Enzyme composition for saccharifying hemicellulose

Abstract

To provide an enzyme preparation useful for saccharification of hemicellulose, and a method for saccharification of hemicellulose using the same.SOLUTION: An enzyme composition for saccharification of hemicellulose contains a protein consisting of a specific amino acid sequence or an amino acid sequence at least 95% identical to the amino acid sequence and having arabinofuranosidase activity, and a protein consisting of another specific amino acid sequence or an amino acid sequence at least 90% identical to the amino acid sequence and having xylosidase activity.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to enzyme compositions useful for the saccharification of hemicellulose. [Background technology]

[0002] In recent years, plant biomass, which is generated as a by-product of agricultural production, such as residues after harvesting crops and wood, is expected to be the most abundant renewable energy source on Earth and an alternative resource to petroleum. The main components of plant biomass are polysaccharides such as cellulose and hemicellulose, and lignin.

[0003] To efficiently decompose cellulose and hemicellulose in biomass, both cellulase and hemicellulase are required, and the type and composition of the enzymes have a large effect. For this reason, in recent years, development has been promoted in search of optimal saccharification enzymes, and optimization of hemicellulase in particular is considered important. Hemicellulose is a heteropolysaccharide containing sugars other than glucose, and although it varies depending on the type of plant, most are arabinoxylan. Xylanase (endo-1,4-β-xylanase) and β-xylosidase are required for the hydrolysis of xylan. β-xylosidase is one of the hydrolases involved in the process of hydrolyzing oligosaccharides (xylooligosaccharides) generated by hydrolysis of hemicellulose with xylanase to produce monosaccharides. In addition, arabinoxylan is hydrolyzed by α-L-arabinofuranosidase at the α-1,2 and / or α-1,3-linked arabinose side chains from the non-reducing end side, releasing L-arabinofuranose.

[0004] For example, Patent Document 1 discloses an enzyme composition containing, as hemicellulase, β-xylosidase (BXL1) derived from Trichoderma reesei and arabinofuranosidase (two or more selected from ABF1, ABF2, and ABF3) derived from Trichoderma reesei. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 5690713 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention relates to providing an enzyme composition useful for saccharifying hemicellulose and a method for saccharifying hemicellulose using the same. [Means for solving the problem]

[0007] The inventors have newly isolated two arabinofuranosidases and three xylosidases with different substrate specificities from Bifidobacterium pseudocatenulatum, which is capable of assimilating xylans found in the human intestine, and found that these can be used for the saccharification of hemicellulose.

[0008] That is, the present invention relates to the following (1) to (4). (1) An enzyme composition for hemicellulose saccharification, comprising at least one protein selected from the group consisting of arabinofuranosidases represented by A below, and at least one protein selected from the group consisting of xylosidases represented by B below. A: Arabinofuranosidase (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 95% identity thereto, and having arabinofuranosidase activity. (A2) A protein consisting of the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 95% identity thereto, and having arabinofuranosidase activity. B: Xylosidase (B1) A protein having an amino acid sequence represented by SEQ ID NO:6 or an amino acid sequence having at least 90% identity thereto, and having xylosidase activity. (B2) A protein having an amino acid sequence represented by SEQ ID NO: 8 or an amino acid sequence having at least 90% identity thereto, and having xylosidase activity. (B3) A protein having an amino acid sequence represented by SEQ ID NO: 10 or an amino acid sequence having at least 90% identity thereto, and having xylosidase activity. (2) A method for saccharifying hemicellulose, which uses the enzyme composition described in (1). (3) A protein consisting of the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 95% identity to said amino acid sequence, and having arabinofuranosidase activity. (4) A protein consisting of the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 90% identity to said amino acid sequence, and having xylosidase activity. Effect of the Invention

[0009] According to the present invention, hemicellulose, particularly hemicellulose rich in arabinoxylan, can be efficiently saccharified. [Brief description of the drawings]

[0010] [Figure 1] 4 shows the results of SDS-PAGE analysis of the arabinofuranosidase and xylosidase of the present invention. [Diagram 2] (A) Amounts of xylose and arabinose produced when arabinoxylan decomposition products were reacted with each enzyme. (B) Substrate specificity of the arabinofuranosidase of the present invention. (C) Metabolic activity of the xylosidase of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] In the present specification, the sequence identity of a base sequence and an amino acid sequence is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, the sequence identity is calculated by performing an analysis using the search homology program of the genetic information processing software Genetyx-Win with the unit size to compare (ktup) set to 2.

