WO2017169751A1 - Method for producing enzyme derived from psychrophilic or mesophilic microorganism - Google Patents

Abstract

A method for producing an enzyme derived from a psychrophilic or mesophilic microorganism, said method comprising: a first step for introducing, into a thermophilic microorganism having an optimum growth temperature of 50^oC or higher, a DNA encoding an enzyme derived from at least one kind of microorganism selected from among a psychrophilic microorganism and a mesophilic microorganism, the optimum growth temperature of which is 50^oC or lower and lower by at least 10^oC than the optimum growth temperature of the thermophilic microorganism,to thereby obtain a transformant; a second step for culturing and growing the transformant at a culture temperature of 50^oC or higher; and a third step for, after the second step, changing the culture temperature to 50^oC or lower and lower by at least 10^oC than the culture temperature in the second step to thereby express the enzyme.

Description

Process for producing thermophilic bacteria and enzymes derived from mesophilic bacteria

The present invention relates to a method for producing enzymes derived from thermophilic bacteria and mesophilic bacteria.

Enzymes are widely used in various fields such as foods, pharmaceuticals, cosmetics and functional materials because they can synthesize and decompose organic substances with low energy and do not generate by-products such as chemical synthesis. As a method for producing an enzyme, in addition to a method for extracting an enzyme in various organisms, a method for culturing a transformant in which a DNA encoding a target enzyme is introduced into a host microorganism and expressing the enzyme is known. As the host microorganism, Escherichia coli, yeast and the like have been widely used conventionally. However, in such a method using a host microorganism, in addition to the target enzyme, an enzyme derived from the host microorganism is also expressed and activated at the same time. Therefore, the expression of the target enzyme is controlled to make the enzyme efficient. Development of a method for manufacturing well is desired.

As a method for producing an enzyme using a transformant, for example, in Japanese Patent Application Laid-Open No. 2011-160778 (Patent Document 1), a mycobacteria is transformed using a gene encoding a thermostable enzyme. A production method including a step of obtaining a transformant and a step of obtaining an immobilized thermostable enzyme by heat-treating the transformant is described. However, the method described in Patent Document 1 is a method for producing a thermostable enzyme derived from a hyperthermophilic bacterium or a thermophilic bacterium, and the obtained enzyme has to be reacted at a high temperature.

JP 2011-160778 A

The present invention has been made in view of the above-described problems of the prior art, and separates the growth stage of transformants into which DNAs encoding enzymes derived from psychrophilic and mesophilic bacteria are introduced and the expression stage of the enzyme. It is an object of the present invention to provide a method for producing a thermophilic bacterium and an enzyme derived from a mesophilic bacterium.

As a result of intensive studies to achieve the above-mentioned object, the present inventors have found that thermophilic bacteria having an optimum growth temperature are high (50 ° C. or higher) and that the optimum growth temperature is low (50 ° C. or lower and the thermophilic). A DNA encoding an enzyme derived from at least one microorganism selected from the group consisting of a psychrotrophic bacterium and a mesophilic bacterium (lower than the optimum growth temperature of the bacterium by 10 ° C. or more). The transformant thus obtained is first cultured at a high temperature (50 ° C. or higher), and then cultured at a low temperature (50 ° C. or lower and 10 ° C. or higher). And the expression stage of the enzyme derived from the psychrophilic bacterium and the mesophilic bacterium are found out, and the present invention has been completed.

That is, the method for producing the psychrophilic and mesophilic bacterium-derived enzymes of the present invention comprises:
A group consisting of a thermophilic bacterium having an optimal growth temperature of 50 ° C. or higher, a thermophilic bacterium having an optimal growth temperature of 50 ° C. or lower and a temperature lower by 10 ° C. or more than the optimal growth temperature of the thermophilic bacterium A first step of obtaining a transformant by introducing DNA encoding an enzyme derived from at least one microorganism selected from:
A second step of culturing and growing the transformant at a culture temperature of 50 ° C. or higher;
After the second step, a third step of expressing the enzyme by changing the culture temperature to 50 ° C. or lower and a temperature lower by 10 ° C. or higher than the culture temperature of the second step;
Is included.

In the method for producing a thermophilic bacterium and mesophilic bacterium-derived enzyme of the present invention, the culture temperature in the second step is preferably 55 to 87 ° C., and the culture temperature in the third step is 25 to 45 ° C. It is preferable. Furthermore, in the method for producing a thermophilic bacterium and mesophilic bacterium-derived enzyme of the present invention, when the OD _{600 of} the culture medium for culturing the transformant is 1 or less, the culturing temperature of the second step is changed to the third step. It is preferable to change the culture temperature.

The enzyme reaction method of the present invention is a method in which the enzyme obtained by the above-described method for producing a thermophilic bacterium and a mesophilic bacterium-derived enzyme of the present invention is brought into contact with a substrate of the enzyme and reacted.

In the present invention, the “optimum growth temperature” refers to the temperature at which a microorganism grows most rapidly, and the temperature at which the specific growth rate obtained by culturing the microorganism for a certain period of time at each temperature is the highest. Optimal growth temperature can be achieved.

According to the present invention, a method for producing a psychrophilic bacterium and a mesophilic bacterium-derived enzyme capable of separating a growth stage of a transformant introduced with DNA encoding an enzyme derived from a psychrophilic bacterium and a mesophilic bacterium and an expression stage of the enzyme. Can be provided. Therefore, for example, by producing a transformant so that the enzyme derived from the psychrotrophic and mesophilic bacterium is expressed in the secretory system, after growing at a high temperature, the medium is changed, and then the culture temperature is changed to a low temperature. Since an enzyme derived from a thermophilic bacterium and a mesophilic bacterium can be selectively obtained in a medium, the enzyme can be easily recovered and purified. In addition, since the activity of the enzyme derived from the thermophilic bacterium that is the host microorganism is suppressed near the optimum temperature of the enzyme derived from the thermophilic bacterium and mesophilic bacterium obtained, the culture solution containing the transformant can be used for the enzyme reaction as it is. It becomes possible.

2 is a growth curve obtained in Examples 1 and 2 and Comparative Example 1. It is the photograph which image | photographed the external appearance when (beta) -galactosidase activity evaluation was implemented about the culture solution at the time of the culture | cultivation start of the 3rd process of the comparative example 1. FIG. FIG. 3 is a photograph of the appearance when β-galactosidase activity was evaluated for culture broths cultured for 3 hours from the start of culture in the third step of Examples 1 and 2 and Comparative Example 1. FIG. FIG. 3 is a photograph of the appearance when β-galactosidase activity evaluation was performed on culture broths cultured for 24 hours from the start of culture in the third step of Examples 1 and 2 and Comparative Example 1. FIG. 4 is a growth curve obtained in Examples 3 to 4 and Comparative Example 2. It is the photograph which image | photographed the external appearance when (beta) -galactosidase activity evaluation (incubation time: 1 hour) was implemented about the culture solution at the time of the culture | cultivation start of the 3rd process of the comparative example 2. FIG. FIG. 4 is a photograph of the appearance when β-galactosidase activity evaluation (incubation time: 1 hour) was performed on the culture solution after 3 hours of culture from the start of the third step of Examples 3 to 4 and Comparative Example 2. . FIG. 4 is a photograph of the appearance when β-galactosidase activity evaluation (incubation time: 1 hour) was performed on the culture solution after 6 hours of culture from the start of the third step of Examples 3 to 4 and Comparative Example 2. . It is the photograph which image | photographed the external appearance when (beta) -galactosidase activity evaluation (incubation time: 3 hours) was implemented about the culture solution at the time of the culture | cultivation start of the 3rd process of the comparative example 2. FIG. FIG. 3 is a photograph of the appearance when β-galactosidase activity evaluation (incubation time: 3 hours) was performed on the culture solution after 3 hours of culture from the start of the third step of Examples 3 to 4 and Comparative Example 2. .

