JP2022057665A - Enzyme composition for saccharification of hemicellulose - Google Patents

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

The present invention relates to an enzymatic composition useful for saccharification of hemicellulose.

In recent years, plant biomass produced as a by-product of agricultural production, such as post-harvest residues of crops and timber, is the most abundant renewable energy source on the planet and is expected as an alternative resource to petroleum. The main components of plant biomass are polysaccharides such as cellulose and hemicellulose and lignin.

Both cellulase and hemicellulose are required to efficiently decompose cellulose and hemicellulose in biomass, and the type and composition of the enzyme have a great influence. For this reason, in recent years, development has been promoted in search of an optimal saccharifying enzyme, and optimization of hemicellulase is particularly important. Hemicellulose is a heteropolysaccharide containing sugars other than glucose, and although it depends on the type of plant, most of them are arabinoxylan. Hydrolysis of xylan requires xylanase (endo-1,4-β-xylanase) and β-xylosidase. β-xylosidase is one of the hydrolyzing enzymes involved in the process of hydrolyzing oligosaccharides (xylo-oligosaccharides) produced by the hydrolysis of hemicellulose by xylanase to produce monosaccharides. In arabinoxylan, α-L-arabinofuranosidase hydrolyzes α-1,2 and / or α-1,3 bound arabinose side chains from the non-reducing end side, and L-arabinofranose is released. Will be done.

For example, Patent Document 1 contains β-xylosidase (BXL1) derived from Trichoderma resei and arabinofuranosidase (two or more selected from ABF1, ABF2, and ABF3) derived from Trichoderma resei as hemicellulase. The enzyme composition is disclosed.

Japanese Patent No. 5690713

The present invention relates to an enzyme composition useful for saccharification of hemicellulose and a method for saccharifying hemicellulose using the same.

The present inventors have two types of arabinofuranosidases with different substrate specificities from B. pseudocatenulatum, which can assimilate xylan species found in the human intestine. Three new xylosidases were newly isolated and found to be usable for saccharification of hemicellulose.

That is, the present invention relates to the following (1) to (4).
(1) Hemicellulose containing at least one protein selected from the group consisting of arabinofuranosidase represented by A below and at least one protein selected from the group consisting of xylosidase represented by B. Enzyme composition for saccharification.
A: Arabinofuranosidase (A1) A protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having at least 95% identity with the amino acid sequence and having arabinofuranosidase activity (A2) SEQ ID NO: 4 A protein consisting of the amino acid sequence represented by the above or an amino acid sequence having at least 95% identity with the amino acid sequence and having arabinofuranosidase activity B: xylosidase (B1) the amino acid sequence shown by SEQ ID NO: 6 or the amino acid sequence. And a protein having at least 90% identity with and having xylosidase activity (B2) consisting of the amino acid sequence represented by SEQ ID NO: 8 or the amino acid sequence having at least 90% identity with the amino acid sequence. Protein having xylosidase activity (B3) Protein according to the amino acid sequence represented by SEQ ID NO: 10 or an amino acid sequence having at least 90% identity with the amino acid sequence and having xylosidase activity (2) (1). A method for saccharifying hemicellulose using a composition.
(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 with the amino acid sequence and having arabinofuranosidase activity.
(4) A protein consisting of the amino acid sequence represented by SEQ ID NO: 6 or an amino acid sequence having at least 90% identity with the amino acid sequence and having xylosidase activity.

According to the present invention, hemicellulose, particularly hemicellulose containing a large amount of arabinoxylan, can be efficiently saccharified.

SDS-PAGE analysis results of arabinofuranosidase and xylosidase of the present invention. (A) Amount of xylose and arabinose produced when arabinoxylan degradation products are reacted with each enzyme. (B) Substrate specificity of the arabinofuranosidase of the present invention. (C) Metabolic activity of the xylosidase of the present invention.

As used herein, sequence identity of base sequences and amino acid sequences is calculated by the Lipman-Pearson method (Science, 1985, 227: 1435-1441). Specifically, it is calculated by performing an analysis with Unit size to homology (ktup) set to 2 using a homology analysis (Search homology) program of the genetic information processing software Genetyx-Win.

In the present specification, "at least 90% identity" with respect to an amino acid sequence or a base 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. Further, "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.

