WO2013115305A1

WO2013115305A1 - Method for producing sugar and alcohol from cellulosic biomass, and microorganisms used for same

Info

Publication number: WO2013115305A1
Application number: PCT/JP2013/052170
Authority: WO
Inventors: 渉小笠原; 光中澤; 岡田　宏文; 川口　剛司; 順一炭谷; 修治谷; 康森川; 良則小林
Original assignee: 国立大学法人長岡技術科学大学; 公立大学法人大阪府立大学
Priority date: 2012-01-31
Filing date: 2013-01-31
Publication date: 2013-08-08
Also published as: JP6114200B2; JPWO2013115305A1

Abstract

Obtained are microorganisms belonging to the genus Trichoderma, which express a β-glucosidase gene (bgl1) derived from microorganisms belonging to the genus Aspergillus under the control of a promoter for egl1 of the microorganisms of the genus Trichoderma. It has been discovered that the use of a diastatic enzyme produced by this transformant enables efficient saccharification of cellulosic biomass. It has also been discovered that fermenting the cellulosic biomass thus saccharified enables efficient production of ethanol.

Description

Method for producing sugar and alcohol from cellulosic biomass, and microorganism used in the method

The present invention relates to a method for producing sugar by saccharifying cellulosic biomass, a method for producing ethanol by fermenting sugar produced by the method, and a cellulase-producing bacterium used in these methods.

Numerous attempts have been made to develop ethanol and other chemicals from cellulosic biomass such as agricultural waste and wood that do not compete with food and feed. For example, a method has been developed in which wood is saccharified with sulfuric acid to form a monosaccharide such as glucose or xylose, and then this monosaccharide is fermented with yeast or bacteria to obtain ethanol (Non-Patent Documents 1 to 3). . However, this method has a problem in that it is difficult to recover and reuse the strong acid used, and in terms of the environmental characteristics when treating waste.

Therefore, a method using an enzyme has been developed as a milder and more environmentally friendly saccharification method of biomass. In this method, cellulase and hemicellulase produced by various microorganisms are generally used. Cellulase is composed of enzyme proteins having various mechanisms of action. Enzymes that act on cellulose in biomass include endoglucanase (EG) that acts on amorphous cellulose and breaks the cellulose chain, and sugars in cellobiose units from the ends of crystalline and amorphous cellulose fibers. There is cellobiohydrolase (CBH) to cut out There is also β-glucosidase (BGL) that hydrolyzes cellobiose produced by the action of these enzymes to produce glucose. On the other hand, examples of hemicellulases that act on hemicellulose, which is a biomass component, include xylanase and β-xylosidase. Cellulose biomass saccharifying enzymes include the above-mentioned enzymes, but in reality, each of these enzymes is further composed of a number of components classified by the carbohydrate hydrolase family. Is very complex.

The cellulase-producing bacteria, strains belonging to the genus Trichoderma, e.g., Trichoderma reesei or Trichoderma viride is famous, have been industrially used (Non-patent document 4). However, as research on biomass saccharification using cellulase produced by a strain belonging to the genus Trichoderma progresses, the biggest drawback of this cellulase is that β-glucosidase activity is related to other enzyme activities (endoglucanase activity, cellobiohydrolase activity) Etc.) has been closed up. Accordingly, Cellic CTec, Cellic CTec2 (Novozymes) and Accellerase 1500 (Genencor) have been developed as improved products with enhanced β-glucosidase activity.

However, in order to put bioethanol into practical use, it is necessary to further reduce its production cost. To achieve this purpose, biomass saccharification can be completed in a smaller amount than these improved enzymes. The development of a new high-performance saccharifying enzyme that is possible is strongly desired.

Under such circumstances, the present inventors obtained a gene ( bgl1 ) of β-glucosidase (BGL), which is derived from Aspergillus and exhibits high specific activity on cellobiose, from the gene of xylanase 3 ( xyn3 ) possessed by the microorganism of the genus Trichoderma . A transformant of a microorganism belonging to the genus Trichoderma expressed under the control of a promoter was prepared (Patent Document 1). Traditionally, cellulases produced by Trichoderma microorganisms have been said to have the greatest disadvantage of low BGL activity, but improved cellulases produced by transformants of this Trichoderma microorganism have greatly enhanced BGL activity. In the saccharification of the pretreatment products of various cellulosic biomass, it showed extremely superior saccharification ability compared to various latest commercial cellulases. Moreover, it was shown that the enzyme cost required for saccharification can be reduced by saccharifying various cellulosic biomass using this improved cellulase. Furthermore, the present inventors have also shown that various cellulosic biomasses can be saccharified using the improved cellulase, and ethanol can be produced from the obtained sugars by various microorganisms.

However, in view of the economic principle of pursuing a reduction in ethanol production costs, it is desired to develop a cellulase with higher functionality.

JP 2012-016329 A

This invention is made | formed in view of such a condition, The objective developed the microorganisms which produce the enzyme which has high saccharification ability with respect to cellulosic biomass, and uses the enzyme which the said microorganisms produce. An object of the present invention is to provide a method capable of efficiently saccharifying cellulosic biomass. It is a further object of the present invention to provide a method for producing ethanol by fermenting sugar thus obtained from cellulosic biomass.

In order to develop a cellulase having higher saccharification ability, the present inventors further examined a promoter for expressing a β-glucosidase gene ( bgl1 ) derived from a microorganism belonging to the genus Aspergillus in a microorganism belonging to the genus Trichoderma . . Generally, using the promoter of the enzyme gene such as Trichoderma microorganism of cellobiohydrolase is a major component of the cellulase I (CBHl) or endoglucanase I (EG1), to express foreign genes in Trichoderma microorganism In this case, it is known that the balance of main components is lost and the performance as a cellulase is reduced (Non-patent Document 5).

In order to confirm this point, the present inventors have confirmed that this is a β-glucosidase gene derived from a microorganism belonging to the genus Aspergillus in the promoter of the CBH1 gene ( cbh1 promoter) or the promoter of the EG1 gene ( egl1 promoter) of the Trichoderma reesei PC-3-7 strain. ( Bgl1 ) was ligated, a transformant into which this was introduced was prepared, and the characteristics of the saccharifying enzymes produced by the transformant were compared and evaluated.

As a result, when evaluated by BGL activity was cellobiose as the substrate, whereas the cellulase which is produced using cbh1 promoter, was about 3 times higher compared to production cellulase with egl1 promoter, FPU activity The general cellulolytic activity such as CMC degradation activity and β-xylosidase activity were almost the same (Table 1). Contrary to xylanase activity, those using the egl1 promoter were slightly higher (Table 1).

