Multi-enzyme system for preparing cellobiose as well as preparation method and application of cellobiose

Abstract

The invention provides a multienzyme system for preparing cellobiose as well as a preparation method and application of cellobiose, and belongs to the technical field of bioengineering. The multi-enzyme system disclosed by the invention is prepared from endoglucanase, cellobiose hydrolase and lysed polysaccharide monooxygenase, wherein the endoglucanase is EG5C-1m, the cellobiose hydrolase is TeCel7A T393K, and the lysed polysaccharide monooxygenase is LsAA9A. According to the method disclosed by the invention, a multi-enzyme system for accurately degrading cellulose into cellobiose is constructed by screening high-efficiency endoglucanase, cellobiose hydrolase and lysed polysaccharide monooxygenase. On the basis, the optimal reaction condition of a multi-enzyme system is determined, a method is provided for efficient generation of cellobiose from cellulose, and a basis is provided for subsequent biomass conversion of high-value products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and particularly relates to a multi-enzyme system for preparing cellobiose, and a preparation method and application of cellobiose. BACKGROUND

[0002] Lignocellulosic biomass, as one of the most abundant renewable resources on earth, is derived from straw, wood and energy crops, etc., and is widely recognized as a very potential sustainable source in large-scale sugar platform biorefining, and is a key raw material for solving energy crisis and environmental pollution problems. Lignocellulosic biomass is mainly composed of cellulose, hemicellulose and lignin, among which cellulose accounts for as high as 50% of the main component of lignocellulosic biomass, and can be converted into various high-value-added products such as starch, inositol and hydrogen through appropriate treatment.

[0003] Cellobiose, as a repeating unit of cellulose, is a very important bio-based product. Cellobiose can be acted on by cellobiose phosphorylase to generate an expensive sugar donor glucose-1-phosphate (G-1-P), and G-1-P can be added to different receptors with corresponding disaccharide phosphorylases to generate various novel functional polysaccharides, which have a wide application prospect. At the same time, cellobiose can also be used as a prebiotic, a cosmetic and an animal feed additive, and is widely used in various industrial fields.

[0004] The traditional preparation method of cellobiose is mainly to obtain it from cellulose by acid hydrolysis or hydrothermal liquefaction method, but the raw material pretreatment conditions are harsh, the cost is high, the product composition is complex and pollutes the environment, which is not compatible with the current green and sustainable development principle. Therefore, the biological enzyme method or microbial synthesis method with the advantages of environmental protection, economy and high efficiency is particularly important. In previous studies, Xu et al. (Xu X, Zhang W, You C, et al. Biosynthesis of artificial starch and microbial protein from agricultural residue [J]. Science Bulletin, 2023, 68(2): 214-223.) used endoglucanase (EG) with carbohydrate-binding module (CBM) and cellobiohydrolase (CBH) to specifically recognize and adsorb to the surface of cellulose, which effectively removed the β-glucosidase (BGL) without CBM in commercial cellulase to prepare cellobiose. However, this method requires complex acid treatment of the substrate, which significantly increases energy consumption and cost. Wu et al. (Wu W, Hildebrand A, Kasuga T, et al. Direct cellobiose production from cellulose using sextuple beta-glucosidase gene deletion Neurospora crassa mutants [J]. Enzyme and Microbial Technology, 2013, 52(3): 184-189.) constructed BGL gene deletion strains of Neurospora crassa, which produced 7.7 g / L of cellobiose from 20 g / L of Avicel in four days, but this method is time-consuming and sugar-consuming, and the efficiency is low. Therefore, there is an urgent need in the art to find enzymes that can efficiently degrade natural cellulose and apply them to the pathway of cellulose degradation to cellobiose to improve substrate utilization and product yield. SUMMARY

[0005] The purpose of the present application is to provide a multi-enzyme system for preparing cellobiose and a method and application for preparing cellobiose. A multi-enzyme system for precisely degrading cellulose to cellobiose is constructed to improve the efficiency of directly generating cellobiose from cellulose in one step.

[0006] In order to achieve the above purpose, the present application provides the following technical solutions:

[0007] The present application provides a multi-enzyme system for preparing cellobiose, which is endoglucanase, cellobiohydrolase and lytic polysaccharide monooxygenase, the endoglucanase is EG5C-1 m, the cellobiohydrolase is TeCel7A T393K and the lytic polysaccharide monooxygenase is LsAA9A.

[0008] Preferably, the mass ratio of the endoglucanase, the cellobiohydrolase and the lytic polysaccharide monooxygenase is (0.14-0.18):(0.08-0.12):(0.01-0.02).

[0009] Preferably, the EG5C-1 m The nucleotide sequence of the TeCel7A T393K is shown in SEQ ID NO. 3; and the nucleotide sequence of the LsAA9A is shown in SEQ ID NO. 6.

[0010] The present application also provides a preparation method of the multi-enzyme system, which is prepared by mixing the endoglucanase, the cellobiohydrolase and the lytic polysaccharide monooxygenase.

[0011] The present application also provides an application of the multi-enzyme system in improving the yield of cellobiose.

[0012] Preferably, the application uses cellulose as the enzymatic substrate, and the application improves the yield and / or purity of cellobiose.

[0013] The present application also provides a method for precisely degrading cellulose and significantly improving the yield of cellobiose, which uses the multi-enzyme system to degrade cellulose to produce cellobiose.

[0014] Preferably, the cellulose includes natural cellulose and / or corn stalk cellulose.

[0015] Preferably, the amount of the cellobiohydrolase used in degrading natural cellulose or corn stalk cellulose is 160 μg / mL; the amount of the endoglucanase used in degrading natural cellulose or corn stalk cellulose is 100 μg / mL; and the amount of the lytic polysaccharide monooxygenase used in degrading natural cellulose or corn stalk cellulose is 10 μg / mL.

[0016] Preferably, the optimal pH of the degradation reaction is 6.5; the optimal temperature of the degradation reaction is 45-50℃; any one of metal ions Mg 2+ , Mn 2+ or Ca 2+ is added to promote enzyme activity during the degradation reaction; and the concentration of the natural cellulose or corn stalk cellulose is 5 mg / mL.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] The present application provides a kind of for preparing cellobiose multi-enzyme system, the multi-enzyme system is endoglucanase (EG), cellobiohydrolase (CBH) and cleavage polysaccharide monooxygenase (LPMO).The present application is screened by high-efficiency EG, CBH and LPMO, constructs the multi-enzyme system that will be degraded to cellobiose accurately cellulose.Based on the "one-pot method" reaction system of the synergistic effect of EG, CBH and LPMO, with cellulose as substrate, by synchronously starting LPMO oxidation, EG endo continuous hydrolysis and CBH exo continuous degradation, the three-step cascade reaction of jointly destroying the crystalline region of natural cellulose, cutting long-chain cellulose and releasing cellobiose, finally realizes the release of high-purity cellobiose.The "one-pot method" is used to avoid the repeated addition of enzyme, system switching and intermediate product purification in multi-step reaction, significantly reduce the operation complexity and equipment cost.The multi-enzyme system provided by the present application can efficiently produce cellobiose, significantly improve the yield and yield of cellobiose.The multi-enzyme system constructed by the present application can accurately release cellobiose, significantly improve the purity of the cellobiose obtained by degradation;For cellulose efficiently generating cellobiose provides a new method, and provides a basis for subsequent biomass conversion high-value products. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is the in vitro production pathway schematic diagram of cellulose producing cellobiose in example 1.

[0020] Figure 2 It is the detection result of HPAEC-PAD in example 1, wherein (A) is HPAEC-PAD detection sugar standard, from left to right, glucose, cellobiose, cellotriose;(B) is three enzyme system hydrolysis corn stalk cellulose.

[0021] Figure 3 It is the influence of TeCel7AT393K enzyme dosage on the yield of cellobiose in example 2.

[0022] Figure 4 It is the influence of EG5C-1 m enzyme dosage on the yield of cellobiose in example 2.

[0023] Figure 5 It is the influence of LsAA9A enzyme dosage on the yield of cellobiose in example 2.

[0024] Figure 6 It is the influence of different pH on the yield of cellobiose in example 2.

[0025] Figure 7 It is the influence of different buffer system on the yield of cellobiose in example 2.

[0026] Figure 8 It is the influence of different temperature on the yield of cellobiose in example 2.

