WO2018159573A1

WO2018159573A1 - Method for producing saccharifying enzyme and method for oligosaccharide production

Info

Publication number: WO2018159573A1
Application number: PCT/JP2018/007113
Authority: WO
Inventors: 雄介加川; 成利吉田; 紳吾平松; 山田　勝成
Original assignee: 東レ株式会社
Priority date: 2017-02-28
Filing date: 2018-02-27
Publication date: 2018-09-07
Also published as: JPWO2018159573A1

Abstract

Disclosed are: a method for producing a saccharifying enzyme whereby a saccharifying enzyme suitable for xylooligosaccharide production can be easily produced while preventing a decrease in the degree of saturation of dissolved oxygen in a culture liquid; and a method for oligosaccharide production using the saccharifying enzyme. The method for producing a saccharifying enzyme comprises culturing a fungus belonging to the genus Trichoderma in a medium containing 0.8 w/v% or more, in terms of dry weight, of soybean hulls. The method for oligosaccharide production comprises hydrolyzing a biomass, said biomass containing xylan and cellulose, with the cellulase that is obtained by the aforesaid production method.

Description

Method for producing saccharifying enzyme and method for producing oligosaccharide

The present invention relates to a method for producing a saccharifying enzyme by Trichoderma filamentous fungi using soybean hull and a method for producing an oligosaccharide using the same.

Currently, in order to produce various general-purpose chemicals and biofuels using cellulosic biomass as raw materials, methods for decomposing cellulosic biomass and efficiently releasing useful sugars such as oligosaccharides are being searched. One of the decomposition methods is an enzyme decomposition method in which cellulosic biomass is hydrolyzed using cellulase, which is a saccharifying enzyme, and has attracted attention.

In addition to the characteristics of low sweetness and low calories, oligosaccharides have the function of promoting the growth of enteric bacteria and maintaining the state of the intestine in good condition. Yes. In particular, xylo-oligosaccharides and cellooligosaccharides are less susceptible to degradation by acids and digestive enzymes, and thus exert their effects even in trace amounts compared to other oligosaccharides. Xylooligosaccharides are used not only for human food applications but also as an additive to livestock feed.

Xylooligosaccharide is obtained by hydrolyzing xylan, which is one of the constituent components of plants, with a saccharifying enzyme. However, when cellulase is used as a saccharifying enzyme, it is difficult to efficiently produce xylooligosaccharide because cellulase also contains β-xylosidase that degrades xylooligosaccharide. Has been. For example, in Patent Document 1, as a method for producing xylooligosaccharides, cellulose-containing biomass is hydrolyzed with cellulase derived from Trichoderma filamentous fungi, and recovered cellulase with reduced β-xylosidase activity recovered from the hydrolyzate is used. Yes.

In addition, as a method for producing cellulase, there is a method for producing cellulase using Trichoderma filamentous fungi having high cellulase production ability. In Patent Document 2, a Trichoderma filamentous fungus is subjected to gene mutation treatment to obtain a Trichoderma filamentous fungus mutant having high cellulase production ability. However, there is no description about β-xylosidase activity in cellulase obtained from the mutant of Patent Document 2 or production of xylo-oligosaccharides. In addition, Patent Document 2 describes that soy bean can be used in addition to cellulose as a carbon source in a medium for producing cellulase.

Trichoderma filamentous fungi belong to aerobic filamentous fungi that require oxygen for growth, and when cultured in a liquid medium, the viscosity of the medium increases. As the viscosity increases, the distribution of oxygen and nutrients becomes uneven, so when culturing Trichoderma filamentous fungi, the culture solution is agitated or the oxygen supply rate is increased, so that the dissolved oxygen saturation during the culture is increased. It is necessary to maintain the degree above a certain level.

Also, as the culture tank becomes larger, the oxygen transfer capacity coefficient becomes lower. Therefore, in order to keep the dissolved oxygen saturation during the culture at a certain level or more, it is necessary to further increase the number of stirring and the amount of oxygen supply. However, when the number of stirring is increased, there is a problem that large shear damage is given to the bacterial cells, and there is a problem that more energy is required to increase the oxygen supply amount.

As a method for reducing the viscosity of a culture solution, Patent Document 3 discloses a method in which a mycelial elongation inhibitor is added to a medium to form a mycelium form in a pellet form, and Patent Document 4 is a causative substance of viscosity. Disclosed are mutants of filamentous fungi that suppress protein secretion.

On the other hand, in order to increase the amount of protein production using filamentous fungi, a method of adding an inducer that induces protein production is known. For example, Patent Document 5 discloses the production of cellulase as a protein. In order to improve the property, a method of adding cellulose or xylan or the like as an inducer to the medium is disclosed.

Japanese Patent Application No. 2014-222968 JP 2008-271927 A Japanese Patent Laid-Open No. 7-31467 International Publication No. 2012/0275580 JP 2014-150745 A

As described above, in order to obtain a saccharifying enzyme suitable for the production of xylo-oligosaccharide, there is a method of recovering cellulase from the hydrolyzate after hydrolysis of cellulose-containing biomass with cellulase, but there are many steps. Was an issue. In addition, since Trichoderma filamentous fungi with high productivity of saccharifying enzymes are aerobic organisms, there has been a problem that the dissolved oxygen saturation in the culture solution is lowered during culture in a culture tank.

The present inventor has conducted intensive studies to solve the above-mentioned problems. As a result, by culturing Trichoderma filamentous fungi in a medium containing 0.8% w / v or more of soybean hull as a dry weight, the β-xylosidase ratio The inventors have found that a saccharifying enzyme having a very low activity can be obtained, and that the decrease in dissolved oxygen saturation can be suppressed, and have completed the present invention.