Next, the saccharification ability for various biomass pretreated products was comparatively evaluated using various latest commercial enzymes as controls. As a result, the saccharification enzyme produced using the cbh1 promoter with very high BGL activity was found to have a greater saccharification effect than the commercially available enzyme. Surprisingly, when saccharification enzyme produced using egl1 promoter, contrary to predictions from the conventional technical common sense (non-patent document 5) and various enzymatic activities were measured results (Table 1), cbh1 It showed higher saccharification ability than saccharification enzymes produced using the promoter (Tables 2 to 5). Moreover, the saccharifying enzyme produced using egl1 promoter is produced not only for biomass pretreated products with high hemicellulose content but also for biomass pretreated products and crystalline cellulose with low hemicellulose content using commercially available enzymes and cbh1 promoter. The saccharification enzyme showed higher saccharification ability.

That is, the present invention relates to a transformant expressing a β-glucosidase gene ( bgl1 ) derived from a microorganism belonging to the genus Aspergillus using the promoter of egl1 of the microorganism belonging to the genus Trichoderma, and a highly functionalized saccharification produced from the transformant The present invention relates to a method for saccharifying cellulosic biomass using an enzyme, and a method for producing ethanol from the sugar thus produced as a raw material, and more specifically, provides the following inventions.
(1) the genus Trichoderma in a microorganism belonging to the genus Trichoderma which β- glucosidase gene derived from microorganisms belonging to the genus Aspergillus that are enabled coupled expressed egl1 promoter was introduced in a microorganism belonging.
(2) The microorganism according to (1), wherein the microorganism belonging to the genus Trichoderma is Trichoderma reesei (Hypocrea jecorina).
(3) The microorganism according to (1) or (2), wherein the microorganism belonging to the genus Aspergillus is Aspergillus aculeatus .
(4) A saccharifying enzyme of cellulosic biomass produced from the microorganism according to any one of (1) to (3).
(5) The method for producing a saccharification enzyme according to (4), comprising a step of culturing the microorganism according to any one of (1) to (3).
(6) A method for producing sugar from cellulosic biomass, comprising the step of saccharifying cellulosic biomass with the saccharifying enzyme according to (4).
(7) A method for producing ethanol, comprising a step of fermenting a sugar obtained by carrying out the method according to (6).

Microorganism belonging to the genus Trichoderma expressing β- glucosidase gene derived from a microorganism belonging to the genus Aspergillus under the control of the egl1 promoter of a microorganism belonging to the genus Trichoderma are capable of producing a saccharifying enzyme having excellent saccharification ability. When the saccharifying enzyme produced by the transformant of the present invention is used, saccharification of cellulosic biomass can be completed in a very small amount as compared with conventional saccharifying enzymes. Furthermore, ethanol can be efficiently produced by fermenting the sugar thus obtained from cellulosic biomass.

It is a figure which shows the structure of an expression cassette at the time of expressing the β-glucosidase gene of Aspergillus aculeatus using egl1 promoter. It is an electrophoretic photograph showing the result of subjecting the culture supernatant of Trichoderma reesei introduced with β-glucosidase gene of Aspergillus aculeatus using egl1 promoter to SDS-PAGE. It is a figure which shows the structure of an expression cassette at the time of expressing the β-glucosidase gene of Aspergillus aculeatus using cbh1 promoter. It is an electrophoresis photograph which shows the result of having used the culture supernatant of Trichoderma reesei which introduce | transduced (beta) -glucosidase gene of Aspergillus aculeatus using cbh1 promoter to SDS-PAGE. It is the graph which showed the saccharification rate at the time of processing hydrothermally-treated Napiergrass pretreatment thing with various enzymes with time.

The present invention provides a microorganism belonging to the genus Trichoderma into which a β-glucosidase gene derived from a microorganism belonging to the genus Aspergillus that is linked to the egl1 promoter of a microorganism belonging to the genus Trichoderma and capable of expression is introduced.

As the “microbe belonging to the genus Aspergillus ” derived from the β-glucosidase gene used in the present invention, the specific activity of the produced β-glucosidase with respect to cellobiose is compared with the case of β-glucosidase produced by the microorganism belonging to the genus Trichoderma. As long as it is a high microorganism, there is no particular limitation, but Aspergillus aculeatus is preferable. Aspergillus aculeatus β-glucosidase 1 (BGL1) has a characteristic of extremely high specific activity against cellobiose compared to β-glucosidase 1 (BGL1) derived from Trichoderma reesei (results of measurement by the present inventors, as a result of Trichoderma BGL1 derived from reesei had a specific activity of 19 U / mg, whereas BGL1 derived from Aspergillus aculeatus had a specific activity of 186 U / mg). The β-glucosidase gene used in the present invention may be a gene having a natural sequence as long as it has the desired activity, and one or more mutations (base addition, deletion, substitution, insertion) have been introduced. It may be. The base sequence of cDNA of Aspergillus aculeatus- derived β-glucosidase 1 (BGL1) is shown in SEQ ID NO: 1, and the amino acid sequence encoded by the cDNA is shown in SEQ ID NO: 2.

The “microorganism belonging to the genus Trichoderma ” used as a host in the present invention is not particularly limited as long as it can increase the saccharification ability of cellulosic biomass by introducing a β-glucosidase gene derived from a microorganism belonging to the genus Aspergillus. There is no. Trichoderma reesei is preferable, and PC-3-7 strain that is a mutant derived from Trichoderma reesei and its derivatives are more preferable.

In the transformation of microorganisms belonging to the genus Trichoderma with β-glucosidase gene, the enzyme activity inherent to the microorganisms belonging to the genus Trichoderma can be reduced so that the enzyme produced by the transformant can exhibit excellent saccharification activity. It is necessary to express an exogenous β-glucosidase gene.

For this purpose, an egl1 promoter of a microorganism belonging to the genus Trichoderma is used in the present invention. Microorganisms belonging to the genus Trichoderma that expressed the β-glucosidase gene under the control of the egl1 promoter are higher than microorganisms belonging to the genus Trichoderma that expressed the β-glucosidase gene under the control of the xyn3 promoter or the cbh1 promoter Producing a saccharifying enzyme having saccharifying ability is an unexpected and surprising finding. The microorganism belonging to the genus Trichoderma from which the egl1 promoter is derived is preferably Trichoderma reesei .

The nucleotide sequence of Trichoderma reesei egl1 promoter is shown in SEQ ID NO: 7. The egl1 promoter used in the present invention may have a natural sequence as long as it has an activity of expressing a gene linked downstream thereof, and may have one or more mutations (base addition, deletion, substitution, Insertion) may be introduced.

When the β-glucosidase gene is introduced into the genomic DNA of Trichoderma reesei , the egl1 gene region can be used as the target region. When the egl1 gene region is used as a target region, a recombinant vector can be used, and this recombinant vector can include a structure consisting of “ egl1 gene promoter region-β-glucosidase gene- egl1 gene terminator region”. . As shown in this Example, in “pPegl1-gus”, which is a vector containing 3 kb upstream and downstream of the egl1 gene, by replacing the gus region with a β-glucosidase gene (eg, egl1 cDNA derived from Aspergillus aculeatus ) A recombinant vector can be constructed. For general genetic manipulations such as gene recombination and introduction of a vector into a host, techniques known to those skilled in the art can be used.