Hereinafter, the present invention will be described in detail on the basis of preferred embodiments thereof.

The method for producing the thermophilic bacterium and the mesophilic bacterium-derived enzyme of the present invention comprises:
A group consisting of a thermophilic bacterium having an optimal growth temperature of 50 ° C. or higher, a thermophilic bacterium having an optimal growth temperature of 50 ° C. or lower and a temperature lower by 10 ° C. or more than the optimal growth temperature of the thermophilic bacterium A first step of obtaining a transformant by introducing DNA encoding an enzyme (an enzyme derived from a thermophilic bacterium or an enzyme derived from a mesophilic bacterium) selected from at least one microorganism selected from:
A second step of culturing and growing the transformant at a culture temperature of 50 ° C. or higher;
After the second step, a third step of expressing the enzyme by changing the culture temperature to 50 ° C. or lower and a temperature lower by 10 ° C. or higher than the culture temperature of the second step;
including.

In the present invention, a “thermophilic bacterium” is a microorganism having an optimal growth temperature of 50 ° C. or higher. Examples of such thermophilic bacteria include thermophilic eubacteria, hyperthermophilic eubacteria, thermophilic archaea and hyperthermophilic archaea. From the viewpoint of easy expression, eubacteria are used as host microorganisms. Since it is preferable to express an eubacteria-derived enzyme as an enzyme derived from the following thermophilic bacteria and mesophilic bacteria, it is more preferably a thermophilic eubacteria or a hyperthermophilic eubacteria. In the present invention, thermophilic means that the optimum growth temperature of the microorganism is 50 ° C. or more and less than 80 ° C., and super thermophilicity means that the optimum growth temperature of the microorganism is 80 ° C. or more. Point to.

From the viewpoint that the thermophilic eubacteria and the hyperthermophilic eubacteria are available from public institutions, have a track record of producing transformants, or tend to be relatively easy to produce transformants. , Geobacillus genus, Bacillus genus, Thermus genus, Thermotoga genus, Pseudothermotoga genus, Thermodessulfobacteria genus, Akwifex genus, Geobacillus genus, Bacillus genus, Thermus genus, Thermotoga Particularly preferred is Geobacillus.

Examples of the genus Geobacillus include Geobacillus thermoglucosidasis (optimum growth temperature: 55 ° C.), Geobacillus kaustophilus (optimum growth temperature: 55 ° C.), Geobacillus s. stearothermophilus, optimal growth temperature: 55 ° C., Geobacillus subterraneus (optimal growth temperature: 55 ° C.), Geobacillus uzenensis, optimal growth temperature: 55 ° C. Geobacillus thermocate ulatas, optimal growth temperature: 60 ° C.), Geobacillus thermodenitificans, optimal growth temperature: 60 ° C., Geobacillus thermoreovorans, optimal growth temperature: 60 ° C. Can be mentioned.

Examples of the genus Bacillus include Bacillus thermanticus (Bacillis thermalarticus, optimal growth temperature: 55 ° C.). Examples of the Thermus genus include Thermos thermophilus (Thermus thermophilus, optimal growth temperature: 75 ° C.), Thermus aquaticus (Thermus aquaticus, optimal growth temperature: 70 ° C.).

Further, as the genus Thermotoga, Thermotoga maritima (Thermotoga maritima (optimum growth temperature: 80 ° C.), Thermotoga naphtophyla (Optimum growth temperature: 80 ° C.), Thermotoga neapolitana (therma tomato temperature) : Thermotoga petrophila (Thermotoga petrophila, optimal growth temperature: 80 ° C), and the Pseudothermotoga retingae (Pseudothermotoga lettingae, optimal growth temperature: 65 ° C). Can be mentioned.

Furthermore, examples of the genus Thermodesulfobacterium include Thermodesulfobacterium commune (optimum growth temperature: 70 ° C.). As the genus Aquifex, Aquifex aeolicus (optimus aeolicus) is optimal. Growth temperature: 85 ° C.) and Aquifex pyrophilus (optimum growth temperature: 85 ° C.).

From the viewpoint that the thermophilic archaea and the hyperthermophilic archaea are available from public institutions, have a track record of producing transformants, or tend to be relatively easy to produce transformants. Aeropyram genus, Alcaeoglobus genus, Methanocordococcus genus, Methanothermobacter genus, Pyrococcus genus, and Sulfolobas genus are preferable. Examples of the genus Aeropyrum include Aeropyrum pernicus (optimum growth temperature: 90 ° C.), and examples of the genus Alcaeoglobus include Archaeoglobus fulgidus (optimum growth temperature: 85). The Methanocordococcus genus includes Methanocardococcus jannaschii (optimum growth temperature: 80 ° C.), and the Methanothermobacter genus includes the Methanothermobacter thermoautotro. And Ficus (Methanotherbacter thermotrophicus, optimal growth temperature: 65 ° C.).

Examples of the Pyrococcus genus include Pyrococcus furiosus (Pyrococcus furiosus, optimal growth temperature: 97 ° C.), Pyrococcus horikoshi (Pyrococcus horikoshii, optimal growth temperature: 95 ° C.), Pyrococcus abyssi (Pyrococcus, optimal growth). Examples of the sulfolobus include Sulfolobus solfataricus (Sulfobus solfataricus, optimum growth temperature: 70 ° C.), Sulfolobus tokodaii, optimum growth temperature: 75 ° C. .

The optimum growth temperature of the thermophilic bacterium according to the present invention needs to be 50 ° C. or higher. If the optimal growth temperature of the thermophile is less than the lower limit, it becomes difficult to separate the growth stage of the transformant and the expression stage of the enzyme. Moreover, as the optimal growth temperature of the thermophilic bacterium, the growth stage of the transformant and the expression stage of the enzyme can be further distinguished, and the culture for growing the transformant is easier. In view of the above, it is preferably 55 to 87 ° C, more preferably 55 to 65 ° C.

In the present invention, “thermophilic and mesophilic bacteria” are microorganisms having an optimum growth temperature of 50 ° C. or lower. The thermophilic bacterium refers to a microorganism having an optimal growth temperature of less than 30 ° C., and the mesophilic bacterium refers to a microorganism having an optimal growth temperature of 30 to 50 ° C. Examples of such a thermophilic bacterium and mesophilic bacterium include eubacteria, archaea, and eukaryotes. From the viewpoint of easy expression, an eubacteria is used as a host microorganism, and the following psychrophilic and mesophilic bacterium-derived enzymes are used: Since it is preferable to express an eubacteria-derived enzyme, eubacteria are more preferable.

