KR101577726B1 - - 14--- Novel Enzyme 14--D-glucan glucohydrolase having Exoglucanase and -glucosidase Activity and Method for Preparing Glucose from Cellulose using the Same - Google Patents

Abstract

The present invention provides a novel enzyme having the activity of exoglucanase and beta-glucosidase, and a method for producing glucose from cellulose using the same. The present invention relates to a process for producing cellulodextrin by lowering the degree of polymerization of cellulose using an ionic liquid and a solid acid catalyst to produce a cellulosic product of a single enzyme 1,4-beta-di-glucan having exoglucanase and beta-glucosidase activity Glucohydrolase is treated to produce glucose. The present invention has advantages in that the process is simple and the cost is reduced because glucose can be produced using only one enzyme as compared with a conventional method of producing glucose requiring three types of enzymes which are essentially necessary. Glucose produced by the present invention can be applied to a saccharification fermentation concurrent process or a saccharification fermentation continuous process in connection with a saccharification process and a fermentation process capable of producing various biochemical materials. Glucose prepared by the present invention can be directly used as a raw material for producing various bio-platform chemicals and derivatives.

Description

Beta -D-glucan glucohydrolase having the activity of exoglucanase and beta-glucosidase at the same time, and a method for producing glucose from cellulose using the enzyme, 1,4-beta-di- D-glucan glucohydrolase having Exoglucanase and β-glucosidase Activity and Method for Preparing Glucose from Cellulose using the Same}

The present invention relates to a novel enzyme 1,4-beta-di-glucan glucohydrolase having both exoglucanase and beta-glucosidase activity, and a method for producing glucose from cellulose using the same.

Cellulose accounts for about 40-50% of fiber-based biomass, a renewable natural resource abundant in nature. In particular, the use of cellulose is a virtually unlimited supply, and is recognized as a sustainable raw material that can replace conventional fossil fuels. In order to utilize it efficiently, however, several obstacles must be overcome first. One of them must convert the biomass, called lignocellulosic material, into a fermentable state by biological or chemical processes. The value of fermentable glucose is not only depleted in bio-energy, but also in almost all industries, such as pharmaceuticals, textiles, food, and cosmetics.

To date, known cellulose hydrolysis techniques have mainly used enzymes or liquid acids as catalysts. However, when a liquid acid catalyst is used, a secondary product is generated, environmental problems are caused by acid treatment, and continuous process is impossible. Also, it is known that the treatment with high temperature and high pressure steam is expensive, and the loss is large. Furfural or hydroxymethyl furfural (HMF) It has a problem that an additional step of removing such by-products is required because it acts as a toxin substance. Although a decomposition treatment method using ammonia at a high temperature has been proposed, problems such as generation of harmful components, loss of effective components, and problems of denaturation still exist. Though there is a technology to combine them properly as well as a supercritical process, all methods have advantages and disadvantages, and because of the variety of biomass, the standardization method and the degree of preprocessing are not yet available . This pretreatment method has the function of enlarging the surface area so that the enzyme can act, but it can not depolymerize the cellulose.

To date, enzymatic glycation techniques, known to the academic and research community, have been linked to three enzymes, endoglucanase (EC 3.2.1.4), exoglucanase (or cellobio hydrolases, EC 3.2.1.91), beta glucoside Glucosidase (EC 3.2.1.21) in a continuous or additive manner to finally produce glucose. That is, the endoglucanase randomly cleaves the internal? -1,4-glycoside bond and the exoglucanase cleaves at the non-reducing sugar terminus (at the reducing end of the cellobiol hydrolase) It is cleaved into cellobiose units. The cell biosynthetic enzyme, beta-glucosidase, acts on the resultant cellobiose and finally produces glucose. As a result, three enzymes are necessary to saccharify cellulose into glucose. However, in Korea, not only these enzymes are wholly dependent on imports but also bio-refineries that produce biofuels and chemical raw materials by using biomass raw materials industrially, .

In order to avoid the intellectual property rights that are prevalent in the developed countries and to develop the cellulase, the existing cellulase and the strains producing the cellulase are being developed in Korea and also in the world, , 1,4-β-D-glucan glucohydrolase was developed from a new strain of the genus Penny Bacillus. The enzyme was developed not only for cellobiose having two glucose units but also for 6 consecutive (Cellohexaose), which is a dextrin cell, was all saccharified with glucose, and at the same time, the present invention was completed by having a mass production system. That is, the present invention has developed a technique for producing glucose using only one enzyme without using three different enzymes. To accomplish this, it is necessary to precede polymerization of cellulose oligomer having low polymerization degree by fusion or modification of the conventional pretreatment method.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

As a method for producing glucose from renewable natural cellulose, the present inventors have found that a single enzyme capable of producing glucose more efficiently and economically as compared with a method using three enzymes for producing glucose from cellulose conventionally and a glucose producing method using the same In order to develop the research. As a result, 1,4-beta-di-glucan glucohydrolase, which is a novel enzyme having simultaneously exoglucanase and beta-glucosidase activity, was developed and the enzyme was dissolved in cellulose To thereby produce glucose. Thus, the present invention has been completed.

Accordingly, an object of the present invention is to provide a novel enzyme 1,4-beta-di-glucan glucohydrolase (1,4-dihydroxybenzoic acid) having simultaneously exoglucanase and beta-glucosidase activity. beta-D-glucan glucohydrolase.

It is another object of the present invention to provide a nucleic acid molecule encoding 1,4-beta-di-glucan glucohydrolase.

It is still another object of the present invention to provide a recombinant vector comprising a nucleic acid molecule encoding 1,4-beta-di-glucan glucohydrolase.

It is still another object of the present invention to provide a cell transformed with a recombinant vector comprising a nucleic acid molecule encoding 1,4-beta-di-glucan glucohydrolase.

It is still another object of the present invention to provide a pretreatment method of cellulosic dextrin for facilitating the action of an applied enzyme.

It is another object of the present invention to provide a method for producing glucose from biomass.

