JP2011182675A - Cellulase composition and use of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cellulase composition including the combination of cellobiohydrolase (CBH) and endoglucanase (EG) which is suitable for the decomposition of cellulose. <P>SOLUTION: One or more first cellobiohydrolases (CBHs) belonging to GHF6, one or more second CBHs belonging to GHF7, one or more first endoglucanases (EGs) belonging to GHF5 and further one or more enzymes selected from the group consisting of (a) one or more second EGs belonging to GHF9, (b) one or more pectate lyases, (c) one or more xylanases, (d) one or more arabinofuranosidases, and (e) one or more glucuronoxylan xylanohydrolases are contained. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a cellulase composition for effectively utilizing cellulose contained in biomass and the use thereof.

  In recent years, as an alternative to finite petroleum resources, there is an increasing expectation for biomass derived from the photosynthesis of plants, and various attempts have been made to use biomass for energy and various materials. Among these, the use of cellulose is expected. Cellulose is a polymer compound in which glucose, which is a sugar, is condensed by β-1,4 glycosidic bonds, and constitutes a strong crystal structure by intermolecular hydrogen bonding.

  Currently, an enzyme preparation derived from Trichoderma reesei is known as a cellulase preparation for cellulose degradation. Various hemicellulases that degrade hemicellulose and the like are also known (Non-Patent Documents 1 and 2).

Tung M-Y, et al, Applied Biochem. Biotechnol. 136 (2007), 1-16 K. Nishitani et al., J. Bio. Chem. Vol. 266, No. 10, 65369-6543, 1991

  For practical use of biomass, the cost reduction of the saccharification process for decomposing monosaccharides has become a major bottleneck. In order to efficiently decompose (saccharify) cellulose into monosaccharides, at least three types of cellulose-degrading enzymes (cellulases) are required, and it is considered possible only when they act in concert ( Hereinafter, this effect is called a synergistic effect.) These three types of cellulase are cellobiohydrolase (CBH), which acts on macromolecular cellulose, and endo-type (randomly cleaved) endoglucanase (EG). ) And β-glucosidase (BGL) that decomposes to some extent a substance that has been oligomerized to some extent by these enzymes. In order to realize an efficient saccharification process, it is important to establish a combination of cellulases that exhibits the maximum synergistic effect. Of these, the combination of CBH and EG is important.

  However, the above conventional cellulase preparation is only a protein purified product produced by Trichoderma reesei, and all cellulases are enzyme preparations derived from T. reesei and also contain unnecessary proteins. In addition, since a gene recombination system has not been sufficiently established, T. reesei is difficult to modify by controlling the type and expression level of the enzyme by gene recombination. In addition, celluloses derived from microorganisms other than T. reesei have been studied for biomass degradation, but it is extremely difficult to obtain cellulolytic activity equivalent to or higher than that of conventional cellulase preparations by combining artificial cellulases. there were.

  In addition, since hemicellulose and the like coexist in the actual biomass in addition to cellulose, it may be possible to treat hemicellulose or the like that may be present in the pretreated biomass with hemicellulase or the like. Effectiveness against glycation has not been confirmed.

  Therefore, an object of the present disclosure is to provide a cellulase composition containing a combination of CBH and EG suitable for cellulose degradation and use thereof.

  The present inventors have conducted detailed studies on various cellulases derived from T. reesei and a plurality of organisms other than T. reesei, in particular, combinations of CBH and EG. As a result, it was found that it is important to combine CBH and EG belonging to a specific GHF. Furthermore, it has been found for the first time that a certain kind of hemicellulase is effective for improving the decomposition efficiency of cellulose by cellulase. According to the disclosure of the present specification, the following means are provided based on these findings.

According to the disclosure of the present specification, one or more first cellobiohydrolases belonging to GHF6, one or more second cellobiohydrolases belonging to GHF7, and one or more second cellobiohydrolases belonging to GHF5. And an endoglucanase, and further comprising one or more selected from the group consisting of the following enzymes (a) to (e).
(A) one or two or more second endoglucanases belonging to GHF9 (b) one or two or more pectate lyases (c) one or two or more xylanases (d) one or two or more arabinofuranosidases (e ) One or more glucuronoxylan xylanohydrolase

  The first cellobiohydrolase is derived from one or more organisms selected from the group consisting of Phanerochaete chrysosporium (P. chrysosporium) and Aspergillus oryzae (A. oryzae, Aspergillus oryzae) It is preferable to include cellobiohydrolase. Among these, the first cellobiohydrolase preferably includes cellobiohydrolase derived from P. chrysosporium.

  The second cellobiohydrolase is present in one or more organisms selected from the group consisting of Aspergillus niger (A. niger), Aspergillus aculeatus (A. aculeatus), and A.oryzae. It is preferred to include a derived cellobiohydrolase. Especially, it is preferable that said 2nd cellobiohydrolase contains the cellobiohydrolase derived from A. niger.

  The first endoglucanase preferably contains an endoglucanase derived from an organism belonging to A. oryzae and Trichoderma reesei (T. reesei). The second endoglucanase preferably includes an endoglucanase derived from an organism belonging to Oryza sativa (O. sativa, rice).

  Furthermore, it is preferable to contain one or more third endoglucanases belonging to GHF12. The third endoglucanase is preferably derived from one or more organisms selected from the group consisting of P. chrysosporium, A. oryzae and T. reesei. The composition preferably contains the second endoglucanase and the third endoglucanase.

  In the composition, the cellobiohydrolase is composed of one or more first cellobiohydrolases belonging to GHF6 and one or two or more second cellobiohydrolases belonging to GHF7, and the endoglucanase is 1 Also provided is a composition comprising a first endoglucanase belonging to two or more GHF5, one or more second endoglucanases belonging to GHF9, and one or more third endoglucanases belonging to GHF12. Is done.

  Also provided are such compositions that are substantially free of β-glucosidase.

  According to the disclosure of the present specification, a method for producing an enzyme, comprising one or more selected from the group consisting of a cellulase derived from the genus Phanerochaete, a cellulase derived from the genus Oryza, and a hemicellulase derived from the genus Bacillus. There is provided a method comprising the step of introducing a gene encoding an enzyme into a non-cellulase-producing microorganism and producing the enzyme with the non-cellulase-producing microorganism. The cellulase non-producing microorganism is an Aspergillus bacterium, and the gene is preferably modified based on the codon usage in the Aspergillus genus. The cellulase non-producing microorganism may be yeast.

According to the disclosure herein, a transformant of a cellulase non-producing microorganism comprising:
The following proteins:
One or more cellulases belonging to GHF6 derived from the genus Phanerochaete,
One or more cellulases belonging to GHF7 derived from the genus Phanerochaete,
One or more cellulases belonging to GHF12 derived from the genus Phanerochaete,
One or more cellulases belonging to GHF9 derived from the genus Oryza, and
Hemicellulase derived from the genus Bacillus,
A transformant obtained by transforming with an expression vector for expressing a gene encoding one or more proteins selected from the group consisting of is provided. The cellulase non-producing microorganism may be an Aspergillus genus, and the gene may be a gene modified based on codon usage in the Aspergillus genus, and the cellulase non-producing microorganism may be a yeast. Good. It is preferable that the transformant secretes the protein extracellularly or presents it on the cell surface.

  According to the disclosure of the present specification, there is provided a method for producing a low molecular weight product of cellulose, comprising the step of decomposing the cellulose using any of the cellulase compositions described above.

  According to the disclosure of the present specification, there is provided a method for producing a useful substance by fermentation of microorganisms, and at least using the cellulase composition described above to make cellulose into a carbon source that can be used by the microorganisms. There is provided a method comprising the steps of decomposing and producing the useful substance by fermenting the carbon source with the microorganism. The microorganism is preferably a microorganism that secretes β-glucosidase outside the cell or presents it on the cell surface so that the partial degradation product derived from cellulose can be used as a carbon source. Further, the microorganism may be yeast, and the useful substance may be ethanol.

  The cellulose may be a cellulose fraction obtained by pretreatment of a cellulose-containing material containing at least cellulose, and the pretreatment may be a hydrothermal treatment or a treatment such as immersion in an ionic liquid. Good.

  According to the disclosure of the present specification, it is possible to provide an enzyme composition for cellulolysis containing a combination of CBH and EG having good cellulolytic activity and use thereof.

