JP2016106538A - Trichoderma reesei variant and production method of cellobiohydrolase using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a Trichoderma reesei variant capable of efficiently producing cellobiohydrolase (CBH) from an inexpensive inducer containing xylan and lignin, and to provide a production method of cellobiohydrolase using the same.SOLUTION: The invention provides a Trichoderma reesei variant in which the width of a clear zone formed under the presence of phosphate swollen cellulose and 5 g/L of xylose, with respect to the width of the clear zone formed under the absence of xylose, is 50% or more; and a production method of cellobiohydrolase by culturing the same.SELECTED DRAWING: None

Description

  The present invention relates to a Trichoderma reesei mutant and a method for producing cellobiohydrase using the mutant.

  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 sugar are being searched. One of the decomposition methods is an enzyme decomposition method in which cellulosic biomass is hydrolyzed using an enzyme called cellulase. However, since the cost of the enzyme is high, it is required to reduce this.

  Trichoderma reesei is a filamentous fungus that produces many diverse cellulases and hemicellulase mixtures. These include cellobiohydrase, endoglucanase, xylanase, β-glucosidase and the like. In cellulase produced by Trichoderma reesei, the ratio of cellobiohydrase (CBH) is the highest. CBH is decomposed from the reducing end of crystalline cellulose from CBH I (EC 3.2.1.-) and non-reducing end. There are two types of CBH II (EC 3.2.1.91) that degrade.

  The expression of CBH I in Trichoderma reesei is induced by cellulose, cellobiose, xylose, sophorose, lactose, xylan, and the expression of CBH II is induced by cellulose, cellobiose, xylose, sophorose, xylobiose, and the expression is promoted. (Non-Patent Documents 1 to 3).

Applied and environmental Microbiology, Vol. 76, p. 1,770, 2010 Biotechnology for biofuels, Vol. 6, No. 62, 2013 Current Genomics, Volume 14, Page 230, 2013

  A Trichoderma reesei mutant strain capable of efficiently producing cellobiohydrase (CBH) from an inexpensive inducer containing xylan and lignin, and a method for producing cellobiohydrase using the same are provided.

  The present inventor paid attention to the Trichoderma reesei mutant in which xylose suppression was canceled, and as a result of intensive studies, the width of the clear zone formed in the presence of phosphate-swelling cellulose and xylose 5 g / L is in the absence of xylose. The present inventors have found that the problem can be solved by breeding a mutant strain having a characteristic of 50% or more with respect to the width of the clear zone formed by using the mutant strain.

That is, this invention is comprised by the following (1)-(4).
(1) A Trichoderma reesei mutant in which xylose suppression is canceled, and the width of a clear zone formed when cultured in an agar medium having the following composition under dark conditions at 28 ° C. for 14 days is expressed as Xa (mm), When the width of the clear zone formed when culturing at 28 ° C. for 14 days in an agar medium added with xylose at a final concentration of 5 g / L in the following composition is Xb (mm), (Xb / Xa) × Trichoderma reesei mutant, 100 ≧ 50.
Composition (per agar):
Phosphoric acid swelling cellulose 250mg
Yeast extract 20mg
KH ₂ PO ₄ 4 mg
(NH ₄ ) ₂ SO ₄ 2.8 mg
MgSO ₄ · 7H ₂ O 0.6mg
CaCl ₂ · 2H ₂ O 0.6mg
Triton X-100 20μL
Trace elements solution _{(0.3g / L H 3 BO 3} , 1.3g / L (NH 4) 6 Mo 7 O 24 · 4H 2 O, 5g / L FeCl 3 · 6H 2 O, 2g / L CuSO 4 · 5H ₂ O, 0.4 g / L MnCl ₂ .4H ₂ O, 10 g / L ZnCl ₂ is included.) 20 μL
Agar powder 0.45g
15 mL water
(2) The Trichoderma reesei mutant strain according to (1), wherein the mutant strain is a mutant strain derived from Trichoderma reesei PC3-7 strain (ATCC # 66589) or Trichoderma reesei QM9414 strain (NBRC # 31329).
(3) A method for producing cellobiohydrase, wherein the mutant strain is cultured with biomass containing 15% by weight or more of xylan by dry weight.
(4) A method for producing cellobiohydrase, wherein the mutant strain is cultured with biomass containing 20% by weight of xylan and 8% by weight or more of lignin by dry weight.