The transformant thus produced produces an excellent cellulosic biomass saccharifying enzyme. In general, an enzyme that degrades cellulose is referred to as cellulase, and an enzyme that degrades hemicellulose is referred to as hemicellulase. The “saccharifying enzyme of cellulosic biomass” in the present invention is meant to include both of these enzymes. The saccharifying enzyme produced by the transformant of the present invention is not limited to a biomass pre-treated product having a high hemicellulose content, but also to a biomass pre-treated product or crystalline cellulose having a low hemicellulose content using a commercially available enzyme or cbh1 promoter. It shows higher saccharification performance than the produced saccharifying enzyme. For this reason, the final ethanol production efficiency can be remarkably improved by using the microorganism of the present invention.

Cultivation of transformants for producing saccharifying enzymes can be performed by those skilled in the art under commonly used culture conditions. Examples of the sugar source used for the culture include various celluloses such as Avicel, filter paper powder, cellulose-containing biomass and lactose, and examples of the nitrogen source include ammonium sulfate, polypeptone, gravy, CSL, and soybean meal. In addition, components required for producing the target cellulase can be added to the medium. Further, xylanase can be increased by adding various xylan components to the medium. For the culture of these strains, various culture methods such as shaking culture, stirring culture, stirring and shaking culture, stationary culture, and continuous culture can be adopted, and shaking culture or stirring culture is preferable. The culture temperature is usually 20 ° C. to 35 ° C., preferably 25 ° C. to 31 ° C., and the culture time is usually 4 to 10 days, preferably 4 to 9 days.

Thus, the present invention provides a saccharification enzyme of cellulosic biomass produced from the transformant and a method for producing a saccharification enzyme of cellulosic biomass including a step of culturing the transformant.

The present invention also provides a method for producing sugar from cellulosic biomass, including a step of saccharifying cellulosic biomass with a saccharifying enzyme of cellulosic biomass produced by the transformant of the present invention. The “cellulosic biomass” used in the present invention may be a herbaceous plant, a woody plant, or a processed product or waste thereof. Examples of herbaceous plants include rice, Elianthus, wheat, sugarcane, reeds, Japanese pampas grass, corn, sorghum, napiergrass, switchgrass, Miscanthus, etc., while woody plants include cedars, eucalyptus, Examples include, but are not limited to, cypress, pine, rice eel, poplar, birch, willow, squirrel, oak, oak, shii, beech, acacia, bamboo, sasa, oil palm and sago palm.

Generally, biomass is difficult to undergo saccharification by cellulase as it is. For this reason, before performing saccharification by a cellulase, it is preferable to perform the process for changing a cellulose biomass into the form which is easy to receive the saccharification by an enzyme. The pretreatment method for cellulosic biomass in the present invention is not particularly limited, but is mechanochemical pulverization method, hydrothermal treatment, alkali treatment (for example, caustic soda (NaOH), KOH, Ca (OH) ₂ , Na ₂ SO ₃ , NaHCO ₃ , NaHSO ₃ , Mg (HSO ₃ ) ₂ , Ca (HSO ₃ ) ₂ , ammonia (NH ₃ , NH ₄ OH), etc.), dilute sulfuric acid treatment, steam explosion treatment, solvolysis treatment, microbial treatment, or these The combined processing is mentioned.

By performing such pretreatment, the cellulose and hemicellulose present in the raw biomass remain on the solid residue side or are hydrolyzed and transferred to the solubilized fraction depending on the treatment method and treatment conditions. To do. Here, a method of hydrolyzing cellulose and hemicellulose and collecting it in a solubilized fraction as an oligosaccharide or monosaccharide by setting the pretreatment method and conditions has been studied, but the pretreatment conditions are generally severe. Then, various components in the biomass are subjected to excessive decomposition, and as a result, an inhibitory substance in the alcohol fermentation process such as acetic acid, formic acid, furfural, hydroxymethylfurfural, and lignin decomposition product is generated, which is not preferable.

Since various advantages and disadvantages are assumed in selecting the biomass pretreatment method and conditions in this way, the optimum one is selected depending on the biomass to be used and the manufacturing method.

When hydrolyzing raw material biomass, hydrothermal treatment, caustic soda treatment, dilute sulfuric acid treatment, or ammonia treatment, for example, the slurry concentration is 1 to 30 (w / v)%, preferably 3 to 20 (w / v)%. The reaction vessel (autoclave) is charged and batchwise processed at a predetermined temperature for a predetermined time. Moreover, it is also possible to process continuously on the basis of equivalent conditions with a flow-type apparatus.

Here, in the case of hydrothermal treatment, the temperature is usually 150 to 250 ° C., more preferably 200 to 230 ° C. The treatment time is usually 3 to 60 minutes, more preferably 5 to 30 minutes.

In the case of caustic soda treatment, the caustic soda concentration is usually 0.1 to 3 (w / v)%, more preferably 0.3 to 1 (w / v)%. The treatment temperature is usually 50 to 230 ° C, more preferably 80 to 210 ° C. The treatment time is usually 3 minutes to 1 hour, more preferably 5 to 30 minutes. In the case of dilute sulfuric acid treatment, the sulfuric acid concentration is usually 0.3 to 3 (w / v)%, more preferably 0.5 to 1 (w / v)%. The treatment temperature is usually 100 to 200 ° C, more preferably 150 to 180 ° C. The treatment time is usually 3 to 30 minutes, more preferably 3 to 15 minutes.

In the case of ammonia treatment, the ammonia concentration is usually 1 to 10 (w / v)%, more preferably 3 to 5 (w / v)%. The treatment temperature is usually from room temperature to 170 ° C., more preferably from 50 to 170 ° C. The treatment time is usually 5 minutes to 14 days, more preferably 3 to 14 days.

The conditions for steam explosion are usually 3 to 30 minutes, preferably 5 to 15 minutes at 1.25 MPa. In the case of 2.33 MPa, it is usually 3 to 20 minutes, more preferably 5 to 10 minutes. In the case of 2.8 MPa, it is usually 1 to 15 minutes, more preferably 3 to 10 minutes. In the case of 3.35 MPa, it is usually 1 to 10 minutes, more preferably 3 to 10 minutes.