That is, the present invention provides the following (1) to (5).
(1) A method for producing a saccharifying enzyme, comprising culturing Trichoderma filamentous fungi in a medium containing 0.8 w / v% or more of soybean hull as a dry weight.
(2) The method for producing a saccharifying enzyme according to (1), wherein the saccharifying enzyme is cellulase.
(3) The β-xylosidase specific activity of the cellulase is 0.1 U / mg protein or less per mg of the cellulase protein as an enzyme activity for degrading 4-nitrophenyl-β-D-xylopyranoside. A method for producing a saccharifying enzyme.
(4) The method for producing a saccharification enzyme according to any one of (1) to (3), wherein the culture is submerged culture.
(5) A method for producing an oligosaccharide, comprising hydrolyzing a biomass containing xylan and cellulose with a cellulase obtained by the production method according to any one of (2) to (4).
(6) The method for producing an oligosaccharide according to (5), wherein the oligosaccharide is a xylooligosaccharide and / or a cellooligosaccharide.

In the present invention, when Trichoderma filamentous fungi are cultured in a medium to which soybean hull is added at 0.8 w / v% or more, oligosaccharides can be efficiently produced from biomass by using a saccharifying enzyme obtained by the culture. Become. Further, by using the method of the present invention, it is possible to maintain a high dissolved oxygen saturation in the culture solution when culturing Trichoderma filamentous fungi.

The Trichoderma filamentous fungus used in the present invention is also referred to as the genus Hypocrea, but is referred to as Trichoderma in the present specification. The Trichoderma filamentous fungi used in the present invention may be any species of the genus Trichoderma, but Trichoderma filamentous fungi having high saccharifying enzyme productivity are preferred. Specifically, Trichoderma reesei, Trichoderma viride, Trichoderma atroviride and Trichoderma longibratitam are preferred. .

The soybean hull used in the present invention is a thin film-like seed coat covering each soybean seed wrapped in soybean straw and is a part of the soybean seed. Soybean hull is contained in about 10 g (dry weight) per 100 g (dry weight) of soybean seeds. Soybean hulls may be produced directly from soybeans or indirectly from okara or defatted soybeans that are discarded during the production of soybean foods. Soybean hull is also referred to as soy bean mill feed or soy hull, and is referred to as soy hull in this specification.

In the present invention, if a medium containing 0.8% w / v or more of soybean hull as a dry weight can be prepared, a soybean-derived material containing soybean hull or a mixture of soybean and soybean-derived material in addition to soybean hull Can be used to prepare a medium. Specific examples of the soybean-derived material include okara, defatted soybean, soybean seed and the like. The dry weight is a value obtained by removing moisture from the fresh weight. Specifically, the soybean hull or the soybean-derived material is left in a dryer at 90 ° C. for 12 hours, and the weight when returned to normal temperature is dried. Weight.

The dry weight of soybean hull contained in soybean or soybean-derived material can be quantified as insoluble dietary fiber contained in soybean or soybean-derived material. In the present invention, the value determined according to the Prosky method is used as the dry weight of soybean hull. A specific method for determining the dry weight of soybean hull by the Prosky method is as follows.

The soybean or soybean-derived material is treated with heat-resistant α-amylase for 30 minutes at pH 6.0 and 95 ° C., and then treated with protease for 30 minutes at pH 7.5 and 60 ° C. Next, this enzyme-treated soybean is treated with amyloglucosidase at pH 4.5 and 60 ° C. for 30 minutes. Subsequently, 95% ethanol corresponding to a volume equivalent to four times the amount used is added and allowed to stand for 1 hour to form a precipitate, and then the precipitate is collected by suction filtration. The collected precipitate is washed with ethanol and acetone, and the washed precipitate is dried at 90 ° C. for 12 hours and then returned to room temperature, and then the dry weight is measured. Since the dried precipitate contains protein derived from soybean, enzyme-derived protein, organic matter, etc. that remains without being decomposed, the protein and ash are separately quantified and subtracted from the dry weight of the precipitate to insoluble food. The fiber amount is calculated. The protein is quantified using the Kjeldahl method for the dried precipitate, and the ash is quantified as the dry weight of soybean hull after the dried precipitate is ashed at 600 ° C.

In the present invention, since soybean hull is used as a saccharifying enzyme inducer, the medium contains nutrients necessary for culturing Trichoderma fungi in addition to soybean hull. In the present invention, the medium composition other than soybean hull for culturing Trichoderma filamentous fungi is not particularly limited as long as the medium composition is such that Trichoderma filamentous fungi can produce saccharifying enzymes. Usually, the medium contains water as a medium, and contains a nitrogen source and trace elements useful for survival or growth of Trichoderma filamentous fungi. Examples of the nitrogen source include at least one selected from corn steep liquor, polypeptone, gravy, and soybean meal. Examples of trace elements include sulfur, calcium, magnesium, zinc, iron, copper, manganese, boron, molybdenum, and the like. Salts of these trace elements (sulfate for sulfur, boric acid for boron, etc.) ) Can be included in the medium. Specific examples include ammonium sulfate, calcium chloride, magnesium sulfate, zinc chloride, iron chloride, copper sulfate, manganese chloride, boric acid, hexaammonium heptamolybdate and the like, and hydrates thereof can also be used. . In addition, since soybean hull functions also as a carbon source, it is not necessary to add a carbon source separately, but carbon sources other than soybean hull may be included. In this case, examples of the carbon source include saccharides. Examples of the saccharide include at least one selected from glucose, xylose, galactose, fructose, cellobiose, lactose, and sucrose. The concentration of the saccharide is preferably 200 g / L or less, more preferably 100 g / L or less, and particularly preferably 50 g / L or less. Further, if desired, an antifoaming agent or a surfactant that is well-known to be added to culture of microorganisms may be added to the medium.

