JP2010227031A - Method for producing protein by continuous culture of fungus and apparatus therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To produce protein by continuous culture of fungus. <P>SOLUTION: The method for producing protein includes a continuous culture of fungus by filtering a culture solution of fungus by a synthetic polymer nonwoven fabric, recovering protein being a product from a filtrate, holding a non-filtrate in the culture solution or returning the non-filtrate to the culture solution and further adding a culture raw material of the fungus to the culture solution. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for producing a protein by continuous culture of filamentous fungi.

  Filamentous fungi are microorganisms that have a high protein production ability and produce secretory proteins such as cellulase, amylase, protease, and lipase in the culture solution. In general, a filamentous fungus culture solution contains a plurality of these proteins, and protein components such as proteases, lipases, and cellulases having desired properties are recovered from them and used industrially.

  One specific example of the protein produced by the filamentous fungus in the culture solution is a cellulase mixture. Filamentous fungi produce a mixture of cellulases composed of a plurality of species having different degradation mechanisms in the culture solution, and the efficient cellulose degradation is enabled by the cooperative action of cellulases having different degradation mechanisms. In particular, Trichoderma spp. Is one type of filamentous fungus that produces a large amount of a cellulase mixture having high cellulolytic activity among the filamentous fungi that produce cellulase.

  Protein production methods by culturing filamentous fungi are roughly classified into (1) batch culture method (Batch culture method) and fed-batch culture method (Fed-Batch culture method), and (2) continuous culture method. be able to.

  The batch culture method and fed-batch culture method of the above (1) have the advantage that they are simple in terms of equipment, complete the culture in a short time, and have little damage due to contamination with bacteria. In addition, it is known that in fed-batch culture, nutrient sources necessary for the growth of filamentous fungi or high production of cellulase can be added as needed, and cellulase can be produced at a higher yield than batch culture (Non-patent Document 1). However, generally, in the batch culture or fed-batch culture of microorganisms, the physiological activity of microorganisms decreases with the passage of time due to accumulation of growth inhibitors and the like in the culture solution. Moreover, in order to efficiently produce proteins in batch culture, it is necessary to carefully perform initial settings of the culture solution such as glucose concentration, nitrogen concentration, and the amount of cellulase inducer added.

  It has been reported that the continuous culture method (2) can produce cellulase more highly than fed-batch culture (Non-patent Document 2). The reason for this is that, in addition to being able to add necessary nutrient sources at any time, as in fed-batch culture, at the same time, factors that inhibit cellulase production can be continuously removed from the culture solution. Factors that inhibit cellulase production are growth inhibitors such as organic acids produced in the culture solution, or excessive amounts of glucose in the culture solution that inhibits cellulase production, or cellulase-degrading enzymes such as nitrogen sources and peptidases. Etc.

  In order to produce a protein by continuous culture of filamentous fungi, it is necessary to continuously extract the culture solution from the culture tank. Filamentous fungi tend to clump under liquid culture, and in the prior art, a special apparatus is not installed to separate the bacterial cells and the culture solution, and the medium is pulled out through the connecting tube in the culture tank or the filamentous fungi are separated. During the separation, continuous culture was performed using a culture apparatus using a metal filter mesh (Non-patent Document 3). However, in the method of continuous cultivation of filamentous fungi without using a separation membrane or using a rough metal filter, fine fungi of filamentous fungi, spores, conidia, etc. are also collected at the same time as the culture solution. However, there was a problem that the bacterial cells could not be sufficiently concentrated and productivity in continuous culture was lowered.

  As a means for solving the above-described problems, there is a method of using a separation membrane having micropores on the surface for separating microorganisms and their culture solutions. For example, in continuous culture of E. coli, a method of culturing while blocking E. coli using a ceramic filter having a pore size of 0.2 μm (Non-patent Document 4), or in the production of L-carnitine by continuous culture of Proteus bacteria, A method of culturing while blocking bacteria using a polyvinylidene fluoride (PVDF) membrane having a pore diameter of 0.1 μm or a polysulfone hollow fiber membrane having a thickness of 0.1 μm is known (Non-patent Document 5). As for filamentous fungi, a polyethylene hollow fiber membrane having a pore diameter of 0.4 μm was used, and the dye was decomposed by continuously cultivating while preventing bamboo shoots (Non-patent Document 6), while the pore diameter was 0.1 μm. A method for producing a protein (Patent Document 1) is known, in which microorganisms (including filamentous fungi) having the ability to secrete and produce proteins are continuously cultured using the PVDF membrane, and the product protein is efficiently separated and recovered. It has been.

  In the above-described separation membrane, particularly a sieve-type filtration using a membrane having fine pores on the membrane surface, there is an advantage that the filtration accuracy is high and microorganisms and objects having a pore diameter larger than that can be efficiently prevented. However, especially in continuous culture of filamentous fungi, mycelium or protein components of filamentous fungi adhere to the micropores of the separation membrane, and the actually functioning micropores become smaller with the culture period, resulting in clogging. There was a problem. In particular, PVDF is a polymer material with extremely high protein adsorptivity, so that proteins produced by filamentous fungi are adsorbed, which causes a problem that the membrane is likely to be clogged. In addition, general proteins or enzymes are monomers and their functions have been analyzed. Many of these proteins exist in various forms such as dimers, multimers, and complexes, and express biological functions. Yes. On the other hand, since protein or enzyme is a high molecular compound, an aggregate is formed when present at a high concentration. That is, in the production of protein by continuous culture of filamentous fungi, a separation membrane having a pore size of about 0.1 μm is a pore size that can be sufficiently recovered as a filtrate when one protein molecule is seen, Depending on the clogging or the form of the protein in the culture solution, there is a problem that the protein as a product cannot be sufficiently recovered as the filtrate.