It is a figure which shows the saccharification result of the rice straw origin cellulose after a hydrothermal treatment. It is a figure which shows the saccharification result of the eucalyptus origin cellulose after an ionic liquid process. It is a figure which shows the comparative evaluation result of the kind of EG in a cellulase composition. It is a figure which shows the comparative evaluation result of the amount of CBH in a cellulase composition. It is a figure which shows the comparative evaluation result of the kind of CBH in a cellulase composition. It is a figure which shows the addition effect of glucuronoxylan xylan hydrolase. It is a figure which shows the addition effect of EG derived from O. sativa and commercial xylanase. It is a figure which shows the addition effect of xylanase derived from A. oryzae and glucuronoxylan xylanohydrolase derived from B. subtilis. It is a figure which shows the addition effect of the xylanase derived from A. oryzae. It is a figure which shows the addition effect of the pectate lyase derived from A. oryzae. It is a figure which shows the addition effect of arabinofuranosidase derived from A. oryzae. It is a figure which shows the addition effect of EG derived from O. sativa. It is a figure which shows the codon usage frequency of Aspergillus oryzae, and the codon used for modification. It is a figure which shows the codon usage frequency of Aspergillus oryzae, and the codon used for modification. It is a figure which shows the codon usage frequency of Aspergillus oryzae, and the codon used for modification.

  The disclosure of the present specification relates to a cellulase composition, a method for producing an enzyme such as cellulase, a transformant, a method for producing a degradation product of cellulose, and a method for producing a useful substance. The cellulase composition disclosed herein includes one or more first CBHs belonging to GHF6, one or more second CBHs belonging to GHF7, and a first belonging to one or more GHF5s. And also contains one or more enzymes selected from a range of cellulose and hemicellulose. Since such a composition is artificially combined for the decomposition of cellulose, particularly for the decomposition of cellulose in a cellulose-containing material that has been subjected to a predetermined pretreatment with hydrothermal treatment or ionic liquid, In an industrial saccharification process of a cellulose-containing material, cellulose can be efficiently decomposed. Therefore, in the saccharification of the cellulose-containing material, it is possible to easily obtain a cellulose degrading activity equivalent to or higher than that of a conventionally provided enzyme preparation for degrading cellulose.

  In the production of cellulase and the like, a gene encoding one or more enzymes selected from the group consisting of cellulase derived from Phanerochaete, cellulase derived from Oryza and hemicellulase derived from Bacillus It is preferable that the enzyme is produced by introduction into a non-producing microorganism and the cellulase non-producing microorganism.

  Hereinafter, various embodiments included in the disclosure of the present specification will be described in detail. As used herein, “GHF (Glycoside Hydrolase Family)” means glycoside hydrolysis provided on the homepage of CAZy (Carbohydrate active Enzymes) (http://www.cazy.org/fam/acc_GH.html). It is a classification of degrading enzymes.

(Cellulase composition)
The composition contains at least one or more first CBHs belonging to GHF6, one or more second CBHs belonging to GHF7, and one or more first EGs belonging to GHF5. ing. Furthermore, 1 or 2 or more 3rd EG which belongs to GHF12 may be included.

(First cellobiohydrolase (CBH))
The first CBH is a CBH belonging to GHF6. CBH belonging to GHF6 is generally considered to be type II (CBH II) that produces cellobiose by cleaving cellulose from its non-reducing end. As CBH belonging to GHF6, those derived from various microorganisms are known (http://www.cazy.org/fam/GH6.html). Examples of the first CBH include CBH derived from P. chrysosporium, A. oryzae, and T. reesei.

  In this specification, for example, “CBH derived from P. chrysosporium” refers to CBH produced by a microorganism classified into P. chrysosporium (which may be a wild strain or a mutant strain) or the microorganism. CBH obtained by a genetic engineering technique using a gene encoding a protein produced by Therefore, CBH, which is a recombinant protein produced by a transformant into which a gene encoding CBH (or a modified gene thereof) obtained from P. chrysosporium has been introduced, also corresponds to CBH derived from P. chrysosporium. Therefore, “CBH derived from P. chrysosporium” includes CBH that is the same genera as P. chrysosporium and is a heterogeneous strain or the same species and obtained from another strain. The same applies to “CBH derived from A. oryzae”. Further, similar expressions disclosed in the present specification are defined in the same manner as described above.

  For example, PcCBH2 consisting of the amino acid sequence represented by SEQ ID NO: 1 derived from P. chrysosporium can be mentioned. Moreover, AoCBH2A which consists of an amino acid sequence represented by sequence number 2 derived from A. oryzae is mentioned. Furthermore, as the first CBH, CBHs of other modes that can be obtained based on such known sequence information can be included. Such CBH will be described later. 1st CBH can be used combining 1 or 2 or more from the above various CBH.

(Second cellobiohydrolase (CBH))
The second CBH is a CBH belonging to GHF7. CBH belonging to GHF7 is generally considered to be type I (CBHI) that produces cellobiose by cleaving cellulose from its reducing end. As CBH belonging to GHF7, those derived from various microorganisms are known (http://www.cazy.org/fam/GH7.html). Among these, one or more selected from CBH derived from A. niger, A. aculeatus, P. chrysosporium and T. reesei can be used. Furthermore, examples of the second CBH include AncbhA composed of the amino acid sequence represented by SEQ ID NO: 3 derived from A. niger and AncbhB composed of the amino acid sequence represented by SEQ ID NO: 4. Moreover, AaCBHI which consists of an amino acid sequence represented by sequence number 5 derived from A. aculeatus is mentioned. Moreover, PcCBH7C which consists of an amino acid sequence represented by sequence number 6 derived from P. chrysosporium is mentioned. Among these, CBH derived from A. niger can be preferably used. Furthermore, as the second CBH, CBH of another aspect that can be acquired based on such known sequence information can be included. Such CBH will be described later. 2nd CBH can be used combining 1 or 2 or more from the above various CBH.

(First endoglucanase (EG))
The first EG is an EG belonging to GHF5. As the first EG belonging to GHF5, those derived from various microorganisms are known (http://www.cazy.org/fam/GH5.html). Especially, it can be set to 1 or 2 or more selected from EG derived from T. reesei and A. oryzae. Furthermore, as the first EG, for example, AocelE derived from A. oryzae consisting of the amino acid sequence represented by SEQ ID NO: 7 and TrEG II derived from T. reesei consisting of the amino acid sequence represented by SEQ ID NO: 8 are used. Can be mentioned. Further, the first EG can include an EG of another aspect that can be acquired based on such known sequence information. Such EG will be described later. 1st EG can be used combining 1 or 2 or more from the above various EGs.

(Additional enzyme)
The present composition may contain one or more enzymes selected from the enzymes sequentially described below. By containing any of these enzymes, the decomposition activity of cellulose by the combination of the first CBH, the second CBH, and the first EG can be effectively enhanced. Many enzymes that contribute to the degradation of cellulose, hemicellulose, and pectin, which are constituents of plant cell walls, are known. The enzymes added to the cellulase composition disclosed in the present specification all contribute to the degradation of cellulose, hemicellulose, etc., but with respect to the first CBH, the second CBH and the first EG. There are no reports on the combination and effects of the combination. Accordingly, each of these enzymes and combinations of two or more thereof have uses as effective enhancers for enzyme compositions comprising the first CBH, the second CBH and the first EG.

(Second endoglucanase (EG) belonging to GHF9)
The second EG is an EG belonging to GHF9. As the second EG belonging to GHF9, those derived from various organisms are known (http://www.cazy.org/fam/GH9.html). The second EG is a preferred enhancer among degrading enzymes such as cellulose. The first CBH, the second CBH, the first EG, and further the third EG can exert a synergistic effect directly upon cellulose degradation. The second EG is preferably derived from a plant, more preferably an EG derived from a plant of the genus Rice (O. sativa, etc.). As 2nd EG, EG derived from O. sativa which consists of an amino acid sequence represented by sequence number 9 is mentioned, for example. Further, the second EG can include an EG of another aspect that can be acquired based on such known sequence information. Such EG will be described later. The second EG can be used by combining one or two or more of the above EGs.

  Moreover, when the composition disclosed by this specification contains 2nd EG, it is preferable to contain 3rd EG mentioned later collectively. When the present composition contains the first to third EGs, a more preferable synergistic effect with the first CBH and the second CBH can be exhibited.

(Pectate lyase)
Pectate lyase (EC4.2.2.2.) Cleaves (1,4) -α-D-galacturonan contained in pectin and releases 4-deoxy-α-D-galacto at the non-reducing end. -4-Enuronosyl group-producing enzyme, an enzyme included in pectin-degrading enzymes. Pectate lyase is known to be derived from various organisms, among which pectate lyase derived from A. oryzae is mentioned. Examples of pectate lyase include AoPL19 derived from A. oryzae consisting of the amino acid sequence represented by SEQ ID NO: 10. The pectate lyase can include other forms of enzymes that can be obtained based on known sequence information. Such an enzyme will be described later. The pectate lyase can be used in combination of one or more of the above various pectate lyases.