[0027] Figure 9 Effects of different metal ions and their addition amounts on the multi-enzyme system in Example 2; wherein (A) is the effect of different metal ions on the multi-enzyme system when Avicel is the substrate; (B) is the effect of Mn 2+ addition amount optimization; (C) is the effect of different metal ions on the multi-enzyme system when corn stalk cellulose is the substrate; (D) is the effect of Mg 2+ addition amount optimization.

[0028] Figure 10 Effects of substrate concentration on cellobiose yield in Example 2, wherein (A) is the effect of Avicel as the substrate on cellobiose yield; (B) is the effect of corn stalk cellulose as the substrate on cellobiose yield.

[0029] Figure 11 Time curve of the multi-enzyme system producing cellobiose under the optimal conditions in Example 2. DETAILED DESCRIPTION

[0030] The present application provides a multi-enzyme system for preparing cellobiose, the multi-enzyme system being endoglucanase, cellobiohydrolase and lytic polysaccharide monooxygenase, the endoglucanase being EG5C-1 m , the cellobiohydrolase being TeCel7A T393K and the lytic polysaccharide monooxygenase being LsAA9A.

[0031] In the present application, the mass ratio of the endoglucanase, the cellobiohydrolase and the lytic polysaccharide monooxygenase is preferably (0.14-0.18):(0.08-0.12):(0.01-0.02), further preferably (0.15-0.17):(0.08-0.11):(0.01-0.015), and more preferably 0.16:0.1:0.01. m , the cellobiohydrolase TeCel7A T393K and the lytic polysaccharide monooxygenase LsAA9A are respectively constructed by EG5C-1 m gene, TeCel7A T393K gene and LsAA9A gene to form a recombinant vector, and then the recombinant vector is transformed into microorganisms to express and ferment to obtain.

[0032] In the present application, the nucleotide sequence of the EG5C-1 m is preferably as shown in SEQ ID NO. 2; the nucleotide sequence of the TeCel7A T393K is preferably as shown in SEQ ID NO. 3; and the nucleotide sequence of the LsAA9A is preferably as shown in SEQ ID NO. 6.

[0033] The present invention also provides a method for preparing the multi-enzyme system, wherein the multi-enzyme system is prepared by mixing the endoglucanase, cellobiase and polysaccharide-lysing monooxygenase.

[0034] In this invention, endoglucanase EG5C-1 m The preparation method includes the following steps: (1) constructing an expression of EG5C-1 m Genetically engineered bacteria; (2) Step (1) prepares EG5C-1 m EG5C-1 was obtained by fermentation culture of genetically engineered bacteria. m Fermentation broth; (3) EG5C-1 prepared in step (2) m After inducing expression in the fermentation broth, centrifuge and discard the supernatant to collect EG5C-1. m Bacterial cells; (4) EG5C-1 collected in step (3) m The bacterial cells were resuspended, lysed, and centrifuged to obtain the supernatant, yielding EG5C-1. m Crude enzyme solution; (5) Using Ni 2+ Affinity chromatography was used to analyze the EG5C-1 obtained in step (4). m The crude enzyme solution was purified to obtain EG5C-1. m (6) Select the purified eluent with a molecular weight cutoff of 10 kDa obtained in step (5), add it to an ultrafiltration tube, collect the filtrate, obtain concentrated and desalted enzyme solution, add 50 mM pH 6.0 Na2HPO4-KH2PO4 buffer, centrifuge again to remove salt, and after ultrafiltration, obtain EG5C-1 m Enzymes.

[0035] In this invention, the preparation method of cellobiose hydrolase TeCel7AT393K includes the following steps: (1) constructing a recombinant strain of Pichia pastoris expressing the TeCel7AT393K gene; (2) fermenting the recombinant strain of Pichia pastoris prepared in step (1) to obtain a fermentation broth, and collecting the cell bodies by centrifugation; (3) resuspending the cell bodies from step (2) and adding 0.5% methanol for culture, and collecting the supernatant by centrifugation after culture to obtain crude TeCel7AT393K enzyme solution; (4) using Ni 2+ The crude TeCel7AT393K enzyme solution obtained in step (4) was purified by affinity chromatography to obtain the purified eluent of the crude TeCel7AT393K enzyme solution; (5) the purified eluent with a molecular weight cutoff of 10kDa obtained in step (4) was selected, added to an ultrafiltration tube, the filtrate was collected, and the concentrated and desalted enzyme solution was obtained. 50mM pH 6.0 Na2HPO4-KH2PO4 buffer was added, and the solution was centrifuged again to remove salt. After ultrafiltration, the TeCel7AT393K enzyme was obtained.

[0036] In the present application, the preparation method of the polysaccharide lytic monooxygenase LsAA9A comprises the following steps: (1) constructing a genetically engineered bacterium expressing the LsAA9A gene; (2) fermenting the genetically engineered bacterium prepared in step (1) to obtain LsAA9A fermentation broth; (3) centrifuging the LsAA9A fermentation broth prepared in step (2) after induced expression to discard the supernatant and collect the LsAA9A bacterial cells; (4) resuspending the LsAA9A bacterial cells collected in step (3), breaking the cells, centrifuging to obtain the supernatant, and obtaining the LsAA9A crude enzyme solution; (5) purifying the LsAA9A crude enzyme solution obtained in step (4) by affinity chromatography to obtain the LsAA9A crude enzyme solution purification eluate; (6) selecting the purified eluate with a molecular weight cutoff of 10 kDa obtained in step (5), adding an ultrafiltration tube, collecting the filtrate, obtaining the concentrated and desalted enzyme solution, adding 50 mM pH 6.0 Na2HPO4-KH2PO4 buffer, centrifuging again to remove the salt, and obtaining the LsAA9A enzyme after the ultrafiltration is completed. 2+

[0037] The present application also provides the use of the multi-enzyme system or the method in improving the yield of cellobiose. The use takes cellulose as the enzyme hydrolysis substrate, and improves the yield and / or purity of cellobiose; the cellulose includes natural cellulose and / or corn stalk cellulose.

[0038] The present application also provides a method for precisely degrading cellulose and significantly improving the yield of cellobiose, which degrades cellulose to produce cellobiose by using the multi-enzyme system. The cellulose is preferably natural cellulose and / or corn stalk cellulose. The dosage of the cellobiose hydrolase in degrading natural cellulose or corn stalk cellulose is 160 μg / mL; the dosage of the endoglucanase in degrading natural cellulose or corn stalk cellulose is 100 μg / mL; and the dosage of the polysaccharide lytic monooxygenase in degrading natural cellulose or corn stalk cellulose is 10 μg / mL. In the present application, the optimal pH of the degradation reaction is 6.5; the optimal temperature of the degradation reaction is 45-50°C; any one of the metal ions Mg 2+ , Mn 2+ or Ca 2+ is added to promote the enzyme activity during the degradation reaction; and the concentration of the natural cellulose or corn stalk cellulose is 5 mg / mL. As an optional embodiment, the optimal temperature of the multi-enzyme system in the substrate reaction of Avicel is 50°C, the yield of cellobiose reaches 0.7 g / L, and the yield rate of cellobiose is 7%; as an optional embodiment, the optimal temperature of the multi-enzyme system in the substrate reaction of corn stalk cellulose is 45°C, the yield of cellobiose reaches 0.694 g / L, and the yield rate of cellobiose is 12.66%. ​

[0039] In the present application, when the multi-enzyme system degrades corn stalk cellulose, 1 mM Co 2+ and Mg 2+ The enzyme activity of the multi-enzyme system can reach more than 100%, and the enzyme activity of the multi-enzyme system is significantly improved with the increase of Mg 2+ concentration, and the enzyme activity of the multi-enzyme system reaches 111% under the condition of 10 mM Mn 2+ As an alternative embodiment, when the multi-enzyme system uses corn stalk cellulose as the substrate, 10 mM Mg 2+ is added, the cellobiose yield reaches 0.825 g / L, and the cellobiose yield is 15.04%.

[0040] In the present application, when Avicel is used as the substrate, the final concentration of LsAA9A is 10 μg / mL, the temperature is 45.343°C, and the substrate concentration is 5 mg / mL, the cellobiose yield is 18.035%; when corn stalk cellulose is used as the substrate, it is predicted that when the final concentration of LsAA9A is 10 μg / mL, the temperature is 45°C, and the substrate concentration is 5 mg / mL, the cellobiose yield is 28.397%.

