Construction of a high-temperature-resistant cellulase complex system and its application in synergistic degradation

Abstract

This invention provides a method for constructing and synergistically degrading a thermostable cellulase complex system. The invention provides a cellulase composition comprising: (1) a thermostable endoglucanase, comprising at least one enzyme selected from the GH5 family, GH9 family, GH12 family, and GH45 family; (2) a thermostable cellobiase, comprising at least one enzyme selected from the GH6 family and GH7 family; and (3) a thermostable β-glucosidase, comprising at least one enzyme selected from the GH1 family and GH3 family.

Description

Technical Field

[0001] This invention belongs to the field of enzymes, and more specifically, this invention relates to the construction and synergistic degradation application of a high-temperature resistant cellulase complex system. Background Technology

[0002] Lignocellulosic biomass is one of the most abundant renewable carbon resources on Earth, and can be used to produce biofuels, bio-based chemicals, and more. Cellulase, which hydrolyzes cellulose into fermentable sugars, is a key enzyme system in biomass conversion. A typical cellulase system mainly consists of three types of enzymes working together: endoglucanase (EG), which is responsible for cleaving the glycosidic bonds inside cellulose; exoglucanase / cellobiose hydrolase (CBH), which is responsible for continuously releasing cellobiose from the chain ends; and β-glucosidase (BGL), which further hydrolyzes cellobiose into glucose.

[0003] In industrial biorefining processes, many pretreatment steps require high-temperature conditions, such as steam explosion and hot water pretreatment, typically exceeding 70°C. High-temperature enzymatic hydrolysis offers several advantages: improved substrate accessibility, reduced slurry viscosity, accelerated reaction rate, reduced risk of microbial contamination, and reduced cooling energy consumption. Therefore, developing a high-temperature resistant cellulase system capable of stable operation above 70°C is of significant industrial value.

[0004] Current research focuses primarily on screening individual thermostable cellulases or improving their stability, while systematic studies on multi-enzyme synergy, the construction and optimization of thermostable cellulase systems, and long-term stable saccharification under high-temperature conditions remain limited. Therefore, there is currently a lack of thermostable cellulase systems that can operate stably at temperatures above 70°C, exhibit good synergistic effects, and be suitable for the high-temperature saccharification of complex lignocellulose substrates. Summary of the Invention

[0005] The purpose of this invention is to provide a high-temperature resistant cellulase complex system for construction and synergistic degradation application.

[0006] A first aspect of the present invention provides a cellulase composition comprising: (1) Thermoresistant endoglucanase, comprising at least one enzyme selected from the GH5 family of endoglucanases, the GH9 family of endoglucanases, the GH12 family of endoglucanases and the GH45 family of endoglucanases; (2) A thermostable cellobiose hydrolase, comprising at least one enzyme selected from the GH6 family of cellobiose hydrolases and the GH7 family of cellobiose hydrolases; and (3) Thermoresistant β-glucosidase, including at least one enzyme selected from the GH1 family β-glucosidase and the GH3 family β-glucosidase.

[0007] In one or more embodiments, the thermostable endoglucanase is a GH12 family endoglucanase, the thermostable cellobiase is a GH6 family cellobiase, and the thermostable β-glucosidase is a GH3 family β-glucosidase.

[0008] In one or more embodiments, the thermostable endoglucanase is TpEG or a variant thereof.

[0009] In one or more embodiments, the TpEG is a GH12 family TpEG, more preferably derived from... Thermotoga petrophila Further preferably, the amino acid sequence of the TpEG is shown in SEQ ID NO:1.

[0010] In one or more embodiments, variations of the TpEG are selected from: (A1) A protein whose amino acid sequence has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:1. (A2) A protein having an addition, deletion, or substitution of amino acids at one or more positions in the amino acid sequence shown in SEQ ID NO:1. (A3) A protein formed by fusing an additional amino acid sequence into this protein sequence. (A4) A fragment of TpEG with an amino acid sequence as shown in SEQ ID NO:1.

[0011] In one or more embodiments, the thermostable cellobiose hydrolase is HmCel6A or a variant thereof.

[0012] In one or more embodiments, the HmCel6A is a GH6 family HmCel6A, more preferably derived from it. 未培养的绿弯菌门细菌 Further preferably, the amino acid sequence of HmCel6A is shown in SEQ ID NO:2.

[0013] In one or more embodiments, variants of the HmCel6A are selected from: (B1) A protein whose amino acid sequence has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:2. (B2) A protein having an addition, deletion, or substitution of amino acids at one or more positions in the amino acid sequence shown in SEQ ID NO:2. (B3) A protein formed by fusing an additional amino acid sequence into this protein sequence. (B4) The amino acid sequence is a fragment of HmCel6A as shown in SEQ ID NO:2.

[0014] In one or more embodiments, the thermostable β-glucosidase is TnBglB or a variant thereof.

[0015] In one or more embodiments, the TnBglB is a GH3 family TnBglB, more preferably derived from... 嗜岩石栖热袍菌 Further preferably, the amino acid sequence of TnBglB is shown in SEQ ID NO:3.

[0016] In one or more embodiments, variants of TnBglB are selected from: (C1) A protein whose amino acid sequence has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:3. (C2) A protein having an addition, deletion, or substitution of amino acids at one or more positions in the amino acid sequence shown in SEQ ID NO:3. (C3) The protein is formed by fusing an additional amino acid sequence into this protein sequence. (C4) The amino acid sequence is a fragment of TnBglB as shown in SEQ ID NO:3.

[0017] In one or more embodiments, the cellulase composition comprises (1) a thermostable endoglucanase selected from TpEG or a variant thereof as described in any embodiment of the present invention, (2) a thermostable cellobiose hydrolase selected from HmCel6A or a variant thereof as described in any embodiment of the present invention, and (3) a thermostable β-glucosidase selected from TnBglB or a variant thereof as described in any embodiment of the present invention, or a combination of the three.

[0018] In one or more embodiments, the mass ratio of the thermostable endoglucanase, thermostable cellobiase, and thermostable β-glucosidase is (1.5~4):(4~6):(1.5~3), preferably (2~3):(5~6):(2~3), (1.5~2.5):(5.5~6.5):(1.5~2.5), (1.7~2.3):(5.7~6.3):(1.7~2.3), (1.8~2.2):(5.8~6.2):(1.8~2.2), or (1.9~2.1):(5.9~6.1):(1.9~2.1), and more preferably 2:6:2.

[0019] A second aspect of the present invention provides a nucleic acid molecule, and a nucleic acid construct comprising the nucleic acid molecule, the nucleic acid molecule having: (D1) Encoding the polynucleotide sequence of the cellulase composition described in any embodiment of the present invention, TpEG or a variant thereof described in any embodiment of the present invention, HmCel6A or a variant thereof described in any embodiment of the present invention, or TnBglB or a variant thereof described in any embodiment of the present invention; and (D2) (D1) The complementary sequence of the polynucleotide sequence.