JP 2009-39074 A

Mclean et al., The Can. J. et al. Chem. Eng. 64, 588-597. 1986 Estebauer et al., Bioresource. Tehcnol. 36.51-65, 1991 Hendy et al., Enzyme Microb. Technol. 73-77, 1984. Li et al. Membr. Sci. 110, 203-210, 1996 Canovas et al. Membr. Sci. , 214, 101-111, 2003 Hai et al. Membr. Sci. , 325, 395-403, 2008

  An object of the present invention is to provide a method for producing a protein by continuous culturing of filamentous fungi, which can continuously cultivate filamentous fungi and stably produce a protein produced by the filamentous fungus in a culture solution for a long period of time. And

  In continuous culturing of filamentous fungi, the protein produced in the culture solution can be obtained without clogging for a long time by using a synthetic polymer nonwoven fabric as a filter medium for separating the filamentous fungi in culture and the protein contained in the culture solution. The present invention was completed by finding that it can be efficiently recovered without loss. That is, this invention is comprised by the following (1)-(6).

  (1) Filamentous fungus culture fluid is filtered through a synthetic polymer nonwoven fabric, the protein as a product is recovered from the filtrate, and the unfiltrated fluid is retained or refluxed in the culture fluid, and the filamentous fungal culture raw material is A method for producing a protein by continuous cultivation of filamentous fungi, which is added to a culture solution.

(2) The method for producing a protein according to (1), wherein filtration is performed using a synthetic polymer nonwoven fabric having an air permeability of 1 to 300 cc / cm ² / sec.

  (3) The method for producing a protein according to (1) or (2), wherein the protein is a cellulase mixture.

  (4) The method for producing a protein according to any one of (1) to (3), wherein the filamentous fungus is a genus Trichoderma.

  (5) The method for producing a protein according to any one of (1) to (4), wherein the filamentous fungus continuous culture is aeration airlift mixing.

  (6) including a culture tank for culturing filamentous fungi, a separation membrane unit comprising a synthetic polymer nonwoven fabric, a recovery tank for recovering proteins, a raw material tank for supplying culture raw materials, and a lower vent for aeration airlift mixing, Protein production device by continuous culture of filamentous fungi.

  ADVANTAGE OF THE INVENTION According to this invention, continuous culture | cultivation stable for a long period of time is attained, concentrating a filamentous fungal body in a culture tank with a synthetic polymer nonwoven fabric. Further, it is possible to separate and recover the protein produced in the filamentous fungus culture solution from the culture solution without loss.

FIG. 1 is a schematic side view for explaining one embodiment of a protein production apparatus using continuous culture of filamentous fungi used in the present invention. FIG. 2 is a schematic perspective view for explaining one embodiment of a separation membrane element used in the present invention.

  Hereinafter, the present invention will be described in more detail.

  The present invention filters a filamentous fungus culture solution with a synthetic polymer nonwoven fabric, collects the product protein from the filtrate, holds or refluxs the unfiltered solution in the culture solution, and uses the filamentous fungus culture raw material as a starting material. This is a method for producing a protein by continuous cultivation of filamentous fungi added to the culture solution.

  The filamentous fungus used in the present invention includes all flagellate fungi, zygomycetes, ascomycetes, basidiomycetes, and incomplete fungi. Preferably, the fungus body is filamentous and forms asexual spores called conidia. It is a filamentous fungus that grows. In addition, filamentous fungi whose biological functions have been altered or improved by manipulation such as mutation treatment or gene recombination can also be used.

  Specific examples of the filamentous fungus used in the present invention include the genus Monascus, the genus Penicillium, the genus Periconia, the genus Nigrospora, the genus Cephalos, and the genus Cephalos. Genus Acremonium, Genus Mucor, Genus Rhizopus, Genus Fusarium, Genus Emerica, Trichoderma (Genus Trilus, Genus Trigul) Genus Claviceps Examples include filamentous fungi belonging to the genera Genus Humicola, Genus Trametes, Genus Fomitopusis, and Genus Phanerochaete, preferably Trichoderma spp. A filamentous fungus belonging to the genus Kawaratake or Fanerocaete, more preferably a fungus belonging to the genus Trichoderma.

  Specific examples of the preferred filamentous fungi include Trichoderma reesei, Aspergillus niger, Aspergillus oryzae, Aspergillus asperas, Monium Cellulolyticus (Acremonium sp.), Aspergillus kawachii, Aspergillus flavus, Trametes versicor et et al. sordida), and the like can be exemplified Phanerochaete chrysosporium di um (Phanerochaete chrysospodium).

  Furthermore, in the Trichoderma reesei, Trichoderma reesei QM9414 (Trichoderma reesei QM9414), Trichoderma reesei QM9123 (Trichoderma reesei QM9123), Trichoderma reesei Rut C-30 (Trichoderma reesei RuteC-30) 847), Trichoderma reesei MCG77 (Trichoderma reesei MCG77), Trichoderma reesei MCG80 (Trichoderma reeseiMCG80), Trichoderma reesei PC-3-7 (Trichoderma reesei PC-3-7), Trichoderma reesei PC-3-7 Ride QM9123 (Trichoderma viride9123) can be preferably used, such as.

  The filamentous fungus used in the present invention contains proteins, particularly protease, lipase, cellobiohydrase, exoglucanase, endoglucanase, β-glucosidase, xylanase, mannase, galactase, amylase (for example, α-amylase), glucose in the culture medium. It produces enzymes such as amylase, peroxidase (eg lignin peroxidase, manganese peroxidase), laccase. Further, since the filamentous fungus produces a plurality of types of proteins in the culture solution, the protein produced according to the present invention preferably contains a plurality of types of protein components. There are no particular restrictions on the composition and mixing ratio of the components.

