Biocatalyst production method

Abstract

To suppress, in a substance conversion process employing a biocatalyst, leakage of proteins from cells after metabolic enzymes are inactivated by heat treatment.SOLUTION: A method comprises: introducing, into a psychrophile, an enzyme gene functioning in a temperature range higher than a temperature range in which the psychrophile can grow, thereby obtaining a transformed psychrophile; making the transformed psychrophile into a cell suspension using a solution containing at least one selected from the group consisting of monovalent or divalent cations; and heat-treating the cell suspension at a temperature higher than a temperature range in which the psychrophile can grow.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to methods for producing biocatalysts. [Background technology]

[0002] Material conversion processes using biocatalysts that express various enzymes within cells are expected to be used in a variety of fields as environmentally friendly processes. Conventional microbial catalysts have a problem in that because various enzymes are expressed within the cells, the substrate is used not only to produce the target substance but also for the metabolic functions of the host, resulting in the generation of various metabolic by-products, which reduces the yield of the target substance.

[0003] Therefore, the inventors developed a method that utilizes the temperature characteristics of enzyme reactions to realize a substance conversion process that can obtain target substances in higher yields. Specifically, as described in Patent Document 1 and Non-Patent Document 1, a gene encoding a mesophilic enzyme was introduced into a psychrophilic bacterium that can grow at 10 to 30°C, and the metabolic enzymes of the psychrophilic bacterium were inactivated by heat treatment at 40 to 50°C, allowing only the mesophilic enzymes to function, thereby suppressing the production of by-products and obtaining target substances in high yields. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2020-000167 [Non-patent literature]

[0005] [Non-Patent Document 1] Takahisa Tajima, Akiko Hida, Junichi Kato: Efficient material conversion using simple enzyme catalysts from psychrophilic bacteria, Journal of Environmental Biotechnology, 21(1), 9-16, 2021 (Journal of the Society for Environmental Biotechnology, Vol. 21, No. 1) Summary of the Invention [Problem to be solved by the invention]

[0006] This method not only inactivates metabolic enzymes through heat treatment, but also partially damages the cell structure. Initially, it was thought that partially damaging the cell structure would have the advantage of improving membrane permeability to substrates, but subsequent research revealed a problem in that depending on the type of enzyme (protein) required for substance conversion, it could leak out of the cell through the damaged area. There were concerns that the leakage of enzymes would reduce reaction efficiency and make sustainable use difficult, so a solution to this problem was required.

[0007] The present disclosure has been made in view of the above points, and its purpose is to suppress protein leakage from cells after metabolic enzymes have been inactivated by heat treatment in a method for producing a biocatalyst. [Means for solving the problem]

[0008] In order to achieve the above object, in the present disclosure, a cell suspension is prepared using a predetermined solution for psychrophilic bacteria, and the cell suspension is then heat-treated.

[0009] Specifically, the method for producing a biocatalyst disclosed herein comprises: a step of introducing into a psychrophilic bacterium an enzyme gene that functions in a temperature range higher than the temperature range at which the psychrophilic bacterium can grow, thereby obtaining a transformed psychrophilic bacterium; preparing a cell suspension of the transformed psychrophilic bacterium using a solution containing at least one selected from the group consisting of monovalent and divalent cations; and heat-treating the cell suspension at a temperature higher than the temperature range in which psychrophilic bacteria can grow.

[0010] According to the method for producing a biocatalyst disclosed herein, a cell suspension is prepared by suspending psychrophilic bacteria in a specified solution, and the cell suspension is then heat-treated. This makes it possible to suppress the leakage of proteins from the cells after metabolic enzymes are inactivated by heat treatment, thereby improving reaction efficiency and enabling sustainable use.

[0011] The cations are preferably calcium ions, potassium ions, sodium ions, magnesium ions, manganese ions and zinc ions, and more preferably magnesium ions, manganese ions and zinc ions.

[0012] The solution used for the cell suspension preferably contains at least one selected from the group consisting of calcium chloride, sodium chloride, magnesium chloride, manganese chloride, zinc chloride, sodium sulfate, and magnesium sulfate. The solution used for the cell suspension may be seawater or artificial seawater, or an aqueous solution containing five components (sodium chloride, magnesium chloride, calcium chloride, anhydrous sodium sulfate, potassium chloride, and sodium bicarbonate) that are present in artificial seawater at 100 mg / L or more.

[0013] The solution used for the cell suspension more preferably contains at least one selected from the group consisting of magnesium chloride, magnesium sulfate, manganese chloride, and zinc chloride.

[0014] In one embodiment, the temperature range in which the psychrophilic bacteria can grow is lower than 30°C, and the heat treatment is performed at a temperature of 40°C or higher and 50°C or lower.

[0015] The psychrophilic bacterium is preferably Shewanella frigidimarina or Shewanella livingstonensis. [Effects of the Invention]