  By using a mutant having the characteristics shown by the present invention, cellobiohydrase can be efficiently produced from an inexpensive inducer containing xylan and lignin.

It is the photograph which inoculated the spore of Trichoderma reesei to the agar medium containing phosphoric acid swelling cellulose, and was cultured on dark conditions. In addition, A is a colony which is a bacterial mass of Trichoderma reesei, and B is a clear zone which is a region where phosphate-swelling cellulose is decomposed by cellulase secreted from Trichoderma reesei. C indicates the width of the clear zone.

  Cellobiohydrase (CBH) is an enzyme that occupies the main component of cellulase made by Trichoderma reesei, and produces cellobiose by degrading β-1,4-linked cellulose chain from the terminal to exo form, The activity can be measured using p-nitrophenyl β-D-lactopyranoside (pNP-Lac) as a substrate. As cellobiohydrase, cellobiohydrase I (CBH I) that degrades the cellulose chain from the reducing end and cellobiohydrase II (CBH II) that degrades the cellulose chain from the non-reducing end are known. . The Trichoderma reesei mutant strain of the present invention is a Trichoderma reesei mutant strain that has produced cellobiohydrase higher than the parent strain by eliminating the suppression of cellulase production by xylose. Also referred to as “xylose-inhibited mutant strain”.

  When biomass containing Trichoderma reesei and hemicellulose mainly composed of xylan is cultured, xylose is liberated into the culture solution, and usually the production of CBH of Trichoderma reesei is suppressed by the liberated xylose. The xylose suppression release mutant is characterized in that CBH production is not suppressed even in the presence of xylose.

  The Trichoderma reesei mutant strain (xylose suppression release strain) has a clear zone formed around a colony grown on a phosphate-swelled cellulose mixed agar medium not added with xylose as shown in Table 1 below. When the clear zone formed around the colonies grown on the agar medium in which xylose is added to the phosphate-swelling cellulose medium shown to a final concentration of 5 g / L is Xbmm, (Xb / Xa) × 100 is 50 or more. It is a mutant strain having the ability to show.

  The clear zone is a transparent region (FIG. 1) in which phosphate-swelling cellulose contained in the agar medium is decomposed by cellulase secreted from a colony (FIG. 1-A) which is a Trichoderma cell mass grown on the agar medium. -B). The width of the clear zone refers to the distance (FIG. 1-C) from the outer peripheral edge of the Trichoderma cell mass grown on the agar medium to the outer peripheral edge of the clear zone. The width of the clear zone is an indicator of the activity of cellulases, particularly CBH and endoglucanase (EG).

The clear zone formation test is also called a halo assay. In the halo assay, 20 mL of an agar medium having the composition shown in Table 1 subjected to autoclaving at 121 ° C. for 20 minutes was solidified in a plastic petri dish having a diameter of 90 mm and a height of 15 mm, and 2 μL of spore (2.0 × 10 ⁵ ) physiological saline. Spot in the center of the agar medium in a suspended state in water, and culture in a constant temperature bath at 28 ° C. for 14 days under dark and humid conditions to measure the clear zone.

  As the phosphate-swelling cellulose used in the halo assay, for example, “Cellulose microcrystalline line” (Merck), which is microcrystalline cellulose, is used. Add 300 mL of phosphoric acid to 10 g of “Cellulose microcrystalline line” in small portions, grind it in a mortar so that the microcrystalline cellulose does not become balls, and then treat at 4 ° C. overnight. Subsequently, microcrystalline cellulose ground to 4.5 L of distilled water is gradually added and mixed well, and then centrifuged at 5,000 × g for 10 minutes at 4 ° C. to recover the precipitated cellulose. . Subsequently, this operation is repeated until the pH of the supernatant obtained when centrifuging is 5 and the washed product is defined as phosphate-swelled cellulose. Moreover, what suspended cellulose in 300 mL distilled water finally is made into a phosphoric acid swelling cellulose solution.

