JP6153341B2 - Method for producing saccharification solution - Google Patents

Description

  The present invention relates to a method for producing a saccharified solution.

  There is known a method for producing ethanol by using lignocellulosic biomass such as rice straw as a substrate, saccharifying the substrate with a saccharifying enzyme, and fermenting the obtained sugar.

  The lignocellulosic biomass has a structure in which lignin is firmly bound to cellulose or hemicellulose. Therefore, in the saccharification, first, the lignocellulosic biomass is pretreated, and a saccharification pretreated product in which the lignin contained in the lignocellulosic biomass is dissociated or the lignocellulosic biomass is swollen is obtained.

  In the present application, the term “dissociation” means that at least a part of the bond between cellulose or hemicellulose and lignin is broken. Further, the term “swelling” means that a void is formed in cellulose or hemicellulose constituting the crystalline cellulose by intrusion of the liquid, or a void is formed inside the cellulose fiber to expand the crystalline cellulose. .

  The pretreatment of the lignocellulosic biomass is performed, for example, by mixing ammonia water with the lignocellulosic biomass as a substrate to form a substrate mixture, and holding the substrate mixture at a temperature of, for example, 25 ° C. for 100 hours. After completion of the pretreatment, the substrate mixture is heated to a temperature equal to or higher than the boiling point of ammonia, for example, 80 ° C., thereby vaporizing and separating ammonia to obtain the pre-saccharification product.

  In saccharification of the pre-saccharification product, it is desirable to obtain not only glucose by saccharification of cellulose but also xylose by saccharification of hemicellulose in order to obtain more sugar. Therefore, conventionally, a method of obtaining a saccharified solution using a thermostable xylanase and a thermostable β-xylosidase as a saccharifying enzyme for saccharification of the hemicellulose is known (see Patent Document 1).

JP 2012-10638 A

  However, a sufficient amount of xylose cannot be obtained by adding a thermostable xylanase and a thermostable β-xylosidase simultaneously to the saccharification pretreatment product obtained by vaporizing and separating ammonia after the pretreatment. There is an inconvenience.

  An object of the present invention is to provide a method for producing a saccharified solution capable of eliminating such disadvantages and obtaining xylose in a high yield from the lignocellulosic biomass.

  The present inventors diligently studied why a sufficient amount of xylose cannot be obtained when a thermostable xylanase and a thermostable β-xylosidase are simultaneously added to the pre-saccharification product after ammonia separation. As a result, the saccharification pretreatment product has a temperature at which ammonia can be vaporized and separated immediately after completion of the pretreatment, and the saccharification pretreatment product at the temperature has the heat resistant β -It was found that when xylosidase was added, the glycation inhibition reaction proceeded rapidly. And it is thought that a part of said substrate is lost as a result of the said saccharification inhibition reaction advancing rapidly.

  Further, in order to avoid the saccharification inhibition reaction, after the temperature of the pre-saccharification product is lowered to a temperature at which the influence of the saccharification inhibition reaction can be ignored, the thermostable xylanase and the thermostable β-xylosidase are added simultaneously. It is possible. However, when this is done, the thermostable xylanase cannot function sufficiently, and a sufficient amount of xylose cannot be obtained.

  Therefore, in order to achieve the above-mentioned object, the present invention maintains a substrate mixture obtained by mixing ammonia water with lignocellulosic biomass as a substrate at a predetermined temperature for a predetermined time, and then vaporizes ammonia. A method for producing a saccharification solution comprising: a step of obtaining a pre-saccharification product by separation; and a step of obtaining a saccharification solution by saccharifying a substrate / saccharification enzyme mixture obtained by adding a saccharification enzyme to the pre-saccharification product In the first temperature range after the ammonia is separated, until the temperature of the saccharification pretreatment product falls to a temperature at which the saccharification inhibition reaction due to the addition of thermostable β-xylosidase falls to a negligible temperature. A thermostable xylanase is added to the pre-saccharification product to obtain a first substrate / saccharification enzyme mixture, and then at least a part of the substrate is saccharified to generate an oligosaccharide. As long as the temperature of the first substrate / saccharifying enzyme mixture falls within a second temperature range from a temperature at which the saccharification inhibiting reaction can be ignored to a temperature at which the thermostable β-xylosidase becomes incapable of functioning. Then, after adding the thermostable β-xylosidase to the first substrate / saccharifying enzyme mixture to obtain a second substrate / saccharifying enzyme mixture, the substrate and the oligosaccharide are saccharified to produce a monosaccharide. And a step of causing the step to occur.