[0016] As described above, according to the present disclosure, it is possible to suppress protein leakage from cells after metabolic enzymes have been inactivated by heat treatment. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 is a schematic diagram showing the structure of plasmid pHA12-gfp. [Figure 2]1 is a graph showing the relationship between the presence or absence of heat treatment of the plasmid pHA12-gfp and protein leakage. [Figure 3] 1 shows the results of SDS-PAGE showing the molecular weight assessment of the leaked proteins. [Figure 4] FIG. 1 is a schematic diagram showing the structure of the plasmid pHA12-gfp-cad. [Figure 5] FIG. 1 is a schematic diagram showing the structure of the plasmid pHA12-gfp-lacZ. [Figure 6] 1 is a graph showing the results of measuring leaked proteins according to Experimental Examples 1 and 2. [Figure 7] 1 is a graph showing the rate of GFP leakage from GFP-expressing strains cultured in NaCl-supplemented medium. [Figure 8] 1 is a graph showing the rate of GFP leakage from GFP-expressing strains cultured in a medium supplemented with artificial seawater. [Figure 9] 1 is a graph showing the rate of GFP-CAD leakage from a GFP-CAD-expressing strain cultured in a medium supplemented with artificial seawater. [Figure 10] 1 is a graph showing the type of medium and the rate of leakage of GFP-CAD. [Figure 11] 1 is a graph showing the relationship between the amount of liquid during heat treatment and the leakage rate of GFP-CAD. [Figure 12] 1 is a graph showing the relationship between the OD value during heat treatment and the leakage rate of GFP-CAD. [Figure 13] 1 is a graph showing the relationship between heat treatment time and the leakage rate of GFP-CAD. [Figure 14] 1 is a graph showing the relationship between the type of cell suspension and the rate of GFP leakage. [Figure 15] 1 is a graph showing the relationship between the type of cell suspension and the rate of GFP leakage. [Figure 16] 1 is a graph showing the relationship between the type of cell suspension and the rate of GFP leakage. [Figure 17] 1 is a graph showing the relationship between the type of cell suspension and the rate of GFP leakage. [Figure 18] 1 is a graph showing the relationship between the type of cell suspension and the rate of GFP leakage. [Figure 19] 1 is a graph showing the relationship between the type of cell suspension and the rate of GFP leakage. [Figure 20] 1 is a graph showing the relationship between the type of cell suspension and the rate of GFP leakage. [Figure 21] 1 is a graph showing the relationship between the type of cell suspension and the rate of GFP leakage. DETAILED DESCRIPTION OF THE INVENTION

[0018] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present disclosure will be described in detail below with reference to the accompanying drawings. The following description of the preferred embodiments is merely exemplary in nature and is not intended to limit the present invention, its applications, or uses.

[0019] The method for producing a biocatalyst of the present disclosure comprises a transformation step in which an enzyme gene that functions in a temperature range higher than the temperature range at which the psychrophilic bacterium can grow is introduced into the psychrophilic bacterium to obtain a transformed psychrophilic bacterium; a cell suspension preparation step in which the transformed psychrophilic bacterium is made into a cell suspension using a solution containing at least one ion selected from the group consisting of calcium ions, potassium ions, sodium ions, magnesium ions, manganese ions, and zinc ions; and a heat treatment step in which the cell suspension is heat-treated at a temperature higher than the temperature range at which the psychrophilic bacterium can grow.

[0020] Psychrotrophs are bacteria that can grow at temperatures below 30°C, and include psychrophilic bacteria. Psychrotrophs cannot grow in environments above 30°C, as the metabolic enzymes within their cells become inactivated. Various psychrophilic bacteria can be used in the method for producing a biocatalyst of the present disclosure. Usable psychrophilic bacteria include, for example, bacteria of the genus Shewanella, including, but not limited to, Shewanella frigidimarina (optimum growth temperature: 20°C) and Shewanella livingstonensis (optimum growth temperature: 18°C).

[0021] The enzyme gene introduced into the psychrophilic bacterium is an enzyme that functions in a temperature range higher than the temperature range at which the psychrophilic bacterium can grow, and is not inactivated by heat treatment, which will be described later. In this embodiment, enzyme genes expressing cis-aconitate dehydrogenase and β-galactosidase are used as the enzyme genes, but this is not limited to these. The enzyme gene may be derived from, for example, Escherichia coli. The enzyme gene may be of one type, or two or more types. Furthermore, the enzyme genes may be derived from the same species or different species. The method for introducing such an enzyme gene into the psychrophilic bacterium is not particularly limited, and may be, for example, a method using a conjugatively transferable plasmid.

[0022] The transformed psychrophilic bacteria are suspended in a predetermined solution to form a cell suspension, which is then heat-treated. The solution used for the cell suspension may be used for collecting or washing the psychrophilic bacteria after cultivation. The solution used for the cell suspension contains at least one selected from the group consisting of monovalent or divalent cations. More specifically, the solution used for the cell suspension contains at least one selected from the group consisting of calcium ions, potassium ions, sodium ions, magnesium ions, manganese ions, and zinc ions. From the viewpoint of enhancing the protein leakage inhibitory effect, the solution used for the cell suspension preferably contains at least one selected from the group consisting of magnesium ions, manganese ions, and zinc ions. The anions for these cations are not particularly limited, but examples include chloride ions and sulfate ions. The solution used for the cell suspension preferably contains at least one selected from the group consisting of calcium chloride, sodium chloride, magnesium chloride, manganese chloride, zinc chloride, sodium sulfate, and magnesium sulfate. From the viewpoint of enhancing the protein leakage inhibitory effect, the solution used for the cell suspension more preferably contains at least one selected from the group consisting of magnesium chloride, magnesium sulfate, manganese chloride, and zinc chloride. The calcium chloride, sodium chloride, and magnesium chloride are preferably present at concentrations similar to those found in general artificial seawater. For example, calcium chloride is preferably about 1.5 g / L, sodium chloride is preferably about 22 g / L, and magnesium chloride is preferably about 10 g / L. The solution used for the cell suspension may be seawater or artificial seawater, or may be an aqueous solution containing the main components of seawater, namely, sodium chloride, magnesium chloride, calcium chloride, sodium sulfate, potassium chloride, and sodium bicarbonate, at concentrations similar to those found in artificial seawater.

[0023] Before adding a substrate to the transformed psychrophilic bacteria to initiate the reaction, the cell suspension is heat-treated at a temperature higher than the temperature range in which the psychrophilic bacteria can grow. In this embodiment, the heat treatment temperature is 40°C or higher and 50°C or lower, but is not limited as long as it is a temperature that can inactivate the metabolic enzymes of the psychrophilic bacteria itself. Furthermore, the heat treatment time is not limited.