Another object of the present invention is to provide glucose produced by the method of the present invention.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a method for producing 1,4-D-glucosidase, which has an amino acid sequence as set forth in SEQ ID NO: 2 and simultaneously has exoglucanase activity and beta-glucosidase activity. Beta-D-glucan glucohydrolase. ≪ / RTI >

As a method for producing glucose from renewable natural cellulose, the present inventors have found that a single enzyme capable of producing glucose more efficiently and economically as compared with a method using three enzymes for producing glucose from cellulose conventionally and a glucose producing method using the same As a result, 1,4-beta-di-glucan glucohydrolase, a novel enzyme having both exoglucanase activity and beta-glucosidase activity, was developed. A novel method of producing glucose by treating the resulting cellulosic dextrin has been developed.

The novel enzyme of the present invention, 1,4-beta-di-glucan glucohydrolase, has an activity of cleaving the bond between glucose in 2 to 6 polymerized glucose units, and is composed of 6 glucose units of cellohexaose (cellohexaose) to glucose monomers.

Conventionally, in order to obtain glucose from cellulose, a three-step enzyme saccharification mechanism has been accepted as an orthodox method in which three enzymes such as endo, exo cellulase, and glucosidase must be hydrolyzed continuously. However, A new concept has been established in that glucose can be produced from cellulose even if only the enzymes are used. In the case of using the conventional technology system, there is a method of using only two enzymes such as exocellulase and glucosidase. However, the role of exocellulase and glucosidase in the present invention is not limited to glucohydrolase (1,4-β-D - glucan glucohydrolase), it became possible to produce glucose as an enzyme. In addition, it was possible to over-express and over-express the enzyme using a transformant, and 3-D structure analysis revealed cellulose and glucose-binding ligands. However, it did not react with cellulose oligomers having a degree of polymerization of at least 6 glucose. As a result of the hydrolysis of cellulose with two to six polymerized glucose, it was judged that glucose was cut one by one regardless of degree of polymerization. That is, it is considered that seven or more oligomers can be hydrolyzed with glucose only if they can be dissolved in a solution in which the enzyme activity is not inhibited. On the other hand, it was possible to immobilize the enzyme by using the surface - modified silica carrier as an enzyme recycling method, and the activity did not decrease during 10 times of recycling. As a result, three enzymes can be reduced to one, followed by immobilization, so that the enzyme can be utilized more than 10 times, and thus the enzyme price can be greatly reduced to about 1/30.

According to the present invention, the 1,4-beta-di-glucan glucohydrolase used in the present invention has the amino acid sequence of the second sequence of the sequence listing.

The 1,4-beta-di-glucan glucohydrolase of the present invention is also interpreted to include an amino acid sequence that exhibits substantial identity to the above-mentioned amino acid sequence. The above-mentioned substantial identity is determined by aligning the amino acid sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using an algorithm commonly used in the art. Homology, more preferably at least 90% homology, and most preferably at least 95% homology.

The biological functional equivalent that can be included in the 1,4-beta-di-glucan glucohydrolase of the present invention is an amino acid equivalent to the biological activity equivalent to the 1,4-beta-di-glucan glucohydrolase of the present invention It will be apparent to those skilled in the art that the present invention will be limited to variations of the sequence.

Such amino acid variations are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are both positively charged residues; Alanine, glycine and serine have similar sizes; Phenylalanine, tryptophan and tyrosine have similar shapes. Thus, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

In introducing the mutation, the hydrophobic index of the amino acid can be considered. Each amino acid is assigned a hydrophobic index according to its hydrophobicity and charge: isoruicin (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).

The hydrophobic amino acid index is very important in imparting the interactive biological function of proteins. It is known that substitution with an amino acid having a similar hydrophobicity index can retain similar biological activities. When a mutation is introduced with reference to a hydrophobic index, substitution is made between amino acids showing a hydrophobic index difference preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

On the other hand, it is also well known that the substitution between amino acids having similar hydrophilicity values leads to proteins with homogeneous biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Aspartate (+ 3.0 ± 1); Glutamate (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ± 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoru Isin (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4).

When a mutation is introduced with reference to the hydrophilicity value, the amino acid is substituted preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

Amino acid exchange in proteins that do not globally alter the activity of the molecule is known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thr / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly.

Considering the mutation having the above-mentioned biological equivalent activity, the 1,4-beta-di-glucan glucohydrolase of the present invention or the nucleic acid molecule encoding the 1,4-beta-glucan hydrolase of the present invention has a sequence substantially identical to the sequence described in the sequence listing And the like. The above substantial identity is determined by aligning the sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using algorithms commonly used in the art, ≪ / RTI > Alignment methods for sequence comparison are well known in the art. Various methods and algorithms for alignment are described by Smith and Waterman, Adv. Appl. Math. 2: 482 (1981) ; Needleman and Wunsch, J. Mol. Bio. 48: 443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988); Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc. Acids Res. 16: 10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8: 155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24: 307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215: 403-10 (1990)) is accessible from NCBI (National Center for Biological Information) It can be used in conjunction with sequencing programs such as blastx, tblastn and tblastx. BLSAT is available at http://www.ncbi.nlm.nih.gov/BLAST/. A method for comparing sequence homology using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

According to one embodiment of the present invention, the 1,4-beta-di-glucan glucohydrolase of the present invention is 1,4-beta-di-glucan glucohydrolase coded by the nucleotide sequence of SEQ ID No. 1 It is Ajay.

According to another aspect of the present invention, there is provided a nucleic acid molecule encoding 1,4-beta-di-glucan glucohydrolase having the amino acid sequence set forth in SEQ ID NO: 2.

As used herein, the term " nucleic acid molecule " has the meaning inclusive of DNA (gDNA and cDNA) and RNA molecules. In the nucleic acid molecule, the nucleotide which is a basic constituent unit is not only a natural nucleotide, Also included are analogues (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990)).

