Method and apparatus for producing enzymes, and method for saccharifying biomass resources

Abstract

To enable the inexpensive and efficient production of cellulase.SOLUTION: An enzyme production method is an enzyme production method in which an enzyme-producing bacterium is inoculated into a substrate containing cellulose to produce an enzyme, the method comprising: molding a granular substrate containing cellulose; and inoculating the substrate with an enzyme-producing bacterium, to produce an enzyme while solid-culturing the enzyme-producing bacterium under aerobic conditions.SELECTED DRAWING: Figure 7

Description

[Technical field]

[0001] The present invention relates to a method and apparatus for producing an enzyme, and a method for saccharifying a biomass resource. [Background technology]

[0002] The disposal of organic residues from food, etc., generated during the production of coffee, tea, etc., has become an issue. Many of these residues are mainly composed of cellulose fibers, and this type of cellulose-containing biomass cannot be directly put into existing treatment plants such as sewage treatment plants or microbial decomposition plants. For this reason, it is necessary to dispose of them as industrial waste, which is very costly and time-consuming.

[0003] It is known that glucose (grape sugar) can be obtained by hydrolyzing cellulose with cellulase. Cellulase is a general term for a group of enzymes that catalyze the enzymatic reaction system that hydrolyzes cellulose into glucose, cellobiose, and cellooligotose, and there are enzymes called by various names such as FPase, CMCase, and cellobiase depending on their mode of action.

[0004] Cellulose processing using cellulase has attracted attention because the use of such cellulase to solubilize cellulose-containing biomass into glucose can be introduced into existing processing facilities that use microorganisms, etc. To achieve this, it is necessary to produce cellulase efficiently at low cost.

[0005] Conventionally, for the production of cellulase used in the saccharification of lignocellulosic biomass resources consisting mainly of lignin and cellulose, Trichoderma reesei (see Non-Patent Document 1), Trichoderma viride (see Non-Patent Document 2), and microorganisms belonging to the genera Aspergillus and Penicillium have been used as cellulase-producing fungi. However, these strains have the drawback of being unable to obtain sufficient cellulase productivity.

[0006] Saccharification reactions using cellulase are essential for the industrial use of biomass resources. For example, when using biomass resources as a basic starting material for the production of bioethanol as an alternative fuel to petroleum, or other carbon-containing compounds, it is necessary to produce cellulase inexpensively. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2008-271927 A [Non-patent literature]

[0008] [Non-Patent Document 1] Biotechnol. Bioeng., 23, 1837-1849 (1981) [Non-Patent Document 2] Appl. Biochem. Biotechnol., 57-58, 349-360 (1996) Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention has been made in view of the above background, and one of its objects is to provide an enzyme production method and enzyme production apparatus capable of producing cellulase inexpensively and efficiently, and a method for saccharifying biomass resources including cellulose.

[0010] The method for producing an enzyme according to the first embodiment of the present invention is a method for producing an enzyme by inoculating a substrate containing cellulose with an enzyme-producing bacterium, and includes the steps of forming a granular substrate containing cellulose, and inoculating the substrate with the enzyme-producing bacterium to produce an enzyme while culturing the enzyme-producing bacterium under aerobic solid culture conditions. In the step of forming the granular substrate, in addition to the granular substrate, 1% or more of a needle-shaped or fibrous substrate is included in order to increase voids between the granular substrate so that air can be distributed throughout the accumulated granular substrate. The bulk density of the molded substrate is 5.0 g / 100 mL to 25.0 g / 100 mL in a dry state. This allows oxygen to be supplied to the gaps between the granular substrates to promote the growth of the fungus, making it possible to obtain enzymes with activity comparable to that of commercially available enzymes. In addition, the presence of long fibrous substrates makes it easier for spaces to form between the substrates, allowing air to easily enter and promoting the growth of the mycelium.

[0011] Furthermore, according to the method for producing an enzyme according to the second embodiment of the present invention, in addition to the above, the enzyme can be cellulase.

[0012] Furthermore, according to the method for producing an enzyme according to the third embodiment of the present invention, in addition to any of the above, the enzyme-producing fungus inoculated with the substrate can be a filamentous fungus.

[0013] Furthermore, according to the enzyme production method of the fourth embodiment of the present invention, in addition to any of the above, the step of shaping the substrate can include a step of shaping and / or pulverizing the substrate into granules, thereby making it possible to ensure breathability and facilitate the supply of oxygen by making the substrate into granules rather than powder, thereby improving the growth of the bacterial cells.

[0015] Furthermore, according to the method for producing an enzyme according to the sixth embodiment of the present invention, in addition to any of the above, the average particle size of the substrate can be set to 0.8 μm to 1000 μm, thereby increasing the surface area and enabling an efficient reaction.

[0016] Furthermore, according to the method for producing an enzyme according to the seventh embodiment of the present invention, in addition to any of the above, the substrate can have a cellulose ratio of 30% to 70%, a protein content of 1% to 10%, and an ash content of 0% to 10%.

[0017] Furthermore, according to the method for producing an enzyme according to the eighth embodiment of the present invention, in addition to any of the above, the bulk density of the molded substrate can be set to 5.0 g / 100 mL to 25.0 g / 100 mL in a dry state.

[0018] Furthermore, according to the method for producing an enzyme according to the ninth embodiment of the present invention, in addition to any of the above, the moisture content of the substrate after molding can be set to 20% to 60%.

[0019] Furthermore, according to the method for producing an enzyme according to the tenth embodiment of the present invention, in addition to any of the above, the step of solid-state culturing the enzyme on the substrate can be carried out without stirring, thereby preventing the grown mycelium from being cut.

[0020] Furthermore, according to the enzyme production method of the eleventh embodiment of the present invention, in addition to any of the above, the step of shaping the substrate can include a step of treating the substrate with high-temperature steam and then sterilizing it with dry heat, thereby promoting the uptake of the medium by the fungus cells by loosening the cellulose fibers with high-pressure steam treatment and then sterilizing it with dry heat.

[0021] Furthermore, according to the method for producing an enzyme according to the twelfth embodiment of the present invention, in addition to any of the above, the enzyme-producing fungus may be at least one of the genera Trichoderma, Aspergillus, Lamicola, Acremonium, and Humicola, or a combination thereof. Among them, Trichoderma spp. is preferred because it has a high growth rate and produces a large amount of enzyme. Aspergillus spp. is also preferred because it can produce cellulase with high β-glucosidase activity.

[0022] Furthermore, according to the enzyme production method of the thirteenth embodiment of the present invention, in addition to any of the above, the step of solid-state culturing the enzyme produced by the enzyme-producing bacteria can include a pre-culture step of inoculating the enzyme-producing bacteria into a Mandel medium and culturing the bacteria, and a main culture step of centrifuging the Mandel medium to separate the bacteria obtained in the pre-culture, and inoculating the obtained bacteria into the molded substrate and culturing the bacteria. This ensures reliable culture by first obtaining a sufficient bacterial solution by liquid culture using the Mandel medium, and then inoculating the substrate containing cellulose.

