WO2016013570A1 - Procédé de production de putrescine par culture bactérienne mixte - Google Patents

Procédé de production de putrescine par culture bactérienne mixte Download PDF

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WO2016013570A1
WO2016013570A1 PCT/JP2015/070812 JP2015070812W WO2016013570A1 WO 2016013570 A1 WO2016013570 A1 WO 2016013570A1 JP 2015070812 W JP2015070812 W JP 2015070812W WO 2016013570 A1 WO2016013570 A1 WO 2016013570A1
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microorganism
arginine
culture
putrescine
pathway
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Japanese (ja)
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雄祐 北田
松本　光晴
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協同乳業株式会社
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    • 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
    • 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
    • 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
    • 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
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the present invention relates to a method for producing putrescine using a microorganism having an arginine-dependent acid resistance mechanism and a microorganism having an AgDI pathway (agmatin deiminase metabolic pathway).
  • the present invention also relates to a method for producing putrescine using an ornithine-producing microorganism and a microorganism having an ornithine decarboxylase (iODC) that works inductively with an ornithine putrescine antiporter.
  • iODC ornithine decarboxylase
  • Polyamine is a general term for aliphatic hydrocarbons having two or more primary amino groups
  • putrescine (1,4-diaminobutane) is one of the simplest polyamines.
  • Polyamines produced by intestinal bacteria are strongly related to the health of the host, such as having an intestinal barrier function enhancing action, antimutagenicity, and antioxidant action. Elucidation of the route is an important theme.
  • research on the polyamine biosynthesis system of intestinal bacteria has been conducted on individual bacterial species, but the polyamine biosynthesis pathway has been specified only for some pathogenic bacteria.
  • the mechanism of polyamine release to the outside of the fungus body (inside the intestinal lumen) and its physiological significance (why it is released outside the fungus body) have not been elucidated in those fungus species.
  • the polyamine-rich food material present in the current market is an extract from plants (Patent Document 1, Patent Document 2) and yeast (Patent Document 3).
  • Patent Document 1 Patent Document 2
  • Patent Document 3 yeast
  • methods for producing putrescine using microorganisms include an example using one kind of microorganism (Patent Document 4) and an example using a mutant microorganism subjected to genetic recombination (Patent Document 5).
  • Patent Document 4 an example using one kind of microorganism
  • Patent Document 5 an example using a mutant microorganism subjected to genetic recombination
  • the present invention provides a new method for producing putrescine satisfying such conditions.
  • the inventor of the present case noted that the in vivo study confirmed that the most abundant putrescine present in the human intestinal tract was induced in a concentration-dependent manner by oral administration of the precursor arginine. Started the research. As a result of intensive studies, we found that mixed culture produced more putrescine than single strains, and hypothesized that putrescine was biosynthesized and released in the intestinal tract by the joint action of multiple bacteria rather than a single bacterium. Stood up. They found a combination of bacteria that produced putrescine from arginine. Based on this finding, the present invention has been completed.
  • the present invention may be as follows. [1] A method for producing putrescine, wherein a microorganism having an arginine-dependent acid resistance mechanism and a microorganism having an AgDI pathway (agmatin deiminase metabolic pathway) are mixed and cultured in the presence of arginine under acidic conditions. Said method.
  • the step of mixed culture includes (A) culturing a microorganism having an arginine-dependent acid-resistant mechanism under acidic conditions in the presence of arginine; and (B) A microorganism having an AgDI pathway (agmatin deiminase metabolic pathway) is added to the culture of (A) and mixed and cultured; The method according to [1], wherein [3] The method according to [1] or [2], wherein the microorganism having an arginine-dependent acid resistance mechanism is a microorganism belonging to the family Enterobacteriaceae.
  • the microorganism having the AgDI pathway is a microorganism belonging to the family Enterococcuaceae, Streptococcaceae, Lactobacillaceae, Pseudomonadaceae, any one of [1] to [4] The method according to item.