The eubacteria as the thermophilic bacterium and the mesophilic bacterium are not particularly limited. Bacterial glutamium (Corynebacterium glutamicum, optimal growth temperature: 28 ° C), Lactococcus lactis (Lactococcus lactis, optimal growth temperature: 30 ° C), Clostridium acetobutylicum (Crostridium acetobutylicum, optimal temperature 37 ° C) Pseudomonas mevaloni (Pseudomonas mevalonii, optimal growth temperature: 30 ° C.). The archaea as the thermophilic bacterium and the mesophilic bacterium are not particularly limited, and examples thereof include Methanosarcina mazei (optimum growth temperature: 37 ° C.). Further, eukaryotes as the above-mentioned thermophilic bacteria and mesophilic bacteria are not particularly limited, and examples thereof include Saccharomyces cerevisiae (optimum growth temperature: 25 ° C.). Among these, as the above-mentioned thermophilic bacterium and mesophilic bacterium, Escherichia coli, Bacillus subtilis, Clostridium acetobutylicum, and Saccharomyces cerevisiae are preferable, and Escherichia coli is particularly preferable from the viewpoint that there is a tendency to use many enzymes derived from the microorganism.

The optimum growth temperature of the thermophilic bacterium and mesophilic bacterium according to the present invention is 50 ° C. or lower, and from the growth temperature of thermophilic bacterium used as a host cell into which DNA encoding the enzyme derived from the psychrophilic bacterium and mesophilic bacterium is introduced. Also, it must be 10 ° C. or more lower. If the optimum growth temperature of the thermophilic bacterium and the mesophilic bacterium exceeds the upper limit, it becomes difficult to separate the growth stage of the transformant from the expression stage of the psychrophilic bacteria and the mesophilic bacterium-derived enzyme. In addition, as the optimum growth temperature of the above-mentioned thermophilic bacterium and mesophilic bacterium, the growth stage of the transformant and the expression stage of the enzyme can be further distinguished, and the expression level of the enzyme can be increased. In view of the above, it is preferably 25 to 45 ° C, more preferably 25 to 40 ° C.

Additional thermophilic bacteria, cold bacteria and mesophilic bacteria, e.g., BGSC (Bacillus Genetic Stock Center), ATCC (The American Type Culture Collection), DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures)) It can be obtained from public institutions such as NPMD (National Institute of Technology and Evaluation, Patent Biological Depositary Center) and private sales companies.

The "Pyrogen and mesophilic bacterium-derived enzyme" according to the present invention is an enzyme derived from at least one microorganism selected from the group consisting of the psychrophilic bacterium and the mesophilic bacterium. Examples of such enzymes include, but are not limited to, for example, sugar metabolism enzymes derived from E. coli, glycolytic enzymes, non-mevalonate pathway enzymes, butanol fermentation enzymes, fatty acid synthetases; glycolytic enzymes derived from Bacillus subtilis, Non-mevalonate pathway enzyme, butanol fermentation enzyme; Butanol fermentation enzyme derived from Corynebacterium glutamine, Shikimate pathway enzyme; Butanol fermentation enzyme derived from Lactococcus lactis; Acetone-butanol-ethanol fermentation enzyme derived from Clostridium acetobutylicum; Pseudomonas -Mevalonate pathway enzyme derived from mevaloni; mevalonate pathway enzyme derived from methanosarkina mazei; glycolytic enzyme derived from Saccharomyces cerevisiae, ethanol fermentation enzyme, and mevalonate pathway enzyme.

As the sugar metabolism enzyme derived from E. coli, β-galactosidase (lacZ) is preferable. The glycolytic enzymes derived from the Escherichia coli, Bacillus subtilis, or Saccharomyces cerevisiae include hexokinase, glucose-6-phosphate isomerase, phosphofructokinase-1, fructose 1,6-bisphosphate aldolase, triose phosphate. Preferred are isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, phosphopyruvate hydratase, and pyruvate kinase.

Non-mevalonate pathway enzymes derived from the E. coli or Bacillus subtilis include DOXP synthase, DOXP reductoisomerase, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase, 4-diphosphoditidyl-2-C-methyl-D. -Erythritol kinase, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, HMB-PP synthase, HMB-PP reductase, isopentenyl diphosphate isomerase are preferred.

As the butanol-fermenting enzyme derived from E. coli, ketol acid reductoisomerase and dihydroxy acid dehydratase are preferable. As the butanol-fermenting enzyme derived from Bacillus subtilis, acetolactate synthase is preferable, butanol-fermenting enzyme derived from Corynebacterium glutamicum. As ketol acid reduct isomerase, dihydroxy acid dehydratase,
Alcohol dehydrogenase is preferred, and the butanol fermentation enzyme derived from Lactococcus lactis is preferably keto acid decarboxylase.

As the fatty acid synthase derived from E. coli, acetyl-CoA carboxylase, ACP-acetyltransferase, ACP-malonyltransferase, β-ketoacyl-ACP synthase, β-ketoacyl ACP reductase, 3-hydroxyacyl ACP dehydrase, and enoyl ACP reductase are preferable. .

Examples of the shikimate pathway enzyme derived from Corynebacterium glutamicum include 7-phospho-2-dehydro-3-deoxyarabinoheptonic acid aldolase, 3-dehydroquinic acid synthase, 3-dehydroquinic acid dehydratase, shikimate dehydrogenase, shikimate Kinase, 3-phosphoshikimate 1-carboxyvinyltransferase, chorismate synthase, chorismate mutase are preferred.

Examples of the acetone-butanol-ethanol fermentation enzyme derived from Clostridium acetobutylicum include pyruvate synthase, thiolase, 3-hydroxybutyryl-CoA dehydrogenase, crotonyl CoA hydratase, butyryl CoA dehydrogenase, acetaldehyde dehydrogenase, ethanol dehydrogenase, butyraldehyde dehydrogenase, butanol Dehydrogenase and acetoacetate decarboxylase are preferred.

As the mevalonate pathway enzyme derived from Pseudomonas mevaloni, HMG-CoA reductase is preferable, and as the mevalonate pathway enzyme derived from Methanosarcina mazei or Saccharomyces cerevisiae, acetyl CoA synthase, acetyl CoA-acetyltransferase, HMG-CoA synthase HMG-CoA reductase, mevalonate kinase, 5-phosphomevalonate kinase, diphosphomevalonate decarboxylase, isopentenyl diphosphate Δ-isomerase are preferred. As the ethanol fermentation enzyme derived from Saccharomyces cerevisiae, pyruvate decarboxylase and alcohol dehydrogenase are preferable.

As the enzymes derived from thermophilic bacteria and mesophilic bacteria according to the present invention, one of the above may be expressed alone, or two or more of them may be expressed in combination. Among these, as the enzyme derived from the psychrotrophic bacterium and mesophilic bacterium, β-galactosidase, pyruvate decarboxylase, and alcohol dehydrogenase are preferable, and β-galactosidase is particularly preferable from the viewpoint of tending to be used.

DNA sequence information encoding the above-mentioned enzymes derived from thermophilic and mesophilic bacteria should be obtained from publicly available databases such as DDBJ (DNA Data Bank of Japan), GenBank, EMBL (European Molecular Biology Laboratory). Can do.

The first step according to the production method of the present invention is a step of obtaining a transformant by introducing DNA encoding the enzyme derived from the thermophilic bacterium and the mesophilic bacterium into the thermophilic bacterium. The method for obtaining the transformant is not particularly limited, and a known method or conditions obtained by appropriately modifying the known method can be appropriately employed. For example, DNA encoding the target thermophilic bacteria and mesophilic bacteria-derived enzyme is isolated from the target thermophilic bacteria and / or mesophilic bacteria by conventional methods, and an expression vector capable of self-replication containing the isolated DNA is prepared. The target transformant can be obtained by introduction into a thermophilic bacterium. Moreover, the target transformant can also be obtained by introducing an expression vector containing the isolated DNA into the hyperthermic bacterium and integrating the DNA into the genome of the hyperthermic bacterium by conjugation transfer or the like.