[0024] The present invention will be explained in more detail below by way of examples, but the present invention is not limited to these examples. [Example]

[0025] <Experimental Example 1: Confirmation of leakage using green fluorescent protein (GFP) expressing strain> First, to confirm whether proteins leaked from psychrophilic bacteria after heat treatment, we constructed psychrophilic bacteria expressing green fluorescent protein (GFP) and evaluated their fluorescence intensity after heat treatment.

[0026] The strains and plasmids used in Experimental Example 1 are shown below. Escherichia coli DH5α: Host for plasmid construction Escherichia coli S17-1: Host for conjugation Shewanella livingstonensis Ac10: Psychrotrophic, Rifampicin-resistant strain Shewanella frigidimarina DSM 12253: Psychrotrophic, Rifampicin-resistant strain pHA12: A broad-host-range enzyme expression plasmid containing an ampicillin resistance marker and lac repressor. pRCII-GFPmut2: GFPmut2 expression plasmid pHA12-gfp: A plasmid in which the gfp gene was inserted into the EcoRI and KpnI sites of pHA12 1. Method for producing GFP-transfected psychrophilic bacteria (1-1) Construction of a plasmid for introducing GFP Specifically, the gfp (GFP mut2) gene was amplified by polymerase chain reaction (PCR) using primers designed with reference to the restriction enzyme site of the pHA12 described above (Arai et al., Construction of Novel Expression Vectors Effective in Pseudomonas Cells. Agricultural and Biological Chemistry. 1991, 55(9), 2431-2432) and the pRCII-GFPmut2 plasmid as a template. The PCR primers used (gfp mut2 (SEQ ID NO: 1), gfp Fw (SEQ ID NO: 2), gfp Rv (SEQ ID NO: 3)) are listed in Table 1, and the PCR reagents and conditions are listed in Tables 2 and 3. Amplification of a DNA fragment of the intended size (717 bp) was confirmed by agarose gel electrophoresis. The fragment was then gel-extracted using the Fast Gene® Gel / PCR Extraction Kit (Nippon Genetics). The expression plasmid pHA12 and the extracted and purified solution of gfp were subjected to restriction enzyme digestion with EcoRI (TOYOBO) and KpnI (TOYOBO) (37°C, 2 hours).

[0027] [Table 1]

[0028] [Table 2]

[0029] [Table 3]

[0030] The reaction composition for restriction enzyme treatment is shown in Table 4. The reaction solution after restriction enzyme treatment was electrophoresed on an agarose gel, and bands of sizes corresponding to gfp and pHA12 were excised. Three gfp fragments and one pHA12 fragment were placed in a 1.5 mL tube, and DNA was extracted from the gel using a Fast Gene® Gel / PCR Extraction Kit. 10 μL of the extract was mixed with 10 μL of Ligation High ver. 2 (TOYOBO) and incubated at 16°C for 30 minutes for ligation reaction, constructing a plasmid for introducing gfp.

[0031] [Table 4]

[0032] (1-2) Transformation of E. coli by heat shock 30 μL of competent E. coli S17-1 cells and 3 μL of the GFP-introducing plasmid were mixed and placed on ice. After 20 minutes, the mixture was heat-shocked at 42°C for 45 seconds and then placed on ice for 1 minute. 300 μL of LB medium (10 g / L tryptone (Nacalai Tesque), 10 g / L NaCl (Fujifilm Wako Pure Chemical Industries, Ltd.), 5 g / L dried yeast extract (Nacalai Tesque), 16 g / L agar powder (Nacalai Tesque)) was added and the mixture was placed at 37°C. After 1 hour, the mixture was plated on LB agar medium (Amp) containing 100 mg / L ampicillin (prepared with Bixillin for Injection, Meiji Seika Pharma) and cultured overnight (approximately 16 hours) at 37°C to allow colony formation. Transformants were isolated.

[0033] (1-3) Transformation of psychrophilic bacteria by conjugation The resulting transformant of E. coli S17-1 was inoculated into 4 mL of LB medium (Amp) and cultured overnight at 37°C with shaking. S. livingstonensis Ac10 and S. frigidimarina DSM 12253 were inoculated into 4 mL of TSB (Tryptic Soy Broth; BD) medium (Rif) containing 50 mg / L rifampicin (Rif; Tokyo Kasei) and cultured at 18°C ​​for approximately 48 hours with shaking. One mL of each culture was placed in a 1.5 mL tube and centrifuged (10,000 rpm, 2 min). The supernatant was removed and the resulting mixture was suspended in 1 mL of TSB medium. This process was repeated, and the resulting mixture was then suspended in 100 μL of TSB medium. Fifty microliters of the E. coli S17-1 transformant cell suspension were added to each psychrophilic bacterial suspension, pipetted, and then 150 μL of the resulting solution was spotted onto antibiotic-free TSB agar medium and incubated at 18°C ​​for 2–3 days. 2 mL of TSB medium was added to the spotted area, and the cells were scraped and suspended. This stock solution was then diluted 10-fold, 100-fold, and 1000-fold with TSB liquid medium. It was then plated onto TSB agar medium (Amp, Rif) and incubated at 18°C ​​for 5 days to allow colony formation. The transformed psychrophilic strains (Ac10 / pHA12-gfp, DSM / pHA12-gfp) were isolated. A schematic diagram of the gfp expression plasmid pHA12-gfp is shown in Figure 1.