  In the method for producing a saccharified solution of the present invention, first, after pre-treatment, ammonia is vaporized and separated to obtain a pre-saccharification product. The pre-saccharification product has a temperature at which ammonia can be vaporized and separated, and the temperature is a temperature at which the saccharification inhibition reaction proceeds rapidly when the heat-resistant β-xylosidase is added, The thermostable xylanase can function without causing the saccharification inhibition reaction.

  Therefore, after separating the ammonia, while the temperature of the pre-saccharification product is in the first temperature range until the saccharification inhibition reaction due to the addition of the thermostable β-xylosidase falls to a temperature at which it can be ignored, A thermostable xylanase is added to the pre-saccharification product to obtain a first substrate / saccharification enzyme mixture. The thermostable xylanase can saccharify the substrate without causing the saccharification inhibiting reaction even in a high temperature range while the temperature of the pre-saccharification product is in the first temperature range. In addition, since the thermostable xylanase can saccharify the substrate more efficiently at a higher temperature as long as it is within a functional temperature range, the temperature of the pre-saccharification product can ignore the saccharification inhibition reaction. The time and heat during the temperature drop can be used effectively.

  As a result, at least a part of the substrate can be saccharified with the thermostable xylanase to efficiently generate oligosaccharides.

  Next, while the temperature of the first substrate / saccharifying enzyme mixture is in the second temperature range from the temperature at which the saccharification inhibiting reaction can be ignored to the temperature at which the thermostable β-xylosidase becomes incapable of functioning In addition, the thermostable β-xylosidase is added to the first substrate / saccharifying enzyme mixture to obtain a second substrate / saccharifying enzyme mixture.

  The second substrate / saccharifying enzyme mixture contains the remainder of the substrate together with the oligosaccharide, while the saccharifying enzyme includes both the thermostable xylanase and the thermostable β-xylosidase. Here, the thermostable xylanase can also function in the second temperature range.

  Therefore, in the second substrate / saccharifying enzyme mixture, the thermostable β-xylosidase saccharifies the oligosaccharide produced by the thermostable xylanase in the first temperature range to produce xylose as a monosaccharide. . The thermostable xylanase saccharifies the remainder of the substrate to produce a new oligosaccharide, and the oligosaccharide is also saccharified by the thermostable β-xylosidase to produce xylose as a monosaccharide.

  As a result, according to the production method of the present invention, xylose can be obtained in high yield from the lignocellulosic biomass.

  In the production method of the present invention, the first temperature range is, for example, 80 to 50 ° C, and the second temperature range is, for example, 50 to 30 ° C.

  In the production method of the present invention, as the thermostable xylanase, for example, a thermostable xylanase derived from a thermophilic bacterium Thermoascus aurantiacus can be used. In addition, as the thermostable β-xylosidase, for example, a thermostable β-xylosidase derived from a thermophilic bacterium Thermotoga maritima can be used.

The graph which shows the density | concentration of the saccharification solution obtained by saccharifying xylan using a thermostable xylanase and a thermostable beta-xylosidase. The graph which shows the density | concentration of the saccharification solution obtained by the manufacturing method of this invention.

  Next, embodiments of the present invention will be described in more detail with reference to the accompanying drawings.

  In the method for producing a saccharified solution of the present embodiment, first, rice straw is pulverized to a size that passes through a mesh with an opening of 3 mm to obtain lignocellulosic biomass as a substrate.

  Next, ammonia water having a concentration of 25% by mass is mixed with the dry mass of the lignocellulosic biomass in a mass ratio of 1: 1 to obtain a substrate mixture. And the said lignocellulosic biomass is pre-processed by hold | maintaining the said substrate mixture at the temperature of 25 degreeC for 100 hours, for example. As a result of the pretreatment, lignin contained in the lignocellulosic biomass is dissociated or the lignocellulosic biomass is swollen.

  When the pretreatment is completed, the substrate mixture is then heated to a temperature not lower than the boiling point of ammonia, for example, a temperature of 80 ° C., thereby vaporizing and separating the ammonia to obtain a pre-saccharification product. The pre-saccharification product contains the substrate at a concentration of, for example, 10 to 26% by mass with respect to the total amount.