Examples of the DNA isolation method include PCR using primers prepared based on the base sequences of the target psychrotrophic and mesophilic bacterium-derived enzymes and the genomic DNA of the target psychrophilic and / or mesophilic bacterium as a template. To isolate the desired genomic DNA by ligating the amplified DNA fragment with an appropriate vector; extracting genomic DNA or mRNA from the target psychrotrophic and / or mesophilic bacterium and synthesizing based on this The prepared cDNA is ligated with an appropriate vector to prepare a DNA library or cDNA library, and desired from the library by hybridization using a probe prepared based on the base sequences of the target thermophilic and mesophilic bacteria. A method for isolating the genomic DNA or cDNA; and a method for artificial chemical synthesis.

The expression vector is a vector containing a protein encoded by the polynucleotide sequence in an expressible state, and is preferably replicable in the thermophilic bacterium. For example, the expression vector is constructed based on a plasmid, phage, or cosmid. be able to. The plasmid serving as the parent of the expression vector can be appropriately selected according to the type of thermophile into which the expression vector is introduced and the method of introduction. Specifically, for example, pNW33N (GenBank ID: AY237122.1), pUB110 (GenBank ID: M19465.1), pSTK1 (GenBank ID: D29989.1), pTB19 (GenBank ID: M63891.1), Hirokazu Suzuki et al., Appl Environ Microb. October 2012, 78 (20), p. Examples thereof include plasmids such as the pGAM46 plasmid described in 7376-7383 and derivatives thereof.

As the expression vector, in addition to DNA encoding the enzyme derived from the thermophilic bacterium and the mesophilic bacterium, a polynucleotide sequence for controlling the expression in order to express the enzyme by actually introducing the enzyme into the thermophilic bacterium Or a genetic marker for selecting a transformant. Further, it preferably further comprises a purification tag sequence for purifying the enzyme derived from the thermophilic bacterium and the mesophilic bacterium, and in the case of secreting the enzyme derived from the psychrophilic and mesophilic bacterium outside the thermophilic bacterium, a secretory signal sequence. It is preferable that it is further included.

Examples of the polynucleotide sequence that controls the expression include a polynucleotide sequence encoding a promoter, a terminator, or a signal peptide. The gene marker can be appropriately selected according to the selection method of the transformant. For example, a gene encoding drug resistance or a gene complementary to auxotrophy can be used.

The promoter can be appropriately selected according to the target psychrophilic and mesophilic bacterium-derived enzymes. For example, Hirokazu Suzuki et al., Appl Environ Microbiol. September 2013, 79 (17), p. 5151-5158, Geobacillus kaustophilus, Pgk704 (putative amylose metabolic gene promoter), Pgk1859 (putative serbiose metabolic gene promoter), Pgk1894 (myoinositol metabolic gene promoter), Pgk1899 (myoinositol metabolic gene promoter), Pgk1907 (Putative L-arabinose metabolic gene promoter), Pgk2150 (putative D-galactose metabolic gene promoter), PsigA (dnaG gene, sigA gene promoter); Lin et al., Metab. Eng. July 2014, 24, p. 192-199, Geobacillus thermoglucosidasis, Pglk (hexokinase gene promoter), Ppgi (glucose-6-phosphate isomerase gene promoter), PpfkA (phosphofructokinase-1 gene promoter), Pgap (glyceraldehyde) -3-phosphate dehydrogenase gene promoter), Ppgk (phosphoglycerate kinase gene promoter), Pgpm (phosphoglycerate mutase gene promoter), Peno (phosphopyruvate hydratase gene promoter), Pldh (lactate dehydrogenase gene promoter), PglpD (glycerol-phosphate dehydrogenase gene promoter) and Bacillus subtili Like P43 of. Such a promoter is generally located upstream of the DNA encoding the enzymes derived from the psychrotrophic and mesophilic bacterium.

In the present invention, in the expression vector, it is preferable that the DNA encoding the enzyme derived from the psychrotrophic and mesophilic bacterium is operably linked to a promoter, and the thermophilic bacterium into which the expression vector is introduced; It is preferable that the microorganism from which the promoter is derived is closely related. For example, when the thermophilic bacterium into which the expression vector is introduced is of the genus Geobacillus, the promoter is preferably a promoter derived from a Gram-positive eubacteria, and a promoter derived from an eubacteria included in the genus Geobacillus and Bacillus More preferably, it is at least one promoter selected from the group consisting of promoters derived from Bacillus subtilis included in the genus.

The production method of the expression vector is not particularly limited, and a known method or a method obtained by appropriately modifying or modifying the known method can be appropriately employed. For example, the expression vector is obtained by ligating the promoter, the DNA encoding the enzyme derived from the psychrotrophic bacterium and the mesophilic bacterium, and the terminator, if necessary, into the expression cassette, and introducing the expression cassette into the vector. I can do it. Moreover, the enzyme and conditions for producing the expression vector are not particularly limited, and commercially available products can be appropriately selected and used.

The transformation method for introducing the expression vector into the thermophilic bacterium is not particularly limited, and a known method can be appropriately employed. For example, a microinjection method, an electroporation method, a polyethylene glycol method, a particle gun Method, protoplast fusion method, junction transfer method, and calcium phosphate method can be used. In addition, the thermophilic bacterium into which the expression vector is introduced may be, if necessary, one that has already been transformed so as to lack a specific function or a mutant.

The second step according to the present invention is a step in which the transformant is cultured and grown at a culture temperature of 50 ° C. or higher. If the culture temperature in the second step is lower than the lower limit, it becomes difficult to separate the growth stage of the transformant from the expression stage of the psychrophilic and mesophilic bacterium-derived enzymes. Further, as the culture temperature in the second step, it is possible to further distinguish between the growth stage of the transformant and the expression stage of the enzyme, and the viewpoint that the culture for growing the transformant is easier. Therefore, the temperature is preferably 55 to 87 ° C, more preferably 55 to 65 ° C.

In the third step according to the present invention, after the second step, the culture temperature is changed to a temperature lower than 50 ° C. and lower than the culture temperature of the second step by 10 ° C. It is a process of making it express. When the culture temperature in the third step exceeds the above upper limit, it becomes difficult to separate the growth stage of the transformant from the expression stage of the psychrophilic and mesophilic bacterium-derived enzymes. In addition, the culture temperature in the third step is 25 to 45 ° C. from the viewpoint of further distinguishing between the growth stage of the transformant and the expression stage of the enzyme and suppressing the killing of the transformant. It is preferably 30 to 45 ° C, more preferably 40 to 45 ° C. In the present invention, the culture temperature in the third step is not necessarily required to coincide with the optimum growth temperature of the above-mentioned thermophilic bacterium and mesophilic bacterium from which the enzyme to be expressed is derived. Enzymes derived from psychrophilic bacteria and mesophilic bacteria can be sufficiently expressed when the culture temperature in the step is within the above temperature range.

In the present invention, when the transformant is in the logarithmic growth phase, more specifically, the OD _{600 of} the culture medium for culturing the transformant is 1 or less, more preferably 0.1 to 0.9. In this case, it is preferable to change the culture temperature from the culture temperature in the second step to the culture temperature in the third step. If the OD ₆₀₀ when changing the culture temperature is less than the lower limit, the amount of expression of the enzyme tends to decrease because of insufficient growth of the transformant. On the other hand, if the OD ₆₀₀ exceeds the upper limit, Tends to be difficult to express. In the present invention, the OD ₆₀₀ value refers to the absorbance at 600 nm of a culture solution (including all components contained in the culture system such as culture medium and microorganisms) in which the transformant is cultured.