  • a method for producing putrescine comprising the following steps: (1) culturing a microorganism having an arginine-dependent acid resistance mechanism under acidic conditions in the presence of arginine; (2) obtaining a culture supernatant from the culture of step (1); (3) A microorganism having an AgDI pathway (agmatin deiminase metabolic pathway) is added to the culture supernatant of step (2) and cultured; and (4) putrescine is separated from the culture solution of step (3); Said method.
  • a method for producing putrescine comprising the step of mixing and culturing a microorganism producing ornithine and a microorganism having iODC which is ornithine decarboxylase.
  • the ornithine-producing microorganism is a microorganism having an ADI pathway (arginine deiminase metabolic pathway), and the mixed culture is performed in the presence of arginine.
  • the microorganism having iODC is Escherichia coli having iODC.
  • the conditions of the mixed culture are the following (a) to (c): (A) pH 4.5 to pH 7.5; (B) arginine is present in the range of 0.1 mM to 500 mM; and (c) aerobic conditions;
  • putrescine By using the joint action of two or more types of microorganisms, putrescine can be produced more efficiently than when one type of microorganism is used. Further, since the method of the present invention does not use a gene recombinant, it is highly safe from the viewpoint of production of food materials.
  • FIG. 1 is a schematic view of a method for producing putrescine by two types of microorganisms, a microorganism having an arginine-dependent acid resistance mechanism and a microorganism having an AgDI pathway.
  • E. coli was exemplified as a microorganism having an arginine-dependent acid resistance mechanism
  • En. Faecalis was exemplified as a microorganism having an AgDI pathway.
  • FIG. 2 is a schematic view of a method for producing putrescine by a microorganism having an ADI pathway and a microorganism having iODC.
  • En. Faecalis was exemplified as a microorganism having an ADI pathway, and E.
  • FIG. 3 is a graph showing the difference between strains in the ability to produce putrescine from ornithine of E. coli.
  • FIG. 4 is a graph showing the results of evaluating the amount of putrescine produced from arginine when combining E. coli and En. Faecalis strains.
  • A The result when combining En. Faecalis JCM5803 and any of four strains of E. coli (JCM5491, JCM1649, JCM1246 or MG1655) is shown.
  • FIG. 5 is a graph showing the effect of ammonia concentration on putrescine production from ornithine of E. coli.
  • FIG. 6 is a graph showing the effect of magnesium hydrogen phosphate on the production of putrescine from arginine during E. coli and En. Faecalis mixed culture.
  • FIG. 7 is a graph showing changes in the concentrations of arginine, ornithine and putrescine during E. coli and En. Faecalis mixed culture in a 1.2 M magnesium hydrogen phosphate-added medium.
  • the present invention relates to a method for producing putrescine using the joint action of two or more kinds of microorganisms.
  • the present application provides a method for producing putrescine using the cooperative action of different microorganisms, wherein one of the different microorganisms has one of the metabolic pathways and the other has the other metabolic pathway.
  • the present invention relates to a method for producing putrescine using a microorganism having an arginine-dependent acid tolerance mechanism and a microorganism having an AgDI pathway (agmatin deiminase metabolic pathway).
  • the arginine-dependent acid resistance mechanism is a mechanism possessed by some microorganisms, which absorbs arginine and releases agmatine under acidic conditions.
  • the AgDI pathway is an agmatine metabolic pathway possessed by some microorganisms, and is a pathway that, after absorbing agmatine, decomposes agmatine using agmatine deiminase and produces putrescine via carbamoylputrescine.
  • the inventors of the present invention have found that a microorganism having an AgDI pathway biosynthesizes and releases putrescine using a metabolite produced by a microorganism having an arginine-dependent acid-resistant mechanism. That is, as illustrated in FIG. 1, it has been found that a microorganism having an arginine-dependent acid-resistant mechanism produces agmatine from arginine, and a microorganism having an AgDI pathway produces putrescine using the produced agmatine. It was.