[0041] As an alternative embodiment, the multi-enzyme hydrolysis conditions when microcrystalline cellulose Avicel is used as the substrate are: EG5C-1 m , the addition amount is 100 μg / mL, the addition amount of TeCel7AT393K is 160 μg / mL, the addition amount of LsAA9A is 10 μg / mL, the pH of 50 mM Tris-HCl is 6.5, the reaction temperature is 45.343°C, 10 mM Mn 2+ , and the substrate concentration is 5 mg / mL. As an alternative embodiment, the multi-enzyme hydrolysis conditions when corn stalk cellulose is used as the substrate are: EG5C-1 m , the addition amount is 100 μg / mL, the addition amount of TeCel7AT393K is 160 μg / mL, the addition amount of LsAA9A is 10 μg / mL, the pH of 50 mM Tris-HCl is 6.5, the reaction temperature is 45°C, 10 mM Mg2+, and the substrate concentration is 5 mg / mL.

[0042] In the present application, when Avicel is used as the substrate, the reaction time is 6 h, the cellobiose yield is the highest, and the efficiency of converting cellulose into cellobiose is the highest. The cellobiose yield reaches 0.961 g / L, the cellobiose yield is 19.21%, and the space-time conversion rate is 161 mg / L / h. When corn stalk cellulose is used as the substrate, the optimal reaction time is 12 h, the cellobiose yield reaches 0.773 g / L, the cellobiose yield is 28.19%, and the space-time conversion rate is 65 mg / L / h.

[0043] The present application realizes efficient degradation of cellulose to generate cellobiose and improves the yield thereof by utilizing raw cellulose and corn stalk cellulose to construct a multi-enzyme system, establishes a multi-enzyme system for accurate degradation of cellulose to cellobiose, and the system can realize space-time conversion rates of 161 mg / L / h and 116 mg / L / h for Avicel and corn stalk cellulose within 6 hours only by using three enzymes, thereby improving the efficiency of one-step generation of cellobiose from natural cellulose.

[0044] The present application refers to the following documents:

[0045] [1] Bose S, Barnes C, Petrich J. Enhanced stability and activity of cellulase in an ionic liquid and the effect of pretreatment on cellulose hydrolysis [J]. Biotechnology and Bioengineering, 2012, 109(2): 434-443.

[0046] [2] Sun Y, Cheng J J, Himmel M E, et al. Expression and characterization of Acidothermus cellulolyticus E1 endoglucanase in transgenic duckweed Lemna minor 8627 [J]. Bioresource Technology, 2007, 98(15): 2866-2872.

[0047] [3] Lv K M, Zhou Y Q, Wu B, et al. Modification and enzymatic properties of persistent endoglucanase EG5C-1 [J]. Bio-processing, 2022, 20(5): 500-506.

[0048] [4] Taylor L E, Knott B C, Baker J O, et al. Engineering enhanced cellobiohydrolase activity [J]. Nature Communications, 2018, 9(1): 1186.

[0049] [5]Zong Z, Li Q, Hong Z, et al. Lysine mutation of the claw-arm-like loop accelerates catalysis by cellobiohydrolases [J]. Journal of the American Chemical Society, 2019, 141(36): 14451-14459.

[0050] [6]Xu Y, Dong F, Wang R, et al. Alternative splicing analysis of lignocellulose-degrading enzyme genes and enzyme variants in Aspergillus niger [J]. Applied Microbiology and Biotechnology, 2024, 108(1): 302.

[0051] In the present application, the Avice is selected from Avicel PH-101 (Sigma-Aldrich, USA).

[0052] In the following examples of the present application, the Buffer A refers to a 1L system containing 10mL glycerol, 0.0154g dithiothreitol, 37.275g KCl, and 0.1M potassium phosphate buffer.

[0053] In the following examples of the present application, the pET28a is purchased from Novagen Company.

[0054] In the following examples, the test methods used are conventional test methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0055] The technical solutions in the present application will be described clearly and completely in combination with the examples in the present application.

[0056] Example 1

[0057] 1. Screening of efficient EG, CBH and LPMO

[0058] In order to efficiently degrade cellulose into cellobiose, a one-pot reaction system based on the synergistic effect of endoglucanase (EG), cellobiohydrolase (CBH) and lytic polysaccharide monooxygenase (LPMO) is proposed (see FIG. 1). Figure 1). This system uses microcrystalline cellulose and slightly alkaline treated corn stalks as substrates, through the simultaneous initiation of LPMO oxidation, EG endo continuous hydrolysis and CBH exo continuous degradation, a three-step cascade reaction to destroy the crystalline region of natural cellulose, cut off long-chain cellulose and release cellobiose: LPMO first oxidizes and cleaves the crystalline region of cellulose to generate C1 or C4 oxidized short-chain oligosaccharides and destroy the hydrogen bond network; EG then randomly cuts the β-1,4 glycosidic bond in the oxidized amorphous region to generate short-chain oligosaccharides; CBH then gradually cuts cellobiose units from the non-reducing end or reducing end of the oligosaccharide chain, ultimately achieving the release of high-purity cellobiose. Further expand the adaptability to high lignin content raw materials (such as corn stalks), and provide a new strategy for low energy consumption biorefining.

[0059] The advantages of this approach mainly include: (1) LPMO destroys the crystalline region of cellulose in real time, EG synchronously and rapidly cuts, and CBH continuously releases cellobiose, forming a continuous reaction chain of "oxidation-endocellulase-exocellulase"; (2) One-pot method avoids repeated addition of enzymes, system switching and purification of intermediates in multi-step reactions, significantly reducing operation complexity and equipment cost. (3) LPMO reduces the dependence on mechanical or chemical pretreatment (such as steam explosion or strong acid treatment), further saving energy and chemical reagent consumption. At the same time, this approach directly acts on microcrystalline cellulose or simple alkali treated lignocellulosic raw materials (such as corn stalks), without complex pretreatment, is environmentally friendly and sustainable.

[0060] 1.1 Screening of high-efficiency EG

[0061] By consulting literature and BRENDA database, the enzymes with smaller k m values, larger k cat values and higher optimal temperature were selected as the screening criteria. AnEndo from Aspergillus niger, AcEndo from Acidothermus cellulolyticus and EG5C-1m from Bacillus subtilis BS-5 were selected as high-efficiency EG candidates for further analysis and comparison. The enzyme catalytic properties of the high-efficiency EG candidate enzymes selected in this embodiment and the selection criteria are shown in Table 1.

[0062] Table 1. Sources of high-efficiency EG, enzyme catalytic properties and selection criteria

[0063]

[0064]

[0065] Note: G2 and G3 in this table represent cellobiose and cellotriose, respectively; NF indicates that no data was found

[0066] Since AnEndo and AcEndo used in the present embodiment are commercial enzymes, only EG5C-1 was prepared by expression m .

[0067] Construction of recombinant plasmid pET28a-EG5C-1 m :

[0068] Obtaining EG5C-1 m The amino acid sequence of the enzyme was added with a 6×His tag at the C-terminus. The amino acid sequence of the EG5C-1 m with a 6×His tag is as follows:

[0069] MAGTKTPVAKNGQLSIKGTQLVNRDGKAVQLKGISSHGLQWYGEYVNKDSLKWLRDDWGIT

[0070] VFRAAMYTAQGGYIDNPSVKNKVKEAVEAAKELGIYVIIDWHILNDGNPNQNKEKAKEFFKEMSSL

[0071] YGNTPNVIYEIANEPNGDVNWKRDIKPYAEEVISVIRKNDPDNIIIVGTGTWSQDVNDAADDQLKDA

[0072] NVMYALHFYAGTHGQFLRDKANYALSKGAPIFVTEWGTSDAWGNGGVFLDQSREWLKYLDSKTIS

[0073] WVNWNLSDKQESSSALKPGASKTGGWRLSDLSASGTFVRENLLGHHHHHH (SEQ ID NO. 1).

[0074] The full gene sequence of EG5C-1 m was obtained according to the codon bias of E. coli (as shown in SEQ ID NO. 2), and was submitted to Suzhou Jinyu Zhi Biological Technology Co., Ltd. for full synthesis of the DNA sequence of EG5C-1 m , and was assembled to the Nco I site after the RBS sequence of the E. coli pET28a vector plasmid to obtain the recombinant plasmid pET28a-EG5C-1 m .

