JP4890816B2 - Immobilization of bacterial cells on hydrophobic carrier - Google Patents

Description

  The present invention relates to immobilization of bacterial cells. More specifically, the present invention relates to a method for immobilizing bacterial cells on a hydrophobic carrier.

  Since enzymes can catalyze various reactions under mild conditions, they are useful for building energy-saving and environmentally friendly processes. However, since the enzyme is usually prepared by separation and purification from the microorganism itself or its culture solution, the cost is high. On the other hand, in recent years, the use of microbial cells themselves as enzyme agents has been studied. This technique of whole cell biocatalyst can simplify the process and reduce the cost.

  In a biological reaction process using cells such as microorganisms, it is desired to immobilize cells by some means.

  Methods for immobilizing cells are broadly classified into the following three methods: 1) a carrier binding method for binding cells to an insoluble carrier, 2) a crosslinking method using a crosslinking agent, and 3) a polymer gel matrix lattice. Comprehensive method of enclosing or encapsulating in semipermeable membrane microcapsules. In order to ensure immobilization, the entrapment method is preferable, but various carrier binding methods using a porous carrier have been studied in consideration of the mass transfer around the immobilized cells. In particular, in the case of cells having aggregating properties or adhesiveness, immobilization is generally carried out very easily by a carrier binding method by a simple operation of mixing easily available porous carriers and cells. Yes. For example, the flocculating yeast can be easily fixed simply by mixing with a polyurethane foam piece (see Non-Patent Documents 1 and 2). Adherent animal cells could be immobilized simply by incubation with a porous cellulose carrier (see Non-patent Document 3), and a porous cellulose carrier into which polyethyleneimine having a positively charged group or a cell adhesion factor was introduced In (2), it was reported that high concentration of floating cells could be immobilized (see Non-Patent Document 4 and Non-Patent Document 5). Patent Document 1 discloses a porous medium in which a cell adhesion factor and a cell adhesion factor are bound via polyethyleneimine having a positively charged group as a carrier for binding these cells to a carrier for active culture of animal cells. Cellulose cellulose carriers are described. However, immobilization on a carrier has been difficult in the case of cells that are not so strongly aggregated or adherent.

  In the past, the present inventors have cultivated cells together with porous particles called Biomass Support Particles (BSP) so that the cells can naturally adhere to the cells due to the adhesion and cohesion of the cells. We have developed a technology for immobilization at a high concentration. This immobilization method is characterized in that the immobilization operation and aseptic operation are simple, and the immobilization carrier is physically strong. Therefore, this method is suitable for use on an industrial scale, and has been applied to immobilization of microorganisms such as filamentous fungi and aggregating yeast and animal / plant cells. However, since bacteria having various useful enzymes have very small cells, there is a problem that immobilization is difficult by the conventional method using BSP.

The present inventors have developed a method for immobilizing bacterial cells on a hydrophobic carrier treated with a polymer having a plurality of pendant amines (see Patent Document 2). By this method, it was possible to immobilize cells on a carrier simply and at high density.
JP-A-5-252941 JP 2004-129572 A H. Furuta, et al., “Journal of Fermentation and Bioengineering”, 1997, Vol. 84, No. 2, p. 169-171 Y. Liu, et al., “Biochemical Engineering Journal”, 1998, Vol. 2, No. 3, p. 229-235 Tatsuya Ogawa, five others, "Journal of Fermentation and Bioengineering", 1992, Vol. 74, No. 1, p. 27-31 Shuji Terashima, 5 others, “Biotechnology Journal”, 1993, Vol. 71, No. 3, p. 165-170 Shuji Terashima, 5 others, “Journal of Fermentation and Bioengineering”, 1994, Vol. 77, No. 1, p. 52-56

  The object of the present invention is to make it easier and more efficient to support bacterial cells, particularly cells that are not very cohesive or adherent, or bacteria that have been difficult to immobilize due to the small size of the bacterial cells. It is to provide a way to immobilize well.

  The present invention provides a method for immobilizing bacterial cells, comprising a step of treating a hydrophobic carrier with polyethyleneimine, and a step of bringing the treated carrier into contact with bacterial cells.

  In a preferred embodiment, the polyethyleneimine has a viscosity average molecular weight of 10,000 to 150,000.

  In a preferred embodiment, the hydrophobic carrier is polyethylene, polypropylene, polyethylene terephthalate, polyurethane, polyvinyl chloride, polyacrylamide, polystyrene, polyvinylidene difluoride, polycarbonate, polyether, polyester, nylon, polyvinyl formal, acetal resin, It is selected from the group consisting of phenol resin, silicon resin, fluororesin, glass, ceramics, and metal.