The arabinofuranosidase of the present invention is a protein represented by the following A1 and A2.
(A1) Amino acid sequence represented by SEQ ID NO: 2 or a protein consisting of an amino acid sequence having at least 95% identity with the amino acid sequence and having arabinofuranosidase activity (A2) Amino acid sequence represented by SEQ ID NO: 4 or A protein consisting of an amino acid sequence having at least 95% identity with the amino acid sequence and having arabinofuranosidase activity.

The protein consisting of the amino acid sequence represented by SEQ ID NO: 2 of A1 is arabinofuranosidase RS01610 (hereinafter, also referred to as “RS01610”) derived from the B. pseudocatenaltum YIT 4072 strain, A2. The protein consisting of the amino acid sequence shown in SEQ ID NO: 4 of the above is arabinofuranosidase RS02375 (hereinafter, also referred to as “RS02375”) derived from the same microbial strain.
RS01610 is a bifidobacteria addresscentis (B) described in Document 1 (Lagaert S, et al. Biochem Biophys Res Commun. 2010; 402 (4): 644-650.) As a result of homology analysis of amino acid sequences. It shows 99% sequence identity with AXH-d3 from (.adolescentis). RS02375 also exhibits 91% sequence identity with AbfA described in the same document.

"Arabinose furanosidase activity" means cleaving the α-1,2 or α-1,3 bound arabinose side chain and the α-1,2 and α-1,3 bound arabinose side chains in the arabinose-containing polysaccharide. It refers to the activity of hydrolyzing α-L-arabinose residues.
As shown in Examples described later, RS01610 hydrolyzes the arabinose side chain in which two molecules of arabinose are bound to α-1,2 and α-1,3 at the non-reducing end, but α-1,3 at the non-reducing end. The arabinose side chain bound to 2 or α-1,3 has a substrate specificity that it does not hydrolyze. On the other hand, RS02375 has a substrate specificity of hydrolyzing an arabinose side chain having α-1,2 or α-1,3 bound to a non-reducing end.
Therefore, the arabinofuranosidase represented by A1 is preferably one having an activity of hydrolyzing the α-L-arabinose residue of the arabinose side chain bound to α-1,2 and α-1,3, and is shown by A2. The arabinofuranosidase is preferably one having an activity of hydrolyzing the α-L-arabinose residue of the arabinose side chain bound to α-1,2 or α-1,3.

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

The protein consisting of the amino acid sequence represented by SEQ ID NO: 6 of B1 is xylosidase RS01595 (hereinafter, also referred to as “RS01595”) derived from Bifidobacterium pseudocatenuratum YIT 4072 strain, and the amino acid represented by SEQ ID NO: 8 of B2. The protein consisting of the sequence is xylosidase RS02300 (hereinafter, also referred to as “RS02300”) derived from the same microorganism strain, and the protein consisting of the amino acid sequence represented by SEQ ID NO: 10 of B3 is the xylosidase RS02400 (hereinafter, “RS02400”) derived from the same microorganism strain. Also called).
RS01595 and XylC derived from Bifidobacterium addresscentis described in the literature (Lagaert S, et al. Appl Microbiol Biotechnol. 2011; 92 (6): 1179-1185.) As a result of homology analysis of amino acid sequences. 81%, Bifidobacterium animalis subsp., Described in the literature (Viborg AH, et al. AMB Express. 2013; 3 (1): 56.). It shows 84% sequence identity with BXA43 from lactis BB-12.
In addition, RS02300 shows 77% sequence identity with XylC and 79% sequence identity with BXA43.
In addition, RS02400 shows 98% sequence identity with XylC and 78% sequence identity with BXA43.

Here, "β-xylosidase activity" means an activity that catalyzes the reaction of hydrolyzing xylooligosaccharides to xylose.

The enzyme composition of the present invention contains arabinofuranosidase and xylosidase, but contains two types of arabinofuranosidases, A1 and A2, and one or more enzymes selected from B1 to B3 xylosidase. preferable.