(Xylanase)
Xylanase (EC3.2.1.8.) Is an enzyme that hydrolyzes the β-1,4 bond of β-1,4-D-xylan, a component of hemicellulose, and is an enzyme included in hemicellulase. . As xylanases, those derived from various organisms are known, and among them, xylanases derived from A. oryzae are mentioned. Examples of the xylanase include Aoxyn887 derived from A. oryzae consisting of the amino acid sequence represented by SEQ ID NO: 11, and Aoxyn139 derived from A. oryzae consisting of the amino acid sequence represented by SEQ ID NO: 12. As a xylanase, the enzyme of the other aspect which can be acquired based on well-known sequence information can be included. Such an enzyme will be described later. A xylanase can be used combining 1 or 2 or more from the above various xylanases.

(Arabinofuranosidase)
Arabinofuranosidase (EC.3.2.1.55) is an enzyme that cleaves arabinofuranoside at the non-reducing end of arabinoside, such as arabinoxylan and arabinan, which are components of hemicellulose, and is included in hemicellulase. It is. Although arabinofuranosidase derived from various organisms is known, among them, arabinofuranosidase derived from A. oryzae can be mentioned. Examples of the arabinofuranosidase include arabinofuranosidases (Aoabf20, Aoabf22, Aoabf18) derived from A. oryzae having the amino acid sequences represented by SEQ ID NOs: 13, 14, and 15, respectively. As arabinofuranosidase, the enzyme of the other aspect which can be acquired based on well-known sequence information can be included. Such an enzyme will be described later. The arabinofuranosidase can be used in combination of one or more of the above various arabinofuranosidases.

(Glucuronoxylan xylanohydrolase)
Glucuronoxylan xylanohydrolase is an enzyme that hydrolyzes β-1,4 bonds of glucuronoxylan, which is a constituent of hemicellulose, and is an enzyme included in hemicellulase. Glucuronoxylan xylanohydrolase is known to be derived from various organisms, and among them, those derived from B. subtilis are mentioned. Examples of glucuronoxylan xylanohydrolase include those derived from B. subtilis consisting of the amino acid sequence represented by SEQ ID NO: 16. The glucuronoxylan xylanohydrolase can include other forms of enzymes that can be obtained based on known sequence information. Such an enzyme will be described later. Glucuronoxylan xylanohydrolase can be used in combination of one or two or more of the above-mentioned various glucuronoxylan xylanohydrolases.

(Third endoglucanase (EG))
The present composition may contain a third endoglucanase belonging to GHF12 in addition to the first CBH, the second CBH and the first EG. As the third EG belonging to GHF12, those derived from various organisms are known (http://www.cazy.org/fam/GH12.html). Among these, one or more selected from EG derived from A. oryzae, EG derived from T. reesei, and EG derived from P. chrysosporium can be used. Preferably, it is EG derived from A. oryzae. Furthermore, as the third EG, for example, AocelA derived from A. oryzae consisting of the amino acid sequence represented by SEQ ID NO: 17, TrEG III derived from T. reesei consisting of the amino acid sequence represented by SEQ ID NO: 18, and P. chrysosporium comprising an amino acid sequence represented by SEQ ID NO: 19 (SEQ ID NO: 2 in JP-A-2009-247434, which may be an EG comprising the amino acid sequences of SEQ ID NOS: 4-8 disclosed in the publication) PcEG III derived from Further, the third EG can include an EG of another aspect that can be acquired based on such known sequence information. Such EG will be described later. The third EG can be used by combining one or two or more of the various EGs described above.

  A preferred enzyme to be used in the present composition is a protein having a certain relationship with known or new sequence information such as a certain amino acid sequence specified for the enzyme, and having a specific enzyme activity. May be.

  As one embodiment of such an enzyme, for example, a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in a specific amino acid sequence disclosed for a certain enzyme can be mentioned. Any one type of amino acid mutation, ie, deletion, substitution or addition, for each amino acid sequence may be used, or two or more types may be combined. The total number of these mutations is not particularly limited, but is preferably about 1 or more and 10 or less. More preferably, it is 1 or more and 5 or less. As an example of amino acid substitution, conservative substitution is preferable, and specific examples include substitution within the following groups. (Glycine, alanine) (valine, isoleucine, leucine) (aspartic acid, glutamic acid) (asparagine, glutamine) (serine, threonine) (lysine, arginine) (phenylalanine, tyrosine).

  Another embodiment includes a protein having an amino acid sequence having 70% or more identity to a specific amino acid sequence disclosed for an enzyme and having cellulase activity. The identity is preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, and still more preferably 95% or more. Most preferably, it is 98% or more.

  As used herein, identity or similarity is a relationship between two or more proteins or two or more polynucleotides determined by comparing sequences, as is known in the art. In the art, “identity” means between protein or polynucleotide sequences, as determined by alignment between protein or polynucleotide sequences, or in some cases by alignment between a series of such sequences. Means the degree of sequence invariance. Similarity is also the correlation between protein or polynucleotide sequences, as determined by alignment between protein or polynucleotide sequences, or in some cases by alignment between a series of partial sequences. Means the degree of More specifically, it is determined by sequence identity and conservation (substitutions that maintain specific amino acids in the sequence or physicochemical properties in the sequence). The similarity is referred to as Similarity in the BLAST sequence homology search result described later. The method for determining identity and similarity is preferably a method designed to align the longest between the sequences to be compared. Methods for determining identity and similarity are provided as programs available to the public. For example, BLAST (Basic Local Alignment Search Tool) program by Altschul et al. (Eg Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. Biol., 215: p403-410 (1990), Altschyl SF, Madden TL, Schaffer AA, Zhang J, Miller W, Lipman DJ., Nucleic Acids Res. 25: p3389-3402 (1997)). The conditions for using software such as BLAST are not particularly limited, but it is preferable to use default values.

  In yet another aspect, the present invention is encoded by a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to a DNA consisting of a base sequence encoding a specific amino acid sequence disclosed for a certain enzyme. Examples include proteins having cellulase activity. The stringent condition refers to, for example, a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. For example, a nucleic acid having a high base sequence identity, that is, a DNA comprising a base sequence having 80% or more, preferably 85% or more, more preferably 90% or more, and most preferably 95% or more identity with a predetermined base sequence In which the complementary strands of the nucleic acids hybridize and the complementary strands of nucleic acids having lower homology do not hybridize. More specifically, the sodium salt concentration is 15 to 750 mM, preferably 50 to 750 mM, more preferably 300 to 750 mM, the temperature is 25 to 70 ° C., preferably 50 to 70 ° C., more preferably 55 to 65 ° C., formamide The condition is that the concentration is 0 to 50%, preferably 20 to 50%, more preferably 35 to 45%. Furthermore, under stringent conditions, the filter washing conditions after hybridization are usually such that the sodium salt concentration is 15 to 600 mM, preferably 50 to 600 mM, more preferably 300 to 600 mM, and the temperature is 50 to 70 ° C., preferably It is 55-70 degreeC, More preferably, it is 60-65 degreeC. In addition, from the above, as another embodiment, it has a base sequence having 80% or more, preferably 85% or more, more preferably 90% or more, and most preferably 95% or more identity with a predetermined base sequence. Examples include proteins encoded by DNA and having cellulase activity.

  This composition may contain only 1st CBH, 2nd CBH, 1st EG, and 2nd EG, and contains only one each of these enzymes. Also good. Moreover, it may contain only 1st CBH, 2nd CBH, 1st EG, 2nd EG, and 3rd EG, and it contains only each of these each 1 type. It may be. According to this composition, since these CBH and EG can exhibit a good synergistic effect on cellulose degradation, cellulose can be effectively decomposed despite such a minimal cellulase composition. This can contribute to cost reduction of the saccharification process.

  In the present composition, the first CBH, the second CBH, the first EG, and the third EG may all be derived from T. reesei. Typically, these enzymes may be commercially available cellulase formulations from T. reesei. The various additional enzymes in the present composition can also act effectively on cellulase preparations derived from T. reesei.

  This composition may contain (beta) -glucosidase (BGL), and does not need to contain substantially. When the enzyme preparation of the present invention does not substantially contain BGL, glucose produced by BGL is preferable in that it does not cause product inhibition with respect to other cellulases such as CBH. Therefore, if it is an enzyme preparation which does not substantially contain BGL, it is possible to reliably avoid product inhibition and obtain the synergistic effect of cellulose degradation confirmed by the inventors in the evaluation system even in the composition. Moreover, the partial decomposition products, such as a cellobiose, can be efficiently produced from a cellulose. Furthermore, in particular, when using the present composition for CBP (consolidated bioprocess (simultaneous progress of saccharification and fermentation)) for simultaneously proceeding saccharification and fermentation, for example, when using a fermenting microorganism displaying BGL on the surface, It is advantageous that the composition is not contained.