  Moreover, the protein produced by the present invention may contain a heterogeneous protein component that is not secreted and produced by the filamentous fungus. That is, a recombinant filamentous fungus is prepared by inserting a gene encoding a heterologous protein into a filamentous fungus, and a protein containing the heterologous protein can be obtained by culturing the recombinant filamentous fungus. Furthermore, in order to improve secretion production efficiency, it is also possible to modify a gene encoding a desired protein. Specifically, the modification is to modify a gene so that a secretory signal peptide is functionally added to a desired protein. The secretory signal peptide is desirably added to the amino terminus of the desired protein. By adding a secretory signal peptide, the secretory production ability can be improved. Further, in the present invention, it is desirable to add a secretion signal peptide that enhances the cellulase secretion production ability of the filamentous fungus used in the present invention. It is also possible to improve protein production performance by linking a gene encoding a desired cellulase under the control of a promoter highly expressed in the filamentous fungal cell used in the present invention.

  It does not specifically limit regarding the composition of the component of the protein manufactured by this invention, and a mixing ratio. Moreover, although the density | concentration of protein is not specifically limited, Usually, it exists in the range of 0.0001-100 mg / mL.

  The protein produced according to the present invention can be further subjected to operations such as isolation of a specific protein component or increase of the protein concentration according to a known method. For example, if you want to isolate and purify specific protein components contained in protein, isolate or purify by salting out using ammonium sulfate, column chromatography, liquid chromatography, ion exchange chromatography, gel filtration, etc. Can do. In addition, as a method for increasing the protein concentration, the protein concentration can be concentrated by an operation such as ultrafiltration or lyophilization.

  A protein that is preferably produced according to the present invention is a cellulase mixture. In general, cellulase is an enzyme that catalyzes the hydrolysis of cellulose or hemicellulose, which is a sugar polymer, and specifically, cellulase mixture is an enzyme mainly composed of cellulase and other useful enzymes for degrading cellulose or hemicellulose. Is a mixture containing

  Examples of the cellulase that is the main component of the cellulase mixture include endo-type cellulase and exo-type cellulase. Endo cellulase is also called exoglucanase, and is an enzyme that catalyzes hydrolysis mainly from the center of the cellulose molecular chain. Exo-type cellulase is also called as cellobiohydrase, and is an enzyme that catalyzes hydrolysis from the reducing end or non-reducing end of a cellulose molecular chain.

  The subcomponent of the cellulase mixture is not particularly limited as long as it does not inhibit the enzyme activity of the main component cellulase. However, it is an enzyme other than cellulase secreted by filamentous fungi, and is used for the hydrolysis of cellulose or cellulosic biomass. It is preferable that the enzyme acts as an auxiliary in the degradation. Specific examples of such subcomponents include β-glucosidase, hemicellulase, xylanase, mannase, galactase, amylase, glucoamylase, peroxidase, laccase, amylase, peptidase, protease, etc., but β-glucosidase, hemicellulase, xylanase , One or more selected from the group consisting of mannase, galactase, amylase, glucoamylase, peroxidase and laccase.

  β-glucosidase is an enzyme that has specificity for cellobiose (disaccharide) produced by cellulose degradation and catalyzes hydrolysis of cellobiose to glucose.

  Hemicellulase is an enzyme that catalyzes the hydrolysis of hemicellulose, such as xylan, arabinan, mannan, and galactan. Since the cellulase mixture containing the enzyme can simultaneously decompose the hemicellulose component forming a complex with cellulose, particularly when the cellulosic biomass is hydrolyzed, the cellulose component in the cellulosic biomass can be hydrolyzed. Decomposition efficiency can be increased. Specific examples of such hemicellulase include enzymes such as xylanase and mannanase. Xylanase is an enzyme that catalyzes the hydrolysis of xylan (a sugar polymer in which xylose is β-1,4-linked) to xylose, which is a hemicellulose component. Mannanase is an enzyme that catalyzes the hydrolysis of mannan (a sugar polymer containing mannose), which is a hemicellulose component.

  Peroxidase (preferably manganese peroxidase or lignin peroxidase) or laccase is often contained when basidiomycetes, white rot basidiomycetes, brown rot basidiomycetes are used as filamentous fungi. Cellulase mixtures containing such enzymes are particularly cellulose. Since the lignin component forming a complex with cellulose can be simultaneously decomposed during hydrolysis of the biomass, it is possible to increase the hydrolysis efficiency of the cellulose component in the cellulose biomass. Enzymes other than cellulase contained in such culture filtrate may be removed according to a known method when it is necessary to remove them depending on the intended use of cellulase.

  As described above, when the cellulase mixture is produced according to the present invention, the filamentous fungus is preferably a filamentous fungus having a high cellulase secretion-producing ability. Examples of such a filamentous fungus include Trichoderma spp., Aspergillus sp., Sporotrichum sp. Monium genus, Kawaratake genus, Fanerocaete genus and the like can be preferably used. Such filamentous fungi may be isolated from the natural environment or may have enhanced cellulase productivity or cellulase activity by mutation. The filamentous fungus used in the present invention is also a filamentous fungus into which one or more genes encoding a desired heterogeneous or homologous cellulase are introduced in order to enhance the cellulolytic activity of the cellulase mixture. Included in filamentous fungi.

  By using the cellulase mixture produced by the present invention, it can be used for hydrolysis reaction of cellulosic biomass and can be carried out according to known literature. For example, cellulosic biomass is converted into cellulose components obtained by acid treatment, concentrated sulfuric acid treatment, dilute sulfuric acid treatment, steam explosion treatment, hot water treatment, subcritical water treatment, alkali treatment, ammonia treatment, ammonia explosion treatment, etc. On the other hand, the cellulose can be hydrolyzed by adding the cellulase obtained by the production method of the present invention and incubating at a temperature of 40 to 70 ° C. for 1 to 7 days. Can be manufactured.