[0020] A third aspect of the present invention provides a host cell comprising a nucleic acid molecule as described in any embodiment of the present invention, or comprising a nucleic acid construct as described in any embodiment of the present invention, or expressing a cellulase composition as described in any embodiment of the present invention.

[0021] A fourth aspect of the present invention provides an enzyme preparation or a kit containing said enzyme preparation, which contains the cellulase composition described in any embodiment of the present invention, or the nucleic acid molecule described in any embodiment of the present invention, a nucleic acid construct containing said nucleic acid molecule, or the host cell described in any embodiment of the present invention.

[0022] In one or more embodiments, the enzyme preparation may also contain other enzymes besides the cellulase composition described in any embodiment of the present invention.

[0023] In one or more embodiments, the enzyme preparation further comprises a buffer solution; preferably, the buffer solution is a phosphate buffer solution; preferably, the pH of the buffer solution is 5-10, more preferably 6-9, and even more preferably 7-8.

[0024] In one or more embodiments, the enzyme preparation is a powder, such as a lyophilized powder.

[0025] In one or more embodiments, the enzyme preparation is the supernatant obtained by lysing host cells expressing the cellulase composition as described in any embodiment of the present invention, or a concentrate of the supernatant.

[0026] A fifth aspect of the invention provides the use of the cellulase composition described in any embodiment of the invention, or the nucleic acid molecule described in any embodiment of the invention, a nucleic acid construct comprising the nucleic acid molecule, or the host cell described in any embodiment of the invention, or the enzyme preparation or kit comprising the enzyme preparation described in any embodiment of the invention, in the catalytic conversion of a cellulose-containing substance, or in the preparation of a glucose-containing substance from a cellulose-containing substance.

[0027] In one or more embodiments, the cellulose-containing material includes filter paper, sawdust, rice straw, bagasse, stalks, cotton fiber, microcrystalline cellulose, lignocellulose, corn cob, corn seed coat, beet pulp, eucalyptus, oak, birch, and cassava.

[0028] In one or more embodiments, the glucose-containing substance includes a saccharified composition or a derivative thereof, wherein the saccharified composition is a glucose-containing food or pharmaceutical, and the derivative includes biofuels, food, feed, and chemicals.

[0029] In one or more embodiments, the catalytic conditions or preparation conditions are high temperature. In one or more embodiments, the high temperature conditions are above 70°C, more preferably 70-110°C, 75-100°C, or 80-95°C.

[0030] A sixth aspect of the present invention provides a method for catalytically converting a cellulose-containing substance, or for preparing a glucose-containing substance from a cellulose-containing substance, the method comprising: catalytically converting a cellulose-containing substance using a cellulase composition described in any embodiment of the present invention, or a nucleic acid molecule described in any embodiment of the present invention, a nucleic acid construct containing the nucleic acid molecule, or a host cell described in any embodiment of the present invention, or an enzyme preparation or a kit containing the enzyme preparation described in any embodiment of the present invention.

[0031] In one or more embodiments, the cellulose-containing material includes filter paper, sawdust, rice straw, bagasse, stalks, cotton fiber, microcrystalline cellulose, lignocellulose, corn cob, corn seed coat, beet pulp, eucalyptus, oak, birch, and cassava.

[0032] In one or more embodiments, the glucose-containing substance includes a saccharified composition or a derivative thereof, wherein the saccharified composition is a glucose-containing food or pharmaceutical, and the derivative includes biofuels, food, feed, and chemicals.

[0033] In one or more embodiments, the catalytic conditions or preparation conditions are high temperature. In one or more embodiments, the high temperature conditions are above 70°C, more preferably 70-110°C, 75-100°C, 80-95°C, or 80-90°C.

[0034] In one or more embodiments, the mass ratio of the cellulase composition to the cellulose-containing substance is 0.1 to 10:100.

[0035] In one or more embodiments, a phosphate buffer solution is used as the reaction solvent, and more preferably, the pH of the reaction system is 5 to 10, and the reaction time is 24 hours or more.

[0036] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0037] Figure 1 Melting temperature tests of different types of endoglucanase, cellobiase and β-glucosidase.

[0038] Figure 2 Table 3 shows the synergistic degradation ability of different cellulase combinations on filter paper.

[0039] Figure 3 Comparison of filter paper degradation effects at different EG:CBH:BGL ratios for 24h (left), 48h (middle), and 72h (right). Detailed Implementation

[0040] Through in-depth research, the inventors have provided a cellulase composition capable of stable and sustained degradation activity at high temperatures. This cellulase composition comprises a thermostable endoglucanase, a thermostable cellobiase, and a thermostable β-glucosidase, with further optimized mass ratios of the three enzymes. The three enzymes in this cellulase composition exhibit significant synergistic degradation activity at specific ratios, maintaining cellulose hydrolytic activity for 72 hours at temperatures exceeding 80°C.

[0041] Cellulase Composition

[0042] This invention provides a cellulase composition comprising: (1) Thermoresistant endoglucanase, comprising at least one enzyme selected from the GH5 family of endoglucanases, the GH9 family of endoglucanases, the GH12 family of endoglucanases and the GH45 family of endoglucanases; (2) A thermostable cellobiose hydrolase, comprising at least one enzyme selected from the GH6 family of cellobiose hydrolases and the GH7 family of cellobiose hydrolases; and (3) Thermoresistant β-glucosidase, including at least one enzyme selected from the GH1 family β-glucosidase and the GH3 family β-glucosidase.

[0043] In some embodiments, the cellulase composition provided by the present invention comprises a thermostable endoglucanase of the GH12 family, a thermostable cellobiase of the GH6 family, and a thermostable β-glucosidase of the GH3 family, or a combination of these three.

[0044] In this invention, "high temperature resistance" refers to the ability to maintain enzyme activity and / or perform enzymatic hydrolysis under high temperature conditions. The high temperature conditions typically refer to temperatures above 70°C, such as above 72°C, 75°C, 78°C, 80°C, 85°C, 90°C, or higher; more specifically, such as 70-110°C, 75-100°C, 80-95°C, 80-90°C, etc. It should be understood that the terms "above" or "below" in this invention include the stated number.

[0045] In the cellulase composition of the present invention, thermostable endoglucanase, thermostable cellobiase and thermostable β-glucosidase can play a synergistic degradation role.

[0046] In this invention, the "endoglucanase," also known as Endoglucanase or EG, refers to an enzyme responsible for cleaving glycosidic bonds within cellulose. The thermostable endoglucanase refers to an enzyme capable of maintaining its activity at high temperatures (e.g., above 70°C) and / or catalyzing the hydrolysis of glycosidic bonds within cellulose. This enzyme typically works synergistically with cellobiase and β-glucosidase. Exemplarily, the thermostable endoglucanase includes TpEG or its variants. "TpEG" refers to enzymes derived from... 嗜岩石栖热袍菌 The GH12 thermostable endoglucanase typically has the amino acid sequence shown in SEQ ID NO:1. Variants of TpEG include fragments, derivatives, and analogs of TpEG. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to proteins that substantially retain the same biological function or activity as the TpEG of the present invention. The protein fragments, derivatives, or analogs of the present invention may be (i) proteins whose amino acid sequence has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:1 (preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity), or (ii) proteins with the addition, deletion, or substitution of amino acids at one or more positions of the amino acid sequence shown in SEQ ID NO:1, wherein such substituted amino acid residues may or may not be encoded by the genetic code, or (iii) proteins formed by fusing additional amino acid sequences to this protein sequence (such as leader sequences, secretory sequences, sequences used to purify this protein, proteoglycan sequences, or fusion proteins), or (iv) fragments of TpEG with an amino acid sequence as shown in SEQ ID NO:1. Typically, the TpEG fragments, derivatives, and analogs described herein have the same or similar thermostable endoglucanase activity as TpEG shown in SEQ ID NO:1.