Further, in the present invention, the culture time after changing the culture temperature from the temperature of the second step to the temperature of the third step, that is, the culture time of the third step is that of the thermophile that is the host microorganism. Depending on the type and culture conditions, for example, when Geobacillus genus (more preferably, Geobacillus thermoglucosidashius) is used as the thermophilic bacterium, it is preferably 1 to 48 hours, and preferably 3 to 48 hours. More preferably, it is 3 to 24 hours, more preferably 3 to 15 hours. When the incubation time after the temperature change is less than the lower limit, the expression level of the enzyme tends to decrease. On the other hand, when the upper limit is exceeded, the expression rate decreases and the expression level of the enzyme becomes unstable. There is a tendency.

In the second step and the third step, other conditions for culturing the transformant (medium composition, medium pH, gas (oxygen, carbon dioxide, etc.) concentration, etc.) are not particularly limited, and the host Depending on the type of thermophile that is a microorganism, it can be selected from known culture conditions or conditions obtained by appropriately modifying or modifying known culture conditions.

According to the production method of the present invention, in the third step, the target psychrophilic and mesophilic bacterium-derived enzymes can be preferentially expressed while suppressing the growth of transformants using the thermophilic bacterium. For example, when a vector containing a secretory signal sequence is used as the expression vector, the medium is changed after the second step, and the enzyme derived from the psychrophilic and mesophilic bacterium is expressed in the third step. Since it can be selectively obtained in a medium, this medium can be used as a crude enzyme in an enzyme reaction. Even when a vector containing a secretory signal sequence is not used, the activity of an enzyme derived from a thermophilic bacterium as a host microorganism is suppressed in the vicinity of the optimum temperature of the obtained psychrotrophic and mesophilic bacterium-derived enzymes. The culture solution containing the body can be used as it is in the enzyme reaction as a crude enzyme. Furthermore, as the enzyme, after completion of the culture of the transformant, a solution obtained by recovering the transformant by centrifugation or filtration and disrupting the cells can be used as the crude enzyme. Further, these crude enzymes can be concentrated by an ultrafiltration method or the like, and a preservative or the like can be added to obtain a concentrated enzyme. The crude enzyme or the concentrated enzyme may be purified by using, for example, a salting-out method, an organic solvent precipitation method, a membrane separation method, or a chromatographic separation method alone or in combination of two or more. Furthermore, when a vector containing a purification tag sequence is used as the expression vector, the enzyme to which the purification tag is added may be purified using a column for purification of the tagged protein.

The enzymes derived from psychrophilic and mesophilic bacteria obtained by the production method of the present invention can be used for various enzyme reactions depending on the kind of the enzyme. Although it does not restrict | limit especially as said enzyme reaction, The method of contacting the said enzyme, its substrate, and the coenzyme of the said enzyme as needed is mentioned. About the density | concentration of each component in such an enzyme reaction, the kind of solvent, temperature conditions, reaction temperature, etc., it can adjust suitably according to the kind of said thermophilic bacteria and mesophilic bacteria origin enzyme.

Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples.

Example 1
<Isolation of DNA (lacZ)>
First, genomes were extracted from BL21 (DE3) (Merck) using DNeasy Blood & Tissue Kit (Qiagen). Using the extracted genome as a template, Escherichia coli β using HM48 primer (nucleotide sequence described in SEQ ID NO: 1), HM49 primer (nucleotide sequence described in SEQ ID NO: 2) and PCR enzyme (KOD Fx Neo, Toyobo Co., Ltd.) A sequence encoding galactosidase (lacZ, nucleotide sequence set forth in SEQ ID NO: 3, GeneBank ID: CP001509.3 (335840-332766)) was amplified. The amplified PCR product was purified using QIAquick Spin Gel Extraction Kit (Qiagen), and the purified PCR product and pET28a + (Merck) were respectively obtained with NcoI (Takara Bio) and NotI (Takara Bio). It cut | disconnected and refine | purified using QIAquick Spin Gel Extraction Kit (Qiagen).

Subsequently, the PCR product (68 ng) and pET28a + (233 ng) after cleavage and purification were ligated using DNA Ligation Kit (Mighty Mix, Takara Bio Inc.) and introduced into 200 μl of E. coli JM109 chemical competent cell. Transformants were selected on an LB medium plate (25 μg / ml kanamycin), inoculated into 3 ml of LB medium (25 μg / ml kanamycin) and grown. The pESG23 plasmid containing lacZ was extracted from the grown transformant using QIAprep spin miniprep kit (Qiagen).

<Preparation of expression vector>
First, pNW33N plasmid (nucleotide sequence described in SEQ ID NO: 4, obtained from Bacillus Genetic Stock Center) was cleaved with HindIII (Takara Bio) and purified using QIAquick Spin Gel Extraction Kit (Qiagen). Also, Kenji Tsuge et al., Nucleic Acids Res. November 2003, 31 (21), the oligomers (VsfiKpn-1F (5 ′ terminal phosphorylation) and VsfiKpn-1R (5s terminal phosphorylation) phosphorylated at each 5 ′ end of the synthetic DNA oligomers VsfiKpn-1F and VsfiKpn-1R ( 5 ′ terminal phosphorylation)) was heated at 98 ° C. for 5 minutes, and then naturally cooled at room temperature to produce an insert. The cleaved and purified pNW33N and the insert were mixed, ligated using DNA Ligation Kit (Mighty Mix, Takara Bio Inc.), and introduced into 200 μl of E. coli JM109 chemical competent cell. Transformants were selected on an LB medium plate (25 μg / ml chloramphenicol), inoculated into 3 ml of LB medium (25 μg / ml chloramphenicol) and grown. The pNW (SfiI) plasmid containing the SfiI site was extracted from the grown transformant using QIAprep spin miniprep kit (Qiagen).

Also, Paul P.M. Lin et al., Metabolic Engineering, July 2014, 24, p. The lactobacillus thermodenitrificans lactate dehydrogenase gene promoter (ldh) described in 192-199 was artificially synthesized to obtain a plasmid containing the same. Using this plasmid as a template, HM05b primer (nucleotide sequence described in SEQ ID NO: 5), HM06-minus primer (nucleotide sequence described in SEQ ID NO: 6) and PCR enzyme (KOD plus ver. 2, Toyobo) were used. Ldh was amplified (PCR1).

Furthermore, using the pESG23 plasmid containing lacZ obtained above as a template, HM51 primer (nucleotide sequence described in SEQ ID NO: 7), HM52H primer (primer for adding His6 tag, nucleotide sequence described in SEQ ID NO: 8) And lacZ was amplified using PCR enzyme (KOD plus ver.2, Toyobo Co., Ltd.) (PCR2).

PCR products amplified by PCR1 and PCR2 were each subjected to agarose gel electrophoresis to confirm the length, and then purified using QIAquick Spin Gel Extraction Kit (Qiagen). Using purified PCR1 product (100 ng) and PCR2 product (100 ng), HM05b primer, HM52H primer, and PCR enzyme (KOD plus ver. 2, Toyobo), Step 1: 98 ° C. for 10 seconds, Step 2: 60 ° C. Fusion PCR was performed under the conditions of 30 seconds, Step 3: 68 ° C. for 4 minutes, and the temperature increase rate from Step 2 to Step 3: 0.1 ° C./second, 35 cycles to prepare an expression cassette in which ldh and lacZ were fused.