  In addition, it does not contain BGL substantially means that it may contain the amount of BGL of the range which can avoid or suppress the product inhibition by BGL besides containing BGL. The composition preferably does not contain BGL. The present composition which does not substantially contain BGL includes, for example, a microorganism that does not retain an endogenous BGL gene (such as yeast), a microorganism that has an endogenous BGL gene, but whose expression of the gene is substantially low, or a microorganism that has been destroyed. It can be easily obtained from a culture (culture supernatant) of a transformant into which genes encoding various enzymes disclosed in the present specification have been introduced.

  The present composition may contain the aforementioned various enzymes as purified ones, or may contain other proteins or other components as unpurified proteins such as culture supernatant. The form of the preparation is not particularly limited, and may be a solid preparation (powder (lyophilized body, etc.), tablet, granule, etc.) or a solution (a frozen body during distribution). ).

  The composition disclosed herein is for cellulose degradation. In the present specification, examples of cellulose include a polymer obtained by polymerizing glucose through β-1,4-glucoside bonds and derivatives thereof. The degree of polymerization of glucose is not particularly limited. In addition, examples of the derivative include derivatives such as carboxymethylation, aldehyde formation, or esterification. Moreover, the cellulose may contain the partial decomposition product. Further, the cellulose may be a complex with β-glucoside, which is a glycoside, lignocellulose, which is a complex with lignin and / or hemicellulose, and pectin. The cellulose may be crystalline cellulose or non-crystalline cellulose. The origin of cellulose is not particularly limited, and may be naturally derived or artificially synthesized.

  Cellulose is usually subjected to decomposition as a cellulose-containing material containing a coexisting component in addition to cellulose. Cellulose-containing materials need only contain at least cellulose, natural fiber products such as cotton and hemp, recycled fiber products such as rayon, cupra, acetate and lyocell, lignocellulosic agricultural products such as rice straw, rice husk and wood chips. So-called real biomass such as waste may be used, or a pre-processed product thereof. The cellulose may be a pretreated cellulose or a cellulose-containing material. The pretreatment includes, for example, a treatment that relaxes or cancels the composite state or a treatment that lowers the crystallinity of crystalline cellulose with respect to a material in which lignin and hemicellulose coexist with cellulose. It is done. Examples of such treatment include hydrothermal treatment and treatment with an ionic liquid. Examples of the hydrothermal treatment include treating at a temperature of 180 ° C. to 240 ° C. for about 30 minutes to 90 minutes. Examples of the treatment with the ionic liquid include a treatment such as immersion in a hydrophobic or hydrophilic ionic liquid at 60 ° C. to 150 ° C. for about 30 minutes to 2 hours.

  The composition is suitable for a biomass material that has been subjected to hydrothermal treatment or pretreatment with an ionic liquid. The enzyme composition of the present composition acts synergistically on the cellulose material in which the composite form is relaxed to some extent, and the decomposition activity of cellulose can be enhanced.

(Method for producing cellulase composition)
The production method of the present composition is not particularly limited. For example, various enzymes may be prepared and mixed, respectively, or may be produced from a culture such as a culture supernatant obtained by culturing a transformant that co-expresses two or more enzymes. Furthermore, the form which combines these suitably may be sufficient.

  These various enzymes can be synthesized by known protein synthesis methods or genetic engineering. For example, it can be expressed in a transformant obtained by introducing a gene encoding the above enzyme into a suitable host microorganism. Such genes are, for example, PCR using primers extracted based on a base sequence encoding a specific enzyme and using DNA extracted from a predetermined yeast, nucleic acids derived from various cDNA libraries or genomic DNA libraries as a template. By performing amplification, it can be obtained as a nucleic acid fragment. Further, it can be obtained as a nucleic acid fragment by performing hybridization using a nucleic acid derived from the above-mentioned library or the like as a template and a DNA fragment which is a part of a gene of various enzymes as a probe. Alternatively, an enzyme gene may be synthesized as a nucleic acid fragment by various nucleic acid sequence synthesis methods known in the art such as chemical synthesis methods. Furthermore, the gene corresponding to the enzyme of the embodiment into which the mutation has been introduced may be, for example, a DNA that encodes the amino acid sequence of the enzyme by molecular mutation using conventional mutagenesis, site-directed mutagenesis, or error-prone PCR. It can be obtained by modifying it by a technique or the like. Examples of such methods include known methods such as the Kunkel method or the Gapped duplex method, or a method similar thereto, such as a mutation introduction kit (for example, Mutant-K (Takara Bio Inc.) using site-directed mutagenesis). Or Mutant-G (manufactured by TAKARA)), or the TAKARA LA PCR in vitro Mutagenesis series kit. In addition, those skilled in the art should refer to Molecular Cloning (Sambrook, J. et al., Molecular Cloning: a Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, 10 Skyline Drive Plainview, NY (1989)). Thus, various types of genes can be obtained based on the nucleotide sequence disclosed for the enzyme.

  In obtaining transformants for genetic engineering of various aspects of the enzyme, appropriately refer to the methods described in the aforementioned Molecular Cloning, etc., various conventionally known methods such as transformation methods, Transfection methods, conjugation methods, protoplast methods, electroporation methods, lipofection methods, lithium acetate methods and the like can be used.

  The host of the transformant is not particularly limited, and various prokaryotic microorganisms and eukaryotic microorganisms can be used. However, yeasts and koji molds that have established a genetic recombination system and fermentation technology can be preferably used. When a host produces BGL, its expression may be suppressed. That is, a host that has an endogenous BGL gene but the gene has been destroyed can be used, or a host that does not have an endogenous BGL gene (for example, a cellulase-nonproducing microorganism described later) can be used. This is because BGL suppresses cellulose degradation by inhibiting the product as already described. Such a transformant is preferable for the present composition, particularly for the present composition for reducing the molecular weight of cellulose to an oligomer. Those skilled in the art can appropriately destroy the specific gene.

  The host is preferably a cellulase non-producing microorganism. A cellulase non-producing microorganism is a substance that does not have a cellulase gene on its genome, and even if it has such a gene, its expression level and expression form (conditions for inducing expression, etc.) are considered substantially. To the extent that it can be said that cellulase is not produced. Cellulase non-producing microorganisms have the advantage that they are easily expressed at high levels by adjusting the expression level of CBH or EG introduced as a foreign gene. That is, since no cellulase other than the preferred combination is produced, only the cellulase suitable for the degradation of cellulose intended in the present specification can be expressed as much as possible. Such a transformant is advantageous for producing a cellulase composition capable of exerting a strong synergistic effect.

  Examples of microorganisms that do not produce cellulase include yeasts of the genus Saccharomyces such as Saccharomyces cerevisiae, yeasts of the genus Schizosaccharomyces such as Schizosaccharomyces pombe, yeasts of the genus Candida such as Candida shehatae, yeasts of the genus Pichia such as Pichia stipitis, yeasts of the genus Pichia such as Hansenula And yeast of the genus Trichosporon, yeast of the genus Brettanomyces, yeast of the genus Pachysolen, yeast of the genus Yamadazyma, yeast of the genus Kluveromyces such as Kluyveromyces marxianus, Kluveromyces lactis. Yeast is a microorganism that does not have a cellulase gene on its genome.

  As the cellulase non-producing microorganism, it is preferable to use various koji molds used industrially such as Aspergillus oryzae. Examples of the genus Aspergillus including Aspergillus oryzae include Aspergillus aculeatus, Aspergillus niger, Aspergillus oryzae, Aspergillus awamori, Aspergillus nidulans, Aspergillus kawachi, Aspergillus saitoi and the like. Neisseria gonorrhoeae are microorganisms that have a cellulase gene on the genome but whose expression is suppressed or only expressed under special inducing conditions. Therefore, in producing the cellulase disclosed herein, there is substantially no influence of the expression of the endogenous cellulase gene of Neisseria gonorrhoeae. Even if the endogenous cellulase gene is expressed depending on the conditions, the endogenous cellulase gene may be destroyed as necessary.

  Thus, in the transformant producing cellulase and the like disclosed in the present specification, it is preferable that the expression of endogenous cellulase genes other than the combinations suitable in the present disclosure is suppressed. However, the expression of the gene is allowed as long as it does not inhibit the synergistic effect of the combination such as cellulase disclosed in the present specification.

  In the above transformant, such a combination of cellulase and the like may be expressed intracellularly, or may be constructed so as to be retained on the cell surface or secreted extracellularly. The transformant or the culture supernatant can be directly used as a cellulase composition as long as the form is retained on the cell surface or secreted to the outside of the cell. The form secreted extracellularly is also advantageous for obtaining the enzyme from the culture supernatant.