[0075] The nucleotide sequence of EG5C-1 m is as follows:

[0076] ATGGCAGGCACCAAAACTCCAGTAGCGAAAAACGGTCAGCTGTCTATTAAAGGCACTCAGC

[0077] TGGTAAACCGTGACGGTAAAGCGGTACAGCTGAAAGGTATCTCCAGCCACGGTCTGCAGTGGTA

[0078] CGGCGAATACGTAAATAAGGACAGCCTGAAATGGCTGCGTGATGACTGGGGTATCACGGTTTTT

[0079] CGTGCAGCAATGTACACCGCGCAGGGCGGCTACATCGATAATCCGAGCGTCAAAAACAAGGTCA

[0080] AGGAAGCCGTGGAGGCTGCGAAAGAGCTGGGCATCTATGTGATCATCGACTGGCACATCCTGAA

[0081] TGACGGCAACCCGAATCAGAACAAAGAGAAGGCTAAAGAGTTCTTTAAGGAAATGTCTTCCCT

[0082] GTACGGTAACACCCCGAACGTGATCTACGAAATTGCGAACGAACCGAACGGTGACGTGAACTG

[0083] GAAACGTGATATCAAGCCGTACGCAGAAGAGGTTATCTCTGTTATCCGCAAGAACGACCCGGAC

[0084] AATATCATTATTGTCGGCACCGGCACTTGGTCTCAGGACGTTAACGATGCGGCAGACGATCAGCT

[0085] GAAAGACGCTAACGTTATGTACGCACTGCACTTCTACGCAGGTACCCACGGCCAGTTTCTGCGT

[0086] GACAAAGCTAACTACGCCCTGTCTAAAGGTGCTCCGATCTTCGTTACCGAGTGGGGTACCTCTG

[0087] ACGCGTGGGGTAACGGCGGTGTGTTTCTGGACCAATCTCGTGAGTGGCTGAAATATCTGGACAG

[0088] CAAAACCATTTCCTGGGTTAACTGGAATCTGTCCGACAAACAGGAAAGCTCTTCTGCACTGAAA

[0089] CCGGGCGCAAGCAAAACCGGTGGTTGGCGTCTGTCCGACCTGTCTGCTAGCGGTACTTTTGTTC

[0090] GTGAAAACCTGCTGGGCCACCATCACCACCATCATTGA (SEQ ID NO. 2).

[0091] Expression of EG5C-1m: Recombinant plasmid pET28a-EG5C-1m was transformed into E. coli competent cells BL21 (DE3) (Shanghai Generay Biotech, China), and a single colony was picked and cultured at 37°C to prepare a seed liquid. The seed liquid was inoculated into TB liquid medium containing 50 pg / mL kanamycin at a volume ratio of 1 mL: 100 mL, and cultured at 37°C and 220 rpm for 3-4 h until the OD600 of the bacterial liquid was 0.6-0.8. Then, IPTG (isopropyl-beta-D-thiogalactoside) was added to a final concentration of 1 mM, and the liquid was cultured at 25°C and 200 rpm overnight for 12 h. The bacterial cells after induction were placed in a pre-cooled 500 mL centrifuge bottle and centrifuged at 4°C and 6000 rpm for 6 min. The supernatant was discarded, and the bacterial cells were collected.

[0092] Preparation of EG5C-1m crude enzyme liquid: The mass of the bacterial cells was measured, 5 times the mass of the bacterial cells of pre-cooled Buffer A was added, and the bacterial cells were resuspended using a homogenizer. Then, the bacterial cells were crushed using a cell crusher at a pressure of 1000 MPa. The crushed liquid was centrifuged at 4°C and 12000 rpm for 20 min, and the supernatant was the crude enzyme liquid of EG5C-1m.

[0093] Purification of EG5C-1m: Ni 2+ Affinity chromatography: 1.0 mL of Ni-NTA resin particles were taken, and the resin particles were washed with sterilized deionized water and Buffer A. Then, the resin particles were transferred to the EG5C-1 mThe crude enzyme solution was placed in a 4°C environment on a decolorizing shaker for 1 h. Then it was transferred to a chromatography column (15.6*83 mm), and the supernatant was allowed to flow out, and then 5 mM, 20 mM, and 40 mM imidazole solutions were used in sequence to wash away impurities (which could be confirmed by 1* Coomassie brilliant blue G250 detection), and finally 250 mM imidazole solution was used to elute the target protein. The eluate was subjected to SDS-PAGE electrophoresis, and the eluate with uniform electrophoresis was combined.

[0094] EG5C-1 m Concentration and desalination of pure enzyme: an ultrafiltration tube with a molecular weight cut-off of 10 kDa was selected, and the above eluate was added to the ultrafiltration tube, which was centrifuged at 4°C, 4000* g for 20 min to obtain concentrated and desalted enzyme solution. Then 50 mM pH 6.0 Na2HPO4-KH2PO4 buffer was added, and the solution was centrifuged again to remove salt. After the completion of ultrafiltration, the enzyme solution was aliquoted into 1.5 mL EP tubes and stored at -80°C.

[0095] Microcrystalline cellulose Avicel and carboxymethyl cellulose sodium CMC-Na were selected as substrates to compare the enzyme activity of each EG enzyme.

[0096] Avicel, as a high-crystallinity cellulose, can evaluate its degradation ability on natural recalcitrant substrates; CMC-Na, as a soluble amorphous cellulose, can directly reflect the basic activity of endoglucanase.

[0097] The results of enzyme activity determination are shown in Table 2. It can be seen that EG5C-1 m has high catalytic activity on both substrates, indicating that it has higher efficiency in dealing with cellulose with different crystallinity, and thus can provide important reference for subsequent construction of dual enzyme system. Therefore, in the following study, EG5C-1 m was selected to construct a multi-enzyme system in order to construct a high-efficiency multi-enzyme system.

[0098] Table 2 Enzyme activity analysis of different EGs

[0099]

[0100] 1.2 Screening of high-efficiency CBH

[0101] By consulting literature and BRENDA database, the CBH with smaller k m value and k catBased on the criteria of higher value and higher optimum temperature, TrCel7A from Trichoderma reesei, TeCel7AT393K from Talaromyces emersonii and MtCel6A from Myceliophthora thermophila were selected as high efficiency CBH candidates for further analysis and comparison. The catalytic properties of the selected high efficiency CBH candidates and the selection criteria are shown in Table 3.

[0102] Table 3. High efficiency CBH sources, catalytic properties and selection criteria

[0103]

[0104]

[0105] Note: G2 in the table refers to cellobiose; NF means not found

[0106] Since TrCel7A is a commercial enzyme among the CBH candidates used in this example, TeCel7AT393K and MtCel6A need to be prepared by expression.

[0107] Construction of recombinant plasmid pPICZαA-TeCel7AT393K: The amino acid sequence of TeCel7AT393K was obtained from the literature, and its DNA sequence was obtained according to the codon bias of Pichia pastoris. A 6×His tag was added at the C-terminus, and the full gene sequence of TeCel7AT393K is shown in SEQ ID NO. 3. The sequence was synthesized by Suzhou Jinyuzhi Biotechnology Co., Ltd., and assembled into the Kpn I site of the Pichia pastoris expression plasmid pPICZαA to obtain the recombinant plasmid pPICZαA-TeCel7AT393K.

[0108]

[0109] Construction of recombinant plasmid pPICZαA-MtCel6A: The amino acid sequence of MtCel6A (GenBank accession number MZ826702) was obtained from the NCBI website, and its DNA sequence was obtained according to the codon preference of Pichia pastoris, and a 6×His tag was added at the C-terminus. The full gene sequence of MtCel6A is shown as SEQ ID NO. 4. It was submitted to Suzhou Jinyu Zhi Biological Technology Co., Ltd. for full synthesis of the gene sequence, and assembled into the Nco I site of the Pichia pastoris expression plasmid pPICZαA to obtain the recombinant plasmid pPICZαA-MtCel6A.

[0110]

[0111] Construction of TeCel7A T393K and MtCel6A expression strains: linearize the recombinant plasmid pPICZαA-TeCel7A T393K, pPICZαA-MtCel6A with restriction enzyme Sac I, and recover the linearized product by agarose gel electrophoresis, and the concentration is best at 500 ng / μL-1000 ng / μL. Take 10 μL of linearized recombinant plasmid and 80 μL of competent cells of Pichia pastoris X33 (Beijing Huayueyang, China), mix evenly, and transfer to a pre-cooled sterile electroporation cup, and place on ice for 30 min; place in an electroporator for electroporation, and then quickly add 1 mL of pre-cooled 1M sterile sorbitol, mix evenly, and transfer to a centrifuge tube, and stand at 30°C for 1 h, and centrifuge at 3000 rpm for 5 min; aspirate 800 μL of supernatant, blow evenly, and plate to YPDS solid medium containing Zeocin (working concentration 100 μg / mL), and cultivate at 30°C in the dark for 3 days. Pour 2-3 mL of sterile water onto the YPDS plate with transformants, and suspend the colonies in sterile water under sterile conditions, and then dilute to 10 4 , take 50-100 μL of the diluent and spread on a YPDS plate containing high-concentration Zeocin (corchyme D1, working concentration 1000 μg / mL), and cultivate at 30°C in the dark for 3 days, and select the colonies with large size as the Pichia pastoris recombinant strain with high-copy expression of foreign CBH.