The pNW (SfiI) plasmid obtained above was cleaved with HindIII, mixed with the expression cassette, purified using QIAquick Spin Gel Extraction Kit (Qiagen), and then Gibson Assembly Master Mix (2x) And introduced into 200 μl of E. coli JM109 chemical competent cell. Transformants were selected on an LB medium plate (25 μg / ml chloramphenicol), inoculated into 3 ml of LB medium (25 μg / ml chloramphenicol) and grown. A pESG21H plasmid (nucleotide sequence described in SEQ ID NO: 9) containing ldh and lacZ was extracted from the grown transformant using QIAprep spin miniprep kit (Qiagen).

<Transformation>
As a host microorganism, a thermophilic bacterium belonging to the genus Geobacillus ordered from Bacillus Genetic Stock Center (Geobacillus thermoglocosidius DSM2542, optimum growth temperature: 55 ° C.) was used. Production and transformation of thermophilic bacteria competent cells is described in Mark P. et al. Taylor et al., Plasmid, July 2008, 60 (1), p. According to the method described in 45-52:

(Production of thermophilic bacteria competent cells)
The thermophile was added to a TGP medium plate (tryptone: 17 g / l, soyton: 3 g / l, K ₂ HPO ₄ : 2.5 g / l, NaCl: 5 g / l, sodium pyruvate: 4 g / l, glycerol: 4 ml / l l, Agar: 15 g / l) and after overnight culture at 60 ° C., the obtained colony was treated with 1 ml of TGP medium (tryptone: 17 g / l, soyton: 3 g / l, K ₂ HPO ₄ : 2.5 g / l, NaCl: 5 g / l, sodium pyruvate: 4 g / l, glycerol: 4 ml / l), cultured at 60 ° C. and 180 rpm, and then inoculated into 50 ml of TGP medium. After culturing until OD ₆₀₀ = 1.6, the mixture was cooled on ice for 10 minutes. Thereafter, the cells were collected by centrifugation at 8000 rpm for 5 minutes, suspended in an electroporation buffer (0.5 M sorbitol, 0.5 M mannitol, 10% glycerol), and centrifuged at 8000 rpm for 5 minutes to collect the cells four times. Repeated. Thereafter, the suspension was suspended in 1.5 ml of electroporation buffer, dispensed in 60 μl aliquots, and stored at −80 ° C.

(Transformation-Production of transformants-)
The thermophilic bacterium competent cell (60 μl) obtained above and the pESG21H plasmid (500 ng) were mixed, put into a 0.1 cm cuvette, and then using a Gene Pulser Xcell (BioRad), 2500 V, 10 μF, 600Ω. Electrical stimulation was given. Thereafter, 1 ml of TGP medium was added, transferred to a 14 ml polypropylene round bottom tube (FALCON), shaken at 60 ° C. for 2 hours, and then applied to a TGP medium plate (10 μg / ml chloramphenicol) at 60 ° C. Cultured overnight. The obtained colonies (4 colonies) were each inoculated into 3 ml of LB medium and cultured at 60 ° C. overnight, and then an equal amount of 80% glycerol was added to prepare a glycerol stock of the transformant. Saved with.

<Culture of transformant>
First, a part of the glycerol stock of the transformant obtained above was placed in LB medium (25 μg / ml chloramphenicol) and cultured overnight. Next, 120 ml of LB medium (25 μg / ml chloramphenicol) was placed in a 500 ml Erlenmeyer flask and inoculated with 1 ml of an overnight culture. Using the incubator (BR-43FL, Taitec Co., Ltd.), the second step of culture was performed at 60 ° C. and 180 rpm until OD ₆₀₀ = 0.785. Thereafter, 30 ml of the culture solution was placed in a 200 ml Erlenmeyer flask, and the culture temperature was changed to 42 ° C. to perform the third step of culture. After starting the culture in the third step (after changing the culture temperature to 42 ° C.), OD ₆₀₀ of the culture solution after culturing for 0, 1.5, 3, 24 hours was measured to obtain a growth curve.

<Evaluation of β-galactosidase activity-Enzyme reaction->
After culturing for the third step (after changing the culture temperature), after culturing for 0, 3, and 24 hours, take 3 ml of the culture solution in a 14 ml polypropylene round bottom tube and dissolve in DMSO (dimethyl sulfoxide). 50 μl of 2% 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) was added and incubated at 42 ° C. for 1 hour with shaking to determine the activity of β-galactosidase. evaluated. The evaluation was performed by visually observing the appearance (color) of the culture solution after incubation, and the following criteria:
0: Orange (no blue color is confirmed (the color of the medium is the same as that without X-gal))
1: Yellow (slightly blue but almost the same as when X-gal was not added)
2: Yellowish green (blue is confirmed)
3: Green 4: Performed based on blue. In addition, it can be judged that the enzyme is expressing more, so that the numerical value of evaluation is large.

(Example 2)
In culturing the transformant, the culture was performed in the same manner as in Example 1 except that the culture temperature in the third step was 37 ° C. After starting the culture in the third step (after changing the culture temperature to 37 ° C.), OD ₆₀₀ of the culture solution after culturing for 0, 1.5, 3, 24 hours was measured to obtain a growth curve. Further, β-galactosidase activity was evaluated in the same manner as in Example 1 except that the incubation temperature after adding X-gal was 37 ° C.

(Comparative Example 1)
In the culture of the transformant, the culture was performed in the same manner as in Example 1 except that the culture temperature in the third step was kept at 60 ° C. following the second step. Also, OD after culturing at −3.5, −1, 0, 1.5, 3, 24 hours, assuming that the culture start time of the second step is −5 hours and the culture start time of the third step is 0 hours. ₆₀₀ is measured to obtain a growth curve. Further, β-galactosidase activity was evaluated in the same manner as in Example 1 except that the incubation temperature after addition of X-gal was 60 ° C.

The growth curves obtained in Examples 1 and 2 and Comparative Example 1 are shown in FIG. The evaluation results of β-galactosidase activity are shown in Table 1. Furthermore, FIG. 2A is a photograph showing the appearance when the β-galactosidase activity evaluation is performed on the culture solution at the start of the culture in the third step of Comparative Example 1 (after 0 hour culture). FIG. 2B is a photograph showing the external appearance when β-galactosidase activity evaluation was performed on the culture solution after 3 hours of culture from the start of the culture in the third step of Example 1, and the third example of Examples 1-2 and Comparative Example 1 was used. The photographs showing the external appearance when the β-galactosidase activity evaluation is performed on the culture solution after 24 hours of cultivation from the start of the cultivation in the step are shown in FIG. 2C, respectively.

(Example 3)
<Isolation of DNA (lacZ)>
In the same manner as in Example 1, a pESG23 plasmid containing lacZ was obtained.

<Preparation of expression vector>
First, Hirokazu Suzuki et al., Appl Environ Microbiol. October 2012, 78 (20), p. The pGAM46 plasmid described in 7376-7383 was cleaved with HindIII (Takara Bio Inc.) and purified using QIAquick Spin Gel Extraction Kit (Qiagen Inc.). The synthetic DNA oligomers VsfiKpn-1F (5 ′ end phosphorylation) and VsfiKpn-1R (5 ′ end phosphorylation)) were heated at 98 ° C. for 5 minutes, and then naturally cooled at room temperature to produce an insert. The cut and purified pGAM46 and the insert were mixed, ligated using DNA Ligation Kit (Mighty Mix, Takara Bio Inc.), and introduced into 200 μl of E. coli JM109 chemical competent cell. Transformants were selected on an LB medium plate (100 μg / ml ampicillin), inoculated into 3 ml of LB medium (100 μg / ml ampicillin) and grown. A pGAM46 (SfiI) plasmid containing the SfiI site was extracted from the grown transformant using QIAprep spin miniprep kit (Qiagen).