[0034] (1-4) Plasmid extraction In the case of E. coli, 4 mL of LB medium (Amp) was used and cultured overnight at 37°C with shaking. In the case of psychrophilic bacteria, 4 mL of TSB medium (Amp, Rif) was used and cultured with shaking for 48 hours at 18°C. 4 mL of the culture medium was placed in a 5 mL tube, and DNA was purified using a Fast Gene® Plasmid Mini Kit. Plasmids were extracted from the transformants using the above method, and the target gene was amplified by PCR using the extracted plasmid as a template. Introduction of the target gene into the plasmid was confirmed by agarose gel electrophoresis.

[0035] (1-5) Cultivation of psychrophilic bacteria One loopful of each of the DSM and Ac10 transformants was inoculated into 4 mL of TSB medium (Amp, Rif) and cultured at 18°C ​​for 24 hours with shaking. The culture was diluted 100-fold and 1000-fold with TSB medium, plated on TSB agar medium (Amp, Rif), and cultured stationary at 18°C ​​for 48 hours. The resulting single colony was inoculated into 4 mL of TSB medium (Amp, Rif) using a toothpick and cultured at 18°C ​​for 48 hours with shaking. This culture was then inoculated into 100 mL of TSB medium (Amp, Rif) at a concentration of 1% and cultured at 18°C ​​in a 300 mL Erlenmeyer shake flask. After 24 hours, 400 μL of 0.1 M IPTG was added, and the culture was cultured for a total of 48 hours with shaking.

[0036] (1-6) Collection and washing of psychrophilic bacteria After incubation, the culture medium was transferred to a 50 mL centrifuge tube and centrifuged (6,000 rpm, 10 min, 4°C) using a TOMY MX-307 to harvest the cells. The precipitate was suspended in 50 mM phosphate buffer (pH 6.5), and the cells were washed twice (5,000 × g, 10 min, 4°C). The supernatant was then discarded. The 50 mM phosphate buffer was prepared by diluting 0.2 M phosphate buffer (pH 6.5) prepared as follows: 31.21 g / L sodium dihydrogen phosphate dihydrate (Nacalai Tesque) and 28.39 g / L disodium hydrogen phosphate (Nacalai Tesque). While measuring the pH, the 0.2 M phosphate buffer (pH 6.5) was adjusted to pH 6.5 by adding sodium dihydrogen phosphate dihydrate solution to the disodium hydrogen phosphate solution.

[0037] (1-7) Microscopic observation The collected and washed cells were again added to 50 mM phosphate buffer (pH 6.5) and diluted to OD 600 = 5 (10-fold dilution) 600= 0.5). 3 μL of the prepared cell suspension was dropped onto a glass slide and a cover glass was placed on top. Images of the prepared sample were taken using a HS all-in-one fluorescence microscope BZ-9000 (Keyence) with a 100x objective lens, using phase contrast and fluorescence observations. The psychrophilic bacteria cells emitted fluorescence, indicating that the GFP expression plasmid constructed was successfully expressed and could be used for analysis.

[0038] (1-8) Fluorescence intensity measurement To obtain detectable fluorescence intensity, the prepared samples were diluted 10-fold or 100-fold with 50 mM phosphate buffer (pH 6.5) and the fluorescence intensity was measured using a spectrofluorometer (FP-6500 Spectrofluorometer, JASCO). The measurement conditions were: maximum excitation wavelength of GFP mut2 at 481 nm, maximum emission wavelength at 507 nm, response time of 1 sec, sensitivity at Medium, and repetition rate of 3.

[0039] (1-9) Confirmation of thermal stability The collected and washed cells were again added to 50 mM phosphate buffer (pH 6.5) to obtain OD 600 The suspension was diluted 10-fold to an OD of 5. 600 = 0.5). 500 μL of the prepared cell suspension was placed in a 1.5 mL tube and heat-treated at various temperatures (30, 40, 45, 50, 60, 70°C). After heat treatment, the sample was left to stand on ice for a short time. The fluorescence intensity of the sample that had been left on ice and the heat-treated sample was then measured, and the following equation (1) was used: Relative fluorescence intensity = Fluorescence intensity of heat-treated sample / Fluorescence intensity of sample placed on ice (1) The fluorescence intensity was compared using a fluorochrome platelet assay. The results showed that the fluorescence intensity of samples from the Ac10 and DSM strains heat-treated at 60°C was approximately 90% of that of samples left on ice, with no significant decrease observed. The GFP expressed in both the Ac10 and DSM strains was not inactivated at moderate temperatures (30°C to 60°C), demonstrating its utility for evaluating the fluorescence of the expressing strains before and after heat treatment.

[0040] (1-10) Measurement of protein leakage rate The harvested and washed cells were diluted in 50 mM phosphate buffer (pH 6.5) to obtain OD 600 = 10 (OD value of 100-fold diluted is OD 600 = 0.1). 7 mL of the prepared cell suspension was placed in a 15 mL centrifuge tube and heat-treated at 45°C for 30 minutes (500 μL placed in a 1.5 mL tube was heat-treated at 45°C for 15 minutes) to prepare a sample, which was then left to stand on ice for approximately 15 minutes. If necessary, a control sample was also prepared by leaving it to stand on ice. The prepared sample was centrifuged in a TOMY MX-307 (10,000 rpm, 10 min, 4°C), and the supernatant was collected and subjected to fluorescence intensity measurement. The measured fluorescence intensity was compared with the following equation (2). Protein leakage rate = Fluorescence intensity of supernatant / Fluorescence intensity of cell suspension (2) The percentage of protein leakage was calculated using

[0041] When comparing the sample that had been left on ice with the heat-treated sample, an increased rate of protein leakage was confirmed in the heat-treated sample, as shown in Figure 2. This suggests that heat treatment promotes protein leakage.

