Enzyme combination for degrading lignocellulose and application thereof

Abstract

The invention discloses an enzyme composition for degrading lignocellulose and application of the enzyme composition, and relates to the technical field of biology. The enzyme combination comprises at least two of StGH5-9 protein, StGH11-5 protein and StAA9-11 protein, and the StGH11-5 protein and the StAA9-11 protein are used as the enzyme combination. The amino acid sequence of the StGH5-9 protein is as shown in SEQ ID NO. 1; the amino acid sequence of the StGH11-5 protein is as shown in SEQ ID NO. 3; and the amino acid sequence of the StAA9-11 protein is as shown in SEQ ID NO. 5. According to the present invention, the research results show that the combination of two or three of the recombinant proteins StGH5-9, StGH11-5 and StAA9-11 can produce the synergistic effect so as to effectively improve the degradation rate of lignocellulose; the invention provides a new enzyme resource for improving the utilization rate of lignocellulose, and has important practical application value.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to an enzyme combination for degrading lignocellulose and application thereof. Background Art

[0002] As the main component of plant cell walls, lignocellulose is the most abundant biomass resource on earth. A large amount of waste from agricultural, forestry and industrial activities is wasted every year. Therefore, the efficient and rational utilization of lignocellulose is the current research focus. Lignocellulose is composed of cellulose, hemicellulose and lignin. Many microorganisms in nature degrade the above lignocellulose components by producing a variety of carbohydrate-active enzymes (CAZymes) such as endoglucanase, exoglucanase, β-glucosidase and xylanase.

[0003] When plant pathogenic fungi infect plants and cause diseases, they secrete plant cell wall degrading enzymes to promote their invasion and colonization. Setosphaeria turcica is a semi-living parasitic ascomycete that can infect corn leaves and cause plant diseases. Studies have found that it secretes a variety of enzymes such as cellulase and laccase during infection to degrade the lignocellulose structure of plant cell walls, assisting the fungus in invading the host and expanding throughout the leaves. Given the specific degradation of maize cell wall cellulose, hemicellulose and other structures by maize pathogenic fungi such as Setosphaeria turcica, it is of great significance to explore the key genes that play a role in degradation and study their application value in degrading lignocellulose resources. Summary of the invention

[0004] The purpose of the present invention is to provide an enzyme combination for degrading lignocellulose and its application to solve the problems existing in the above-mentioned prior art. The enzyme combination can effectively improve the degradation rate of lignocellulose.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an enzyme combination for degrading lignocellulose, comprising at least two of StGH5-9 protein, StGH11-5 protein and StAA9-11 protein;

[0007] The amino acid sequence of the StGH5-9 protein is shown in SEQ ID NO.1;

[0008] The amino acid sequence of the StGH11-5 protein is shown in SEQ ID NO.3;

[0009] The amino acid sequence of the StAA9-11 protein is shown in SEQ ID NO.5.

[0010] Furthermore, when the enzyme combination includes two proteins, the mass ratio of the two proteins in the enzyme combination is 1:1.

[0011] Furthermore, when the enzyme combination includes the StGH5-9 protein, the StGH11-5 protein and the StAA9-11 protein; the mass ratio of the StGH5-9 protein, the StGH11-5 protein and the StAA9-11 protein is (1-3): (1-3): (1-3).

[0012] Preferably, the mass ratio of the StGH5-9 protein, the StGH11-5 protein and the StAA9-11 protein is 1:1:(2-3).

[0013] More preferably, the mass ratio of the StGH5-9 protein, the StGH11-5 protein and the StAA9-11 protein is 1:1:3.

[0014] The present invention also provides the use of the enzyme combination in preparing a saccharifying enzyme preparation of lignocellulose.

[0015] The present invention also provides a saccharifying enzyme preparation for lignocellulose, wherein the active ingredients include the enzyme combination.

[0016] The present invention also provides the use of the above enzyme combination or saccharifying enzyme preparation in degrading lignocellulose.

[0017] The present invention also provides a method for degrading lignocellulose, comprising the step of utilizing the above enzyme combination or saccharifying enzyme preparation to perform enzymatic degradation on lignocellulose.

[0018] Furthermore, the temperature of the enzyme-catalyzed degradation is 50° C. and the pH is 5.0.