[0012] As used herein, "at least 90% identity" with respect to an amino acid sequence or a nucleotide sequence means 90% or more, preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and more preferably 99% or more identity. Also, "at least 95% identity" means 95% or more, more preferably 97% or more, more preferably 98% or more, and more preferably 99% or more identity.

[0013] The arabinofuranosidase of the present invention is a protein represented by A1 and A2 below. (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 95% identity thereto, and having arabinofuranosidase activity. (A2) A protein consisting of the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 95% identity thereto, and having arabinofuranosidase activity.

[0014] The protein consisting of the amino acid sequence shown in sequence number 2 of A1 is arabinofuranosidase RS01610 (hereinafter also referred to as "RS01610") derived from Bifidobacterium pseudocatenulatum YIT 4072 strain, and the protein consisting of the amino acid sequence shown in sequence number 4 of A2 is arabinofuranosidase RS02375 (hereinafter also referred to as "RS02375") derived from the same microbial strain. As a result of amino acid sequence homology analysis, RS01610 shows 99% sequence identity with AXH-d3 derived from B. adolescentis described in reference 1 (Lagaert S, et al. Biochem Biophys Res Commun. 2010;402(4):644-650.), and RS02375 shows 91% sequence identity with AbfA described in the same reference.

[0015] The term "arabinofuranosidase activity" refers to the activity of cleaving α-1,2 or α-1,3-linked arabinose side chains, or α-1,2 and α-1,3-linked arabinose side chains in arabinose-containing polysaccharides, and hydrolyzing α-L-arabinose residues. As shown in the Examples below, RS01610 has substrate specificity of hydrolyzing an arabinose side chain having two arabinose molecules bonded to the non-reducing terminal via α-1,2 and α-1,3 bonds, but not hydrolyzing an arabinose side chain having α-1,2 or α-1,3 bonds to the non-reducing terminal, whereas RS02375 has substrate specificity of hydrolyzing an arabinose side chain having α-1,2 or α-1,3 bonds to the non-reducing terminal. Therefore, the arabinofuranosidase represented by A1 is preferably one having the activity of hydrolyzing α-L-arabinose residues in α-1,2 and α-1,3 linked arabinose side chains, and the arabinofuranosidase represented by A2 is preferably one having the activity of hydrolyzing α-L-arabinose residues in α-1,2 or α-1,3 linked arabinose side chains.

[0016] The xylosidase of the present invention is a protein represented by the following B1 to B3. (B1) A protein having an amino acid sequence represented by SEQ ID NO:6 or an amino acid sequence having at least 90% identity thereto, and having xylosidase activity. (B2) A protein having an amino acid sequence represented by SEQ ID NO: 8 or an amino acid sequence having at least 90% identity thereto, and having xylosidase activity. (B3) A protein having an amino acid sequence represented by SEQ ID NO: 10 or an amino acid sequence having at least 90% identity thereto, and having xylosidase activity.

[0017] The protein consisting of the amino acid sequence shown in SEQ ID NO: 6 in B1 is the xylosidase RS01595 (hereinafter also referred to as "RS01595") derived from Bifidobacterium pseudocatenulatum YIT 4072 strain, the protein consisting of the amino acid sequence shown in SEQ ID NO: 8 in B2 is the xylosidase RS02300 (hereinafter also referred to as "RS02300") derived from the same microbial strain, and the protein consisting of the amino acid sequence shown in SEQ ID NO: 10 in B3 is the xylosidase RS02400 (hereinafter also referred to as "RS02400") derived from the same microbial strain. Based on amino acid sequence homology analysis, RS01595 shows 81% sequence identity with XylC from Bifidobacterium adolescentis described in the literature (Lagaert S, et al. Appl Microbiol Biotechnol. 2011;92(6):1179-1185.) and 84% sequence identity with BXA43 from B. animalis subsp. lactis BB-12 described in the literature (Viborg AH, et al. AMB Express. 2013;3(1):56.). RS02300 also shows 77% sequence identity with XylC and 79% with BXA43. RS02400 also shows 98% sequence identity with XylC and 78% with BXA43.

[0018] Here, "β-xylosidase activity" means the activity of catalyzing the reaction of hydrolyzing xylooligosaccharides to xylose.

[0019] The enzyme composition of the present invention contains arabinofuranosidase and xylosidase, and preferably contains two types of arabinofuranosidases, A1 and A2, and one or more types of enzymes selected from xylosidases B1 to B3.

