WO2017018060A1 - Procédé de production d'un dérivé de cystéine et d'un dérivé de cystéine sulfoxyde - Google Patents

Procédé de production d'un dérivé de cystéine et d'un dérivé de cystéine sulfoxyde Download PDF

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WO2017018060A1
WO2017018060A1 PCT/JP2016/066974 JP2016066974W WO2017018060A1 WO 2017018060 A1 WO2017018060 A1 WO 2017018060A1 JP 2016066974 W JP2016066974 W JP 2016066974W WO 2017018060 A1 WO2017018060 A1 WO 2017018060A1
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cystathionine
yeast
synthase
formula
group
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山本　英樹
誠吾 菅原
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味の素株式会社
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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
    • C12N9/88—Lyases (4.)
    • 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
    • C12N15/09—Recombinant DNA-technology
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/12—Methionine; Cysteine; Cystine

Definitions

  • the present invention relates to a method for producing a cysteine derivative and a cysteine sulfoxide derivative.
  • Cysteine derivatives and cysteine sulfoxide derivatives are expected as raw materials for pharmaceutical intermediates and food additives, and various inexpensive industrial production methods have been studied.
  • methods for producing cysteine derivatives there are reports of methods for producing S-alkylcysteine or S-alkenylcysteine using cysteine desulfhydrase (Patent Documents 1 and 2) and tryptophan synthase (Patent Documents 3 and 4). The amount is not enough.
  • Non-Patent Documents 1 and 2 Requires removal of the remaining hydrogen peroxide.
  • the present invention relates to cysteine derivatives and cysteine sulfoxide derivatives that are expected as raw materials for pharmaceutical intermediates and food additives, in particular, cysteine derivatives such as S-alkylcysteine and S-alkenylcysteine, and S-alkylcysteine sulfoxide and S
  • cysteine derivatives such as S-alkylcysteine and S-alkenylcysteine
  • An object is to provide a method for producing a cysteine sulfoxide derivative such as alkenyl cysteine sulfoxide in a stable manner on an industrial scale and also in a safe manner when used in foods.
  • the present inventors focused on a method for enzymatically producing a cysteine derivative from a thiol compound and L-serine, and as a result of intensive studies, it has been found that cystathionine ⁇ -synthase can be the responsible enzyme. I found it. Furthermore, paying attention to a method for enzymatically converting a cysteine derivative to a cysteine sulfoxide derivative, and as a result of extensive studies, the present inventors have found a method for efficiently converting a cysteine derivative to a cysteine sulfoxide derivative with oxidase. Obtaining these findings, the present invention was completed by successfully obtaining cysteine derivatives and cysteine sulfoxide derivatives more efficiently and stably than in the past.
  • the manufacturing method of this cysteine sulfoxide derivative including the process (process 2) of manufacturing the cysteine sulfoxide derivative represented by these.
  • the oxidase is at least one selected from the group consisting of glucose oxidase, galactose oxidase, uricase, cholesterol oxidase, choline oxidase and acyl CoA oxidase.
  • R is a 1-propenyl group, a propyl group, a methyl group or an allyl group.
  • cystathionine ⁇ -synthase is derived from a cystathionine ⁇ -synthase producing microorganism.
  • the microorganism is a mutant strain having enhanced cystathionine ⁇ -synthase activity.
  • cystathionine ⁇ -synthase is an enzyme encoded by a CYS4 gene.
  • the manufacturing method of this cysteine sulfoxide derivative including the process (process 2) of manufacturing the cysteine sulfoxide derivative represented by these.
  • the oxidase is at least one selected from the group consisting of glucose oxidase, galactose oxidase, uricase, cholesterol oxidase, choline oxidase and acyl CoA oxidase.
  • R is a 1-propenyl group, a propyl group, a methyl group or an allyl group.
  • yeast is at least one selected from the group consisting of a CYS4 gene high expression mutant; a CYS3 low expression mutant; and a CYS4 high expression and CYS3 low expression mutant To [26].
  • yeast is a Saccharomyces genus yeast.
  • Saccharomyces genus yeast is Saccharomyces cerevisiae.
  • the cystathionine ⁇ -synthase activity of the yeast extract and / or culture is 2500 U or more, 5000 U or more, 10,000 U or more, 20000 U or more, 30000 U or more, or 40000 U or more per 1 g of the extract and / or culture.
  • the cystathionine ⁇ -synthase activity of the yeast extract and / or culture is 1 ⁇ 10 6 U or less, 5 ⁇ 10 5 U or less, 1 ⁇ 10 5 U or less per gram of the extract and / or culture. Or the method according to any one of [22] to [30] above, which is 5 ⁇ 10 4 U or less.
  • the cystathionine ⁇ -lyase activity of the yeast extract and / or culture is 100 U or less, 50 U or less, 10 U or less, 1 U or less, or substantially free per gram of the extract and / or culture, or The method according to any one of [22] to [31] above, which is 0 U.
  • a cysteine derivative and a cysteine sulfoxide derivative can be produced more efficiently and stably.
  • FIG. 3 is a graph showing the results of measuring the amount of S-allylcysteine (ALC) produced in a mutant yeast strain in which genes encoding various enzymes were deleted.
  • Wild strains Saccharomyces cerevisiae BY4742, which is a laboratory yeast, ⁇ CYS3: CYS3 gene deletion mutant, ⁇ CYS4: CYS4 gene deletion mutant, ⁇ TRP5: TRP5 gene deletion mutant, ⁇ MET17: MET17 gene deletion mutant.
  • the vertical axis indicates the concentration (ppm) of ALC in the reaction solution.
  • 3 is a graph showing the results of quantifying the amount of S-allylcysteine (ALC) produced by reacting S-allyl mercaptan with L-serine in the presence of purified cystathionine ⁇ -synthase over time.
  • the vertical axis indicates the concentration (ppm) of ALC in the reaction solution.