[0042] (1-11) Ultrasonic disruption The harvested and washed cells were diluted in 50 mM phosphate buffer (pH 6.5) to obtain OD 600 = 10 (100-fold dilution) 600 The cells were suspended to a pH of 0.1. The resulting cell suspension was sonicated using a Digital Sonifier (BRANSON) at an amplitude of 20%, a pulse duration of 10 min, pulse on = 1.0 sec, and pulse off = 1.0 sec. The cells were then centrifuged (10,000 rpm, 4°C, 10 min) and the supernatant was collected.

[0043] (1-12) Molecular weight evaluation of proteins by SDS-PAGE To confirm whether there is a correlation between the molecular weight of the protein and the leakage rate, the amount of leaked protein was evaluated by SDS-PAGE. The results are shown in Figure 3.

[0044] Reagents other than TEMED (N,N,N',N'-tetramethylethylenediamine, Nacalai Tesque) were mixed according to Tables 5 and 6 to prepare 10% separating gel solution and stacking gel solution, respectively. TEMED was added to the separating gel, and the separating gel was poured into the gap of a gel preparation plate and quickly overlaid with approximately 150 μL of butanol. The mixture was left at 28°C for approximately 30 minutes to allow the gels to bind. After removing the butanol, TEMED was added to the stacking gel, which was then poured onto the separating gel. A comb was inserted and the mixture was left at room temperature for 30 minutes to polymerize the gel. The comb was then removed and the gel was placed in the electrophoresis chamber. The inside of the electrophoresis chamber and gel plate were filled with electrophoresis buffer, and 5 μL of a marker (Precision Plus Protein Standards, BIO-RAD) and a sample (mixed with an equal volume of 2x sample buffer (Nacalai Tesque)) were applied to the wells. Electrophoresis was performed at approximately 15 mA until the marker reached the bottom of the gel.

[0045] [Table 5]

[0046] [Table 6]

[0047] After electrophoresis, the gel was removed from the gel plate, immersed in staining solution CBB Strain One Super (Nacalai Tesque), heated in a microwave oven until boiling, removed, and shook vigorously several times. The gel was then immersed in ion-exchanged water, heated in a microwave oven, boiled, removed, and shook vigorously several times. Finally, the gel was again filled with ion-exchanged water, immersed from above with Kimwipes, and shaken overnight for further destaining.

[0048] For the samples, the collected and washed cells were added to 50 mM phosphate buffer (pH 6.5) at OD 600 The following three types of samples were prepared: (1) Sample of ultrasonically disrupted cell suspension (total protein) (2) 1 mL of the sample was placed in a 1.5 mL tube and heat-treated at 45°C for 15 minutes (leaked protein). (3) Samples placed on ice (leaked proteins) As shown in Figure 3, when the cell lysate and the supernatant after heat treatment were compared, the large molecular weight bands that were detected in the cell lysate were difficult to detect in the supernatant after heat treatment. This result suggests that the amount of leaked protein may depend on its molecular weight.

[0049] <Experimental Example 2: Confirmation of leakage from fusion protein expression strain> Plasmids expressing fusion proteins in which CAD (cis-aconitic acid decarboxylase) and LacZ (β-galactosidase) were fused to GFP were prepared and introduced into S. livingstonensis Ac10 and S. frigidimarina DSM 12253 to construct strains expressing these proteins. These strains were then used to evaluate the leakage of fusion proteins of different molecular weights from psychrophilic cells using GFP fluorescence as an indicator. Note that in the following explanation, the same production and evaluation methods as those described in Experimental Example 1 were used, and therefore overlapping details will be omitted.

[0050] 2. Method for producing psychrophilic bacteria with fusion protein introduced (2-1) Construction of a plasmid for introducing a fusion protein The plasmid (pHA12-gfp-lacZ) used in Experimental Example 2 is a plasmid in which the gfp-lacZ gene has been inserted between the EcoRI and HindIII sites of pHA12, and the plasmid used in Experimental Example 3 is a plasmid in which the gfp-cad gene has been inserted between the EcoRI and HindIII sites of pHA12.

[0051] Each gene (including the linker portion) was amplified by PCR. The PCR primers used (cad (SEQ ID NO: 4), lacZ (SEQ ID NO: 5), Infusion_gfp_Fw (SEQ ID NO: 6), Infusion_gfp_Rv (SEQ ID NO: 7), Infusion_cad_Fw (SEQ ID NO: 8), Infusion_cad_Rv (SEQ ID NO: 9), Infusion_lacZ_Fw (SEQ ID NO: 10), Infusion_lacZ_Rv (SEQ ID NO: 11)) are shown in Tables 7 to 9, and the PCR reaction composition and conditions are shown in Tables 10 and 11. Agarose gel electrophoresis was used to confirm whether DNA fragments of the intended sizes (717 bp for gfp, 1473 bp for cad, and 3075 bp for lacZ) had been amplified.

[0052] [Table 7]

[0053] [Table 8]

[0054] [Table 9]

[0055] [Table 10]

[0056] [Table 11]

[0057] After confirming that the target DNA fragment had been amplified, gel extraction was performed using the Fast Gene (registered trademark) Gel / PCR Extraction Kit. The extracted sample was subjected to PCR to ligate and amplify DNA fragments containing gfp and cad, and gfp and lacZ. The PCR primers used are shown in Tables 7 to 9, and the PCR reaction composition is shown in Table 12. The reaction conditions were the same as those in Table 11. Agarose gel electrophoresis was used to confirm whether DNA fragments of the intended size had been amplified. After confirming that the target DNA fragment had been amplified, gel extraction was performed using the Fast Gene (registered trademark) Gel / PCR Extraction Kit.

