invention.
• the lipase-producing bacterium used in the present invention is a bacterium belonging to the genus Staphylococcus' cavitas and having a lipase-producing ability. Any bacterium having such properties can be used in the method of the present invention. For example, American Type Culture Collection (12301 No., one-crown, drive, rock-building, 20852 ⁇ United States; (12301 Parklawn Drive, Rockville, Maryland 20852, USA)), and easily available Staphylococcus cavitais can be used. Such bacteria include, for example, Staphylococcus cavities ATCC 27840, ATCC 27841, ATCC 27842, and ATCC 27843.
• the present inventors can use the Staphylococcus cavitiens strain T-111 (SAM 0001) isolated from human scalp for the first time. .
• This strain has the following mycological properties.
• Inorganic nitrogen source utilization capacity Uses nitrate, but does not use ammonium salt.
• Acid, gas generation (-); D-galactose, D-xylose, L-arabinose, maltose, lactose, trenpero , D-Sonorebit, Inosit, Starch.
• a lipase-producing bacterium belonging to the aforementioned Staphylococcus Cavities is cultured in a medium.
• any medium that allows the microorganism to grow well can be used.
• glucose, sucrose, molasses, fats and oils and the like can be used alone or in combination.
• Various types of nitrogen sources Tonnes, soybean flour, corn precaution, yeast extract, etc. can be used alone or in combination.
• salts such as Mg 2 + , Caz + , and Na + , various vitamins, and an antifoaming agent can be added to the medium in accordance with the culture.
• liquid culture or solid culture can be used, but aerobic liquid culture is usually preferred. Aerobic conditions can be achieved by shaking culture or aeration and stirring culture.
• the culture conditions in the present invention slightly vary depending on the strain used and the composition of the medium, and the most advantageous conditions for the production of the target lipase are selected.
• the T-1-1 strain is preferably cultured at a temperature of 20 to 40, particularly preferably at 30 to 37, and at a pH of 5 to 9, particularly preferably at a pH of 6 to 7. Good lipase production is achieved.
• a novel lipase when a novel lipase is produced by culturing a lipase-producing bacterium belonging to Staphylococcus caveitus in a medium, an oil or fat such as olive oil or lecithin is added to the medium.
• the productivity increases markedly by the addition of such phosphorus fats.
• the addition amount of these lipids is appropriately 0.01 to 2% based on the culture medium.
• the cultivation period varies depending on the strain, and rivase production is completed in 1 to 3 days.
• the liquid containing the lipase can be easily separated from the cells by ordinary centrifugation or filtration.
• techniques commonly used for the collection and purification of enzymes such as ammonium sulfate Salt precipitation using ethanol, etc., solvent precipitation using ethanol, acetate, etc., isoelectric point precipitation, physical adsorption using activated carbon, calcium phosphate, etc., ion exchange chromatography, gel filtration Enzyme standards of various purities can be obtained by one or a combination of a plurality of methods or affinity chromatography.
• a clear enzyme solution can be obtained by filtering or centrifuging the culture obtained by the method of the present invention. After concentrating this enzyme solution and folding it through 20 ⁇ Tris-ECU buffer solution (pH 7.0), the concentrated solution that has been made transparent is transferred to DEAE-cell mouth which has been equilibrated with the same buffer solution. Adsorb and elute gradient with NaC £. This lipase is eluted at a NaC concentration of 0.4-0.5M. After concentration as appropriate, electrophoresis is performed by gel filtration using Ultrogel AcA34 (manufactured by LKB) equilibrated with 100 mM MTris—HC buffer (pH 8.0) ′.
• the above culture solution is concentrated using an ultrafiltration membrane, and the concentrated solution is adjusted to pH 3.9 to 4.0 using dilute hydrochloric acid and kept at a low temperature (preferably 4) for several hours. After that, the sediment is recovered by centrifugation or filtration, and the enzyme preparation can be obtained by freeze-drying.
• Extensive western fats and oils such as olive oil, coconut oil, castor oil It hydrolyzes vegetable oils such as beef, animal fats such as beef tallow, and water-soluble lipids such as triptyline.
• FIG. 1 shows the lipase activity of the present enzyme when various fats and oils were used as substrates, as a relative activity to the lipase activity (100%) when using olive oil as the substrate.
• FIG. 2 shows the lipase activity of this enzyme when glyceride was used as a substrate, relative to the lipase activity (100%) when triolein was used as a substrate.
• this lipase shows activity in the range of ⁇ H 4.5 to 10.5, and the optimal pH is 7.5 to 8.0, which is on the slightly alkaline side.