  In a preferred embodiment, the hydrophobic carrier is porous.

  In a preferred embodiment, the microbial cell is a microbial cell that is weakly clumping or adherent.

  In a preferred embodiment, the microbial cell is a bacterium.

  The present invention also provides an immobilized microbial cell immobilized on a hydrophobic carrier pretreated with polyethyleneimine.

  According to the method for immobilizing bacterial cells of the present invention, the bacterial cells can be immobilized on a carrier simply and at a high density. In particular, cells that are not very strong in cohesiveness or adherence, or bacteria that are small in size, can be more easily and densely immobilized on a carrier at a high density.

(Carrier)
The carrier used in the present invention is not particularly limited as long as it is a hydrophobic solid. The hydrophobic carrier is preferably porous in that it can hold many cells. In that case, the pore diameter is not particularly limited, and can be appropriately determined according to the size of the bacterial cells. The pore diameter is preferably 0.1 to 1,000 μm, more preferably 1 to 100 μm.

  As a material for the hydrophobic carrier, a material usually used as a porous carrier for immobilizing aggregating bacterial cells is preferable. Specifically, polyethylene, polypropylene, polyethylene terephthalate, polyurethane, polyvinyl chloride, polyacrylamide, polystyrene, polyvinylidene difluoride, polycarbonate, polyether, polyester, nylon, polyvinyl formal, acetal resin, phenol resin, silicone resin, fluorine Resins, glass, ceramics, and metals are included. Polyvinyl formal is a product obtained by subjecting hydrophilic polyvinyl alcohol to a formalization treatment to make it hydrophobic, preferably having a high degree of formalization having a high hydrophobicity. Polyester, polyurethane, nylon, polypropylene, and polyvinyl formal are preferable from the viewpoints of availability and ease of handling. Particularly preferred hydrophobic carriers include polyvinyl formal sponge, polyester foam, polyurethane foam, and polypropylene foam.

  The shape and size of such a carrier are not particularly limited. For example, the shape may be a block shape, a sheet shape, or the like. Also, in terms of ease of handling, a few mm square strips are most often used.

(Polyethyleneimine)
The polyethyleneimine used in the present invention can efficiently immobilize cells in particles by electrostatic interaction between the amino group in polyethyleneimine adsorbed on the surface of the immobilization carrier and the negative charge on the cell surface. . The polyethyleneimine is not particularly limited as long as it does not adversely affect the activity of the bacterial cells in the culture solution and has the ability to bind to other hydrophobic substances by hydrophobic interaction. As such polyethyleneimine, commercially available polyethyleneimine can be used, and it can also be prepared by synthetic techniques commonly used in the art. The polyethyleneimine is preferably a large molecule considering the ability of hydrophobic interaction, and the viscosity average molecular weight is preferably 10,000 or more, more preferably 70,000 or more and 150,000 or less. In the present invention, for example, polyethyleneimine (viscosity average molecular weight 70,000) commercially available from Wako Pure Chemical Industries, Ltd. can be suitably used.

(Bacteria)
In the present invention, the cells that can be immobilized refer to minute single-cell organisms, and include bacteria, yeast, single-cell algae, cyanobacteria, and the like. Such cells preferably retain various useful enzymes. The immobilization method of the present invention can be used for multicellular organisms such as aggregating fungi and fungi, but can be particularly suitably used for fungi that have poor aggregability or adhesion. Here, the weak cohesiveness or adherence means that there is little formation of aggregates and adhesion to the container during the culture, and there is a tendency to float in the culture solution.

  The immobilization method of the present invention can also be particularly suitably used for minute cells, such as bacteria, which have been difficult to immobilize by conventional methods using BSP. Examples of such bacteria include Escherichia coli, Bacillus subtilis, lactic acid bacteria, photosynthetic bacteria, chemically synthesized bacteria, and thermophilic bacteria. These may be natural or recombinant.

(Immobilization method)
The method for immobilizing bacterial cells of the present invention includes a step of treating a hydrophobic carrier with polyethyleneimine, and a step of bringing the treated carrier into contact with bacterial cells.

  First, the treatment of the hydrophobic carrier is performed by immersing the carrier in an aqueous solution of polyethyleneimine. The concentration of the aqueous polyethyleneimine solution is not particularly limited. A concentration of 0.01 to 10 mg / ml is preferred. The immersion time of the carrier can be appropriately determined according to the concentration of the polyethyleneimine aqueous solution, the type of carrier, and the like. Usually, it is immersed for 5 minutes to 24 hours with shaking or stirring.