  • the horizontal axis indicates the reaction time (hr).
  • 3 is a graph showing the results of quantifying the amount of S-methylcysteine produced over time by reacting S-methyl mercaptan with L-serine in the presence of purified cystathionine ⁇ -synthase.
  • the vertical axis represents the concentration (ppm) of S-methylcysteine in the reaction solution.
  • the horizontal axis indicates the reaction time (hr).
  • 3 is a graph showing the results of quantifying the amount of S-propylcysteine produced by reacting S-propyl mercaptan with L-serine in the presence of purified cystathionine ⁇ -synthase over time.
  • the vertical axis represents the concentration (ppm) of S-propylcysteine in the reaction solution.
  • the horizontal axis indicates the reaction time (hr).
  • 3 is a graph showing the results of quantifying the amount of S-allyl cysteine sulfoxide (ALCSO) produced by reacting glucose with S-allyl cysteine in the presence or absence of glucose oxidase over time.
  • ALCSO S-allyl cysteine sulfoxide
  • the upper row shows the results for test zone 1, the middle row shows the results for test zone 2, and the lower row shows the results for test zone 3.
  • the vertical axis indicates the concentration (ppm) of gluconic acid, ALC, and ALCSO in the reaction solution.
  • the horizontal axis indicates the reaction time (hr). It is a chart figure which shows the method of preparing a crude enzyme liquid from a strain. Each reaction condition in the ALC generation test is shown. It is the graph which showed the ALC accumulation
  • shaft shows the amount of ALC accumulation
  • the “C 1-6 alkyl group” means a linear or branched alkyl group having 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms, and specifically includes methyl, ethyl, n- Examples include propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl, pentyl, hexyl and the like. Preferred are methyl and propyl.
  • the “C 2-6 alkenyl group” means a straight or branched alkenyl group having 2 to 6 carbon atoms, preferably 2 or 3, and one or more divalent alkenyl groups in the alkyl group. The thing etc.
  • cysteine derivative (II) is enzymatically produced.
  • the method comprises formula (I) in the presence of cystathionine ⁇ -synthase:
  • R is preferably a 1-propenyl group, a propyl group, a methyl group or an allyl group.
  • Cystathionine ⁇ -synthase refers to an enzyme that catalyzes a reaction for synthesizing cystathionine from homocysteine and serine, and is classified into EC 4.2.1.22.
  • the structural gene is known as, for example, CYS4 gene (GenBank NM_001181284) in Saccharomyces cerevisiae.
  • the origin of cystathionine ⁇ -synthase used in the present invention is not particularly limited as long as it can produce cysteine derivative (II) from thiol compound (I) and L-serine. It may be a natural product, chemically or biochemically synthesized, or may be produced by genetic engineering.
  • cystathionine ⁇ -synthase producing microorganism is not particularly limited as long as it can accumulate cystathionine ⁇ -synthase in cells.
  • Specific examples include yeast, filamentous fungi, basidiomycetes and the like, and yeast is preferred.
  • Yeast refers to a unicellular fungus, and examples include spore yeast, basidiomycetous yeast, and incomplete yeast.
  • Saccharomyces yeasts include laboratory yeast, sake yeast, shochu yeast, wine yeast, beer yeast, baker's yeast, and the like. More preferred are Saccharomyces cerevisiae and Candida utilis, and most preferred is Saccharomyces cerevisiae. These strains are commercially available or can be obtained and prepared based on known literature.
  • the American Type Culture Collection (address 12301 Parklawn Drive, Rockville, Maryland 20852 PO Box 1549, Manassas, VA 20108, United States of America) has a registration number corresponding to each strain. Can be sold (see http://www.atcc.org/). The registration number corresponding to each strain is described in the catalog of American Type Culture Collection. Specific examples of Saccharomyces cerevisiae include BY4742 strain (ATCC204508) and S288C strain (ATCC26108). As Candida utilis, specifically, Candida utilis ATCC22023 strain can be used. Particularly preferred is Saccharomyces cerevisiae.
  • the cystathionine ⁇ -synthase-producing microorganism may be a wild strain or a strain that has been modified to have a mutation in an enzyme involved in the synthesis of cysteine derivative (II), that is, a mutant strain.
  • the enzyme involved in the synthesis of the cysteine derivative (II) in the cystathionine ⁇ -synthase-producing microorganism the cystathionine ⁇ -synthase is a positively regulated enzyme, as has been clarified by the present inventors in the examples described later.
  • cystathionine ⁇ -lyase can be mentioned as an enzyme that is negatively regulated.
  • Cystathionine ⁇ -lyase refers to an enzyme that catalyzes a reaction that degrades cystathionine into cysteine and ⁇ -ketobutyric acid, and is classified as EC 4.4.1.1.
  • the structural gene is known as, for example, CYS3 gene (GenBank NM_001178157) in Saccharomyces cerevisiae.
  • the “mutant strain” refers to a strain in which the enzyme activity is enhanced or reduced (including complete loss of activity) as compared to a wild strain or a strain before modification (parent strain). Means that. Examples of mutant strains preferably used in the present invention include Saccharomyces cerevisiae mutant strains.
  • CYS4 high expression mutant strain that highly expresses a gene encoding cystathionine ⁇ -synthase and a cystathionine ⁇ -lyase.
  • Examples include “CYS3 gene low expression mutant” in which gene expression is suppressed.
  • the expression of the CYS4 gene is significantly enhanced in the “CYS4 gene high expression mutant”, specifically 1.5 times or more, more preferably 2 times. More preferably, it is 3 times or more, particularly preferably 5 times or more, and most preferably 10 times or more.
  • the expression of the CYS3 gene is significantly reduced in the “CYS3 gene low expression mutant”, specifically 50% or less, more preferably 30% or less, and further It is preferably reduced to 20% or less, particularly preferably 10% or less, and most preferably 5% or less. Further, it is preferable that the expression of the cystathionine ⁇ -lyase gene is substantially lost (that is, less than the detection limit).