[0112] Expression and purification of TeCel7A T393K and MtCel6A: streak the Pichia pastoris recombinant strain preserved in a glycerol tube on a YPDS plate containing Zeocin, inoculate a single transformant into BMGY medium, cultivate overnight at 30°C at 240 rpm to OD 600 = 2-3, centrifuge at 4000 x g for 15 min to collect the bacterial cells, replace the flask with new BMGY medium, resuspend the bacterial cells with BMMY medium, and pour back into the flask for continued cultivation, and add 0.5% methanol every 12 h, cultivate at 30°C at 240 rpm for 3 days. After cultivation, centrifuge at 4°C at 4000 x g for 30 min to collect the supernatant, which is the crude enzyme solution secreted and expressed by Pichia pastoris. Purify by Ni 2+ affinity chromatography: mix the crude enzyme solution of TeCel7A T393K and MtCel6A with 1.0 mL of Ni-NTA resin particles, and shake on a 4°C shaker for 1 h, then transfer to a chromatography column (15.6*83 mm), wash off the impurities with 20 mM imidazole solution, and elute the target protein with 250 mM imidazole solution, and perform SDS-PAGE electrophoresis on the eluate, and combine the eluate with uniform electrophoresis, to obtain purified TeCel7A T393K and MtCel6A.

[0113] The enzyme activity of each CBH was determined.

[0114] CBH enzyme activity determination selected Avicel and p-nitrophenyl cellobioside (pNPC) as substrates, CBH gradually releases cellobiose from the end of the cellulose chain, and Avicel is used to determine its actual degradation efficiency; pNPC is connected to the chromogenic group by β-glycosidic bond, and p-nitrophenol (pNP) is released after hydrolysis, which can specifically reflect the activity of CBH. The results of enzyme activity determination are shown in Table 4. It can be seen that the enzymes from the three sources all have the ability to hydrolyze Avicel, in addition, TrCel7A and TeCel7AT393K also have the ability to release cellobiose. However, MtCel6A as CBH II type cannot bind and catalyze the reaction because the non-reducing end of the substrate is blocked by the nitrophenyl group of pNPC, so no activity is detected.

[0115] Based on the above analysis of the enzyme activity of CBH from three different sources, it is found that they can all degrade Avicel, and at the same time, due to the high crystallinity of Avicel and the lack of EG cooperation during the reaction, CBH can contact fewer ends, and it is possible that it does not fully exert its catalytic potential. Therefore, it is decided to combine the three enzymes with EG5C-1 m in the subsequent construction of a dual enzyme system, and through comparison and analysis, an efficient dual enzyme system is obtained.

[0116] Table 4 Enzyme activity analysis of different CBHs

[0117]

[0118] Note: ND in this table means not detected

[0119] 1.3 Screening of high-efficiency LPMO

[0120] By consulting the literature and BRENDA database, for LPMO, enzymes with high synergistic efficiency with cellulase are selected as the screening standard, TcAA9A from Talaromyces cellulolyticus, LsAA9A from Lentinus similis and EGLD-AS from Aspergillus niger are selected as high-efficiency LPMO candidates for further analysis and comparison. The enzyme catalytic properties and selection basis of the selected high-efficiency LPMO candidate enzymes in this embodiment are shown in Table 5.

[0121] Table 5 Sources of high-efficiency LPMO, enzyme catalytic properties and selection basis

[0122]

[0123] Note: NF in this table means not found.

[0124] The sequences of TcAA9A, LsAA9A and EGLD-AS were optimized according to the codon bias of E. coli, the synthetic gene sequence was synthesized, and the synthetic gene fragment was inserted into the pCold-TF plasmid to construct the plasmids pCold-TF-TcAA9A, pCold-TF-LsAA9A and pCold-TF-EGLD-AS. The specific steps are as follows.

[0125] Construction of pCold-TF-TcAA9A recombinant plasmid: The amino acid sequence of TcAA9A (GenBank accession number MK359134) was queried from the NCBI website, the DNA sequence thereof was obtained according to the codon bias of E. coli, and a 6×His tag was added at the C-terminus. The TcAA9A full gene sequence is shown as SEQ ID NO. 5. It was submitted to Suzhou Jinyu Zhi Biological Technology Co., Ltd. for full synthesis of the gene sequence, and it was assembled into the Trigger Factor sequence of the E. coli expression plasmid pCold-TF, thereby expressed in frame with the Trigger Factor, to obtain the recombinant plasmid pCold-TF-TcAA9A.

[0126] TcAA9A full gene sequence is GAATTCATGCATGGCTTTGTGCAGAACATTGTGATTGATGGCAAAAG CTATAGCGGCTATCTGGTGAATCAGTTTCCGTATGAAAGCAACCCGCCGGCGGTTATTGGCTGGGCGACCACCGCGACCGATCTGGGCTTTGTGGCGCCGAGCGAATATACCAACGCGGATATTATTTGCCATAAAAATGCCACGCCGGGCGCGCTGAGTGCGCCGGTGGCCGCGGGTGGCACCGTGGAACTGCAGTGGACCACCTGGCCGGATAGCCATCATGGCCCGGTGATTAGCTATCTGGCGAACTGCAACGGCAACTGCAGCACCGTGGATAAAACCAAACTGAACTTTGTGAAAATTGATCAAGGCGGCCTGATTGATGATACCACCCCGCCGGGCACCTGGGCGAGCGATAAACTGATTGCGGCGAACAACAGCTGGACCGTGACCATTCCGAGCACCATTGCGCCGGGCAACTATGTGCTGCGCCATGAAATTATTGCGCTGCATAGCGCGGGCAACGCGGATGGCGCGCAGAACTATCCGCAGTGCATTAACCTGGAAATTACCGGCAGCGGTACCGCGGCGCCGAGCGGCACCGCGGGCGAAAAACTGTATACGAGCACCGATCCGGGCATTCTGGTGAACATTTATCAGAGCTTTGGTGCGGCGAACGGCGCCGTTGCGACCGGCAGCGCGACCGCGGTTGCCACCACCGCCGCGGCGAGTGCGACGGCGACCCCGACGACGTTAGTGACGAGTGTGGCCCCGGCGAGTAGCACGAGCGCCACCGCCGTTGTGACCACCGTGGCCCCGGCGGTGACCGATGTGGTGACGGTTACCGATGTTGTGACCGTGACGACCGTGATTACGACCACCGTGCTGCATCACCATCACCATCATTAACTCGAG (SEQ ID NO. 5).

[0127] Construction of pCold-TF-LsAA9A recombinant plasmid: The amino acid sequence of LsAA9A was obtained from the NCBI website (GenBank accession number KT368674), and its DNA sequence was obtained according to the codon bias of E. coli, and a 6xHis tag was added at the C-terminus. The full gene sequence of LsAA9A is shown in SEQ ID NO. 6. It was sent to Suzhou Jinyizhi Biotechnology Co., Ltd. to synthesize the gene sequence, and assemble it into the Trigger Factor sequence of the E. coli expression plasmid pCold-TF, so as to be expressed in frame with the Trigger Factor, thereby obtaining the recombinant plasmid pCold-TF-LsAA9A.