[0058] [Table 12]

[0059] Furthermore, pHA12 was digested with restriction enzymes EcoRI (TOYOBO) and HindIII (TOYOBO) at its restriction enzyme sites. The digested pHA12 was confirmed by agarose gel electrophoresis, and gel extraction was performed using a Fast Gene (registered trademark) gel / PCR extraction kit.

[0060] The DNA concentrations of the prepared insert and linear vector were confirmed, and they were mixed using the reaction composition for In-Fusion Cloning (Takara) shown in Table 13. In-Fusion Cloning was performed (reacted at 50°C for 15 minutes, then allowed to stand on ice) to construct a plasmid for introducing the fusion protein. Figure 4 shows a schematic diagram of the gfp_cad expression plasmid pHA12-gfp-cad, and Figure 5 shows a schematic diagram of the gfp_lacZ expression plasmid pHA12-gfp-lacZ.

[0061] [Table 13]

[0062] The nucleotide sequence of the expressed protein was confirmed using the same method as in Experimental Example 1. The primers used were a primer containing a pHA12-derived sequence, a primer containing a cad-derived sequence, and a primer containing a lacZ-derived sequence, and the presence or absence of mutations was confirmed. The lacZ-derived primers are shown in Table 14 (gfp_309_Fw (SEQ ID NO: 12), lacZ_263_Fw (SEQ ID NO: 13), lacZ_648_Fw (SEQ ID NO: 14), lacZ_1260_Fw (SEQ ID NO: 15), lacZ_1803_Fw (SEQ ID NO: 16), and lacZ_2360_Fw (SEQ ID NO: 17)). To confirm the construction of the fusion protein expression strain, microscopic observation was performed to observe fluorescence from psychrophilic cells. Fluorescence due to GFP was observed in both the GFP_CAD-expressing strain and the GFP_LacZ-expressing strain, indicating successful expression of the fusion protein.

[0063] [Table 14]

[0064] (2-2) Measurement of protein leakage rate in fusion protein expression strains The rate of protein leakage after heat treatment was measured for the GFP-expressing strain constructed in Experimental Example 1 and the fusion protein-expressing strains (GFP_CAD-expressing strain and GFP_LacZ-expressing strain) constructed in Experimental Example 2. The results are shown in Figure 6. The rate was approximately 78% for the GFP-expressing strain, approximately 26-39% for the GFP-CAD-expressing strain, and approximately 11-16% for the GFP-LacZ-expressing strain, suggesting that the amount of extracellular leakage decreased as the protein expressed intracellularly increased.

[0065] <Experimental Example 3: Inhibition of protein leakage> In Experimental Example 3, the method for culturing psychrophilic bacteria, collection and washing of psychrophilic bacteria, and measurement of fluorescence intensity were carried out as follows.

[0066] (3-1) Cultivation method of psychrophilic bacteria Psychrotrophic bacteria that had been subcultured on TSB agar medium (Amp, Rif) were inoculated into approximately 300 μL of TSB medium using a platinum loop, plated on TSB agar medium (Amp, Rif), and cultured statically at 18°C ​​for 24 hours. The psychrotrophic bacteria grown on the agar medium were suspended in 300 μL of TSB medium placed on ice using a toothpick, and the solution was diluted 10-fold to obtain OD . 600 The OD value was measured. 600 The OD of this culture was 0.1 and the resulting suspension was inoculated to a concentration of 1% and cultured at 18°C ​​for 40 hours with shaking. 600 OD values ​​were measured and added to 100 mL of TSB medium (Amp, Rif). 600 The cells were inoculated into a 1% culture medium (1 mL) at 18°C ​​in a 300 mL Erlenmeyer shake flask. After 24 hours, 200 μL of 0.1 M IPTG (isopropyl-β-D-thiogalactopyranoside, Nacalai Tesque) was added, and the cells were cultured with shaking for a total of 48 hours.

[0067] (3-2) Collection and washing of psychrophilic bacteria After incubation, the culture medium was transferred to a 50 mL centrifuge tube and centrifuged using a TOMY MX-307 (6,000 rpm, 10 min, 4°C) to harvest the cells. The precipitate was suspended in 50 mM phosphate buffer (pH 6.5), and the cells were washed twice (5,000 × g, 10 min, 4°C), after which the supernatant was discarded. During harvesting and washing, the cells were suspended by pipetting 15 times using a 5 mL pipette set to 5 mL.

[0068] (3-3) Fluorescence intensity measurement The prepared cell suspension was diluted with 50 mM phosphate buffer (pH 6.5) to OD 600 = 10 (10-fold diluted OD 600The cells were suspended so that the value was 1). One mL of the cell suspension was placed in a 1.5 mL centrifuge tube and heat-treated at 45°C for 15 minutes. The sample was then left on ice for approximately 15 minutes. A control sample was also prepared by leaving the tube on ice. These samples were centrifuged in a TOMY MX-307 (10,000 rpm, 10 min, 4°C) and the supernatant was collected. These samples were diluted 10-fold or 100-fold with 50 mM phosphate buffer (pH 6.5) and their fluorescence intensity was measured using a spectrofluorometer (FP-6500 Spectrofluorometer, JASCO). To stabilize the measurement, measurements were started at least 20 minutes after the instrument was turned on. Because the fluorescence intensity value fluctuated even after the sample was set, measurements were continued for approximately 1 minute after setting the sample. Once the fluorescence intensity value stabilized, the measurement was confirmed.

[0069] (3-4) Examination of medium composition In Experimental Example 3, since the culture temperature, medium components, etc. may affect membrane fluidity, we attempted to suppress protein leakage by examining conditions such as medium composition.

