CN1238501C - Novel externally tangent-beta-1,4-glucanase/internally tangent-beta-1,4-xylanase and application thereof - Google Patents

Abstract

The present invention provides a new exo-beta-1,4-glucanase/endo-beta-1,4-xylanase, a polynucleotide encoding the enzyme and the production of the enzyme by DNA recombination technology an enzymatic method. The present invention also discloses the carrier and host cell containing the exo-β-1,4-glucanase/endo-β-1,4-xylanase, and its effect on the production of simple sugars and glucose use.

Description

Exo-β-1,4-glucanase/endo-β-1,4-xylanase and its application

technical field

The present invention relates to the field of biology. More specifically, the present invention relates to a novel exo-β-1,4-glucanase/endo-β-1,4-xylanase, a polynucleotide encoding the enzyme, and Techniques for producing such enzymes. The present invention also relates to vectors and host cells containing the exo-β-1,4-glucanase/endo-β-1,4-xylanase and its use in the production of simple sugars and glucose.

Background technique

Cellulose is nature's most abundant renewable energy source. Bioconverting cellulose without any chemical treatment into simple sugar or glucose as a fermentative carbon source to produce ethanol is one of the most ideal and effective methods for utilizing natural cellulose resources.

It is generally believed that the biotransformation of cellulose to produce glucose requires the synergy of at least three different enzymes: (1) endoglucanase (Endo-1, 4-β-D-glucanase, E.C.3.2.1.4, also known as for endocellulase). It acts on the non-crystalline region of cellulose, randomly hydrolyzes β-1,4-glycosidic bonds to generate shorter oligosaccharides with non-reducing ends, (2) exoglucanase (Exo-1,4-β- D-glucanase, E.C.3.2.1.91), it acts on the non-reducing end of the cellulose molecule to hydrolyze the β-1,4-glucosidic bond to produce cellobiose, (3) β-glucosidase (β-D-glucosidase, E.C. 3.2.1.21), which hydrolyzes cellobiose to glucose.

At present, the research is more concentrated on the cellulase system of fungi and bacteria. The cellulase systems of species such as Trichoderma reesei (T.reesei) and Trichoderma viride (T.viride), Clostridium thermocellum (C.thermocellum) and cellulomonas fimi in filamentous fungi are relatively complex, with a variety of sub Class endonuclease, exonuclease and glucosidase [ThomasM.Wood, Biochemical.SocietyTransactions.1992,20,46-53], for example Trichoderma viride has 6 subtypes of endonuclease [Beldman, G.et al Eur. J. Biochem. 1985, 146: 301-308]. The cellulase of Clostridium thermocellum needs to form a large molecular weight fiber body (Cellulosome) with multiple proteins to perform the catalytic reaction [Felix C.R and L.G.Ljiungdahl, Annu.Rev.Microbiol.1993, 47:791-819], obviously Complex enzyme systems will inevitably bring great difficulties to research and application. On the other hand, purified single-component endo- and exo-enzymes either cannot hydrolyze untreated natural cellulose to produce simple sugars, or have very low hydrolytic activity, such as the fiber of Trichoderma reesei (T.reesei) The synergistic action of at least 14 types of cellulase is needed in the enzyme system to hydrolyze the untreated plant fiber.

The traditional view is that animals do not have their own cellulase system, and need to rely on the cellulase of symbiotic bacteria to hydrolyze cellulose into simple sugars for the needs of life activities. However, in the late 1990s, endocellulase in termites and crayfish was discovered [Hirofumi, W.Gaku, T, nathan, L Nature, 1998, 394: 330-331; Keren A.B. et al., Gene, 1999, 239 , 317-324]. In addition, 12 endonuclease genes were also found in Arabidopsis [del Compillo, Curr. Top. Dev. Boil. 1999, 46: 39-61]. Animal cellulase may become a hotspot of new applied research.

On the earth, more than 10 billion tons of dry plant matter can be formed by photosynthesis of fixed _CO2 every year, and more than half of these materials are composed of cellulose, followed by hemicellulose (mainly composed of xylan) ). If we add the discarded cellulose caused by human activities, such as rice straw, wheat straw, etc., the amount of its existence will be calculated in astronomical figures. Cellulase and xylanase (hemicellulase) play a key role in how to comprehensively utilize plant dry matter or waste cellulose by biotechnology. Efficiently converting these native celluloses into simple sugars is key to the use of cellulose as a renewable energy source. Current cellulase enzymes are far from meeting the needs of converting plant cellulose into simple sugars as renewable energy sources. Therefore, there is an urgent need in this field to develop new cellulase enzymes from different sources that can hydrolyze cellulose with high efficiency, and a new process for producing glucose.

Contents of the invention

The purpose of the present invention is to provide a new exo-beta-1,4-glucanase/endo-beta-1,4-xylanase and its fragments, analogs and derivatives, and its coding sequence.

Another object of the present invention is to provide a method for producing exo-β-1,4-glucanase/endo-β-1,4-xylanase and its use.

Another object of the present invention is to provide a new process for the production of simple sugars and glucose.