[0023] Furthermore, according to the enzyme production method of the 14th embodiment of the present invention, in addition to any of the above, the process of solid-state culturing of cellulase enzyme with the substrate can be carried out in a tank having a breathable filter, wire mesh demister, punched plate, or perforated plate disposed on the bottom surface.

[0024] Furthermore, according to the fifteenth embodiment of the method for producing an enzyme of the present invention, in addition to any of the above, it can further comprise a step of extracting the solid-cultured cellulase enzyme from the substrate.

[0025] Furthermore, according to the method for producing an enzyme according to the sixteenth embodiment of the present invention, in addition to any of the above, the substrate can contain wheat straw, thereby obtaining an enzyme with excellent FPU activity.

[0026] Furthermore, according to the enzyme producing method of the seventeenth embodiment of the present invention, in addition to any of the above, the substrate can contain paper sludge, which has the advantage that PS, which is an industrial waste, can be effectively used as a medium for enzyme-producing bacteria.

[0027] Furthermore, according to the enzyme producing method of the 18th embodiment of the present invention, in addition to any of the above, the substrate can be a mixture of paper sludge and wheat straw. By this means, although the bulk density of paper sludge alone is too high, by mixing a component such as wheat straw, which has a low bulk density to begin with, it is possible to provide a suitable medium for the enzyme-producing bacteria.

[0028] Furthermore, according to the enzyme production method of the 19th embodiment of the present invention, in addition to any of the above, the maximum FPU activity value of the extracted cellulase enzyme can be made to be 0.900 FPU / g-dry paper sludge or more.

[0029] Furthermore, a 20th embodiment of the present invention provides a method for saccharifying a biomass resource, which comprises using cellulase produced by any one of the above enzyme production methods to saccharify and decompose a biomass resource containing cellulose fibers.

[0030] Furthermore, according to the method for saccharifying a biomass resource according to a twenty-first embodiment of the present invention, in addition to any of the above, the biomass resource can be food waste.

[0031] Furthermore, according to a twenty-second embodiment of the enzyme production apparatus of the present invention, there is provided an enzyme production apparatus for producing cellulase by applying an enzyme-producing bacterium to a substrate containing cellulose, the enzyme production apparatus comprising a tank having a filter-shaped bottom surface for introducing a granular substrate containing cellulose, the granular substrate introduced into the tank, and a substrate containing 1% or more of a needle-shaped or fibrous substrate in order to increase the gaps between the granular substrate so that air can be distributed throughout the accumulated granular substrate. The bulk density is 5.0g / 100mL to 25.0g / 100mL in a dry state. The tank can be provided with a production bacteria application section for applying enzyme-producing bacteria to the substrate, and a duct for supplying oxygen to the filter-like bottom surface of the tank. With the above configuration, cellulase can be produced by solid culture using the substrate placed in the tank as the culture medium. As a result, unlike conventional liquid culture, there is no need for aeration power to shake the heavy culture medium, and production with low energy consumption is realized. In addition, rupture of mycelium due to shaking can be avoided, and high enzyme production can be expected. [Brief description of the drawings]

[0032] [Figure 1] This is a photograph showing the measurement state of wheat straw and PS measuring cylinders. [Diagram 2] This is a photograph showing dry wheat straw and PS placed in a flask. [Diagram 3] This is a photograph showing hydrated wheat straw and PS placed in a flask. [Figure 4] FIG. 1 is a schematic diagram showing an enzyme production apparatus. [Diagram 5] This is a photograph with a schematic enlarged view showing PS. [Figure 6] FIG. 1 is a schematic diagram showing the FPU measurement method. [Figure 7] 1 is a graph showing the relationship between the elapsed time for each culture temperature and enzyme activity. [Figure 8] 1 is a graph showing the relationship between the elapsed time and the enzyme activity for each moisture content of PS. [Figure 9] This is a photograph of PS with a moisture content of 50-60% on the 12th day. [Figure 10] This is a photograph of PS with a moisture content of 80% on the 6th day. [Figure 11] 1 is a graph showing the relationship between the elapsed time and enzyme activity for each C / N ratio at the start of culture. [Figure 12] This is a photo of PS on the 6th day. [Figure 13] 1 is a graph showing the relationship between the elapsed time and enzyme activity for each bacterial species. [Figure 14] This is a photo of the PS on the 6th day after mixing of the bacterial species. [Figure 15] 1 is a graph showing the relationship between the elapsed time for each PS pretreatment and enzyme activity. [Figure 16] FIG. 1 is a schematic diagram showing the procedure of an enzymatic saccharification experiment. [Figure 17] 1 is a graph showing the relationship between the elapsed time and the glucose concentration when Bemcot is used as a substrate. [Figure 18] 1 is a graph showing the relationship between time and glucose concentration when untreated coffee grounds are used as a substrate. [Figure 19] 1 is a graph showing the relationship between elapsed time and glucose concentration for each coffee grounds processing method. [Figure 20] 1 is a graph showing the relationship between the elapsed time and the saccharification rate for a commercially available enzyme and a produced enzyme. [Figure 21] 1 is a photograph showing PS with an average particle size of 250 to 500 μm. [Figure 22] 1 is a photograph showing wheat straw having an average particle size of 100 to 250 μm. [Figure 23] 1 is a graph showing the relationship between the elapsed time and the enzyme activity with or without stirring. [Figure 24] This is a photo of PS with stirring. [Diagram 25] This is a photograph of PS without stirring. [Figure 26] 1 is a graph showing the relationship between the elapsed time and the enzyme activity for PS and WS. [Figure 27] This is a photo from PS. [Figure 28] This is a photo of WS. [Figure 29] This is a photograph showing an Erlenmeyer flask stoppered with silicon. [Diagram 30] This is a photograph showing an Erlenmeyer flask covered with aluminum foil. [Diagram 31] 1 is a graph showing the relationship between elapsed time and enzyme activity when silicosene and aluminum foil are used. [Diagram 32] This is a photo of a WS plugged with silicone. [Diagram 33] This is a photo of a WS covered with aluminum foil. [Diagram 34] 1 is a graph showing the relationship between reaction time and glucose for each pretreatment for 6.7 FPU / g-substrate. [Diagram 35] 1 is a graph showing the relationship between reaction time and glucose for each pretreatment for 11 FPU / g-substrate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Hereinafter, the embodiments of the present invention will be described with reference to the drawings. However, the embodiments shown below are merely examples for embodying the technical idea of ​​the present invention, and the present invention is not limited to the following. In addition, this specification never specifies the members shown in the claims to the members of the embodiments. In particular, the dimensions, materials, shapes, and relative positions of the components described in the embodiments are merely explanatory examples, and are not intended to limit the scope of the present invention, unless otherwise specified. Note that the size and positional relationship of the members shown in each drawing may be exaggerated to clarify the explanation. Furthermore, in the following explanation, the same name and symbol indicate the same or similar members, and detailed explanation will be omitted as appropriate. Furthermore, each element constituting the present invention may be configured as a form in which multiple elements are composed of the same member, and one member serves multiple elements, or conversely, the function of one member can be shared by multiple members. [Embodiment 1]

[0034] The enzyme production method according to the first embodiment of the present invention produces an enzyme by applying an enzyme-producing bacterium to a substrate containing cellulose. Specifically, the method includes a step of forming a granular substrate containing cellulose, a step of inoculating the substrate with an enzyme-producing bacterium to solid-state culture the cellulase enzyme, and a step of extracting the solid-cultured cellulase enzyme from the substrate. This allows cellulase to be produced by solid culture, and unlike conventional liquid culture, there is no need for aeration power to shake a heavy medium, realizing production with low energy consumption. In addition, the breakage of mycelium due to shaking can be avoided, and high enzyme production can be expected. Here, solid culture refers to a nutrient source itself in a solid state, not a liquid nutrient solidified with agar or gelatin.