After pretreatment of the raw material biomass in this way, solid-liquid separation is performed by filtration, centrifugation, etc. to obtain a pretreated solid and a filtrate. Here, various methods can be adopted for quantitative analysis of cellulose and hemicellulose in raw biomass and pretreated solids, but the Laboratory Analytical of NREL (National Renewable Energy Laboratory) in the United States, which is used as a standard formulation worldwide. Procedure (LAP) Determination of Structural Carbohydrates and Lignin in Biomass is preferred. In this method, raw material biomass and pretreated solids are hydrolyzed with sulfuric acid, and then the content of constituent sugars such as glucose, xylose, mannose, galactose, etc. contained in these samples are analyzed and quantified by the HPLC method to determine the content rate. It is. In this method, the obtained glucose is determined to be derived from cellulose, and other components such as xylose, mannose and galactose are derived from hemicellulose, and the respective contents are determined.

The conditions for saccharifying the cellulosic biomass thus pretreated with the saccharifying enzyme produced from the transformant of the present invention are as follows. The solid concentration when the residual solid content of the pretreated product is saccharified is usually about 1 to 20 (w / v)%, preferably about 5 to 10 (w / v)%. The pH is usually in the range of 3-9, preferably 4-6. The temperature is 10 to 80 ° C, preferably 40 to 60 ° C. The enzyme concentration is 1 to 20 mg / g-biomass weight, preferably 1 to 10 mg / g-biomass weight, based on the amount of protein per dry matter weight of the biomass pretreatment product. Under these conditions, for example, the saccharification reaction can proceed under shaking or standing. In this saccharification reaction, a bactericidal agent such as sodium azide may be added for the purpose of preventing contamination with various bacteria. In this case, it is preferable to select a compound and a concentration that do not adversely affect the subsequent fermentation process of the saccharified solution. The saccharification rate can be determined by analyzing the product in the saccharified solution by various reducing sugar quantitative methods or HPLC methods.

Depending on the pretreatment method and conditions, xylose, which is a constituent sugar of hemicellulose in the biomass component, may be obtained as a monosaccharide or oligosaccharide in the soluble fraction. In this case, if the saccharide in the soluble fraction is a monosaccharide, if it is an oligosaccharide, various hemicellulases and the like are allowed to act on it, and after hydrolysis to a monosaccharide, use it as a saccharide for ethanol raw materials. Can do.

The present invention also provides a method for producing ethanol, comprising a step of fermenting sugar obtained by carrying out the method for producing sugar from the above-mentioned cellulosic biomass.

A general method can be applied to the production of ethanol from the saccharified solution. Examples of the microorganism used for producing ethanol include, but are not limited to, microorganisms belonging to the genera Saccharomyces , Zymomonas , Pichia , Zymobacter , Corynebacterium , Kluyveromyces , and Escherichia . It is also possible to use a microorganism incorporating a gene related to a metabolic system for converting sugar to ethanol, or a mutant thereof. By inoculating and culturing these microorganisms in a saccharified solution, ethanol can be produced. The saccharified solution used preferably has a sugar concentration of 3 to 15% and a pH of 3 to 7. The culture temperature is preferably 20 to 40 ° C. Ethanol fermentation can be carried out either batchwise or continuously. Ethanol fermentation can be performed simultaneously with the saccharification reaction of the above-mentioned cellulosic biomass (parallel double fermentation). In this case, the saccharification reaction conditions and the ethanol fermentation conditions may be combined, and conditions such as sugar concentration, temperature, pH, etc. that will give the highest productivity as a whole may be appropriately selected.

Hereinafter, the present invention will be described more specifically based on examples and comparative examples, but the present invention is not limited to the following examples.

[Example 1]
The oligonucleotide primers "e1Saabgl1 + 9: 5'-cttagtccttcttgttgtcccaaaATGAAGCTCAGTTGGCTTGAG-3 '(SEQ ID NO: 3)" and "e1aaabgl1 + 9: 5'-acagaccagaggcaagtcaacgctTCATTGCACCTTCGGGAGCG-3' (SEQ ID NO: 4)" are used. PCR was performed using the plasmid vector “pBxyn3Aabgl1” into which cDNA of BGL1 of Aspergillus aculeatus was inserted as a template.

In order to replace the gus gene part of pPegl1-gus and the PCR product bgl1 gene from Aspergillus aculeatus using In-fusion PCR reaction, in the primer, pPegl1-gus and 24 bp are added to both ends of the PCR product. The homologous region was designed to be added (Fig. 1).

PCR reaction conditions were 98 ° C for 10 seconds, 55 ° C for 5 seconds, and 72 ° C for 3 minutes. One cycle was repeated for 30 cycles. In addition, PrimeSTAR HS DNA polymerase (Takara Bio Inc.) was used for the PCR reaction.

[Example 2]
The obtained PCR amplified fragment ( bgl1 cDNA, 2583 bp) was subjected to oligonucleotide primer “E1AB1inverts: 5′-AGCGTTGACTTGCCTCTGGTCTGTC-3 ′ (SEQ ID NO: 5)” and “E1AB1inverta: 5′-TTTGGGACAACAAGAAGGACTAAGATAGGGG-3 ′ ( SEQ ID NO: 6) ”and pegl1-gus were used as templates and ligated to the amplified vector.

Inverse PCR was performed at 98 ° C. for 1 minute, followed by a two-stage reaction of “98 ° C. for 10 seconds and 68 ° C. for 10 minutes” as one cycle, and this was repeated 40 cycles. PrimeSTAR GXL DNA polymerase (Takara Bio Inc.) was used for the PCR reaction.

An In-fusion kit (TaKaRa) was used for linking the PCR amplified fragment to the vector. In-fusion reaction was performed by mixing 200 ng PCR product, 400 ng amplification vector, 5 × In-fusion buffer 2 μL, water 1 μL, and In-fusion enzyme 2 μL, incubating at 37 ° C. for 30 minutes, and then at 50 ° C. for 15 minutes. Performed by keeping warm. The base sequence of egl1 upstream sequence (SEQ ID NO: 7), bgl1 cDNA sequence and marker sequence ( amdS : 3089bp / SEQ ID NO: 8) of the obtained T. reesei expression cassette was determined to confirm that there were no PCR errors. . CEQTM2000XL DNA Analysis System (BECKMAN COULTER) was used to determine the base sequence, and the details of the operation were in accordance with the attached instruction manual.

[Example 3]
The vectors constructed in Example 2 were introduced into Trichoderma reesei PC-3-7 strain (WT), respectively. The introduction was performed by the protoplast PEG method. The transformant was selected with a selective medium for acetamide utilization ability. The composition of the medium is as follows.

2% glucose, 1.1M sorbitol, 2% agar, 0.2% KH ₂ PO ₄ (pH 5.5), 0.06% CaCl ₂ · 2H ₂ O, 0.06% CsCl ₂ , 0.06% MgSO ₄ · 7H ₂ O, 0.06% Acetamide solution, in order to confirm the expression of BGL1 transformants three strains obtained 0.1% Trace element, 10 ⁶ spores were inoculated into φ20 tubes containing liquid medium 5mL for Trichoderma reesei. The composition of the medium is as follows.