In a first aspect of the present invention, there is provided a novel isolated exo-β-1,4-glucanase/endo-β-1,4-xylanase comprising: having SEQ ID NO : a polypeptide of 2 amino acid sequences, or a conservative variant polypeptide thereof, or an active fragment thereof, or an active derivative thereof. Preferably, the enzyme is selected from the group consisting of: (a) a polypeptide having the amino acid sequence of SEQ ID NO: 2; (b) the amino acid sequence of SEQ ID NO: 2 undergoes substitution, deletion or addition of one or more amino acid residues The polypeptide derived from (a) formed and having exoglucan and/or endo-xylan functions.

In a second aspect of the present invention there is provided a polynucleotide encoding an isolated exo-β-1,4-glucanase/endo-β-1,4-xylanase comprising a A nucleotide sequence having at least 70% identity to a nucleotide sequence selected from the group consisting of: (a) encoding the aforementioned exo-β-1,4-glucanase/endo - a polynucleotide of beta-1,4-xylanase; and (b) a polynucleotide complementary to polynucleotide (a). Preferably, the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID NO:2. More preferably, the sequence of the polynucleotide is one selected from the following group: having the sequence of 77-1261 in SEQ ID NO: 1; having the sequence of 1-1293 in SEQ ID NO: 1.

In the third aspect of the present invention, there are provided vectors containing the above-mentioned polynucleotides, and host cells transformed or transduced by the vectors or host cells directly transformed or transduced by the above-mentioned polynucleotides.

In a fourth aspect of the present invention, a method for preparing exo-β-1,4-glucanase/endo-β-1,4-xylanase is provided, the method comprising: (a) suitable for Under the condition of expressing the exo-β-1,4-glucanase/endo-β-1,4-xylanase, cultivate the above-mentioned transformed or transduced host cells; (b) from the culture The exo-β-1,4-glucanase/endo-β-1,4-xylanase was isolated from

In the fifth aspect of the present invention, the use of exo-β-1,4-glucanase/endo-β-1,4-xylanase, its coding sequence, vector or host cell of the present invention is provided , they can be used for the production of simple sugars and glucose, especially from plant dry matter such as straw.

In the sixth aspect of the present invention, a new process for producing glucose is provided, comprising the steps of: (a) using the above-mentioned exo-β-1,4-glucanase/endo-β-1 of the present invention , 4-xylanase or transformed or transduced host cells process the cellulosic material, thereby producing simple sugars; (b) isolating said simple sugars. Preferably, said cellulosic material is cellulosic material without any chemical pretreatment.

Other aspects of the invention will be apparent to those skilled in the art from the technical disclosure herein.

Description of drawings

Figure 1 is the SDS-polymerization of the hydrolyzed peptide fragments generated by endo-Glu C proteolytic enzyme hydrolysis of apple snail exo-β-1,4-glucanase/endo-β-1,4-xylanase Acrylamide gel electrophoresis (SDS-PAGE) pattern.

Figure 2 shows that the host cells transformed with the apple snail exo-β-1,4-glucanase/endo-β-1,4-xylanase cDNA sequence have the activity of hydrolyzing xylan.

Figure 3 shows the effect of chloride ion on apple snail exo-β-1,4-glucanase/endo-β-1,4-xylanase [exo-β-1,4-glucanase] effect on activity.

Detailed ways

After extensive and in-depth research, the inventors first obtained a cellulase system from Ampullarium Crossean that can effectively hydrolyze rice straw that has not undergone any chemical treatment to produce simple sugars, and isolated exo-cellulase and endo-cellulase and β-glucosidase. Wherein, a kind of isolated apple snail exocellulase also has xylohydrolase (hemicellulase) activity, that is, a bifunctional enzyme with two kinds of enzymatic activities. According to the principle of enzyme taxonomy, the The enzyme should belong to the 10th glycoside hydrolase family (family 10). Therefore, the enzyme can be called exo-β-1,4-glucanase/endo-β-1,4-xylanase (exo-β-1,4-glucanase/endo-β-1, 4-xylanase), the exonuclease activity of this enzyme is not inhibited by high-concentration products, the bifunctional enzyme with uniform SDS-PAGE can hydrolyze rice straw to generate simple sugars, and also has the activity of hemicellulase with high specific activity, It is one of the most active hemicellulases found so far. The present invention has been accomplished on this basis.

In the present invention, the terms "Piaphylla exocellulase", "Piaphylla exoglucanase", "Piaphylla exo-1,4-β-D-glucanase", or "Exo-β -1,4-glucanase/endo-β-1,4-xylanase"are used interchangeably, both refer to -1,4-xylanase amino acid sequence (SEQ ID NO: 2) protein or polypeptide. They include exo-beta-1,4-glucanases/endo-beta-1,4-xylanases with or without an initial methionine. Here endo-β-1,4-xylanase may also be referred to as hemicellulase.

As used herein, "isolated" means that the material is separated from its original environment (if the material is native, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist together in the natural state .

As used herein, "isolated exo-β-1,4-glucanase/endo-β-1,4-xylanase protein or polypeptide" refers to exo-β-1,4-glucanase Glycanases/endo-beta-1,4-xylanases are substantially free of other proteins, lipids, carbohydrates or other substances with which they are naturally associated. Those skilled in the art can purify the exo-β-1,4-glucanase/endo-β-1,4-xylanase using standard protein purification techniques. Substantially pure polypeptides yield a single band on non-reducing polyacrylamide gels.