  One aspect of the method for producing cellulase disclosed in the present specification is a method for producing a protein such as cellulase, in which a gene encoding one or more proteins is introduced into a cellulase-nonproducing microorganism, And the step of producing the enzyme. Cellulase non-producing microorganisms can be good hosts for producing foreign proteins. It is particularly suitable for the production of cellulose-derived organism-derived proteins that metabolize plant cell wall components such as cellulase, hemicellulase, and pectin-degrading enzyme, and in particular contributes to the degradation of cellulose such as cellulase, hemicellulase, and pectin-degrading enzyme. It is preferable that it is a protein. The cellulose-producing microorganism is not particularly limited, and examples thereof include P. chrysosporium, B. subtilis and O. sativa. Moreover, although the kind of cellulase suitable for expression is not particularly limited, xylanase can be mentioned in addition to EGH belonging to GHF6, GHF7, EG belonging to GHF5, 9, 12, and the like. For example, there are CBH I, CBH II belonging to P. chrysosporium-derived GHF6 or 7, glucuronoxylan xylanohydrolase derived from B. subtilis, EG derived from O. sativa belonging to GHF9, and EG derived from P. chrysosporium belonging to GHF12. Can be mentioned.

  As the cellulase non-producing microorganisms, the microorganisms already described can be used, but Aspergillus bacteria and yeasts are preferably used. According to this production method, an exogenous cellulase can be efficiently produced in order to use Aspergillus bacteria as a host. In addition, although the proteins derived from the above various genera have a small amount of production in Aspergillus genus with the codons as they are, the GC content and the ATGC ratio are adjusted for the frequently used codons based on the codon usage of the host Aspergillus genus. The production amount can be remarkably increased by using it. As for the codon usage frequency of the genus Aspergillus, it is preferable to use a codon suitable for Aspergillus genus bacteria as a host. For example, Aspergillus oryzae is disclosed on the site of the Codon Usage Database (http://www.kazusa.or.jp/codon/) of the Kazusa DNA Laboratory Things can be used. Also,

  From the above, as an embodiment of the transformant of the cellulase non-producing microorganism disclosed in the present specification, the following foreign protein: one or two or more cellulases such as CBH belonging to GHF6 derived from the genus Phanerochaete, the genus Phanerochaete Cellulase such as 1 or 2 or more CBH belonging to GHF7 derived from genus, Cellulase such as 1 or 2 or more EG belonging to GHF12 derived from genus Phanerochaete, Cellulase such as EG belonging to GHF9 derived from genus Oryza, and Bacillus genus A transformant transformed with an expression vector for expressing a gene encoding one or more proteins selected from the group consisting of hemicellulases derived from is provided. The cellulase non-producing microorganism may be a yeast or an Aspergillus genus. When using Aspergillus sp., Transformants obtained by transforming with expression vectors for expressing modified genes using codons with high usage frequency based on codon usage in Aspergillus sp. It is preferable.

(Production method of cellulose low molecular weight product)
The method for producing a low molecular weight product of cellulose disclosed in the present specification can comprise a step of decomposing cellulose using the cellulase composition disclosed in the present specification. According to this method, it is possible to efficiently reduce the molecular weight of cellulose and produce cellulose oligomers or glucose. In addition, it is preferable to use β-glucosidase to reduce the molecular weight to glucose.

  In order to efficiently decompose cellulose into cellulose oligomers in the molecular weight reduction step, it is preferable to decompose cellulose in the substantial absence of BGL. By carrying out like this, the influence of the product inhibition by BGL can be avoided or suppressed. Note that “in the substantial absence of BGL” means that BGL may be present within a range in which BGL is not present and product inhibition by BGL can be avoided or suppressed. In order to obtain a cellulose oligomer, it is preferable that BGL does not exist in this enzyme reaction system.

  The molecular weight reduction step can be a step in which BGL is supplied and decomposed with respect to a decomposition product obtained using the present composition substantially free of BGL in the absence of BGL. By doing so, cellulose can be efficiently decomposed into glucose.

  The combination of each enzyme contained in the present composition used in the production method of the present invention may be provided as the present composition, or secrete and express such enzymes extracellularly (including cell surface display form) 1 or A combination of two or more transformants may be provided, or a combination thereof may be provided. The transformant may co-express two or more enzymes.

  The cellulose may be in the form of a cellulose-containing material containing a coexisting component other than cellulose. Cellulose may be pretreated with hydrothermal treatment or ionic liquid.

(Useful substance production method)
Disclosed herein is a method for producing a useful substance by fermentation of a microorganism, wherein at least the composition is used to decompose cellulose to a carbon source usable by the microorganism, and the carbon And fermenting a source with the microorganism to produce the useful substance. According to this production method, since cellulose is efficiently decomposed by a composition suitable for the decomposition of cellulose, a saccharification step can be performed at low cost, and as a result, useful substances can be produced from cellulose at low cost. .

  The decomposition step may be performed in the substantial absence of BGL when cellulose is reduced to an oligomer. On the other hand, as already explained, when the molecular weight is reduced to glucose, it is carried out in the presence of BGL. As will be described later, when yeast or the like that displays BGL on the surface is used in the production process of useful substances, the decomposition process is preferably carried out in the substantial absence of BGL. In addition, the said decomposition | disassembly process includes the various aspects in the decomposition | disassembly process in the production method of the low molecular weight thing of the cellulose disclosed by this specification.

  The microorganism used in the production process of the useful substance is not particularly limited, and examples thereof include ethanol-producing microorganisms such as yeast and organic acid-producing microorganisms such as lactic acid bacteria. Any of these may be artificially obtained microorganisms. For example, it may be genetically engineered so that it can produce a compound that is not the original metabolite obtained by replacing or adding one or more enzymes in the metabolic system from glucose by genetic recombination. Good. By using such microorganisms, for example, biochemicals such as the production of fine chemicals (coenzyme Q10, vitamins and their raw materials, etc.) by adding an isoprenod synthesis pathway, the production of glycerin by modification of glycolysis, and the raw materials for plastics and chemical products. Applicable to refinery technology. Although it does not specifically limit as a useful substance, The thing which can produce | generate microorganisms using glucose is preferable. As noted above, materials across biorefinery technology can be targeted. In the present specification, the “organic acid” is an organic compound that exhibits acidity, and is a free acid or a salt thereof. The acidic group included in the “organic acid” is preferably a carbonoxyl group. Examples of such “organic acids” include lactic acid, butyric acid, acetic acid, pyruvic acid, succinic acid, formic acid, malic acid, citric acid, malonic acid, propionic acid, ascorbic acid, and adipic acid. These “organic acids” may be DL form in addition to D form and L form. The “organic acid” is preferably lactic acid.

  The microorganism used in the production process of the useful substance is preferably a microorganism that expresses BGL extracellularly so that a partial degradation product derived from cellulose by the composition can be used as a carbon source. By carrying out like this, the production process of this useful substance can be implemented as the same decomposition process. That is, saccharification and fermentation can be performed in the same process. By avoiding product inhibition by glucose, it is possible to produce useful substances by microorganisms by fermentation while efficiently saccharifying cellulose. The microorganism that expresses BGL extracellularly preferably presents BGL on the cell surface. Examples of the partially decomposed product derived from cellulose include cellulose oligomers and cellobiose.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these Examples. The gene recombination operation described below was performed according to the previously described Molecular Cloning.

(Construction of Neisseria gonorrhoeae expression plasmid)
<Incorporation of niaD gene into E. coli vector>
Aspergillus genomic DNA is obtained using primer A (SEQ ID NO: 21) and primer B (SEQ ID NO: 22) so that the nitrate reductase gene niaD (SEQ ID NO: 20) derived from Aspergillus oryzae becomes a Pst I-Hind III fragment. Using LA-Taq (Takara Bio Inc.) as a template, PCR (96 ° C (5 minutes) for 1 cycle, 96 ° C (20 seconds), 60 ° C (30 seconds) and 72 ° C (5 minutes) for 30 cycles, Amplification was performed at 72 ° C. (7 minutes).

  The obtained PCR amplification product was treated with the restriction enzyme Pst I-Hind III at 37 ° C. and then cut out by agarose gel electrophoresis. For the cutting, QIAquick Gel Extraction Kit (QIAGEN) was used. E. coli plasmid pUC119 (Takara Bio Inc.) was ligated using DNA ligation Kit (Takara Bio Inc.) and transformed into E. coli JM109 strain. As a result, plasmid pNIA2 in which the niaD marker was subcloned was obtained. The plasmid pNIA2 is constructed so that the gene can be introduced into both PstI and SalI sites using the gene as a unique site.

<Incorporation of glaB terminator>
An attempt was made to insert the gene of gonococcal glaB terminator (SEQ ID NO: 23) into pNI2. One cycle of PCR (96 ° C. (5 minutes)) using LA-Taq (Takara Bio Inc.) using Primer C (SEQ ID NO: 24) and Primer D (SEQ ID NO: 25) and DNA of Aspergillus genome as a template , 96 ° C. (20 seconds), 60 ° C. (30 seconds) and 72 ° C. (5 minutes) by 30 cycles, and 72 ° C. (7 minutes) by 1 cycle).