  • mutant strains may exist naturally (natural mutation) or may be artificially created. Examples of methods for artificial production include self-cloning methods, conventional breeding methods (for example, drug mutation, hybridization), and the like. Each method for “modification that reduces enzyme activity” or “modification that enhances enzyme activity” described later may be used.
  • Enzyme activity is reduced means that the target enzyme activity (in the present invention, cystathionine ⁇ -lyase activity) is reduced compared to an unmodified strain such as a wild strain or a parent strain. Including the case where is completely disappeared.
  • the cystathionine ⁇ -lyase activity is preferably 50% or less, more preferably 30% or less, still more preferably 20% or less, particularly preferably 10% or less, and most preferably 5%. % Or less. Further, it is preferable that the cystathionine ⁇ -lyase activity substantially disappears.
  • the modification that reduces the enzyme activity can be performed by, for example, a mutation treatment or a gene recombination technique.
  • Mutation treatment is usually performed by ultraviolet irradiation or mutation treatment such as N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ethylmethanesulfonate (EMS), methylmethanesulfonate (MMS), etc. Treatment with the mutagen used is included.
  • MNNG N-methyl-N′-nitro-N-nitrosoguanidine
  • EMS ethylmethanesulfonate
  • MMS methylmethanesulfonate
  • Examples of gene recombination techniques include known techniques (FEMS Microbiology Letters 165 (1998) 335-340, JOURNAL OF BACTERIOLOGY, Dec. 1995, p7171-7177, Curr Genet 1986; 10 (8): 573-578, WO 98/14600 etc.) can be used.
  • modification that reduces enzyme activity can be performed by, for example, destroying a gene encoding the enzyme.
  • Gene disruption can be achieved, for example, by deleting part or all of the coding region of the gene encoding the target enzyme on the chromosome. Furthermore, the entire gene including the sequences before and after the gene on the chromosome may be deleted.
  • gene disruption can be performed, for example, by introducing an amino acid substitution (missense mutation) into the coding region of the gene encoding the target enzyme on the chromosome, introducing a stop codon (nonsense mutation), or 1-2 bases. It can also be achieved by introducing a frame shift mutation that adds or deletes.
  • gene disruption can be achieved, for example, by inserting another sequence into the coding region of the gene encoding the target enzyme on the chromosome.
  • Enzyme activity is enhanced means that the target enzyme activity (in the present invention, cystathionine ⁇ -synthase activity) is enhanced as compared with a non-modified strain such as a wild strain or a parent strain.
  • the cystathionine ⁇ -synthase activity is preferably 1.5 times or more, more preferably 2 times or more, still more preferably 3 times or more, particularly preferably 5 times or more, most preferably when compared with the wild type strain (Saccharomyces cerevisiae BY4742). Is enhanced more than 10 times.
  • the modification that enhances the enzyme activity can be achieved, for example, by increasing the expression of a gene encoding the target enzyme. Increased gene expression can be achieved, for example, by replacing the promoter of the gene on the chromosome with a stronger promoter. In addition, increase in gene expression can be achieved by increasing the copy number of the gene, for example, by introducing the gene of interest onto the chromosome. Alternatively, a gene may be incorporated into a transposon and transferred to introduce multiple copies of the gene into the chromosome. Furthermore, the increase in the copy number of the gene can also be achieved by introducing a vector containing the target gene into the host. Moreover, the modification that enhances the enzyme activity can be achieved, for example, by increasing the specific activity of the target enzyme.
  • Enzyme activity is enhanced means that not only the target enzyme activity is enhanced in a microorganism originally having the target enzyme activity but also the target enzyme activity in a strain not originally having the target enzyme activity. This includes a case where a gene encoding the enzyme is newly introduced so as to be given and the enzyme is expressed.
  • the host When the target gene is introduced into the host, the host may be the same or different from the strain from which the target gene is derived.
  • Confirmation that the target enzyme activity has been enhanced or decreased can be performed by measuring the activity of the enzyme.
  • Examples of the method for measuring the activity of cystathionine ⁇ -synthase include the method of Kraus et al. (Jan P. Kraus, Methods ENZYMOLOGY 143 (1987) 388-394) and the like. More specifically, the activity of cystathionine ⁇ -synthase is determined by reacting with a substrate (3 mM homocysteine, 3 mM serine, 100 ⁇ M pyridoxal phosphate) for 1 hour at 45 ° C. in a buffer solution (50 mM potassium phosphate buffer, pH 9). Can be measured.
  • the amount of enzyme required to produce 1 ⁇ mol of cystathionine per minute by the reaction is defined as 1 U.
  • Examples of the method for measuring the activity of cystathionine ⁇ -lyase include the method of Yamagata et al. (Journal of Bacteriology, Aug. 1993, p.4800-4808) and a method analogous thereto. More specifically, the activity of cystathionine ⁇ -lyase is determined in the buffer (50 mM potassium phosphate buffer, pH 7.4) by the substrate [3 mM cysteine (when cystathionine is used as a substrate, ⁇ -lyase coexists.
  • the amount of enzyme required to produce 1 ⁇ mol of pyruvic acid per minute by the reaction is defined as 1 U.
  • Confirming that the target enzyme activity has been enhanced or decreased is to confirm that the amount of transcription of the gene encoding the target enzyme has increased or decreased, and that the amount of target enzyme has been increased or decreased. This can be done by confirming this.
  • the transcription amount of the gene encoding the target enzyme has increased or decreased can be confirmed by comparing the amount of mRNA transcribed from the same gene with that of the unmodified strain.
  • methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR, and the like (Molecular cloning (Cold spring spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)).
  • the amount of transcription is decreased, the amount of mRNA is, for example, 50% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 0% compared to the unmodified strain. It is preferable that the number is decreased.