  Next, the first pre-saccharification product temperature is lowered from the temperature of 80 ° C. to a temperature at which saccharification-inhibiting reaction caused by addition of heat-resistant β-xylosidase can be ignored, for example, 50 ° C. While in the temperature range, a thermostable xylanase is added to the pre-saccharification product to obtain a first substrate / saccharification enzyme mixture. The first substrate / saccharifying enzyme mixture contains the thermostable xylanase at a concentration of, for example, 0.015 to 0.5% by mass with respect to the total amount thereof.

  As the thermostable xylanase, for example, those derived from a thermophilic bacterium Thermoascus aurantiacus can be used. Moreover, as said thermostable (beta) -xylosidase, the thing derived from a thermophilic bacterium Thermotoga maritima can be used, for example.

  Since the thermostable xylanase functions more actively as the temperature is higher than the functional temperature range, when the temperature of the pre-saccharification product is as high as possible in the first temperature range, It is preferable to add to the pre-saccharification product.

  Next, the first substrate / saccharifying enzyme mixture is held in the first temperature range for a time in the range of 12 to 36 hours, for example, 24 hours. By doing so, a part of the substrate contained in the first substrate / saccharifying enzyme mixture is saccharified with the thermostable xylanase to generate xylobiose as an oligosaccharide.

  Next, when the temperature of the first substrate / saccharifying enzyme mixture falls to a temperature at which the saccharification inhibiting reaction can be ignored, for example, 50 ° C., the thermostable β- Xylosidase is added to obtain a second substrate / saccharifying enzyme mixture. The thermostable β-xylosidase is in a second temperature range until the temperature of the first substrate / saccharifying enzyme mixture falls to a temperature at which the thermostable β-xylosidase becomes inoperable, for example, a temperature of 30 ° C. In the meantime, it is added to the first substrate / saccharifying enzyme mixture.

  Next, the second substrate / saccharifying enzyme mixture is held in the second temperature range for a time in the range of 24 to 72 hours, for example, 48 hours. By doing so, xylobiose as an oligosaccharide contained in the second substrate / saccharifying enzyme mixture is saccharified by the thermostable β-xylosidase to produce xylose as a monosaccharide.

  In addition, the second substrate / saccharifying enzyme mixture includes the remainder of the substrate that has not been saccharified into oligosaccharides by the thermostable xylanase among the substrates contained in the first substrate / saccharifying enzyme mixture, Contains xylanase. Therefore, the remainder of the substrate is saccharified into oligosaccharides by the thermostable xylanase while the second substrate / saccharifying enzyme mixture is maintained in the second temperature range, and the oligosaccharides are further converted into the thermostable compounds. Saccharified by sex β-xylosidase to produce xylose as a monosaccharide.

  Therefore, according to the method for producing a saccharified solution of the present embodiment, xylose can be obtained in a high yield from the lignocellulosic biomass, and a saccharified solution containing a high concentration of xylose can be obtained.

  Next, examples and comparative examples of the present invention are shown.

[Example 1]
In this example, a saccharification solution was produced using a 5% by mass xylan (Sigma Aldrich) aqueous solution as a model of the pre-saccharification product.

  In this example, first, the aqueous xylan solution is heated to a temperature of 75 ° C. corresponding to the first temperature range, and a thermostable xylanase derived from a thermophilic bacterium Thermoascus aurantiacus is added so that the final concentration becomes 0.1% by mass. Added and held at temperature for 24 hours. The thermostable xylanase was prepared by concentrating a culture solution of xylanase-producing koji mold constructed in-house. The thermostable xylanase was constructed as follows.

  First, using the endoxylanase gene derived from the thermophilic bacterium Thermoascus aurantiacus of SEQ ID NO: 1 and the endoxylanase gene derived from the thermophilic bacterium Thermoascus aurantiacus (manufactured by Takara Bio Inc.) as a template, the primer of SEQ ID NO: 2 and DNA polymerase ( PCR amplification was performed using Toyobo Co., Ltd. (trade name: KOD-plus). The obtained PCR amplification product was purified using a PCR purification kit (trade name: QIAquick PCR purification kit, manufactured by QIAGEN) to obtain an endoxylanase gene EX.

  Next, a nitrate reductase gene derived from Aspergillus oryzae was PCR-amplified using the genomic DNA gene of Aspergillus oryzae RIB40 strain as a template and the primers of SEQ ID NOs: 4 and 5 and the DNA polymerase. The obtained PCR amplification product and plasmid vector pBR322 (manufactured by Takara Bio Inc.) were treated with restriction enzymes AvaI and NdeI at 37 ° C., and then cut out by agarose gel electrophoresis. The cutting was performed using a gel extraction kit (trade name: QIAquick Gel Extraction Kit, manufactured by QIAGEN).