[0128] The full gene sequence of LsAA9A is: GAATTCATGAAATATAGCATTCTGGGCCTGACCGCGCTGAGCTTTGTGGCGAGCGCGGCCGCGCATACCCTGGTGTGGGGCGTGTGGGTGAACGGCGTGGATCAAGGCGATGGCCGCAACATTTATATTCGCAGCCCGCCGAACAATAACCCGGTGAAAAACCTGACGAGCCCGGATATGACCTGCAACGTGGATAACCGCGTGGTGCCGAAAAGCGTGCCGGTGAACGCGGGCGATACCCTGACCTTTGAATGGTATCATAACACCCGCGATGACGATATTATTGCGAGCAGCCATCATGGCCCGATTGCGGTGTATATTGCGCCGGCGGCGAGCAACGGCCAAGGCAACGTGTGGGTTAAATTATTTGAAGATGCGTATAACGTGACCAACAGCACCTGGGCGGTGGATCGCCTGATTACCGCGCATGGTCAGCATAGCGTGGTTGTGCCGCATGTGGCGCCGGGCGATTATCTGTTTCGCGCGGAAATTATTGCGCTGCATGAAGCGGATAGCCTGTATAGTCAGAACCCGATTCGCGGCGCGCAGTTTTATATTAGCTGCGCGCAGATTACCATTAACAGCAGCGATGACAGCACCCCGCTGCCGGCGGGCGTGCCGTTTCCGGGCGCGTATACCGATAGCACCCCGGGTATTCAGTTTAACATTTACACGACGCCGGCGACGAGCTACGTGGCGCCGCCACCGAGTGTGTGGAGCGGCGCGCTGGGCGGTAGCATTGCGCAAGTGGGCGATGCGAGCCTGGAACATCACCATCACCATCATTAACTCGAG (SEQ ID NO. 6).

[0129] Construction of pCold-TF-EGLD-AS recombinant plasmid: the amino acid sequence of EGLD-AS was obtained from the literature, and a 6×His tag was added at the C-terminus. The amino acid sequence of EGLD-AS is shown in SEQ ID NO. 7.

[0130] The amino acid sequence of the EGLD-AS is: MAMKTTTYSLLALAAASKLASAHTTVQAVWINGEDQGLGNSAD GYIRSPPSNSPVTDVTSTDMTCNVNGDQAASKTLSVKAGDVVTFEWHHSDRSDSDDIIASSHKGPVQVYMAPTAKGSNGNNWVKIAEDGYHKSSDEWATDILIANKGKHNITVPDVPAGNYLFRPEIIALHEGNREGGAQFYMECVQFKVTSDGSSELPSGVSIPGVYTATDPGILFDIYNSFDSYPIPGPDVWDGSSSGSSSGSSSAAAAATTSAAVAATTPATQAAVEVSSSAAAVVESTSSAAAATTEAAAPVVSSAAPVQQATSAVTSQAQAPTTFATSSKSSKTACKNKTKSKSKVAASSTEAVVAPAPTSSVVPAVSASASASAGGVAKMYERCGGINHTGPTTCESGSVCKKWNPYYYQCVASQLEHHHHHH (SEQ ID NO. 7).

[0131] The nucleotide sequence of the EGLD-AS was obtained according to the codon bias of E. coli (as shown in SEQ ID NO. 8). It was submitted to Suzhou Jinyizhi Biotechnology Co., Ltd. for full synthesis of the gene sequence, and assembled to the Trigger Factor sequence of the E. coli expression plasmid pCold-TF, so as to be expressed in frame with the Trigger Factor, to obtain the recombinant plasmid pCold-TF-EGLD-AS.

[0132]

[0133] Expression of TcAA9A, LsAA9A and EGLD-AS: Recombinant plasmids pCold-TF-TcAA9A, pCold-TF-LsAA9A and pCold-TF-EGLD-AS were transformed into E. coli competent cells BL21 (DE3) respectively, and single colonies were picked and small-scale seed liquid was prepared. The seed liquid was transferred into TB liquid medium containing 50 μg / mL ampicillin at an inoculation ratio of 1:100 (volume ratio of seed liquid to TB liquid medium), and cultured at 37°C and 220 rpm for 3-4 h. When the OD600 of the bacterial liquid was measured to be 0.6-0.8, the bacterial liquid was removed. IPTG was added at a final concentration of 1 mM, and the liquid was cultured at 25°C and 200 rpm overnight for 12 h. The bacterial cells after induction and expression were placed in a 500 mL centrifuge bottle pre-cooled at 4°C, and centrifuged at 6000 rpm for 6 min. After centrifugation, the supernatant was discarded, and the bacterial cells were collected.

[0134] Preparation of TcAA9A, LsAA9A and EGLD-AS crude enzyme solution: The mass of the bacterial cells was measured, and 5 times the mass of the bacterial cells of pre-cooled Buffer A was added. The bacterial cells were resuspended using a vortex mixer, and then a cell disrupter was used to disrupt the cells at a pressure of 1000 MPa. The disrupted liquid was centrifuged at 4°C and 12000 rpm for 20 min, and the supernatant was the TcAA9A, LsAA9A and EGLD-AS crude enzyme solution.

[0135] Purification of TcAA9A, LsAA9A and EGLD-AS: Ni 2+ Affinity chromatography: 1.0 mL of Ni-NTA resin particles was taken, and the resin particles were washed with sterilized deionized water and Buffer A. The resin particles were then transferred into the TcAA9A, LsAA9A and EGLD-AS crude enzyme solution, and affinity was performed on a decolorization shaker at 4°C for 1 h. Then the resin particles were transferred into a chromatography column (15.6*83 mm), and the supernatant was allowed to flow out. 5 mM, 20 mM and 40 mM imidazole solutions were used in sequence to wash away impurities (which could be confirmed by 1x Coomassie Brilliant Blue G250 detection). Finally, 250 mM imidazole solution was used to elute the target protein, and the eluate was subjected to SDS-PAGE electrophoresis. The eluate with uniform electrophoretic bands was combined.

[0136] Concentration and desalting of TcAA9A, LsAA9A and EGLD-AS pure enzyme: An ultrafiltration tube with a molecular weight cut-off of 10 kDa was selected, and the above eluate was added to the ultrafiltration tube. The tube was centrifuged at 4°C and 4000 x g for 20 min to obtain the concentrated and desalted enzyme solution. 50 mM Na2HPO4-KH2PO4 buffer at pH 6.0 was added, and the tube was centrifuged again to remove the salt. After ultrafiltration was completed, the enzyme solution was aliquoted into 1.5 mL EP tubes and stored at -80°C.

[0137] Avicel was chosen as the substrate to compare the enzyme activity of each LPMO, and the results are shown in Table 6. It can be seen that the three LPMOs can all degrade Avicel. Considering that LPMO is an auxiliary enzyme, if the influence of the enzyme activity on the multi-enzyme system is analyzed, it may not be accurate, because it plays a supporting role more when acting together. Therefore, in the subsequent study, TcAA9A, LsAA9A and EGLD-AS were added to the double-enzyme system respectively, in order to obtain the best combination that can precisely degrade cellulose to cellobiose.

[0138] Table 6 Enzyme activity analysis of different LPMOs

[0139]

[0140] 2 Construction of multi-enzyme system for precise degradation of cellulose to cellobiose

[0141] Substrate preparation:

[0142] One substrate is commercial microcrystalline cellulose Avicel PH-101 (Sigma product), and the second substrate is corn stalk cellulose.

[0143] The treatment method of corn stalk cellulose is as follows: corn stalk is crushed and passed through a 40-mesh sieve. 10.0 g of corn stalk is weighed, 4% NaOH is added, the solid-liquid ratio is 1:30, and the mixture is soaked in a water bath at 80°C for 2 h. After filtration, the filter residue is the corn stalk cellulose with partially removed lignin. The contents of the three elements (cellulose, hemicellulose and lignin) are shown in Table 7. It can be seen that after low-concentration alkali treatment, the lignin content decreases from 33.93% to 20.24%, and the cellulose content increases from 33.16% to 54.85%.

[0144] Table 7 Contents of three elements (cellulose, hemicellulose and lignin) of NaOH pretreated corn stalk

[0145]

[0146] Construction of double-enzyme system:

[0147] EG5C-1 m was used to combine with different CBHs to determine the ability of different double-enzyme systems to produce cellobiose.

[0148] In a total reaction volume of 1 mL, 10 mg / mL Avicel or corn stalk cellulose prepared above, 80 μg / mL EG5C-1 mThe reaction was carried out at 45°C, 1200rpm for 12h in a metal bath. After reaction, the samples were centrifuged at 12000rpm for 5min at 4°C, and the supernatant was collected and boiled for 10min. After boiling, the samples were filtered through a 0.22μm needle filter into 1.5mL vials, and then analyzed by HPLC for cellobiose production and HPAEC-PAD for reaction products.

[0149] The cellobiose yield in this study was calculated according to the following formula:

[0150]

[0151] wherein:

[0152] Cellobiose yield: %;

[0153] C cellobiose : Cellobiose production, g / L;

[0154] C cellulose : Cellulose content, g / L.