When the soybean hull is added to the medium, the soybean hull may be pretreated. Specific pretreatment methods include drying, steaming, heating, and pulverization.

When the amount of Trichoderma filamentous fungi to be cultured is small, the Trichoderma filamentous fungus is cultured and grown under normal conditions not containing soybean hull, and then the Trichoderma filamentous fungus is grown in a medium containing soybean hull. The main culture may be performed in the medium.

The saccharifying enzyme obtained by the method of the present invention includes cellulase, amylase, invertase, chitinase and the like. In the present invention, cellulase can be produced particularly efficiently.

Cellulase includes various hydrolases, including enzymes having a degrading activity on xylan, cellulose, and hemicellulose. Specific examples include cellobiohydrase (EC 3.2.1.91) that produces cellobiose by hydrolysis of cellulose chains, and endoglucanase (EC 3.2.1.4) that hydrolyzes from the central part of cellulose chains. ), Β-glucosidase that hydrolyzes cellooligosaccharide or cellobiose (EC 3.2.121), xylanase (EC 3.2.1.8) characterized by acting on hemicellulose or especially xylan, xylooligosaccharide And β-xylosidase (EC 3.2.1.37) that hydrolyzes.

By using the method of the present invention, a cellulase having a reduced specific activity of β-xylosidase can be obtained. β-xylosidase is an enzyme that hydrolyzes xylobiose in which two molecules of xylose are β1,4-linked to xylose. That is, when a xylan-containing biomass is hydrolyzed using a saccharifying enzyme with reduced β-xylosidase activity obtained by the method of the present invention, xylobiose hydrolysis is suppressed, and xylo-oligosaccharide can be produced efficiently. Xylooligosaccharides are those having a chain length in which two or more sugars are linked by β-1,4 bonds, and specifically, xylobiose, xylotriose, xylotetraose, xylopentaose, and the like.

In order to further reduce the specific activity of β-xylosidase and increase the amount of xylooligosaccharide accumulated, 4.0 (w / v)% or more of soybean hull is contained in the medium used for culturing Trichoderma filamentous fungi Preferably, it is more preferably 8.0 (w / v)% or more. The upper limit of the content of soybean hull in the medium is not particularly limited, but usually the content of soybean hull is 30 (w / v)% or less, preferably 20 (w / v)% or less, particularly Preferably, it is 10 (w / v)% or less.

The specific activity of β-xylosidase is that p-nitrophenyl β-D-xylopyranoside (4-nitrophenyl β-D-xylopyranoside) is used as a substrate, and 4-nitrophenol produced by enzymatic degradation is increased by increasing the absorbance at 405 nm. taking measurement. Specifically, 10 μL of a culture supernatant diluted 20-fold with distilled water was added to 90 μL of a substrate solution (1 mM 4-nitrophenyl β-D-xylopyranoside, 0.1 M sodium acetate (pH 5.0)) at 30 ° C. Incubate for exactly 30 minutes. Subsequently, 10 μL of a reaction stop solution (2M Na ₂ CO ₃ ) is added and mixed well to stop the reaction, and absorbance at 405 nm is measured. One unit (U) of β-xylosidase activity represents the enzyme activity that liberates 1 μmol of 4-nitrophenol per minute under the reaction conditions of 30 ° C. for 30 minutes.

β-xylosidase specific activity is preferably 0.1 U / mg protein or less, more preferably 0.02 U / mg protein or less, per 1 mg of cellulase protein as activity against p-nitrophenyl β-D-xylopyranoside, More preferably, it is 0.01 U / mg protein or less.

Of the hydrolases contained in the cellulase obtained by the method of the present invention, the specific activity of β-glucosidase is preferably low. β-Glucosidase (BGL) is an enzyme that hydrolyzes cellobiose with β-1,4-linked glucose into glucose. When cellulolyse is hydrolyzed using cellulase with low specific activity of β-glucosidase, hydrolysis of cellooligosaccharide is performed. Since the decomposition reaction does not proceed, cellooligosaccharide can be produced efficiently. Cellooligosaccharides are those having a chain length in which two or more sugars are linked by β1,4 glucoside bonds, and are made of cellulose, which is a constituent component of plants. Because it is not easily affected by acids and enzymes, it reaches the intestines and is used as an additive to health foods for the purpose of improving bowel movements. By using the cellulase of the present invention having a low β-glucosidase specific activity, an oligosaccharide composed of xylooligosaccharide and cellooligosaccharide can be obtained.

The specific activity of β-glucosidase is that p-nitrophenyl β-D-glucopyranoside (4-nitrophenyl β-D-glucopyranoside) is used as a substrate, and 4-nitrophenol produced by enzymatic degradation is increased by increasing the absorbance at 405 nm. taking measurement. Specifically, 10 μL of a culture supernatant diluted 20-fold with distilled water was added to 90 μL of a substrate solution [1 mM 4-nitrophenyl β-D-glucopyranoside, 0.1 M sodium acetate (pH 5.0)] at 30 ° C. Incubate for exactly 10 minutes. Subsequently, 10 μL of a reaction stop solution (2M Na ₂ CO ₃ ) is added and mixed well to stop the reaction, and absorbance at 405 nm is measured. One unit (U) of BGL activity represents the enzyme activity that liberates 1.0 μmol of 4-nitrophenol per minute under the reaction conditions of 30 ° C. for 10 minutes.