1% Avicel, 0.14% (NH ₄ ) ₂ SO ₄ , 0.2% KH ₂ PO ₄ , 0.03% CaCl ₂ · 2H ₂ O, 0.03% MgSO ₄ · 7H ₂ O, 0.1% Bacto Polypepton, 0.05% Bacto Yeast extract, 0.1% Tween 80, 0.1% Trace element, 50mM Tartrate buffer at pH4.0 final concentration
The composition of the trace element is as follows.

6 mg H ₃ BO ₃ , 26 mg (NH ₄ ) ₆ MO ₇ O ₂₄・ 4H ₂ O, 100 mg FeCl ₃・ 6H ₂ O, 40 mg, CuSO ₄・ 5H ₂ O, 8 mg MnCl ₂・ 4H ₂ O, 200 mg ZnCl ₂ 100ml with distilled water
The culture solution cultured at 120 spm for 3 days at 28 ° C. was centrifuged at 10,000 rpm for 5 minutes at 4 ° C., and 10 μL of the culture supernatant was collected and subjected to SDS-PAGE. The result is shown in FIG. No significant difference was observed between transformants No. 1 and No. 3 from the wild type, but transformant No. 2 had a slightly smear with a molecular weight of about 130 kDa that was not found in PC-3-7. From the fact that a protein band was observed, it was found that BGL1 derived from A. aculeatus was expressed.

[Example 4]
Trichoderma reesei PC-3-7 strain (WT) and 10 ⁷ spores of transformant No. 2 prepared in Example 3 were placed in a 300 mL Erlenmeyer flask containing 50 mL of Trichoderma reesei liquid medium (similar to Example 3). Inoculated. After culturing at 220 rpm and 28 ° C. for 7 days, the culture solution was centrifuged at 3000 rpm for 15 minutes, the separated culture supernatant was further filtered with Sartolab RF1000 filtersystem (Sarutorius), and the obtained filtrate was used as an enzyme solution.

[Example 5]
10 ⁷ spores of transformant No. 2 prepared in Example 3 were inoculated into a 300 mL Erlenmeyer flask containing 50 mL of a medium in which 0.5% birchwood xylan was added to a liquid medium for Trichoderma reesei (similar to Example 3). did. After culturing at 220 rpm and 28 ° C. for 7 days, the culture solution was centrifuged at 3000 rpm for 15 minutes, the separated culture supernatant was further filtered with Miracloth (Cosmo Bio), and the obtained filtrate was used as an enzyme solution.

[Comparative Example 1]
The oligonucleotide primers “Saabgl1-cbh + 9: 5′-atagtcaaccgcggactgcgcatcATGAAGCTCAGTTGGCTTGAG-3 ′” (SEQ ID NO: 9) and “Aaabgl1-cbh + 9: 5′-aggctttcgccacggagcactagtTCATTGCACCTTCGGGAGCG-3 ′” (sequence number: PCR was performed using the plasmid vector “pABG7” into which BGLI cDNA of Aspergillus aculeatus was inserted as a template.

An In-fusion PCR reaction using to replace the bgl1 gene from Aspergillus aculeatus is cbh1 gene portion and PCR product pcbh1-sv, in the primer, at both ends of the PCR products, pcbh1-sv and 24bp The homologous region was designed to be added (Fig. 3).

[Comparative Example 2]
The obtained PCR amplified fragment ( bgl1 cDNA, 2583bp) was used as oligonucleotide primers "cbh1vector prom: 5'-GATGCGCAGTCCGCGGTTGACTATTG-3 '(SEQ ID NO: 11)" and "cbh1vector term: 5'-ACTAGTGCTCCGTGGCGAAAGCCT-3' ( SEQ ID NO: 12) "and pcbh1-sv as a template were linked to a vector amplified by Inverse PCR.

An In-fusion kit (TaKaRa) was used for linking the PCR amplified fragment to the vector. In-fusion reaction was performed by mixing 200 ng PCR product, 400 ng amplification vector, 5 × In-fusion buffer 2 μL, water 1 μL, and In-fusion enzyme 2 μL, incubating at 37 ° C. for 30 minutes, and then at 50 ° C. for 15 minutes. Performed by keeping warm. The nucleotide sequence of the cbh1 upstream sequence, bgl1 cDNA sequence, and marker sequence ( amdS : 3089 bp) of the obtained T. reesei expression cassette was determined, and it was confirmed that there was no PCR error. CEQTM2000XL DNA Analysis System (BECKMAN COULTER) was used to determine the base sequence, and the details of the operation were in accordance with the attached instruction manual.

[Comparative Example 3]
The vectors constructed in Comparative Example 2 were introduced into Trichoderma reesei PC-3-7 strain (WT), respectively. The introduction was performed by the protoplast PEG method. The transformant was selected with a selective medium for acetamide utilization ability. The composition of the medium is as follows.

2% glucose, 1.1M sorbitol, 2% agar, 0.2% KH ₂ PO ₄ (pH 5.5), 0.06% CaCl ₂ · 2H ₂ O, 0.06% CsCl ₂ , 0.06% MgSO ₄ · 7H ₂ O, 0.06% Acetamide solution, 0.1% Trace element
To confirm the expression of BGL1 transformants 5 strains obtained, 10 ⁶ spores were inoculated into φ20 tubes containing liquid medium 5mL for Trichoderma reesei. The composition of the medium is as follows.

6 mg H ₃ BO ₃ , 26 mg (NH ₄ ) ₆ MO ₇ O ₂₄・ 4H ₂ O, 100 mg FeCl ₃・ 6H ₂ O, 40 mg, CuSO ₄・ 5H ₂ O, 8 mg MnCl ₂・ 4H ₂ O, 200 mg ZnCl ₂ 100ml with distilled water
The culture solution cultured at 120 spm for 3 days at 28 ° C. was centrifuged at 10,000 rpm for 5 minutes at 4 ° C., and 10 μL of the culture supernatant was collected and subjected to SDS-PAGE. The results are shown in FIG. In the transformant, a slightly smeared protein band with a molecular weight of about 130 kDa that was not found in the PC-3-7 strain was observed, indicating that BGL1 derived from A. aculeatus was expressed.

[Comparative Example 4]
10 ⁷ spores of transformant No. 3 prepared in Comparative Example 3 were inoculated into a 300 mL Erlenmeyer flask containing 50 mL of a liquid medium for Trichoderma reesei (as in Example 3). After culturing at 220 rpm and 28 ° C. for 7 days, the culture solution was centrifuged at 3000 rpm for 15 minutes, and the separated culture supernatant was further filtered through Sartolab RF1000 filtersystem (Sarutorius), and the obtained filtrate was used as an enzyme solution. .