  • the transcription amount is increased, the amount of mRNA is, for example, 1.5 times or more, 2 times or more, 3 times or more, 5 times or more, or 10 times or more compared to the unmodified strain. It is preferable to increase.
  • the amount of the target enzyme is decreased, the amount is, for example, 50% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 0 compared to the unmodified strain. It is preferable to reduce to%.
  • the amount of the target enzyme is, for example, 1.5 times or more, 2 times or more, 3 times or more, 5 times or more, or 10 times or more compared to the unmodified strain. It is preferable that the number is increased.
  • cystathionine which is a product of the enzymatic reaction of cystathionine ⁇ -synthase, is degraded by cystathionine ⁇ -lyase
  • the degree of variation in cystathionine ⁇ -synthase activity according to the above measurement method do not necessarily coincide.
  • the cystathionine ⁇ -synthase activity is lower than that in the absence of cystathionine ⁇ -lyase.
  • cystathionine ⁇ -synthase activity When measuring cystathionine ⁇ -synthase activity in the presence of cystathionine ⁇ -lyase, it is preferable to calculate and correct the amount of cystathionine consumed by cystathionine ⁇ -lyase activity in advance.
  • the cystathionine ⁇ -synthase-producing microorganism preferably used in the present invention includes (A) a mutant strain in which cystathionine ⁇ -synthase activity is enhanced, (B) a mutant strain in which cystathionine ⁇ -lyase activity is reduced, or ( C) a mutant strain having enhanced cystathionine ⁇ -synthase activity and reduced cystathionine ⁇ -lyase activity, more preferably (D) a CYS4 gene high expression mutant strain, (E) a CYS3 gene low expression mutant strain, or (F) CYS4 gene high expression and CYS3 gene low expression mutant yeast.
  • a preferred embodiment of the mutant strain (E) includes a CYS3 gene disruption mutant strain (E ′).
  • a CYS4 gene high expression and CYS3 gene disruption mutant strain (F ′) can be mentioned.
  • the cystathionine ⁇ -synthase used in the present invention is a microorganism obtained by culturing a microorganism that produces it (for example, yeast having cystathionine ⁇ -synthase activity, CYS4 gene high expression and / or CYS3 gene disruption mutant strain of the yeast). From the culture. For example, a method for culturing a cystathionine ⁇ -synthase-producing microorganism is shown below.
  • the medium for culturing the cystathionine ⁇ -synthase-producing microorganism is appropriately selected depending on the type of cystathionine ⁇ -synthase-producing microorganism to be used.
  • Either a synthetic medium or a natural medium can be used as long as it contains a carbon source, a nitrogen source, an inorganic substance, and if necessary, a small amount of micronutrients. Any kind of carbon source and nitrogen source may be used as long as the microorganism used can be used.
  • the carbon source various carbohydrates such as glucose, glycerol, fructose, sucrose, maltose, mannose, starch, starch hydrolyzate and molasses can be used.
  • Nitrogen sources include various inorganic and organic ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate, and ammonium acetate, or meat extract, yeast extract, corn steep liquor, casein hydrolyzate, fishmeal or its Natural organic nitrogen sources such as digests, defatted soybean meal or digests thereof can be used. Many natural organic nitrogen sources are both nitrogen sources and carbon sources.
  • the inorganic substance potassium monohydrogen phosphate, potassium dihydrogen phosphate, potassium chloride, magnesium sulfate, sodium chloride, ferrous sulfate and the like can be used as necessary.
  • the YPD medium described in the examples can be suitably used.
  • Cultivation is performed under aerobic conditions such as shaking culture or aeration-agitation deep culture.
  • the culture temperature is preferably in the range of 20 to 50 ° C, particularly preferably 28 to 37 ° C.
  • the pH of the medium during the culture may be appropriately adjusted according to the growth of the microorganism, and examples thereof include pH 5-7.
  • the culture time is usually 12 hours to 10 days, more preferably 24 hours to 5 days, still more preferably 36 hours to 3 days.
  • the cystathionine ⁇ -synthase is recovered from the culture containing the microorganisms obtained as described above.
  • the cystathionine ⁇ -synthase used in the present invention does not necessarily have to be isolated and purified as long as the enzyme activity is maintained. That is, a cystathionine ⁇ -synthase-producing microorganism (preferably a yeast having cystathionine ⁇ -synthase activity) as it is, or a living cell body collected from the microorganism by a method such as centrifugation, or a dried bacterium thereof The body can be used in the method of the present invention.
  • an extract (bacteria) obtained by crushing, self-digesting, sonicating, or the like, living cells of cystathionine ⁇ -synthase-producing microorganisms (preferably yeast having cystathionine ⁇ -synthase activity) or dried cells thereof can be used in the method of the present invention.
  • a crudely purified product containing cystathionine ⁇ -synthase obtained from the treated bacterial cell can be used in the method of the present invention.
  • these immobilized enzymes or immobilized cells can also be used in the production method of the present invention.
  • the extract and / or culture of a cystathionine ⁇ -synthase-producing microorganism preferably a yeast having cystathionine ⁇ -synthase activity
  • the enzyme activity is usually 2500 U or more per gram, preferably 5000 U or more, 10000 U or more, 20000 U or more, 30000 U or more, or 40000 U or more. If the enzyme activity is too low, a sufficient enzyme reaction cannot be obtained.
  • the enzyme activity of the extract and / or culture is usually 1 ⁇ 10 6 U or less per gram, preferably 5 ⁇ 10 5 U or less, preferably 1 ⁇ 10 5 U or less, Or it is 5 ⁇ 10 4 U or less.
  • the extract and / or culture Preferably have low cystathionine ⁇ -lyase activity. Accordingly, the cystathionine ⁇ -lyase activity of the extract and / or culture is usually 100 U or less, 50 U or less, 10 U or less, 1 U or less, or substantially 0 U per gram.