[0047] In this invention, the "cellobiose hydrolase," also known as exoglucanase, cellobiohydrolase, or CBH, is an enzyme responsible for the continuous release of cellobiose from the chain ends. The cellobiose hydrolase cleaves 2 to 4 units from the ends of exposed chains produced by endocellulase to obtain cellobiose. The thermostable cellobiose hydrolase refers to an enzyme capable of maintaining its activity at high temperatures (e.g., above 70°C) and / or catalyzing the release of cellobiose. This enzyme typically also works synergistically with endoglucanase and β-glucosidase. Exemplarily, the thermostable cellobiose hydrolase includes HmCel6A or a variant thereof. "HmCel6A" refers to enzymes derived from… 未培养的 绿弯菌门细菌 The superheat-resistant GH6 cellobiose hydrolase typically has the amino acid sequence shown in SEQ ID NO:2. Variants of HmCel6A include fragments, derivatives, and analogs of HmCel6A. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to proteins that substantially retain the same biological function or activity as the HmCel6A of the present invention. The protein fragments, derivatives, or analogs of the present invention may be (i) proteins whose amino acid sequence has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:2 (preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity), or (ii) proteins with the addition, deletion, or substitution of amino acids at one or more positions of the amino acid sequence shown in SEQ ID NO:2, wherein such substituted amino acid residues may or may not be encoded by the genetic code, or (iii) proteins formed by fusing additional amino acid sequences to this protein sequence (such as leader sequences, secretion sequences, sequences used to purify this protein, proteomic sequences, or fusion proteins), or (iv) fragments of HmCel6A with an amino acid sequence as shown in SEQ ID NO:2. Typically, the fragments, derivatives, and analogs of HmCel6A have the same or similar thermostable cellobiose hydrolase activity as HmCel6A shown in SEQ ID NO:2.

[0048] In this invention, the "β-glucosidase," also known as β-Glucosidase or BGL, is an enzyme that hydrolyzes cellobiose into glucose. The thermostable β-glucosidase refers to an enzyme that can maintain its activity at high temperatures (e.g., above 70°C) and / or catalyze the hydrolysis of cellobiose into glucose. This enzyme typically works synergistically with endoglucanase and cellobiose hydrolase. Exemplarily, the thermostable β-glucosidase includes TnBglB or a variant thereof. "TnBglB" refers to enzymes derived from...嗜岩石栖热袍菌 The GH3 ultrathermal β-glucosidase typically has the amino acid sequence shown in SEQ ID NO:3. Variants of TnBglB include fragments, derivatives, and analogs of TnBglB. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to proteins that substantially retain the same biological function or activity as the TnBglB of the present invention. The protein fragments, derivatives, or analogs of the present invention may be (i) proteins whose amino acid sequence has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:3 (preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity), or (ii) proteins with the addition, deletion, or substitution of amino acids at one or more positions of the amino acid sequence shown in SEQ ID NO:3, wherein such substituted amino acid residues may or may not be encoded by the genetic code, or (iii) proteins formed by fusing additional amino acid sequences to this protein sequence (such as leader sequences, secretory sequences, sequences used to purify this protein, proteomic sequences, or fusion proteins), or (iv) fragments of TnBglB with an amino acid sequence as shown in SEQ ID NO:3. Typically, the TnBglB fragments, derivatives, and analogs described herein have the same or similar thermostable β-glucosidase activity as TnBglB shown in SEQ ID NO:3.

[0049] The term "identity" refers to the similarity between two amino acid sequences. The degree of identity was determined using the EMBOSS Water pairwise sequence alignment program from EBI (European Bioinformatics Institute) http: / / www.ebi.ac.uk / Tools / psa / emboss_water / , with the following parameters: BLOSUM62, Gap open 10, Gap extend 0.5. The algorithm is described in Smith and Waterman (1981).

[0050] Besides originating from 嗜岩石栖热袍菌 TpEG, derived from 未培养的绿弯菌门 细菌 HmCel6A, originating from 嗜岩石栖热袍菌 Besides TnBglB, TpEG, HmCel6A, and TnBglB derived from other fungi, bacteria, or metagenomics are all within the scope of protection of this invention, as long as they also have the same or similar biological functions or activities as TpEG, HmCel6A, and TnBglB.

[0051] It should be understood that the aforementioned TpEG variant, HmCel6A variant, and TnBglB variant can be obtained through AI screening, targeted modification, and other methods. After obtaining these variants, experiments can be conducted to further verify whether the aforementioned TpEG variant, HmCel6A variant, and TnBglB variant possess heat-resistant properties, such as detecting the melting temperature of the enzyme protein through protein thermal stability testing, and detecting the enzyme's degradation effect under high-temperature conditions.

[0052] In the cellulase composition, the mass ratio of the thermostable endoglucanase, thermostable cellobiase, and thermostable β-glucosidase can be (1.5~4):(4~6):(1.5~3), for example, the thermostable endoglucanase is 1.5~4 parts by weight, the thermostable cellobiase is 4~6 parts by weight, and the thermostable β-glucosidase is 1.5~3 parts by weight. In some more preferred embodiments, the mass ratio of thermostable endoglucanase, thermostable cellobiase, and thermostable β-glucosidase can be (2~3):(5~6):(2~3), (1.5~2.5):(5.5~6.5):(1.5~2.5), (1.7~2.3):(5.7~6.3):(1.7~2.3), (1.8~2.2):(5.8~6.2):(1.8~2.2), or (1.9~2.1):(5.9~6.1):(1.9~2.1), and more preferably 2:6:2.

[0053] In a preferred embodiment, the present invention provides a cellulase composition comprising TpEG or a variant thereof, HmCel6A or a variant thereof, and TnBglB or a variant thereof, or a combination thereof. This cellulase composition exhibits a significant synergistic effect at 80°C, with a synergistic index reaching 5.13 after 72 hours of reaction. In some embodiments, the mass ratio of TpEG or a variant thereof, HmCel6A or a variant thereof, and TnBglB or a variant thereof may be (2~4):(4~6):(1.5~3), for example, 2~4 parts by weight of TpEG or a variant thereof, 4~6 parts by weight of HmCel6A or a variant thereof, and 1.5~3 parts by weight of TnBglB or a variant thereof. In some preferred embodiments, the mass ratio of TpEG or a variant thereof, HmCel6A or a variant thereof, and TnBglB or a variant thereof can be (2~3):(5~6):(1.5~2.5), (2±0.5):(6±0.5):(2±0.5), (2±0.3):(6±0.3):(2±0.3), (2±0.2):(6±0.2):(2±0.2), or (2±0.1):(6±0.1):(2±0.1). A mass ratio of 2:6:2 yields the best results.