[0035] The step of forming the substrate preferably includes a step of pulverizing the substrate into granules. By doing so, the substrate is made granular rather than powdery, ensuring breathability and facilitating the supply of oxygen, thereby improving the growth of the bacterial cells. Of course, a substrate already formed into granules may be introduced.

[0036] In addition, the granular substrate preferably contains 1% or more of needle-shaped or fibrous materials. This makes it easier for spaces to form between the substrates due to the presence of long fiber substrates, allowing air to easily enter and promoting the growth of mycelium. The needle-shaped or fibrous materials may be of the same material as the other substrates, or other materials may be added. For example, other needle-shaped or fibrous fibers, such as polyurethane foam (PUF) or glass fiber filters, may be added to substrates such as PS or wheat straw.

[0037] The average particle size of the substrate is preferably 0.8 μm to 1000 μm. This increases the surface area, enabling efficient reactions. The average particle size can be measured by calculating the average particle size from an enlarged photograph of the particles observed with an SEM. For example, when wheat straw is used as the substrate (details will be described later), the average particle size is 100 μm to 4 mm, as it contains needle-shaped particles. When paper sludge is used as the substrate, the same SEM observation revealed that not only granular particles but also fibrous particles were observed, and the average particle size was 800 nm to 800 μm.

[0038] The step of forming the substrate preferably includes a step of treating the substrate with high-temperature steam and then dry-heat sterilizing the substrate, which promotes the uptake of the medium by the fungus cells by loosening the cellulose fibers with high-pressure steam and then dry-heat sterilizing the substrate.

[0039] The substrate formed as described above is used as a medium, and enzyme-producing bacteria are applied to solid-state culture the enzyme. Cellulase is a suitable example of the enzyme. A granular substrate containing cellulose is used as a pseudo-grain, and enzymes such as cellulase are produced in this medium. The enzyme-producing bacteria obtain the nitrogen source and carbon source contained in the medium and grow filamentous fungi. The process of solid-state culture of cellulase enzymes on the substrate includes a process of adjusting the moisture content of the substrate, a process of inoculating the solid medium with the cells of the enzyme-producing bacteria, and a process of supplying air to the solid medium at an appropriate level while adjusting the temperature and humidity. Hereinafter, the process of solid-state culture of cellulase enzymes on the substrate is referred to as the cellulase culture process.

[0040] Unlike liquid culture, solid culture has the advantage of not requiring the power to shake a heavy medium. Also, depending on the species of bacteria, high production of products (enzymes) can be expected.

[0041] The cellulase culture step is preferably carried out in an environment that maintains ventilation, which allows oxygen to be supplied to the gaps between the granular substrates, promoting the growth of the cells.

[0042] In addition, the cellulase culture step is preferably carried out without stirring, which can prevent the grown mycelia from being torn apart.

[0043] Furthermore, the cellulase culture step preferably includes a pre-culture step in which the cellulase-producing fungus is inoculated into a Mandel medium and cultured, and a main culture step in which the fungus obtained in the pre-culture step is centrifuged from the Mandel medium, and the fungus obtained is inoculated into a substrate and cultured. This ensures reliable culture by first obtaining a sufficient fungus liquid by liquid culture using the Mandel medium, and then inoculating the substrate containing cellulose. (Cellulase-producing bacteria)

[0044] The cellulase-producing fungus is preferably at least one of the genera Trichoderma, Aspergillus, Lamicola, Acremonium, and Humicola. Among them, Trichoderma spp. is preferred because it has a high growth rate and produces a large amount of cellulase. Aspergillus spp. is also preferred because it can produce cellulase with high β-glucosidase activity. More preferably, a combination of Trichoderma and Aspergillus is used as the cellulase-producing fungus. (substrate)

[0045] The substrate constituting the medium to which the enzyme-producing bacteria is applied is a carbon source. As the carbon source of such a medium, it is possible to use filter paper, general paper, waste paper, textile products such as used clothing and their shredded materials, wood, bran, wheat straw, rice straw, rice husk, bamboo, sawdust, crustacean shells, dried corn stover, dried bagasse (sugarcane pomace), soybean meal, soybean okara, dead grass, crushed or pulverized materials such as leaves, coffee grounds, rice bran, lactose, lactose hydrate, whey, dairy products, hydrolysis residue, powdered cellulose, Avicel, cellobiose, and mixtures thereof. In this embodiment, it is particularly effective for biomass substrates containing cellulose, and among them, wheat straw and paper sludge can be preferably used.

[0046] By using wheat straw as a substrate, an enzyme with excellent FPU activity can be obtained, as described below.

[0047] On the other hand, paper sludge (PS) is a product of concentrating and dehydrating short fibers and inorganic matter that are not turned into paper and are washed away in wastewater when recycled paper such as toilet paper is produced from waste paper. Its moisture content is 55-60%, and its ash content is 45-52%. In Japan, about 4.4 million tons are generated annually, and most of it is incinerated or disposed of in landfills. As such, PS is an industrial waste, and disposal is difficult, especially in the paper industry. This PS has the advantage that it is not seasonal and can be supplied all year round, so it is easy to obtain in large quantities. Another advantage of PS is that, unlike other substrates such as wheat straw, rice straw, husks, bagasse, and corn stover, it does not require a separate crushing process or the crushing process can be simplified because it is originally granular.

[0048] According to the enzyme production method of this embodiment, such unused PS can be effectively utilized by using it as a substrate for enzyme production by solid culture, thereby promoting the utilization of waste materials and contributing to the realization of a sustainable circular society.

[0049] The substrate preferably has a cellulose content of 30% to 70%, a protein content of 1% to 10%, and an ash content of 0% to 10%.

[0050] For example, when the substrate is PS, a moisture content of 55 to 60% and an ash content of 8% are preferable.

[0051] Furthermore, when the substrate is bagasse or rice straw, it is preferable that the cellulose content is 38% to 52%, the ash content is 3% to 8%, and the crude protein content is 2% to 8%, although this depends on the quality.

[0052] In the case of bagasse, cellulose: 38%-45%; ash: 2%-5%; crude protein: 2.4%.

[0053] Furthermore, the ratio of the carbon source to the nitrogen source in the substrate is preferably 39 to 53%.