  In the present invention, filamentous fungi are cultured, and the culture solution of the filamentous fungus is filtered with a synthetic polymer nonwoven fabric, so that the filamentous fungus cells contained in the filamentous fungus culture solution are blocked with the synthetic polymer nonwoven fabric, and only the culture solution is used. It is characterized by permeation and filtration separation.

  In the culture solution of the filamentous fungus of the present invention, in addition to the protein which is the object of the production method of the present invention, preferably a cellulase mixture, the fungus body (or mycelium) and spores (or conidia) of the fungus and the growth thereof A medium containing a component selected from carbon sources, nitrogen sources, inorganic salts, inducers, surfactants, antifoaming agents, pH adjusting agents, etc. Is filtered through a synthetic polymer nonwoven fabric, and the product protein is recovered from the filtrate. On the other hand, filamentous fungi or mycelia, or some spores (conidia) are blocked in the culture solution by the synthetic polymer nonwoven fabric, so that the filamentous fungus concentration in the culture tank can be maintained or increased. Is possible.

  The concentration of filamentous fungi in the culture solution should be maintained at a high level in order to obtain efficient productivity and within a range where the culture environment is inappropriate for the growth of the filamentous fungus and the rate of killing does not increase. Is preferred. Moreover, the upper limit of the density | concentration of a filamentous fungus is not specifically limited unless the malfunction on the driving | operation of a continuous culture apparatus or the fall of production efficiency is caused.

  The non-woven fabric refers to a cloth-like material formed into a sheet shape without weaving fibers, and the synthetic polymer non-woven fabric refers to a non-woven fabric using a synthetic polymer as a fiber constituting the non-woven fabric. Synthetic polymers such as polyester, polypropylene, polyolefin, polyamide, polystyrene, and polyvinyl chloride are used as the raw material for the fibers constituting the synthetic polymer nonwoven fabric. The synthetic polymer nonwoven fabric used in the present invention is preferably a material that can be steam sterilized when washing the filamentous fungus attached to the surface, because it is used for the purpose of filtering the filamentous fungus culture solution. Polypropylene is preferred. Polypropylene is a polymer obtained by polymerizing propylene. Polyester is a copolymer of polycarboxylic acid and polyalcohol. Depending on the structure of polycarboxylic acid and polyalcohol used, polyethylene terephthalate resin, polytrimethylene terephthalate resin, polybutylene terephthalate resin, polyethylene naphthalate Examples thereof include resins, polybutylene naphthalate resins, and copolymers thereof.

  The average fiber diameter of the fibers constituting the synthetic polymer nonwoven fabric used in the present invention is preferably 2 to 30 μm, and more preferably 7 to 25 μm. If the average fiber diameter is smaller than 2 μm, the nonwoven fabric may be excessively densified, leading to a decrease in the air flow rate, or the spinnability during the production of the nonwoven fabric may be deteriorated, leading to troubles such as yarn breakage. On the other hand, when the average fiber diameter is larger than 30 μm, spot weight spots are generated on the obtained nonwoven fabric, density spots and rigidity spots are likely to be generated, and surface smoothness tends to be inferior. Here, an average fiber diameter means what was measured by the method as described in a postscript reference example (1).

  In the fibers constituting the synthetic polymer nonwoven fabric used in the present invention, crystal nucleating agents, matting agents, lubricants, pigments, antifungal agents, antibacterial agents, flame retardants, hydrophilicity, as long as the effects of the present invention are not impaired. You may add chemical | medical agents, such as an agent. In particular, when used as a separation membrane for filamentous fungi, which is the main purpose of the present invention, there is no limitation as long as the growth of the filamentous fungus is not inhibited and the product protein is not adversely affected. In some cases, a drug having an effect of suppressing adhesion of filamentous fungi to the synthetic polymer nonwoven fabric during continuous culture may be added as a drug.

  The fibers constituting the synthetic polymer nonwoven fabric used in the present invention may be composed of a single component resin, or may be a composite fiber composed of two or more kinds of resins. Examples of the composite fibers include concentric core-sheath fibers, eccentric core-sheath fibers, sea-island fibers, and split fibers. Regardless of whether they are single-component fibers or composite fibers, the shapes thereof include, for example, a circular cross section, a flat cross section, and a multiple cross section. Examples include a square cross section, a multileaf cross section, and a hollow cross section.

The synthetic polymer nonwoven fabric of the present invention preferably has an air permeability of 1 to 300 cc / cm ² / sec, and preferably ^{2 to} 260 cc / cm ² / sec. If the aeration rate is less than 1 cc / cm ² / sec, it may be difficult to continuously filter the culture solution during continuous culture. On the other hand, if the aeration rate is greater than 300 cc / cm ² / sec, the density of the nonwoven fabric is lost, which may lead to membrane breakage in long-term continuous culture. In addition, the ventilation | gas_flowing amount of a nonwoven fabric says what was measured by the method etc. as described in the postscript reference example (3) etc., for example.

  The production method of the synthetic polymer nonwoven fabric used in the present invention is limited to the spunbond method, melt blow method, flash spinning method, needle punch method, hydroentanglement method, airlaid method, thermal bond method, resin bond method, wet method, etc. Although it is not made, since it is composed of long fiber filaments, the fibers do not fall off when used as a filter medium in the present invention and are not mixed into the filtrate, and the strength and rigidity of the resulting nonwoven fabric, and also the production The spunbond method is preferred because of its excellent cost.

  The spunbond method is a method of extruding molten polymer from a nozzle and drawing it with a high-speed suction gas, collecting the fibers on a moving conveyor to form a web, and then applying thermal bonding and entanglement continuously. This is a method for producing a non-woven fabric that is integrated into a sheet. From the viewpoint of obtaining sufficient strength of the nonwoven fabric when used as a filter medium in the present invention, the spinning speed is preferably 2000 m / min or more in order to cause the constituent fibers to be highly oriented and crystallized. It is more preferable that it is 4000 m / min or more.