Variations in nucleotides do not cause changes in the protein. Such nucleic acids include functionally equivalent codons or codons that encode the same amino acid (e.g., by codon degeneration, six codons for arginine or serine), or codons that encode biologically equivalent amino acids ≪ / RTI >

Also, variations in the nucleotides may result in changes in the 1,4-beta-di-glucan glucohydrolase itself. Beta -D-glucan glucuronidase can be obtained that exhibits almost the same activity as the 1,4-beta-di-glucan glucuronhydrolase of the present invention even in the case of a mutation that causes change in the amino acid of 1,4-beta-di- have.

According to another aspect of the present invention, the present invention provides a recombinant vector comprising said nucleic acid molecule.

The vector system of the present invention can be constructed through various methods known in the art, and specific methods for this are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001) This document is incorporated herein by reference.

The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed using prokaryotic cells as hosts. The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression.

For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (e.g., T7 promoter, tac promoter, lac promoter, lac UV5 promoter, lpp promoter, p it is common to include _L ^λ promoter, p _R ^λ promoter, rac 5 promoter, amp promoter, recA promoter, SP6 Pro meoteo and trp promoters and the like), Lai bojom binding site and a transcription / translation termination sequences for the initiation of the decryption. When E. coli is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol. , 158: 1018-1024 (1984)) and the left promoter of phage l _L ^? Promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet. , 14: 399-445 (1980)).

The vectors that can be used in the present invention include plasmids (for example, pIVEX, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pUC19, pET etc.), phage (for example, λgt4λB, ,?? z1, and M13) or a virus (e.g., SV40 or the like).

On the other hand, the vector of the present invention includes, as a selection marker, an antibiotic resistance gene commonly used in the art, and includes, for example, ampicillin, gentamycin, carbinicillin, chloramphenicol, streptomycin, kanamycin, And resistance genes for tetracycline.

According to another aspect of the present invention, there is provided a cell transformed with said recombinant vector.

Any host cell known in the art can be used as a host cell capable of continuously cloning and expressing the vector of the present invention in a stable manner. For example, E. coli JM109, E. coli BL21 (DE3), E. coli RR1 , E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus tulifene, and Salmonella typhimurium, Serratia marcesensus and various Pseudomonas species And enterobacteria and strains such as the species.

Methods for delivering the vector of the present invention into a host cell can be carried out using a CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA , 9: 2110-2114 SN et al, Proc Natl Acac Sci USA, 9: 2110-2114 (1973); and Hanahan, D., J. Mol Biol, 166:....... 557-580 (1983)) and electroporation method (Dower, WJ et al., Nucleic Acids Res. , 16: 6127-6145 (1988)).

The vector injected into the host cell may be expressed in host cells, in which case a large amount of glucosidase is obtained. For example, when the expression vector comprises a lac promoter, the host cell may be treated with IPTG to induce gene expression.

According to a preferred embodiment of the present invention, the host cell used in the present invention is E. coli (BL21).

According to the present invention, the 1,4-beta-di-glucan glucohydrolase of the present invention can produce the enzyme in large quantities using a transformant. The 1,4-beta-di-glucan glucohydrolase of the present invention cuts glucose one by one into cellulodextrin polymerized with up to six glucose. That is, it is considered that seven or more oligomers can be hydrolyzed with glucose only if they can be dissolved in a solution in which the enzyme activity is not inhibited. On the other hand, when an enzyme is immobilized using a silica carrier whose surface is modified by an enzyme recycling method, the production cost of the enzyme can be greatly reduced.

According to another aspect of the present invention there is provided a pretreatment method of cellulose for facilitating the action of an applied enzyme comprising the steps of:

(a) adding an ionic liquid and a solid acid catalyst to the cellulose obtained from the biomass; And

(b) treating the result of step (a) with chloroform to induce crystallization and obtaining cellooligomer and cellodextrin as a precipitate.

Cellulose has the property that the monomer, glucose, does not dissolve in general organic solvent or water because it forms supramolecular structure strongly connected through hydrogen bonding. In order to produce various chemical products from cellulose, which is a polymer raw material, dissolution of cellulose must be preliminarily performed. In the present invention, ionic liquid and solid acid catalyst are used for cellulose dissolution.

The cellulodextrin obtained by pretreating cellulose can be used for various processes such as glucose production. When the solvent used for pretreatment of cellulose remains, the activity of the enzyme to be applied in the next step can not be exerted. Therefore, it is very important to completely remove the solvent used after the cellulose pretreatment. In the present invention, a method of simply removing the solvent by treating the pretreated product with chloroform to precipitate the dissolved product has been developed.

According to one embodiment of the present invention, the enzyme applied to the cellulodextrin obtained through the treatment with chloroform does not inhibit the activity.

According to another aspect of the present invention, there is provided a process for producing glucose from biomass comprising the steps of:

(a) adding an ionic liquid and a solid acid catalyst to the cellulose obtained from the biomass;

(b) treating the product of step (a) with chloroform to induce crystallization and obtaining cellooligomer and cellodextrin as a precipitate; And

(c) 1,4-beta-di-glucan glucohydrolase (1,4-dihydroxyvitamin), which is a single enzyme having the activity of exoglucanase and beta-glucosidase in the cellulodextrin, β-D-glucan glucohydrolase) to obtain glucose as a result.

The present inventors have fundamentally improved the conventional method of using endoglucanase, exoglucanase and beta-glucosidase as a method of producing glucose from renewable natural cellulose . As shown in FIG. 1, a technique capable of separating various cellulose oligomers by modifying the degree of polymerization of cellulose by treating a solid acid catalyst in the state of dissolving cellulose using an ionic liquid has been developed. Among these, 6 glucose By combining cell dextrin with less than hexaose and oligomer with more than 7 cells, the entire process could be unified. In this process, we emphasized recycling basically, including ionic liquids, solvents, and enzymes. In addition, nonreacted materials can be recycled based on continuous reactions. The volatile solvent, which dissolves the ionic liquid well but dissolves the sugar, such as chloroform or dichloromethane, was selected for the separation of the cellulose oligomer depolymerized by the ionic liquid and the solid acid catalyst, and was recycled through distillation. Subsequently, of the crystallized cellulose oligomers, cellulose hexaose polymerized with 6 glucose was dissolved, so that it was used as a substrate of the enzyme reaction and oligomers were reused as unreacted products in glucose of 7 or more cells.