[0019] The present invention discloses the following technical effects:

[0020] The present invention analyzes the genes encoding CAZymes in the genome of Setosphaeria turcica, and uses transcriptome sequencing to analyze the expression of CAZymes gene transcription under corn stalk induction, and performs heterologous expression and enzymatic property analysis on some genes in the GH5, GH11 family and the auxiliary active enzyme AA9 family that are strongly induced by corn stalks, preliminarily detects the degradation effect of the recombinant protein on the lignocellulose substrate, and develops its application value. The present invention has found that combining two or three of the recombinant proteins StGH5-9, StGH11-5 and StAA9-11 can produce a synergistic effect, thereby effectively improving the degradation rate of lignocellulose. The present invention provides a new enzyme resource for improving the utilization rate of lignocellulose, and has important practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 It is the PCR agarose gel electrophoresis diagram of the prokaryotic expression vector of the endoglucanase gene; wherein, A: the PCR agarose gel electrophoresis result of the target fragment StGH5-9; B: the agarose gel electrophoresis result of the bacterial liquid PCR verification of the recombinant transformant;

[0023] Figure 2 The transparent hydrolysis zone of the recombinant protein StGH5-9 expression strain on the Congo red screening plate (A) and SDS-PAGE detection diagram (B);

[0024] Figure 3 The results of the experiment on optimizing the induction conditions of StGH5-9 recombinant protein are shown in Figure 1. A: The effect of induction time on the enzyme activity of the recombinant protein StGH5-9; B: The effect of IPTG concentration on the enzyme activity of the recombinant protein StGH5-9;

[0025] Figure 4 The figures are the detection results of the enzyme activity of StGH5-9 recombinant protein under different conditions; A: the statistical graph of the cellulase activity of StGH5-9 at different temperatures; B: the statistical graph of the cellulase activity of StGH5-9 at different temperatures and times; C: the statistical graph of the cellulase activity of StGH5-9 at different pH values; D: the statistical graph of the cellulase activity of StGH5-9 at different pH values ​​and times;

[0026] Figure 5 The PCR agarose gel electrophoresis diagram of the prokaryotic expression vector of the xylanase gene; wherein, A: the PCR agarose gel electrophoresis result of the target fragment StGH11-5; B: the agarose gel electrophoresis result of the PCR verification of the recombinant transformant;

[0027] Figure 6 This is the SDS-PAGE detection diagram of the recombinant protein StGH11-5;

[0028] Figure 7 The results of the experiment on optimizing the induction conditions of StGH11-5 recombinant protein are shown in Figure 1. A: The effect of induction time on the enzyme activity of the recombinant protein StGH11-5; B: The effect of IPTG concentration on the enzyme activity of the recombinant protein StGH11-5;

[0029] Figure 8 The figures are the detection results of the enzyme activity of StGH11-5 recombinant protein under different conditions; A: the statistical graph of the cellulase activity of StGH11-5 at different temperatures; B: the statistical graph of the cellulase activity of StGH11-5 at different temperatures and times; C: the statistical graph of the cellulase activity of StGH11-5 at different pH values; D: the statistical graph of the cellulase activity of StGH11-5 at different pH values ​​and times;

[0030] Fig. 9 It is the PCR agarose gel electrophoresis diagram of the polysaccharide monooxygenase prokaryotic expression vector; A: PCR agarose gel electrophoresis result of the target fragment StAA9-11; B: agarose gel electrophoresis result of PCR verification of the recombinant transformant;

[0031] Fig.10 This is the SDS-PAGE detection diagram of the recombinant protein StAA9-11;

[0032] Fig.11 The figures are the detection results of the enzyme activity of StAA9-11 recombinant protein under different conditions; A: a statistical graph of the cellulase activity of StAA9-11 at different temperatures; B: a statistical graph of the cellulase activity of StAA9-11 at different temperatures and times; C: a statistical graph of the cellulase activity of StAA9-11 at different pH values; D: a statistical graph of the cellulase activity of StAA9-11 at different pH values ​​and times. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] Example 1

[0039] 1. Materials and Methods

[0040] 1.1 Test materials

[0041] The pathogen of corn leaf blight, Setosphaeria turcica 01-LNXB, was isolated by the Mycotoxin and Plant Molecular Pathology Laboratory of Hebei Agricultural University and was deposited in the General Microbiology Center of China Microbiological Culture Collection on November 17, 2014, with the deposit number CGMCC No.9857.

[0042] The genome data of Setosphaeria turcica was obtained by denovo sequencing of the genomic DNA extracted from the hyphae of Setosphaeria turcica 01-LNXB using Illumina / Solexa next-generation sequencing technology. The genome sequencing, assembly, gene prediction and function prediction of Setosphaeria turcica were all commissioned to BGI-Shenzhen.