The exo-β-1,4-glucanase/endo-β-1,4-xylanase of the present invention may be recombinant, natural, synthetic, preferably recombinant. The exo-β-1,4-glucanase/endo-β-1,4-xylanase of the present invention may be a natural purified product, or a chemically synthesized product, or obtained from prokaryotic Or produced in eukaryotic hosts (eg, bacteria, yeast, higher plants, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated, or may be non-glycosylated. Polypeptides of the invention may or may not include an initial methionine residue.

The present invention also includes fragments, derivatives and analogs of the exo-beta-1,4-glucanase/endo-beta-1,4-xylanase. As used herein, the terms "fragment", "derivative" and "analogue" refer to the native exo-beta-1,4-glucanase/endo-beta-1,4-glucanase/endo-beta-1,4-glucanase of the present invention A polypeptide having the same biological function or activity as xylanase. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide in combination with another compound (such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol), or (iv) an additional amino acid sequence fused to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence). Such fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

In the present invention, the term "Pipa snail exo-β-1,4-glucanase/endo-β-1,4-xylanase" refers to Enzyme/endo-beta-1,4-xylanase activity of the polypeptide of SEQ ID NO: 2 sequence. The term also includes variant forms of the sequence of SEQ ID NO: 2 having exo-β-1,4-glucanase/endo-β-1,4-xylanase. These variations include (but are not limited to): deletions and insertions of several (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of apple snail exo-β-1,4-glucanase/endo-β-1,4-xylanase.

The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and exo-β-1,4- Protein encoded by the DNA of the glucanase/endo-beta-1,4-xylanase DNA hybrid. The present invention also provides other polypeptides, such as fusion proteins comprising apple snail exo-β-1,4-glucanase/endo-β-1,4-xylanase or fragments thereof. In addition to the nearly full-length polypeptide, the present invention also includes soluble fragments of apple snail exo-β-1,4-glucanase/endo-β-1,4-xylanase. Typically, the fragment has at least about 30 consecutive amino acids, preferably at least about 50 consecutive amino acids of the apple snail exo-β-1,4-glucanase/endo-β-1,4-xylanase sequence Amino acids, more preferably at least about 80 contiguous amino acids, most preferably at least about 100 contiguous amino acids.

The invention also provides analogues of apple snail exo-β-1,4-glucanase/endo-β-1,4-xylanase or polypeptide. The difference between these analogues and natural apple snail exo-β-1,4-glucanase/endo-β-1,4-xylanase may be the difference in amino acid sequence, or it may not affect the sequence A difference in the form of modification, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

In the present invention, "the conservative variant polypeptide of apple snail exo-β-1,4-glucanase/endo-β-1,4-xylanase" refers to the amino acid sequence of SEQ ID NO:2 In contrast, at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table A.

Table A initial residue representative replacement preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala His(H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Leu

A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes a protein with SEQ ID NO: 2, but differs from the sequence of the coding region shown in SEQ ID NO: 1.

The polynucleotide encoding the mature polypeptide of apple snail exo-β-1,4-glucanase/endo-β-1,4-xylanase includes: the coding sequence that only encodes the mature polypeptide; the coding sequence of the mature polypeptide + Various additional coding sequences; the coding sequence (and optional additional coding sequences) of the mature polypeptide + non-coding sequences.

The term "polynucleotide encoding apple snail exo-β-1, 4-glucanase/endo-β-1, 4-xylanase" may include only encoding apple snail exo-β-1,4 - A glucanase/endo-beta-1,4-xylanase polynucleotide, which may also comprise additional coding and/or non-coding sequences.

The present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides or polypeptide fragments, analogs and derivatives having the same amino acid sequence as the present invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides without substantially altering the function of the polypeptide it encodes .

The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 95%. Also, the polypeptide encoded by the hybridizable polynucleotide has exo-beta-1,4-glucanase activity and endo-beta-1,4-xylanase activity.

The present invention also relates to nucleic acid fragments that hybridize to the above-mentioned sequences. As used herein, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 30 nucleotides in length, more preferably at least 50 nucleotides in length, most preferably at least 100 nucleotides in length. Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR) to determine and/or isolate polysaccharides encoding apple snail exo-β-1,4-glucanase/endo-β-1,4-xylanase polynucleotide.

The apple snail exo-β-1,4-glucanase/endo-β-1,4-xylanase nucleotide full-length sequence or its fragments of the present invention can usually be amplified by PCR or recombination or obtained by artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the apple snail cDNA library prepared by conventional methods is used as a template to amplify to obtain the relevant sequences.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short.

Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The present invention also relates to vectors comprising polynucleotides of the present invention, as well as vectors or exo-β-1,4-glucanase/endo-β-1,4-xylanase coding sequences of the present invention Transformed host cells, and methods for producing the polypeptides of the invention via recombinant techniques.

By conventional recombinant DNA technology (Science, 1984; 224:1431), the polynucleotide sequence of the present invention can be used to express or produce recombinant exo-β-1,4-glucanase/endo - beta-1,4-xylanase. Generally speaking, there are the following steps:

(1). Use the polynucleotide (or variant) encoding exo-β-1,4-glucanase/endo-β-1,4-xylanase of the present invention, or use the polynucleotide (or variant) containing the The recombinant expression vector of the polynucleotide transforms or transduces a suitable host cell;

(2). Host cells cultured in a suitable medium;

(3). Isolate and purify protein from culture medium or cells.