In addition, a HindIII-promoter-fw primer (described in SEQ ID NO: 11) using a plasmid containing a putative amylose metabolic gene promoter (Pgk704 promoter (Pgk704), nucleotide sequence described in SEQ ID NO: 10) derived from the Geobacillus kaustophilus HTA426 genome as a template. Pgk704 was amplified using a promoter-rv primer (nucleotide sequence described in SEQ ID NO: 12) and a PCR enzyme (KOD Fx Neo, Toyobo Co., Ltd.) (PCR1).

Furthermore, using the pESG23 plasmid containing lacZ obtained above as a template, promoter-bGalE-fw primer (nucleotide sequence described in SEQ ID NO: 13), HindIII-H6-bGalE-rv primer (described in SEQ ID NO: 14) LacZ was amplified using (nucleotide sequence) and PCR enzyme (KOD Fx Neo, Toyobo Co., Ltd.) (PCR2).

PCR products amplified by PCR1 and PCR2 were each subjected to agarose gel electrophoresis to confirm the length, and then purified using QIAquick Spin Gel Extraction Kit (Qiagen). The fusion PCR was performed using PCR1 product and PCR2 product after purification, HindIII-promoter-fw primer, HindIII-H6-bGalE-rv primer, PCR enzyme (KOD Fx Neo, Toyobo Co., Ltd.), and Pgk704 and lacZ were fused. A cassette was made.

The pNW (SfiI) plasmid was cleaved with HindIII, treated with CIAP (Alkaline Phosphatase (Calf intestine), Takara Bio), purified with QIAquick Spin Gel Extraction Kit (Qiagen), and purified with H in III. It mixed with the said expression cassette refine | purified similarly, it connected using DNA Ligation Kit (Mighty Mix, Takara Bio Inc.), and introduce | transduced into 200 microliters E. coli JM109 chemical competent cell. Transformants were selected on an LB medium plate (34 μg / ml chloramphenicol), inoculated into 3 ml of LB medium (34 μg / ml chloramphenicol) and grown. A pESG28 plasmid containing Pgk704 and lacZ was extracted from the grown transformant using QIAprep spin miniprep kit (Qiagen).

Next, using the obtained pESG28 plasmid as a template, HM58 primer (nucleotide sequence described in SEQ ID NO: 15), HM60 primer (nucleotide sequence described in SEQ ID NO: 16) and PCR enzyme (KOD plus Ver. 2, Toyobo Co., Ltd.) ) Was used to amplify the sequence in which Pgk704 and lacZ were linked (PCR3).

The PCR3 product amplified by PCR3 was subjected to agarose gel electrophoresis to confirm the length, and then purified using a QIAquick Spin Gel Extraction Kit (Qiagen). In addition, the pGAM46 (SfiI) plasmid obtained above was cleaved with HindIII, purified using QIAquick Spin Gel Extraction Kit (Qiagen), mixed with the PCR3 product purified in the same manner, and In-Fusion HD Cloning. The cells were ligated using Kit (Takara Bio Inc.) and introduced into 200 μl of E. coli JM109 chemical competent cell. Transformants were selected on an LB medium plate (100 μg / ml ampicillin), inoculated into 3 ml of LB medium (100 μg / ml ampicillin) and grown. A pESG32 plasmid containing Pgk704 and lacZ was extracted from the grown transformant using QIAprep spin miniprep kit (Qiagen).

<Transformation>
As host microorganisms, Suzuki Hirokazu et al., Appl Environ Microbiol. January 2015, 81 (1), p. The thermophile of the genus Geobacillus described in 149-158 (Geobacillus kaustophilus MK242, optimal growth temperature: 55 ° C.) was used. Geobacillus kaustophilus MK242 was obtained from Dr. Hirokazu Suzuki, Graduate School of Engineering, Tottori University. First, Suzuki Hirokazu et al., J Microbiol Biotechnol. September 2012, 22 (9), p. The pESG32 plasmid was introduced into E. coli according to the method described in 1279-1287.

That is, Suzuki Hirokazu et al., J Microbiol Biotechnol. September 2012, 22 (9), p. After culturing Escherichia coli BR408 described in 1279-1287 in LB medium, the operation of suspending and centrifuging the cells in ultrapure water and collecting the cells was repeated three times. Thereafter, the cells were suspended in 10% glycerol to obtain E. coli competent cells. Next, the obtained Escherichia coli competent cell (60 μl) and the pESG32 plasmid were mixed, put into a 0.1 cm cuvette, and 1800 V, 25 μF, and 200 Ω electrical stimulation were given using Gene Pulser Xcell (BioRad). . Thereafter, 1 ml of LB medium was added and shaken at 37 ° C. for 1 hour, and then applied to an LB medium plate (100 μg / ml ampicillin, 34 μg / ml chloramphenicol, 25 μg / ml kanamycin) overnight at 37 ° C. After culturing, colonies of E. coli BR408 (pESG32) into which the pESG32 plasmid was introduced were obtained.

Then, Hirokazu Suzuki et al., Appl Environ Microbiol. October 2012, 78 (20), p. The pESG32 plasmid was introduced into Geobacillus kaustophilus MK242 by conjugation transfer according to the method described in 7376-7383.

That is, first, the colonies of the E. coli BR408 (pESG32) were transformed into LB medium (100 μg / ml ampicillin, 34 μg / ml chloramphenicol, 25 μg / ml kanamycin) at 37 ° C., and Geobacillus kaustophilus MK242 was added to LB medium (no antibiotics added). ) At 60 ° C. until OD ₆₀₀ = 0.6. Next, E. coli BR408 (pESG32) and Geobacillus kaustophilus MK242 were mixed at a ratio of 2: 8 (mass ratio), and the bacteria were adsorbed on a membrane filter (pore size: 0.2 μm, Omnipore, Merck Millipore) by filtration under reduced pressure. This was placed on an LB agar medium and cultured at 37 ° C. for 4 to 6 hours.

Then, this was added to the minimum medium (K ₂ SO ₄ : 0.3 g / l, Na ₂ HPO ₄ · 12H ₂ O: 2.5 g / l, NH ₄ Cl: 1 g / l, Casamino Acids (BD Bacto Casano Acids, Vitamin Assay): 1 g / l, MgSO ₄ : 0.4 g / l, MnCl ₂ · 4H ₂ O: 3 mg / l, CaCl ₂ · 2H ₂ O: 5 mg / l, FeCl ₃ · 6H ₂ O: 7 mg / l, Tris HCl (pH 7.6): 10 mM, D-glucose: 10 g / l, Trace Element: 0.10% vol / vol (Trace Element: ZnSO ₄ .7H ₂ O: 400 mg / l, H ₃ BO ₃ : 10 mg / L, CoCl ₂ · 6H ₂ O: 50 mg / l, CuSO ₄ : 200 mg / l, NiCl _{2 · 6H 2 O: 10mg /} l, EDTA: 250mg / l)) were transferred to the plate, 60 and cultured overnight at ° C., colonies of Geobacillus kaustophilus MK242 which PESG32 plasmid sequences integrated into the genome (MK242 (pESG32)) Got.