  Next, the PCR amplification product and the plasmid vector pBR322 were ligated using a DNA ligation kit (manufactured by Takara Bio Inc.). E. coli strain JM109 was transformed. As a result, plasmid pBR-niaD was obtained.

  Next, the agdA terminator gene derived from Aspergillus was PCR amplified using the genomic DNA gene of Aspergillus oryzae RIB40 strain as a template and using the primers of SEQ ID NOs: 6 and 7 and the DNA polymerase. The obtained PCR amplification product and the plasmid pBR-niaD were treated with restriction enzymes SalI and AvaI at 37 ° C., and then cut out by agarose gel electrophoresis. The excision was performed using the gel extraction kit.

  Next, the PCR amplification product and the plasmid pBR-niaD were ligated using the DNA ligation kit. E. coli strain JM109 was transformed. As a result, plasmid pBR-TagdA-niaD was obtained.

  Next, the enoA promoter gene derived from Neisseria gonorrhoeae was PCR amplified using the genomic DNA gene of Neisseria gonorrhoeae RIB40 as a template and using the primers of SEQ ID NOs: 8 and 9 and the DNA polymerase. The obtained PCR amplification product and the plasmid pBR-TagdA-niaD were treated with restriction enzymes NheI and SalI at 37 ° C., and then cut out by agarose gel electrophoresis. The excision was performed using the gel extraction kit.

  Next, the PCR amplification product and the plasmid pBR-TagdA-niaD were ligated using the DNA ligation kit. E. coli strain JM109 was transformed. As a result, plasmid pBR-PenoA-TagdA-niaD was obtained.

  Next, the plasmid pBR-PenoA-TagdA-niaD was treated with the restriction enzyme SmaI at 30 ° C. and then cut out by agarose gel electrophoresis. The excision was performed using the gel extraction kit. As a result, a restriction enzyme SmaI-treated fragment of the plasmid pBR-PenoA-TagdA-niaD was obtained.

  Next, the restriction enzyme SmaI-treated fragment of the plasmid pBR-PenoA-TagdA-niaD and the endoxylanase gene EX were ligated using a directional cloning kit (Clontech, trade name: In-Fusion HD Cloning Kit). As a result, the plasmid pBR-PenoA-EX-TagdA-niaD of SEQ ID NO: 10 was obtained as a vector for expressing the endoxylanase bacilli. Next, the plasmid pBR-PenoA-EX-TagdA-niaD was transformed into Stellar Competent Cells (manufactured by Clontech) to obtain an endoxylanase Escherichia coli transformed strain. Next, the obtained endoxylanase E. coli transformed strain was cultured overnight at 37 ° C. and 180 rpm in an LB medium containing 100 μg / mL ampicillin, and a miniprep kit (trade name: QIAquick, manufactured by QIAGEN) was obtained from the resulting culture. The plasmid pBR-PenoA-EX-TagdA-niaD was prepared in a large amount using Miniprep Kit).

  Next, the Aspergillus oryzaenia D300 strain obtained from the National Liquor Research Institute was transformed using the plasmid pBR-PenoA-EX-TagdA-niaD according to a standard method by the PEG-calcium method. Next, a Czapek-Dox medium (3 mass / volume% dextrin, 0.1 mass / volume% potassium dihydrogen phosphate, 0.2 mass / volume% potassium chloride, 0.05 mass / volume%) A strain capable of growing with magnesium sulfate (0.001 mass / volume% iron sulfate, 0.3 mass / volume% sodium nitrate) was selected to obtain an endoxylanase bacilli transformed strain.

Next, the endoxylanase bacilli transformed strain was sporulated in the tourpex medium, and the spores were collected with sterile water. Next, in a 500 mL Erlenmeyer flask, PD liquid medium (2 mass / volume% dextrin, 1 mass / volume% polypeptone, 0.1 mass / volume% casamino acid, 0.5 mass / volume% potassium dihydrogen phosphate,. (05 mass / volume% magnesium sulfate, 0.1 mass / volume% sodium nitrate) (100 mL) was taken, and the spores were inoculated to a final spore concentration of 1 × 10 ⁴ / mL. Next, liquid culture was performed at 30 ° C. for 3 days to obtain a culture solution of xylanase-producing gonococci in which endoxylanase, the target gene, was secreted and expressed in the medium.