[Comparative Example 5]
Inoculate 107 spores of transformant No. 3 prepared in Comparative Example 3 into a 300 mL Erlenmeyer flask containing 50 mL of a medium containing 0.5% birchwood xylan in a liquid medium for Trichoderma reesei (same as Example 3). did. After culturing at 220 rpm and 28 ° C. for 7 days, the culture solution was centrifuged at 3000 rpm for 15 minutes, the separated culture supernatant was further filtered with Miracloth (Cosmo Bio), and the obtained filtrate was used as an enzyme solution.

[Example 6]
For each of the enzyme solutions obtained in Examples 4 and 5 and Comparative Examples 4 and 5, FPU activity, CMC degradation activity, BGL activity using cellobiose as a substrate, xylanase activity, and β-xylosidase activity were measured. As comparative examples, Accellerase 1500 (Genencor) and Cellic CTec2 (Novozymes) were similarly measured for activity. Here, FPU activity was measured in accordance with NREL (National Renewable Energy Laboratory: USA) Measurment of Cellulase Activities Laboratory Analytical Procedure (LAP). CMC degradation activity uses 3,5-dinitrosalicylic acid as a reducing sugar produced by reaction with Sigma's carboxymethylcellulose (Low viscosity) at a substrate concentration of 1 (w / v)%, pH 5.0, 50 ° C. for 15 minutes. Quantitative measurement using glucose (DNS method) with glucose as standard. BGL activity was measured by quantifying glucose produced by the reaction of substrate cellobiose concentration 20 mM, pH 5.0, 50 ° C. for 10 minutes by the enzymatic method (Wako Pure Chemical Industries: Glucose CII Test Wako). The xylanase activity was measured using a Sigma Birchwood xylan, and the xylose produced by the reaction at a substrate concentration of 1 (w / v)%, pH 5.0, 37 ° C. for 10 minutes was quantified by the DNS method using xylose as a standard. . β-xylosidase activity was determined by quantifying 4-Nitrophenyl produced by a reaction at a substrate concentration of 1 mM, pH 5.0, 50 ° C. for 10 minutes using Sigma 4-Nitrophenyl β-D-xylopyranosaide. The amount of protein was quantified using Biovine Laboratories, Inc.'s Quick Start Bradford Protein Assay Kit with Bovine Gamma Globulin as a standard. Specific activity (U / mg) was determined from these enzyme activity values and protein content. The obtained results are shown in Table 1.

[Example 7]
21g of rice straw (multi-harvest rice K226) pulverized product (100-200μm) was suspended in 700ml of 0.5 (w / v)% caustic soda solution to a slurry concentration of 3% (w / v), and batch type pretreatment equipment ( And heat-treated at 100 ° C. for 5 minutes. Thereafter, the treated product was filtered and washed with water. The composition of the processed product thus obtained (Sample No. K209) is as follows: cellulose 50.1 (w / w)%, hemicellulose 24.9 (w / w)%, lignin 7.1 (w / w)%, ash content 5.4 (w / w) %Met.

Using this rice straw pre-treatment product K209 as a substrate, a reaction system with a substrate concentration of 5 (w / v)%, final sodium azide concentration of 0.02 (w / v)%, and final acetate buffer concentration of 100 mM (pH 5) was constructed. did. To this reaction system, the enzyme solutions prepared in Examples 4 and 5 and Comparative Examples 4 and 5, and enzymes Accellerase 1500 (DANISCO), Cellic CTec, Cellic CTec2 (Novozymes) were added at various concentrations, While shaking, the saccharification reaction was performed at 50 ° C. for 72 hours. Then, produced | generated glucose and xylose were quantified by HPLC method and the saccharification rate was calculated | required. Cellulose and hemicellulose contained in the biomass pre-treatment product as a substrate were converted to monosaccharides, and the saccharification rate was calculated as the ratio of free product sugar to them. Next, the saccharification rate determined in this way was plotted against the amount of enzyme protein per g of pretreated biomass used in the reaction, and the amount of enzyme protein showing an 80% saccharification rate was calculated. The results are shown in Table 2.

In alkali-treated rice straw where most hemicellulase remains, the saccharification enzyme produced from the transformant of the present invention is 2.4 to 2.9 times that of the latest commercial enzyme (Cellic CTec2). It was found to be 1.3 to 1.6 times more powerful than

The analysis of the pretreated material obtained here was performed by the following method based on the NREL LAP method. The pretreated product was dried with a constant temperature dryer so that the water content was about 10% or less, and pulverized with a mill mixer or the like to pass through a 100 μm mesh. The pulverized product was subjected to a moisture content meter to measure moisture. About 100 mg of this pulverized product was weighed into a pressure-resistant glass tube, 1 mL of 72% sulfuric acid was added, mixed well with a glass rod for 1 minute, and incubated at 30 ° C. for 60 minutes in a constant temperature water bath. During incubation, occasionally mixed with a glass rod. After 60 minutes, 28 mL of pure water was added and mixed well, and the mixture was treated in an autoclave at 121 ° C. for 1 hour. After cooling well, the mixture was filtered through an absolutely dry glass filter (Whatman, pore size: 1.0 mm), and the residue was transferred to an absolutely dry weighing bottle together with the glass filter. The residue was once dried at 70 to 80 ° C. to evaporate water, then dried at 105 ° C. for 3 hours or more, and the dry weight was measured. Calcium carbonate was added little by little to the filtrate, and the pH was adjusted to 5-6 while checking with pH test paper. This was centrifuged (2000 rpm, 5 minutes), the supernatant was passed through a 0.2 μm syringe filter, and a HPLC method (column: Asahipak NH2P-50 4E, 4 mm x 250 mm, Lot: 080806) using a post-column fluorescence detector. The saccharides in the sample were quantitatively analyzed by Shodex). From the obtained quantitative value, glucose was derived from cellulose and xylose, mannose, galactose, and the like were regarded as derived from hemicellulase, and the content (w / w) was determined. Quantification of cellulose and hemicellulose in the following examples was also performed by this method.

[Example 8]
21 g of eucalyptus pulverized product was suspended in 700 ml of water so as to have a slurry concentration of 3% (w / v), and heat-treated at 210 ° C. for 15 minutes with a batch type pretreatment device (manufactured by Toyo Koatsu Co., Ltd.). Thereafter, the treated product was filtered and washed with water. The composition of the pre-treated product thus obtained (sample No. Y134) was cellulose 60.9 (w / w)%, hemicellulose 0.8 (w / w)%, lignin 33.6 (w / w)%, ash content 0.4 (w / w). )%Met.