  • the present application provides a method for producing putrescine, comprising a step of mixing and culturing a microorganism having an arginine-dependent acid resistance mechanism and a microorganism having an AgDI pathway under acidic conditions in the presence of arginine.
  • Microorganisms having an arginine-dependent acid resistance mechanism include, but are not limited to, microorganisms belonging to the family Enterobacteriaceae.
  • the microorganism having an arginine-dependent acid resistance mechanism is a microorganism belonging to the genus Escherichia or Salmonella.
  • Escherichia coli and Salmonella enterica are particularly preferable, and any one of these may be appropriately selected.
  • Salmonella enterica is closely related to E. coli and is known to have a metabolic pathway similar to that of E. coli (Brenneman, K. E., et al., (2013) J. Bacteriol., 195 (13) : 3062-3072).
  • Microorganisms having the AgDI pathway include, but are not limited to, microorganisms belonging to the family Enterococcuaceae, Streptococcaceae, Lactobacillaceae, Pseudomonadaceae Not. These microorganisms are known to have metabolic pathways similar to Enterococcus faecalis (Ladero, V., et al., (2011) Appl. Envion. Microbiol., 77 (18): 6409-6418; Nakada, Y., et al., (2001) J. Bacteriol., 183 (22): 6517-6524).
  • the microorganism having the AgDI pathway may be selected from Enterococcus faecalis, Lactococcus lactis, Lactobacillus brevis, and Pseudomonas aeruginosa.
  • the microorganism having the AgDI pathway is a microorganism belonging to the family Enterococcus.
  • the microorganism having the AgDI pathway is Enterococcus faecalis.
  • the microorganism is cultured in the presence of arginine.
  • the arginine concentration in the culture solution is not particularly limited, but may be 0.1 mM or more, 0.5 ⁇ mM or more, or 1.0 mM or more.
  • the upper limit of the arginine concentration is not particularly limited, but is 1.0 M or less, 800 mM or less, 600 mM or less, 400 mM or less, 200 mM or less, 150 mM or less, 100 mM or less, 75 mM or less, or 50 mM or less Good.
  • the upper and lower limits of the arginine concentration can be appropriately selected from the above ranges.
  • the concentration of arginine in the culture solution is 0.1 ⁇ m to 200 ⁇ m, 0.1 ⁇ m to 500 ⁇ m, 1.0 ⁇ m to 50 ⁇ m.
  • the microorganism is cultured under acidic conditions.
  • the acidic condition is not particularly limited as long as it is in the range of pH 4.0 or more and less than pH 7.0.
  • the lower limit of the acidic condition is pH 4.0 or more, pH 4.5 or more, pH 5.0 or more, or pH 5.5 or more, and the upper limit is less than pH 7.0, pH 6.5 or less, or pH 6.0. It is as follows.
  • the lower limit and the upper limit of the acidic condition can be appropriately selected from the above ranges.
  • the acidic conditions are pH 4.0 or higher and pH 6.0 or lower.
  • the microorganism is preferably cultured under anaerobic conditions, but is not limited thereto.
  • mixed culture may be performed using any medium available to those skilled in the art.
  • the method of the present invention it has been observed that the amount of putrescine production increases as the number of bacteria in the culture increases. From this point of view, a medium in which a medium component sufficiently contains components necessary for the growth of microorganisms is preferable.
  • the mixed culture may be performed by batch culture (that is, a method in which medium components are not added during culture) or fed-batch culture (that is, a method in which medium components are continuously added during culture). Good. Batch culture has the advantage that it can be industrialized relatively easily.
  • fed-batch culture since the culture medium components are continuously added during the culture, the number of bacteria in the culture solution generally increases. For this reason, increase in putrescine production can be expected by using fed-batch culture.
  • a person skilled in the art can appropriately select the type of medium, the amount of medium components, the culture method, and the like in consideration of various situations.
  • the time for performing the mixed culture is not particularly limited, and those skilled in the art can appropriately set by sampling the mixed culture and measuring the amount of putrescine in the culture.
  • the culture time of the mixed culture may be, for example, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours.