  The obtained PCR amplification product was treated with the restriction enzyme SalI-XhoI at 37 ° C. and then cut out by agarose gel electrophoresis. For the cutting, QIAquick Gel Extraction Kit (QIAGEN) was used. The SalI site of E. coli plasmid pNIA2 was ligated using DNA ligation Kit (Takara Bio Inc.) and transformed into E. coli JM109 strain. As a result, plasmid pNIAT in which the slaB terminator was subcloned was obtained. The plasmid pNIAT is constructed so that the gene can be introduced into both PstI and SalI sites as a unique site.

<Incorporation of sodM promoter>
An attempt was made to insert a gene of the sodM promoter (SEQ ID NO: 26) derived from Aspergillus into pNIA2. One cycle of PCR (96 ° C. (5 minutes)) using LA-Taq (Takara Bio Inc.) using Primer E (SEQ ID NO: 27) and Primer F (SEQ ID NO: 28), and DNA of Aspergillus genome as a template , 96 ° C. (20 seconds), 60 ° C. (30 seconds) and 72 ° C. (5 minutes) by 30 cycles, and 72 ° C. (7 minutes) by 1 cycle).

  The obtained PCR amplification product was treated with the restriction enzyme SalI-PstI at 37 ° C. and then cut out by agarose gel electrophoresis. For the cutting, QIAquick Gel Extraction Kit (QIAGEN) was used. The PstI-SalI site of the E. coli plasmid pNIAT was ligated using a DNA ligation Kit (Takara Bio Inc.) and transformed into E. coli JM109 strain. As a result, a plasmid pNMB in which the sodM terminator was subcloned was obtained. Plasmid pNMB is constructed such that a gene encoding a protein to be expressed in Aspergillus oryzae can be introduced into SalI site as a unique site.

<Incorporation of target gene>
(1) Preparation of vector
After treating pNMB with restriction enzyme SalI at 37 ° C., dNTP was added to a final concentration of 10 mM and treated with T4 DNA polymerase (Takara Bio) at 37 ° C. for 1 hour. Furthermore, it was made to react at 50 degreeC for 30 minutes using alkaline phosphatase derived from bacteria (Takara Bio Inc.). The obtained reaction product was processed and eluted with a PCR cleanup column (Promega) to obtain a vector.
(2) Preparation of Insert The gene encoding the protein to be expressed in Aspergillus oryzae shown in Table 1 was used as an insert for subcloning into the above vector. Using the primer of the sense strand 30 bp downstream from the start codon of the target gene (phosphorylation at the 5 ′ end) and the primer of the antisense strand 30 bp upstream from the stop codon of the target gene (phosphorylation at the 5 ′ end) PCR (96 ° C. (5 minutes), 1 cycle, 96 ° C. (20 seconds), 60 ° C. (30 seconds) and 72 ° C. using pfu Taq polymerase (Toyobo Co., Ltd.) using genomic DNA derived from the gene origin organism as a template. (5 minutes) for 30 cycles and 72 ° C. (7 minutes) for 1 cycle). The obtained PCR fragment was processed and eluted with a PCR cleanup column (Promega) to obtain an insert.

(3) Ligation of vector and insert Add in a molar ratio of vector: insert = 1: 20, ligate using DNA ligation Kit (Takara Bio), and transform into E. coli JM109 strain. Converted. A plasmid was prepared from the transformant, and a gene fragment in which the reading frame of the target gene was subcloned in the forward direction with the sodM promoter was prepared as a target gene expression plasmid.

(Preparation of target gene expression strain)
In this example, enzyme gene expression strains shown in Table 1 were prepared. A. oryzae niaD Mutant Co., Ltd. (Independent Administrative Institution, National Institute of Advanced Industrial Science and Technology) It has been deposited as FERM P-17707 at the Research Institute Patent Biological Deposit Center). Czapek-Dox medium using nitric acid as a single nitrogen source (2% glucose, 0.1% dipotassium hydrogen phosphate, 0.05% potassium chloride, 0.05% magnesium sulfate, 0.001 By selecting a strain capable of growing in (iron sulfate, 0.3% sodium nitrate), a plurality of transformants carrying the target gene expression plasmid were obtained.

(Preparation of target gene product)
The transformant expressing the enzyme gene prepared in Example 2 was sporulated with potato dextrose medium, and the spores were collected with sterile water. 100 ml GPY liquid medium (2% glucose, 1% polypeptone, 0.5% yeast extract, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, 0.001% sulfuric acid in a 500 ml Erlenmeyer flask Iron, 0.3% sodium nitrate) was inoculated to a final spore concentration of 1 × 10 ⁶ / ml. The target gene product was secreted and expressed in the medium in liquid culture at 30 ° C. for 3 days, and the culture solution was used as an enzyme sample. Table 2 shows the results of estimating the protein expression level by SDS-PAGE using the enzyme sample solution thus obtained.

(Degradation evaluation of commercially available enzyme preparation of cellulose derived from rice straw after hydrothermal treatment and this composition)
50 mM acetic acid buffer (pH 5.0) and Composition 1 were added to the cellulose fraction of rice straw after hydrothermal treatment heated at 200 ° C. for 30 minutes so as to be 12 mg / g biomass, and after 0 to 48 hours at 45 ° C. The glucose concentration in the solution was measured by liquid chromatography (HPLC). As a positive control, a commercially available enzyme preparation (Sigma Crust C2730 manufactured by Sigma) added to 12 mg / g biomass was used.
(Composition 1)
CBH 5% from Phanerochaete chrysosporium belonging to GHF6
CBH 50% derived from Aspergillus niger belonging to GHF7
10% EG derived from Aspergillus oryzae belonging to GHF5
30% EG from Aspergillus oryzae belonging to GHF12
5% EG from Oryza sativa belonging to GHF9

  In order to measure glucose as a final product by liquid chromatography, 1/5 amount of BGL (Novo188), which is the manufacturer's recommendation, was added to the commercially available enzyme preparation, and all of the oligomerized cellulose was converted to glucose. Similarly, the same BGL was added to the reaction solution of the enzyme composition, and the amount of glucose was measured. The saccharification result is shown in FIG. As shown in FIG. 1, it was found that the enzyme composition had a saccharification effect equivalent to that of a commercially available enzyme preparation.

(Degradation evaluation of eucalyptus-derived cellulose after ionic liquid treatment with commercially available enzyme preparation and enzyme composition)
Pretreatment of eucalyptus with ionic liquid was performed as follows. 1.0 g of ionic liquid 1-Ethyl-3-methylimidazoliumacetate (hereinafter referred to as [Emim] [Ac]) (Kanto Chemical Co., Inc.) was collected in a vial, and 30 mg of a biomass sample was added thereto. As biomass, eucalyptus powder having a particle size of 250 μg, which was crushed by a cutter mill, was used. The sample was treated under static conditions at 120 ° C. for 1 hour, and then washed with 15 ml of sterilized water. Further, it was washed with 10 ml of 50 mM acetate buffer pH 5.0 and used for the following experiments. To the cellulose fraction derived from eucalyptus after the ionic liquid treatment, 50 mM acetate buffer pH 5.0, enzyme composition was added to 10 mg / g biomass, and the glucose concentration of the solution was adjusted after reaction at 50 ° C. for 0 to 24 hours. Measured by liquid chromatography. As a positive control, a commercially available enzyme preparation (Sigma Crust C2730 from Sigma) added to 10 mg / g biomass was used. The enzyme composition was as follows.
(Composition 1)
CBH 5% from Phanerochaete chrysosporium belonging to GHF6
CBH 50% derived from Aspergillus niger belonging to GHF7
10% EG derived from Aspergillus oryzae belonging to GHF5
30% EG from Aspergillus oryzae belonging to GHF12
5% EG from Oryza sativa belonging to GHF9
(Composition 2)
CBH 5% from Phanerochaete chrysosporium belonging to GHF6
CBH 50% derived from Aspergillus niger belonging to GHF7
40% EG from Trichoderma reesei belonging to GHF5
5% EG from Oryza sativa belonging to GHF9

  In order to measure glucose as a final product by HPLC, 1/5 amount of BGL (Novo188), which is the manufacturer's recommended amount, was added to the commercially available enzyme preparation and the amount of glucose was measured. Similarly, the same amount of BGL (Novo188) was also added to the enzyme composition reaction solution, and the amount of glucose was measured. As shown in FIG. 2, when the enzyme composition was added, the amount of glucose produced was high in the early stage (up to the seventh hour) after the addition, and the composition was compared with the commercially available enzyme preparation at the fourth hour. The product 1 showed high saccharification ability about 2 times, and the composition 2 about 1.5 times. The amount of glucose produced at 24 hours was equivalent to the commercially available enzyme preparation. Since saccharification is efficiently performed at an early stage after addition, eucalyptus treated with ionic liquid is more susceptible to cellulase attack than cellulosic hydrothermally treated rice straw. Was guessed. In addition, when compositions 1 and 2 are compared, composition 1 has a higher activity, indicating that EG of Aspergillus oryzae-derived GHF5,12 has a higher effect of addition than EG from Trichoderma reesei of GHF5. It was.