[0054] The moisture content of the substrate is preferably 20% to 60%. By adjusting the initial moisture content in this way, breathability can be ensured. In particular, the culture of the enzyme proceeds as the initial moisture evaporates and the moisture content of the molded substrate decreases. It is believed that initially, growth proceeds in a two-dimensional direction on the surface of the molded substrate, and then, as the moisture in the surface layer decreases, the growth of the bacteria proceeds toward the inside. If there is too much moisture here, the growth is completed only on the surface layer and the growth toward the inside weakens, while if there is too little moisture, the absolute amount of the bacteria decreases, resulting in a decrease in the amount of enzyme, which is the product. Taking these into consideration, by adjusting the moisture content of the substrate to the above-mentioned range, an environment suitable for the culture of the enzyme can be realized. (bulk density)

[0055] The bulk density of the substrate is preferably 5.0g / 100mL to 25.0g / 100mL in a dry state. By keeping the bulk density low in this way, a medium with good aeration can be obtained. As the substrate used in the examples described later, wheat straw in a dry state had a bulk density of 13.5g / 100mL and a PS of 11.5g / 100mL.

[0056] Wheat straw contains needle-shaped samples and does not absorb water easily, so it does not clump or form lumps even when moistened. In contrast, PS tends to form lumps when it absorbs water, and does not expand in volume much in the flask.

[0057] In addition, the bulk density of the hydrated state was calculated as 17.7g / 100mL, because the measuring volume of the measuring cylinder was 57mL for 5.06g of wheat straw and 5g of distilled water. The measuring volume of the measuring cylinder was 38mL for 5.08g of PS and 5g of distilled water, so the bulk density of the hydrated state of PS was 26.5g / 100mL. Figure 1 shows the measuring state of wheat straw and PS in the measuring cylinder, Figure 2 shows the results of the flasks in the dry state, and Figure 3 shows the results of the flasks in the wet state. In these figures, the left side shows wheat straw and the right side shows PS. As shown in these figures, in the dry state before adding water, the apparent volume of the flasks does not change much. On the other hand, in the wet state after adding water, when they are well mixed, it can be seen that there is a large difference in appearance between wheat straw and PS.

[0058] This is thought to be because when the medium substrate contains moisture, the PS becomes clogged and dense. Even if the bulk density is the same as that of wheat straw in a dry state, when it becomes hydrated, the density increases and the breathability decreases, which is thought to result in a decrease in cellulase production. In other words, a medium suitable for cellulase production preferably has a suitable amount of gaps between the fibers that make up the substrate, which is thought to allow oxygen to be supplied to the gaps and facilitate the proliferation of the bacteria. A large surface area is also preferable, as this will increase the concentration of the bacteria and the enzyme concentration.

[0059] On the other hand, granular wheat straw is difficult for water to penetrate and is thought to be water repellent. In addition, mycelium grows on the surface of the cellulose, which has a high oxygen concentration, and wheat straw, which has a large surface area, has a higher mycelium concentration than PS. As a result, wheat straw is preferable because it produces more enzymes than PS.

[0060] Furthermore, as mentioned above, it is effective to mix acicular fibers into cellulose raw materials such as PS to reduce the bulk density in a wet state.

[0061] Instead of bulk density, the specific surface area [m -1 ] can also be used to select substrate characteristics. (Enzyme production equipment)

[0062] The process of solid-state culturing of cellulase enzymes with a substrate is preferably carried out in a tank with a breathable filter placed on the bottom. An enzyme production apparatus 100 for producing cellulase is shown in Fig. 4. The enzyme production apparatus 100 shown in this figure includes a tank 1, a production bacteria application section 2, and a duct 3.

[0063] The tank 1 is a member for introducing a granular substrate containing cellulose. The bottom surface of the tank 1 is in the form of a filter.

[0064] The producer bacteria application section 2 is a member for applying the enzyme-producing bacteria to the substrate placed in the tank 1.

[0065] The duct 3 is a member for supplying oxygen to the filter-like bottom surface of the tank 1.

[0066] Such an enzyme production apparatus 100 can produce cellulase by solid culture using the substrate placed in the tank 1 as the culture medium. In particular, by configuring the enzyme production apparatus 100 as a powder bed gas phase flotation type filamentous fungus culture apparatus, it becomes possible to culture filamentous fungi using powdered food waste. As a result, unlike conventional liquid culture, there is no need for aeration power to shake the heavy culture medium, and production with low energy consumption is realized. In addition, rupture of mycelium due to shaking can be avoided, and high enzyme production can be expected.

[0067] In particular, when using PS as the substrate, there is a problem that the bulk density becomes large in a wet state, as mentioned above. Therefore, when growing filamentous fungi, it seems effective to use air to float the bed of mycelium and fly it up into powder form while growing it. However, in this case, air is required to be supplied, so it is necessary to efficiently exhaust the respiratory metabolic gases that are generated. Therefore, as mentioned above, a filter is installed on the floor of tank 1, and the gas components are replaced while the powder is retained inside through this filter. This makes it possible to supply fresh air while preventing exhaust gas from accumulating, and to efficiently grow filamentous fungi.

[0068] Using the cellulase thus obtained, biomass resources containing cellulose fibers can be saccharified and decomposed. Examples of such biomass resources include food waste. By saccharifying and decomposing food waste, which is organic waste, with cellulase, processing using microorganisms becomes easier. Another example of such biomass resources is coffee grounds.

[0069] Furthermore, the cellulase culture step preferably includes a pre-culture step in which the enzyme-producing bacteria is inoculated into a Mandel medium and cultured, and a main culture step in which the bacteria obtained in the pre-culture step is centrifuged from the Mandel medium, and the bacteria obtained are inoculated into a substrate and cultured. This ensures reliable culture by first obtaining a sufficient bacterial solution by liquid culture using the Mandel medium, and then inoculating the substrate containing cellulose. [Example 1: PS]

[0070] Hereinafter, as Example 1, an enzyme production method using PS as a substrate will be described. Here, cellulase was produced by solid culture using PS, and culture conditions showing high production (high activity value) were explored. Specifically, preferable temperature conditions, moisture content, bacterial species, etc. for the cellulase culture process were examined. In addition, to evaluate the characteristics of the produced cellulase, saccharification experiments were carried out using coffee grounds, which are surplus food waste, as a substrate, and the cellulase was compared and evaluated with commercially available enzymes.

[0071] Enzyme-producing fungi that produce cellulase include Trichoderma spp., Aspergillus spp., Ramicola spp., Acremonium spp., Humicol spp., etc. Here, we used the Trichoderma spp., which has a high growth rate and produces a large amount of enzymes, and the Aspergillus spp., which produces cellulase with high β-glucosidase activity. (preculture)

[0072] Using this enzyme-producing bacterium, cellulase enzymes are cultured on a substrate in solid state. Here, the cellulase culture process is divided into pre-culture and main culture. First, in the pre-culture, Mandel medium and a carbon source are molded as the pre-culture medium and placed in a flask. The Mandel medium here contains 1.4 g / L (NH4)SO4, 2.0 g / L KH2PO4, 0.4 g / L CaCl2·2H2O, 0.005 g / L MgSO47H2O, 0.0037 g / L CoCl·6H2O, 0.0016 g / L MnSO4·H2O, 0.005 g / L FeSO4·7H2O, 0.0014 g / L ZnSO4·7H2O, 0.3 g / L urea, and 0.1 g / L peptone. The concentrations here are those when mixed with the carbon source. The carbon source contained 5.0 g / L glucose and 10.0 g / L Avicel. The concentrations were measured when mixed with Mandel medium. The Mandel medium and carbon source were placed in a 1:1 ratio in a flask with a culture volume of 100 mL, and the enzyme-producing bacteria were inoculated and stirred at 180 rpm at 28°C for 48 hours. The enzyme-producing bacteria used were Trichoderma reesei ATCC 26921, Trichoderma reesei ATCC 56765, and Aspergillus oryzae ATCC 12892. (Main culture)

[0073] The bacterial cells thus obtained by pre-culture were extracted from the pre-culture medium. Here, the bacterial cells were taken out by centrifugation. Nutrients were added to the bacterial cells to make a bacterial liquid. As the nutrient source, 5.0 mL of high-concentration Mandel medium (10 times the concentration of the above Mandel medium components) was added as a nitrogen source.