[0070] In this study, the following media were used to evaluate the rate of protein leakage. 1) TSB medium with added NaCl: 30 g of Tryptic Soy Broth (BD) and 30 g of NaCl (Fujifilm Wako Pure Chemical Industries) dissolved in 1 L of distilled water. 2) TSB medium with artificial seawater added: 12.5 L of distilled water was added to one 25 L packet of Marine Art SF-1 (Tomita Pharmaceuticals) artificial seawater to make double-concentration artificial seawater. For TSB, 60 g of Tryptic Soy Broth was dissolved in 1 L of distilled water to make a double-concentration TSB solution, which was then mixed in equal parts with double-concentration TSB medium and double-concentration artificial seawater. The composition of the artificial seawater is shown in Table 15.

[0071] [Table 15]

[0072] Figure 7 shows the percentage of GFP leakage in the GFP-expressing strain cultured in TSB medium supplemented with NaCl. The percentage of GFP leakage in the NaCl-supplemented medium was nearly 80%, which was similar to that in the NaCl-free medium. This suggests that increasing the salt concentration by adding NaCl to the medium does not affect the percentage of protein leakage.

[0073] Figures 8 and 9 show the protein leakage rates of the GFP-expressing and GFP-CAD-expressing strains in medium supplemented with artificial seawater. As shown in Figure 8, when the leakage rate of the GFP-expressing strain was assessed using medium supplemented with artificial seawater, GFP leakage was nearly 80%, which was similar to that observed when cultured in medium without NaCl or artificial seawater. This suggests that the addition of artificial seawater to the medium does not significantly affect the GFP leakage rate. Furthermore, as shown in Figure 9, when the leakage rate of the GFP-CAD-expressing strain was assessed using medium supplemented with artificial seawater, the Ac10 strain showed approximately 16% leakage and the DSM 12253 strain showed approximately 11% leakage. Considering that the leakage rates in TSB medium were approximately 26% for the Ac10 strain and approximately 39% for the DSM 12253 strain, the DSM 12253 strain showed an improvement in the leakage rate. This suggests that the addition of artificial seawater to the medium somehow affects the membrane and reduces the GFP-CAD leakage rate. Furthermore, considering that there was no change in the leakage rate in the GFP-expressing strain, it is possible that the size of the gaps in the cell membrane of psychrophilic bacteria cultured in a medium containing artificial seawater is smaller than that of heat treatment.

[0074] (3-5) Study of artificial seawater components Next, we measured the leakage rate of GFP-CAD using a medium prepared based on the components of artificial seawater and the DSM 12253 strain, which was significantly affected by artificial seawater. The following media were used for this study. The results are shown in Figure 10. (1) TSB medium only (2) TSB medium containing artificial seawater (3) A medium containing sodium chloride and the main components of artificial seawater (five components contained in artificial seawater at 100 mg / L or more: magnesium chloride hexahydrate (Kanto Chemical), calcium chloride dihydrate (Fujifilm Wako Pure Chemical), anhydrous sodium sulfate (Katayama Chemical), potassium chloride (Fujifilm Wako Pure Chemical), and sodium bicarbonate (Nacalai Tesque)) in TSB. (4) TSB medium supplemented with sodium chloride and calcium chloride (5) TSB medium supplemented with sodium chloride and magnesium chloride In the media (3) to (5), sodium chloride, magnesium chloride, calcium chloride, anhydrous sodium sulfate, potassium chloride, and sodium bicarbonate were prepared as aqueous solutions with the same concentrations as those of the components of artificial seawater shown in Table 15. Aqueous solutions containing these at twice the concentrations were prepared, and then the same amounts were mixed with twice the concentration of TSB medium to prepare the media.

[0075] The leakage rate was slightly reduced in the medium containing artificial seawater (2) compared to the TSB medium alone (1). However, the leakage rates in the media (3) to (5) were almost unchanged or increased compared to the TSB medium alone (1). This reaffirmed that the addition of artificial seawater had a slight effect, but the significance of the effect of adding specific inorganic salts was not confirmed.

[0076] (3-6) Examination of heat treatment conditions In an attempt to suppress protein leakage, we investigated the GFP-CAD expressing strain by changing only the heat treatment conditions while keeping the culture conditions the same as before. The conditions were varied: the liquid volume during heat treatment was 100 μL, 500 μL, and 1000 μL; the turbidity during heat treatment was 1, 5, 10, and 30; and the heat treatment time was also varied: 5, 10, 30, and 60 min; and the leakage rate was evaluated. The results are shown in Figures 11 to 13. The liquid volume during heat treatment did not affect the leakage rate, but it was confirmed that the leakage rate fluctuated when the turbidity and time during heat treatment were changed. Therefore, in subsequent experiments, the heat treatment conditions were set at OD 1000 with a liquid volume of 1 mL. 600The experiment was conducted with the value set to 10 (10 times dilution = 1) and the heat treatment time set to 15 minutes.

[0077] (3-7) Study of cell suspension during heat treatment In Experiments 1 and 2, 50 mM phosphate buffer was used to prepare the cell suspension for heat treatment. In this study, we measured the protein elution rate using a GFP-expressing strain when heat treatment was performed using 50 mM phosphate buffer as in previous experiments and when artificial seawater was used. The results are shown in Figure 14. When phosphate buffer was used to prepare the cell suspension, protein leakage occurred as a result of heat treatment, as in previous experiments. However, the leakage rate was lower in samples heat-treated using artificial seawater than in samples that were not heat-treated using phosphate buffer and placed on ice. This suggests that suspending in artificial seawater may be able to suppress leakage, even for low-molecular-weight proteins such as GFP.