The importance of ionic liquids is widely known, but one of the reasons why they can not be universally used is that they are expensive. However, the cost is not a problem because the process can be very easily recycled. Since ionic liquids can be synthesized in various ways, various ionic liquids other than the ionic liquids used in this study may be applied. This technology is not limited to cellulose but can be applied to all sugar polymers contained in mannan biomass such as xylose polymer, xylan, mannose polymer.

The method for producing glucose from the cellulose of the present invention will be described in detail as follows:

Step (a): Cellulose depolymerization step

First, depolymerization and dissolution of the cellulose obtained from the biomass yields cellodextrin.

As used herein, the term biomass is derived from living or recently dead organisms as a renewable energy source. Biomass is based on carbon, hydrogen and oxygen and can be used directly or converted to other energy products such as biofuels. Biomass is a resource produced by photosynthesis of plants, and its variety and composition vary. However, biomass is a source of sugars and thinning materials including starch, corn, potatoes, sugarcane, sugar beet, etc., woody materials including waste wood, . The biomass used to obtain the cellodextrin in the present invention is starchy, carbohydrate and / or woody biomass including cellulose.

According to one embodiment of the present invention, the cellulose of step (a) is produced from a variety of biomass present in nature. It is also possible to recycle used paper after it has already been used for printed paper such as a newspaper or a book or various packaging containers.

Methods for depolymerizing and dissolving cellulose can be carried out by various methods known in the art and preferably include adding an ionic liquid, sodium hydroxide (NaOH) and Urea, an acid solution, a base solution, In the present invention, after dissolving in an ionic liquid, depolymerization is carried out using a solid acid catalyst, and the ionic liquid is recycled.

As used herein, the term ionic liquid refers to a liquid consisting solely of ions, generally consisting of a large cation containing nitrogen and a smaller anion. This structure reduces the lattice energy of the crystal structure and consequently has a low melting point. The ionic liquid may include one or more compounds represented by the following formula (1).

[Chemical Formula 1]

[A] ⁺ [B] ^-

In the above formula (1), [A] ⁺ is at least one compound selected from the group consisting of compounds having the structural formula shown in Table 1.

R ₁ , R ₁ , R ₂ , R ₃ and R ₄ in Table 1 above may each independently be selected from the group consisting of hydrogen, C ₁ -C ₁₅ alkyl and C ₂ -C ₂₀ alkene, Sulfone, sulfoxide, thioester, ether, amide, hydroxyl, and amine.

In Formula 1, [B] ^- is ^{Cl -, Br -, I -} , OH -, NO 3 -, SO 4 -, CF 3 CO 2 -, CF 3 SO 3 -, BF 4 -, PF 6 -, (CF ₄ SO ₂ ) ₂ N ^- , AlCl ₄ ^- and Cl ^- / AlCl ₃ . For example, such compounds include 1-butyl-3-methyl imidazolium tetrachloroaluminate, 1-ethyl-3-methyl imidazolium tetrachloroaluminate, 1-ethyl-3-methyl imidazolium hydrogensulfate Butylimidazolium acetate, 1-butyl-3-methylimidazolium acetate, tris-2 (hydrolyzed) Ethyl-3-methylimidazolium methanesulfonate, methyl-tri-n-butylammonium methylsulfate, 1-butyl-3-methylimidazolium ethylsulfate, 3-methyl imidazolium thiocyanate, 1-butyl-3-methyl imidazolium thiocyanate, 1-ethyl-3-methyl imidazolium thiocyanate, Aryl-3-methyl imidazolium chloride, And the mixtures and composites, and the like.

As the above-mentioned normal temperature ionic liquid, commercially available products may be used, for example, 1-ethyl-3-methylimidazolium acetate, 1-butyl- Butyl-3-methylimidazolium chloride, 1-methylimidazolium chloride, 1-ethyl-2,3-dimethylimidazolium ethylsulfate, 1,2,3-trimethylimidazolium ethylsulfate, 3-methylimidazolium chloride, 1-hexyl-3-methylimidazolium chloride, 1-ethyl-3-methylimidazolium chloride, 1- Methyl-3-methylimidazolium bromide, 1-ethyl-3-methylimidazolium bromide, 1- Butyl-1-methylpyrrolidinium chloride, 1-butyl-2,3-dimethylimidazolium chloride, N- Methylimidazolium bromide, 1-methylimidazolium chloride, 1-ethylimidazolium bromide, 1-methylimidazolium bromide, 1-methylimidazolium bromide, 1-ethylimidazolium chloride, and 1,2-dimethylimidazolium chloride (Table 2), but are not limited thereto.

As used herein, the term "cellulose" is a renewable natural resource abundant in the natural world, which accounts for about 40% of the fibrous biomass. However, since cellulose is a crystalline substance having a? -1,4-glycosidic bond and is not easily dissolved in water or an organic solvent, it is difficult to utilize it as a fuel or a chemical substance.

The ionic liquid interferes with the complicated hydrogen bonding between the hydroxyl groups of the cellulose and the anion in the ionic liquid bonds with the hydrogen of the hydroxyl group of the cellulose and the cation binds with the oxygen of the hydroxyl group of the cellulose, . The amount of the ionic liquid added is not particularly limited and may be 5 to 20 times the amount of the solid component.

According to another embodiment of the present invention, in order to depolymerize the cellulose dissolved in the ionic liquid, a solid acid catalyst which does not limit the kind is treated to obtain cellulodextrin as a result. The degree of polymerization of the cellulose to be depolymerized can be controlled by adjusting the treatment concentration, temperature and pressure, and short-length cell dextrin can be prepared.