(Comparative evaluation of EG types in enzyme composition)
50 mM acetate buffer pH 5.0, Compositions 1 and 2 used in Example 5 were added to the cellulose fraction of rice straw after hydrothermal treatment to give 10 mg / g biomass, and the mixture was heated at 50 ° C. for 0 to 24 hours. After the reaction, the glucose concentration of the solution was measured by HPLC. As a positive control, a commercially available enzyme preparation (Sigma Crust C2730 from Sigma) added to 10 mg / g biomass was used.

  In order to measure glucose as a final product by HPLC, 1/5 amount of BGL (Novo188), which is the manufacturer's recommended amount, was added to the commercially available enzyme preparation and the amount of glucose was measured. Similarly, the same amount of BGL (Novo188) was also added to the enzyme composition reaction solution, and the amount of glucose was measured. As shown in FIG. 3, when the enzyme cocktail was added, the amount of glucose produced was slightly lower than that of the commercially available enzyme preparation in the early stage after the addition (up to the 7th hour). The amount of glucose produced by the eyes was higher than that of the commercially available enzyme preparation. Further, comparing Compositions 1 and 2, it can be seen that EG of Aspergillus oryzae-derived GHF5,12 has a higher additive effect than Trichoderma reesei-derived EG of GHF5 because composition 1 is more active. It was.

(Comparative evaluation of the amount of CBH in the enzyme composition)
To the cellulose fraction of rice straw after hydrothermal treatment, 50 mM acetate buffer pH 5.0, the following compositions 1 and 3 were added so as to be 12 mg / g biomass, and the solution was reacted at 45 ° C. for 0 to 24 hours. The glucose concentration of was measured by HPLC. As a positive control, a commercially available enzyme preparation (Cell Crust C2730 from Sigma) added to 12 mg / g biomass was used. The composition of the enzyme composition was as follows.
(Composition 1)
CBH 5% from Phanerochaete chrysosporium belonging to GHF6
CBH 50% derived from Aspergillus niger belonging to GHF7
10% EG derived from Aspergillus oryzae belonging to GHF5
30% EG from Aspergillus oryzae belonging to GHF12
5% EG from Oryza sativa belonging to GHF9
(Composition 3)
10% CBH from Phanerochaete chrysosporium belonging to GHF6
CBH 50% derived from Aspergillus niger belonging to GHF7
10% EG derived from Aspergillus oryzae belonging to GHF5
EG from Aspergillus oryzae belonging to GHF12 25%
5% EG from Oryza sativa belonging to GHF9

  In order to measure glucose as a final product by HPLC, 1/5 amount of BGL (Novo188), which is the manufacturer's recommended amount, was added to the commercially available enzyme preparation and the amount of glucose was measured. Similarly, the same amount of BGL (Novo188) was also added to the enzyme composition reaction solution, and the amount of glucose was measured. As a result, as shown in FIG. 4, in composition 1, the amount of glucose produced is larger than that in composition 3, so that the glycation ability is higher when the proportion of CBH derived from P. chrysosporium in GHF6 is 10% than 5%. Was found to be expensive.

(Comparative evaluation of types of CBH in enzyme composition)
(Comparative evaluation of the amount of CBH in the enzyme composition)
To the cellulose fraction of rice straw after hydrothermal treatment, 50 mM acetate buffer pH 5.0, the following composition 4 was added to give 22, 44, and 90 mg / g biomass, and composition 5 was added to 90 mg / g biomass. After the reaction at 45 ° C. for 0 to 48 hours, the glucose concentration of the solution was measured by HPLC. As a positive control, a commercially available enzyme preparation (Cell Crust C2730 from Sigma) added to 12 mg / g biomass was used. The composition of the enzyme composition was as follows.
(Composition 4)
CBH 5% from Phanerochaete chrysosporium belonging to GHF6
CBH 40% derived from Aspergillus niger belonging to GHF7
55% EG from Trichoderma reesei belonging to GHF5
(Composition 5)
CBH 5% from Aspergillus oryzae belonging to GHF6
CBH 40% derived from Aspergillus niger belonging to GHF7
55% EG from Trichoderma reesei belonging to GHF5

  In order to measure glucose as a final product by HPLC, 1/5 amount of BGL (Novo188), which is the manufacturer's recommended amount, was added to the commercially available enzyme preparation and the amount of glucose was measured. Similarly, the same amount of BGL (Novo188) was also added to the enzyme composition reaction solution, and the amount of glucose was measured. As shown in FIG. 5, when composition 4 is added, the amount of glucose produced increases as the amount of enzyme increases, and when compositions 4 and 5 are compared at 90 mg / g addition, In addition, the glucose production amount of composition 5 (when 90 mg / g was added) was equivalent to that of composition 4 (when 44 mg / g was added), and composition 5 was about half that of composition 4 It was found that there was only a certain degree of activity. Compositions 4 and 5 differed in CBH of GHF6, and it was found that the cellulolytic activity when Phanerochaete chrysosporium-derived CBH was added was higher than that of Aspergillus oryzae-derived CBH.

(Additional effect of glucuronoxylan xylanohydrolase)
To the cellulose fraction of rice straw after hydrothermal treatment, 50 mM acetate buffer pH 5.0, the following compositions 6 and 7 were added to 12 mg / g biomass, and the solution was reacted at 45 ° C. for 0 to 24 hours. The glucose concentration of was measured by HPLC. As a positive control, a commercially available enzyme preparation (Cell Crust C2730 from Sigma) added to 12 mg / g biomass was used. The composition of the enzyme composition was as follows.
(Composition 6)
10% CBH from Phanerochaete chrysosporium belonging to GHF6
CBH 40% derived from Aspergillus niger belonging to GHF7
15% EG derived from Aspergillus oryzae belonging to GHF5
30% EG from Aspergillus oryzae belonging to GHF12
5% EG from Oryza sativa belonging to GHF9
(Composition 7)
10% CBH from Phanerochaete chrysosporium belonging to GHF6
CBH 40% derived from Aspergillus niger belonging to GHF7
15% EG derived from Aspergillus oryzae belonging to GHF5
EG from Aspergillus oryzae belonging to GHF12 25%
5% EG from Oryza sativa belonging to GHF9
5% Glucuronoxylan Xylanohydrolase from B. subtilis belonging to GHF30

  In order to measure glucose as a final product by HPLC, 1/5 amount of BGL (Novo188), which is the manufacturer's recommended amount, was added to the commercially available enzyme preparation and the amount of glucose was measured. Similarly, the same amount of BGL (Novo188) was also added to the enzyme composition reaction solution, and the amount of glucose was measured. As shown in FIG. 6, the results show that the amount of glucose produced in the composition 7 is larger than that in the composition 6, so that the addition of GHF30 Bacillus subtilis-derived glucuronoxylan xylanohydrolase further improves the saccharification efficiency. It was done.

(Additional effect of various hemicelluloses)
To the cellulose fraction of rice straw after hydrothermal treatment, a commercially available enzyme preparation (Sercrust C2730 from Sigma) was added to 200 mg / g biomass, and the peptate lyase derived from Aspergillus oryzae listed in Table 1 Culture supernatants of various Aspergillus oryzae transformants expressing various enzymes were added to 2% (w / v), and the glucose concentration of the solution was measured by HPLC after reaction at 50 ° C.

  In order to measure glucose as a final product by HPLC, 1/5 amount of BGL (Novo188), which is the manufacturer's recommended amount, was added to the commercially available enzyme preparation and the amount of glucose was measured. Similarly, as a positive control, a commercially available enzyme preparation, BGL and 2% commercially available xylanase (Hampton from Trichoderma reesei) added, and a commercially available enzyme preparation and BGL added were used. The results are shown in FIGS.

  As shown in FIGS. 7 to 11, those having a large glucose production amount were OsEG, Aoxyn139, BsGX, Aoxyn887, AoPL19, Aoabf18, 20, and 22. In addition, for OsEG (rice-derived EG belonging to GHF9) that had the highest additive effect, the results were compared with commercially available enzyme preparations (200 mg / g and 400 mg / g biomass) and PcCBH2, which the inventors have already confirmed the additive effect. Is shown in FIG. As shown in FIG. 12, by adding 2% OsEG, it was found that the amount of glucose produced showed the same effect as that obtained by adding 400 mg / g biomass of twice the amount of commercially available enzyme preparation. This was equivalent to the effect when PcCBH2 was added.