[0043] 1.2 Bioinformatics analysis of CAZymes of Setosphaeria turcica

[0044] 1.2.1 Prediction of CAZymes-encoding genes in the genome

[0045] The genome data of Setosphaeria turcica obtained by sequencing was searched in CAZy database (http: / / www.cazy.org), and BLASTP was used to perform homology comparison of protein sequences to obtain CAZymesn protein sequences with homology in Setosphaeria turcica (E value ≤ 10 and sequence identity ≥ 30%). At the same time, CAT (CAZymes Analysis Toolkit, http: / / cricketornlgov / cgi-bin / cat.cgi) was used to predict Setosphaeria turcica CAZymes online, and CAZymes with secretory protein properties were annotated and analyzed, including glycoside hydrolases (GH), glycosyltransferases (GT), polysaccharide lyases (PL), auxiliary active enzymes (AA), carbohydrate esterases (CE) and carbohydrate binding modules (CBM).

[0046] 1.2.2 Bioinformatics analysis of GH5 / 6 / 11 and AA9 families

[0047] The above-obtained GH5 / 6 / 11 and AA9 family proteins of Setosphaeria turcica were analyzed in detail, and the numbers were compiled according to the order of the above-mentioned family group genes in the genome. The following analyses were performed on the GH5 / 6 / 11 and AA9 family proteins of Setosphaeria turcica: signal peptide prediction was performed using the online software SignalP (http: / / www.cbs.dtu.dk / services / SignalP-2.0 / ), protein relative molecular mass, isoelectric point (pI) and protein hydrophobicity (GRAVY) were predicted using ProtParam (http: / / web.expasy.org / protparam / ), and protein localization was performed using ProtComp9.0.

[0048] The present invention identified endoglucanase StGH5-9, xylanase StGH11-5 and polysaccharide monooxygenase StAA9-11, and the sequence information is as follows:

[0049] The amino acid sequence of endoglucanase StGH5-9 (SEQ ID NO.1):

[0050] MLFNNIFLAAALASGASSSSTSASTTASGKMKWFGINESGPEFGKANIPGLYNKDYIWPDLNKVDQFIGQGMNMFRINFLMERLTPGSLTGSFDANYLSNLTAQVNHVTSKGAYAMIQPHNYGRFNGQIMTDTAGFEKWWTNVAGQFKDNSLVVFDINNEFHDMDQTLVVNLNQAGINGIRAAGAKSQIITPEGNAWTGAWSWVSSGNAASMANLKDPENKLVYQMHQYLDSDSSGTNANCVSTTIFQERLVAATQWLKDNKKLGVIGEYGAGSNAQCVAALQGGLEYMNKNSDVWQGAIWWAAGPWWGSYMYSMEPTDGPAWTGILPKIKSYFV。

[0051] Nucleotide sequence of the StGH5-9 gene (SEQ ID NO.2):

[0052]

[0053] Amino acid sequence of xylanase StGH11-5 (SEQ ID NO.3):

[0054] MVSFTSIITAAVAATGALAAPATDIAARAPSDLVARQSTPNGEGTHNGCFYSWWSDGGARATYTNGAGGSYSVSWGTGGNLVGGKGWNPGTARTITYSGQYNPNGNSYLAIYGW TRNPLVEYYVVENFGTYDPSSQAQNKGTVTSDGSSYKIAQSTRTNQPSIDGTRTFQQYWSVRQNKRSSGSVNMKTHFDAWASKGMNLGSHYYQIVATEGYFSSGSASITVNCP.

[0055] The nucleotide sequence of StGH11-5 gene (SEQ ID NO.4):

[0056] ATGGTTTCTTTCACCTCCATCATCACTGCTGCTGTTGCGGCCACTGGCGCTCTCGCCGCCCCCGCTACCGACATCGCTGCCCGCGCTCCCAGCGACCTCGTTGCCCGCCAGAGCACTCCCAACGGCGAGGGAACCCACAACGGCTGCTGTAAGTTGTTTCTCAGAGCCATACAAACACACTAGCTGACCCATCTCTCTAGTCTACTCCTGGTGGTCTGATGGCGGTGCCCGGGCTACCTACACTAACGGAGCCGGAGGCAGCTACAGCGTAAGCTGGGGAACTGGCGGCAACCTCGTCGGTGGAAAGGGCTGGAACCCAGGAACTGCCCGGTAAGCCACCCTTTTGCTGCAGCATGTATAACGCATTGCAGCCCTGACTAACACACTCAAAGCACCATCACCTACTCGGGCCAGTACAACCCCAACGGCAACTCTTACCTCGCCATCTACGGCTGGACCCGCAACCCGCTCGTCGAGTACTACGTCGTCGAGAACTTTGGCACGTACGACCCGTCGTCGCAGGCGCAGAACAAGGGCACCGTCACCTCGGACGGCTCCTCGTACAAGATTGCCCAGTCCACCCGCACCAACCAGCCGTCCATCGACGGCACCAGGACCTTTCAGCAGTACTGGTCTGTTCGTCAGAACAAGCGCTCGTCCGGCTCCGTCAACATGAAGACGCACTTTGACGCCTGGGCCAGCAAGGGCATGAACCTCGGTTCTCACTACTACCAGATTGTCGCTACTGAGGGCTACTTCTCCTCTGGCAGCGCTTCCATCACTGTCAACTGCCCGTAA。