  The parent strain of the Trichoderma reesei mutant of the present invention is not particularly limited, but is preferably QM9414 strain (NBRC # 31329) or PC3-7 strain (ATCC # 66589).

Specific examples of the Trichoderma reesei mutant obtained using the QM9414 strain as a parent strain include the XDR1 strain (deposit number: NITE P-01940) described in the Examples of the present specification as an example. PC3-7 strain As specific examples of the Trichoderma reesei mutant obtained as a parent strain, the XDR2 strain (deposit number: NITE P-01941), XDR3 strain, XDR4 strain, XDR5 strain, XDR6 strain described in the Examples of the present specification, An example is the XDR7 strain.

  Cellobiohydrase can be produced by culturing with the Trichoderma reesei mutant and biomass containing xylan.

  In the present invention, biomass is a renewable organic organic resource. Specific examples include herbaceous biomass such as pulp, bagasse, switchgrass, napiergrass, Eliansus, corn stover, corn hull, rice straw, and straw, and woody biomass such as trees and waste building materials. . These cellulose-containing biomasses are also called lignocellulose because they contain lignin and cellulose / hemicellulose which are aromatic polymers.

  In the case of producing cellobiohydrase, the biomass needs to contain xylan that induces cellobiohydrase production of the Trichoderma reesei mutant. Xylan is preferably contained in an amount of 15% by weight or more, more preferably 20% by weight or more, based on the total dry weight of biomass. By including 15% by weight or more of xylan with respect to the total dry weight of biomass, it is possible to prevent a decrease in the relative activity of the cellobiohydrase of the mutant strain in the present invention. Moreover, although the upper limit of the xylan weight ratio contained in biomass is not particularly limited, it is usually 50% by weight or less.

  It is preferable that the biomass further contains lignin because it is proportional to the lignin content of the biomass and prevents the relative activity of the cellobiohydrase of the mutant strain in the present invention from decreasing. Lignin is preferably contained in an amount of 8% by weight or more, preferably 20% by weight or more, based on the total dry weight of biomass. By including 8% by weight or more of lignin with respect to the total dry weight of biomass, it is possible to prevent a decrease in the relative activity of the cellobiohydrase mutant of the present invention. Moreover, although the upper limit of the lignin weight ratio contained in biomass is not particularly limited, it is usually 30% by weight or less.

  Cellulose contained in the biomass should contain at least 30% by weight in order to induce cellobiohydrase expression in the range where the total weight ratio of xylan and lignin to the total dry weight of biomass does not exceed 100% by weight. Is preferred. The upper limit of the weight ratio of cellulose contained in the biomass is not particularly limited, but the higher the weight ratio, the more preferable, but it is usually 80% by weight or less.

  The method for producing cellobiohydrase is as follows. The spores of the XDR1 strain, the XDR2 strain, the XDR3 strain, the XDR4 strain, the XDR5 strain, the XDR6 strain, and the XDR7 strain, which are the xylose suppression release mutant strains described in the Examples of the present specification, are cultured in the preculture solution. The preculture is inoculated into the main culture and cultured. The culture is a flask culture, stirred aeration culture, or the like. Moreover, in order to induce the expression of cellobiohydrase by adding a nitrogen source or a sulfur source as a nutrient source, culture is performed using biomass. Let the liquid fraction of the culture solution after culture be a cellobiohydrase solution.

The contents of xylan, lignin and cellulose contained in biomass can be determined by the methods described in [1] to [3] below.
[1] Add 3 mL of 72% sulfuric acid to 0.3 g of biomass dried at 105 ° C., and leave it for 1 hour with stirring at 30 ° C. Distilled water 84mL is added to this reaction liquid, and it heat-disassembles by an autoclave at 120 degreeC for 1 hour.
[2] After the thermal decomposition, the decomposition solution and the residue are separated by filtration, and the solution obtained by adding the residue washing to the filtrate and making up to 100 mL is quantified by high performance liquid chromatography to calculate the cellulose and xylan content. .
[3] The residue obtained by the constituent sugar analysis described in [1] and [2] is dried at 105 ° C. and weighed to calculate the decomposition residue rate. Furthermore, the residue is heated at 600 ° C., the remaining ash content is measured, and the lignin content is calculated by subtracting the ash content from the decomposition residue rate.