Using the eucalyptus pre-treated product Y134 prepared in this way, a reaction system with a substrate concentration of 5 (w / v)%, a final sodium azide concentration of 0.02 (w / v)%, and a final acetate buffer concentration of 100 mM (pH 5) Built. To this reaction system, the enzyme solutions prepared in Examples 4 and 5 and Comparative Examples 4 and 5, and enzymes Accellerase 1500 (DANISCO), Cellic CTec, Cellic CTec2 (Novozymes) were added at various concentrations, While shaking, the saccharification reaction was performed at 50 ° C. for 72 hours. The saccharification rate was calculated in the same manner as in Example 7, and the amount of enzyme protein showing an 80% saccharification rate was calculated. The results are shown in Table 3.

In hydrothermal treatment eucalyptus, the saccharification enzyme produced from the transformant of the present invention is 1.2 to 1.3 times higher than the latest commercial enzyme (Cellic CTec2), 1.1 to 1.6 times higher than the saccharification enzyme using Cbh1 promoter. It turned out to be a function.

[Example 9]
150g of Elianthus pulverized product (100-200μm) is suspended in 5L of 0.5% (w / v) caustic soda solution to a slurry concentration of 3% (w / v). Heat treatment was performed at 120 ° C. for 5 minutes. Thereafter, the treated product was filtered and washed with water. The composition of the pre-treated product thus obtained (sample No. E71) was: cellulose 56.0 (w / w)%, hemicellulose 22.2 (w / w)%, lignin 6.2 (w / w)%, ash content 1.6 (w / w) )%Met.

Using the Erianthus pretreated E71 prepared in this way, a reaction with a substrate concentration of 5 (w / v)%, a final sodium azide concentration of 0.02 (w / v)%, and a final acetate buffer concentration of 100 mM (pH 5). A system was constructed. To this reaction system, the enzyme solutions prepared in Examples 4 and 5 and Comparative Examples 4 and 5, and enzymes Accellerase 1500 (DANISCO), Cellic CTec, Cellic CTec2 (Novozymes) were added at various concentrations, While shaking, the saccharification reaction was performed at 50 ° C. for 72 hours. The saccharification rate was calculated in the same manner as in Example 7, and the amount of enzyme protein showing an 80% saccharification rate was calculated. The results are shown in Table 4.

In the caustic soda-treated Elianthus, the saccharifying enzyme produced from the transformant of the present invention is 1.3 to 1.4 times that of the latest commercial enzyme (Cellic CTec2), 1.2 to 1.3 times that of the saccharifying enzyme utilizing the Cbh1 promoter, It turned out to be highly functional.

[Example 10]
200 g of a 2 liter steam explosion apparatus (manufactured by Tsukishima Kikai Co., Ltd.) was filled with cedar chips, and then heat-treated with steam at 240 ° C. (3.35 MPa) for 10 minutes. Thereafter, a pretreatment product was prepared by blasting treatment. The composition of this pre-treated product (sample No. EC11) is cellulose 40.1 (w / w)%, hemicellulose 0.4 (w / w)%, lignin 53.4 (w / w)%, ash content 0.1 (w / w)% there were.

Using the cedar pretreatment EC11 prepared in this way, a reaction system having a substrate concentration of 5 (w / v)%, a final sodium azide concentration of 0.02 (w / v)%, and a final acetate buffer concentration of 100 mM (pH 5). Built. To this reaction system, the enzyme solutions prepared in Examples 4 and 5 and Comparative Examples 4 and 5, and the enzymes Accellerase 1500 (DANISCO) and Cellic CTec2 (Novozymes) were added at various concentrations, and shaken. The saccharification reaction was performed at 50 ° C. for 72 hours. The saccharification rate was calculated in the same manner as in Example 7, and the amount of enzyme protein showing an 80% saccharification rate was calculated. The results are shown in Table 5.

In the blast-treated cedar pretreated product, the saccharification enzyme produced from the transformant of the present invention is 1.9 to 2.1 times that of the latest commercial enzyme (Cellic CTec2), and 1.1 to that of the saccharification enzyme using the Cbh1 promoter. 1.3 times higher performance.

[Example 11]
Using microcrystalline cellulose (Theoras PH-101 (Asahi Kasei)) as a substrate, substrate concentration 5 (w / v)%, final sodium azide concentration 0.02 (w / v)%, final acetate buffer concentration 100 mM (pH 5) The reaction system of was constructed. To this reaction system, the enzyme solutions prepared in Examples 4 and 5 and Comparative Examples 4 and 5, and enzymes of Accellerase 1500 (DANISCO) and Cellic CTec2 (Novozymes) were added at various concentrations, and shaken. The saccharification reaction was performed at 50 ° C. for 72 hours. The saccharification rate was calculated in the same manner as in Example 7, and the amount of enzyme protein showing an 80% saccharification rate was calculated. The results are shown in Table 6.

In microcrystalline cellulose, the saccharifying enzyme produced from the transformant of the present invention is 2.3 to 2.8 times compared to the latest commercial enzyme (Cellic CTec2), 1.4 to 1.6 times compared to the saccharifying enzyme utilizing Cbh1 promoter, It turned out to be highly functional.

[Example 12]
10 g (based on dry weight) of the rice straw caustic soda pretreatment product K209 described in Example 7 was weighed into a 500 ml plastic bottle. In a plastic bottle, 160 ml of 50 mM acetate buffer (pH 5.0), 2 ml of 2 (w / v)% sodium azide solution, T.reesei BGL recombinant strain enzyme prepared in Example 4 with a protein amount of 80 mg A considerable amount was added, and water was further added so that the total amount of water was 200 ml. This reaction solution was reacted at 50 ° C. with 100 stroke reciprocal shaking for 72 hours. This reaction solution was heated in boiling water for 5 minutes and then centrifuged (13000 rpm, 5 minutes) to obtain 180 ml of a supernatant. About this sample, when the product was quantified by HPLC method, glucose was 26.8 mg / ml and xylose was 12.2 mg / ml. From this result, 4.82 g of glucose and 2.20 g of xylose could be prepared from 10 g (dry weight basis) of rice straw pre-treated product K209.

[Example 13]
7 g (based on dry weight) of Eucalyptus hydrothermal pretreatment Y134 described in Example 8 was weighed into a 200 ml plastic bottle. In a plastic bottle, 80 ml of 50 mM acetate buffer (pH 5.0), 1 ml of 2 (w / v)% sodium azide solution, 50 mg of T. reesei BGL recombinant strain enzyme prepared in Example 4 A considerable amount was added, and water was further added so that the total amount of water was 100 ml. This reaction solution was reacted at 50 ° C. with 100 stroke reciprocal shaking for 72 hours. This reaction solution was heated in boiling water for 5 minutes and then centrifuged (13000 rpm, 5 minutes) to obtain 90 ml of a supernatant. About this sample, when the product was quantified by HPLC method, glucose became 40.1 mg / ml. From this result, 3.61 g of glucose could be prepared from 7 g of the eucalyptus pretreated product.