In the present invention, the exo-β-1,4-glucanase/endo-β-1,4-xylanase polynucleotide sequence can be inserted into the recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, or other vectors well known in the art. Vectors suitable for use in the present invention include, but are not limited to: T7-based expression vectors for expression in bacteria (Rosenberg, et al. Gene, 1987, 56:125). In short, any plasmid and vector can be used as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, marker genes, and translational control elements.

Methods well known to those skilled in the art can be used to construct DNA sequences encoding exo-β-1,4-glucanase/endo-β-1,4-xylanase and appropriate transcription/translation controls Signal expression vector. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989). Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: E. coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, reverse LTRs of transcription viruses and other promoters known to control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, and neomycin resistance, Or tetracycline, kanamycin or ampicillin resistance for E. coli.

Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.

The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell. Escherichia coli is preferred. Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, etc.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

The enzymes of the present invention also include immobilized enzymes immobilized on solid phase supports. Various immobilized enzyme techniques well known in the art can be used to prepare the immobilized exo-β-1,4-glucanase/endo-β-1,4-xylanase of the present invention.

In addition, the present invention provides the use of exo-β-1,4-glucanase/endo-β-1,4-xylanase, its coding sequence, vector or host cell, which are used to produce The craft of simple sugars, especially glucose. The simple sugars include cellobiose, simple pentoses, glucose and mixtures thereof.

The process for producing simple sugars of the present invention comprises the steps of: (a) using the above-mentioned exo-β-1,4-glucanase/endo-β-1,4-xylanase of the present invention or transforming or transforming processing the cellulosic material by the induced host cells to produce simple sugars; (b) isolating said simple sugars. The enzyme of the invention can directly produce simple sugars such as cellobiose and simple pentose. A preferred process also includes the addition of other enzymes, such as β-glucosidase (which hydrolyzes cellobiose to glucose), so that glucose can be produced directly.

A representative technique comprises the steps of: (a) mixing (by weight/volume) 20-30% of mechanically crushed rice straw with 0.05M pH5.2 acetate buffer containing 0.1M sodium chloride at 45° C. Heat for 10-15 minutes. (b) Then add 0.05-0.2% exo-β-1,4-glucanase/endo-β-1,4-xylanase and β-glucosidase, stir slowly, and place in a water bath at 45°C After reacting for 12-24 hours, collect 90% supernatant of natural sedimentation. Evaporate to dryness to obtain crude glucose. (C) Add 0.05M pH5.2 acetic acid buffer solution containing 0.1M sodium chloride to the original volume of the precipitated part, stir slowly and react in a water bath at 45°C for 24 hours, and collect 90% of the supernatant part of natural sedimentation. Evaporate to dryness to obtain crude glucose.

The main advantage of exoglucanase of the present invention is:

(1) High activity. Exo-β-1,4-glucanase/endo-β-1,4-xylanase of the present invention has the highest activity of cellulosic exonuclease (hydrolyzing microcrystalline cellulose) and hemicellulose One of the enzymes (the specific activities of the two enzymes are 37 and 1300 IU per mg protein).

(2) A single enzyme can hydrolyze rice straw without any chemical treatment to produce simple sugars, which is not possessed by the reported cellulase enzymes. This property is of great commercial development value in hydrolyzing natural cellulose .

(3) In the presence of chloride ions, it has dual activity of hydrolyzing cellulose or hemicellulose; in the absence of chloride ions, it only hydrolyzes hemicellulose. That is, the selectivity of chloride ions in the catalytic reaction of the cellulase to the substrate can be switched on and off. This helps to expand its range of applications.

(4) The activity of exonuclease to hydrolyze cellulose is not inhibited by the product (cellobiose), which is extremely beneficial for improving the conversion rate of natural cellulose into sugar and reducing costs.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggested conditions.

Example 1:

Purification of apple snail exo-β-1,4-glucanase/endo-β-1,4-xylanase:

Apple snails are widely distributed in Fujian, Guangdong, Guangxi, Zhejiang, Jiangsu and other provinces in China. Apple snails were introduced from South America 20 years ago as food. They like to eat seedlings. Even if the water quality is poor, apple snails can multiply and grow, becoming rivers. and a scourge in rice fields. The apple snails purchased in the market were used as experimental materials.

(1) Extract gastric juice from apple snails and precipitate with 0.5 saturation degree ammonium sulfate.

(2) Collect the precipitate, dissolve it with 10mM phosphate buffer (containing 0.1M sodium chloride and 1mM EDTA), and dialyze.

(3) Purify by DEAE-Sephadex A-50 column chromatography, and collect the passing peaks.

(4) Purified by Bio-gel P-100 column chromatography, and collected the hydrolysis activity peak of pNPC (p-Nitrophenyl β-D-cellobioside).

(5) Purify by phenyl-Sepharose CL-4B column chromatography, collect and combine the hydrolysis activity peaks of pNPC, and obtain the apple snail exo-β-1,4-glucanase/endo-β-1,4-wood Glycanase pure enzyme preparation.