  The present invention will be specifically described below with reference to examples. However, the present invention is not limited to this.

(Example 1) Trichoderma reesei strain Trichoderma reesei QM9414 strain (NBRC # 31329), RutC-30 strain (ATCC # 56765), PC3-7 strain (ATCC # 66589), and Trichoderma reesei mutant strain XDR1 strain, XDR2 strain, XDR3 strain, XDR4 strain, XDR5 strain, XDR6 strain, XDR7 strain were used.

  XDR1 strain was obtained by subjecting Trichoderma reesei QM9414 strain to random mutagenesis by nitrosoguanidine and ultraviolet irradiation, and adding xylose to a medium containing phosphate-swelled cellulose shown in Table 1 to 5 g / L. The mutant derived from the strain QM9414 selected for its ability to form a large clear zone.

  XDR2 strain, XDR3 strain, XDR4 strain, XDR5 strain, XDR6 strain, XDR7 strain were subjected to random mutation introduction to Trichoderma reesei PC3-7 strain by nitrosoguanidine and UV irradiation, and the phosphate swelling cellulose shown in Table 1 was also used. It is a mutant strain of PC3-7 strain selected for the ability to form a large clear zone on an agar medium in which xylose is added to the medium containing 5 g / L.

(Example 2) Collection of spores Various Trichoderma reesei strains of Example 1 were cultured on potato dextrose agar medium at 28 ° C for 10 days in the dark to form spores. Subsequently, 4 mL of a glycerol stock solution (0.8% NaCl, 0.025% Tween 20, 20% glycerol) is added to each agar medium, and the spores are scraped off with a conical rod and miracloth ^(R) autoclaved. (Merck Millipore). The filtrate was used as a spore solution.

(Example 3) Clear zone formation test A medium containing phosphate-swelling cellulose (xylose-free medium) shown in Table 1 and a medium containing phosphate-swelling cellulose shown in Table 1 so that the final concentration is 5 g / L. Trichoderma reesei QM9414 strain, RutC-30 strain, PC3-7 strain, XDR1 strain, XDR2 strain, XDR3 strain, XDR4 strain, XDR5 strain, XDR6 strain, XDR7 strain, respectively, to the medium supplemented with xylose (medium containing medium) 0.05 × 10 ⁵ cells were suspended in 2 μL of physiological saline and inoculated into the center of the agar medium and cultured at 28 ° C. for 14 days under dark conditions. At that time, the clear zone formed around the colony over time was measured.

  On the 14th day from the start of the culture, the width Xamm of the clear zone formed in the xylose-free medium was measured. On the 14th day from the start of the culture, the width Xbmm of the clear zone formed in the xylose-containing medium was measured. The ratio of the clear zone was determined by (Xb / Xa) × 100 (Tables 2 and 3). The value obtained by (Xb / Xa) × 100 was 0 in the QM9414 strain, whereas it was 56 in the XDR1 strain that imparted the ability to release xylose suppression to the QM9414 strain (Table 2). In addition, in the case of the PC3-7 strain, which was 26, in the mutant strain to which the PC3-7 strain was given the ability to release xylose suppression, there were 3 strains about 3 times higher than the PC3-7 strain, about 3 strains. There were 3 strains more than twice as high (Table 3). In addition, for the RutC-30 strain, a clear zone was not formed in the xylose-free medium, so the value obtained by (Xb / Xa) × 100 was set to 0.

(Example 4) Flask culture evaluation using biomass by a QM9414 strain and a QM9414 strain-derived xylose suppression release mutant strain (XDR1 strain) (preculture)
Trichoderma reesei QM9414 strain and XDR1 strain spores were diluted with physiological saline to 1.0 × 10 ⁷ / mL, and 0.1 mL of the diluted spore solution was placed in a 50 mL baffled flask. Inoculated into 10 mL of preculture solution composed of composition. The inoculated preculture was cultured for 3 days at 28 ° C. and 160 rpm.