  • Cysteine derivative (II) can be produced by reacting thiol compound (I) with L-serine in the presence of the above-mentioned cystathionine ⁇ -synthase.
  • the raw materials L-serine and thiol compound (I) are both commercially available and can be easily obtained.
  • the reaction between the thiol compound (I) and L-serine is usually carried out in an aqueous solvent.
  • the aqueous solvent is not particularly limited as long as it does not inhibit the enzyme reaction, but may include physiological saline, potassium phosphate buffer, Tris-hydrochloric acid buffer, glycine-sodium hydroxide buffer, boric acid-sodium hydroxide buffer, and the like. Can be mentioned.
  • the concentration of L-serine in the reaction solution is not particularly limited, but is preferably 1 to 20% by weight, particularly preferably 1 to 10% by weight.
  • the concentration of the thiol compound (I) in the reaction solution varies depending on the type of compound used and is not particularly limited as long as it does not inhibit the enzyme reaction, but is preferably used at a concentration of 10% by weight or less. In addition, you may add thiol compound (I) sequentially so that it may become the density
  • the amount of cystathionine ⁇ -synthase used in the reaction can be appropriately adjusted depending on the substrate concentration, reaction time, and other conditions, but is preferably 0.1 U to 1000 U, preferably 1 U to 500 U, more preferably 10 U to 1 U per gram of reaction solution. Add in the range of 250U.
  • the reaction is carried out in the range of pH 7 to 9.5, preferably pH 8 to 9.
  • the reaction temperature is usually 4 ° C. to 60 ° C., but is preferably 20 ° C. to 55 ° C., more preferably 35 ° C. to 50 ° C. in terms of enzyme stability and reaction rate.
  • the reaction time is appropriately set according to the synthesis amount of the desired cysteine derivative (II), but is usually 0.5 to 24 hours, preferably 1 to 10 hours, particularly preferably 1 to 5 hours, most preferably 1 ⁇ 3 hours.
  • Any known method can be used to separate and recover the cysteine derivative (II) from the reaction solution obtained by the above reaction.
  • TCA trichloroacetic acid
  • ultrafiltration concentration method e.g., ultrafiltration concentration method
  • ammonium sulfate precipitation method salting out
  • organic solvent eg, acetone, ethanol, propanol, methanol, etc.
  • water-soluble polymer e.g, polyethylene) Glycol, dextran, etc.
  • Cysteine sulfoxide derivative (III) can also be produced by reacting cysteine derivative (II) with oxidase and its substrate without separating and recovering cysteine derivative (II).
  • a method for enzymatically producing a cysteine sulfoxide derivative represented by the formula (hereinafter also simply referred to as cysteine sulfoxide derivative (III)) is provided. This method is characterized in that cysteine derivative (II) is reacted with oxidase and its substrate. Cysteine derivative (II) is converted to cysteine sulfoxide derivative (III) by hydrogen peroxide produced by the reaction of oxidase and its substrate.
  • R is preferably a 1-propenyl group, a propyl group, a methyl group or an allyl group.
  • the oxidase used in the method is not particularly limited as long as it produces hydrogen peroxide by reacting with a substrate.
  • Specific examples include glucose oxidase, galactose oxidase, uricase, cholesterol oxidase, choline oxidase, and acyl CoA oxidase. At least one of these enzymes is used. Glucose oxidase is preferable.
  • Glucose oxidase is a dimeric protein and is classified as EC 1.1.3.4.
  • Glucose oxidase is an enzyme that catalyzes a reaction that produces gluconolactone (gluconolactone is non-enzymatically hydrolyzed to gluconic acid) and hydrogen peroxide in the presence of oxygen using glucose as a substrate.
  • the origin of the glucose oxidase used in the present invention is not particularly limited as long as it has the enzyme action described above. It may be a natural product, chemically or biochemically synthesized, or may be produced by genetic engineering. In the present invention, commercially available products can be suitably used.
  • glucose oxidase commercially available from Shin Nippon Chemical Industry Co., Ltd. under the trade name “Sumiteam PGO” can be mentioned.
  • the activity of glucose oxidase can be measured using a reaction that produces a quinoneimine dye by allowing peroxidase to act on the produced hydrogen peroxide in the presence of aminoantipyrine and phenol.
  • the amount of the produced quinoneimine dye is obtained from a calibration curve, and the enzyme activity is calculated.
  • the amount of enzyme required to oxidize 1 ⁇ mol of glucose per minute is defined as 1 U.
  • Galactose oxidase is classified into EC 1.1.3.9 and is an enzyme that catalyzes the reaction of producing D-galacto-hexodialdose and hydrogen peroxide in the presence of oxygen using D-galactose as a substrate.
  • Uricase is classified as EC 1.7.3.3 and is an enzyme that catalyzes the reaction of producing allantoin and hydrogen peroxide in the presence of oxygen using uric acid as a substrate.
  • Cholesterol oxidase is classified as EC 1.1.3.6 and is an enzyme that catalyzes the reaction of producing 3-oxosteroid and hydrogen peroxide in the presence of oxygen using 3 ⁇ -hydroxysteroid as a substrate.
  • Choline oxidase is an enzyme classified as EC 1.1.3.17. Choline oxidase is a reaction that produces betaine aldehyde and hydrogen peroxide in the presence of oxygen using choline as a substrate (first reaction), and a reaction that produces betaine and hydrogen peroxide in the presence of oxygen using betaine aldehyde as a substrate (second reaction). Reaction).
  • Acyl CoA oxidase is classified as EC 1.3.3.6, and is an enzyme that catalyzes a reaction of producing trans-2,3-dehydroacyl CoA and hydrogen peroxide in the presence of oxygen using acyl CoA as a substrate.