The preferred specific activity of β-glucosidase of the present invention is preferably 1.2 U / mg protein or less per mg of cellulase protein as the enzyme activity for degrading p-nitrophenyl β-D-glucopyranoside.

The cellulase obtained by the method of the present invention preferably contains cellobiohydrase, endoglucanase, and xylanase as enzyme components other than those described above.

The protein concentration of cellulase is measured as follows. The culture solution obtained by culturing Trichoderma filamentous fungi by the method of the present invention is centrifuged at 15,000 × g for 10 minutes, and the supernatant is used as a cellulase solution. 5 μL of a cellulase solution diluted to 250 μL of Quick Start Bradford protein assay (Bio-Rad) is added, and the absorbance at 595 nm after standing at room temperature for 15 minutes is measured. Using the bovine serum albumin solution as a standard solution, the protein concentration contained in the saccharifying enzyme solution is calculated based on the calibration curve.

The culture method for culturing Trichoderma filamentous fungi in the present invention is not particularly limited. For example, the culture can be performed by liquid culture using a centrifuge tube, flask, jar fermenter, tank, or solid culture using a plate. it can. Trichoderma filamentous fungi are preferably cultured under aerobic conditions. Among these culture methods, jar fermenters and deep culture in which aeration and agitation are performed in a tank are particularly preferable. Cultivation is performed under conditions where saccharifying enzyme is produced until a recoverable amount of saccharifying enzyme is accumulated. The culture conditions are not particularly limited, but the culture temperature is usually about 25 ° C to 35 ° C, preferably about 25 ° C to 31 ° C. The pH is usually about 3.0 to 7.0, preferably about 4.0 to 6.0. The culture time is usually about 24 to 96 hours, preferably about 36 to 72 hours. The amount of ventilation is usually about 0.1 vvm to 2.0 vvm, preferably about 0.3 vvm to 1.5 vvm, and particularly preferably about 0.5 vvm to 1.0 vvm.

In the method of the present invention, since the soy hull is contained in the medium used for culturing Trichoderma filamentous fungi in an amount of 0.8 w / v% or more as a dry weight, the viscosity during the culturing does not increase. Can be suppressed and the dissolved oxygen saturation can be kept high. When cultivating on a large scale, maintaining a high dissolved oxygen saturation in the medium is effective because it reduces the capacity of the blower, stirring motor, and stirring energy required for aeration.

The dissolved oxygen saturation in the culture solution can be calculated by measuring the oxygen utilization rate in the culture solution. The oxygen utilization rate (mM / L / hr) in the present invention refers to the oxygen consumption rate per liter of culture solution per unit time 24 hours after the start of culture. The specific calculation method is to maintain the culture conditions constant, stop the supply of oxygen 24 hours after the start of culture, and plot the dissolved oxygen (mg / L) value (DO value) every 10 seconds. Then, the slope (A) (unit: DO / sec) is obtained for a plot of three or more points that logarithmically decrease in the curve. The formula for calculating the oxygen utilization rate is as follows.

Oxygen utilization rate (mM / L / hr) = (− A) × (1/32) × 60 × 60 (Formula 1). *

A commercially available DO meter can be used for measuring the DO value. There is no restriction | limiting in particular in the DO meter to be used, What is necessary is just what can measure DO value correctly. Examples include a sealed DO electrode (Able Co., Ltd.) and a dissolved oxygen sensor (Mettler Toledo Co., Ltd.). The DO meter is preliminarily subjected to zero point calibration and span calibration. Zero point calibration is performed using 2% sodium sulfite solution. Span calibration is performed with aeration and agitation in the absence of bacterial cells under the actual culture conditions, and waits until the dissolved oxygen is saturated.After that, confirm that the indicated value of the instrument is stable, and Calibrate according to saturated dissolved oxygen. Moreover, when performing DO measurement by pressurizing the culture tank, it is necessary to perform pressure correction. Furthermore, when the culture tank is large, it is necessary to perform hydrostatic pressure correction. The calculation formula for correction is as follows.

D = DO (1 + α + β) (Formula 2)
D: corrected saturated dissolved oxygen DO: 1 atm, saturated dissolved oxygen in pure water α: gauge pressure (kg / cm ² )
β: Hydrostatic pressure (liquid depth (m) / 10 at the DO meter mounting position).

The dissolved oxygen saturation is determined during the culture period for saturated dissolved oxygen when the pH and temperature are set as culture conditions using a medium that does not contain bacteria, and the saturated state of dissolved oxygen is 100% when aerated with air. The ratio of dissolved oxygen is calculated as dissolved oxygen saturation. Dissolved oxygen (mg / L) represents the concentration of oxygen dissolved in water. Saturated dissolved oxygen refers to dissolved oxygen in a state in which dissolved oxygen is kept constant by aeration and agitation in the absence of bacterial cells under the culture conditions for actual culture. When calculating the dissolved oxygen saturation, the culture conditions such as the aeration conditions are not changed during the culture period. As oxygen demand decreases, dissolved oxygen saturation increases. The method for calculating the dissolved oxygen saturation is as follows.