  Next, the xylan aqueous solution to which the thermostable xylanase was added was cooled, and the temperature was lowered from 75 ° C. to 30 ° C. corresponding to the second temperature range over 30 minutes. Then, a thermostable β-xylosidase derived from the thermophilic bacterium Thermotoga maritima is added at the temperature of 30 ° C. so that the final concentration is 0.1% by mass, and the saccharified solution is maintained at the temperature for 47.5 hours. Got.

  The thermostable β-xylosidase was prepared by treating a culture solution of β-xylosidase-producing Escherichia coli constructed in-house with a protein extraction reagent (trade name: BugBuster, manufactured by Merck) and concentrating the obtained protein extract. . The thermostable β-xylosidase was constructed as follows.

  First, a β-xylosidase gene BX derived from the thermophilic bacterium Thermotoga maritima of SEQ ID NO: 11 was used as a template with the genomic DNA of the thermophilic bacterium Thermotoga maritima obtained from American Type Culture Collection (ATCC), and primers of SEQ ID NOS: 12 and 13; PCR amplification was performed using DNA polymerase (manufactured by Toyobo Co., Ltd., trade name: KOD-plus). The obtained PCR amplification product was purified using a PCR purification kit (QIAGEN, trade name: QIAquick PCR purification kit).

  Next, the obtained PCR amplification product and plasmid vector pET28 (a) (manufactured by Novagen) were treated with restriction enzymes NcoI and NotI at 37 ° C., and then cut out by agarose gel electrophoresis. The cutting was performed using a gel extraction kit (trade name: QIAquick Gel Extraction Kit, manufactured by QIAGEN). As a result, plasmid pET28 (a) -BX of SEQ ID NO: 14, which is a restriction enzyme-treated fragment of the plasmid vector pET28 (a) and the β-xylosidase gene BX derived from the thermophilic bacterium Thermotoga maritima, was obtained.

  Next, the plasmid pET28 (a) -BX was ligated using DNA ligation Kit (manufactured by Takara Bio Inc.). E. coli strain BL21 was transformed. As a result, a transformant was obtained in which the β-xylosidase gene BX derived from the thermophilic bacterium Thermotoga maritima was introduced between the restriction enzymes NcoI and NotI of the plasmid pET28 (a) -BX-pET28 (a).

  Next, the transformant was cultured overnight in an LB medium containing 30 μg / mL kanamycin and 50 μg / mL chloramphenicol, and 100 μL of the obtained culture was inoculated into the LB medium, and 2 ° C. at 37 ° C. Incubate for hours. Next, isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM and further cultured for 2 hours to obtain a culture solution of the β-xylosidase-producing Escherichia coli.

  Next, the concentrations of xylobiose and xylose in the saccharified solution obtained in this example were quantified by high performance liquid chromatography. The results are shown in FIG.

[Comparative Example 1]
In this comparative example, except that the xylan aqueous solution was heated to a temperature of 75 ° C. corresponding to the first temperature range, the thermostable xylanase and the thermostable β-xylosidase were added simultaneously, and the temperature was maintained for 72 hours. A saccharification solution was obtained in exactly the same manner as in Example 1.

  Next, the concentrations of xylobiose and xylose in the saccharified solution obtained in this comparative example were quantified by high performance liquid chromatography. The results are shown in FIG.

[Comparative Example 2]
In this comparative example, except that the aqueous xylan solution was heated to a temperature of 30 ° C. corresponding to the second temperature range, the thermostable xylanase and the thermostable β-xylosidase were added simultaneously, and the temperature was maintained for 72 hours. A saccharification solution was obtained in exactly the same manner as in Example 1.

  Next, the concentrations of xylobiose and xylose in the saccharified solution obtained in this comparative example were quantified by high performance liquid chromatography. The results are shown in FIG.

  FIG. 1 clearly shows that according to the production method of this example, xylose can be obtained in a high yield and a saccharification solution containing a high concentration of xylose can be obtained. In contrast, in the production method of Comparative Example 1 in which the thermostable xylanase and the thermostable β-xylosidase were simultaneously added at a temperature of 75 ° C. corresponding to the first temperature range, the saccharification solution obtained in Example 1 In contrast, the xylose concentration is clearly lower. This is presumably because the heat-resistant β-xylosidase was added at a temperature corresponding to the first temperature range, so that a saccharification inhibition reaction occurred and a part of the substrate (xylan) was lost.