[0020] The arabinofuranosidase or xylosidase of the present invention can be produced, for example, from a transformant into which a polynucleotide encoding the arabinofuranosidase or xylosidase of the present invention has been introduced. That is, the arabinofuranosidase or xylosidase of the present invention is produced by introducing a polynucleotide encoding the arabinofuranosidase or xylosidase of the present invention, or a vector containing the same, into a host to obtain a transformant, and then culturing the transformant in an appropriate medium. The arabinofuranosidase or xylosidase of the present invention can be obtained by isolating or purifying the produced arabinofuranosidase or xylosidase from the culture.

[0021] Polynucleotides encoding the arabinofuranosidase or xylosidase of the present invention include polynucleotides encoding the proteins represented by A1 to A2, or the proteins represented by B1 to B3, but preferably include polynucleotides having a base sequence selected from the following (a1) to (a2) and (b1) to (b3). (a1) a nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence having at least 95% identity to said nucleotide sequence (a2) a nucleotide sequence shown in SEQ ID NO: 3 or a nucleotide sequence having at least 95% identity thereto (b1) a base sequence shown in SEQ ID NO: 5 or a base sequence having at least 90% identity thereto (b2) a nucleotide sequence shown in SEQ ID NO: 7 or a nucleotide sequence having at least 90% identity thereto (b3) a base sequence shown in SEQ ID NO: 9 or a base sequence having at least 90% identity thereto

[0022] The polynucleotide encoding the arabinofuranosidase or xylosidase of the present invention can be isolated by any method used in the art from Bifidobacterium pseudocatenulatum YIT 4072, etc. For example, the polynucleotide can be obtained by extracting the total genomic DNA of Bifidobacterium pseudocatenulatum YIT 4072, selectively amplifying a target gene by PCR using primers designed based on the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, or 9, and purifying the amplified gene.

[0023] The type of vector containing a polynucleotide encoding the arabinofuranosidase or xylosidase of the present invention is not particularly limited, and examples thereof include vectors commonly used for gene cloning, such as plasmids, cosmids, phages, viruses, YACs, BACs, etc. Among these, plasmid vectors are preferred, and for example, commercially available plasmid vectors for protein expression, such as shuttle vectors pHY300PLK, pUC119, and pBR322 (all manufactured by Takara Bio Inc.), can be suitably used.

[0024] The above-mentioned vector may contain a DNA replication origin region or a DNA region containing a replication origin, and a control sequence such as a promoter region or a terminator region for initiating transcription of the gene may be operably linked upstream of the polynucleotide encoding the arabinofuranosidase or xylosidase of the present invention. In addition, the vector may further contain a marker gene for selecting a host into which the vector has been appropriately introduced.

[0025] Examples of hosts for transformants into which the vectors are introduced include microorganisms such as bacteria, filamentous fungi, yeasts, etc. Examples of bacteria include Escherichia coli, bacteria belonging to the genera Staphylococcus, Enterococcus, and Bacillus, and among these, Escherichia coli is preferred.

[0026] The vector can be introduced into the host by a method commonly used in the art, such as the protoplast method, electroporation, etc. A strain into which the vector has been appropriately introduced can be selected based on the expression of a marker gene, nutritional requirements, etc., to obtain a desired transformant into which the vector has been introduced.

[0027] When the thus obtained transformant into which the polynucleotide encoding the arabinofuranosidase or xylosidase of the present invention or the vector containing the same has been introduced is cultured in an appropriate medium, the arabinofuranosidase or xylosidase of the present invention is produced. The medium used for culturing the transformant can be appropriately selected by those skilled in the art depending on the type of microorganism of the transformant.

[0028] The arabinofuranosidase and xylosidase of the present invention produced in the above culture can be isolated or purified by a general method used for protein purification, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., either alone or in appropriate combination. The arabinofuranosidase recovered from the culture may be further purified by known means.

[0029] The enzyme composition of the present invention which combines arabinofuranosidase and xylosidase can be used for hemicellulose saccharification. Furthermore, the enzyme composition can be used for biomass saccharification by further combining it with other saccharification enzymes, such as cellulase or hemicellulase other than the enzyme of the present invention.

[0030] Here, "hemicellulose" refers to a polymeric component of plant substances that contains sugar monomers other than glucose. Hemicellulose can contain xylose, mannose, galactose, rhamnose, and arabinose in addition to glucose, with xylose being the most common sugar monomer. Hemicellulose includes galactan, mannan, xylan, arabanan, arabinoxylan, glucomannan, galactomannan, etc., but in the present invention, those containing xylan and arabinoxylan are preferred.