According to the above steps, the exo-β-1,4-glucanase/endo-β-1,4-xylanase of apple snail with uniform SDS-PAGE can be obtained.

Example 2

Amino acid sequence determination of exo-β-1,4-glucanase/endo-β-1,4-xylanase

2.1 Main reagents and instruments

PVDF membranes were purchased from Millipore Corporation. Endo-Glu C protease was purchased from Promega. DTT was purchased from Boehringer Corporation. Iodoacetic acid (ICH ₂ COOH) was purchased from Sigma. The electrotransfer system uses 2117-250 NOVABLOT Electrophoretic Transfer Kit from LKB Company.

2.2 Exo-β-1,4-glucanase/endo-β-1,4-xylanase native N-terminal amino acid sequence determination

Exo-β-1,4-glucanase/endo-β-1,4-xylanase pure enzyme preparation is loaded for SDS-PAGE, in which the stacking gel is 5%, the separating gel is 10%, each well 70ug of sample was loaded, the electrophoresis voltage was 70V for 30 minutes, and then 125V for 90 minutes.

After electrophoresis, the separation gel was transferred to the membrane by semi-dry method. After transfer, stain the PVDF membrane with 0.2% Ponceau S (dissolved in 3% trichloroacetic acid) for 1 minute, decolorize with double distilled water, and cut out the exo-β-1,4-glucanase/endo - Beta-1,4-xylanase protein band. Entrusted the Shanghai Institute of Biochemistry to determine the amino acid sequence of the protein.

The measured exo-β-1,4-glucanase/endo-β-1,4-xylanase natural N-terminal amino acid sequence is:

NH ₂ -Ala-Ala-Gly-Ala-Gly-Val-Thr-Ser-Glu-Ile (SEQ ID NO: 3)

2.3 Determination of proteolytic fragments containing exo-β-1,4-glucanase/endo-β-1,4-xylanase N-terminal amino acid sequence

Endo-Glu C protease hydrolyzes exo-β-1,4-glucanase/endo-β-1,4-xylanase, SDS-PAGE electrophoresis separates the proteolysis product (Figure 1), electrophoresis ends Afterwards, the separating gel was transferred to the membrane by a semi-dry method. Transfer at a current of 0.2 mA/ ^cm2 for 12 hours. After the membrane transfer, stain the PVDF membrane with 0.2% Ponceau S (dissolved in 3% trichloroacetic acid) for 1 minute, decolorize it with double distilled water, cut out the component I shown by the arrow in Figure 1, and perform protein amino acid sequence determination (consigned Shanghai Institute of Biochemistry).

The N-terminal amino acid sequence of component I was determined as: NH ₂ -Leu-Phe-Arg-Ile-Ala-His-Ala-Ala-Asp-Pro (SEQ ID NO: 4)

Example 3

cDNA cloning of exo-β-1,4-glucanase/endo-β-1,4-xylanase

3.1 Instrument

The PCR instrument is the SingleBlock ^TM system from Ericomp Company of the United States.

3.2 RNA extraction and reverse transcription

Total RNA was extracted from fresh apple snail stomach tissue with Trizol, and cDNA was reverse-transcribed using oligo(dT) ₁₈ as a primer to serve as a PCR template.

3.3 The first step of PCR

According to the two amino acid sequences obtained by protein sequencing, the following PCR degenerate primers were synthesized:

Primer 1A:

5′-GCA(T/G/C) GCA(T/G/C) GGA(T/G/C) GCA(T/G/C) GGA(T/G/C)

GTA(T/G/C) AC-3' (SEQ ID NO: 5)

Primer 1B:

5′-GG A(G)TC A(T/G/C)GC A(T/G/C)GC A(G)TG A(T/G/C)GCA(G)AT-3′(SEQ ID NO: 6)

Using the cDNA obtained by reverse transcription as a template, the DNA amplification reaction was carried out to obtain a DNA product with a length of about 600 bp. The DNA product was loaded into pBluescript II SK(+)T-vector, cloned and sequenced to obtain the middle fragment of the gene.

3.4 The second step of PCR

According to the obtained gene fragments, design and synthesize nested PCR primers,

Primer 2 (outer primer):

5'-ACC AGC ATC AAC TGA ATG-3' (SEQ ID NO: 7)

Primer 3 (inner primer):

5′-ATG ACA ATG GCT ACA AC-3′ (SEQ ID NO: 8)

In the first step of nested PCR, the outer primer Primer 2 and oligo(dT) ₁₈ are used as a primer pair, and in the second step, the inner primer Primer 3 and oligo(dT) ₁₈ are used as a primer pair, and finally a DNA fragment of about 900 bp can be obtained. The DNA product was loaded into pBluescript II SK(+)T-vector, cloned and sequenced to obtain the 3' end fragment of the gene.