[0054] Nucleic acid

[0055] The present invention also provides multinucleotide sequences and their complementary sequences encoding TpEG or variants thereof, HmCel6A or variants thereof, and TnBglB or variants thereof as described in the present invention.

[0056] The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand.

[0057] The polynucleotide molecule encoding the mature protein of the mutant includes: a coding sequence that encodes only the mature protein; a coding sequence for the mature protein and various additional coding sequences; a coding sequence for the mature protein (and optional additional coding sequences) and a non-coding sequence.

[0058] A “polynucleotide molecule encoding a protein” can be a polynucleotide that includes the protein, or it can include polynucleotides that also include additional coding and / or non-coding sequences.

[0059] The polynucleotide molecules of this invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in this invention, especially the open reading frame sequences, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art as templates. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0060] Nucleic acid constructs

[0061] The present invention also relates to nucleic acid constructs, such as expression cassettes, that include one or more regulatory sequences operatively linked to the polynucleotide molecule described herein.

[0062] In this text, "operable linkage" or a similar description refers to the arrangement of elements, wherein the components are arranged in a specific shape to perform their required functions. Thus, a given promoter operably linked to a coding sequence enables the efficient expression of that coding sequence in the presence of the correct transcription factors, etc. The promoter does not need to be adjacent to the coding sequence, as long as it serves to guide the expression of that sequence. Therefore, sequences that do not participate in translation but are transcribed can exist between the promoter sequence and the coding sequence, just like transcribed introns; and the promoter sequence can still be considered "operable linked" to the coding sequence.

[0063] The polynucleotide encoding the present invention can be manipulated in various ways to ensure the expression of TpEG or its variants, HmCel6A or its variants, and TnBglB or its variants. The manipulation of the polynucleotide molecule prior to its insertion into the expression vector may be desired or necessary depending on the expression vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0064] The regulatory sequence can be a suitable promoter sequence, a nucleotide sequence recognized by a host cell expressing the polynucleotide molecule described in this invention. The promoter sequence contains a transcriptional regulatory sequence attached to polypeptide expression. The promoter can be any nucleotide sequence exhibiting transcriptional activity in the selected host cell, including mutant, truncated, and heterozygous promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that of the host cell.

[0065] In Escherichia coli, suitable promoters include, but are not limited to, lac (lactose promoter), trp (tryptophan promoter), and tac (a hybrid promoter of lactose and tryptophan).

[0066] The regulatory sequence can also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operatively linked to the 3′ end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in a selected host cell can be used in this invention. In some embodiments, the transcription terminator sequence is a functional terminator in *E. coli*.

[0067] In some implementations, the nucleic acid construct is an expression cassette containing a promoter and a transcription terminator operatively linked to the polynucleotide molecule described herein.

[0068] carrier

[0069] The nucleic acid constructs described in this article can also be vectors, including but not limited to expression vectors and cloning vectors.

[0070] In expression vectors, various nucleic acid molecules and regulatory sequences can be linked together to produce recombinant expression vectors comprising one or more restriction sites that allow insertion of the polynucleotide molecules or nucleic acid constructs described herein at such sites. During the fabrication of the expression vector, the polynucleotide molecules described herein are located within the vector to enable their operative linking to appropriate regulatory sequences.

[0071] The term "recombinant expression vector" includes bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors such as pET vectors (more specifically, pET-28a), pCold vectors, pGEX vectors, and E. coli expression vectors (more specifically, pET-28a). 大肠杆菌Expression vectors include Shuffle T7, yeast expression vectors, Bacillus expression vectors, and fungal expression vectors. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0072] Recombinant expression vectors can be any vector (such as plasmids or viruses) that can readily undergo recombinant DNA methods and result in the expression of nucleotide sequences of interest. The choice of vector generally depends on its compatibility with the host cell in which it is introduced. The vector can be a linear or closed circular plasmid.

[0073] The vector can be a self-replicating vector, that is, a vector that exists as an extrachromosomal entity and whose replication does not depend on chromosome replication, such as a plasmid, extrachromosomal element, mini-chromosome, or artificial chromosome. The vector can contain any means necessary to ensure self-replication. Alternatively, the vector can be a vector that, when introduced into a host cell, integrates into the genome and replicates along with the chromosome into which it has already been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids, or transposons, containing the total DNA to be introduced into the host cell's genome, can be used.

[0074] The vector of the present invention may contain one or more selectable markers that allow for easy selection of cells for transformation, transfection, transduction, etc. The selectable markers are genes whose products provide resistance to antibiotics or viruses, resistance to heavy metals, prototrophic to auxotrophic traits, etc.

[0075] The vector of the present invention may include elements that allow the vector to integrate into the host cell genome or to replicate autonomously in the cell independently of the genome.

[0076] One or more copies of the polynucleotide of the present invention can be inserted into a host cell to increase the yield of the gene product. The increase in polynucleotide copy number can be achieved by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selector gene and the polynucleotide. Cells containing an amplified selector gene and thereby containing additional copies of the polynucleotide can be screened by culturing the cells in the presence of a suitable selector.

[0077] Cloning vectors containing the polynucleotide sequences of this invention can be used to replicate a sufficient number of target plasmids. Therefore, the cloning vectors of this invention possess strong self-replication elements, such as replication initiation sites. Typically, the cloning vectors of this invention do not contain expression elements.

[0078] host cells

[0079] This invention also includes a host cell genetically engineered using the vector of this invention, or a cellulase composition (e.g., those containing the coding sequence of TpEG or variants thereof, HmCel6A or variants thereof, and TnBglB or variants thereof), and a method for generating the protein described in this invention via recombinant technology. The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Representative examples include: *Escherichia coli*, *Bacillus subtilis*, *Streptomyces*, and *Agrobacterium*; eukaryotic cells such as yeast and plant cells. In a specific embodiment of this invention, *Escherichia coli* is used as the host cell.

[0080] Recombinant cellulase compositions (e.g., those containing TpEG or variants thereof, HmCel6A or variants thereof, and TnBglB or variants thereof) can be expressed or produced using conventional recombinant DNA techniques and the polynucleotide sequences of this invention. Generally, the following steps are involved: (1) Transform or transduce suitable host cells with the cellulase-encoding composition of the present invention (e.g., those containing TpEG or variants thereof, HmCel6A or variants thereof, and TnBglB or variants thereof), or with a recombinant expression vector containing such polynucleotides; (2) Host cells cultured in a suitable culture medium; (3) Isolate and purify proteins from culture media or cells.

[0081] Methods well known to those skilled in the art can be used to construct expression vectors containing cellulase compositions (e.g., those containing TpEG or variants thereof, HmCel6A or variants thereof, and TnBglB or variants thereof) encoding DNA sequences and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. The expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting transformed host cells.