  Furthermore, in order to control the characteristics such as the air flow rate in order to obtain a nonwoven fabric suitably used as the synthetic polymer nonwoven fabric of the present invention, sheet integration by thermal bonding is preferable. Examples of the thermal bonding method include a method having a pair of uneven rolls, a partial thermal bond by thermal bonding using a roll having a concavo-convex and a flat roll, and a whole surface thermal bonding using a pair of flat rolls. In addition, in order to control the properties of the nonwoven fabric more precisely, a partial thermal bond is applied after a partial thermal bond, a partial thermal bond is applied after a full thermal bond, or a full thermal bond is applied after a full thermal bond. A two-step thermal bonding method such as applying can also be preferably used. In the case of two-stage thermal bonding, the first-stage and second-stage thermal bonding may be performed continuously, or after the first-stage thermal bonding is performed, the nonwoven fabric is wound up once, and then the second-stage thermal bonding is performed again. Adhesion may be performed.

  As temperature of this roll, it is preferable that it is 120-20 degreeC lower than melting | fusing point of the fiber which comprises a nonwoven fabric, and it is more preferable that it is 100-30 degreeC lower. Moreover, in the case of the composite fiber which consists of 2 or more types of resin from which melting | fusing point differs, it is preferable that it is 120-20 degreeC lower than melting | fusing point of the resin with the lowest melting | fusing point which exists on the fiber surface, and it is more preferable that it is 100-30 degreeC lower. On the other hand, the linear pressure of the roll is preferably 20 kg / cm or more, and more preferably 50 kg / cm or more.

  In addition, when the whole surface thermal bonding is applied to the first stage of the two-stage thermal bonding, it becomes easier to control the characteristics of the nonwoven fabric in the second stage thermal bonding. It is preferable to perform thermal bonding at a lower temperature and / or lower linear pressure than thermal bonding at the stage.

  In this invention, the separation membrane element which installs a synthetic polymer nonwoven fabric is used. The member constituting the separation membrane element is preferably a member resistant to high-pressure steam sterilization operation. If the inside of the continuous culture apparatus can be sterilized, the risk of contamination by undesired filamentous fungi during continuous culture can be avoided, and more stable continuous culture can be achieved. The members constituting the separation membrane element are preferably resistant to 15 minutes at 121 ° C., which is the condition of the high-pressure steam sterilization operation. Examples of the separation membrane element member include metals such as stainless steel and aluminum, polyamide resins, fluorine resins, polycarbonate resins, polyacetal resins, polybutylene terephthalate resins, PVDF, modified polyphenylene ether resins, and polysulfone resins. These resins can be preferably selected.

  As the separation membrane element, one shown in FIG. 2 is preferably used as described later. In addition, the separation membrane element may be installed in the culture tank or outside the culture tank. However, if the separation membrane element is installed in the culture tank as shown in 2 of FIG. 1, the entire culture apparatus can be simplified. Therefore, it is preferable.

  The starting material for filamentous fungi in the present invention is not limited as long as it contains components such as carbon source, nitrogen source, inorganic salt, metal salt and vitamin necessary for the growth of filamentous fungus and protein production. Absent. Further, if necessary, it may contain an antifoaming agent, a surfactant and the like. In particular, with regard to the antifoaming agent, a liquid level sensor and a control device may be provided, and bubbles generated on the liquid level may be sensed to add the antifoaming agent.

  Moreover, when manufacturing a cellulase mixture by this invention, it is preferable to contain a cellulase inducer as a culture raw material. Examples of the cellulase inducer include cellulose (solid), oligosaccharide, sophorose, cellobiose, gentiobiose, laminaribiose, lactose, sorbose and the like.

  In order to adjust the pH of the filamentous fungus culture solution, a neutralizing solution may be added to the culture solution. The neutralizing solution is not limited as long as it is an acidic solution or an alkaline solution. Moreover, you may adjust the pH of a culture solution using two types of neutralization solutions of an acidic solution and an alkaline solution. Further, it is preferable to use an aqueous ammonia solution as the alkaline solution used as the neutralizing solution because the ammonia used for neutralization is used as the nitrogen source.

  In the present invention, the method for stirring the filamentous fungus culture solution includes stirring and mixing using a stirring blade or airlift mixing by lower surface aeration, but aeration airlift mixing is preferable. Filamentous fungi are known to cause severe mycelial breakage when vigorously stirred and mixed, which may reduce protein productivity. The protein productivity of filamentous fungi is also improved.

  Of the continuous culture apparatus used in the present invention, a typical example in which the separation membrane element is installed inside the culture tank is shown in the schematic diagram of FIG. FIG. 1 is a schematic side view for explaining one embodiment of protein production by continuous culture of filamentous fungi of the present invention.

  In FIG. 1, the continuous culture apparatus basically includes a culture tank 1 for culturing filamentous fungi, a separation membrane element 2, a culture raw material tank 11, and a recovery tank 14 therein. Here, a synthetic polymer nonwoven fabric is incorporated in the separation membrane element 2. In the present invention, since the culture solution from the culture tank 1 can be filtered while the filamentous fungus is maintained in the culture tank 1, the filamentous fungus is continuously cultured to ensure sufficient growth. Later, it is possible to efficiently produce the target protein by changing the ingredients of the culture raw material. As an example, Enz. Microb. Technol. 739-743, 9, 1987 (Turker et al.), As a carbon source, filamentous fungi are grown for several days in a medium containing glucose, and then glucose is included as a medium for the production of cellulase mixtures. It is possible to produce a cellulase mixture by refluxing no culture raw material solution.