[0031] In the enzyme composition of the present invention, the protein ratio between arabinofuranosidase and xylosidase (arabinofuranosidase / xylosidase) is not particularly limited, but is preferably 1:1 to 1:4, and more preferably 1:1 to 1:2.

[0032] The enzyme composition of the present invention is used for saccharification of hemicellulose. That is, the saccharification method of the present invention includes a step of saccharifying hemicellulose with the enzyme preparation of the present invention. When saccharifying hemicellulose, from the viewpoint of improving pulverization efficiency, saccharification efficiency, and production efficiency, a pretreatment step such as alkali treatment, pulverization treatment, or hydrothermal treatment of the hemicellulose may be further included prior to the saccharification step.

[0033] The conditions for the saccharification treatment are not particularly limited as long as the arabinofuranosidase and xylosidase of the present invention are not inactivated. For example, the enzyme composition of the present invention may be added to a suspension containing hemicellulose, and reacted at a predetermined pH and temperature for a certain period of time. The content of hemicellulose in the suspension may be appropriately adjusted from the viewpoint of improving saccharification efficiency or sugar production efficiency. The amount of the enzyme composition used may also be appropriately determined. EXAMPLES

[0034] Example 1 Preparation of enzyme (1) Preparation of recombinant proteins B. pseudocatenulatum YIT 4072 TUsing the genome of as a template, the ORFs of the genes (SEQ ID NOs: 1, 3, 5, 7, and 9) encoding the enzyme proteins consisting of the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, and 10 were amplified using Fw and Rv primers and PrimeSTAR MAX DNA Polymerase (Takara) or KOD-plus- (TOYOBO). After purification, the amplified products were introduced into the pCold I vector (Takara) using the restriction enzymes NdeI and XhoI, or using the In-Fusion HD Cloning Kit (Clontech). The E. coli JM 109 strain was used as the host for cloning. The primers used and the construction method of each plasmid are shown in Table 1.

[0035] [Table 1]

[0036] (2) Purification of His-tagged proteins Each expression plasmid was introduced into E. coli BL21 strain by the heat shock method to obtain a transformant. The resulting strain was cultured in 10 ml of LB medium containing 100 μg / ml of ampicillin. OD 600 When the ratio reached 0.5, the culture was incubated at 15°C for 30 minutes. After that, 100 μl of 100 mM IPTG was added, and the culture was further incubated at 15°C for 24 hours. The culture was centrifuged at 8000 rpm for 10 minutes to recover the cells. The recovered cells were subjected to B-PER. TM Bacterial Protein Extraction Reagent (Thermo Fisher Scientific), B-PER required TMProteins were extracted by adding 2 μl of Recombinant DNase I (RNase-free) (Takara) and 2 μl of Lysozyme from chicken egg white (50 mg / ml) (Sigma-Aldrich) per 1 ml of Bacterial Protein Extraction Reagent. The His-tagged recombinant protein of interest was purified from the protein extract obtained from the bacterial cells using a Ni-NTA Spin Column (QIAGEN). If necessary, the purified protein was concentrated using a Vivaspin 500-50K (GE Healthcare). The results of SDS-PAGE analysis of the purified recombinant enzyme are shown in FIG.

[0037] Example 2 Measurement of enzyme activity (1) Measurement of xylose and arabinose produced by metabolism of arabinoxylan decomposition products To confirm whether each enzyme was a xylosidase or an arabinofuranosidase, the amount of xylose and arabinose produced when the arabinoxylan decomposition product was metabolized was measured. To obtain the arabinoxylan decomposition product, 50 ml of 2% wheat-derived arabinoxylan (Megazyme, P-WAXYL) was reacted with Cellvibrio mixtus-derived xylanase (Megazyme, E-XYNBCM) at 40°C for 16 hours. The reaction solution was incubated in boiling water in a container for 10 minutes and allowed to cool to room temperature. 200 ml of ethanol was added, and the supernatant obtained by centrifugation was dried in an evaporator to obtain the arabinoxylan decomposition product. The obtained powder was dissolved in water to a final concentration of 10%, and 12.5 μl of the solution was mixed with 37.5 μl of McIlvaine buffer (pH 6.3) and 2 μl of each enzyme obtained in Example 1, and incubated at 37°C for 16 hours. After the reaction, the mixture was incubated at 94°C for 5 minutes, and a glucose solution was added to a final concentration of 1 mM, after which the sugar composition was analyzed by HPLC. For HPLC, sugars were separated using a KS-802 column (Showa Denko KK) using a Shimadzu Corporation system, and detected with a Showa Denko RI detector RI-101.