  • the origin of the galactose oxidase, uricase, cholesterol oxidase, choline oxidase, and acyl CoA oxidase used in the present invention is not particularly limited as long as they have the above enzyme action. It may be a natural product, chemically or biochemically synthesized, or may be produced by genetic engineering. In the present invention, commercially available products can be suitably used.
  • the cysteine derivative (II), which is a raw material, may be prepared by any method. It is a compound prepared by the manufacturing method of a cysteine derivative.
  • the reaction between the cysteine derivative (II) and glucose as a substrate is usually carried out in an aqueous solvent in the presence of oxygen. Preferably, it is carried out by circulating air in the reaction vessel.
  • the aqueous solvent is not particularly limited as long as it does not inhibit the enzyme reaction, but may include physiological saline, potassium phosphate buffer, Tris-hydrochloric acid buffer, glycine-sodium hydroxide buffer, boric acid-sodium hydroxide buffer, and the like. Can be mentioned.
  • the concentration of glucose in the reaction solution is not particularly limited, but is usually 0.1 to 10% by weight, preferably 0.1 to 1% by weight. If desired, glucose can be added during the reaction, and is preferably added.
  • the concentration of the cysteine derivative (II) in the reaction solution varies depending on the type of compound used, but is not particularly limited as long as it does not inhibit the enzyme reaction. Usually, it is 0.1 to 10% by weight, preferably 0.1 to 1% by weight.
  • the amount of glucose oxidase used in the reaction can be appropriately adjusted depending on the substrate concentration, reaction time, and other conditions, but is preferably 0.001 U to 100 U, preferably 0.01 U to 50 U, more preferably 0.8. Add in the range of 1U to 10U.
  • the reaction is carried out at a pH of 5 to 7, preferably 5.5 to 6.5.
  • the reaction is preferably carried out at 4 ° C. to 60 ° C., but is particularly preferably carried out at 30 ° C. to 50 ° C., more preferably 35 to 45 ° C. in terms of enzyme stability and reaction rate.
  • the reaction time is appropriately set according to the synthesis amount of the desired cysteine sulfoxide derivative (III), but is usually 0.5 to 24 hours, preferably 1 to 10 hours, and more preferably 1 to 5 hours.
  • a known method can be used as a method for separating and recovering the cysteine sulfoxide derivative (III) from the reaction solution obtained by the above reaction.
  • a known method can be used.
  • TCA trichloroacetic acid
  • ultrafiltration concentration method e.g., ultrafiltration concentration method
  • ammonium sulfate precipitation method salting out
  • organic solvent eg, acetone, ethanol, propanol, methanol, etc.
  • water-soluble polymer eg, polyethylene) Glycol, dextran, etc.
  • Example 1 Identification of Enzyme Involved in S-Allylcysteine (ALC) Production
  • AAC S-Allylcysteine
  • a 50 mM potassium phosphate buffer (pH 7.4) was used as a reaction buffer, and S-allyl mercaptan and ammonium sulfate / pyruvic acid were used as substrates.
  • the produced ALC was precipitated with 10% TCA and quantified by LC / MS.
  • (3) Results The results of the amount of ALC produced after the reaction for 2 hours are shown in FIG. No ALC was produced in the ⁇ CYS4 mutant. ALC production was significantly increased in the ⁇ CYS3 mutant. From these results, the CYS4 gene was considered to be a gene encoding an enzyme involved in ALC generation. Furthermore, it was considered that the CYS3 gene is a gene encoding an enzyme involved in ALC degradation, or that CYS4 expression may be negatively affected.
  • Example 2 Production of S-allyl cysteine by purified cystathionine ⁇ -synthase
  • CYS4 gene was assumed to be a gene encoding an enzyme involved in ALC production, in this example, CYS4 gene was highly expressed. It was verified whether purified cystathionine ⁇ -synthase obtained from the mutant strain produced S-allylcysteine. 1. Preparation of purified cystathionine ⁇ -synthase First, a Saccharomyces cerevisiae strain that highly expresses CYS4 was produced by the following procedure.
  • Saccharomyces cerevisiae BY4742 was used, including the CYS3 gene disruption strain BY4742 cys3 ⁇ , obtained from Thermo Scientific, Yeast Knockout Clones and Collections.
  • the CYS4 gene was augmented by introducing an integration type plasmid. The method for preparing the integration plasmid was as follows.
  • An integration-type plasmid for highly expressing the CYS4 gene is prepared by first preparing a multi-copy plasmid expressing the CYS4 gene from the TDH3 gene (Glyceraldehyde-3-phosphate dehydrogenase) promoter (TDH3p), which is a constitutive high expression promoter.
  • TDH3p Glyceraldehyde-3-phosphate dehydrogenase promoter
  • the TDH3p-CYS4 fragment amplified using as a template was cloned into the pUC19 plasmid (Invitrogen).
  • DH5- ⁇ or JM109 strain (Takara Bio) was used as an E. coli competent cell in the process of plasmid preparation.
  • a wild-type Saccharomyces cerevisiae S288C (ATCC 204508) genomic DNA was used as a template using a primer represented by SEQ ID NO: 1 having a Sma I restriction enzyme recognition sequence and a primer represented by SEQ ID NO: 2 having an Xba I restriction enzyme recognition sequence.
  • SEQ ID NO: 1 having a Sma I restriction enzyme recognition sequence
  • SEQ ID NO: 2 having an Xba I restriction enzyme recognition sequence.
  • PCR was performed to obtain a DNA fragment having a TDH3 promoter.
  • PCR conditions were denaturation (94 ° C., 10 sec), annealing (60 ° C., 10 sec), extension (72 ° C., 4 min), 25 cycles.
  • this DNA fragment was cloned into the Sma I- Xba I site of pYES2 to obtain plasmid pYES2-TDH3p (all restriction enzymes used in this example were obtained from Takara Bio Inc.).