(Improvement of production by converting heterogeneous proteins into frequently used codons in the genus Aspergillus)
It is known that heterologous protein expression by Aspergillus is difficult compared to the case of expressing the same type of protein. When CBH derived from Phanerochaete chrysosporium was expressed in koji molds with the base sequence of Phanerochaete chrysosporium, the protein expression level of the culture supernatant was estimated by SDS-PAGE. In the following, CBH of the same origin of GHF7 was about 1 mg / L. Regarding CBH derived from Phanerochaete chrysosporium belonging to GHF6, in the genus Aspergillus obtained from the Kazusa DNA Laboratory's gene code usage database (http://www.kazusa.or.jp/codon/) shown in FIG. Codons selected in consideration of the codon usage and GC content used (about 50% or more and about 55% or less) and the A / T / G / C ratio (each about 25 ± 4%) (colored in FIG. 13A) As a result of expressing a heterologous protein using DNA (SEQ ID NO: 29) having a base sequence modified with the codon shown in the part, a yield of 29 mg / L was shown. In other words, the production amount was improved about 290 times. In addition, for EG derived from O. sativa belonging to GHF6 and glucuronoxylan xylanohydrolase derived from B. subtilis, codons selected in the same manner as codons used in the genus Aspergillus (colored portions in FIGS. 13B and 13C). As a result of expressing the protein using DNA (SEQ ID NO: 30, SEQ ID NO: 31, respectively) having a base sequence modified with the codons shown in FIG. 1, the production amounts were 134 mg / L and 89 mg / L, respectively. From these results, it was found that for this type of protein, modification of the base sequence based on the codon usage in the genus Aspergillus is effective.

(Cellulase production by yeast)
CBH2 derived from Phanerochaete chrysosporium was amplified by PCR and subcloned into pRS436GAPSSRG, a yeast secretion expression vector. pRS436GAPSSRG has a secretion signal downstream of the TDH3 promoter and can secrete the enzyme outside the cell. This vector is transformed into yeast (MT8-2 strain), SD-URA agar (yeast nitrogen base without amino acids without ammonium sulfate 1.7g, casamino acid 5g, amino acid mix, glucose 20g, agar 20g, deionized water 1000ml ) At 30 ° C. for 3 days. The grown colonies were inoculated into SD-URA liquid medium (yeast nitrogen base without amino acids without ammonium sulfate 1.7 g, casamino acid 5 g, -URA amino acid mix 0.77 g, glucose 20 g, deionized water 1000 ml), 30 ° C., 20 The bacterial solution that had been cultured for a period of time was used as a preculture solution. The main culture was performed using a fermenter while maintaining the pH at 5.5. The preculture was inoculated into 500 ml of SD-URA liquid medium so that OD600 = 0.1, and cultured at 25 ° C. for 3 days. The culture supernatant was collected and precipitated with ammonium sulfate at a concentration of 70%. The protein after the ammonium sulfate precipitation was dissolved in a buffer (1M ammonium sulfate, 0.1 M Tris (pH 7.0)) and completely buffer-substituted by ultrafiltration was used as a sample for purification. An Avicel column was prepared by packing 2 ml of an Avicel solution (Avicel 10 g, buffer 40 ml) swollen with the same buffer. Samples were run through the column using a peristaltic pump at a flow rate of 1 ml / min. Then, it was washed with 20 ml of the same buffer (flow rate 1 ml / min) and eluted with sterilized water (flow rate 1 ml / min). As a result of confirming the fraction collected by 1 ml by SDS-PAGE and CMC halo assay, active PcCBH2 could be concentrated and purified to almost a single band. The amount of protein was measured using a protein assay kit manufactured by Bio-Rad. The specific activity of PSC degradation was almost equivalent to PcCBH2 produced by Aspergillus. It is also found that GHF5 EG derived from T. reesei, EG12 of GHF12, CBH of GHF6, CBH of GHF7, EG of GHF12 derived from P. chrysosporium, CBH of GHF7, etc. can also be secreted in an active form in yeast. It was.

  Sequence number 21, 22, 24, 25, 27, 28: Primer

Claims (25)

One or more first cellobiohydrolases belonging to GHF6; one or more second cellobiohydrolases belonging to GHF7; one or more first endoglucanases belonging to GHF5;
And the following enzymes:
(A) one or more second endoglucanases belonging to GHF9,
(B) one or more pectate lyases,
(C) one or more xylanases,
(D) one or more arabinofuranosidases,
(E) A cellulase composition comprising one or more enzymes selected from the group consisting of one or more glucuronoxylan xylanohydrolases.   The first cellobiohydrolase is derived from one or more organisms selected from the group consisting of Phanerochaete chrysosporium (P. chrysosporium) and Aspergillus oryzae (A.oryzae, Aspergillus oryzae) The composition of claim 1 which is a cellobiohydrolase.   The composition according to claim 1 or 2, wherein the first cellobiohydrolase is a cellobiohydrolase derived from P. chrysosporium.   The second cellobiohydrolase may be one or more organisms selected from the group consisting of Aspergillus niger (A. niger), Aspergillus aculeatus (A. aculeatus) and A.oryzae. The composition according to any one of claims 1 to 3, which is derived from cellobiohydrolase.   The composition according to any of claims 1 to 4, wherein the second cellobiohydrolase comprises cellobiohydrolase derived from A. niger.   The composition according to any one of claims 1 to 4, wherein the first endoglucanase is an endoglucanase derived from an organism belonging to A. oryzae and Trichoderma reesei (T. reesei, Trichoderma reesei).   The composition according to any one of claims 1 to 6, wherein the second endoglucanase is an endoglucanase derived from an organism belonging to Oryza sativa (O. sativa, rice).   Furthermore, the composition in any one of Claims 1-7 containing 1 or 2 or more 3rd endoglucanase which belongs to GHF12.   The third endoglucanase is an endoglucanase derived from one or more organisms selected from the group consisting of P. chrysosporium, A. oryzae and T. reesei. Composition.   The composition according to any one of claims 1 to 9, comprising the second endoglucanase and the third endoglucanase. The cellobiohydrolase comprises one or more of the first cellobiohydrolase belonging to GHF6 and one or more second cellobiohydrolase belonging to GHF7,
The endoglucanase consists of the first endoglucanase belonging to one or more GHF5 and the one or more second endoglucanases belonging to GHF9. Composition. The cellobiohydrolase comprises one or more of the first cellobiohydrolase belonging to GHF6 and one or more of the second cellobiohydrolase belonging to GHF7,
The first endoglucanase belonging to one or more GHF5, the one or more second endoglucanases belonging to GHF9, and one or more third endoglucanases belonging to GHF12. The composition according to claim 1, comprising:   The composition in any one of Claims 1-12 which does not contain (beta) -glucosidase substantially. An enzyme production method,
A gene encoding one or more enzymes selected from the group consisting of a cellulase derived from the genus Phanerochaete, a cellulase derived from the genus Oryza, and a hemicellulase derived from the genus Bacillus is introduced into a non-cellulase producing microorganism. Producing the enzyme with a production microorganism;
A method comprising:   The method according to claim 14, wherein the non-cellulase-producing microorganism is an Aspergillus genus, and the gene is modified based on codon usage in the Aspergillus genus.   The method according to claim 14, wherein the non-cellulase-producing microorganism is yeast. A transformant of a cellulase non-producing microorganism,
The following proteins:
One or more cellulases belonging to GHF6 derived from the genus Phanerochaete,
One or more cellulases belonging to GHF7 derived from the genus Phanerochaete,
One or more cellulases belonging to GHF12 derived from the genus Phanerochaete,
One or more cellulases belonging to GHF9 derived from the genus Oryza, and
Hemicellulase derived from the genus Bacillus,
A transformant obtained by transforming with an expression vector for expressing a gene encoding one or more proteins selected from the group consisting of:   The transformant according to claim 17, wherein the cellulase non-producing microorganism is an Aspergillus genus, and the gene is a gene modified based on codon usage in the Aspergillus genus.   The transformant according to claim 17, wherein the cellulase non-producing microorganism is yeast.   The transformant according to any one of claims 17 to 19, wherein the protein is secreted extracellularly or presented on a cell surface. A method for producing a low molecular weight product of cellulose,
Decomposing the cellulose using the cellulase composition according to any one of claims 1 to 13,
A method comprising: A method for producing useful substances by fermentation of microorganisms,
Using at least the cellulase composition according to any one of claims 1 to 13 to decompose cellulose into a carbon source usable by the microorganism;
Fermenting the carbon source with the microorganism to produce the useful substance;
A method comprising:   The method according to claim 22, wherein the microorganism is a microorganism that secretes β-glucosidase outside the cell or presents it on the cell surface so that the partial decomposition product of cellulose can be used as a carbon source.   The method according to claim 22 or 23, wherein the cellulose is a fraction containing cellulose obtained by pretreating a cellulose-containing material containing at least cellulose.   The method according to claim 24, wherein the pretreatment is a hydrothermal treatment or a treatment with an ionic liquid.

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