[0082] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express proteins.

[0083] Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0084] Enzyme preparations and kits

[0085] The present invention also provides an enzyme preparation containing the cellulase composition described in any embodiment of the present invention (e.g., those containing TpEG or variants thereof, HmCel6A or variants thereof, and TnBglB or variants thereof). It should be understood that the enzyme preparation may also contain other enzymes, such as amylase, ligninase, other cellulases, etc.

[0086] In some embodiments, the enzyme preparation is a buffer solution containing the cellulase composition. Preferably, the buffer solution is a phosphate buffer, such as sodium phosphate buffer, with a pH of 5-10, more preferably 6-9, and even more preferably 7-8.

[0087] In some embodiments, the enzyme preparation is a powder, such as a lyophilized powder.

[0088] In some embodiments, the enzyme preparation is the supernatant obtained after lysis of the host cells of the cellulase expression composition described herein, such as *E. coli*. Lysis can be performed using methods well known in the art. For example, the bacterial culture can be collected by centrifugation and resuspended in lysis buffer (25 mM Tris-HCl, 500 mM NaCl, pH 7.4), followed by sonication to disrupt the cells. Centrifugation of the sonicated sample yields the supernatant, which can be further purified by affinity chromatography. In some embodiments, the enzyme preparation can be a concentrate of the supernatant, and the solvent in the supernatant can be removed using conventional methods. In some embodiments, the enzyme preparation can be a lyophilized version of the supernatant.

[0089] The nucleic acids, nucleic acid constructs (e.g., vectors), and host cells of the cellulase compositions of the present invention can be formulated in industrially acceptable vectors to obtain compositions suitable for catalytic reactions or for storage.

[0090] The cellulase composition, its nucleic acid, nucleic acid construct, host cell, or enzyme preparation of the present invention can also be included in a kit for industrial use or commercialization. Furthermore, the kit may also contain cellulose-containing substances participating in the reaction, as well as auxiliary reagents such as reaction buffers and reaction termination reagents. Generally, the kit also includes an instruction manual.

[0091] application

[0092] The cellulase composition of the present invention can be used (e.g., under high temperature conditions) to catalyze the conversion of cellulose-containing substances (e.g., catalytically converting cellulose in cellulose-containing substances into glucose); or to prepare glucose-containing substances (including saccharification compositions and their derivatives) from cellulose-containing substances as raw materials (e.g., under high temperature conditions). Therefore, the present invention also provides applications of the cellulase composition, its nucleic acids, nucleic acid constructs, host cells, enzyme preparations, or kits (e.g., under high temperature conditions) in catalyzing the conversion of cellulose-containing substances (e.g., catalytically converting cellulose in cellulose-containing substances into glucose); or (e.g., under high temperature conditions) in preparing glucose-containing substances (including saccharification compositions and their derivatives) from cellulose-containing substances as raw materials.

[0093] In this invention, the "cellulose-containing substances" include, but are not limited to: filter paper (more specifically, for example, Whatman No. 1 filter paper), sawdust, straw, bagasse (e.g., sugarcane bagasse), straw (including wheat straw, rice straw, and corn straw), cotton fiber, microcrystalline cellulose, lignocellulose (e.g., pretreated lignocellulose), corn cob, corn seed coat, beet pulp, eucalyptus, oak, birch, and cassava. In addition to the plants listed above, the cellulose-containing substances also include industrial fiber waste. It should be understood that any substance that can be catalyzed and converted by the cellulase composition under high-temperature conditions (e.g., above 70°C) falls under the category of cellulose-containing substances described in this invention and is within the scope of protection of this invention. In some embodiments, the weight percentage of cellulose in the cellulose-containing substances is 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or higher than the total weight.

[0094] In this invention, the "glucose-containing substance" can be, for example, a saccharified composition or a derivative thereof. The saccharified composition can be a glucose-containing food (e.g., a sugar solution). The derivatives include, but are not limited to: biofuels, foods (e.g., alcoholic beverages, sweeteners, seasonings), feed (e.g., as a carbon source), and chemicals (e.g., detergents, astaxanthin). In some embodiments, the glucose-containing substance contains glucose at a weight percentage of 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or higher than the total weight. It should be understood that glucose in the glucose-containing substance can be detected by various methods, such as, but not limited to, DNS method, HPLC method, glucose oxidase method, ion chromatography, biosensor detection, and PAHBAH method.

[0095] During catalytic conversion, the mass ratio of the cellulase composition to the cellulose-containing substance can be 0.1 to 10:100, for example, 0.5:100, 0.8:100, 0.9:100, 1:100, 1.1:100, 1.2:100, 1.5:100, 2:100, 4:100, 5:100, 6:100, 8:100, 10:100, or any combination thereof. In some embodiments, when the total reaction volume is 1 mL, the mass of the cellulase composition can be 50-200 μg (e.g., 60-190 μg, 70-180 μg, 80-150 μg, 80-120 μg, 90-110 μg, 95-105 μg, 98-102 μg, 99-101 μg, etc.), and the mass of the cellulose-containing substance can be 1-20 mg (e.g., 2-18 mg, 3-17 mg, 4-16 mg, 5-15 mg, 6-14 mg, 7-13 mg, 8-12 mg, 9-11 mg, etc.).

[0096] During catalytic conversion, a phosphate buffer solution (e.g., sodium phosphate buffer) can be used as the reaction solvent. The pH of the reaction system can be 5-10, preferably 6-9, and more preferably 7-8. The reaction time can be 24 hours or more, for example, 28 hours or more, 30 hours or more, 32 hours or more, 34 hours or more, 36 hours or more, 40 hours or more, 42 hours or more, 44 hours or more, 46 hours or more, 48 hours or more, 50 hours or more, 52 hours or more, 55 hours or more, 58 hours or more, 60 hours or more, 62 hours or more, 65 hours or more, 68 hours or more, 70 hours or more, 72 hours or more, or longer.

[0097] In this invention, the high temperature conditions generally refer to temperatures above 70°C, such as above 72°C, above 75°C, above 78°C, above 80°C, above 85°C, above 90°C, or higher, and more specifically, such as 70-110°C, 75-100°C, 80-95°C, 80-90°C, etc.

[0098] method

[0099] The present invention also provides a method for catalytically converting cellulose-containing substances (e.g., under high temperature conditions) or preparing glucose-containing substances (including saccharified compositions and their derivatives) from cellulose-containing substances as raw materials (e.g., under high temperature conditions), the method comprising: catalytically converting cellulose-containing substances using the cellulase composition, its nucleic acid, nucleic acid construct, host cell, enzyme preparation or kit described in the present invention.