3.5 The third step of PCR

Design and synthesize the following 5′-RACE reverse transcription primers and nested PCR primers according to the obtained gene fragments:

GSP1:

5'-ACA TCC CAG TGC T-3' (SEQ ID NO: 9)

GSP2:

5'-GAG CCT TGA CCC AGT TCT G-3' (SEQ ID NO: 10)

GSP3:

5'-ACC GCC CAG TTG TAG TGC TGG T-3' (SEQ ID NO: 11)

Anchor:

5'-GGC CTG CAG TCG ACT AGT ACT TTT TTT TTT TTT TTT T-3' (SEQ ID NO: 12)

UAPs:

5'-GGC CTG CAG TCG ACT AGT AC-3' (SEQ ID NO: 13)

GSP1 was used as primer to reverse transcribe the total RNA of apple snail stomach tissue to generate cDNA template, which was hydrolyzed by RNaseH and poly(dA) tail was added to the 3′ end of cDNA, and the cDNA was used as template for nested PCR amplification to obtain the target DNA fragment. The DNA fragment was loaded into pBluescript II SK(+)T-vector, cloned and sequenced. The target DNA fragment is the 5' end fragment of the exo-β-1,4-glucanase/endo-β-1,4-xylanase gene.

3.6 The fourth step of PCR

According to the 5' end fragment and the 3' end fragment of the exo-β-1,4-glucanase/endo-β-1,4-xylanase gene, the following primers were synthesized:

Primer 4A:

5'-CAG GCT GAC CAG AAT CCA CTA-3' (SEQ ID NO: 14)

Primer 4B:

5'-TTC AAC TTT ATT GCC CTC TG-3' (SEQ ID NO: 15)

Using oligo(dT) ₁₈ as a primer to reverse transcribe the total RNA of the apple snail stomach tissue to generate a cDNA template, select Primer 4A and Primer 4B as primers to amplify the full-length fragment of the gene, and load the DNA fragment into pBluescript II SK(+) T-vector, cloned and sequenced. The resulting fragment has a full length of 1293bp (SEQ ID NO: 1), which comprises an open reading frame of 1188bp, a 5' untranslated region of 77bp and a 3' untranslated region of 30bp, encoding an exoglucanase (SEQ ID NO: 395) containing 395 amino acid residues NO: 2).

Sequence alignment shows that the exo-β-1,4-glucanase/endo-β-1,4-xylanase cDNA of apple snail and the deduced amino acid sequence are similar to those of the aerobic bacteria cellulomonas fimi Cellulose exo-glucanase/xylanase (exo-glucanase/xylanase) are very different.

Example 4

Expression of exo-β-1,4-glucanase/endo-β-1,4-xylanase cDNA of apple snail in methanolic yeast

4.1 Construction of methanol yeast expression strain

The exo-beta-1,4-glucanase/endo-beta-1,4-xylanase gene obtained in Example 3, after being cut by EcoRI and NotI, was loaded into the same enzyme cut The methanol yeast secretory expression plasmid pPIC9K was transformed into Escherichia coli E.coli DH12S to obtain a monoclonal strain containing the secretory recombinant expression plasmid pPIC9K-EGX.

The secretory recombinant expression plasmid pPIC9K-EGX was extracted, and 5 μg was single-cut and linearized with the restriction endonuclease Bpu1102I.

Take 5ug of the linearized plasmid pPIC9K-EGX to electrotransform methanolic yeast GS115 cells, and the electrotransformed cells are screened for His ⁺ clones on MD plates.

Pick 20-30 His ⁺ clones, extract DNA, and confirm the clone containing exo-β-1,4-glucanase/endo-β-1,4-xylanase gene by PCR method .

Clones containing exo-β-1,4-glucanase/endo-β-1,4-xylanase genes were screened for Mut ⁺ phenotype on MM plates.

4.2. Expression of methanol yeast expression strain in liquid medium

Inoculate the clone containing the exo-β-1,4-glucanase/endo-β-1,4-xylanase gene in 1ml of BMG medium and culture at 220rpm at 30°C for 24 hours.

Inoculate the MG culture solution into 5ml of BMGY medium at a ratio of 1:200, culture at 30°C and 220rpm until OD ₆₀₀ =2-6, transfer the bacteria to BMMY medium, and make the concentration of the bacteria solution reach OD ₆₀₀ =1.0 , 30° C. 220 rpm for 7 days, supplemented with methanol to 0.5% every 24 hours.

The cells were collected by centrifugation, and the activities of exo-β-1,4-glucanase/endo-β-1,4-xylanase inside and outside the cells were determined.

4.3. Expression of methanol yeast expression strain in solid medium

Inoculate the clone containing exo-β-1,4-glucanase/endo-β-1,4-xylanase gene in 1ml MG medium (1.34% yeast basic nitrogen source, 0.1% glycerol, 0.1M pH6.0 phosphate buffer, 20mM sodium chloride, biotin 4×10 ^-5 %), and cultured at 220 rpm at 30°C for 24 hours.

The MG culture solution was inoculated into 5ml BMGY medium (1.34% yeast basic nitrogen source, 0.1% glycerol, 0.1M pH6.0 phosphate buffer, 20mM sodium chloride, biotin 4×10 ^-5 %) at 220 rpm at 30°C until OD ₆₀₀ = 2-6, spot 0.5 μl of the culture on BMMY plate medium, culture at 30°C for 7 days, and add 100 μl of methanol to the upper cover of the culture dish every 24 hours.