Dissolved oxygen saturation (%) = (dissolved oxygen during culture) / (saturated dissolved oxygen before start of culture) × 100 (Equation 3)

A commercially available digital rotational viscometer can be used to measure the viscosity of the culture solution. There is no restriction | limiting in particular in the viscometer to be used, What is necessary is just to be able to measure the viscosity of a culture solution correctly. Examples include a digital rotational viscometer DV2T (BROOKFIELD), spindle LV-1 (BROOKFIELD), and the like. The digital rotational viscometer is previously calibrated at 0 point. The viscosity of the culture solution is determined by placing the culture solution in the middle of the culture in a specified container, immersing the spindle in the culture solution and rotating it, and measuring the torque, which is the viscous resistance acting on the spindle at this time. Can be measured. The unit of viscosity is centipoise (cP). One poise is defined as the viscosity at which there is a velocity gradient of 1 cm / sec per cm in the fluid, producing a stress in the magnitude of 1 dyne per cm ² of velocity in a plane perpendicular to the direction of the velocity gradient. The

The saccharifying enzyme obtained by culturing Trichoderma filamentous fungi is used for the hydrolysis reaction of biomass containing xylan. The method for preparing the saccharifying enzyme from the culture solution of Trichoderma filamentous fungi is not particularly limited, but it is preferable to remove the Trichoderma filamentous fungi or treat them so that they do not grow. This is to prevent the glucose produced when hydrolyzing the biomass containing xylan with a saccharifying enzyme from being consumed by the cells. Examples of the method for removing the cells include centrifugation, membrane separation and the like. Examples of the treatment method for preventing the bacterial cells from growing include heat treatment, chemical treatment, acid / alkali treatment, and UV treatment. Thus, the culture solution processed so that a microbial cell may not be removed or grown can be used for hydrolysis of biomass as it is. When it is desired to purify or partially purify the saccharifying enzyme, it can be purified or partially purified by, for example, a filter press method, a centrifugal separation method, a filter filtration method, or the like.

The biomass containing xylan and cellulose of the present invention is not particularly limited, and in addition to plants such as seed plants, fern plants, moss plants, algae and aquatic plants, waste building materials and the like can also be used. Seed plants are classified into gymnosperms and angiosperms, and both can be preferably used. Angiosperms are further classified into monocotyledonous plants and dicotyledonous plants. Specific examples of monocotyledonous plants include bagasse, switchgrass, napiergrass, eliansus, corn stover, corn cob, rice straw, and straw. As specific examples of dicotyledonous plants, beet pulp, eucalyptus, oak, birch and the like are preferably used. Particularly preferred in the present invention is bagasse.

In addition, pretreated biomass may be used as the biomass containing xylan and cellulose. The pretreatment method is not particularly limited, and known methods such as acid treatment, sulfuric acid treatment, dilute sulfuric acid treatment, alkali treatment, hydrothermal treatment, subcritical treatment, fine pulverization treatment, and steaming treatment can be used. Pulp may be used as biomass containing such pretreated xylan.

The reaction conditions for hydrolyzing biomass containing xylan and cellulose by the saccharifying enzyme obtained in the present invention are not particularly limited, but the reaction pH is preferably 3 to 7, more preferably 4 to 6, Preferably it is around 5. The reaction temperature is not particularly limited, but is preferably 40 ° C to 70 ° C.

Oligosaccharide can be efficiently produced by hydrolyzing biomass containing xylan and cellulose with the saccharifying enzyme obtained in the present invention. Examples of the oligosaccharide produced in the present invention include xylooligosaccharides, cellooligosaccharides, fructooligosaccharides, and isomalouloligosaccharides, preferably xylooligosaccharides and / or cellooligosaccharides.

The oligosaccharide produced can be collected by a conventional method, for example, a filtration method using a membrane.

Hereinafter, the present invention will be specifically described with reference to examples.

Reference Example 1 Protein Concentration Measuring Method A commercially available protein concentration measuring reagent (Quick Start Bradford protein assay, manufactured by Bio-Rad) was used. 5 μL of the filamentous fungus-derived cellulase solution diluted to 250 μL of the protein concentration measuring reagent returned to room temperature was added, and the absorbance at 595 nm after standing at room temperature for 5 minutes was measured with a microplate reader. BSA was used as a standard product, and the protein concentration was calculated against a calibration curve.

Reference Example 2 Method for Measuring β-xylosidase Activity 10 μL of enzyme dilution solution was added to 90 μL of 50 mM acetate buffer containing 1 mM p-nitrophenyl-β-xylopyranoside (Sigma Aldrich Japan) and reacted at 50 ° C. for 30 minutes. Thereafter, 10 μL of 2M sodium carbonate was added and mixed well to stop the reaction, and the increase in absorbance at 405 nm was measured. The activity to release 1 μmol of p-nitrophenol per minute was defined as 1U.

Reference Example 3 Method for Measuring β-Glucosidase Activity 10 μL of enzyme diluent was added to 90 μL of 50 mM acetate buffer containing 1 mM p-nitrophenyl-β-glucopyranoside (Sigma Aldrich Japan), and reacted at 50 ° C. for 10 minutes. . Thereafter, 10 μL of 2M sodium carbonate was added and mixed well to stop the reaction, and the increase in absorbance at 405 nm was measured. The activity to release 1 μmol of p-nitrophenol per minute was defined as 1U.

Reference Example 4 Calculation of Dissolved Oxygen Saturation Oxygen supply was stopped 24 hours after the start of culture, and the change over time in the dissolved oxygen content (DO value) of the culture solution was measured every 10 seconds and plotted. The slope of three or more plots that are logarithmically reduced in the plotted curve was determined, the oxygen utilization rate was determined from the slope, and the dissolved oxygen saturation (%) was calculated using Equation 3. As the DO meter, a sealed dissolved oxygen electrode SDOC-12F-L120 (manufactured by Able Co., Ltd.) was used.