  • PCR was performed using the wild-type Saccharomyces cerevisiae S288C (ATCC 204508) genomic DNA as a template, using the primer represented by SEQ ID NO: 3 and the primer represented by SEQ ID NO: 4 to which a base sequence encoding 6 copies of histidine was added.
  • a CYS4 DNA fragment having 6 copies of His-tag at the C-terminus of the gene was obtained.
  • PCR conditions were denaturation (94 ° C., 10 sec), annealing (60 ° C., 10 sec), extension (72 ° C., 4 min), 25 cycles.
  • this DNA fragment was digested with the restriction enzyme Xba I and linearized into pYES2-TDH3p by the in-fusion cloning method (Clontech, In-Fusion (registered trademark) HD Cloning Kit), the plasmid pYES2-TDH3p- CYS4_6xHis was obtained.
  • the TDH3p-CYS4_6xHis fragment was amplified using the pYES2-TDH3p-CYS4_6xHis plasmid as a template with the primer shown in SEQ ID NO: 7 and the primer shown in SEQ ID NO: 8.
  • Each DNA fragment was introduced into the SphI-EcoRI site of pUC19-URA3 by the in-fusion cloning method to prepare an integration type plasmid, pUC19-TDH3p-CYS4-URA3.
  • CYS4 high expression expression type plasmid pUC19-TDH3p-CYS4-URA3 was linearized by digestion with BglII, and BY4742 or BY4742 cys3 ⁇ , which is a uracil-requiring strain, was transformed to produce a CYS4 high expression strain . Transformation was performed using the Frozen-EZ Yeast Transformation II Kit (Zymo Research), and the cells into which the integration plasmid was introduced were selected by growing the cells on a plate medium not containing uracil.
  • the amount of ALC produced from S-allyl mercaptan was measured using the obtained purified cystathionine ⁇ -synthase (2.64 mg / mL; 200 ⁇ L). 200 ⁇ L of enzyme solution, serine solution (0.158 g / 10 mL, 1760 ⁇ L), 10 mM pyridoxal phosphate (20 ⁇ L) and S-allyl mercaptan (20 ⁇ L) were reacted at 30 ° C. and 50 rpm. A 50 mM potassium phosphate buffer (pH 7.4) was used as a reaction buffer.
  • the cystathionine ⁇ -synthase activity was 109 U per 1 g of the reaction solution.
  • Example 3 Production of S-methylcysteine by purified cystathionine ⁇ -synthase Using purified cystathionine ⁇ -synthase (0.23 mg / mL; 100 ⁇ L) obtained in Example 2, S-methylcysteine from S-methyl mercaptan The amount produced was measured. 100 ⁇ L of enzyme solution, serine solution (0.158 g / 10 mL, 1760 ⁇ L), 10 mM pyridoxal phosphate (20 ⁇ L) and 15% methyl mercaptan (120 ⁇ L) were reacted at 30 ° C. and 50 rpm. A 50 mM potassium phosphate buffer (pH 7.4) was used as a reaction buffer.
  • the cystathionine ⁇ -synthase activity was 19 U per 1 g of the reaction solution.
  • the amount of S-methylcysteine produced was evaluated.
  • the produced S-methylcysteine was stopped by adding 10% TCA, and quantified by LC / MS. The results are shown in FIG. It was confirmed that S-methylcysteine was produced from S-methyl mercaptan and L-serine by cystathionine ⁇ -synthase.
  • Example 4 Production of S-propylcysteine by purified cystathionine ⁇ -synthase Using purified cystathionine ⁇ -synthase (0.23 mg / mL; 100 ⁇ L) obtained in Example 2, S-propylcysteine from S-propyl mercaptan The amount produced was measured. 100 ⁇ L of enzyme solution, serine solution (0.158 g / 10 mL, 1760 ⁇ L), 10 mM pyridoxal phosphate (20 ⁇ L) and propyl mercaptan (20 ⁇ L) were reacted at 30 ° C. and 50 rpm.
  • a 50 mM potassium phosphate buffer (pH 7.4) was used as a reaction buffer.
  • the cystathionine ⁇ -synthase activity was 19 U per 1 g of the reaction solution.
  • the amount of S-propylcysteine produced was evaluated.
  • the produced S-propylcysteine was stopped by adding 10% TCA, and quantified by LC / MS. The results are shown in FIG. It was confirmed that S-propylcysteine was produced from S-propyl mercaptan and L-serine by cystathionine ⁇ -synthase.
  • Example 5 Production of S-allyl cysteine sulfoxide (ALCSO) with glucose oxidase (GO)
  • ALCSO glucose oxidase
  • the reaction for producing ALCSO from glucose and S-allyl cysteine (ALC) was investigated for the three test zones described in Table 2.
  • the ALC prepared in Example 1 was used.
  • As the GO a food additive “Sumiteam PGO” (Shin Nihon Chemical Co., Ltd.) was used.
  • the reaction was carried out at 40 ° C., pH 6.0 (50 mM potassium phosphate buffer), 300 rpm for 5 hours. Further, air was aerated from the nozzle having a nozzle diameter of 10 to 50 ⁇ m at an air flow rate of 1000 mL / min in the reaction vessel.
  • Test Zone 1 only aeration was performed without adding GO.
  • test group 2 GO was added.
  • test group 3 0.23% of GO and 0.16% of glucose were added 1 hour after the reaction. The results are shown in FIG. In test section 1 to which no GO was added, almost no ALCSO was produced. In test group 2 to which GO was added, gluconic acid was produced, but reached a plateau in 1 hour of reaction. A similar trend was seen in ALC consumption and ALCSO production. It was estimated that the decrease in hydrogen peroxide supply due to the decrease in reactivity had an effect. However, by adding GO and glucose 1 hour after the reaction (test group 3), the conversion of ALC to ALCSO proceeded again and could be terminated.