Wash away the BMMY plate culture medium (1.34% yeast basic nitrogen source, 0.1M pH6.0 phosphate buffer, 20mM sodium chloride, 0.1% soluble xylan, 2% agar powder, biotin 4×10 ^-5 %, methanol 0.5%) surface methanol yeast colonies, add 10ml 0.1% Congo red staining for 40 minutes, then add 1N sodium chloride solution for decolorization, the golden yellow hydrolysis spot shows that exo-β-1,4-glucose Glycanase/endo-β-1,4-xylanase hydrolytic activity produced.

The results are shown in Figure 2. The big spot is the methanolic yeast expression strain containing cellulase, which releases cellulase to the outside of the cell during its growth, so that the cellulose in the solid medium is hydrolyzed into simple sugars. The control bacteria not transfected with the apple snail exoglucanase gene had no obvious spots.

4.4. Isolation and purification of recombinant exoglucanase

1) 4.2. Centrifuge the supernatant part of the culture broth fermented for 7 days, and precipitate it with 0.5 saturated ammonium sulfate, and the precipitate is the crude product of the recombinant enzyme.

(2) Dissolve the crude product in 10 mM phosphate buffer (containing 0.1 M sodium chloride and 1 mM EDTA), and dialyze.

(3) Purify by DEAE-Sephadex A-50 column chromatography, and collect the passing peaks.

(4) Purified by Bio-gel P-100 column chromatography, and collected the hydrolysis activity peak of pNPC (p-Nitrophenyl β-D-cellobioside).

(5) Purify by phenyl-Sepharose CL-4B column chromatography, collect and combine the hydrolysis activity peaks of pNPC, and obtain apple snail exo-β-1,4-glucanase/endo-β-1,4-wood Glycanase pure enzyme preparation.

According to the above steps, the exo-β-1,4-glucanase/endo-β-1,4-xylanase of apple snail with uniform SDS-PAGE can be obtained.

Example 5

Exo-β-1,4-glucanase/endo-β-1,4-xylanase activity assay

5.1 Determination of hydrolysis activity (exocellulase) of p-nitrophenol cellobioside (pNPC)

450μl 1mg/ml pNPC (dissolved in 0.1M pH5.2 acetate buffer containing 0.1M sodium chloride)+50μl 10mg/ml D-Gluconi Acid Lactone (dissolved in 0.1M pH5.2 acetate buffer)→45 ℃ water bath for 5 minutes→add appropriate amount of enzyme solution→react in 45℃ water bath for 8 minutes→add 525μl 2% Na ₂ CO ₃ to stop the reaction, and develop color→read the light absorption value at 410nm. The extinction coefficient is 1.76×10 ^-5 pM ^-1

The hydrolysis activity of pNPC is defined as 1 unit (unit) of the enzyme required to hydrolyze 1 pmol of p-nitrophenol (pNP) per minute under the above conditions.

5.2 Determination of hydrolysis activity of microcrystalline cellulose (Sigmacell)

500μl 1% Sigamcell (suspended in 0.1M pH5.2 acetate buffer containing 0.1M sodium chloride)→preheat in 45℃ water bath for 5 minutes→add appropriate amount of enzyme solution, react in 45℃ water bath for 8 minutes→add 40μg/ml Glucose DNS reagent 0.5ml → boil water bath for 5 minutes, cool in cold water, add 0.5ml double distilled water → read the light absorption value at 540nm.

The hydrolytic activity of microcrystalline cellulose (Sigmacell) is defined as 1 unit (unit) of enzyme required to hydrolyze 1 μmol of glucose reducing equivalent per minute under the above conditions.

5.3 Determination of xylan hydrolysis activity

The determination of xylan hydrolysis activity uses soluble xylan as the substrate. After the xylan is added with water and fully stirred, a xylan suspension is obtained, the suspension is centrifuged, and the supernatant is freeze-dried to obtain the soluble xylan.

500 μl 1% soluble xylan (dissolved in 0.1M pH5.2 acetate buffer containing 0.1M sodium chloride)? Preheat in water bath at 45°C for 5 minutes → add appropriate amount of enzyme solution, react in water bath at 45°C for 8 minutes → add 0.5ml of DNS reagent containing 40μg/ml glucose → bath in boiling water for 5 minutes, cool in cold water, add 0.5ml of double distilled water → read Absorption value at 540nm.

The hydrolysis activity of xylan is defined as 1 unit (unit) of enzyme required to hydrolyze 1 μmol of glucose reducing equivalent per minute under the above conditions.

5.4 Determination of endo-β-1,4-glucanase hydrolysis activity

200μl 1% sodium carboxymethylcellulose (dissolved in 0.05M pH5.2 acetate buffer containing 0.1M sodium chloride)→preheat in water bath at 45°C for 5 minutes→add appropriate amount of enzyme solution, react in water bath at 45°C for 8 minutes→ Add 0.5ml of DNS reagent containing 40μg/ml glucose → bath in boiling water for 5 minutes, cool in cold water, add 0.5ml of double distilled water → read the absorbance at 540nm.

5.5 Determination of Vitality of Hydrolyzed Dried Rice Straw

500μl dry rice straw suspension (containing 150mg dry rice straw, suspended in 0.05M pH5.2 acetate buffer containing 0.1M sodium chloride) → preheat in water bath at 45°C for 5 minutes → add 18μg exo-β-1,4-glucose Glycanase/endo-β-1,4-xylanase, react in water bath at 45°C for 40 minutes → add 0.5ml of DNS reagent containing 40μg/ml glucose → bath in boiling water for 5 minutes, cool in cold water, add 0.5ml double Distilled water Read the absorbance at 540nm. Then calculate the amount of reducing sugar produced.