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, if a transformant is obtained by introducing a polynucleotide encoding arabinofuranosidase or xylosidase of the present invention or a vector containing the same into a host, and then culturing the transformant in an appropriate medium, the present invention can be obtained. The arabinofuranosidase or xylosidase of the invention is produced. The arabinofuranosidase or xylosidase of the present invention can be obtained by isolating or purifying the produced arabinofuranosidase or xylosidase from the culture.

Examples of the polynucleotide encoding the arabinofuranosidase or xylosidase of the present invention include the polynucleotides encoding the proteins represented by A1 to A2 and the proteins represented by B1 to B3, and the following (preferably). Examples thereof include polynucleotides having a base sequence selected from a1) to (a2) and (b1) to (b3).
(A1) Nucleotide sequence represented by SEQ ID NO: 1 or a base sequence having at least 95% identity with the nucleotide sequence (a2) Nucleotide sequence represented by SEQ ID NO: 3 or having at least 95% identity with the nucleotide sequence. Nucleotide sequence (b1) Nucleotide sequence represented by SEQ ID NO: 5 or a nucleotide sequence having at least 90% identity with the nucleotide sequence (b2) Nucleotide sequence represented by SEQ ID NO: 7 or at least 90% identity with the nucleotide sequence (B3) The base sequence represented by SEQ ID NO: 9 or the base sequence having at least 90% identity with the base sequence.

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

The type of vector containing the polynucleotide encoding the arabinofuranosidase or xylosidase of the present invention is not particularly limited, and vectors usually used for gene cloning such as plasmids, cosmids, phages, viruses, YAC, BAC and the like can be used. Can be mentioned. Of these, a plasmid vector is preferable, and for example, a commercially available plasmid vector for protein expression, for example, shuttle vectors pHY300PLK, pUC119, pBR322 (all manufactured by Takara Bio Inc.) and the like can be preferably used.

The vector can contain a DNA replication initiation region or a DNA region containing an origin of replication, and is a promoter for initiating transcription of the gene upstream of the polynucleotide encoding the arabinofuranosidase or xylosidase of the present invention. Control sequences such as regions and terminator regions may be operably linked. In addition, the vector may further incorporate a marker gene for selecting a host into which the vector has been appropriately introduced.

Examples of the host of the transformant into which the vector is introduced include microorganisms such as bacteria, filamentous fungi, and yeast. Examples of the bacterium include Escherichia coli, Staphylococcus genus, Enterococcus genus, Bacillus genus, and the like, of which Escherichia coli is preferable.

As a method for introducing the vector into the host, a method usually used in the art such as a protoplast method and an electroporation method can be used. By selecting a strain that has been appropriately introduced using the expression of the marker gene, auxotrophy, etc. as indicators, the target transformant into which the vector has been introduced can be obtained.

If the transformant thus obtained into which the polynucleotide encoding the arabinofuranosidase or the xylosidase of the present invention or the vector containing the same is introduced is cultured in an appropriate medium, the arabinofuranosidase of the present invention or the arabinofuranosidase of the present invention or Xylan cidase is produced. The medium used for culturing the transformant can be appropriately selected by those skilled in the art according to the type of microorganism of the transformant.

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

The enzyme composition in which the above-mentioned arabinofuranosidase and xylosidase of the present invention are combined can be used for hemicellulose saccharification.
Further, the enzyme composition can be used for biomass saccharification by further combining with other saccharifying enzymes such as cellulase and hemicellulase other than the enzyme of the present invention.

Here, "hemicellulose" is a polymer component of a plant substance containing a monomer of a sugar other than glucose. Hemicellulose can contain xylose, mannose, galactose, rhamnose and arabinose in addition to glucose, with xylose being the most common sugar monomer. Examples of hemicellulose include galactan, mannan, xylan, arabanan, arabinoxylan, glucomannan, galactomannan and the like, but in the present invention, those containing xylan and arabinoxylan are preferable.

The protein content ratio of arabinofuranosidase to xylosidase (arabinofuranosidase / xylosidase) in the enzyme composition of the present invention is not particularly limited, but is preferably 1: 1 to 1: 4, more preferably 1. 1: 1 to 1: 2.