• FIG. 4 shows the residual lipase activity when kept in various buffers at 4 for 24 hours. This enzyme is inactivated on the acidic side of pH 4 and on the alkaline side of PH 11 or more, and is stably maintained in the range of PH 5 to ⁇ 10.
• this lipase exhibits activity in the range of 20 to 45'c, and the optimal temperature is 30 to 35.
• the residual lipase activity after treatment at each temperature for 30 minutes is as shown in FIG. It is stable up to 45 when treated for 30 minutes.
• the molecular weight determined by SDS-Boliacrylamide gel electrophoresis and gel filtration is about 128,000.
• the isoelectric point obtained by using a carrier antenna (LKB) has an isoelectric point of 3.9 ⁇ 0.1.
• the unit of lipase activity (Unit) is defined as the activity to release 1 mole of fatty acids per minute under the above reaction conditions.
• AFF Peptone Soybean protein hydrolyzate; Ajinomoto K.K.
• 500 mg of a liquid medium (pH 6.3) containing 10% and 0.5% NaC was dispensed into 500 mg of Yosaka Lofrasco, sterilized, and this medium was inoculated with 1% by volume of the inoculum.
• the cells were shake-cultured at 30 ° C for 2 days using a reciprocating shaker for 300 times.
• the supernatant of the culture obtained in the same manner as described in Example 1, was subjected to ultrafiltration (Pellicon Lab Casette, manufactured by Nippon Millipore; molecular weight 10,000, ultrafiltration membrane PTGC 0LC05). After concentration, the concentrate was subjected to chromatography using a DEAE-cellulose column equilibrated with a 20-m MTris-HC buffer (pH 7.0), followed by the method described in Example 1 below. Purification was performed, and a single enzyme sample was obtained electrophoretically.
• Liquid medium consisting of 5% AFF peptone and 0.5% NaC (pH 6.3) 7 lbs. The medium was then sterilized in a Brunswick Scientific (manufactured by NEW BRUNSWICK SCIENTIFIC) to prepare a medium.
• Inoculated to the above medium was 200 m < 0 > of the inoculum [Staphylococcus cavities T-1-1 (FERM P-7723)] cultured in the same manner as in Example 1; The culture was performed for 24 hours at a stirring speed of 300 r.p.nt. and an aeration rate of 2.0 vvm. The culture solution was centrifuged to remove the cells, and concentrated 4 times by ultrafiltration (Nippon Millipore, Pellicon Labcasset molecular weight 10,000, ultrafiltration membrane, PTGC 0LC05).
• the concentrated solution is adjusted to pH 3.9 with hydrochloric acid, left at room temperature (preferably 4) for 2 hours, and the resulting precipitate is collected by centrifugation or filtration, dissolved, and lyophilized. As a result, a purified enzyme product was obtained.
• the specific activity (activity per mg protein) in the above purification process was 87.0 U nom mg, and the recovery was 28%.
• Example 1 25 Add 5 ⁇ m MTris-EC £ buffer (pH 8.0), 10 mM CaC & z 2 £, substrate triolein (99% purity, semi-chemical) and 10 ⁇ & The enzyme preparation obtained in Example 1 was added thereto so that the total activity of the enzyme was 3 U, and the reaction was carried out at 30'C. For comparison, a commercially available Rhizopus alhidus
• the thin-layer chromatographic system was as follows.
• Example 2 Using the enzyme preparation obtained in Example 1, an enzymatic reaction was carried out using trio-lein, zio-lein and mono-olein as substrates, and the reaction product was subjected to silica gel thin-layer chromatography. Identification In this identification, three triolane (7) were added to three reaction media consisting of I mi of 25 mM MTris-HC £ buffer (PH 8.0) and 10 mM CaC & 2 , respectively. 99% pure), 5 £ zeolane (99% pure), and 3 monolane (99% purity), and then an enzyme solution was added to each medium to make the total activity 3 U.
• the enzyme solution had the following lipase titer.
• the present invention is useful for industrially producing rivase, which substantially completely decomposes triglyceride into fatty acids and glycerin. This rivase hydrolyzes fats and oils to economically produce fatty acids. It is useful for manufacturing.

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• 1984
  • 1984-07-20 JP JP14958584A patent/JPS6128387A/ja active Granted
• 1985
  • 1985-07-19 WO PCT/JP1985/000409 patent/WO1986000925A1/ja not_active Ceased
  • 1985-07-19 US US06/844,389 patent/US4791059A/en not_active Expired - Fee Related
  • 1985-07-19 EP EP19850903698 patent/EP0187869A4/en not_active Withdrawn

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