[0057] Amino acid sequence of polysaccharide monooxygenase StAA9-11 (SEQ ID NO.5):

[0058] MKYSTILLATAATVSAHSTWQQLWVGSTDKASTCVRTVKDNSPIDSLSSSDMFCGRGPASSSGVCEVAAGGSLTVEMHAQPGARSCSQPAIGGNHYGPVLIYMAKVSDAKTATSGSFFKVAEDGYTGTTASWGTEILNANCGKRSFTVPRSLAAGDYLVRAEAIALHAGAGSPQPYVSCFQVKVTGGGSATPSGVSFPGAYKISDALFSKSIYDSSFKYVSPGPAVYSG。

[0059] Nucleotide sequence of the StAA9-11 gene (SEQ ID NO.6):

[0060] ATGAAGTACTCTACCATTCTCCTCGCCACGGCCGCCACCGTCTCCGCCCACTCGACATGGCAGCAACTGTGGGTTGGCAGTACAGACAAGGCCAGCACCTGTGTGCGCACCGTCAAGGACAACAGCCCCATTGACAGCCTCTCTTCCTCGGACATGTTCTGTGGCCGTGGCCCGGCCTCGTCCTCGGGAGTCTGTGAAGTCGCCGGTATGTTTTCACTCCGCATTCCCGAAGCACGAAACCAGGCCTATCCCTTCCCACTCACTCACTCACTCACTCGCCCACTCATCCACTGTCTCTGTGCAAGAAAGCTAATCCATTTCACAGCCGGCGGCTCCCTCACCGTCGAAATGCACGCGCAGCCCGGCGCGCGCTCGTGCTCGCAGCCCGCCATCGGCGGCAACCACTACGGCCCCGTGCTCATCTACATGGCCAAAGTCAGCGACGCCAAAACCGCAACGTCCGGCTCCTTCTTCAAGGTGGCCGAGGACGGGTACACGGGCACCACGGCGTCATGGGGCACCGAGATCCTCAACGCAAACTGCGGCAAACGCTCCTTCACGGTGCCGCGCTCGCTGGCCGCCGGCGACTATCTCGTGCGCGCCGAGGCCATCGCCCTGCATGCTGGTGCCGGAAGCCCGCAGCCCTACGTCAGTTGCTTCCAGGTCAAGGTTACCGGTGGAGGCAGCGCCACGCCTTCGGGTGTCAGTTTCCCCGGCGCATACAAGATTAGCGATGCGCTCTTCTCCAAGTCGATTTATGACTCCAGCTTCAAGTATGTGAGCCCTGGACCCGCTGTGTACTCTGGTTAG。

[0061] 1.3 Heterologous expression of genes related to lignocellulose degradation

[0062] 1.3.1 Construction of recombinant vector and transformation of expression host

[0063] Based on the sequence multiple cloning site of the vector pet32a, the restriction sites contained in the cDNA sequence of the target gene were analyzed and compared, and finally XhoⅠ and EcoRⅠ were selected for the connection between the vector and the target fragment StAA9-11 to construct the recombinant vector pet32a-StAA9-11. Xho I and BamH I were selected for the connection between the vector and the target fragment StGH5-9 to construct the recombinant vector pet32a-StGH5-9. NotI and BamHI were selected for the connection between the vector and the target fragment StGH11-5 to construct the recombinant vector pet32a-StGH11-5. The specific construction steps are as follows:

[0064] (1) Obtaining the linearized pET32a vector

[0065] Escherichia coli carrying an empty pet32a vector was cultured and propagated overnight at 37°C and 220 r / min. The pet32a vector was extracted using a plasmid extraction kit, and then double-digested using the restriction endonucleases designed for the corresponding gene vectors. The double-digested products were detected by 1% agarose gel electrophoresis and recovered by gel cutting. For specific experimental steps, see the instructions for use of the plasmid extraction kit and gel recovery kit, and the gel recovery products were stored at -20°C.

[0066] (2) PCR amplification and recovery of target genes

[0067] Using the genomic cDNA of the wild-type strain 01-LNXB of Setosphaeria turcica as a template, the following seamless cloning primers for the upstream and downstream homologous arms of the target gene were designed according to the linearization vector pet32a and the principle of homologous recombination (Table 1) to amplify the cDNA sequence of the target gene with both ends of the vector homologous arms.