[0100] In this invention, the "cellulose-containing substances" include, but are not limited to: filter paper (more specifically, for example, Whatman No. 1 filter paper), sawdust, straw, bagasse (e.g., sugarcane bagasse), straw (including wheat straw, rice straw, and corn straw), cotton fiber, microcrystalline cellulose, lignocellulose (e.g., pretreated lignocellulose), corn cob, corn seed coat, beet pulp, eucalyptus, oak, birch, and cassava. In addition to the plants listed above, the cellulose-containing substances also include industrial fiber waste. It should be understood that any substance that can be catalyzed and converted by the cellulase composition under high-temperature conditions (e.g., above 70°C) falls under the category of cellulose-containing substances described in this invention and is within the scope of protection of this invention. In some embodiments, the weight percentage of cellulose in the cellulose-containing substances is 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or higher than the total weight.

[0101] In this invention, the "glucose-containing substance" can be, for example, a saccharified composition or its derivatives. The saccharified composition can be a glucose-containing food (e.g., a sugar solution). The derivatives include, but are not limited to: biofuels, foods (e.g., alcoholic beverages, sweeteners, flavorings), feed (e.g., as a carbon source), and chemicals (e.g., detergents, astaxanthin). In some embodiments, the glucose-containing substance contains glucose at a weight percentage of 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or higher than the total weight. It should be understood that glucose in the glucose-containing substance can be detected by various methods, such as, but not limited to, the DNS method (e.g., detecting the absorbance of reducing sugars at 540 nm), HPLC, glucose oxidase method, ion chromatography, biosensor detection, and PAHBAH method.

[0102] In this invention, the "catalytic conversion" includes catalytic degradation, catalytic hydrolysis, catalytic lignocellulose saccharification, catalytic conversion into biofuels, biorefining, industrial enzyme catalysis, and high-temperature fermentation pretreatment.

[0103] During catalytic conversion, the mass ratio of the cellulase composition to the cellulose-containing substance can be 0.1 to 10:100, for example, 0.5:100, 0.8:100, 0.9:100, 1:100, 1.1:100, 1.2:100, 1.5:100, 2:100, 4:100, 5:100, 6:100, 8:100, 10:100, or any combination thereof. In some embodiments, when the total reaction volume is 1 mL, the mass of the cellulase composition can be 50-200 μg (e.g., 60-190 μg, 70-180 μg, 80-150 μg, 80-120 μg, 90-110 μg, 95-105 μg, 98-102 μg, 99-101 μg, etc.), and the mass of the cellulose-containing substance can be 1-20 mg (e.g., 2-18 mg, 3-17 mg, 4-16 mg, 5-15 mg, 6-14 mg, 7-13 mg, 8-12 mg, 9-11 mg, etc.).

[0104] In this method, a buffer solution can be used as the reaction solvent to ensure the reaction proceeds under specific pH conditions. For catalytic conversion, a phosphate buffer solution (e.g., sodium phosphate buffer) can be used as the reaction solvent. The pH of the reaction system can be 5-10, preferably 6-9, and more preferably 7-8. The reaction time can be 24 hours or more, for example, 28 hours or more, 30 hours or more, 32 hours or more, 34 hours or more, 36 hours or more, 40 hours or more, 42 hours or more, 44 hours or more, 46 hours or more, 48 hours or more, 50 hours or more, 52 hours or more, 55 hours or more, 58 hours or more, 60 hours or more, 62 hours or more, 65 hours or more, 68 hours or more, 70 hours or more, 72 hours or more, or longer.

[0105] In this invention, the high temperature conditions generally refer to temperatures above 70°C, such as above 72°C, above 75°C, above 78°C, above 80°C, above 85°C, above 90°C, or higher, and more specifically, such as 70-110°C, 75-100°C, 80-95°C, 80-90°C, etc.

[0106] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0107] Experimental materials

[0108] All cellulase-encoding genes were synthesized by Sangon Biotech after codon optimization in E. coli, and the protein expression host was Shuffle T7. 大肠埃希氏菌(Weidi Biotechnology).

[0109] The actual substrate material used for cellulose degradation in this invention includes Whatman No. 1 filter paper (90 mm, Cytiva). It was treated with 0.25 mol / L NaOH at 60°C for 1 h at a solid-liquid ratio of 1:10 (w / v), followed by thorough washing and drying for later use.

[0110] The protein expression and purification reagents used in this invention include IPTG (Meryer), Kanamycin (Sangon Biotech), Ni-NTA Resin (Sangon Biotech), Amicon Ultrafiltration centrifuge tubes (30kDa MWCO, Millipore), and SYPRO Orange dye.

[0111] The protein lysis buffer (Lysis buffer, 25 mM Tris-HCl, 500 mM NaCl, pH 7.4), wash buffer (Wash buffer, 25 mM Tris-HCl, 500 mM NaCl, 50 mM Imidazole, pH 7.4), elution buffer (Elution buffer, 25 mM Tris-HCl, 500 mM NaCl, 250 mM Imidazole, pH 7.4), and protein preservation buffer (Storage buffer, 25 mM Tris-HCl, 500 mM NaCl, 10% Glycerol, pH 7.4) used in this invention are as follows:

[0112] The pH stability assay buffers used in this invention include Glycine-HCl (50 mM, pH 2.5-3.5), Sodium acetate buffer (50 mM, pH 4.5-5.5), and Sodium phosphate buffer (50 mM, pH 6.5-7.5). DNS reagent is used for the detection of reducing sugars. 10 g of 3,5-dinitrosalicylic acid, 10 g of NaOH, 300 g of potassium tartrate, 2 g of phenol, and 0.5 g of sodium sulfite are diluted to 1000 mL with distilled water and stored in a brown bottle protected from light at room temperature.

[0113] The main instruments and equipment used in this invention include a real-time quantitative PCR instrument (LightCycler 480 Instrument II, Roche) and an ELISA reader (SpectraMax). ® iD5, Molecular Devices).

[0114] The sequence information of TpEG enzyme, HmCel6A enzyme, and TnBglB enzyme in this invention, including NCBI number, species origin, and complete amino acid sequence, is shown in Table 1.

[0115] Table 1

[0116] Experimental methods

[0117] (I) Obtaining and recombinantly expressing cellulase genes

[0118] 1. Obtaining the target gene

[0119] All cellulase genes involved in this invention were obtained through artificial synthesis. After codon optimization of the target sequences based on the *E. coli* expression system, they were synthesized by Sangon Biotech. The optimized gene fragments were cloned into the pET-28a expression vector via BamHI and HindIII restriction sites. The vector contains an N-terminal 6×His tag for subsequent protein purification.

[0120] 2. Recombinant protein expression

[0121] Transformation of recombinant plasmids to 大肠杆菌 Expression was performed in Shuffle T7 Express competent cells. The strain containing the recombinant plasmid was inoculated in LB medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking until the OD600 reached 0.8–1.0. Then, IPTG was added to a final concentration of 1 mM to induce protein expression, and the cells were cultured at 16°C for 16–20 h.

[0122] 3. Recombinant protein purification

[0123] After centrifugation, the bacterial cells were collected and resuspended in lysis buffer (25 mM Tris-HCl, 500 mM NaCl, pH 7.4) for lysis by sonication. The supernatant was collected after centrifugation and purified by Ni-NTA affinity chromatography. The purified protein was concentrated by ultrafiltration and buffer replaced before being stored at -80°C for later use.