  Further, in the production method of Comparative Example 2 in which the thermostable xylanase and the thermostable β-xylosidase were simultaneously added at a temperature of 30 ° C. corresponding to the second temperature range, the saccharification concentration obtained in Comparative Example 1 was used as the xylose concentration. Although it is high compared with the solution, it is clear that it does not reach the saccharification solution obtained in Example 1. This is presumably because the thermostable xylanase did not function sufficiently because the thermostable xylanase was added at a temperature corresponding to the second temperature range, although the saccharification inhibition reaction could be avoided.

[Example 2]
In this example, first, rice straw was pulverized to a size passing through a mesh with an opening of 3 mm to prepare lignocellulosic biomass as a substrate. Next, with respect to the dry mass of the lignocellulosic biomass, ammonia water having a concentration of 25% by mass was mixed at a mass ratio of 1: 1 to obtain a substrate mixture. Then, the substrate mixture is kept at a temperature of 25 ° C. for 100 hours to pretreat the lignocellulosic biomass, dissociate lignin contained in the lignocellulosic biomass, or swell the lignocellulosic biomass. It was.

  Next, the substrate mixture after the pretreatment was heated to a temperature of 80 ° C. that is equal to or higher than the boiling point of ammonia to vaporize and separate the ammonia to obtain a pre-saccharification product. The saccharification pretreatment product contained the substrate at a concentration of, for example, 20% by mass relative to the total amount.

  Next, when the temperature of the substrate mixture drops to a temperature of 75 ° C. corresponding to the first temperature range, the same as that used in Example 1 so that the final concentration is 0.15% by mass. Thermostable xylanase was added to obtain a first substrate / saccharifying enzyme mixture. The first substrate / saccharifying enzyme mixture was held at the temperature of 75 ° C. for 24 hours.

  Next, the first substrate / saccharifying enzyme mixture was cooled, and the temperature was lowered from 75 ° C. to 30 ° C. corresponding to the second temperature range over 30 minutes. Then, at the temperature of 30 ° C., the same thermostable β-xylosidase as that used in Example 1 was added so that the final concentration was 0.15% by mass. At the same time, a culture solution of Acremonium cellulolyticus was added as a cellulolytic enzyme so that the final concentration was 0.5% by mass to obtain a second substrate / saccharifying enzyme mixture. And the saccharification solution which hold | maintained the said 2nd substrate and saccharifying enzyme mixture for 47.5 hours at the said 30 degreeC temperature was obtained.

  Next, the cellulose and xylose concentrations in the saccharified solution obtained in this example were quantified by high performance liquid chromatography. The results are shown in FIG.

  From FIG. 2, it is clear that according to the production method of this example, a saccharification solution containing high concentration of xylose together with glucose as a monosaccharide can be obtained.

Claims (1)

A step of obtaining a pre-saccharification product by vaporizing and separating ammonia after pre-treating a substrate mixture obtained by mixing ammonia water with lignocellulosic biomass as a substrate at a predetermined temperature for a predetermined time;
In a method for producing a saccharification solution, comprising a step of saccharifying a substrate / saccharification enzyme mixture obtained by adding a saccharification enzyme to the pre-saccharification product,
After the ammonia is separated, the temperature of the pre-saccharification product is within the first temperature range until the saccharification inhibition reaction due to the addition of thermostable β-xylosidase falls to a temperature at which it can be ignored. Adding a thermostable xylanase to the treated product to obtain a first substrate / saccharifying enzyme mixture, and then saccharifying at least a part of the substrate to produce an oligosaccharide;
While the temperature of the first substrate / saccharifying enzyme mixture is in a second temperature range from a temperature at which the saccharification inhibiting reaction can be ignored to a temperature at which the thermostable β-xylosidase becomes inoperable, Adding the thermostable β-xylosidase to the first substrate / saccharifying enzyme mixture to obtain a second substrate / saccharifying enzyme mixture, and then saccharifying the substrate and the oligosaccharide to produce a monosaccharide; equipped with a,
The first temperature range is in the range of 80-75 ° C, the second temperature range is in the range of 50-30 ° C,
The thermostable xylanase is derived from a thermophilic bacterium Thermoascus aurantiacus,
The heat-resistant β- xylosidase method of manufacturing a glycosylated solution characterized in origin der Rukoto thermophile Thermotoga maritima.