The enzyme composition of the present invention is used for saccharification from hemicellulose. That is, the saccharification method of the present invention includes a step of saccharifying hemicellulose with the above-mentioned enzyme preparation of the present invention.
When the hemicellulose is saccharified, from the viewpoint of improving crushing efficiency, saccharification efficiency and production efficiency, the hemicellulose is further included in a pretreatment step such as alkaline treatment, crushing treatment or hydrothermal treatment before the saccharification treatment step. be able to.

The conditions for the saccharification treatment are not particularly limited as long as the arabinofuranosidase and xylansidase 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 is also appropriately determined.

Example 1 Preparation of enzyme (1) Preparation of recombinant protein B. The ORF of the gene (SEQ ID NO: 1, 3, 5, 7 and 9) encoding each enzyme protein consisting of the amino acid sequences represented by SEQ ID NOs: 2, 4, 6, 8 and 10 using the genome of pseudocantunulatum YIT 4072 ^T as a template. Was amplified with Fw and Rv primers and PrimeSTAR MAX DNA Polymerase (Takara) or KOD-plus- (TOYOBO). After purification of the amplified product, it was introduced into a pCold I vector (Takara) using restriction enzymes NdeI, XhoI or using an In-Fusion HD Cloning Kit (Clontech). The host of cloning is E.I. The coli JM 109 strain was used. Table 1 shows the primers used and the method for constructing each plasmid.

(2) Purification of His-tag-added protein E.I. It was introduced into the coli BL21 strain by the heat shock method to obtain a transformant. The obtained strain was cultured in 10 ml of LB medium containing 100 μg / ml ampicillin. When OD ₆₀₀ = 0.5, the culture broth was incubated at 15 ° C. for 30 minutes. Then, 100 μl of 100 mM IPTG was added, and the mixture was further incubated at 15 ° C. for 24 hours. The obtained culture broth was centrifuged at 8000 rpm for 10 minutes, and the cells were collected. B-PER ^TM Bactial Protein Extraction Reagent (Thermo Fisher Scientific), required B-PER ^TM Bactial Protein Extraction Reagent 1 ml with 2 μl Protein was extracted by adding chiken egg white (50 mg / ml) (Sigma-aldrich). The desired His-tagged recombinant protein was purified from the protein extract obtained from the cells using Ni-NTA Spin Colon (QIAGEN). If necessary, the purified protein was concentrated using Vivaspin 500-50K (GE Healthcare).
The SDS-PAGE analysis results of the purified recombinant enzyme are shown in FIG.

Example 2 Measurement of enzyme activity (1) Measurement of xylose and arabinose when arabinoxylan degradation products are metabolized When arabinoxylan degradation products are metabolized to confirm whether each enzyme is xylosidase or arabinofuranosidase. The amount of xylose and arabinose was measured. To obtain arabinoxylan degradation products, 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 with the container in boiling water for 10 minutes and allowed to cool to room temperature. 200 ml of ethanol was added, and the supernatant obtained by centrifugation was dried by an evaporator to obtain an arabinoxylan decomposition product. The obtained powder was dissolved in water to a final concentration of 10%, of which 12.5 μl was mixed with 37.5 μl of McIlvaine buffer (pH 6.3) and 2 μl of each enzyme obtained in Example 1. , 37 ° C. for 16 hours. After the reaction, the mixture was incubated at 94 ° C. for 5 minutes, a glucose solution was added to a final concentration of 1 mM, and the sugar composition was analyzed by HPLC. For HPLC, sugar was separated by KS-802 volume (Showa Denko KK) using a system of Shimadzu Corporation, and detected by RI director RI-101 of Showa Denko KK.

(2) Measurement of xylosidase activity 465 μl of MacIlvine buffer (pH = 6.3) and 10 μl of 100 mM xylotetraose were mixed and preincubated at 37 ° C. for about 10 minutes. To the reaction solution, 25 μl of each enzyme obtained in Example 1 diluted to 1.6 μM was added, and incubation was performed at 37 ° C. For analysis by TLC, 50 μl sampling was performed after 0 minutes, 5 minutes, 15 minutes, 30 minutes and 90 minutes from the similarly reacted solution, and incubated at 94 ° C. for 5 minutes to inactivate the enzyme. 6 μl was spotted on TLC silica gel 60 (Merck, 1.05721.0001). With reference to the literature (Nong G, Rice JD, Chow V, Preston JF. 2009. Appl Environ Microbiol 75: 4410-4418), it was developed twice at CH ₃ COOH: Chloroform: H ₂ O = 7: 6: 1. The color was developed by heating with 2% Orcinol (in H ₂ SO ₄ : MeOH = 1: 9) and detected.