As used herein, the term " cellodextrin " refers to a cellulosic degradation product wherein the glucose molecule is less polymerized than the cellulose oligomer.

Step (b): Step of obtaining cellrodextrin

In step (a), the volatile organic solvent which dissolves the ionic liquid but does not dissolve the sugar (glucose, xylose, mannode, etc.) oligomers is treated to precipitate and separate all the cellulose oligomers including cellodextrin. Followed by the addition of water to dissolve only the cellodextrin. All of the undissolved cellulose oligomers are recycled in the first reaction vessel.

The method of the present invention can be applied regardless of the kind of the fibrinolytic biomass. When the separated cellulose is freeze-dried, it can be made into powder and can be stored for a long time. The cellulosic depolymerization product or the cellulodextrin can be efficiently separated through pretreatment of the separated cellulose with appropriate strength and the final product glucose (cello) can be separated by treating 1,4-beta-di-glucan glucohydrolase with the separated cellulodextrin -sugar.

According to an embodiment of the present invention, chloroform is added to the resultant product of step (a) and stirred, and then cellulose oligomer and cellodextrin are obtained as a precipitate.

Step (c): Step of obtaining glucose

According to one embodiment of the present invention, a single enzyme having the activity of exoglucanase and beta-glucosidase in dissolved cellulodextrin is treated to obtain glucose.

According to a specific embodiment of the present invention, glucose is obtained by treating 1,4-beta-di-glucan glucohydrolase with the activity of exoglucanase and beta-glucosidase in dissolved cellulodextrin.

According to one embodiment of the present invention, the conditions of the 1,4-beta-di-glucan glucohydrolase treatment are carried out under conventional enzyme reaction conditions. For example, 1,4-beta-di-glucan glucohydrolase is added to the cellodextrin at a reaction temperature of 30-45 ° C, preferably 39-41 ° C for 10-50 hours, preferably 20-30 hours To obtain glucose. In addition, the reaction temperature, the treatment time, the treatment concentration, and the amount of water can be controlled arbitrarily.

According to an embodiment of the present invention, the step (c) may further include the step of concentrating the glucose solution.

The glucose obtained in the step (c) can be directly used as a fermentation sugar by direct dehydration condensation using a membrane or as a raw material for producing a bio-platform chemical substance. Although the sugar concentration of the enzymatic glycosylation product of cellulose is about 3%, it is necessary to use it as a fermentation sugar, or to convert it into a biosubstrate chemical such as sorbitol through a dehydration reaction, In principle, distillation is used. However, when the distillation method is used, a lot of energy is needed. It can be concentrated by using the energy of 1/5 by using the membrane separation method, and the caramelization reaction which occurs when the distillation method is used can be excluded. Membranes that can be used in this device can utilize seawater desalination membranes and can detect sugar concentrations up to 30%.

The glucose production method of the present invention excludes the effects of excessive decomposition products and impurities which may be generated in a complicated conventional pretreatment process, and also simplifies the process, reduces the cost, eliminates environmental pollution, and is performed at room temperature. And a reduction in the production cost of saccharifying enzymes.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a novel enzyme 1,4-β-D-glucan glucohydrolase having the activity of exoglucanase and β-glucosidase, and a method for producing glucose from cellulose using the same.

(a) The present invention relates to a process for producing cellulose by lowering the degree of polymerization of cellulose by using an ionic liquid and a solid acid catalyst, and then producing a single enzyme 1,4-β-glucosidase having exoglucanase activity and beta-glucosidase activity, D-glucan glucohydrolase to prepare glucose, and in the production of glucose from cellulose, a process for recycling an ionic liquid, a solid acid catalyst, a volatile solvent, etc., and a technique for concentrating glucose are provided.

(c) Compared with the conventional method for producing glucose, which requires essentially three types of enzymes, the present invention can produce glucose with only one enzyme, so that the process is simple and the cost is greatly reduced. .

(d) Glucose produced by the present invention can be applied to a saccharification fermentation concurrent process or a saccharification fermentation continuous process in conjunction with a saccharification process and a fermentation process capable of producing various biochemical substances.

(e) Glucose prepared by the present invention can be directly used as a raw material for producing various bio-platform chemicals and derivatives.

1 is a whole process diagram of the new concept proposed in the present invention.
2 is a schematic diagram of a new concept of saccharification mechanism proposed in the present invention.
FIG. 3 is a diagram showing the three-dimensional structure of 1,4-β-D-glucan glucohydrolase and cellulose and glucose ligands.
4 is a depolymerization diagram of cellulose using an ionic liquid and a solid acid catalyst.
5 is a graph showing the results of HPLC analysis of glucose produced from various cellodextrins.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Production of cellodextrin through cell depolymerization

The ionic liquid treatment was carried out by dissolving the ionic liquid in a bath of 95-100 ° C in a hot water bath with a 32 ml (g) ionic liquid (BMIMCl) (Sigma Aldrich) in a 150 ml test tube, adding 1.0 g of cellulose And the mixture was dispersed as uniformly as possible, and then sufficiently dissolved at 97 ° C for about 3 hours using a Stirring bar. The amount of cellulose in the cellulose / ionic liquid solution added with various types of solid acid catalysts such as Amberlyst 15 DRY (Aldrich) or Graphene Oxide-Sulfonate (self-synthesized) %, Followed by stirring for 5 hours while dispersing the mixture as uniformly as possible. 100 ml of chloroform was added to the reaction solution, stirred, and filtered with a filter paper. Through this process, only naturally dissolved cellulose oligomer and cellulose remain as a precipitate with recrystallized sieve. The sugar precipitate was washed with a small amount of chloroform and the degree of polymerization of the recrystallized product was measured. The polymerization degree was measured by Ferdi (Angewante Chemie, 2008) method. The dried cellulose oligomer was dissolved in an ionic liquid (BMIMCl) (Sigma Aldrich) and then substituted with phenylisocyanate to obtain cellulose tricalcium Rate, dissolved in THF and analyzed using GPC (gel permeation chromatography). The analysis conditions were measured at 236 nm with a UV-VIS detector and a polystyrene standard curve was used. The other treatment methods can utilize ordinary treatment methods available in the art and can not be described in detail because they can be carried out variously according to the kind and treatment method of biomass.