[0078] Next, to narrow down the cause of the protein leakage inhibitory effect, we evaluated the effects of osmotic pressure and charge using a solution containing artificial seawater and its main inorganic salts, as well as sodium chloride and sorbitol solutions with osmotic pressures similar to those of seawater. Specifically, we prepared artificial seawater, a solution containing six inorganic salts (sodium chloride, magnesium chloride, calcium chloride, anhydrous sodium sulfate, potassium chloride, and sodium bicarbonate) added at concentrations of 100 mg / L or more as the main components of artificial seawater, a 0.5 M NaCl solution with an osmotic pressure similar to that of seawater (2.5 MPa), and a 1 M sorbitol (Fujifilm Wako Pure Chemical Industries) solution. The results are shown in Figure 15. Similar leakage inhibitory effects were observed with the main components of artificial seawater, and a slight effect was observed with sodium chloride solution, but no leakage inhibitory effect was observed with sorbitol. This suggests that the leakage inhibitory effect is not due to osmotic pressure, but is largely due to the influence of inorganic salts contained in the main components of artificial seawater and charge.

[0079] Furthermore, we investigated the effects of solutions containing the main inorganic salts in artificial seawater, individually or in combination, on leakage. Specifically, we prepared a 0.5 M MgCl2 + NaCl solution (Mg + NaCl) containing artificial seawater, magnesium chloride, and sodium chloride at an osmotic pressure similar to seawater (2.5 MPa), a 0.5 M CaCl2 + NaCl solution (Ca + NaCl) containing calcium chloride and sodium chloride at an osmotic pressure similar to seawater (2.5 MPa), and a 0.5 M MgCl2 solution (Mg) containing magnesium chloride at an osmotic pressure similar to seawater (2.5 MPa) were added to psychrophilic bacteria to prepare cell suspensions. As shown in Figures 16 and 17, the addition of magnesium chloride and calcium chloride to sodium chloride, respectively, significantly suppressed leakage compared to the sodium chloride solution shown in Figure 15. The addition of magnesium chloride was particularly effective. Magnesium chloride alone also significantly suppressed leakage.

[0080] (3-8) pH of cell suspension Because the difference in pH between the phosphate buffer and artificial seawater used in the above experiments may have affected leakage, we investigated the pH of the cell suspension. The cell suspension was adjusted to 6.7, the same pH as the phosphate buffer where leakage was significant, using magnesium chloride (9.9 g / L) and calcium chloride (1.5 g / L). The effect on GFP leakage was examined using the same method as in the above experiment. As shown in Figure 18, magnesium chloride and calcium chloride solutions at pH 6.7 inhibited GFP leakage to the same extent as artificial seawater, indicating that the inhibitory effect on protein leakage is not affected by pH. To examine the concentration dependence of magnesium chloride and calcium chloride, we also compared the leakage rates in solutions adjusted to 5, 10, 25, and 49 mM. As shown in Figure 19, calcium chloride and magnesium chloride exhibited similar inhibitory effects on protein leakage, but the leakage rate tended to decrease with increasing magnesium chloride concentration.

[0081] (3-9) Study of inorganic salts The following study was conducted to determine whether there are any inorganic salts in artificial seawater that have inhibitory effects other than magnesium chloride and calcium chloride. Cell suspensions prepared with 10 mM and 49 mM sodium sulfate, sodium chloride, and potassium chloride were examined for their effects on GFP leakage using the same method as in the previous experiment. The same phosphate buffer and artificial seawater were used as in the previous experiment. As shown in Figure 20, the cell suspension prepared with 49 mM sodium sulfate, sodium chloride, and potassium chloride exhibited a lower leakage rate than the phosphate buffer, demonstrating a protein leakage inhibitory effect, although not as pronounced as in artificial seawater.

[0082] Furthermore, we examined the effect of manganese chloride, zinc chloride, and magnesium sulfate heptahydrate, which are divalent cations like magnesium chloride and calcium chloride, on GFP leakage using a cell suspension prepared at 49 mM. As shown in Figure 21, the cell suspension containing manganese chloride, zinc chloride, and magnesium sulfate had a lower leakage rate than calcium chloride, and exhibited a significant inhibitory effect on protein leakage comparable to that of the cell suspension containing magnesium chloride.

Claims

1. a step of introducing into a psychrophilic bacterium an enzyme gene that functions in a temperature range higher than the temperature range at which the psychrophilic bacterium can grow, thereby obtaining a transformed psychrophilic bacterium; preparing a cell suspension of the transformed psychrophilic bacterium using a solution containing at least one selected from the group consisting of monovalent and divalent cations; and heat-treating the cell suspension at a temperature higher than the temperature range in which psychrophilic bacteria can grow.

2. 2. The method for producing a biocatalyst according to claim 1, wherein the cations are calcium ions, potassium ions, sodium ions, magnesium ions, manganese ions and zinc ions.

3. 3. The method for producing a biocatalyst according to claim 2, wherein the cations are magnesium ions, manganese ions and zinc ions.

4. 4. The method for producing a biocatalyst according to claim 3, wherein the solution used for the cell suspension contains at least one selected from the group consisting of calcium chloride, sodium chloride, magnesium chloride, manganese chloride, zinc chloride, sodium sulfate, and magnesium sulfate.

5. 5. The method for producing a biocatalyst according to claim 4, wherein the solution used for the cell suspension contains at least one selected from the group consisting of magnesium chloride, magnesium sulfate, manganese chloride, and zinc chloride.

6. 6. A method for producing a biocatalyst according to claim 1, wherein the temperature range in which the psychrophilic bacteria can grow is lower than 30°C, and the heat treatment is performed at a temperature of 40°C or higher and 50°C or lower.

7. 7. The method for producing a biocatalyst according to claim 6, wherein the psychrophilic bacterium is Shewanella frigidimarina or Shewanella livingstonensis.

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