[0074] Furthermore, the bacterial solution thus obtained is applied to a substrate containing cellulose for the main culture. PS was used as the substrate here. PS contains 52.2% cellulose and 8.0% ash. The particle size of the PS pellets is 2.5-3.5 mm in diameter and 15-30 mm in length, as shown in Figure 5. 5.0 g of this PS in a dry state and 5.0 mL of bacterial solution to which a nutrient source has been added are placed in each flask. The water content of the PS was adjusted with sterile water. The flasks were cultured at a constant temperature, and the progress was observed. Specifically, one of the flasks was extracted every day, 0.05 M citrate buffer was added, and the flasks were shaken for 2-3 hours. The flasks were then centrifuged at 3500 rpm for 15 minutes, and only the supernatant was separated, which was used as the enzyme solution, and the filter paper digestion activity (FPU) of the obtained enzyme solution was measured. (FPU measurement method)

[0075] Here, we explain how to measure FPU (Filter Paper Unit). FPU is the amount of enzyme that produces 1 μmol of glucose per minute. As shown in Figure 6, 250 μL of the enzyme solution is first placed in a microtube. 1 mL of 0.05 M citrate buffer (pH 5.0) is also placed in the microtube. A roll of filter paper (Whatman No. 1 filter paper) with a width of 1 cm, length of 6 cm, and weight of 55 mg is rolled up and immersed in the liquid in this microtube. In this state, it is left at 50°C for 1 hour, and then boiled for 10 minutes. The amount of reducing sugar is then measured by the Somogyi-Nelson method. Specifically, 100 μL of the enzyme solution taken out of the microtube is placed in another microtube, and 100 μL of Somogyi copper reagent is added. Then, it is boiled for 10 minutes and rapidly cooled, after which 100 μL of Nelson reagent is added and left for 15 minutes. In this state, 1.2 mL of distilled water is added and the absorbance at 660 nm is measured. (1: Comparison by culture temperature)

[0076] First, a comparison was made based on the culture temperature. Here, the culture temperature was 30°C or 36°C. Specifically, the strains used were Trichoderma reesei ATCC 56765 and Trichoderma reesei ATCC 26921, and the water content of PS at the start of culture was 50-60%. The culture temperature was (Condition 1) 30°C all day, (Condition 2) 30°C from the start of culture to the second day, and 36°C from the third day onwards. The pretreatment of PS was only dry heat sterilization. Dry heat sterilization was performed until the PS was completely dried, which took about 1 to 2 hours (same below). The C / N ratio at the start of culture was 53.3.

[0077] The results of such comparative tests of the culture temperature in enzyme production using PS as a substrate are shown in the graph in Figure 7. This graph shows the enzyme activity [FPU / g-dryPS] versus time. The culture data at the maximum FPU activity value are shown in Table 1.

[0078] [Table 1]

[0079] As shown in Figure 7 and Table 1, Trichoderma reesei ATCC 56765 cultured at 30°C showed high enzyme activity on the fifth day. Visual observation of the PS also revealed that the entire sample was ochre-colored with thin white hyphae on the surface. Therefore, this combination of culture temperature and strain used was deemed preferable, and these conditions were used for culture in the next experiment.

[0080] In addition, other publications, "Applied Biochemistry and Biotechnology, 161, 382-394, 2010" and "Production of cellulase from Kraft paper mill sludge by Trichoderma Reesei Rut C-30", report that pre-treatment of PS improves the FPU activity of the production enzyme, although it is difficult to make a pure comparison since it was an experiment using liquid culture. Furthermore, when PS is not treated, the production bacteria hardly grow. Furthermore, washing with water results in an FPU of about 1 to 1.5. From these, it can be said that it is a favorable result that the same level of FPU activity was obtained by solid culture. (2: Comparison of water content of PS at the start of culture)

[0081] In order to examine the moisture content from the viewpoint of solid culture, a comparison was made based on the moisture content of PS at the start of culture. Here, the moisture content of PS at the start of culture was set to 50-60% or 80%. Specifically, the strain used was Trichoderma reesei ATCC 56765. The moisture content of PS at the start of culture was set to (Condition 1) 50-60% and (Condition 2) 80%. The culture temperature was 30°C, and the pretreatment of PS was high-pressure steam treatment and dry heat sterilization. The high-pressure steam treatment was performed at 121°C for 20 minutes (same below). The C / N ratio at the start of culture was also set to 53.3.

[0082] The results are shown in the graph of FIG. 8. In this figure, the horizontal axis indicates the elapsed time, and the vertical axis indicates the enzyme activity. The maximum FPU value for the moisture content of 50-60% was 0.926 FPU / g-dryPS on the 12th day, while the maximum FPU value for the moisture content of 80% was 0.910 FPU / g-dryPS on the 6th day, and no significant difference was observed. In addition, the maximum FPU values ​​for each of conditions 1 and 2 are shown in FIG. 9, which shows the photographs of PS on the 12th day for 50-60% and on the 6th day for 80%. As shown in FIG. 9, the 60% PS was pale yellow on the 12th day, and a thin layer of white hyphae was observed covering the surface. On the other hand, as shown in FIG. 10, the 80% PS was ochre-colored on the 6th day, and a thick layer of white hyphae was observed covering the surface. (3: Comparison based on C / N ratio at the start of culture)

[0083] Next, to compare the C / N ratio at the start of the culture, the ratio of carbon source to nitrogen source at the start of the main culture was set to C / N = 53.3 or C / N = 17.8. Specifically, the strain used was the same Trichoderma reesei ATCC 56765, the moisture content of PS at the start of the culture was 50-60%, and the culture temperature was 30°C. The pretreatment of PS was high-pressure steam treatment and dry heat sterilization. The C / N ratio at the start of the culture was set to 53.3 (condition 1) and 17.8 (condition 2).

[0084] The results are shown in the graph in Figure 11. As shown in this figure, there was no significant difference in the FPU activity value when the C / N ratio was changed, between condition 1 (C / N ratio = 53.3) and condition 2 (17.8). Moreover, a photograph of the PS on the 6th day, when the maximum FPU value was observed, is shown in Figure 12 (3 times the concentration of the main culture medium). As shown in this figure, it was confirmed that some of the PS had begun to turn pale yellow, and that the surface was thinly covered with white hyphae. (4: Synergistic effects of combining bacterial species)

[0085] Furthermore, to confirm the synergistic effect of combining bacterial species, Trichoderma reesei ATCC56765 and Aspergillus oryzae ATCC12892 were used and compared when used alone and when used in combination. Here, the moisture content of PS at the start of cultivation was set to 50-60%, and the cultivation temperature was set to 30°C. Pretreatment of PS was performed by high-pressure steam treatment and dry heat sterilization, and the C / N ratio at the start of cultivation was set to 53.3.