  • the mixed culture may be performed in any order as long as the microorganism having an arginine-dependent acid-resistant mechanism and the microorganism having the AgDI pathway are both present during the culture. Absent.
  • a microorganism having an arginine-dependent acid resistance mechanism is first cultured, and a microorganism having an AgDI pathway is added later and mixed and cultured.
  • the mixed culture process is performed as follows: (A) culturing a microorganism having an arginine-dependent acid-resistant mechanism under acidic conditions in the presence of arginine; and (B) A microorganism having an AgDI pathway (agmatin deiminase metabolic pathway) is added to the culture of (A) and mixed and cultured; May be performed.
  • a microorganism having an AgDI pathway agmatin deiminase metabolic pathway
  • the time for culturing in the above step (A) is not particularly limited, and may be, for example, 6 hours, 12 hours, 24 hours, or 48 hours.
  • the time which performs the mixed culture of the said process (B) is not specifically limited, For example, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours may be sufficient.
  • the method for producing putrescine of the present invention may further include a step of separating putrescine from the culture solution of the mixed culture.
  • step of separating putrescine putrescine is separated and / or purified from the culture solution obtained in the mixed culture step.
  • Putrescine is contained in the culture solution, and the cells may be separated from the culture solution as a pretreatment for purification, and the separated cells may be reused.
  • Separation of the bacterial cells from the culture solution may be performed by any method including known methods. For example, the separation may be performed by centrifugation, a filtration filter, a bacterial cell immobilization technique, or the like. Separation of putrescine from the culture solution may be performed by any method including known methods.
  • UPLC ultra high performance liquid chromatography
  • HPLC high performance liquid chromatography
  • GC gas chromatography
  • capillary electrophoresis capillary electrophoresis
  • ion chromatography gel filtration, centrifugation, etc.
  • purified putrescine may be obtained as a result of the step of separating putrescine, or a culture solution from which the cells have been removed may be obtained as a crude putrescine purified product.
  • the application provides the following steps: (1) culturing a microorganism having an arginine-dependent acid resistance mechanism under acidic conditions in the presence of arginine; (2) obtaining a culture supernatant from the culture of step (1); (3) A microorganism having an AgDI pathway is added to the culture supernatant of step (2) and cultured; and (4) putrescine is separated from the culture solution of step (3); A method for producing putrescine is provided.
  • the microorganisms having an arginine acid resistance mechanism the microorganisms having an AgDI pathway, the culture conditions such as the amount of arginine and acidic conditions, and the range intended as a method for separating putrescine, As described in the embodiment.
  • the culture time is not particularly limited, and may be, for example, 6 hours, 12 hours, 24 hours, 48 hours.
  • the time for culturing in the step (3) of the second aspect is not particularly limited.
  • a person skilled in the art can appropriately set by sampling the mixed culture solution and measuring the amount of putrescine in the culture solution. For example, it may be 6 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours.
  • the method for obtaining the culture supernatant may be performed by any method including a known method.
  • it can be performed by centrifugation, filter sterilization, a technique using a cell immobilization technique, or the like.
  • the present invention relates to a method for producing putrescine using a microorganism producing ornithine and a microorganism having iODC which is ornithine decarboxylase. Specifically, the method includes a step of culturing a microorganism that produces ornithine and a microorganism having iODC.
  • the ornithine-producing microorganism is not particularly limited as long as it has the characteristic that ornithine is produced, but it may be a wild-type microorganism that produces ornithine, or a microorganism that has been modified to improve the production of ornithine (for example, And microorganisms described in JP-A-2013-544532).
  • Wild-type microorganisms that produce ornithine include microorganisms having the ADI pathway described later, microorganisms belonging to the genus Corynebacterium, Saccharomyces, Candida, or Pichia. included.
  • the present invention relates to a method for producing putrescine using a microorganism having an ADI pathway (arginine deiminase metabolic pathway) and a microorganism having iODC which is an ornithine decarboxylase. That is, as exemplified in FIG. 2, ornithine is produced from arginine by a microorganism having an ADI pathway, and a microorganism having iODC produces putrescine using the produced ornithine.