[0068] Table 1 Primers required for amplifying gene cDNA

[0069]

[0070] Template cDNA was amplified using upstream and downstream primers carrying homology arms, and DNA polymerase 2×M5 Taq HiFiPCRmix was used for amplification. The PCR reaction program was 95℃3min; 94℃25s, 55℃25s, 72℃1min, 34 cycles, and 72℃5min extension. The reaction system is shown in Table 2.

[0071] Table 2 PCR amplification reaction system of target gene

[0072] Reagents Addition amount cDNA 1μL Primer 1 1μL Primer 2 1μL DNA polymerase 25μL <![CDATA[ddH2O]]> 22μL Total 50μL

[0073] The PCR products were detected by 1% agarose gel electrophoresis and recovered by gel excision. The PCR recovered products were stored at -20°C for future use.

[0074] (3) Construction of recombinant prokaryotic expression vector

[0075] The recombinant prokaryotic expression vector was constructed using seamless cloning technology, and the PCR product was recovered by gel recovery kit and connected with pET32a after enzyme digestion. The reaction conditions were 50℃ for 45min, and the reaction system was shown in Table 3.

[0076] Table 3 Ligation reaction system

[0077] Reagents Addition amount cDNA fragment 2μL Linearized vector pET32a 3μL 2×SeamlessCloningMix 5μL Total 10μL

[0078] (4) E. coli transformation and verification of positive clones

[0079] The recombinant plasmid constructed above was mixed with 50 μL of E. coli DH5α competent cells, and the bacterial solution after 1 hour of culture was evenly spread on 50 μg / mL ampicillin-resistant LB solid medium at 37°C and 200 r / min, and cultured at 37°C overnight. Single colonies grown in the resistant medium were picked and inoculated into 0.5 mL of LB liquid medium and cultured at 37°C and 220 r / min for 3 hours. PCR amplification was performed using the universal primers T7 and T7 ter on the pet32a vector to screen positive clones.

[0080] The PCR product was verified by 1% agarose gel electrophoresis, and the bacterial solution of the positive clone with the correct fragment size was selected and sent to Beijing Huada Company for sequencing. The above-mentioned bacterial solution with the correct sequencing was cultured and propagated overnight at 37°C and 220r / min, and the plasmid was extracted and transformed into Escherichia coli BL21, and passed the ampicillin resistance screening and PCR detection.

[0081] 1.3.2 Histone induction expression and condition optimization

[0082] (1) Screening of positive clones expressing active recombinant proteins

[0083] In order to screen the positive clone strains expressing recombinant proteins with endocellulase activity and exocellulase activity, 1 μL of the positive clone suspension screened by PCR was inoculated on Congo red agar plates with CMC or Avicel as the sole carbon source and 0.2 mM / L IPTG, and inverted at 37°C for 48 hours. The positive clones expressing active recombinant proteins were screened by observing the size of the transparent hydrolysis circle. At the same time, the protein expression of the positive clones screened was detected by SDS-PAGE. The SDS-PAGE protein gel configuration method can be found in the SDS-PAGE gel rapid preparation kit (Biosharp). The positive clones expressing highly active recombinant proteins finally obtained were preserved.

[0084] In order to screen the positive clone strains expressing recombinant proteins with xylanase and β-glucosidase activities, the positive clones identified by the above PCR were induced to express proteins. The bacterial suspension was cultured at 37°C in LB medium until the OD of 600 When the concentration reaches about 0.6-0.8, add IPTG with a final concentration of 0.2mM and induce expression at 25℃ and 200r / min for 6-9h. After enrichment and expression, the bacteria are precipitated and resuspended in PBS. After ultrasonic disruption, the supernatant is centrifuged and tested by SDS-PAGE. At the same time, the enzyme activities of xylanase and β-glucosidase are tested respectively, and the positive clones expressing highly active recombinant proteins are finally obtained and preserved.

[0085] (2) Optimization of recombinant protein induction expression conditions

[0086] The positive clones obtained above were inoculated into LB medium containing 50 μg / mL ampicillin resistance and cultured at 37°C and 220 r / min until OD 600 The concentration of 0.6-0.8, after adding 0.2mmol / L IPTG, continue to shake and culture at 25℃200-220r / min, and sample and detect protein expression at 6h, 9h, and 12h. Centrifuge the sampled bacterial solution at 12000r / min for 2min, mix and resuspend the bacterial precipitate with 30μL protein loading buffer and 120μL ddH2O, place it in a boiling water bath or a 100℃ metal bath for 10min, centrifuge it at 12000r / min for 10min, and take the supernatant for SDS-PAGE detection. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with a separation gel concentration of 12% is used to detect protein expression. At the same time, the corresponding enzyme activity of the obtained supernatant is determined, and the optimal induction time is determined by the content and activity of the induced recombinant protein, which is used to optimize the subsequent protein expression conditions.