  The obtained carrier is preferably sterilized immediately before contacting the cells. The sterilization treatment may be performed by any method, and a normal autoclave treatment is preferable because it is easy.

  Next, the treated carrier is brought into contact with the bacterial cells. This step is performed by adding the treated carrier to a medium containing bacterial cells and culturing with shaking or stirring in a culture tank. The medium has a normal composition and does not require a special composition. The amount of the bacterial cells, the amount of the medium, the amount of the carrier and the like can be appropriately determined according to the type of the bacterial cell, the type of the carrier, and the like. The contact time is usually several hours to several days. After contacting cells with a medium containing cells and seeding the cells on a carrier, the carriers can be taken out and added to a fresh medium, and further cultured for several days, so that the cells can be immobilized on the carrier at a high density. . If necessary, the medium can be replaced with fresh medium at the appropriate time.

  The number of cells or the concentration of cells immobilized on the carrier can be measured by various methods.

  For example, by the MTT assay (Tim Mosmann, “Journal of Immunological Methods”, 1983, 65, p. 55-63) The concentration can be accurately measured. Specifically, the MTT assay is a dehydrogenase in which yellow MTT (3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide) is present in mitochondria in living cells. And the resulting amount of purple MTT formazan is based on the number of viable cells. Since the produced MTT formazan is insoluble in water, the absorbance is usually measured after dissolving in 2-propanol.

  Alternatively, the cells can be physically peeled from the carrier in a predetermined solution, and the amount of the cells immobilized on the carrier can be measured from the difference in the dry weight or wet weight of the carrier before and after that. Further, after removing the cells, the turbidity of the obtained suspension may be measured, or the number of cells in the suspension may be counted using a hemocytometer. The concentration of immobilized cells can also be measured by measuring the activity of an enzyme that is constantly expressed in the cells, such as lactate dehydrogenase.

(Immobilized cells)
Immobilized cells can be obtained by the above method. The immobilized cells can be stored in a medium or dried.

  The obtained immobilized microbial cells are used for an enzyme reaction according to each microbial cell, and can be used in an appropriate reaction system. Since it can be immobilized on the carrier at a high density, the reaction efficiency is good. It can also be used repeatedly.

  EXAMPLES Hereinafter, although this invention is demonstrated based on an Example, this invention is not restrict | limited to this Example.

(Example 1: Immobilization of E. coli on a carrier treated with polyethyleneimine)
Escherichia coli K12 was used as the microbial cell. This Escherichia coli cell suspension medium was prepared as follows. Competent cells (NovaBlue, Merck) were applied to a plate medium (tryptone (10 g / l), yeast extract (5 g / l), NaCl (5 g / l), agar (15 g / l)) at 37 ° C. And static culture. One colony formed is taken and transferred to a test tube containing 4 ml of liquid medium (tryptone (10 g / l), yeast extract (5 g / l), NaCl (5 g / l)), and reciprocal shaking culture at 37 ° C. for 12 hours. did. This cell suspension medium was diluted 10 times with a fresh liquid medium.

  Polyvinyl alcohol (PVA) sponge sheet DA (A) (pore size 60 μm) (thickness 2 mm, Aion Co., Ltd.) having a high degree of formalization as 2 × 2 × 2 mm particles as immobilized carrier particles (BSP) I used it after chopping it.

A fixed-capacity flask having a capacity of 100 ml was charged with 250 immobilized carrier particles and about 20 ml of pure water, and sterilized by autoclaving at 121 ° C. for 20 minutes. After removing pure water, a polyethyleneimine solution having a concentration of 0.1 mg / ml (30% polyethyleneimine P-70 solution (viscosity average molecular weight 70,000, Wako Pure Chemical Industries, Ltd.) dissolved in pure water) 15 ml was added and shaken back and forth for 4 hours at room temperature with a shaking speed of 100 times / minute. After removing the polyethyleneimine solution, 15 ml of Escherichia coli cell suspension medium was added, and reciprocal shaking culture was started in a 37 ° C. water bath at a shaking speed of 100 times / minute. Ten carrier particles were sampled about 1, 3, 6, 9, and 11 hours after the start of the culture. After destroying the cells by treatment with an ultrasonic homogenizer for 20 seconds, the activity of lactate dehydrogenase (LDH) in the cells of Escherichia coli immobilized on the carrier particles is measured by LDH cytotoxicity detection kit (Takara Bio Inc.) It measured using. In addition, E. coli was shaken and cultured using a 100 ml screw-mouthed triangular shake flask in advance, and a proportional relationship between the turbidity (OD ₆₆₀ ) of the E. coli cell suspension medium at a wavelength of 660 nm and the LDH activity in the cells. It was confirmed that Based on the proportional relationship between the two, the concentration of the immobilized cells was evaluated from the measured LDH activity in the immobilized cells. The results are shown in FIG. Here, the vertical axis in FIG displays an immobilized cell concentration as _{OD 660} per particle 1 cm ^3.