Reference Example 5 Quantification of soybean hull by the Prosky method Dry okara (Materis Co., Ltd.) was treated with a heat-resistant α-amylase aqueous solution at pH 6.0 and 95 ° C. for 30 minutes, and then pH 7 with an aqueous protease solution. 5. Treated for 30 minutes at 60 ° C. Next, this enzyme-treated soybean was treated with an amyloglucosidase aqueous solution at pH 4.5 and 60 ° C. for 30 minutes. Subsequently, 95% ethanol corresponding to a volume equivalent to four times the amount used was added and allowed to stand for 1 hour to form a precipitate, and then the precipitate was collected by suction filtration. The obtained precipitate was washed with ethanol and acetone, the washed precipitate was dried at 90 ° C. for 12 hours, and the dry weight was measured. Further, proteins and ash contained in the dried precipitate were quantified, and a value subtracted from the dry weight of the precipitate was used as the dry weight of soybean hull in the following experiment. The protein was quantified by the Kjeldahl method, and the ash was measured by weighing the residue after the dried precipitate was ashed at 600 ° C. From the measured value, 10 g of soybean hull was contained per 100 g of dried okara.

<Reference Example 6> Measurement of viscosity of culture solution In order to measure the viscosity of the sampled culture solution 24 hours after the start of culture, a digital rotational viscometer DV2T and spindle LV-1 (manufactured by BROOKFIELD) were used, and the number of rotations was adjusted. The viscosity (cP) when set to 10 rpm was determined.

Example 1
(1) Pre-culture Trichoderma reesei PC-3-7 strain (ATCC # 66589) spores were diluted with physiological saline to 1.0 × 10 ⁷ / mL, and 1 mL of the diluted spore solution and corn steep liquor : 5.0 (w / v)%, glucose: 2.0 (w / v)%, ammonium tartrate: 0.37 (w / v)%, ammonium sulfate: 0.14 (w / v)%, calcium chloride Dihydrate: 0.03 (w / v)%, magnesium sulfate heptahydrate: 0.03 (w / v)%, zinc chloride: 0.02 (w / v)%, iron (III) chloride Hexahydrate: 0.01 (w / v)%, copper (II) sulfate pentahydrate: 0.004 (w / v)%, manganese chloride tetrahydrate: 0.0008 (w / v) %, Boric acid: 0.0006 (w / v)%, hexaammonium hexamolybdate tetrahydrate: 0.026 ( 100 mL of a preculture solution dissolved in purified water so as to be w / v)%, PE-M (antifoaming agent): 0.01 (w / v)%, Tween 80: 0.01 (w / v)% It put into the flask with a 500 mL baffle, and culture | cultivated for 72 hours on 28 degreeC and 120 rpm conditions with the shaking incubator.

(2) Main culture Soybean hull (manufactured by Shimizu Flour Mills Co., Ltd.) or dried okara of Reference Example 5 (Materis Co., Ltd., containing 10 w / w% soy hull) is added to the following main culture medium, Using a micro jar fermenter (Bio-Jr. 8, manufactured by Biot Co., Ltd.), each of the subcultures was examined. Soybean hull was pulverized as it was with dried okara, using a “home mortar tea powder device whole green tea EU6820P” (manufactured by Panasonic Corporation) and added to the medium.

10 mL of the pre-culture solution of Trichoderma reesei PC-3-7 strain was inoculated into 100 mL of the main culture medium supplemented with soybean hull or dried okara. The final concentration of soybean hull as dry weight in the main culture medium after inoculation with the preculture solution was soybean hull: 8.0 (w / v)%, dried okara: 8.0 (w / v)% (soy hull end) The concentration was 0.8 w / v%). The components of the main culture medium are as follows.

Corn steep liquor: 5.0 (w / v)%, ammonium sulfate: 0.14 (w / v)%, calcium chloride dihydrate: 0.03 (w / v)%, magnesium sulfate heptahydrate: 0.03 (w / v)%, zinc chloride: 0.02 (w / v)%, iron (III) chloride hexahydrate: 0.01 (w / v)%, copper sulfate (II) pentahydrate Japanese: 0.004 (w / v)%, Manganese chloride tetrahydrate: 0.0008 (w / v)%, Boric acid: 0.0006 (w / v)%, Hexammonium hexamolybdate tetrahydrate Japanese: 0.026 (w / v)%, PE-M (antifoaming agent): 0.01 (w / v)%, Tween 80: 0.01 (w / v)%.

As the culture conditions, after inoculating the main culture medium with the preculture solution, deep culture was performed for 96 hours while controlling the pH at 5 at 28 ° C., 900 rpm, and aeration rate of 100 mL / min.

(3) Collection of culture solution 1 mL of the culture solution was collected 96 hours after the start of the culture. The culture was centrifuged at 15,000 × g and 4 ° C. for 10 minutes to obtain a supernatant. The supernatant was filtered through a 0.22 μm filter, and the filtrate was used as a cellulase solution in the following experiments.

(4) Measurement of protein concentration Using the method described in Reference Example 1, the protein concentration of cellulase in the culture solution at 96 hours after the start of culture was measured. As a result, the protein concentration contained in the culture solution obtained using the medium containing soybean hull having a final concentration of 0.8 (w / v)% was 5.60 g / L, and the final concentration was 8.0 (w / v). v) The protein concentration contained in the culture medium obtained by culturing using the medium containing% soybean hull was 4.27 g / L. The results are shown in Table 1.