[0087] In order to determine the optimal IPTG concentration for induction of recombinant protein, the selected positive transformants were inoculated into LB medium containing 50 μg / mL ampicillin resistance and shaken at 37°C and 220 r / min until the bacterial OD reached 0. 600 After the concentration of 0.6-0.8, 0.2, 0.4, 0.8 and 1 mmol / L IPTG were added respectively, and the mixture was shaken at 25°C and 200-220 r / min until the optimal induction time, and then samples were taken. Protein expression was detected by SDS-PAGE with a separation gel concentration of 12% to determine the optimal inducer concentration.

[0088] 1.4 Analysis of enzymatic properties of recombinant proteins

[0089] 1.4.1 Enzyme activity determination

[0090] (1) Preparation of crude enzyme solution of prokaryotic expression recombinant cellulase

[0091] Induce the expression of recombinant protein under the optimal induction conditions, centrifuge 50mL of bacterial suspension to enrich the bacteria, resuspend the bacteria in PBS buffer solution with pH 7.2-7.5, and perform ultrasonic cell disruption in an ice bath. The ultrasonic cell disruption power is 200W, ultrasonic for 3s, interval 5s, and the total duration is 25min. After ultrasonic disruption until the solution is clear or white and transparent, centrifuge at 12000r / min for 10min, collect the supernatant as the crude enzyme solution, and use it for subsequent experiments to determine its corresponding enzyme activity.

[0092] (2) Determination of endoglucanase activity

[0093] Take 1mL of crude enzyme solution and 1mL of 1% CMC-Na (pH4.8) substrate and mix them in a 50℃ water bath for 30min. Determine the reducing sugar content produced by the system after the reaction by DNS method. The enzyme inactivated in a boiling water bath for 10min before the insulation reaction is used as a blank control to calculate its enzyme activity. The endoglucanase activity is defined as the amount of enzyme that produces 1μmol of glucose per minute under the test conditions as 1U.

[0094] (3) Xylanase activity assay

[0095] Take 1mL of crude enzyme solution and 1mL of 1% xylose (pH4.8) substrate and react in a 50℃ water bath for 60min. Determine the reducing sugar content produced by the system after the reaction by DNS method. Use the inactivated enzyme as the blank control. The xylanase activity is defined as the amount of enzyme that produces 1μmol of oligoxylose per minute under the test conditions as 1U.

[0096] (4) Determination of polysaccharide monooxygenase activity

[0097] Take 100 μL of crude enzyme solution and 100 μL of 1% CMC-Na (pH 4.8) substrate and mix them in a 50°C water bath for 30 minutes. The reducing sugar content produced by the reaction system was determined by the DNS method. The enzyme inactivated by boiling water bath for 10 minutes before the reaction was used as a blank control. After the reaction was completed, take 200 μL of each sample and use a microplate reader to measure the OD 540 The international unit of enzyme activity is defined as the amount of enzyme required to produce 1 μmol of glucose per minute (1U).

[0098] 1.4.2 Optimal temperature, pH and stability of recombinant protein

[0099] In order to determine the optimal reaction temperature of each recombinant protein, 1 mL of crude enzyme solution was taken and reacted at 30°C, 40°C, 50°C, and 60°C for 30-60 min according to the reaction system of the above enzyme activity determination. After the reaction, the reducing sugar was detected by the DNS method, and the activity of the recombinant enzyme at different temperatures was calculated, and the relative enzyme activity under other conditions was calculated with the highest enzyme activity as 100%. In order to explore its temperature stability, the recombinant enzyme was placed in a water bath at 30°C, 50°C, and 60°C for 0h, 3h, 6h, and 9h, respectively, and then the enzyme activity was measured respectively, with 0h of storage as 100%, and the enzyme activity stability at different temperatures was calculated.

[0100] In order to determine the pH of the optimal reaction system, the above recombinant enzymes were reacted with their respective substrates in a reaction system of pH 4-11 at the optimal temperature for 30 minutes. The buffer systems used include 0.1M citric acid-sodium citrate buffer (pH 4, pH 5, pH 6 and pH 7) and 0.1M Tris-hydrochloric acid buffer (pH 8, pH 9, pH 10 and pH 11). The highest enzyme activity was taken as 100%, and the relative enzyme activity under other conditions was calculated. In order to explore its pH stability, the recombinant enzymes were placed in buffers of different pH values ​​and stored for 0h, 3h, 6h, and 9h, and then the enzyme activity was measured respectively, and the enzyme activity stability under different reaction system pH was calculated with 0h storage as 100%.