Example 2: Production of 1,4-beta-di-glucan glucohydrolase

The present inventors isolated a gene encoding an enzyme protein having 1,4-beta-di-glucan glucuronidase activity from a strain of Penny Bacillus HPL-001 isolated and identified through previous studies. In the open reading frame (ORF) encoded by NCBI, the blast program of the NCBI was used to isolate seven genes with similarity to the previously reported β-glucosidase gene, Transformed into E. coli JM109 after insertion into the Smith vector. Seven clones showing 1,4-beta-di-glucan glucohydrolase activity were selected through the above experiment. The present inventors selected ORF1 (2160 bp, 719 A / a) (Amersham) overexpression vector and transformed into E. coli BL21.

In order to prepare a transformant overexpressing the novel 1,4-beta-di-glucan glucohydrolase, a translation frame was aligned at both ends of a 2,160 bp sequence including a start codon and a stop codon based on the gene base sequence A pair of oligonucleotides (5'-ACATGCCATATGATGAAAAAA CAAGGG-3 ', 5'-ACATGCGGATCCCTTACTCTAAAATAAACGAAG-3') were prepared by introducing BamH I and Nde I restriction enzyme recognition sites. PCR was performed using these oligonucleotides as bidirectional primers and ORF2-T ejaculatory vector plasmid DNA diluted 1/100 as a template. PCR reactions and reaction conditions were the same as above. The amplified product was purified and digested with restriction enzymes BamH I (NEB) and Nde I (NEB), and inserted between the BamH I and Nde I recognition sites of the protein overexpression vector pGEX (Amersham) to prepare a recombinant plasmid. The recombinant plasmid thus prepared was transformed into E. coli BL21 (RBC) to prepare 1,4-beta-di-glucan glucuronidase.

The plasmid DNA extracted from the transformed E. coli was digested with restriction enzyme recognition sites, and then the vector and the target DNA were successfully recombined through electrophoresis. After incubation for 18 h (37 ° C, 250 rpm, A ₅₉₅ = 1.0), the cells were inoculated again in fresh LB liquid medium and treated with 1 mM IPTG at an absorbance value of A ₅₉₅ of 0.6, Lt; / RTI > for 18 hours and harvested the cells. The harvested cells were suspended and centrifuged at 13,000 g after ultrasonication. The supernatant was separated into supernatant and precipitate, and its molecular weight was confirmed to be about 79 KDa by SDS PAGE.

The pGEX-Peani-PGD? 4 recombinant plasmid containing the gene encoding the novel 1,4-beta-di-glucan glucohydrolase was transformed into E. coli BL21-Gold (DE) (Stratagene, USA) (LB 25 g / L), and the mixture was incubated at 37 ° C with stirring at 150 rpm until the OD ₅₉₅ value became 0.4-0.6. To induce expression of the target protein in E. coli cells, IPTG (isopropyl-D-thiogalactoside) was added to the suspension to a final concentration of 1 mM, followed by 18-hour agitation at 18 ° C and 150 rpm. The culture was centrifuged at 10,000 rpm for 10 minutes, and the precipitate was washed twice with a potassium phosphate buffer solution (PBS). The washed precipitate was resuspended in PBS, and the cells were disrupted using an ultrasonic disrupter (Bio rad), and the supernatant was recovered by centrifugation (12,000 rpm, 10 minutes). The purified 1,4-beta-di-glucan glucuronidylhydrolase pure water in the recovered supernatant was collected by adding the supernatant to the glutathione-S-transferase (hereinafter referred to as " glutathione S-transferase ") equilibrated with a washing buffer solution (50 mM Tris-HCl, (GST binding resin column, Novagen), treated with factor Xa protease (NEB), and purified using a washing buffer solution (50 mM Tris-HCl, 100 mM NaCl; pH 7.0) Respectively.

The activity of 1,4-beta-di-glucan glucohydrolase was measured from each sample recovered in the purification step, and the purification of the enzyme active fraction was confirmed by SDS-PAGE. Protein content was measured using the Bradford method (Bradford, Sigma Aldrich) and BSA (Bovine Serum Albumin) was used as the standard protein. As a result, a large amount of 1,4-beta-di-glucan glucohydrolase could be easily produced by glutathione resin column chromatography. In addition, since 1,4-beta-di-glucan glucuronhydrolase activity was not changed even in the state of 1,4-beta-di-glucan glucuronhydrolase bound to the resin, It can be applied to the high efficiency conversion process.

Using the purified 1,4-beta-di-glucan glucohydrolase, the activity of producing glucose by the kind of cellodextrin (cellobiose, cellotriose, cellotetraose, cellopentose, cellulosanose, etc.) Respectively. Glucose produced by reacting the developed enzyme was analyzed and confirmed by HPLC. The analysis conditions were as follows: Shodex Asahipak column NH ₂ P-50 4E, eluent: H ₂ O / CH ₃ CN = 1: 3, flow rate: 1 ml / Min, detector: RI, column temperature: 30 < 0 > C, and the like. The results are shown in Table 2 and FIG.