  In FIG. 1, the culture raw material is fed from the culture raw material tank 11 to the culture tank 1 by the culture raw material supply pump 12. In FIG. 1, by aeration of gas through the lower vent, the filamentous fungus culture solution in the culture tank can be stirred and oxygen necessary for filamentous fungus growth can be supplied. By arranging the cylindrical rectifying plate 3 in the culture tank 1, the convection of the filamentous fungus culture solution can be controlled in a certain direction in the culture tank. If necessary, the oxygen partial pressure of the gas to be supplied can be increased by the mass flow controller 6 connected to the lower vent. In addition, the DO sensor / control device 7 in the culture solution is used to measure the dissolved oxygen concentration in the culture solution, and this measured value is sent as a signal to the mass flow controller 6 to control the oxygen partial pressure. Can be done automatically. Further, if necessary, the pH of the culture solution can be adjusted by adding the neutralization solution 10 to the culture tank 1 by the pH sensor / control device 8 and the neutralization solution supply pump 9. If necessary, the temperature of the culture solution can be adjusted by the temperature controller 4. The culture solution containing protein is filtered and separated into a culture solution containing filamentous fungi and protein by the separation membrane element 2, and the protein is collected in the collection tank 14 by the filtrate pump 13.

  As shown in FIG. 2, the separation membrane element is configured by arranging a flow path material 17 and a synthetic polymer nonwoven fabric 18 in this order on one surface of a support plate 15 having rigidity. The support plate 15 has a recess 16. The synthetic polymer nonwoven fabric 18 filters the filamentous fungus culture solution. The flow path member 17 is for efficiently flowing the filtrate filtered by the synthetic polymer nonwoven fabric 18 to the support plate 19. The filtrate that has flowed to the support plate 15 passes through the recess 16 of the support plate 15 and is taken out to the collection tank through the water collecting pipe 19 that is a discharge means.

  According to the present invention, a high volumetric production rate can be obtained as compared with the conventional batch culture, and an extremely efficient protein production can be achieved. Here, the protein production rate in the continuous culture is calculated by the following (Equation 3).

  Protein production rate (g / L / day) = protein concentration in the extract (g / L) × culture solution extract rate (L / day) ÷ operating fluid amount of the device (L) (Equation 3).

  The production rate of protein mixture by batch culture is obtained by dividing the protein concentration (g / L) in the culture solution by the culture period (day) and the culture solution amount (L) at that time.

  Further, when the protein produced by the present invention is a cellulase mixture, the amount of cellulase (FPU) in the culture solution is calculated by the following (Equation 4).

  FPU (amount of enzyme that produces 1 micromole of glucose per minute) = enzyme concentration (units / ml) required to produce 0.37 / 2.0 mg of glucose (Formula 4).

  Hereinafter, in order to explain in more detail the method for producing a protein by continuous culture of filamentous fungi of the present invention, examples of continuous culture of filamentous fungi by using the apparatus shown in the schematic diagrams of FIGS. 1 and 2 will be given. explain.

  In Examples relating to a method for producing a protein by continuous culture of filamentous fungi of the present invention, Trichoderma reesei RutC30 strain (Example) of Trichoderma reesei as an example of a filamentous fungus that produces a cellulase mixture as a protein. 2) was used. Moreover, regarding the nutrient sources such as the carbon source, nitrogen source, and inorganic salts that are the culture raw materials, the culture medium described in the following examples was used.

Reference Example 1 Characteristic Analysis of Synthetic Polymer Nonwoven Fabric and Woven Fabric Each characteristic value of the above-described nonwoven fabric and each characteristic value in the following examples are measured by the following methods.

(1) Average fiber diameter (μm)
Ten small sample samples were taken at random from the non-woven fabric, photographed at 500 to 3000 times with a scanning electron microscope, 10 fibers from each sample were measured, and the diameter of 100 fibers in total was measured. Calculated by rounding off the first decimal place.

(2) Weight per unit (g / m ² )
Three nonwoven fabrics of 30 cm × 50 cm were sampled, the weight of each sample was measured, the average value of the obtained values was converted per unit area, and the first decimal place was rounded off.

(3) Aeration rate (cc / cm ² / sec)
Based on the 4.8 (1) Frazier method of JIS L 1906 (2000 edition), measurement was performed on any 45 points in a 30 cm × 50 cm non-woven fabric at a pressure of 125 bar. However, the average value was rounded off to the second decimal place.

(Reference Example 2) Characteristic analysis of porous separation membrane (average pore diameter (μm))
The surface of the porous resin layer of the separation membrane is observed with a scanning electron microscope at a magnification of 10,000 times within a range of 9.2 μm × 10.4 μm, and the diameters of all the observable pores are measured, and the average is calculated. did.

Reference Example 3 Production of Porous Separation Membrane (Part 1)
A porous separation membrane (PVDF membrane 1) of polyvinylidene fluoride (PVDF) resin prepared as described in Reference Example 1 of JP-A-2009-39074 was used as (Comparative Example 1). The surface of the porous resin layer was observed with a scanning electron microscope at a magnification of 10,000 within a range of 9.2 μm × 10.4 μm. The average diameter of all the pores that can be observed was 0.1 μm.

Reference Example 4 Production of Porous Separation Membrane (Part 2)
Polyvinylidene fluoride (PVDF) resin as a resin, polyethylene glycol (PEG) having a molecular weight of about 20,000 as a pore-opening agent, N, N-dimethylacetamide as a solvent, and pure water as a non-solvent, respectively. These were sufficiently stirred at a temperature of 90 ° C. to obtain a stock solution having the following composition.
Polyvinylidene fluoride: 13.0% by weight
Polyethylene glycol: 5.5% by weight
N, N-dimethylacetamide: 78.0% by weight
Pure water: 3.5% by weight.

Next, after cooling the stock solution to 25 ° C., it was applied to a non-woven fabric made of polyester fiber having a density of 0.48 g / cm ³ and a thickness of 220 μm, and after application, immediately immersed in pure water at 25 ° C. for 5 minutes, Furthermore, it was immersed in hot water at 80 ° C. three times to wash out N, N-dimethylacetamide and polyethylene glycol to obtain a separation membrane.