[0038] (2) Measurement of xylosidase activity 465 μl of MacIlvaine buffer (pH=6.3) and 10 μl of 100 mM xylotetraose were mixed and pre-incubated at 37° C. for about 10 minutes. 25 μl of each enzyme obtained in Example 1 diluted to 1.6 μM was added to the reaction solution and incubated at 37° C. For TLC analysis, 50 μl of the solution was sampled after 0 minutes, 5 minutes, 15 minutes, 30 minutes, and 90 minutes from the reaction solution, and the enzyme was inactivated by incubation at 94° C. for 5 minutes, and 6 μl of the sample was spotted on TLC silica gel 60 (Merck, 1.05721.0001). Based on the literature (Nong G, Rice JD, Chow V, Preston JF. 2009. Appl Environ Microbiol 75:4410-4418), the samples were developed twice with CH3COOH:Chloroform:H2O=7:6:1, and then the samples were heated and stained with 2% Orcinol (in H2SO4:MeOH=1:9) for detection.

[0039] (3) Measurement of arabinofuranosidase activity The reaction substrates were four types of AXOS (Megazyme), in which arabinose was bound to xylooligosaccharides in different bond patterns, i.e., 3 2 -α-L-Arabinofuranosyl-xylobiose(AXOS1), 2 3 ,3 3 -di-α-L-Arabinofuranosyl-xylotriose(AXOS2), 2 3 -α-L-Arabinofuranosyl-xylotriose (AXOS3) and 3 3 -α-L-arabinofuranosyl-xylotetraose (AXOS4) was used. 1 μl of 100 mM AXOS and 5 μl of each enzyme obtained in Example 1 diluted to 2 μM were added to 44 μl of McIlvaine buffer (pH 6.3), and the reaction was carried out at 37°C for 2 hours, and then the enzyme was inactivated by incubation at 94°C for 5 minutes. The sugar composition of 6 μl of the reaction solution was analyzed by TLC. (3) Results The results are shown in Figure 2. Figure 2A shows the amount of xylose and arabinose produced when arabinoxylan decomposition products were reacted with each enzyme. RS01610 and RS02375 were found to have arabinofuranosidase activity (=are arabinofuranosidases) because arabinose was produced. Similarly, RS01595, RS02300, and RS02400 were found to have xylosidase activity (=are xylosidases) because xylose was produced. FIG. 2B shows the substrate specificity of the arabinofuranosidase activity of RS01610 and RS02375. Here, AXOS1 to 4 indicate arabinoxylooligosaccharides with different bonding patterns of the arabinose side chain, and XOS2 to 4 indicate disaccharide, trisaccharide, and tetrasaccharide xylooligosaccharides, respectively. From FIG. 2B, it can be seen that RS01610 has substrate specificity in that it hydrolyzes an arabinose side chain having two molecules of arabinose bonded to the non-reducing end via α-1,2 and α-1,3 bonding, but does not hydrolyze an arabinose side chain having α-1,2 or α-1,3 bonding to the non-reducing end. On the other hand, it can be seen that RS02375 has substrate specificity in that it hydrolyzes an arabinose side chain having α-1,2 or α-1,3 bonding to the non-reducing end, but does not hydrolyze an arabinose side chain having two molecules of arabinose bonded to the non-reducing end. By using these two types of arabinofuranosidase, arabinose side chains with various linkages can be separated. Figure 2C shows the xylosidase activity of RS01595, RS02300, and RS02400. Figure 2C shows that each enzyme has xylosidase activity, and that RS01595 in particular has a short decomposition time from xylooligosaccharides to xylose and a strong metabolic activity.

Claims

1. An enzyme composition for hemicellulose saccharification comprising at least one protein selected from the group consisting of arabinofuranosidases represented by A below, and at least one protein selected from the group consisting of xylosidases represented by B below. A: Arabinofuranosidase (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 97% identity thereto, and having arabinofuranosidase activity. (A2) A protein consisting of the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 95% identity thereto, and having arabinofuranosidase activity. B: Xylosidase (B1) A protein having an amino acid sequence represented by SEQ ID NO: 6 or an amino acid sequence having at least 90% identity thereto, and having xylosidase activity. (B2) A protein having an amino acid sequence represented by SEQ ID NO: 8 or an amino acid sequence having at least 90% identity thereto, and having xylosidase activity. (B3) A protein having an amino acid sequence represented by SEQ ID NO: 10 or an amino acid sequence having at least 90% identity thereto, and having xylosidase activity.

2. A method for saccharifying hemicellulose, comprising using the enzyme composition according to claim 1.

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