[0086] The results are shown in Figure 13. As this figure shows, FPU activity rose rapidly just two days after the start of cultivation. The rapid rate of increase suggests that a synergistic effect can be achieved by diversifying the enzyme groups resulting from the combination of bacterial species. Figure 14 shows a photograph of the PS on the sixth day of the example of the bacterial species mixture that showed the maximum FPU value. As this figure shows, it can be seen that the PS is covered with so thick white hyphae that it is no longer visible. (5: Effect of pretreatment with PS)

[0087] Furthermore, the effect of pretreatment of PS was confirmed. Here, the strain used was Trichoderma reesei ATCC 56765, the moisture content of PS at the start of cultivation was 50-60%, and the cultivation temperature was 30°C. Three types of pretreatment were performed in advance for PS: (Condition 1) dry heat sterilization only, (Condition 2) high-pressure steam treatment at 121°C for 20 minutes and dry heat sterilization, and (Condition 3) crushing with a cutter mill for 30 seconds, high-pressure steam treatment at 121°C for 20 minutes, and dry heat sterilization. The C / N ratio at the start of cultivation was 53.3. The results are shown in the graph in Figure 15. As shown in this figure, condition 3, crushing + high-pressure steam treatment + dry heat sterilization, showed good enzyme activity. The reason for this is presumably that the surface area of ​​PS was increased by crushing treatment, and the high-pressure steam treatment had the effect of loosening the PS fibers, resulting in an improvement in FPU activity.

[0088] Regarding FPU activity, many of the literature reports that the activity level becomes constant after about 5 or 6 days of culture. We also looked into the literature, considering the presence of proteases, but found no mention of this, so we think that the influence of proteases is unlikely. (Enzymatic saccharification experiment)

[0089] Next, a saccharification experiment was carried out on the substrate using the produced enzyme, and the results were compared with commercially available enzymes. The produced enzyme was obtained by culturing for 30 days under the following conditions: culture temperature: 30°C, water content of PS at the start of culture: 50%, pretreatment of PS: only dry heat sterilization. Acremonium cellulase manufactured by Meiji Seika Pharma Co., Ltd. was used as the commercially available enzyme. This is derived from Acremonium cellulolyticus, has an optimum temperature of 50-60°C, an optimum pH of 4-5, and an FPU activity of 269 FPU / g-powdered enzyme.

[0090] Using these enzymes, saccharification experiments were carried out using Bemcot (cellulose fiber) and untreated coffee grounds as substrates. Specifically, as shown in FIG. 16, the enzyme solution with 0.05M citrate buffer (pH 5) and the substrate were placed in a bottle and stirred at 50°C and 140 rpm. The reaction supernatant was taken out into a microtube and the glucose concentration was measured by the glucose oxidase method (GOD). The results are shown in the graphs of FIG. 17 and FIG. 18. In these figures, FIG. 17 shows that 87% of the substrate was saccharified when Bemcot was used as the substrate, the substrate concentration was 40 g / L, and 6.6 FPU / g-substrate. FIG. 18 shows that almost no saccharification was observed when untreated coffee grounds were used as the substrate, the substrate concentration was 50 g / L, and 5 FPU / g-substrate. Thus, while Bemcot was saccharified, the coffee grounds were not saccharified, which is thought to be because the cellulose in the coffee grounds was covered with lignin and other materials and the enzymes did not come into contact with the cellulose. Therefore, we investigated pretreatment of coffee grounds below.

[0091] Here, we carried out enzymatic saccharification experiments under various conditions to determine the optimal conditions. First, we compared the enzyme produced by changing the conditions of (1) pretreatment of the substrate (coffee grounds). Here, we carried out saccharification experiments on three types of substrates: (a) untreated; (b) treated with 3 wt% NaOH; and (c) treated with 6 wt% NaOH after ball milling. When the substrate was untreated, the sugar content was 43.8% and the acid insoluble components were 39.9%. When the substrate was ball milled and then treated with 6 wt% NaOH, the sugar content was 65.5% and the acid insoluble components was 15.5%. The substrate concentration was 50 g / L in both cases, and the enzyme activity was 5 FPU / g-substrate. The results are shown in Figure 19. As shown in this figure, by treating the coffee grounds, the saccharification rate was increased compared to the untreated case.

[0092] Next, (2) a comparison was made between the produced enzyme and a commercially available enzyme (Acremonium cellulase). Here, as in (c) above, a saccharification experiment was conducted in which the substrate (coffee grounds) was ball milled and then treated with 6 wt% NaOH. Again, the substrate concentration was 50 g / L, and the enzyme activity was 5 FPU / g-substrate. The results are shown in Figure 20. As shown in this figure, it was confirmed that the produced enzyme can exert more than twice the saccharification power compared to the commercially available enzyme (Acremonium cellulase).

[0093] From the above, it was found that a culture temperature of around 30°C is appropriate for enzyme production in the case of the fungus species Trichoderma reesei ATCC 56765. In addition, it was confirmed that the moisture content at the start of culture was equivalent to 80% even at a moisture content of 50-60%, when compared with the FPU activity value. As a result, the culture volume and weight can be reduced, and the culture equipment can be scaled down.

[0094] It was also confirmed that changes in the C / N ratio at the start of main cultivation had no effect on the FPU activity value. This can reduce the cost of nutrient sources. Furthermore, it was confirmed that the number of days required to reach the maximum FPU activity value was shortened by combining Trichoderma reesei ATCC 56765 and Aspergillus oryzae ATCC12892 as a combination of bacterial species, resulting in a synergistic effect. Furthermore, it was confirmed that high-temperature, high-pressure steam treatment and crushing treatment as pretreatments of PS were effective in improving the FPU activity value. In particular, a maximum FPU activity value of 0.926 FPU / g-dryPS was achieved.

[0095] Furthermore, it was confirmed that pre-treatment of coffee grounds for enzymatic saccharification could advance saccharification more than untreated coffee grounds. In particular, the produced enzyme achieved a saccharification rate that was approximately twice that of a commercially available enzyme (Acremonium cellulase).

[0096] As described above, in the method for saccharification of a substrate, in which the enzyme produced in an enzyme-producing medium (enzyme-producing substrate) is used to enzymatically saccharify the target substrate, it is preferable to include a step of treating the target substrate with alkali. For example, after crushing the substrate, hydrolysis is promoted by dissolving lignin with NaOH. The concentration of NaOH is preferably 3 wt% to 10 wt%. [Example 2: Wheat straw]

[0097] In the above example, PS was used as a substrate for the medium in which cellulase was produced by solid culture. However, the present invention does not limit the substrate to PS, and other substrates can be used. Next, a comparison example will be given between the use of wheat straw (WS) as a substrate and the use of paper sludge (PS).