  • ADI pathway arginine deiminase metabolic pathway
  • iODC which is an ornithine decarboxylase
  • the ADI pathway is a pathway possessed by some microorganisms, and is a metabolic pathway in which arginine is absorbed, then arginine is decomposed using arginine deiminase, and ornithine is produced via citrulline (Simon, J. P. Et al., Journal of Bacteriology, (1982), 150 (3): 1085-1090).
  • IODC is one of the ornithine decarboxylases and is encoded by the SpeF gene.
  • iODC works inductively with ornithine and putrescine antiporter when ornithine is present outside the cells, and biosynthesizes putrescine from the incorporated ornithine. The biosynthesized putrescine is released outside the cells (Kashiwagi, K., et al., The Journal of Biological Chemistry, (1991), 266 (31): 20922-20927).
  • the present application provides a method for producing putrescine, comprising a step of culturing a microorganism having an ADI pathway and a microorganism having iODC in the presence of arginine.
  • Microorganisms having the ADI pathway include microorganisms belonging to the family Enterococcus, Streptococcus, Lactobacillus, Pseudomonas, Vibrionaceae, Streptomycetaceae, or Mycobacteriaceae, It is not limited to this.
  • the microorganism having the ADI pathway is a microorganism belonging to the family Enterococcus, more preferably Enterococcus faecalis.
  • the microorganism having iODC is not particularly limited as long as it is a microorganism expressing iODC.
  • the microorganism having iODC is E. coli having iODC, more preferably E. ⁇ ⁇ ⁇ ⁇ coli JCM5491 (ATCC25922) or E. coli JCM1649 (ATCC11775).
  • the microorganism is cultured in the presence of arginine.
  • concentration of arginine in the culture solution is not particularly limited, but may be 0.1 ⁇ mM or higher, 0.5 ⁇ mM or higher, 1.0 ⁇ mM or higher, 50 ⁇ mM or higher, 100 ⁇ mM or higher, 200 ⁇ mM or higher.
  • the upper limit of the arginine concentration is not particularly limited, but may be 1.0 M or less, 800 mM or less, 600 mM or less, or 400 mM or less.
  • the upper and lower limits of the arginine concentration can be appropriately selected from the above ranges.
  • the concentration of arginine in the culture solution is 0.1 to 500 ⁇ m, 200 to 400 ⁇ m.
  • Culture in the presence of arginine may be performed by culturing in a medium to which arginine has been added, and / or by adding a microorganism that produces arginine and performing mixed culture.
  • arginine may be added to the medium in advance and / or arginine may be appropriately added during the culture.
  • the mixed culture by adding microorganisms that produce arginine means that mixed culture of at least three kinds of microorganisms, that is, microorganisms having an ADI pathway, microorganisms having iODC, and microorganisms that produce arginine is performed.
  • the microorganisms that produce arginine are not particularly limited as long as they produce arginine.
  • the microorganisms include genus Corynebacterium, Brevibacterium, Bacillus, Serratia, Escherichia or Saccharomyces. The microorganism to which it belongs is mentioned.
  • the microorganism is cultured under acidic conditions or under weakly acidic to neutral conditions.
  • the acidic condition is not particularly limited as long as it is in a range of pH 4.0 or more and less than pH 7.0.
  • the mildly acidic to neutral conditions are not particularly limited as long as they are in the range of pH 5.0 or more and pH 7.5 or less.
  • the microorganism is cultured under mildly acidic to neutral conditions, more preferably at a pH in the range of 6.0 to 7.5.
  • the microorganism may be cultured under either aerobic conditions or anaerobic conditions.
  • the mixed culture is performed under aerobic conditions.
  • the type of medium, the amount of medium components, the culture method, etc. in the mixed culture process are described in I. And can be appropriately selected by those skilled in the art in consideration of various situations.
  • the culture time for mixed culture is the same as that described in I. above. As described above.