The obtained colony of MK242 (pESG32) was cultured in 200 ml of LB medium, and 200 μl of the culture solution was inoculated into 200 ml of LB medium and cultured four times, and then 1 ml of the culture solution was centrifuged. The precipitate was suspended in 1 ml of ultrapure water. 50 μl or 250 μl of suspension was cultured for 4-6 hours in 5 ml of selective minimal medium 1 (medium supplemented with 1 μg / ml uracil, 50 μg / ml 5-fluoroorotic acid to the minimal medium). Thereafter, 1 μl or 100 μl of the culture solution is applied to a plate of selective minimal medium 2 (medium supplemented with 10 μg / ml uracil, 50 μg / ml 5-fluoroorotic acid to the minimal medium), and cultured at 60 ° C. for about 1 day. To obtain a colony. The obtained colonies were further applied to the selective minimal medium plate and the LB medium plate, respectively, and the colonies that did not grow on the minimal selective medium were selected to produce glycerol stocks from the corresponding colonies of the LB medium, The following transformants were cultured and used for β-galactosidase activity evaluation. The colonies that did not grow on the minimal selection medium are those that do not contain pGAM46-derived sequences among MK242 (pESG32), that is, Geobacillus kaustophilus MK242 (MK242 (Pgk704 / lacZ)) in which Pgk704 and lacZ are integrated into the genome. It is.

<Culture of transformant>
Using MK242 (Pgk704 / lacZ) obtained above as a transformant, the transformant was treated in the same manner as in Example 1 except that the culture in the second step was performed until OD ₆₀₀ = 0.425. After culturing and starting the culture in the third step (after changing the culture temperature to 42 ° C.), measure the OD ₆₀₀ of the culture after 0, 1.5, 3, ₆ , 24, 30 hours of culture. A growth curve was obtained.

<Evaluation of β-galactosidase activity-Enzyme reaction->
Evaluation of β-galactosidase activity in the same manner as in Example 1 except that the culture solution after culturing for 0, 3, and 6 hours after starting the culture in the third step (after changing the culture temperature) was used. Carried out. In addition, β-galactosidase activity was evaluated in the same manner for those in which the incubation time after addition of X-gal was 3 hours.

(Example 4)
Transformants were cultured in the same manner as in Example 3 except that the culture temperature in the third step was 37 ° C., and a growth curve was obtained. In addition, β-galactosidase activity was evaluated in the same manner as in Example 3 except that the incubation temperature after adding X-gal was 37 ° C.

(Comparative Example 2)
In the culture of the transformant, the transformant was cultured in the same manner as in Example 3 except that the culture temperature in the third step was kept at 60 ° C. following the second step. OD ₆₀₀ after culturing at −2.5, −1, 0, 1.5, 3, 6, 24, and 30 hours, assuming that the culture start time is −2.5 hours and the culture start time of the third step is 0 hours. Was measured to obtain a growth curve. Furthermore, β-galactosidase activity was evaluated in the same manner as in Example 3 except that the incubation temperature after addition of X-gal was 60 ° C.

The growth curves obtained in Examples 3 to 4 and Comparative Example 2 are shown in FIG. Table 2 shows the evaluation results of β-galactosidase activity (incubation time: 1 hour). Further, FIG. 4A shows a photograph showing an external appearance when β-galactosidase activity evaluation (incubation time: 1 hour) is performed on the culture solution at the start of culture (after 0 hour culture) in the third step of Comparative Example 2. FIG. 4B is a photograph showing the external appearance when the β-galactosidase activity evaluation (incubation time: 1 hour) was performed on the culture solution after 3 hours of culture from the start of the third step of Examples 3 to 4 and Comparative Example 2. In addition, photographs showing the appearance when β-galactosidase activity evaluation (incubation time: 1 hour) was performed on the culture solution after 6 hours of culture from the start of the culture in the third step of Examples 3 to 4 and Comparative Example 2. Each is shown in FIG. 4C.

Moreover, the photograph which shows the external appearance when implementing beta-galactosidase activity evaluation (incubation time: 3 hours) about the culture solution at the time of the culture | cultivation start (after 0-hour culture | cultivation) of the 3rd process of the comparative example 2 to FIG. 5A, FIG. 5B is a photograph showing the external appearance when the β-galactosidase activity evaluation (incubation time: 3 hours) was performed on the culture solution after 3 hours of cultivation from the start of the third step in Examples 3 to 4 and Comparative Example 2. Respectively.

As is clear from the above results, in the production method of the present invention (Examples 1 to 4), in the third step, an enzyme (β-galactosidase) derived from psychrophilic bacteria and mesophilic bacteria (E. coli) is encoded. It was confirmed that the growth of the transformant introduced with DNA (lacZ) was suppressed and the enzyme was sufficiently expressed.

As described above, according to the present invention, a psychrophilic bacterium and a mesophilic temperature capable of separating the growth stage of a transformant introduced with DNA encoding an enzyme derived from psychrophilic bacteria and mesophilic bacteria and the expression stage of the enzyme. It is possible to provide a method for producing a bacterium-derived enzyme.

SEQ ID NO: 1
<223> HM48 primer SEQ ID NO: 2
<223> HM49 primer SEQ ID NO: 4
<223> pNW33N plasmid SEQ ID NO: 5
<223> HM05b primer SEQ ID NO: 6
<223> HM06-minus primer SEQ ID NO: 7
<223> HM51 primer SEQ ID NO: 8
<223> HM52H primer SEQ ID NO: 9
<223> pESG21H plasmid SEQ ID NO: 11
<223> HindIII-promoter-fw primer SEQ ID NO: 12
<223> promoter-rv primer SEQ ID NO: 13
<223> promoter-bGalE-fw primer SEQ ID NO: 14
<223> HindIII-H6-bGalE-rv primer SEQ ID NO: 15
<223> HM58 primer SEQ ID NO: 16
<223> HM60 primer

Claims (5)

1. A group consisting of a thermophilic bacterium having an optimal growth temperature of 50 ° C. or higher, a thermophilic bacterium having an optimal growth temperature of 50 ° C. or lower and a temperature lower by 10 ° C. or more than the optimal growth temperature of the thermophilic bacterium A first step of obtaining a transformant by introducing DNA encoding an enzyme derived from at least one microorganism selected from:
   A second step of culturing and growing the transformant at a culture temperature of 50 ° C. or higher;
   After the second step, a third step of expressing the enzyme by changing the culture temperature to 50 ° C. or lower and a temperature lower by 10 ° C. or higher than the culture temperature of the second step;
   A method for producing a thermophilic bacterium and an enzyme derived from a mesophilic bacterium.
2. The method for producing enzymes derived from psychrophilic bacteria and mesophilic bacteria according to claim 1, wherein the culture temperature in the second step is 55 to 87 ° C.
3. The method for producing enzymes derived from psychrophilic bacteria and mesophilic bacteria according to claim 1 or 2, wherein the culture temperature in the third step is 25 to 45 ° C.
4. The culture temperature of the second step is changed to the culture temperature of the third step when the OD _{600 of} the culture medium for culturing the transformant is 1 or less. Process for producing psychrophilic and mesophilic bacteria-derived enzymes.
5. An enzyme reaction method in which an enzyme obtained by the method for producing an enzyme derived from a psychrotrophic bacterium or a mesophilic bacterium according to any one of claims 1 to 4 is brought into contact with a substrate of the enzyme to react.

Images