[Example 14]
10 g of Erianthus caustic soda E71 described in Example 9 (based on dry weight) was weighed into a 500 ml plastic bottle. In a plastic bottle, 160 ml of 50 mM acetate buffer (pH 5.0), 2 ml of 2 (w / v)% sodium azide solution, T. reesei BGL recombinant strain enzyme prepared in Example 5 with a protein amount of 80 mg A considerable amount was added, and water was further added so that the total amount of water was 200 ml. This reaction solution was reacted at 50 ° C. with 100 stroke reciprocal shaking for 72 hours. This reaction solution was heated in boiling water for 5 minutes and then centrifuged (13000 rpm, 5 minutes) to obtain 185 ml of a supernatant. About this sample, when the product was quantified by HPLC method, glucose was 27.5 mg / ml and xylose was 10.5 mg / ml. From these results, 5.09 g of glucose and 1.94 g of xylose could be prepared from 10 g of the Erianthus pretreated product.

[Example 15]
180 ml of enzyme saccharified solution of caustic soda-treated rice straw (K209) prepared in Example 11 was concentrated with a rotary evaporator, and 35 ml of the obtained concentrated solution was sterile filtered with a sterile suction filtration device to obtain 32 ml of sterile filtrate. The sugar composition of this filtrate was 136 mg / ml glucose and 62 mg / ml xylose as determined by HPLC analysis.

A 300 ml Erlenmeyer flask was sterilized with 25 ml of the medium solution, and 30 ml of sterile filtrate was aseptically added thereto. Medium components (final concentration) other than sugar were yeast extract (0.45%) and peptone (0.75%), and the pH of the medium solution was adjusted to 5.0. To this, 5 ml of a 5 ml culture solution of yeast Kluyveromyces cellobiovorus (ATCC60381) separately seeded (2% glucose, 0.45% yeast extract and 0.75% peptone, pH 5.0, 48 hours) was added, and alcohol fermentation (flask 100 rpm) And allowed to shake at 28 ° C. for 72 hours. When the supernatant was analyzed after the culture, glucose and xylose were all disappeared and the ethanol concentration was 4.2% (w / v). The alcohol yield was 84% of theory.

[Example 16]
21g of Elianthus pulverized product (100-200μm) is suspended in 700ml of 1 (w / v)% dilute sulfuric acid solution so that the slurry concentration becomes 3 (w / v)%. At 180 ° C. for 7 minutes. Thereafter, the treated product was filtered and washed with water. The composition of the pre-treatment product thus obtained (Sample No. E-117) was as follows: cellulose 51.6 (w / w)%, hemicellulose 1.2 (w / w)%, lignin 35.4 (w / w)%, ash content 5.7 (w / w)%.

Using the Elianthus pretreatment E-117 prepared in this way, the substrate concentration was 5 (w / v)%, the final sodium azide concentration was 0.02 (w / v)%, and the final acetate buffer concentration was 100 mM (pH 5. The reaction system of 0) was constructed. The enzyme solutions prepared in Examples 4 and 5 and the enzymes Accellerase 1500 (DANISCO) and Cellic® CTec2 (Novozymes) were added to the reaction system at various concentrations and shaken at 50 ° C. for 72 hours. A saccharification reaction was performed. The saccharification rate was calculated in the same manner as in Example 7, and the amount of enzyme protein showing an 80% saccharification rate was calculated. The results are shown in Table 7.

It was found that the saccharification enzyme produced from the transformant of the present invention was 1.7 times more functional than the latest commercial enzyme Cellic CTec2 in the dilute sulfuric acid-treated Elinanthus pretreatment product.

[Example 17]
400 g of pulverized napier grass (100-200 μm) was suspended in 4 L of water and treated with a 5 L batch pretreatment device (manufactured by Toyo Koatsu Co., Ltd.) at 220 ° C. for 15 minutes. Thereafter, the treated product was filtered and washed with water. The composition of the pre-treated product thus obtained (sample No. N-9) was as follows: cellulose 48.2 (w / w)%, hemicellulose 1.6 (w / w)%, lignin 35.3 (w / w)%, ash content 6.2 (w / w)%.

Using the thus prepared Napiergrass pretreatment product N-9, the substrate concentration was 5 (w / v)%, the final sodium azide concentration was 0.02 (w / v)%, and the final acetate buffer concentration was 100 mM (pH 5. The reaction system of 0) was constructed. While adding each enzyme solution prepared in Examples 4 and 5 to this reaction system, Accellerase 1500 (DANISCO), Cellic CTec2 (Novozymes) to become 3 mg protein / g-biomass substrate, shaking, The saccharification reaction was performed at 50 ° C. At 24, 48 and 72 hours, 100 μl was sampled, heated in boiling water for 5 minutes, and then centrifuged to obtain a supernatant. The produced reducing sugar in the supernatant was quantified by the 3,5-dinitrosalicylic acid method (DNS method) using glucose as a standard. Cellulose and hemicellulose contained in the pretreated product used as a substrate were converted into monosaccharides, and the saccharification rate was calculated from the ratio of free product sugars to them. The obtained results are shown in FIG.

In the hydrothermally treated Napiergrass pre-treated product, the saccharification enzyme produced from the transformant of the present invention was found to have higher function than the latest commercial enzyme (Cellic CTec2).

The saccharifying enzyme produced by the transformant of the present invention is produced using not only a biomass pre-treated product with a high hemicellulose content but also a biomass pre-treated product or crystalline cellulose with a low hemicellulose content using a commercially available enzyme or cbh1 promoter. Since the saccharification enzyme exhibits higher saccharification performance than the saccharification enzyme, saccharification of the cellulosic biomass can be completed with a small amount. Furthermore, ethanol can be produced by fermenting the saccharified cellulosic biomass. According to the present invention, it is possible to produce bioethanol at a low price, which can greatly contribute to the industrialization of bioethanol.

SEQ ID NOs: 3-6, 9-12
<223> Artificially synthesized primer sequences

Claims

Microorganism belonging to the genus Trichoderma which β- glucosidase gene derived from microorganisms belonging to the genus Aspergillus that are enabled coupled expressed egl1 promoter microorganisms was introduced belonging to the genus Trichoderma.
The microorganism according to claim 1, wherein the microorganism belonging to the genus Trichoderma is Trichoderma reesei (Hypocrea jecorina).
The microorganism according to claim 1 or 2, wherein the microorganism belonging to the genus Aspergillus is Aspergillus aculeatus .
A saccharification enzyme of cellulosic biomass produced from the microorganism according to any one of claims 1 to 3.
The method for producing a saccharifying enzyme according to claim 4, comprising a step of culturing the microorganism according to any one of claims 1 to 3.
A method for producing sugar from cellulosic biomass, comprising the step of saccharifying cellulosic biomass with the saccharifying enzyme according to claim 4.
A method for producing ethanol, comprising a step of fermenting sugar obtained by carrying out the method according to claim 6.