[0098] Here, cellulase was produced by solid culture using unused cellulosic biomass, and in order to explore the culture conditions that show high activity, the cases of using PS and wheat straw as substrates were compared. As mentioned above, the strain used was Trichoderma reesei ATCC 56765, which has high enzyme productivity among the Trichoderma sp., which produces many highly active cellulases. The experimental conditions were generally similar to those of Example 1 described above. That is, 5 ml of a mixture of Mandel medium and pre-cultured bacteria was added. The C / N ratio was 53.3 for PS and 38.76 for WS. The substrates (PS and WS) were treated with a cutter mill for 30 seconds. The substrates after milling were further sterilized by steam at 121°C for 20 minutes and sterilized by dry heat until completely dry. The dry weight was 5 g. However, the culture temperature was 30°C. A comparison was also made between cases where stirring was performed during culture and cases where it was not performed. Furthermore, as aeration conditions, a comparison was made between a culture vessel (100 mL Erlenmeyer flask) plugged with silicosene and covered with aluminum foil.

[0099] Specifically, in the pre-culture, the same Mandel medium and carbon source as in Example 1 were molded, and the bacterial strain was applied and cultured at 30° C. for 48 hours with shaking at 180 rpm.

[0100] In the main culture, PS with an average particle size of 800 nm to 800 μm (Figure 21) and WS with an average particle size of 100 μm to 4 mm (Figure 22) were used as substrates, which were steam sterilized and dry heat sterilized. Here, PS had a cellulose ratio of 52.2%, protein ratio of 8.0%, and ash ratio of 8.0%. WS had a cellulose ratio of 37.9%, protein ratio of 1.4%, and ash ratio of 7.5%. These substrates were introduced into Erlenmeyer flasks. On the other hand, the pre-cultured solution of 0.97 g / 100 mL of bacterial cells obtained in the pre-culture was centrifuged, the bacterial cells were extracted, and 5 mL of nutrient source was added, and the resultant was introduced into an Erlenmeyer flask. The cells were cultured in this state, and samples were taken every day to measure FPU. The initial moisture content was 62%.

[0101] After the bacteria were grown in the medium in this way, the enzyme was extracted. Here, 0.05M pH5 citrate buffer was added, and 20 mL of PS and 50 mL of WS were shaken for about 1 hour. Then, the mixture was centrifuged at 3500 rpm for 15 minutes, and the filter paper dissolving activity (FPU) of the supernatant enzyme solution was measured. The FPU measurement was performed in the same manner as in Example 1 described above. (1: Comparison with and without stirring)

[0102] First, the results of a comparison of the presence and absence of stirring during the main culture are shown in the graph in Figure 23. The stirring here is performed by stirring the Erlenmeyer flask about five times with a medicine spoon once every 24 hours. The substrate with stirring is shown in Figure 24, and the substrate without stirring is shown in Figure 25. The culture conditions here are PS as the substrate, 30°C as the culture temperature, and a 100mL Erlenmeyer flask as the culture vessel, plugged with silicosene. Figure 23 shows that the FPU value was higher overall without stirring during the main culture. The FPU hardly increased with stirring. The maximum FPU with stirring was 0.25FPU / g-dryPS on the 7th day. The maximum FPU without stirring was 0.79FPU / g-dryPS on the 8th day. Figures 24 and 25 also show that there is almost no difference in the appearance of PS with and without stirring. (2: Comparison by substrate)

[0103] Next, the results of a comparison based on the difference in substrate are shown in the graph in Figure 26. The culture conditions used here were PS or WS as the substrate. The culture temperature was 30°C, and there was no stirring during culture. The culture vessel used was a 100mL Erlenmeyer flask, plugged with silicosene. Photographs of PS and WS are shown in Figure 27 and Figure 28, respectively.

[0104] As shown in Figure 26, the FPU of PS increased from the second to third day, reaching a maximum on the eighth day, but then decreased. As shown in Figure 27, the whole was ochre-colored with a white surface. On the other hand, the FPU of WS increased rapidly from the third to fourth day. As shown in Figure 28, the whole was whitish, and bacterial growth was apparent. Comparing the FPU activity values, WS was higher than PS. (3: Comparison by ventilation conditions)

[0105] Next, a comparison was made under aeration conditions. The culture conditions were WS as the substrate and the culture temperature was 30°C. No stirring was performed during the culture. The culture vessel was a 100mL Erlenmeyer flask, and a comparison was made between one plugged with silicosene as shown in Figure 29 and one covered with aluminum foil as shown in Figure 30. The results are shown in the graph in Figure 31. Figure 32 shows a photograph of a WS plugged with silicosene, and Figure 33 shows a WS covered with aluminum foil. As shown in Figure 31, in the case of aluminum foil, the FPU rose sharply on the fourth day. Overall, the results showed that the FPU value was higher for aluminum foil than for silicosene. Furthermore, as shown in Figures 32 and 33, both silicosene and aluminum foil turned white overall, and the growth of bacteria was observed. (Enzymatic saccharification experiment)

[0106] Furthermore, the saccharification power of the produced enzyme was compared with that of the commercial enzyme to confirm the usability of the produced enzyme. Here, (A) untreated wheat straw and (B) wheat straw treated with 3% NaOH in an autoclave at 121°C for 30 minutes were used as substrates. The untreated wheat straw was 37.9% cellulose and 22.8% lignin, while the NaOH-treated wheat straw was 53.6% cellulose and 6.3% lignin.

[0107] The commercially available enzyme used was Meicelase manufactured by MeijiSeica Pharma Co., Ltd. Meicelase is derived from Trichoderma viride, with an optimum temperature of 50-60°C, an optimum pH of 5.0, and an FPU activity of 126 FPU / g-powdered enzyme. The production enzyme was cultured for 6 days under the following conditions for enzyme production: substrate used was WS, culture temperature was 30°C, no stirring during culture, and aeration was performed using aluminum foil.

[0108] These enzymes were applied to the substrate, and the mixture was shaken at 50° C. and 160 rpm, after which the amount of glucose was quantified using the GOD method.

[0109] In carrying out such enzymatic saccharification experiments, the following combinations of substrates and enzymes were used. (a) Untreated wheat straw was saccharified using a commercial enzyme at 6.7 FPU / g-dry substrate. This confirmed that untreated wheat straw was not saccharified using the commercial enzyme. (b) The produced enzyme was applied to NaOH-treated wheat straw and saccharified at 6.7 or 11 FPU / g-dry substrate. (c) NaOH-treated wheat straw was saccharified using commercial enzymes at 6.7 or 11 FPU / g-dry substrate.

[0110] The results are shown in Figures 34 and 35 as glucose concentration versus reaction time. Figure 34 shows the results for 6.7 FPU / g-substrate with substrate concentrations of 50 g / L (untreated) and 30.7 g / L (NaOH treatment), while Figure 35 shows the results for 11 FPU / g-substrate with substrate concentration of 25 g / L (NaOH treatment). Figure 34 reveals that saccharification of untreated wheat straw does not proceed with either enzyme. In addition, the saccharification rate at 6.7 FPU / g-substrate was inferior to that of the commercial enzyme. On the other hand, as shown in Figure 35, the saccharification rate at 11 FPU / g-substrate, where the amount of enzyme added to the substrate was increased, was comparable to that of the commercial enzyme, confirming that sufficient efficacy was obtained.