Under the above conditions, the specific activity of the enzyme was 3.18 international units with rice straw as the substrate.

The test results are summarized in Table 1.

Table 1: Comparison of activity of apple snail exocellulase with those reported in literature source exonuclease vitality definition Substrate concentration Specific activity (u/mg) T. reesei CBHI μmol glucose 1% Avicel 0.0175 CBHII μmol glucose 1% Avicel 0.0391 PFvar.cellulosa Exo-I μmol glucose 1% Avicel ------ T.viride Exo-I μmol glucose 1% Avicel 0.008 Exo-II μmol glucose 1% Avicel 0.0032 Exo-III μmol glucose 1% Avicel 0.0039 Irpex lacteus exocellulase μmol glucose 1% Avicel 0.0571 Huwicola insolens CBH μmol glucose 1% Avicel 1.60 C.cellulolytium CelA μmol glucose 0.8% Avicel 5.4 CelC μmol glucose 0.8% Avicel 0.8 Apple snails Exo-β-1,4-glucanase/Endo-β-1,4-xylanase μmol glucose 1%Sigmacell ^## 37.2

## Sigmacell and Avicel are both Sigma's trade names for microcrystalline cellulose. After 1998, Avicel no longer appears in the product catalog, only Sigmacell

#Definition: 1 μmol of simple sugar produced by hydrolysis of microcrystalline cellulose (Sigmacell) or polyxylan (birchwood) per minute is 1 U.

Example 6

Effect of Chloride Ions on Exo-β-1,4-Glucanase/Endo-β-1,4-Xylanase Activity

Exo-β-1,4-glucanase/endo-β-1,4-xylanase activity was measured in reaction systems containing different concentrations of chloride ions to study the exo-β-1, Effect of 4-glucanase/endo-β-1,4-xylanase activity.

The results are shown in Figure 3. Chloride ions have no significant effect on the endo-β-1,4-xylanase activity of the enzyme, but have a significant activation effect on the exo-β-1,4-glucanase activity , and is necessary for exo-β-1,4-glucanase activity.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Sequence Listing

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Leu Glu Leu Phe Glu His Arg Trp Met Thr Asp Glu Thr His Asn Leu

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ttcaacttta ttgccctctg 20

Claims (15)

1. An isolated enzyme with exo-beta-1,4-glucanase and/or endo-beta-1,4-xylanase activity, characterized in that it is selected from the group consisting of: (a) a polypeptide having the amino acid sequence of SEQ ID NO: 2; (b) The amino acid sequence of SEQ ID NO: 2 is formed by substitution, deletion or addition of 1-10 amino acid residues, and has the function of exoglucan and/or endo-xylan by (a) Derived peptides. 2. The enzyme of claim 1, wherein the enzyme has a SEQ ID NO: 2 amino acid sequence. 3. An isolated polynucleotide, characterized in that it is selected from the group consisting of: (a) polynucleotide encoding enzyme as claimed in claim 1; (b) A polynucleotide complementary to polynucleotide (a). 4. The polynucleotide according to claim 3, wherein the polynucleotide encodes a polynucleotide having an amino acid sequence shown in SEQID NO:2. 5. The polynucleotide of claim 4, wherein the polynucleotide is selected from the group consisting of: (i) have the nucleotide sequence of positions 77-1261 in SEQ ID NO: 1; (ii) have the nucleotide sequence of 1-1293 in SEQ ID NO:1. 6. A recombinant expression vector, characterized in that it contains the polynucleotide according to claim 3. 7. A transformed host cell, characterized in that it contains the recombinant expression vector of claim 6. 8. a method for preparing an enzyme with exo-beta-1,4-glucanase and/or endo-beta-1,4-xylanase activity according to claim 1, characterized in that , the method contains: (a) cultivating the host cell according to claim 7 under conditions suitable for expressing the enzyme according to claim 1; (b) isolating the enzyme having exo-β-1,4-glucanase and/or endo-β-1,4-xylanase activity according to claim 1 from the culture. 9. A use of an enzyme with exo-β-1,4-glucanase and/or endo-β-1,4-xylanase activity according to claim 1, characterized in that, Process for the production of simple sugars such as cellobiose, glucose and mixtures thereof. 10. The use according to claim 9, wherein said simple sugar is glucose. 11. The use of the host cell according to claim 7, characterized in that it is used in a process for producing simple sugars, and the simple sugars are cellobiose, glucose and mixtures thereof. 12. The use according to claim 11, characterized in that said simple sugar is glucose. 13. A method for producing simple sugars, comprising the steps of: (a) with exo-beta-1,4-glucanase and/or endo-beta-1,4-xylanase activity described in claim 1 or the enzyme described in claim 7 Host cells process cellulose to produce simple sugars; (b) isolating said simple sugars including cellobiose, glucose and mixtures thereof. 14. The method of claim 13, wherein said cellulose is cellulose without any chemical pretreatment. 15. The method of claim 14, wherein said simple sugar is glucose.

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