  Scanning electron microscope observation was performed at a magnification of 10,000 times within the range of 9.2 μm × 10.4 μm of the surface of the porous resin layer on the side of the separation membrane on which the stock solution was applied. The average diameter of all the observable pores was 0.02 μm. This porous separation membrane was used as the PVDF membrane 2 (Comparative Example 2).

(Reference Example 5) Characteristics of Woven Fabric The characteristics of the woven fabric used as a comparative example are shown below.

Woven fabric 1 (Comparative Example 3)
Product Name: Madison Filter PX305-07
Basis weight: 300 g / m ²
Aeration rate: 1.5 cc / cm ² / sec
Material: Polypropylene warp: Monofilament weft: Multifilament weave: satin weave.

Woven fabric 2 (Comparative Example 4)
Product Name: Madison Filter PX515-07
Per unit weight: 510 g / m ²
Aeration rate: 0.2 cc / cm ² / sec
Material: Polypropylene warp: Monofilament weft: Multifilament weave: satin weave.

Woven fabric 3 (Comparative Example 5)
Product Name: Madison Filter PX587-10
Weight per unit: 600 g / m ²
Aeration rate: 0.3 cc / cm ² / sec or less Material: Polypropylene warp: Multifilament weft: Staple span weave: Reversible double weave.

Woven Cloth 4 (Comparative Example 6)
Product Name: Madison Filter PX351-82
Per unit weight: 350 g / m ²
Ventilation rate: 32cc / cm ² / sec
Material: Polypropylene warp: Monofilament Weft: Monofilament weave: Double weave.

  The woven fabrics 1 to 4 were measured by the method for measuring the average pore diameter described in Reference Example 2. However, since the warp and the weft crossed, the pores as seen in the porous separation membrane were used. Could not be confirmed.

(Reference Example 6) Natural Fiber Nonwoven Fabric Properties of the natural fiber nonwoven fabric (filter paper) used as a comparative example are shown below.

Natural fiber nonwoven fabric (Comparative Example 7)
Product name: Whatman No. 1Qualitative / quantitative filter paper material: Cellulose fiber thickness: 0.18mm
Per unit weight: 86 g / m ²
Aeration rate: 0.64 cc / cm ² / sec.

  The above-mentioned natural fiber nonwoven fabric (filter paper) was measured by the average pore diameter measurement method described in Reference Example 2, but the surface has a structure in which long fibers are intertwined in a complicated manner. Such pores could not be confirmed.

(Example 1) Characteristics of synthetic polymer nonwoven fabric In the present invention, a synthetic polymer nonwoven fabric having the following characteristics was used, and protein was produced by continuous culture of filamentous fungi. The synthetic polymer nonwoven fabric used was a polyester long fiber nonwoven fabric “Axter” manufactured by Toray Industries, Inc. These features are summarized in Table 1 with respect to the basis weight (g / m ² ), air flow rate (cc / cm ² / sec), average fiber diameter (μm), and film thickness (mm) of each product number.

  In addition, about the said synthetic polymer nonwoven fabric, although it measured by the measuring method of the pore diameter of the reference examples 3 and 4, since the surface is a structure where the long fiber was entangled intricately, it seems to be seen with a porous separation membrane. No fine pores could be confirmed.

(Example 2) Manufacture of a cellulase mixture by continuous culture of Trichoderma Using a continuous culture apparatus in which the synthetic polymer nonwoven fabric shown in Fig. 1 is installed in a membrane separation unit, it is possible to manufacture proteins, particularly cellulase mixtures, by continuous culture of filamentous fungi. In order to investigate this, a continuous culture test using the same apparatus was conducted. Trichoderma reesei RutC30 strain was used as the filamentous fungus. The medium shown in Table 2 was used as the medium, and it was used after high-pressure steam sterilization at a temperature of 121 ° C. for 15 minutes. As the separation membrane element member, a molded product of stainless steel and polysulfone resin was used.

  Further, as a comparative example, the PVDF membrane 1 (Comparative Example 1) and the PVDF membrane 2 (Comparative Example 2) produced in Reference Examples 3 and 4, and the woven fabric 1 described in Reference Example 5 (Comparative Example) as a comparative example of the woven fabric 3), Woven Fabric 2 (Comparative Example 4), Woven Fabric 3 (Comparative Example 5), Woven Fabric 4 (Comparative Example 6), Natural Fiber Nonwoven Fabric (Comparative Example 7), and Synthetic Polymer Nonwoven Fabric Example 1 Each of the five synthetic polymer nonwoven fabrics described was used. In Comparative Example 8, the separation membrane element was not provided with a separation membrane.

The continuous culture conditions in Example 2 are as follows unless otherwise specified.
・ Culture tank capacity: 2.0 (L)
Separation membrane used: synthetic polymer nonwoven fabric, porous separation membrane (Comparative Examples 1 and 2), woven fabric (Comparative Examples 3 to 6), natural fiber nonwoven fabric (Comparative Example 7)
・ Membrane separation element effective filtration area: 20 cm ²
・ Temperature adjustment: 28 (℃)
-Culture tank aeration: 1.0 (L / min)
・ Sterilization: The culture tank containing the separation membrane element and the medium to be used are all autoclaved at 121 ° C. for 20 min. High pressure steam sterilization. ・ PH adjustment: pH adjusted to 5.0 with 1N NH ₄ OH. L / day).