  • primary magnesium phosphate Mg (H 2 PO 4 ) 2
  • secondary magnesium phosphate magnesium hydrogen phosphate (MgHPO 4 )
  • tertiary magnesium phosphate Mg 3 (PO 4) 2
  • mixed culture may be performed using a medium to which an ammonia removing agent selected from the group consisting of magnesium pyrophosphate (Mg 2 P 2 O 7 ) is added.
  • an ammonia removing agent selected from the group consisting of magnesium pyrophosphate (Mg 2 P 2 O 7 ) is added.
  • An example of a preferred ammonia scavenger is magnesium hydrogen phosphate.
  • the ammonia removing agent is used to remove ammonia generated when ornithine is produced from arginine by a microorganism having an ADI pathway.
  • the concentration of the ammonia removing agent added to the medium is 400 mM or more, preferably 600 mM or more, 700 mM or more, or 800 mM or more.
  • the upper limit of the ammonia removing agent concentration is not particularly limited, but is, for example, 2.0 M or less and 1.5 M or less.
  • the lower limit and the upper limit of the concentration of the ammonia removing agent can be appropriately selected from the above ranges.
  • the concentration of the ammonia removal agent added to the medium is 400 mM to 1.5 M, 600 mM to 1.5 M, or 800 mM to 1.5 M.
  • the above-described method for producing putrescine may further include a step of separating putrescine from the culture solution of the mixed culture.
  • a step of separating putrescine the above-mentioned I.I. As described in.
  • Example 1 Search for combinations of microorganisms that produce putrescine from arginine> A search was made for a combination of two bacterial species having the largest amount of putrescine production among the 14 intestinal bacterial species shown in Table 1.
  • Example 2 Influence of pH of culture solution> Of the eight combinations of bacterial species found in Example 1, the combination of E. coli and Enterococcus faecalis that produced the largest amount of putrescine was used as a model case in the culture solution. Changes in the amount of putrescine production due to the difference in pH were examined.
  • E. coli and En. Faecalis were treated in the same manner as in Example 1 in a culture solution supplemented with arginine at different pH (pH 5.0, pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5).
  • the mixed culture was performed, and the putrescine concentration in the culture medium was measured.
  • the amount of putrescine produced by the mixed culture increased in the weakly acidic range (pH 6.0, pH 5.5, pH 5.0) rather than the neutral range (pH 7.0 and 7.5).
  • Example 3 Two-stage culture> The following experiment was conducted for the purpose of confirming whether metabolites were transported between E. coli and Enterococcus faecalis.
  • Example 4 Putrescine production by mixed culture of two bacterial species> The following experiment was conducted using Escherichia coli MG1655 strain (ATCC700926) and Enterococcus faecalis V583 strain (ATCC700802) (both purchased from ATCC (purchased from American Type Culture Collection)).
  • (1) Medium The following medium was used. The culture conditions are anaerobic culture at 37 ° C. unless otherwise specified.
  • putrescine can be produced in a high yield by using a combination of E. coli and E.cofaecalis regardless of the strain type.
  • Example 5 Increase in yield by examining culture conditions>
  • Example 4 is the same as Example 4 except that GAM bouillon medium (manufactured by Nissui Pharmaceutical Co., Ltd.) adjusted to pH (pH 4.0 to pH 7.0) by adding 50 mM arginine instead of A medium and B medium was used. A similar experiment was conducted. The composition of GAM bouillon is as shown in Table 6.
  • Example 6 Identification of E. coli retaining iODC activity> The E. coli strains (purchased from ATCC or RIKEN) shown in Table 8 were examined to retain iODC activity.
  • Example 7 Putrescine production from arginine by mixed culture of E. coli and En. Faecalis> Using the E. coli and En. Faecalis strains shown in Table 10, production of putrescine from arginine by mixed culture of E. coli and En. Faecalis was examined.