(5) Measurement of β-xylosidase activity Using the method described in Reference Example 2, the specific activity of β-xylosidase (BXL) contained in the cellulase solution 96 hours after the start of culture was measured. As a result, the BXL specific activity in the cellulase solution obtained by culturing using a medium containing soybean hull at a final concentration of 0.8 (w / v)% was 0.02 U / mg protein, whereas soybean hull was BXL activity and specific activity were not detected at all in the cellulase solution obtained by culturing using a medium containing a final concentration of 8.0 (w / v)%. The higher the content of soybean hull in the culture solution, the lower the specific activity of BXL. The results are shown in Table 1.

(6) Measurement of β-glucosidase activity Using the method described in Reference Example 3, the specific activity of β-glucosidase (BGL) contained in the cellulase solution 96 hours after the start of culture was measured. As a result, the BGL specific activity in the cellulase solution obtained by culturing using the medium containing soybean hull at final concentrations of 0.8 (w / v)% and 8 (w / v)% was about 0.3 U / mg protein. The results are shown in Table 1.

(7) Measurement of dissolved oxygen saturation in culture solution Using the method described in Reference Example 4, the dissolved oxygen saturation over time in the culture solution was measured. As a result, the dissolved oxygen saturation was 60% or more when cultured using a medium containing soybean hulls at final concentrations of 0.8 (w / v)% and 8.0 (w / v)%. The results are shown in Table 1.

(8) Measurement of viscosity of culture solution Using the method described in Reference Example 5, the viscosity of the culture solution over time was measured. As a result, when the soybean hull was cultured using a medium containing a final concentration of 8.0 (w / v)%, the viscosity was 21.3 cP. The results are shown in Table 1.

(9) Production of Xylooligosaccharide and Cellooligosaccharide As biomass containing xylan, a pulp [Arbocel (registered trademark) (J. Rettenmeier & Sohne)] and bagasse were used, and a hydrolysis reaction was performed using each cellulase solution. Take 0.1 g of each biomass in a 2 mL tube, dilute each cellulase solution as protein to 1 mg with 0.1 M sodium acetate buffer (pH 5.0), make up to 1 mL, and increase the biomass. Added to. The mixture was rotated and mixed at 50 ° C. for 24 hours using a rotator. The sample after saccharification reaction is centrifuged at 15,000 xg for 10 minutes at 4 ° C, the supernatant is collected, and 1/10 volume of 1N aqueous sodium hydroxide is added to the volume of the supernatant. The saccharification reaction was stopped.

The xylobiose, xylotriose, cellobiose, and cellotriose concentrations contained in the saccharified solution were quantified by comparison with the standard under the following HPLC conditions.

Column: AQUITY UPLC BEH Amide (manufactured by Water)
Mobile phase A: 80% acetonitrile + 0.1% TFA
Mobile phase B: 30% acetonitrile + 0.1% TFA
Flow rate: 0.12 mL / min.

In 10 minutes, the ratio of mobile phase B was gradually increased so as to reach 0 to 40%, and only in mobile phase A in 10.01 minutes, and analysis was performed up to 20 minutes.

Detection method: ELSD (evaporative light scattering detector)
Temperature: 55 ° C.

The results of the pulp hydrolysis reaction are shown in Tables 2 and 3, and the results of the bagasse hydrolysis reaction are shown in Tables 4 and 5. Regarding xylobiose production, the cellulase solution produced using a medium containing 8.0 (w / v) soybean hull is more preferable than the cellulase solution produced using a medium containing 0.8 (w / v) soybean hull. The accumulated amount at the time of hydrolysis of the pulp was about twice as large as 4.0 g / L, whereas the accumulated amount at the time of hydrolysis of the bagasse was about 2.5 times larger than 1.75 g / L. In addition, xylotriose was not accumulated during pulp hydrolysis, but during bagasse hydrolysis, both cellulase solutions produced using a medium containing soybean hulls 0.8 and 8.0 (w / v)% were reduced to 0.0. An accumulation amount of 2 g / L or more could be confirmed. Regarding cellobiose production, both the cellulase solution produced using a medium containing soybean hull 0.8 and 8.0 (w / v)% was about 9.0 g / L at the time of pulp hydrolysis, and 1. at the time of bagasse hydrolysis. Accumulated amount of 0 g / L or more was confirmed. Cellotriose was about 1.5 g / L at the time of pulp hydrolysis, and the accumulated amount could not be confirmed at the time of bagasse hydrolysis.

Comparative Example 1
(1) Precultured in the same manner as in Example 1.

(2) Main culture Main culture medium components similar to those in Example 1 were added to pulp [Arbocel (registered trademark) (J. Rettenmeier & Sohne)], ground bagasse, alkali-treated ground bagasse, ground corn hull, dried okara of Reference Example 5 ( A culture test was conducted in the same manner as in Example 1 except that each medium was added with a material added by Materis Co., Ltd. The crushed bagasse was a crushed bagasse obtained by pulverizing a solid of bagasse so as to have an average particle size of 100 μm. The alkali-treated ground bagasse is a solid content concentration 30 (w / v)% slurry added with 100 mg of sodium hydroxide per gram of bagasse solids at 180 ° C. for 10 minutes, followed by solid-liquid separation. What was thoroughly washed was designated as alkali-treated bagasse. Then, the alkali-treated bagasse was crushed so as to have an average particle size of 100 μm. The pulverized corn hull was obtained by pulverizing corn hull (Qinhuangdao Ryufu Agricultural Products Processing Co., Ltd.) to an average particle size of 100 μm. Note that corn hull indicates the rind of corn seed.