(Biomass pretreatment)
Arbocel ^(R) (J. Rettenmeier & Sohne), Elianthus, which has been pretreated with ammonia, and corn hull (Qinhuangdao Ryugo Agricultural Products Processing Co., Ltd.) were used as the cellulose-containing biomass which is a cellulase inducer.

  Eliansus was treated with ammonia (pretreatment) as follows. Eliansus was put into a small reactor TVS-N230mI (pressure-resistant glass industry) and cooled with liquid nitrogen. Ammonia gas was introduced into the reactor, and the Elianthus was completely immersed in liquid ammonia. The reactor lid was closed and left at room temperature for about 15 minutes. Subsequently, it processed in the 150 degreeC oil bath for 1 hour. After the treatment, the reactor was taken out from the oil bath, and ammonia gas was immediately leaked in the fume hood, and then the reactor was evacuated to 10 Pa with a vacuum pump to dry the ammonia-treated product. This ammonia-treated product (pre-treated product) was wrapped in miraculose, washed with a large amount of pure water, and then air-dried. Next, the ammonia pre-treated Elianthus and Cornhull were pulverized using a “home mortar tea powder device whole green tea EU6820P” (Panasonic Corporation). As a result of measuring the particle size with a sieve after pulverization, the powdered ammonia-treated Eliansus (powdered Elianthus) had an average particle size of 50 μm, and the corn hull (powdered cornhull) had an average particle size of 15 μm.

(Main culture)
Trichoderma reesei QM9414 strain and XDR1 strain 1 mL of each preculture solution was inoculated into 10 mL of the main culture solution shown in Table 5 placed in a flask with a 50 mL baffle, and cultured at 28 ° C. and 160 rpm for 10 days. . Biomass was used Arbocel ^(R), ammonia treated powder erianthus or powder Konharu respectively.

(Collecting culture fluid)
500 μL of the culture solution was collected every 2 days from the start of the culture. Then, centrifugation was performed 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 culture supernatant.

(Measurement of enzyme activity)
Enzyme activity was measured for the culture supernatant on the 10th day after the start of the culture. Cellobiohydrolase activity uses p-nitrophenyl β-D-lactopyranoside (4-nitrophenyl β-D-lactopyranoside) manufactured by Sigma-Aldrich Japan as a substrate, and 4-nitrophenol produced by enzymatic degradation at 405 nm. Quantification was performed by increasing the absorbance. 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-lactopyranoside, 0.1 M sodium acetate (pH 5.0)] at 30 ° C. For exactly 60 minutes. Subsequently, 10 μL of a reaction stop solution (2M Na ₂ CO ₃ ) was added and mixed well to stop the reaction, and absorbance at 405 nm was measured. One unit of CBH activity was expressed as the amount of enzyme that liberates 1 μmol of 4-nitrophenol per minute under the reaction conditions of 30 ° C. for 60 minutes.

  The XDR1 strain was compared with the QM9414 strain in the degradation activity of pNP-Lac. When using “Arbocel” (registered trademark) as an inducer, it was 1.5 times, and when powdered corn hull was used, it was 5 times or more. In the case of using, the activity was 10 times (Table 6). The higher the lignin content in the biomass, the better the relative activity of CBH in the xylose-inhibited mutant.

(Example 5) Flask culture evaluation using biomass by PC3-7 strain and PC3-7 strain-derived xylose suppression release mutant strain In the same manner as in Example 4, PC3-7 strain and PC3-7 strain-derived xylose suppression Flask culture of the demutated mutant strains (XDR2, XDR3, XDR4, XDR5, XDR6, and XDR7 strains) and pNP-Lac degradation activity measurement using the culture supernatant were performed.

  The mutant strain of PC3-7 strain showed a pNP-Lac degradation activity about 1.5 times higher than that of PC3-7 strain when powdered Eliansus was used as a cellulase inducer (Table 7). The higher the lignin content in the biomass, the higher the relative activity of CBH in the xylose-inhibited mutant.