HPLC analysis of glucose produced from various cell dextrins Reaction time
(minute) Detection amount by substrate (area, mV * s) Cellobiose Celrotrios Cellotetraose Cellopentaose Cell nuclearaceus Cell
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Example 3 Production and Concentration of Glucose

Although the sugar concentration of the enzymatic glycosylation product of cellulose is about 3%, it is necessary to use it as a fermentation sugar, or to convert it into sorbitol through dehydration reaction, it should be concentrated to a sugar concentration of 15% or more. do. However, when the distillation method is used, a lot of energy is needed. It can be concentrated by using the energy of 1/5 by using the membrane separation method, and the caramelization reaction which occurs when the distillation method is used can be excluded. Membranes that can be used in this device can utilize seawater desalination membranes and can detect sugar concentrations up to 30%.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> KOREA RESEARCH INSTITUTE OF CHEMICAL TECHNOLOGY <120> Method for Preparing Glucose from Cellulose using Single Enzyme <130> PN130510 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 2160 <212> DNA <213> glucohydrolase nucleotide <400> 1 atggacaacc ggcagttaac cgaactgttg aatcagatga cgctggagga gaagattgcg 60 cagctgctcc agctgaccgc caattttcat gaaggcggcg aaagccaaat taccggaccg 120 cttgaggaaa tggggatcac cgaatcgatg gtgaccgcca gcggctccgt tctggggtta 180 tcgggagcgc agtcggtcat cgacgtgcag aaatcatatt taagcaaaag ccggctcggc 240 attccgctga tgtttatggc cgatgtggtg catggcttca aaacgatctt tccgatcccg 300 ctggcgatcg gatgctcctg ggatacggag ctggccgaga agagcgccga aattgccgcg 360 agagagtcgg cggtatccgg cattcatgta acgttcgcac caatggtcga tttggtgcgc 420 ggccgcggt ggggacgcgt catggagacg accggcgaag atccgtacct gaacagcctg 480 tttgcccgcg cttttgtccg cggctatcaa ggcaagagcc tgaaggatga gccggagcgg 540 ctggctgcct gcgtgaagca cttcgccgcc tatggcgctc ctgaaggagg acgcgactac 600 aacacggtca acatgtcgga acgcaatctg cgggagcagt atctgccggc ctataaagcg 660 gcactggatg aaggctgcga gatggtcatg acgtcgttca ataccgtgga cggcattccg 720 gctaccggca atgagaagct gatgcgcggc atcctccgcg atgagtgggg ttttgacggc 780 gtcctgattt cggactgggc gtcgatccgc gaaatgatcg cgcacggcgc ggcggaggac 840 gattgcgagg ccgcttaccg ggcgatccgg gccggcgtgg atatcgagat gatgacccct 900 tgttacgtac agcatctgcc gggcctcatc cagagcggac gcgtggacga agcgttggtc 960 gacgaagcgg tgctgcggat attgcggctg aaagacaagc tcgggttgtt cgacaatccg 1020 ctgcgaggag ccgatccgga gcgtgagcgc gaagtactgt tctgtcagga ccatcgaaac 1080 gtttcttacg agctggcagc caaatcgagc gtactgctga aaaacgaagg cggcctgctg 1140 ccgcttgcgt ccgggaagaa ggttgcgctg attggaccgt tcgcccagag cgacgacatc 1200 ctcggctggt ggtcctgcgt cggcgtgaag gaagacgccg tgaagctggg aaccgccctg 1260 caggagcggc tggctgaggg acagctggtc attgccgaag gctccggcgt cgtaagcgtg 1320 gagcaatcgc agattgacgc tgccatcgcc gcggctcaag acgcggacgt gatcgtgctc 1380 gcccttggcg aagattcggc catgagcggg gaaggcgggt cccgcacgaa catccgcttg 1440 cctgaagccc agctggagct ggtgcggcag ctgaagaaga ttggcaagcc gatggctgcg 1500 gtaatcttta atggccgccc gctggacctg cacggaatca ttgacgaagc ggacgccgtc 1560 ctggaagcct ggttcccggg cagcgaaggc ggagccgcga tcgccgacat tctgatcggg 1620 cgcgtgaatc cttcggcccg gctgacgatg tccttcccgc attcggtggg acaaattccg 1680 gtctattata accattacaa taccggccgt ccgatgaatc cggacaaaac ggaagagcgg 1740 tatgtgtcca aatatatcga cagtccgaat gatccgctgc ttccgttcgg atacggccta 1800 tcctatacga cgtttgaata cggcgagctg aaattgtccg gaaacgagat aacggccaat 1860 cagccgcttc aggtcagcgt gaacgtaacg aacaccgggg atcgcgcagg catcgaaacg 1920 gttcagctgt atatccgcga cgtggccggt gaagtggtcc gtccgatgcg cgagctgaaa 1980 ggcttcgtac gtctcgagct tgctcccggg gaaagcgcgg atgcggtctt tacgatcacg 2040 gaggaacagc ttcgttatca tcattccgac cttacgcaag ccagcgatcc gggaacgttc 2100 caggtgtttg tcggtccgaa cagccgggac acccttgctg agtcgttccg gctggtttag 2160                                                                         2160 <210> 2 <211> 719 <212> PRT <213> glucohydrolase amino acid <400> 2 Met Asp Asn Arg Gln Leu Thr Glu Leu Leu Asn Gln Met Thr Leu Glu   1 5 10 15 Glu Lys Ile Ala Gln Leu Leu Gln Leu Thr Ala Asn Phe His Glu Gly              20 25 30 Gly Glu Ser Gln Ile Thr Gly Pro Leu Glu Glu Met Gly Ile Thr Glu          35 40 45 Ser Met Val Thr Ala Ser Gly Ser Val Leu Gly Leu Ser Gly Ala Gln      50 55 60 Ser Val Ile Asp Val Gln Lys Ser Tyr Leu Ser Lys Ser Arg Leu Gly  65 70 75 80 Ile Pro Leu Met Phe Met Ala Asp Val Val His Gly Phe Lys Thr Ile                  85 90 95 Phe Pro Ile Pro Leu Ala Ile Gly Cys Ser Trp Asp Thr Glu Leu Ala             100 105 110 Glu Lys Ser Ala Glu Ile Ala Ala Arg Glu Ser Ala Val Ser Gly Ile         115 120 125 His Val Thr Phe Ala Pro Met Val Asp Leu Val Arg Asp Pro Arg Trp     130 135 140 Gly Arg Val Met Glu Thr Thr Gly Glu Asp Pro Tyr Leu Asn Ser Leu 145 150 155 160 Phe Ala Arg Ala Phe Val Arg Gly Tyr Gln Gly Lys Ser Leu Lys Asp                 165 170 175 Glu Pro Glu Arg Leu Ala Cys Val Lys His Phe Ala Ala Tyr Gly             180 185 190 Ala Pro Glu Gly Gly Arg Asp Tyr Asn Thr Val Asn Met Ser Glu Arg         195 200 205 Asn Leu Arg Glu Gln Tyr Leu Pro Ala Tyr Lys Ala Ala Leu Asp Glu     210 215 220 Gly Cys Glu Met Val Met Thr Ser Phe Asn Thr Val Asp Gly Ile Pro 225 230 235 240 Ala Thr Gly Asn Glu Lys Leu Met Arg Gly Ile Leu Arg Asp Glu Trp                 245 250 255 Gly Phe Asp Gly Val Leu Ile Ser Asp Trp Ala Ser Ile Arg Glu Met             260 265 270 Ile Ala His Gly Ala Ala Glu Asp Asp Cys Glu Ala Ala Tyr Arg Ala         275 280 285 Ile Arg Ala Gly Val Asp Ile Glu Met Met Thr Pro Cys Tyr Val Gln     290 295 300 His Leu Pro Gly Leu Ile Gln Ser Gly Arg Val Asp Glu Ala Leu Val 305 310 315 320 Asp Glu Ala Val Leu Arg Ile Leu Arg Leu Lys Asp Lys Leu Gly Leu                 325 330 335 Phe Asp Pro Leu Arg Gly Ala Asp Pro Glu Arg Glu Arg Glu Val             340 345 350 Leu Phe Cys Gln Asp His Arg Asn Val Ser Tyr Glu Leu Ala Ala Lys         355 360 365 Ser Ser Val Leu Leu Lys Asn Glu Gly Gly Leu Leu Pro Leu Ala Ser     370 375 380 Gly Lys Lys Val Ala Leu Ile Gly Pro Phe Ala Gln Ser Asp Asp Ile 385 390 395 400 Leu Gly Trp Trp Ser Cys Val Gly Val Lys Glu Asp Ala Val Lys Leu                 405 410 415 Gly Thr Ala Leu Gln Glu             420 425 430 Glu Gly Ser Gly Val Val Ser Val Glu Gln Ser Gln Ile Asp Ala Ala         435 440 445 Ile Ala Ala Gln Asp Ala Asp Val Ile Val Leu Ala Leu Gly Glu     450 455 460 Asp Ser Ala Met Ser Gly Glu Gly Gly Ser Arg Thr Asn Ile Arg Leu 465 470 475 480 Pro Glu Ala Gln Leu Glu Leu Val Arg Gln Leu Lys Lys Ile Gly Lys                 485 490 495 Pro Met Ala Ala Val Ile Phe Asn Gly Arg Pro Leu Asp Leu His Gly             500 505 510 Ile Ile Asp Glu Ala Asp Ala Val Leu Glu Ala Trp Phe Pro Gly Ser         515 520 525 Glu Gly Gly Ala Ile Ala Asp Ile Leu Ile Gly Arg Val Asn Pro     530 535 540 Ser Ala Arg Leu Thr Met Ser Phe Pro His Ser Val Gly Gln Ile Pro 545 550 555 560 Val Tyr Tyr Asn His Tyr Asn Thr Gly Arg Pro Met Asn Pro Asp Lys                 565 570 575 Thr Glu Glu Arg Tyr Val Ser Lys Tyr Ile Asp Ser Pro Asn Asp Pro             580 585 590 Leu Leu Pro Phe Gly Tyr Gly Leu Ser Tyr Thr Thr Phe Glu Tyr Gly         595 600 605 Glu Leu Lys Leu Ser Gly Asn Glu Ile Thr Ala Asn Gln Pro Leu Gln     610 615 620 Val Ser Val Asn Val Thr Asn Thr Gly Asp Arg Ala Gly Ile Glu Thr 625 630 635 640 Val Gln Leu Tyr Ile Arg Asp Val Ala Gly Glu Val Val Arg Pro Met                 645 650 655 Arg Glu Leu Lys Gly Phe Val Arg Leu Glu Leu Ala Pro Gly Glu Ser             660 665 670 Ala Asp Ala Val Phe Thr Ile Thr Glu Glu Gln Leu Arg Tyr His His         675 680 685 Ser Asp Leu Thr Gln Ala Ser Asp Pro Gly Thr Phe Gln Val Phe Val     690 695 700 Gly Pro Asn Ser Arg Asp Thr Leu Ala Glu Ser Phe Arg Leu Val 705 710 715