  • Example 6 Production of S-allyl cysteine sulfoxide (ALCSO) using a CYS4 gene high-expression yeast mutant strain Using the CYS4 gene high-expression mutant strain prepared in Example 2 in the same manner as in Example 1 (2) Incubate. After culturing, the cells are collected (collected), and the cells are crushed to prepare an extract. Substrates such as L-serine and S-allyl mercaptan are added to this extract (yeast extract: enzyme source) to produce ALC (step I). Glucose oxidase is added to the reaction solution to oxidize glucose, and ALC is converted to ALCSO (step II) to obtain a yeast extract containing ALCSO.
  • ALCSO S-allyl cysteine sulfoxide
  • Example 7 Generation of S-allyl cysteine sulfoxide (ALCSO) using a mutant strain with reduced CYS3 gene activity
  • a wild strain of a cystathionine ⁇ -synthase-producing microorganism was subjected to mutation treatment by irradiation with ultraviolet light.
  • a mutant strain with reduced CYS3 gene activity is obtained by screening a cysteine-requiring strain from among the mutant strains.
  • the yeast extract containing ALCSO is obtained by implementing the process I and the process II like Example 6 using the obtained mutant strain.
  • Example 8 Evaluation of ALC accumulation ability of each gene-disrupted strain and high-expressing strain Using BY4742 as a parent strain, disruption of CYS3 gene (abbreviated as cys3 ⁇ ), high expression of CYS4 gene (abbreviated as CYS4 ⁇ ), disruption of CYS3 gene and The effect of high expression of CYS4 gene (abbreviated as cys3 ⁇ + CYS4 ⁇ ) was verified.
  • the parent strain and each mutant strain were obtained from Thermo Scientific, Yeast Knockout Clones and Collections, or prepared in the same manner as in Example 2. A crude enzyme solution was prepared from each strain by the method shown in FIG. 6, and an ALC generation test was performed under the reaction conditions shown in FIG.
  • the cystathionine ⁇ -synthase activity (per gram of yeast extract solids) of the crude enzyme solution of each strain is 2500 U or more, particularly 5000 U or more in the CYS4 gene high-expression mutant, and the CYS4 gene is further disrupted.
  • cystathionine ⁇ -synthase activity per gram of yeast extract solids
  • cystathionine ⁇ -synthase activity was 23312U.
  • the mutant strain in which the CYS3 gene was disrupted had 0 U of cystathionine ⁇ -lyase activity (per gram of yeast extract solids).
  • the amount of ALC accumulated per dry weight of yeast used was measured.
  • the dry weight (abbreviated as DM) is measured as follows. (Measurement method) The empty weight of the weighing bottle (# 6-743-01) was measured with an electronic balance, and the weight after 1 ml of yeast cell suspension was placed in the weighing bottle was measured with an electronic balance. Thereafter, the weight after drying at 105 ° C. for 1 hour in an oven (DN600 YAMATO) was measured. DM (%) is measured from the weight ratio before and after oven drying.
  • the ALC accumulation ability of each strain is shown in FIG. Compared with the parent strain, ALC accumulated significantly due to CYS3 gene disruption or CYS4 gene high expression. In addition, the accumulation of ALC significantly increased due to the CYS3 gene disruption and the high expression of the CYS4 gene.
  • a cysteine derivative and a cysteine sulfoxide derivative which are expected as raw materials for pharmaceutical intermediates and food additives, can be produced more efficiently and stably.
  • SEQ ID NO: 1 PCR primer SEQ ID NO: 2: PCR primer SEQ ID NO: 3: PCR primer SEQ ID NO: 4: PCR primer SEQ ID NO: 5: PCR primer SEQ ID NO: 6: PCR primer SEQ ID NO: 7: PCR primer SEQ ID NO: 8: PCR primer

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Abstract

La présente invention concerne un procédé de réaction d'un composé de thiol représenté par la formule (I) (R dans la formule est tel que défini dans la description) avec une L-sérine en présence d'une cystathionine β-synthase pour obtenir un dérivé de cystéine représenté par la formule (II) (R dans la formule est tel que défini ci-dessus), et un procédé de réaction du dérivé de cystéine avec une oxydase et un substrat pour celle-ci pour obtenir un dérivé de cystéine sulfoxyde représenté par la formule (III) (R dans la formule est tel que défini ci-dessus). Ces procédés permettent d'obtenir le dérivé de cystéine et le dérivé de cystéine sulfoxyde de manière stable à l'échelle industrielle et en utilisant des moyens qui sont sans danger pour une utilisation dans des produits alimentaires.
PCT/JP2016/066974 2015-07-27 2016-06-07 Procédé de production d'un dérivé de cystéine et d'un dérivé de cystéine sulfoxyde WO2017018060A1 (fr)

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CN110004134A (zh) * 2019-05-21 2019-07-12 福州大学 一种褐藻胶裂解酶突变体及其应用
CN114916561A (zh) * 2022-05-27 2022-08-19 山东大学 一种防治作物病原菌的生物农药
CN115725556A (zh) * 2022-09-20 2023-03-03 青岛硕景生物科技有限公司 一种稳定性提高的突变胱硫醚β裂解酶及其制备方法和应用

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Publication number Priority date Publication date Assignee Title
CN110004134A (zh) * 2019-05-21 2019-07-12 福州大学 一种褐藻胶裂解酶突变体及其应用
CN114916561A (zh) * 2022-05-27 2022-08-19 山东大学 一种防治作物病原菌的生物农药
CN114916561B (zh) * 2022-05-27 2022-11-18 山东大学 一种防治作物病原菌的生物农药
CN115725556A (zh) * 2022-09-20 2023-03-03 青岛硕景生物科技有限公司 一种稳定性提高的突变胱硫醚β裂解酶及其制备方法和应用
CN115725556B (zh) * 2022-09-20 2024-05-03 青岛硕景生物科技有限公司 一种稳定性提高的突变胱硫醚β裂解酶及其制备方法和应用

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