(3) Measurement of arabinofuranosidase activity As a reaction substrate, four types of AXOS (Megazyme) in which arabinose was bound to xylobiose in a different binding manner, that is, 32 ^- α-L-Arabinofuranosyll-xylobiose ( AXOS1), 2 ³ , 3 ³ -di-α-L-Arabinofuranosyl-xylotriose (AXOS2), 2 ³ -α-L-Arabinofuranosyll-xylotriose (AXOS3) and 3 ³ -α-L-arase (AXOS3) Using. To 44 μl of McIlvaine buffer (pH 6.3), 5 μl of each enzyme obtained in Example 1 diluted with 1 μl of 100 mM AXOS to 2 μM was added, reacted at 37 ° C. for 2 hours, and then incubated at 94 ° C. for 5 minutes. And inactivated the enzyme. The sugar composition of 6 μl of the reaction solution was analyzed by TLC.
(3) Results The results are shown in FIG.
FIG. 2A shows the amounts of xylose and arabinose produced when the arabinoxylan degradation product is reacted with each enzyme. Since arabinose was produced in RS01610 and RS02375, it was found that they have arabinofuranosidase activity (= arabinofuranosidase). Similarly, RS01595, RS02300 and RS02400 were found to have xylosidase activity (= xylosidase) because xylose was produced.
FIG. 2B shows the substrate specificity of RS01610 and RS02375 in arabinofuranosidase activity. Here, AXOS1 to 4 show arabinoxylooligosaccharides having different binding modes of arabinose side chains, and XOS2 to 4 show disaccharide, trisaccharide and tetrasaccharide xylooligosaccharides, respectively. From FIG. 2B, RS01610 hydrolyzes the arabinose side chain in which two molecules of arabinose are bound to α-1,2 and α-1,3 at the non-reducing end, but α-1,2 or α-at the non-reducing end. It can be seen that the arabinose side chains bound to 1,3 have a substrate specificity that they do not hydrolyze. On the other hand, RS02375 hydrolyzes the arabinose side chain with α-1,2 or α-1,3 bound to the non-reducing end, but cannot hydrolyze the arabinose side chain with two molecules of arabinose bound to the non-reducing end. It can be seen that it has substrate specificity. By using these two types of arabinofuranosidase, arabinose side chains having various bonds can be separated.
FIG. 2C shows the xylosidase activity of RS01595, RS02300 and RS02400. From FIG. 2C, it can be seen that each enzyme has xylosidase activity, and in particular, it can be seen that the decomposition time of RS01595 from xylooligosaccharide to xylose is short and the metabolic activity is strong.

Claims (4)

An enzyme for hemicellulose saccharification containing at least one protein selected from the group consisting of arabinofuranosidase represented by A below and at least one protein selected from the group consisting of xylosidase represented by B below. Composition.
A: Arabinofuranosidase (A1) A protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having at least 95% identity with the amino acid sequence and having arabinofuranosidase activity (A2) SEQ ID NO: 4 A protein consisting of the amino acid sequence represented by the above or an amino acid sequence having at least 95% identity with the amino acid sequence and having arabinofuranosidase activity B: xylosidase (B1) the amino acid sequence shown by SEQ ID NO: 6 or the amino acid sequence. And a protein having at least 90% identity with and having xylosidase activity (B2) consisting of the amino acid sequence represented by SEQ ID NO: 8 or the amino acid sequence having at least 90% identity with the amino acid sequence. Protein with xylosidase activity (B3) A protein consisting of the amino acid sequence represented by SEQ ID NO: 10 or an amino acid sequence having at least 90% identity with the amino acid sequence and having xylosidase activity. A method for saccharifying hemicellulose using the enzyme composition according to claim 1. A protein consisting of the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 95% identity with the amino acid sequence and having arabinofuranosidase activity. A protein consisting of the amino acid sequence represented by SEQ ID NO: 6 or an amino acid sequence having at least 90% identity with the amino acid sequence and having xylosidase activity.

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