[0155] EG5C-1 m The cellobiose production of different CBH combinations is shown in Table 8. EG5C-1 m EG5C-1 and TeCel7A T393K combinations have a significant advantage in cellobiose production over other combinations, with a yield of 1.91% and 0.64% respectively, and will be used for further construction of triple enzyme systems.

[0156] Table 8 Comparison of different CBH combinations for cellobiose production from Avicel and corn stover cellulose

[0157]

[0158] Construction of triple enzyme systems:

[0159] To screen the LPMO that is beneficial to the production of cellobiose by the dual-enzyme system, 40 μg / mL of different LPMOs were added to the above-mentioned dual-enzyme system, and the production of cellobiose was determined, and the results are shown in Table 9. When Avicel and corn stalk cellulose were used as substrates for the reaction, the production of cellobiose in the presence of LPMO in the dual-enzyme system showed varying degrees of increase, thus confirming that LPMO has a promoting effect on the production of cellobiose. Among them, the three-enzyme system composed of EG5C-1m, TeCel7A T393K and LsAA9A has obvious advantages in the ability to produce cellobiose compared with other combinations. When Avicel was used as the substrate, the cellobiose yield was 2.45%, which was 1.28 times that of the dual-enzyme system, and when corn stalk cellulose was used as the substrate, the cellobiose yield was 4.94%, which was 7.72 times that of the dual-enzyme system.

[0160] Table 9 Comparison of the ability of different three-enzyme combinations to produce cellobiose by hydrolyzing Avicel and corn stalk cellulose

[0161]

[0162] Further HPAEC-PAD detection was performed on the products of the three-enzyme system hydrolyzing corn stalk cellulose Figure 2 , using glucose, cellobiose and cellotriose as standard products, and it was found that in addition to cellobiose, no cellotriose was produced in the conversion system, and in the reaction products of the three-enzyme system hydrolyzing cellulose, glucose as a byproduct accounted for 19% of the free sugars, and the purity of cellobiose in the free sugars was more than 80%. This shows that the multi-enzyme system constructed in this embodiment can accurately release cellobiose and has the potential for further optimization.

[0163] Example 2

[0164] 2 Optimization of catalytic conditions of multi-enzyme system for accurate degradation of cellulose to cellobiose

[0165] 2.1 Enzyme dosage optimization of multi-enzyme system:

[0166] In a 1 mL reaction system, the effect of different enzyme dosages (0.04 mg, 0.08 mg, 0.12 mg, 0.16 mg) of TeCel7AT393K on the production of cellobiose was investigated, and the results are shown in Figure 3, with the increase of enzyme dosage, the yield of cellobiose increased, when the enzyme dosage increased to 0.16 mg, i.e. the final concentration was 160 μg / mL, the yield of cellobiose was the highest, and the efficiency of cellulose conversion to cellobiose was the highest. The yield of cellobiose of the multi-enzyme system with Avicel as the substrate reached 0.281 g / L, and the yield of cellobiose was 2.81%; the yield of cellobiose of the multi-enzyme system with corn stalk cellulose as the substrate reached 0.327 g / L, and the yield of cellobiose was 5.96%.

[0167] In a 1 mL reaction system, the effect of different enzyme dosages (0.02 mg, 0.04 mg, 0.06 mg, 0.08 mg, 0.1 mg, 0.12 mg) of EG5C-1 m on the yield of cellobiose was explored, and the results are shown in Figure 4 , with the increase of enzyme dosage, the yield of cellobiose increased, when the enzyme dosage increased to 0.1 mg, i.e. the final concentration was 100 μg / mL, the yield of cellobiose was the highest, and the efficiency of cellulose conversion to cellobiose was the highest. The yield of cellobiose of the multi-enzyme system with Avicel as the substrate reached 0.304 g / L, and the yield of cellobiose was 3.04%; the yield of cellobiose of the multi-enzyme system with corn stalk cellulose as the substrate reached 0.367 g / L, and the yield of cellobiose was 6.68%.

[0168] In a 1 mL reaction system, the effect of different enzyme dosages (0.01 mg, 0.02 mg, 0.04 mg, 0.06 mg, 0.08 mg, 0.1 mg) of LsAA9A on the yield of cellobiose was explored, and the results are shown in Figure 5 , with the increase of enzyme dosage, the yield of cellobiose decreased, when the enzyme dosage was 0.01 mg, i.e. the final concentration was 10 μg / mL, the yield of cellobiose reached the highest value, and the efficiency of cellulose conversion to cellobiose was the highest. The yield of cellobiose of the multi-enzyme system with Avicel as the substrate reached 0.41 g / L, and the yield of cellobiose was 4.1%; the yield of cellobiose of the multi-enzyme system with corn stalk cellulose as the substrate reached 0.525 g / L, and the yield of cellobiose was 9.57%. Since LsAA9A is an auxiliary enzyme, increasing the dosage in the reaction process does not necessarily improve the yield of cellobiose, therefore, optimizing the ratio and dosage of enzymes is the key to improving the yield of cellobiose.

[0169] 2.2 Optimal pH of multi-enzyme system:

[0170] In order to determine the optimal pH of the multi-enzyme system reaction, pH 5.0, 5.5, 6.0, 6.5, and 7.0 were selected to determine the optimal pH of the multi-enzyme system. The experimental results are shown in Figure 6 When the pH was 6.5, the catalytic activity of the multi-enzyme system reached the highest, indicating that the optimal pH of the multi-enzyme system reaction was 6.5.

[0171] 2.3 Screening for the optimal buffer solution for multi-enzyme system reactions

[0172] Considering that different types of buffer solutions may affect the catalytic effect of enzymes, in order to further determine the optimal buffer solution at the pH of the multi-enzyme system reaction, two points were selected: pH 6.0 and pH 6.5, using three different buffer solutions respectively. The experimental results are shown in [Figure number missing]. Figure 7 The results indicate that the optimal buffer solution for the multi-enzyme system reaction is pH 6.5 Tris-HCl, under which the cellobiose yield is highest, and the corresponding cellulose conversion to cellobiose is most efficient. The multi-enzyme system using Avicel as a substrate achieved a cellobiose yield of 0.662 g / L and a cellobiose yield of 6.62%; the multi-enzyme system using corn straw cellulose as a substrate achieved a cellobiose yield of 0.689 g / L and a cellobiose yield of 12.57%.

[0173] 2.4 Optimal temperature optimization of multi-enzyme systems:

[0174] To determine the optimal temperature for the multi-enzyme system reaction, the optimal temperatures were measured at 45℃, 50℃, 55℃, and 60℃. The experimental results are shown below. Figure 8 Among them, the optimal temperature for the multi-enzyme system with Avicel as substrate was 50℃, and the cellobiose yield reached 0.7 g / L with a cellobiose yield of 7%; the optimal temperature for the system with corn straw cellulose as substrate was 45℃, and the cellobiose yield reached 0.694 g / L with a cellobiose yield of 12.66%.

[0175] 2.5 Optimization of metal ions in multi-enzyme systems:

[0176] To determine the effect of metal ions on a multi-enzyme system, different metal ions, such as Co, were added. 2+ Mg 2+ Mn 2+ Ca 2 + Zn 2+ Fe 3+ To carry out the reaction ( Figure 9 The results showed that the addition of 1 mM Mg2+ to the multi-enzyme system during the degradation of Avicel was effective. 2+ Mn 2+ Ca 2+ The enzyme activity of the multi-enzyme system can all reach over 100%, among which Mn 2+ The best promoting effect was observed, with enzyme activity increased by 18.7% ( Figure 9 A); then Mn 2+ The addition amount was optimized, and the results showed that the enzyme activity of the multi-enzyme system increased with the addition of Mn. 2+ The concentration increases significantly, especially at 10 mM Mn.2+ Under the condition of 10 mM Mn Figure 9 B). Considering the cost and the subsequent reaction system, etc., it was determined that 10 mM Mn 2+ At this time, the cellobiose yield reached 1.18 g / L, and the cellobiose yield was 11.8%.

[0177] When the multi-enzyme system degraded corn stalk cellulose, 1 mM Co 2+ and Mg 2+ The enzyme activity of the multi-enzyme system could reach more than 100%, and the enzyme activity was increased by 11.13% and 4.12% respectively Figure 9 C) ; because Co 2+ is a heavy metal ion, Mg 2+ was selected and its amount was optimized, and the results showed that the enzyme activity of the multi-enzyme system was significantly improved with the increase of Mg 2+ concentration, and under the condition of 10 mM Mn 2+ , the enzyme activity of the multi-enzyme system reached 111% Figure 9 D). Considering the cost and the subsequent reaction system, etc., it was determined that 10 mM Mg 2+ was added when the multi-enzyme system was used as the substrate of corn stalk cellulose, at this time, the cellobiose yield reached 0.825 g / L, and the cellobiose yield was 15.04%.