Claims (11)

Beta -D-glucan glucuronidase, which has an activity of exoglucanase and beta-glucosidase of the amino acid sequence set forth in Sequence Listing Sequence 2, (1,4-β-D-glucan glucohydrolase).
The 1,4-beta-di-glucan glucohydrolase according to claim 1, wherein the 1,4-beta-di-glucan glucohydrolase cleaves the bond between the glucose polymers.
A nucleic acid molecule encoding 1,4-beta-D-glucan glucohydrolase consisting of the amino acid sequence set forth in SEQ ID NO: 2.
A recombinant vector comprising the nucleic acid molecule of claim 3.
A cell transformed by the recombinant vector of claim 4.
delete A method for producing glucose from cellulose comprising the steps of:
(a) adding an ionic liquid and a solid acid catalyst to the cellulose obtained from the biomass;
(b) treating the product of step (a) with chloroform to induce crystallization and obtaining cellooligomer and cellodextrin as a precipitate; And
(c) the cellulodextrin is a single enzyme having the activity of exoglucanase and? -glucosidase, which is composed of the amino acid sequence of the second sequence of Sequence Listing; - treating 1,4-β-D-glucan glucohydrolase to obtain glucose as a result.
delete 8. The method of claim 7, wherein the biomass is a carbohydrate, starch, or woody biomass.
8. The method of claim 7, further comprising concentrating the glucose produced as a result of step (c).
delete

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