(Example 6) Manufacture of cellobiohydrase by a xylose suppression release mutant strain derived from QM9414 strain and a xylose suppression release mutant strain derived from PC3-7 strain (preculture)
Spore collection was carried out in the same manner as in Example 2 from the xylose suppression release mutant strain (XDR1, XDR2, XDR3, XDR4, XDR5, XDR6, and XDR7) in the present invention. Preculture was performed in the same manner as in (Culture).

(Main culture)
XDR1 strain, XDR2 strain, XDR3 strain, XDR4 strain, XDR5 strain, XDR6 strain, XDR7 strain 1 mL each of the preculture solution in a 50 mL flask with baffle was inoculated into 10 mL of the main culture solution shown in Table 8 at 28 ° C. Cultivation was performed for 10 days under a culture condition of 160 rpm. As the biomass, ammonia-treated powder Elianthus or powdered corn hull was used.

(Recovery)
The culture solution cultured for 10 days was transferred to a 50 mL centrifuge tube (Corning), and centrifuged at 8,000 × 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 cellobiohydrase solution.

(Reference Example 1) Ratio of cellulose, xylan and lignin contained in biomass (measurement of cellulose, xylan and lignin content)
Add 3 mL of 72% sulfuric acid to Arbocel ^(R) dried at 105 ° C, powdered ammonia-treated powder Eliansus (powder Eliansus) and 0.3 g of powdered corn hull biomass, and let stand for 1 hour with stirring at 30 ° C. did. Distilled water (84 mL) was added to the reaction solution, and the mixture was thermally decomposed at 120 ° C. for 1 hour in an autoclave. After thermally decomposing, the decomposition solution and the residue were separated by filtration, and the solution obtained by adding the residue washing solution to the filtrate and making up to 100 mL was quantified by high performance liquid chromatography, and the cellulose and xylan contents were calculated. The obtained residue was dried at 105 ° C. and weighed to calculate the decomposition residue rate. Furthermore, the residue was heated at 600 ° C., the remaining ash content was measured, and the lignin content was calculated by subtracting the ash content from the decomposition residue rate (Table 9).

  Cellulase biomass, especially cellulase using cellulase-inducing substances such as Elianthus and corn hulls, can be produced at a high rate and can be used for efficient saccharification reaction of cellulose resources.

Claims (4)

A Trichoderma reesei mutant in which xylose suppression is canceled, the width of the clear zone formed when cultured for 14 days at 28 ° C. in an agar medium having the following composition is Xa (mm), and xylose is terminated in the following composition: When the width of the clear zone formed when cultured for 14 days at 28 ° C. under dark conditions in an agar medium added to a concentration of 5 g / L is Xb (mm), (Xb / Xa) × 100 ≧ 50, a Trichoderma reesei mutant.
Composition (per agar):
Phosphoric acid swelling cellulose 250mg
Yeast extract 20mg
KH ₂ PO ₄ 4 mg
(NH ₄ ) ₂ SO ₄ 2.8 mg
MgSO ₄ · 7H ₂ O 0.6mg
CaCl ₂ · 2H ₂ O 0.6mg
Triton X-100 20μL
Trace elements solution _{(0.3g / L H 3 BO 3} , 1.3g / L (NH 4) 6 Mo 7 O 24 · 4H 2 O, 5g / L FeCl 3 · 6H 2 O, 2g / L CuSO 4 · 5H ₂ O, 0.4 g / L MnCl ₂ .4H ₂ O, 10 g / L ZnCl ₂ is included.) 20 μL
Agar powder 0.45g
15 mL water   The Trichoderma reesei mutant strain according to claim 1, wherein the mutant strain is a mutant strain derived from Trichoderma reesei PC3-7 strain (ATCC # 66589) or Trichoderma reesei QM9414 strain (NBRC # 31329).   A method for producing cellobiohydrase, wherein the mutant strain is cultured with biomass containing 15% by weight or more of xylan by dry weight.   A method for producing cellobiohydrase, wherein the mutant strain is cultured with a biomass containing 20% by weight of xylan and 8% by weight or more of lignin by dry weight.