[0178] 2.6 Substrate concentration optimization of multi-enzyme system:

[0179] In order to further determine the generation of cellobiose by the multi-enzyme system under different substrate concentrations, experiments were carried out under the conditions of 5 mg / mL, 10 mg / mL, 15 mg / mL and 20 mg / mL of substrate concentration, and the results are shown in Figure 10 When Avicel was used as the substrate, with the gradual increase of the substrate concentration, the yield of cellobiose showed a continuous upward trend, but the cellobiose yield gradually decreased, when the Avicel concentration was 5 mg / mL, the yield of cellobiose reached 0.858 g / L, and the cellobiose yield reached 17.16% Figure 10 A) ; while when corn stalk cellulose was used as the substrate, it was also observed that the yield of cellobiose increased with the increase of the substrate concentration, but at this time the cellobiose yield also showed a downward trend, when the corn stalk cellulose concentration was 5 mg / mL, the yield of cellobiose reached 0.775 g / L, and the cellobiose yield reached 28.26% Figure 10 B).

[0180] 2.7 Plackett-Burman test results of multi-factor optimization:

[0181] Based on the above single factor optimization of multi-enzyme system, the main factors affecting the conversion of cellulose to cellobiose were screened by Plackett-Burman test. The factors and levels of Plackett-Burman test design are shown in Table 10.

[0182] Table 10 Factors and levels of Plackett-Burman test design

[0183]

[0184] The significant factors affecting the conversion of cellulose to cellobiose were found from all factors with cellobiose yield as response value, and each group of experiment was repeated three times to obtain the average value as the test result (Table 11). The results showed that LsAA9A, temperature and substrate concentration had significant effects on cellobiose yield, while other factors had no significant effects.

[0185] Table 11 Plackett-Burman test design and results

[0186]

[0187]

[0188] Note: A is TeCel7A T393K; B is EG5C-1 m ; C is LsAA9A; D is pH; E is temperature; F is MnCl2·4H2O / MgSO4·7H2O; G is substrate amount; Cellobiose yield is cellobiose yield (%); Corn straw cellulose is corn straw cellulose.

[0189] 2.8 Box-Behnken test results of multi-factor optimization:

[0190] According to the results of Plackett-Burman test, three factors having greater effects on the conversion of cellulose to cellobiose were selected, and cellobiose yield was taken as response value. LsAA9A, temperature and substrate amount were re-labeled as A, B and C to perform Box-Behnken test. The test design scheme is shown in Table 12, and the results are shown in Table 13.

[0191] Table 12 Box-Behnken test design scheme

[0192]

[0193] Table 13 Box-Behnken test design and results

[0194]

[0195] Note: A is LsAA9A; B is temperature; C is substrate amount; Cellobiose yield is cellobiose yield (%); Corn straw cellulose is corn straw cellulose.

[0196] The response surface curves of LsAA9A, temperature and substrate concentration on the efficiency of the multi-enzyme system to generate cellobiose were drawn using Design Expert 13 software, and the optimal level of the model was predicted. The results were as follows: when Avicel was used as the substrate, the final concentration of LsAA9A was 10 pg / mL, the temperature was 45.343°C, and the substrate concentration was 5 mg / mL, the cellobiose yield was 18.035%. According to this condition, 3 parallel experiments were carried out to verify it, and the actual measured cellobiose yield was 18.01%, which was very close to the predicted value; when corn straw cellulose was used as the substrate, the predicted factors were that the final concentration of LsAA9A was 10 pg / mL, the temperature was 45°C, and the substrate concentration was 5 mg / mL, the cellobiose yield was 28.397%. According to this condition, 3 parallel experiments were carried out to verify it, and the actual measured cellobiose yield was 28.15%, which was very close to the predicted value, confirming the effectiveness of the single-factor optimization result, and also indicating that the model constructed by using the quadratic regression equation could better predict the cellobiose yield of Avicel and corn straw cellulose.

[0197] Under the optimal multi-enzyme catalytic conditions, the multi-enzyme hydrolysis conditions when using microcrystalline cellulose Avicel as the substrate were as follows: EG5C-1m addition amount 100 pg / mL, TeCel7AT393K addition amount 160 pg / mL, LsAA9A addition amount 10 pg / mL, 50 mM pH 6.5 Tris-HCl, reaction temperature 45.343°C, 10 mM Mn2+, substrate concentration 5 mg / mL; the multi-enzyme hydrolysis conditions when using corn straw cellulose as the substrate were as follows: EG5C-1m addition amount 100 pg / mL, TeCel7AT393K addition amount 160 pg / mL, LsAA9A addition amount 10 pg / mL, 50 mM pH 6.5 Tris-HCl, reaction temperature 45°C, 10 mM Mg2+, substrate concentration 5 mg / mL. By adjusting the different reaction times, the time when the cellobiose yield reached the highest was explored, and the samples were taken after 6h, 12h, 18h, 24h, 36h, 48h, 60h of reaction, respectively. The results are shown in Table 3. Figure 11 When Avicel was used as the substrate, the reaction time was 6h, the cellobiose yield was the highest, and the efficiency of cellulose conversion to cellobiose was the highest. The cellobiose yield was 0.961 g / L, the cellobiose yield was 19.21%, and the space-time conversion rate was 161 mg / L / h, and then with the extension of the reaction time, the cellobiose yield decreased Figure 11A); when corn stalk cellulose was used as substrate, the cellobiose production was stable within 12-24h, thus the optimal reaction time of the substrate was determined as 12h, the cellobiose production reached 0.773g / L, the cellobiose yield was 28.19%, and the space-time conversion rate was 65mg / L / h Figure 11 B).

[0198] Although the above embodiments have made a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and people can also obtain other embodiments according to the present embodiments without creativity, which all belong to the protection scope of the present application.

Claims

1. A multi-enzyme system for preparing cellobiose, characterized in that, The multi-enzyme system comprises endoglucanase, cellobiase, and polysaccharide-lysing monooxygenase, wherein the endoglucanase is EG5C-1. m The cellobiose hydrolase is TeCel7A T393K and the polysaccharide cleaving monooxygenase is LsAA9A.

2. The multi-enzyme system according to claim 1, characterized in that, The mass ratio of the endoglucanase, the cellobiase, and the polysaccharide-lysing monooxygenase is (0.14–0.18):(0.08–0.12):(0.01–0.02).

3. The multi-enzyme system according to claim 1, characterized in that, The EG5C-1 m The nucleotide sequence of the first is shown in SEQ ID NO.2; the nucleotide sequence of the second is shown in SEQ ID NO.3; and the nucleotide sequence of the third is shown in SEQ ID NO.

6.

4. The method for preparing the multi-enzyme system according to any one of claims 1 to 3, characterized in that, The multi-enzyme system is prepared by mixing the endoglucanase, cellobiase, and polysaccharide-lysing monooxygenase.

5. The application of the multi-enzyme system according to any one of claims 1 to 3 in increasing the yield of cellulosic disaccharide.

6. The application according to claim 5, characterized in that, The application uses cellulose as an enzymatic hydrolysis substrate, and the application improves the yield and / or purity of cellobiose.

7. A method for precisely degrading cellulose and significantly increasing cellobiose yield, characterized in that, Cellulose is produced by degrading cellulose using any one of the multi-enzyme systems described in claims 1 to 3.

8. The method according to claim 7, characterized in that, The cellulose includes natural cellulose and / or corn stalk cellulose.

9. The method according to claim 7, characterized in that, The amount of cellobiose hydrolase used in degrading natural cellulose or corn stalk cellulose is 160 μg / mL; the amount of endoglucanase used in degrading natural cellulose or corn stalk cellulose is 100 μg / mL; and the amount of polysaccharide monooxygenase used in degrading natural cellulose or corn stalk cellulose is 10 μg / mL.

10. The method according to claim 7, characterized in that, The optimal pH for the degradation reaction is 6.5; the optimal temperature for the degradation reaction is 45–50 °C; and the addition of Mg ions during the degradation reaction is necessary. 2+ Mn 2+ or Ca 2+ The activity of any one of the enzymes is promoted; the concentration of the natural cellulose or corn stalk cellulose is 5 mg / mL.

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