[0124] (II) Methods for detecting the thermal stability of proteins

[0125] This invention uses SYPRO Orange fluorescent dye to detect the thermal stability of proteins. Purified protein was diluted to a final concentration of 0.2 mg / mL, and 19 mL of protein sample + 1 mL of 50× SYPRO Orange dye was added to each reaction system. Detection was performed using a LightCycler 480 Instrument II instrument, with the temperature increased from 35°C to 99°C at a rate of 0.05°C / s, and held at the endpoint for 2 min. The protein melting temperature (Tm) was calculated based on the fluorescence signal change curve to evaluate protein thermal stability.

[0126] (III) Enzyme activity detection methods

[0127] 1. Endoglucanase detection method (DNS method)

[0128] 10 μg of purified enzyme was mixed with 500 μL of 0.5% (w / v) CMC-Na substrate in 50 mM sodium phosphate buffer (pH 7.5). The reaction was carried out at 80°C for 60 min, and then an equal volume of DNS chromogenic reagent was added to terminate the reaction. The mixture was heated at 90°C for 10 min to develop color. After cooling to room temperature, the absorbance was measured at 540 nm (A540) using a SpectraMax® iD5 microplate reader. The amount of reducing sugar produced was calculated based on the glucose standard curve. 1 U of enzyme activity was defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute. All experiments were performed in triplicate and expressed as mean ± standard deviation.

[0129] 2. β-glucosidase activity detection

[0130] 10 μg of purified enzyme was mixed with 100 μL of 20 mM p-nitrophenyl-β-D-glucopyranoside (pNPG) substrate in 50 mM sodium phosphate buffer (pH 7.5). The reaction was carried out at 80°C for 10 min, and then 100 μL of 0.5 M Na2CO3 was added to terminate the reaction. The amount of p-nitrophenol (pNP) released was measured at 410 nm (p-nitrophenyl is generated after hydrolysis and has maximum absorption at 410 nm). The product concentration was calculated according to the pNP standard curve. The amount of enzyme required to release 1 μmol of pNP per minute was defined as 1 U of β-glucosidase activity.

[0131] 3. Detection of cellobiose hydrolase activity

[0132] Cellobiose hydrolase activity was determined using the DNS reducing sugar method. 0.5% (w / v) CMC-Na was used as the substrate. 10 μg of purified enzyme was added to a reaction system containing 50 mM sodium phosphate buffer (pH 7.5) and reacted at 80°C for 24 h. The reaction was then terminated by adding an equal volume of DNS chromogenic reagent, and the mixture was heated at 90°C for 10 min for color development. After cooling to room temperature, the absorbance was measured at 540 nm (A540) using a SpectraMax® iD5 microplate reader. The amount of reducing sugar produced was calculated based on the glucose standard curve. 1 U of enzyme activity was defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute. All experiments were performed in triplicate and the results are expressed as mean ± standard deviation.

[0133] (iv) Cellulase-assisted degradation experiment

[0134] Using Whatman No. 1 filter paper as the substrate, a cellulase synergistic degradation experiment was conducted in a reaction system containing 50 mM sodium phosphate buffer (pH 7.5). The substrate addition was 10 mg, the total reaction volume was 1 mL, and the total enzyme addition was fixed at 100 μg. Single-enzyme systems (EG only, CBH only, BGL only), two-enzyme systems (EG+CBH, EG+BGL, CBH+BGL), and three-enzyme systems (EG+CBH+BGL) were set up. After the reaction was carried out at 80°C for 24 h, the supernatant was collected by centrifugation. The amount of reducing sugar released was determined by the DNS method, and the absorbance was measured at 540 nm. The synergistic effect of the enzymes was evaluated by the synergistic index (DS), where DS = Y(i+j) / [Y(i)+Y(j)] for the two-enzyme system and DS = Y(i+j) / [Y(i)+Y(j)] for the three-enzyme system. Y(i+j+k) / [Y(i)+Y(j)+Y(k)], where Y(i), Y(j), and Y(k) represent the sugar production of the single enzyme system, Y(i+j) and Y(i+j+k) represent the sugar production of the mixed enzyme system, and DS>1 indicates the presence of a synergistic effect.

[0135] Example 1: Construction and Synergistic Degradation Performance Analysis of a High-Temperature Resistant Cellulase Composite System

[0136] To construct a thermostable cellulase system suitable for cellulose degradation under high-temperature conditions, this invention, through extensive research, first screened the thermostability of various cellulases derived from thermophilic or moderately thermophilic microorganisms. Differential scanning fluorescence (DSF) results showed that TpEG, HmCel6A, and TnBglB exhibited high melting temperatures (Tm) and good high-temperature stability in endoglucanase, cellobiase, and β-glucosidase, respectively. Figure 1(Table 2), therefore it was selected as the core component of the high-temperature resistant cellulase cocktail system.

[0137] Although Clocl_4158 and HmCel6A have similar melting temperatures in the cellobiose hydrolase fraction, Clocl_4158 is prone to precipitation during purification; therefore, HmCel6A is preferred as the cellobiose hydrolase fraction. In the β-glucosidase fraction, although DturβGlu and TnBglB have similar melting temperatures, TnBglB has a higher expression level than DturβGlu; therefore, TnBglB is preferred as the β-glucosidase fraction.

[0138] Further analysis using Whatman No. 1 filter paper degradation experiments was conducted to evaluate the synergistic effect between different enzyme combinations. The results showed that the combined use of TpEG and HmCel6A significantly increased the amount of reducing sugar released compared to the single-enzyme system, indicating a significant synergistic effect between the endonuclease and cellobiose hydrolase. Even after adding TnBglB to form a three-enzyme system, the system still maintained a high cellulose degradation capacity. Figure 2 Synergistic index (DS) analysis showed that the DS value of the TpEG+HmCel6A system was 3.56±0.84, while the DS value of the TpEG+HmCel6A+TnBglB system further increased to 5.13±1.29, indicating that the three enzymes have a strong synergistic degradation effect (Table 3).

[0139] Subsequently, the ratios of EG:CBH:BGL were optimized. The results showed that, after reacting at 80°C for 72 h, the 2:6:2 system exhibited the highest reducing sugar release (A540 = 1.709 ± 0.090), significantly higher than the 2:5:3 system. Figure 3 (See Table 4). Therefore, 2:6:2 was chosen as the optimal working ratio for the thermostable cellulase cocktail system.

[0140] Table 2. Melting temperature test of different types of cellulase

[0141] Table 3. Enzyme activities and calculated synergistic index (DS) of different cellulase combinations

[0142] Table 4. Degradation effect of filter paper at different EG:CBH:BGL ratios

[0143] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. A cellulase composition, characterized in that, It includes: (1) Thermoresistant endoglucanase, comprising at least one enzyme selected from the GH5 family of endoglucanases, the GH9 family of endoglucanases, the GH12 family of endoglucanases and the GH45 family of endoglucanases; (2) A thermostable cellobiose hydrolase, comprising at least one enzyme selected from the GH6 family of cellobiose hydrolases and the GH7 family of cellobiose hydrolases; and (3) Thermoresistant β-glucosidase, including at least one enzyme selected from the GH1 family β-glucosidase and the GH3 family β-glucosidase; Preferably, the thermostable endoglucanase is a GH12 family endoglucanase, the thermostable cellobiase is a GH6 family cellobiase, and the thermostable β-glucosidase is a GH3 family β-glucosidase.