  • the basal medium was prepared as described in Example 6. Further, as a medium for main culture, a liquid medium in which 400 mM arginine was added to a basal medium was prepared. Each strain was precultured in basal medium for 24 hours. In the main culture, En. Faecalis is added to the main culture solution in a 1% amount of the pre-culture solution, E. coli is added to the main culture solution in an amount of 0.001%, and the aerobic condition is 37 ° C. It was performed by culturing under 30 hours. And the putrescine density
  • Example 8 Inhibition of iODC activity of E. coli by ammonia>
  • the production of putrescine from arginine by the route of FIG. 2 has been problematic in that the production of putrescine from ornithine in E. coli is suppressed with the progress of ornithine production from arginine by En. Faecalis.
  • En. Faecalis produces 2 moles of ammonia simultaneously when producing 1 mole of ornithine from arginine. It was thought that this ammonia inhibited putrescine production from ornithine. Therefore, the inhibition of iODC activity of E. coli by ammonia was examined using E. coli JCM5491.
  • the basal medium was prepared as described in Example 6.
  • a medium for main culture a liquid medium was prepared by adding 400 mM ornithine hydrochloride and 0 to 200 mM ammonium chloride to the basal medium.
  • the strain is precultured in a basal medium for 24 hours.
  • 1% of the preculture is added to the main culture, and cultured at 37 ° C. under aerobic conditions.
  • Ornithine concentration and putrescine concentration were measured.
  • Results It was found that when the ammonia concentration in the culture solution increased, the amount of decrease in ornithine concentration in the culture solution decreased and the putrescine concentration did not increase (FIG. 5). From this, it was confirmed that the ammonia concentration in the culture solution inhibited the iODC activity of E. coli.
  • Example 9 Effect of addition of magnesium hydrogen phosphate on putrescine production from arginine by mixed culture of E. coli and En. Faecalis>
  • the struvite is a crystal of magnesium ammonium hydrogen phosphate, and has the property that it is extremely difficult to dissolve in neutral to alkaline water. So far, it has been reported that ammonia is recovered as struvite in wastewater treatment (Japanese Patent Laid-Open No. 2006-289168).
  • we tried to improve the inhibition of E. coli iODC activity by adding magnesium hydrogen phosphate to the medium and reacting with the ammonia produced by En. Faecalis to precipitate it as struvite.
  • E. coli JCM1649 and En. Faecalis JCM8726, and E. coli JCM5491 and En. Faecalis JCM5803 are mixed and cultured in the presence of magnesium hydrogen phosphate to reduce the amount of putrescine production.
  • a basal medium was prepared in the same manner as in Example 6.
  • a medium for main culture a medium in which 400 mM arginine and 0, 200, 400, 600, or 800 mM magnesium hydrogen phosphate were added to a basal medium was prepared.
  • Faecalis as a by-product of ornithine for putrescine production from arginine by mixed culture of E. coli and En. Faecalis. As one of them, a method for precipitating and removing ammonia as struvite has been demonstrated.

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  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
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Abstract

L'invention concerne un procédé de production de putrescine utilisant une action coopérative d'au moins deux sortes de micro-organismes. Plus précisément, l'invention concerne un procédé de production de putrescine utilisant un micro-organisme qui a un mécanisme de résistance aux acides arginine-dépendant et un micro-organisme qui a une voie métabolique d'agmatine déiminase (AgDI) et un procédé de production de putrescine utilisant un micro-organisme qui produit de l'ornithine et un micro-organisme qui a de l'ornithine décarboxylase inductible (iODC) susceptible d'agir de manière inductible avec un antiport ornithine-putrescine.
PCT/JP2015/070812 2014-07-22 2015-07-22 Procédé de production de putrescine par culture bactérienne mixte WO2016013570A1 (fr)

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JP2014148824A JP2017158434A (ja) 2014-07-22 2014-07-22 バクテリア混合培養によるプトレッシン製造方法
JP2014-148824 2014-07-22

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CN115948316B (zh) * 2022-12-13 2024-03-22 四川大学 一种提升乳酸菌耐酸性的方法

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