(Comparative Example 1: Immobilization of E. coli on an untreated carrier)
The same procedure as in Example 1 was performed except that the immobilized carrier particles were not treated with polyethyleneimine. The results are also shown in FIG.

(Comparative Example 2: Immobilization of E. coli on a carrier treated with either poly-L-lysine, poly-L-ornithine, poly-L-arginine, or poly-L-histidine)
Instead of polyethylenimine, poly-L-lysine (poly-L-lysine hydrobromide (viscosity average molecular weight> 70,000, Wako Pure Chemical Industries, Ltd.) dissolved in pure water), poly- L-ornithine (poly-L-ornithine hydrobromide (viscosity average molecular weight> 100,000, Sigma Chemical Co.)), poly-L-arginine (poly-L-arginine hydrochloride (viscosity average molecular weight 70,000) -150,000, Sigma Chemical Co.)), or poly-L-histidine (poly-L-histidine hydrochloride (viscosity average molecular weight> 5,000, Sigma Chemical Co.)) .1 mg / ml) was performed in the same manner as in Example 1. The results are also shown in FIG.

(Comparative Example 3: Immobilization of Escherichia coli on a carrier treated with poly-L-glutamic acid or poly-L-asparagine)
Immobilized carrier particles were treated with poly-L-glutamic acid (poly-L-glutamic acid sodium salt (viscosity average molecular weight 50,000-100,000, Sigma Chemical Co.)) or poly-L-asparagine (viscosity average molecular weight 5,000- 15,000, Sigma Chemical Co.)). The results are also shown in FIG.

  When the immobilized carrier particles were used without any treatment (Comparative Example 1), Escherichia coli was hardly immobilized in the particles. On the other hand, when the particles were previously treated with polyethyleneimine (Example 1), a very high bacterial cell concentration was obtained.

  In addition, when treated with poly-L-lysine, poly-L-ornithine, poly-L-arginine, or poly-L-histidine instead of polyethyleneimine (Comparative Example 2), relatively high fixation Chemical cell concentration was obtained, but lower than in the case of polyethyleneimine treatment. When treated with poly-L-asparagine having no charge or poly-L-glutamic acid having a negative charge near neutrality (Comparative Example 3), the concentration of immobilized cells was as low as in the case of no treatment.

(Example 2: Immobilization of photosynthetic bacteria on a carrier treated with polyethyleneimine)
As a bacterial body, a red non-sulfur bacterium Rhodovulum sulfidophilum strain W-1S (hereinafter abbreviated as W-1S strain), which is a facultative anaerobic photosynthetic bacterium, Maeda, et al., “Biotechnology Letters”, 1997 19, Vol. 12, No. 12, pp. 1209-1212). When the average particle size of the bacterium was measured using a laser diffraction / scattering particle size distribution analyzer (LA-300, Horiba, Ltd.), it was 0.84 μm (sphere equivalent diameter was 0.84 μm). It was.

  Polyvinyl alcohol (PVA) sponge sheet DA (A) (pore size 60 μm) (thickness 2 mm, Aion Co., Ltd.) having a high degree of formalization as 2 × 2 × 2 mm particles as immobilized carrier particles (BSP) I used it after chopping it.