2. The cellulase composition according to claim 1, characterized in that, The thermoresistant endoglucanase is TpEG or a variant thereof; Preferably, the TpEG is a GH12 family TpEG, more preferably it is derived from... Thermotoga petrophila Further preferably, the amino acid sequence of the TpEG is as shown in SEQ ID NO:1; and / or, Preferably, the variants of TpEG are selected from: (A1) A protein whose amino acid sequence has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

1. (A2) A protein having an addition, deletion, or substitution of amino acids at one or more positions in the amino acid sequence shown in SEQ ID NO:

1. (A3) A protein formed by fusing an additional amino acid sequence into this protein sequence. (A4) A fragment of TpEG with an amino acid sequence as shown in SEQ ID NO:

1.

3. The cellulase composition according to claim 1 or 2, characterized in that, The thermoresistant cellobiose hydrolase is HmCel6A or a variant thereof; Preferably, the HmCel6A is a GH6 family HmCel6A, more preferably it is derived from... uncultured Chloroflexota bacterium Further preferably, the amino acid sequence of HmCel6A is as shown in SEQ ID NO:2; and / or, Preferably, the variants of HmCel6A are selected from: (B1) A protein whose amino acid sequence has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

2. (B2) A protein having an addition, deletion, or substitution of amino acids at one or more positions in the amino acid sequence shown in SEQ ID NO:

2. (B3) A protein formed by fusing an additional amino acid sequence into this protein sequence. (B4) The amino acid sequence is a fragment of HmCel6A as shown in SEQ ID NO:

2.

4. The cellulase composition according to any one of claims 1-3, characterized in that, The thermostable β-glucosidase is TnBglB or a variant thereof; Preferably, the TnBglB is a GH3 family TnBglB, more preferably it is derived from... Thermotoga petrophila Further preferably, the amino acid sequence of TnBglB is as shown in SEQ ID NO:3; and / or, Preferably, the variants of TnBglB are selected from: (C1) A protein whose amino acid sequence has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

3. (C2) A protein having an addition, deletion, or substitution of amino acids at one or more positions in the amino acid sequence shown in SEQ ID NO:

3. (C3) The protein is formed by fusing an additional amino acid sequence into this protein sequence. (C4) The amino acid sequence is a fragment of TnBglB as shown in SEQ ID NO:

3.

5. The cellulase composition according to any one of claims 1-4, characterized in that, The mass ratio of the thermostable endoglucanase, thermostable cellobiase, and thermostable β-glucosidase is (1.5~4):(4~6):(1.5~3), preferably (2~3):(5~6):(2~3), (1.5~2.5):(5.5~6.5):(1.5~2.5), (1.7~2.3):(5.7~6.3):(1.7~2.3), (1.8~2.2):(5.8~6.2):(1.8~2.2) or (1.9~2.1):(5.9~6.1):(1.9~2.1), and more preferably 2:6:

2.

6. A nucleic acid molecule, and a nucleic acid construct comprising said nucleic acid molecule, characterized in that, The nucleic acid molecule has: (D1) Encoding the polynucleotide sequence of any one of the cellulase compositions of claims 1-5, TpEG or a variant thereof of claim 2, HmCel6A or a variant thereof of claim 3, or TnBglB or a variant thereof of claim 4; and (D2) (D1) The complementary sequence of the polynucleotide sequence.

7. A host cell, characterized in that, It comprises the nucleic acid molecule of claim 6, or comprises the nucleic acid construct of claim 6, or expresses the cellulase composition of any one of claims 1-5.

8. An enzyme preparation or a kit comprising said enzyme preparation, characterized in that, It contains the cellulase composition of any one of claims 1-5, or the nucleic acid molecule of claim 6, a nucleic acid construct containing the nucleic acid molecule, or the host cell of claim 7; Preferably, the enzyme preparation further contains other enzymes besides the cellulase compositions of claims 1-5; Preferably, the enzyme preparation further contains a buffer solution; preferably, the buffer solution is a phosphate buffer solution; preferably, the pH of the buffer solution is 5-10, more preferably 6-9, and even more preferably 7-8. Preferably, the enzyme preparation is a powder, such as a lyophilized powder; Preferably, the enzyme preparation is the supernatant obtained from the lysis of the host cell as described in claim 7, or a concentrated solution of the supernatant.

9. The use of the cellulase composition according to any one of claims 1-5, or the nucleic acid molecule according to claim 6, the nucleic acid construct comprising the nucleic acid molecule, or the host cell according to claim 7, or the enzyme preparation or kit comprising the enzyme preparation according to claim 8 in the catalytic conversion of a cellulose-containing substance, or in the preparation of a glucose-containing substance from a cellulose-containing substance. Preferably, the catalytic conditions or preparation conditions are at high temperature; more preferably, the high temperature conditions are above 70°C; even more preferably, 70-110°C, 75-100°C, or 80-95°C. Preferably, the cellulose-containing material includes filter paper, sawdust, rice straw, bagasse, stalks, cotton fiber, microcrystalline cellulose, lignocellulose, corn cob, corn seed coat, beet pulp, eucalyptus, oak, birch, cassava; and / or, Preferably, the glucose-containing substance includes a saccharified composition or a derivative thereof, wherein the saccharified composition is selected from glucose-containing foods and / or pharmaceuticals, and the derivative is selected from any one or more of biofuels, foods, feed, and chemicals.

10. A method for catalytically converting a cellulose-containing substance, or for preparing a glucose-containing substance from a cellulose-containing substance, characterized in that, The method includes the step of catalytically converting a cellulose-containing substance using the cellulase composition of any one of claims 1-5, or the nucleic acid molecule of claim 6, a nucleic acid construct containing the nucleic acid molecule, or the host cell of claim 7, or the enzyme preparation of claim 8 or a kit containing the enzyme preparation. Preferably, the cellulose-containing material includes filter paper, sawdust, rice straw, bagasse, stalks, cotton fiber, microcrystalline cellulose, lignocellulose, corn cob, corn seed coat, beet pulp, eucalyptus, oak, birch, cassava; and / or, Preferably, the glucose-containing substance includes a saccharified composition or a derivative thereof, wherein the saccharified composition is a glucose-containing food or pharmaceutical, and the derivative includes biofuels, food, feed, chemicals; and / or, Preferably, the catalytic conditions or preparation conditions are high temperature; more preferably, the high temperature conditions are above 70°C, more preferably 70-110°C, 75-100°C, 80-95°C, or 80-90°C; and / or, Preferably, the mass ratio of the cellulase composition to the cellulose-containing substance is 0.1~10:100; and / or, Preferably, phosphate buffer solution is used as the reaction solvent, and more preferably, the pH of the reaction system is 5-10, and the reaction time is more than 24 hours.

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