[0111] From the above, the usefulness of solid-state culture for cellulase production by T. reesei using WS was confirmed by comparing the culture temperature, agitation, substrate, and aeration conditions using solid-state culture. In addition, the maximum FPU activity value obtained by solid-state culture was 2.85 FPU / g-dryWS [μmol / min / g-dryWS].

[0112] For reference, as reported examples of liquid culture, 0.0050 FPU / g-dryPS was obtained for PS from Trichoderma reesei NBRC 31329, and 0.0082 FPU / g-dryWS was obtained for WS from Trichoderma reesei ATCC 56765. In comparison, the enzyme production method using the solid culture method according to this embodiment achieved a significantly higher FPU per dry substrate, confirming its superiority.

[0113] As described above, we focused on filamentous fungi, which often produce strong cellulases, as cellulase-producing fungi. There are liquid culture and solid culture methods for cellulase production by filamentous fungi, and the simple liquid culture method is generally used. In contrast, in this embodiment, cellulase can be efficiently produced using a solid culture method that does not require the power to shake a heavy medium, and is often used to produce high-potency enzymes. Also, unlike conventional liquid culture, there is no need for the power of aeration to shake a heavy medium, and production with low energy consumption is realized. Furthermore, rupture of mycelia due to shaking can be avoided, and high production of enzymes can be expected. In this way, high-quality cellulase enzymes can be produced inexpensively by solid culture. [Industrial Applicability]

[0114] According to the enzyme production method and enzyme production apparatus, and the method for saccharifying cellulosic biomass resources of the present invention, cellulase can be produced using cellulosic biomass resources such as paper sludge and wheat straw, thereby reducing the environmental burden by making effective use of industrial waste. [Explanation of symbols]

[0115] 100...Enzyme production device 1. Tank 2…Production bacteria application section 3…Duct

Claims

1. A method for producing an enzyme, comprising inoculating an enzyme-producing fungus into a substrate containing cellulose to produce an enzyme, comprising the steps of: forming a granular substrate comprising cellulose; a step of inoculating the substrate with an enzyme-producing bacterium and producing the enzyme while culturing the enzyme-producing bacterium under aerobic conditions on a solid state; Including, In the step of forming the granular substrate, in addition to the granular substrate, 1% or more of a needle-like or fibrous substrate is contained in order to increase voids between the granular substrate so that air can be distributed throughout the accumulated granular substrate, The method for producing an enzyme, wherein the bulk density of the molded substrate is 5.0 g / 100 mL to 25.0 g / 100 mL in a dry state.

2. A method for producing the enzyme according to claim 1, comprising the steps of: A method for producing an enzyme, wherein the enzyme is a cellulase.

3. A method for producing the enzyme according to claim 1 or 2, comprising the steps of: The method for producing an enzyme, wherein the step of shaping the substrate comprises shaping and / or grinding the substrate into granules.

4. A method for producing the enzyme according to any one of claims 1 to 3, comprising the steps of: The method for producing an enzyme, wherein the enzyme-producing fungus to be inoculated onto the substrate is a filamentous fungus.

5. A method for producing the enzyme according to any one of claims 1 to 4, comprising the steps of: A method for producing an enzyme, wherein the substrate has an average particle size of 0.8 μm to 1000 μm.

6. A method for producing the enzyme according to any one of claims 1 to 5, The substrate is Cellulose ratio 30% to 70%, Protein 1% to 10%, Ash content 0% to 10% A method for producing an enzyme.

7. A method for producing the enzyme according to any one of claims 1 to 6, comprising the steps of: The method for producing an enzyme, wherein the moisture content of the substrate after molding is 20% to 60%.

8. A method for producing the enzyme according to any one of claims 1 to 7, comprising the steps of: A method for producing an enzyme, wherein the step of solid-state culturing the enzyme with the substrate is carried out without stirring.

9. A method for producing the enzyme according to any one of claims 1 to 8, comprising the steps of: A method for producing an enzyme, wherein the step of molding the substrate comprises a step of treating the substrate with high-temperature steam and then sterilizing it with dry heat.

10. A method for producing the enzyme according to any one of claims 1 to 9, comprising the steps of: A method for producing an enzyme, wherein the enzyme-producing fungus is at least one of the fungi of the genus Trichoderma, Aspergillus, Lamicola, Acremonium and Humicola, or a combination thereof.

11. A method for producing the enzyme according to any one of claims 1 to 10, comprising: The step of solid-state culturing the enzyme produced by the enzyme-producing bacteria comprises: a pre-culture step inoculating the enzyme-producing fungus into a Mandel medium and culturing the fungus; the bacterial cells obtained in the pre-culture are centrifuged from the Mandel medium, and the obtained bacterial cells are inoculated onto the molded substrate and cultured; and A method for producing an enzyme comprising the steps of:

12. A method for producing the enzyme according to any one of claims 1 to 11, The method for producing an enzyme comprises carrying out the step of solid-state culturing of a cellulase enzyme on the substrate in a tank having a breathable filter, wire mesh demister, punched plate or perforated plate disposed on the bottom surface of the tank.

13. A method for producing the enzyme according to any one of claims 1 to 12, further comprising: A method for producing an enzyme comprising the step of extracting the solid-state cultured cellulase enzyme from the substrate.

14. A method for producing the enzyme according to any one of claims 1 to 13, comprising the steps of: A method for producing an enzyme, wherein the substrate comprises wheat straw.

15. A method for producing the enzyme according to any one of claims 1 to 14, comprising the steps of: A method for producing an enzyme, wherein the substrate comprises paper sludge.

16. A method for producing the enzyme according to any one of claims 1 to 15, comprising the steps of: A method for producing an enzyme, wherein the substrate is a mixture of paper sludge and wheat straw.

17. A method for producing the enzyme according to claim 13, comprising the steps of: The method for producing an enzyme, wherein the extracted cellulase enzyme has a maximum FPU activity value of 0.900 FPU / g-dry paper sludge or more.

18. A method for saccharifying a cellulosic biomass resource, comprising saccharifying and decomposing a biomass resource containing cellulose fiber, using an enzyme produced by the method for producing the enzyme according to any one of claims 1 to 17.

19. The method for saccharifying a cellulosic biomass resource according to claim 18, The method for saccharifying a cellulosic biomass resource, wherein the biomass resource is food waste.

20. An enzyme production apparatus for producing cellulase by applying an enzyme-producing bacterium to a substrate containing cellulose, a tank with a filter-like bottom for introducing a granular substrate containing cellulose; a production bacteria application section for applying enzyme-producing bacteria to the granular substrate introduced into the tank, the substrate containing 1% or more of needle-shaped or fibrous substrate in order to increase voids between the granular substrate so that air can be distributed throughout the accumulated granular substrate, and having a bulk density of 5.0 g / 100 mL to 25.0 g / 100 mL in a dry state; a duct for supplying oxygen to the filter-like bottom of the tank; An enzyme production apparatus comprising:

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