[0101] 1.5 Degradation of corn straw by multi-enzyme complex

[0102] The above-obtained recombinant endoglucanase StGH5-9, xylanase StGH11-5, and polysaccharide monooxygenase StAA9-11 were used to treat corn stover powder, and the degradation effect on biomass raw materials was studied by calculating the saccharification rate. The effect of different enzymes mixed on the saccharification rate was examined, and the total amount of enzymes used in each group remained unchanged, only the type and ratio of the enzymes were adjusted.

[0103] According to the optimal reaction conditions of each enzyme, the reaction conditions for degrading corn straw powder were selected as follows: PBS buffer with a pH of 5.0 was used as the enzyme reaction system buffer, 50 g / L corn straw powder and 1 g / L enzyme (single enzyme or compound enzyme) were added, and the mixture was treated at 50°C for 72 h. The addition of PBS buffer was used as a blank control. After the reaction, the supernatant was centrifuged and the reducing sugar content (mg) was determined by the DNS method. The saccharification rate of the enzyme system in corn straw was calculated as follows:

[0104] Saccharification rate = total reducing sugar content (mg) / total biomass mass (mg) × 100%.

[0105] 2. Results

[0106] 2.1 Identification and analysis of CAZy enzymes in the genome of Setosphaeria turcica

[0107] Based on the whole genome sequence of the corn leaf blight fungus obtained in the laboratory earlier, the CAZymesAnalysisToolkit prediction program was used for annotation comparison, showing that the genes encoding CAZy enzymes in the corn leaf blight genome are composed of 649 modules, including 255 glycoside hydrolases (GH), 147 auxiliary active enzymes (AA), 128 glycosyltransferases (GT), 87 sugar esterases (CE), 17 polysaccharide lyases (PL) and 15 carbohydrate binding modules (CBM).

[0108] 2.2 Heterologous expression of endoglucanase and analysis of its enzymatic properties

[0109] 2.2.1 Construction of prokaryotic expression vector of endoglucanase gene

[0110] The present invention heterologously expresses the endoglucanase gene StGH5-9. The cDNA synthesized by reverse transcription of the RNA of corn leaf blight pathogen is used as a template, and the cDNA of the above gene is amplified using specific primers with upstream and downstream homologous arms. The results are as follows: Figure 1 As shown in A. The PCR product size is 957bp, which is the same as the theoretical prediction of the target fragment size, indicating that the above gene cDNA fragment was successfully amplified. The heterologous expression vector pet32a was double-digested with restriction endonucleases Xho I and BamHI, and the enzyme-digested product fragment with a corresponding band size of about 5950bp was recovered by gel cutting to obtain the linearized vector pet32a. The above gene cDNA fragment was seamlessly cloned and fused with its corresponding linearized vector and transformed into Escherichia coli DH5α competent cells. The positive transformants containing the above gene recombinant vector were verified by ampicillin resistance screening and bacterial liquid PCR amplification. The results are shown in Figure 1 As shown in B. Sequencing of the positive transformant bacterial solution confirmed that it was consistent with the cDNA fragment sequence of the above gene, indicating that the prokaryotic expression vector was successfully constructed.

[0111] 2.2.2 Transformation and screening of the recombinant vector pet32a-StGH5-9

[0112] The recombinant vector obtained above was transformed into competent E. coli BL21, and single colonies that grew well on LB solid medium with ampicillin resistance were selected for bacterial liquid PCR verification, and positive transformants E.coil pet32a-StGH5-9 were obtained. The positive clones corresponding to the correct and bright bands detected by agarose gel electrophoresis in bacterial liquid PCR were selected and further screened by Congo red method ( Figure 2The target substrate is CMC, which is used to detect the degradation ability of endocellulase in positive cloned strains. The size of the transparent hydrolysis zone on the Congo red plate can reflect the enzyme activity and degradation ability. The strain with the largest transparent zone was selected. This strain carries the recombinant vector pet32a-StGH5-9 and is further tested by SDS-PAGE ( Figure 2 (B), the observed protein size was consistent with the predicted value, indicating that the recombinant protein StGH5-9 was successfully induced.

[0113] 2.2.3 Analysis of enzymatic properties of recombinant protein StGH5-9

[0114] The recombinant protein StGH5-9 was induced to express for different time periods and its enzyme activity was measured. The results are as follows: Figure 3 As shown in A. It was found that the crude enzyme solution with an induction time of 9h had a higher enzyme activity, so the subsequent experiments were carried out using an induction time of 9h. The recombinant protein StGH5-9 was induced to express with different final concentrations of IPTG (0.2, 0.4, 0.6, 0.8, 1.0mM), and its enzyme activity was measured. The results are shown in Figure 3 As shown in B. When induced at a final IPTG concentration of 0.4 mM, the recombinant protein StGH5-9 had a high enzyme activity, so IPTG at a final concentration of 0.4 mM was used for subsequent induction.