First, 125 carrier particles and about 20 ml of pure water were placed in a 50-ml screw-mouthed conical flask, and sterilized by autoclaving at 121 ° C. for 20 minutes. After removing pure water, 10 ml of a polyethyleneimine solution having a concentration of 0.1 mg / ml was added, and the mixture was reciprocally shaken at a shaking speed of 90 times / minute at room temperature for 4 hours. After removing the polyethyleneimine solution, 10 ml of a medium in which W-1S is suspended (medium prepared so that the turbidity of the culture solution at a wavelength of 660 nm is 1.5) is added, and light irradiation conditions in a water bath at 30 ° C. Then, reciprocal shaking culture was performed at a shaking speed of 90 times / minute for 2 days. The medium to be used is, NaCl (30g / l), MgSO 4 · 7H 2 O (0.25g / l), CaCl 2 · 2H 2 O (0.2g / l), FeSO 4 · 7H 2 O (0.02g / _{l), H 3 BO 4 (} 2.86mg / l), MnCl 2 · 4H 2 O (1.81mg / l), ZnSO 4 · 7H 2 O (0.22mg / l), CuSO 4 · 5H 2 O (0.08mg / _{l), Na 2 MoO 4 (} 0.021mg / l), CoCl 2 · 6H 2 O (0.01mg / l), disodium ethylenediaminetetraacetate (0.05g / l), sodium pyruvate (1g / l), succinic acid To an aqueous solution containing sodium (1 g / l), DL-sodium malate (1 g / l), sodium acetate (1 g / l), and NH ₄ Cl (5 mM), phosphate buffer (thiamine hydrochloride (100 mg / l ), Nicotinic acid (100 mg / l), p-aminobenzoic acid (60 mg / l), biotin (10 mg / l), KH ₂ PO ₄ (8.16 g / l), K ₂ HPO ₄ (99 g / l), pH = 8.0) at a rate of 5 ml / l. After this seeding operation, the cell suspension medium was removed, 10 ml of fresh medium was added, and reciprocal shaking culture was resumed. The medium was replaced with fresh medium in a timely manner, and shaking culture was continued. Five carrier particles were sampled one by one and four days after the start of the culture, and the concentration of the cells immobilized on the carrier particles was evaluated by MTT assay.

  The MTT assay was performed as follows. First, MTT solution was prepared by dissolving MTT (Dojindo Laboratories Co., Ltd.) in a medium so as to have a concentration of 1 g / l and sterilizing by filtration using a filter having a pore size of 0.22 μm. The sampled carrier particles were added to this MTT solution and allowed to stand for 4 hours in a 20 ° C. incubator. After 4 hours, the MTT solution was removed, 2-propanol was added, and MTT formazan formed in the carrier particles was extracted. The absorbance at 560 nm and 700 nm of the extract was measured using a spectrophotometer, and the amount of MTT formazan produced by immobilized cells was calculated from the difference in absorbance using the molar extinction coefficient of MTT formazan.

On the other hand, the conversion reaction of MTT was similarly performed in advance using a W-1S suspension medium, and it was confirmed that there was a proportional relationship between the bacterial cell concentration and the amount of MTT formazan produced. Based on the proportional relationship between the two, the concentration of immobilized cells was determined from the amount of MTT formazan produced by the immobilized cells. Table 1 shows the results of the MTT assay 1 day and 4 days after the start of the culture. Here, the numerical values in Table 1 represent the concentration of immobilized cells [mg dry weight / cm ³ BSP].

(Comparative Example 4: Immobilization of photosynthetic bacteria on a carrier treated with poly-L-lysine)
The same procedure as in Example 2 was performed except that poly-L-lysine was used instead of polyethyleneimine. The results are also shown in Table 1.

(Comparative Example 5: Immobilization of photosynthetic bacteria on an untreated carrier)
The same procedure as in Example 2 was conducted except that the immobilized carrier particles were not treated with polyethyleneimine. The results are also shown in Table 1.

  As can be seen from Table 1, when the carrier particles were previously treated with polyethyleneimine (Example 2), the amount was significantly higher than when the immobilized carrier particles were used without any treatment (Comparative Example 5). An immobilized cell concentration was obtained. In addition, even when treated with poly-L-lysine (Comparative Example 4), a high concentration of immobilized cells was obtained.

  According to the method for immobilizing bacterial cells of the present invention, cells that are not very cohesive or adherent, or bacteria that have a small size and have been difficult to immobilize so far (for example, E. coli) Bacteria can be immobilized on a carrier more easily and at a high density. Since Escherichia coli is widely used as a host for gene recombination, it is advantageous for use of mass-produced gene recombination products. Since the immobilized microbial cell of the present invention is fixed at a high density, an increase in reaction efficiency can be expected as a whole reaction system using an enzyme in the microbial cell. Further, since polyethyleneimine is available at a relatively low cost, it is advantageous in terms of cost.

It is a graph which shows a time-dependent change of the microbial cell density | concentration of colon_bacillus | E._coli fix | immobilized by the fixed support particle | grains which performed various processes.

Claims (5)

  A method for immobilizing a microbial cell, comprising a step of treating a hydrophobic carrier with polyethyleneimine, and a step of bringing the treated carrier into contact with bacterial cells, wherein the material of the hydrophobic carrier is polyvinyl formal.   The method of claim 1, wherein the polyethyleneimine has a viscosity average molecular weight of 10,000 to 150,000.   The method according to claim 1 or 2, wherein the hydrophobic carrier is porous. The cell is a bacterium, the method according to any one of claims 1 to 3.   An immobilized microbial cell immobilized on a hydrophobic carrier pretreated with polyethyleneimine, wherein the material of the hydrophobic carrier is polyvinyl formal.

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