The final concentration of the additive in the main culture medium is as follows: pulp: 8.0 (w / v)%, ground bagasse: 8.0 (w / v)%, alkali-treated ground bagasse: 8.0 (w / v) %, Ground corn hull: 8.0 (w / v)%, dried okara: 0.5 (w / v)% (0.05 w / v% as the final concentration of soybean hull).

(3) Measurement of protein concentration The protein concentration of the cellulase solution obtained from each culture solution was measured in the same manner as in Example 1. As a result, the protein concentration contained in the culture medium obtained by culturing was about 9.6 g / L when pulp was added without adding soybean hull, and about 2.0 g when crushed bagasse was added. / L, about 4.9 g / L when alkali-treated ground bagasse was added, and about 6.3 g / L when ground corn hull was added. When 0.05 (w / v)% is added as soybean hull, it is 0.77 g / L, the production amount of cellulase is very small, and the cell growth of Trichoderma reesei PC-3-7 strain can be almost confirmed. There wasn't. The results are shown in Table 6.

(4) Measurement of β-xylosidase activity The specific activity of β-xylosidase (BXL) contained in the cellulase solution obtained from each culture solution was measured in the same manner as in Example 1. As a result, the specific activity of BXL with respect to the total protein weight in the cellulase solution obtained by culturing was 0.24 U / mg protein when pulp was added, 0 U / mg protein when ground bagasse was added, and alkali-treated grinding. 0.19 U / mg protein when bagasse is added, 0.20 U / mg protein when ground corn hull is added, 0.20 U / mg protein when 0.05 (w / v)% as soybean hull is added there were. The results are shown in Table 4.

(5) Measurement of β-glucosidase activity In the same manner as in Example 1, the specific activity of β-glucosidase (BGL) contained in the cellulase solution obtained from each culture solution was measured. As a result, the specific activity of BGL with respect to the total protein weight in the cellulase solution obtained by culturing was 0.23 U / mg protein when pulp was added, 0.39 U / mg protein when ground bagasse was added, 0.19 U / mg protein when alkali-treated ground bagasse is added, 0.26 U / mg protein when ground corn hull is added, and 0.30 U / mg when 0.05 (w / v)% is added as soybean hull mg protein. The results are shown in Table 6.

(6) Measurement of dissolved oxygen saturation in culture solution The dissolved oxygen saturation over time in each culture solution was measured in the same manner as in Example 1. As a result, when the culture was carried out using a medium containing pulp, powdered bagasse, alkali-treated ground bagasse, and ground corn hull at a final concentration of 8.0 (w / v)%, the dissolved oxygen saturation decreased to 50% or less. Although dissolved oxygen saturation was 90% when cultured using a medium containing 0.05% (w / v) soybean hull, as described above, almost no growth of cells that consume oxygen was confirmed. It was judged that the fall of the dissolved oxygen was suppressed by this. The results are shown in Table 6.

(7) Measurement of Viscosity of Culture Solution By the same method as in Example 1, the viscosity over time in the culture solution when pulp and ground corn hull were added was measured. As a result, it was 66.4 cP when the pulp was added, and 31.3 cP when the pulverized corn hull was added. The results are shown in Table 7.

(8) Production of Xylooligosaccharide Hydrolysis reaction of pulp and bagasse was performed in the same manner as in Example 1 using each cellulase solution. As a result of saccharifying the pulp using a cellulase solution obtained by culturing added with pulp, alkali-treated ground bagasse, and ground corn hull, no xylooligosaccharide was accumulated in any of the cellulase solutions. Further, as a result of hydrolysis of bagasse, when a cellulase solution obtained by culturing with addition of pulp and alkali-treated ground bagasse was used, the amount of xylooligosaccharide accumulated was about 0.2 g / L. Moreover, when the cellulase solution obtained by the culture | cultivation which added corn hull was used, the accumulation amount of xylooligosaccharide was about 0.1 g / L. In the culture to which 0.05 (w / v)% of ground bagasse and soybean hull were added, a sufficient amount of cellulase necessary for the saccharification reaction could not be obtained. The results of the bagasse hydrolysis reaction are shown in Table 8.

From the results of Example 1 and Comparative Example 1, by culturing Trichoderma filamentous fungi in a medium containing 0.8 w / v% or more of soybean hull as a dry weight, a sufficient amount of saccharifying enzyme with reduced β-xylosidase activity was obtained. It has been found that production is further suppressed and a decrease in dissolved oxygen concentration in the culture medium is also suppressed. Moreover, it became possible to produce xylo-oligosaccharide efficiently by using the cellulase obtained in the Example, and to produce cellooligosaccharide simultaneously.

When the present invention is used, a saccharification enzyme suitable for producing oligosaccharides simply and efficiently from biomass containing xylan and cellulose can be obtained.

Claims

A method for producing a saccharification enzyme, comprising culturing Trichoderma filamentous fungi in a medium containing 0.8 w / v% or more of soybean hull as a dry weight.
The method for producing a saccharifying enzyme according to claim 1, wherein the saccharifying enzyme is a cellulase.
The saccharifying enzyme according to claim 2, wherein the specific activity of β-xylosidase of the cellulase is 0.1 U / mg protein or less per mg of the cellulase protein as an enzyme activity for degrading 4-nitrophenyl-β-D-xylopyranoside. Production method.
The method for producing a saccharifying enzyme according to any one of claims 1 to 3, wherein the culture is a submerged culture.
A method for producing an oligosaccharide, comprising hydrolyzing a biomass containing xylan and cellulose with a cellulase obtained by the production method according to any one of claims 2 to 4.
The method for producing an oligosaccharide according to claim 5, wherein the oligosaccharide is a xylooligosaccharide and / or a cellooligosaccharide.