  First, conidia of the RutC30 strain solid-ground cultured on a potato dextrose agar medium were cultured with shaking in a test tube (100 ° C.) shown in Table 2 for 3 days (28 ° C.) (preculture). 100 mL of the obtained preculture solution was inoculated into the culture tank 1 containing 2.0 L of the growth medium shown in Table 2 in FIG. Then, it culture | cultivated on the same conditions as preculture for 3 days. Immediately after completion of the culture, the culture material tank 11 containing the surfactant, antifoaming agent, and cellulase inducer of Table 4 containing sophorose (Sigma-Aldrich Japan) is fed to the culture tank 1 by the culture material feed pump 12. did. At the same time, the culture liquid in the culture tank 1 is filtered through the separation membrane installed in the separation membrane unit using the filtration pump 13, and the culture liquid and the cellulase mixture contained in the culture liquid are recovered in the recovery tank 14. did. The feed rates of the culture raw material feed pump and the filtration pump were set so that 2 L of the cellulase mixture solution could be collected per day (24 hours).

  Table 5 summarizes the culture periods during which continuous cultivation of filamentous fungi was possible when various separation membranes were used in the separation unit using the continuous culture apparatus of FIG. As a result of continuous culture for 25 days, it can be confirmed that a stable cellulase mixture can be produced for a long period of time by using the continuous culture apparatus using the synthetic polymer nonwoven fabric shown in FIG. It was. On the other hand, in the PVDF membranes 1 and 2 and the woven fabrics 1 to 4, it was difficult to carry out protein recovery continuously for a long time due to clogging of the membrane. The natural fiber nonwoven fabric was broken in the first day of continuous culture, and leakage of filamentous fungus bodies into the collection tank was confirmed.

  In the continuous culture of the present invention, it is necessary to continuously recover the protein as a filtrate. Therefore, the protein produced by the filamentous fungus in the culture solution was recovered without loss in each culture day, or the recovery rate was calculated by the following formula 5.

  Recovery rate (%) = protein concentration (mg / mL) in the filtrate on the culture day X / protein concentration (mg / mL) in the culture tank on the culture day X × 100 (formula 5).

  That is, in Formula 5, when the numerical value is 100 or less, a part of the components of the protein produced in the culture solution is blocked by the separation membrane, and the protein is not efficiently recovered as a filtrate in the recovery tank. Represents that. Table 6 summarizes the recovery rates when each separation membrane was used. Since the PVDF membrane or woven fabric as a comparative example could not be cultured for 25 days, the recovery rate at the time of the first day or the fifth day of the culture was calculated. Further, “−: hyphen” is displayed in a period during which continuous culture could not be performed. As shown in Table 6, the synthetic polymer nonwoven fabric used in the present invention maintained a protein recovery rate of 95% or more over the culture period, indicating that efficient protein recovery is possible. ing. On the other hand, it was found that the woven fabric or PVDF membrane used as the comparative example could not recover protein efficiently.

  Next, in order to see the correlation between the recovery efficiency of the protein and its cellulase activity, the results of measuring the cellulase activity in the filtrate on the fifth day of continuous culture are summarized in Table 7. When cellulase activity in the filtrate collected through each separation membrane was measured, cellulase activity was found to be lower in continuous culture using PVDF membrane and woven fabric than when using synthetic polymer nonwoven fabric. I understand. Therefore, those with a low protein recovery rate in the results of Table 6 have a low activity of the cellulase mixture recovered as a filtrate, indicating that there is a correlation between the two. That is, it was shown that some of the enzyme components contained in the cellulase mixture may be blocked by the separation membrane. On the other hand, in the continuous culture using the synthetic polymer nonwoven fabric in the present invention, it was shown that the cellulase mixture is not blocked by the separation membrane and can be efficiently recovered.

  Next, Table 8 summarizes the transition of cellulase activity in the filtrate over time when continuous culture of the present invention was performed. As Comparative Example 8, a separation membrane element without a separation membrane was used as a comparative example. As shown in Table 8, by using a synthetic polymer nonwoven fabric, filamentous fungi can be concentrated in the culture tank, and it can be confirmed that the production efficiency of the cellulase mixture is improved as compared with the example in which no separation membrane element is used. It was.

  From the above examples, it can be confirmed that by using a synthetic polymer nonwoven fabric in continuous culture of filamentous fungi, the target cellulase mixture can be efficiently recovered over a long period of time compared to using other separation membranes. It was.

  The present invention can be used in the production of proteins using filamentous fungi. Examples of the protein that can be produced in the present invention include cellulase, protease, amylase, lipase and the like, and these can be produced with high efficiency.

DESCRIPTION OF SYMBOLS 1 Culture tank 2 Separation membrane element 3 Current plate 4 Temperature controller 5 Aeration nozzle 6 Mass flow controller 7 DO sensor / control device 8 pH sensor / control device 9 Neutralization liquid feed pump 10 Neutralization liquid 11 Culture raw material tank 12 Culture raw material Liquid feed pump 13 Filtration pump 14 Collection tank 15 Support plate 16 Concave portion 17 Channel material 18 Synthetic polymer nonwoven fabric 19 Water collecting pipe

Claims (6)

  Filamentous fungus culture fluid is filtered through a synthetic polymer nonwoven fabric, the protein as a product is recovered from the filtrate, and the unfiltrated fluid is retained or refluxed in the culture fluid, and the filamentous fungus culture raw material is added to the culture fluid. A method for producing a protein by continuous cultivation of filamentous fungi. The method for producing a protein according to claim 1, wherein a filtration treatment is performed using a synthetic polymer nonwoven fabric having an air flow rate of 1 to 300 cc / cm ² / sec.   The method for producing a protein according to claim 1 or 2, wherein the protein is a cellulase mixture.   The method for producing a protein according to any one of claims 1 to 3, wherein the filamentous fungus is Genus Tricoderma.   The method for producing a protein according to any one of claims 1 to 4, wherein the filamentous fungus continuous culture is aeration airlift mixing.   Continuous filamentous fungus, including a culture tank for cultivating filamentous fungi, a separation membrane unit equipped with a synthetic polymer nonwoven fabric, a recovery tank for recovering proteins, a raw material tank for supplying culture raw materials, and a lower vent for aeration airlift mixing Protein production equipment by culture.

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