[0115] pH and reaction temperature are important indicators of enzymatic reactions. The optimal conditions for the enzymatic reaction of StGH5-9 recombinant enzyme were determined using CMC as a substrate when the reaction temperature ranged from 30-60°C and the reaction pH ranged from 4.0 to 11.0. Figure 4 As shown in Figure A, the enzyme activity of StGH11-5 recombinant protein was the highest at 45°C. The temperature stability of StGH5-9 was further determined at 30°C, 45°C, and 60°C. Figure 4 B). After 9 hours of incubation at 45°C, the three recombinant proteins can maintain more than 80% of their activity, and after 3 hours of high temperature at 60°C, they can still maintain more than 50% of their activity, indicating that StGH5-9 has a certain degree of heat resistance. Further, the relative enzyme activity of StGH5-9 at pH 4.0-11.0 in the reaction system was measured at a reaction temperature of 45°C. The results are as follows Figure 4 As shown in Figure C, the recombinant protein StGH5-9 has the highest enzyme activity at pH 4.5, and the enzyme activity decreases significantly when the pH is higher than 5. The stability of the recombinant protein StGH5-9 at pH 4.5 and pH 7 was tested, and the results showed that it could maintain 90% activity within 3 hours at pH 4.5 ( Figure 4 (D) The enzyme activity of StGH5-9 recombinant protein under the optimal conditions was 23.63 U / mL.

[0116] 2.3 Heterologous expression of xylanase and analysis of enzymatic properties

[0117] 2.3.1 Construction of prokaryotic expression vector of xylanase gene

[0118] The xylanase gene StGH11-5 was heterologously expressed. The cDNA synthesized by reverse transcription of RNA extracted from corn leaf blight pathogen was used as a template to amplify the cDNA of the above gene using specific primers with upstream and downstream homologous arms. The results are as follows: Figure 5 As shown in A, the size of the PCR product is consistent with the theoretical prediction of the target fragment size, indicating that the above-mentioned gene cDNA fragment was successfully amplified. The heterologous expression vector pet32a was double-digested with restriction endonucleases XhoI, BamHI and NotI, BamHI, respectively, and the restriction product fragments with a corresponding band size of about 5950bp were recovered by gel cutting to obtain the linearized vector pet32a. The above-mentioned gene cDNA fragment was seamlessly cloned and fused with its corresponding linearized vector and transformed into Escherichia coli DH5α competent cells. The positive transformants of the recombinant vector pet32a-StGH11-5 were verified by ampicillin resistance screening and bacterial liquid PCR amplification. The results are shown in FIG. Figure 5 As shown in B. Transformants with corresponding fragment sizes were amplified respectively, and sequencing of the bacterial liquid of the positive transformants confirmed that the sequence was consistent with the cDNA fragment sequence of the above gene, indicating that the prokaryotic expression vector was successfully constructed.

[0119] 2.3.2 Inducible expression of recombinant protein StGH11-5

[0120] The engineered bacteria carrying the recombinant vector pet32a-StGH11-5 were induced to express. After collecting the bacterial precipitate, it was broken by ultrasound, the enzyme activity of the supernatant was detected, and the expressed recombinant protein was detected by SDS-PAGE ( Figure 6 ). The enzyme activity of the crude enzyme solution was further tested. The results showed that the enzyme activity of StGH11-5 was 2.8U / mL and 13.6U / mL at pH = 5.0. StGH11-5 had a higher enzyme activity, so the StGH11-5 recombinant protein was selected for subsequent experiments.

[0121] 2.3.3 Analysis of enzymatic properties of recombinant enzyme StGH11-5

[0122] The recombinant enzyme StGH11-5 was induced to express for different time periods, and the enzyme activity of the crude enzyme solution was determined. Figure 7 As shown in A, it was found that the crude enzyme activity was higher when the induction time was 9h, so the induction time of 9h was used as the condition for subsequent experiments. The recombinant protein StGH11-5 was induced to express with different final concentrations of IPTG (0.2, 0.4, 0.6, 0.8, 1.0mM), and the enzyme activity of the crude enzyme solution was measured. The results are shown in Figure 7As shown in B. Under different IPTG concentrations, the crude enzyme activity was higher when the final IPTG concentration was 0.8 mM. Since too high IPTG concentration would lead to slow growth of the strain, a final IPTG concentration of 0.4 mM was used in the later induction expression experiment.

[0123] The enzymatic reaction temperature range selected in the present invention is 30-60°C, and the reaction pH is between 4.0-11.0. Using xylose as a substrate, the optimal conditions for the StGH11-5 recombinant enzyme catalyzed reaction are determined. Figure 8 As shown in Figure A, the enzyme activity of StGH11-5 recombinant protein was the highest at 50°C. The temperature stability of StGH11-5 was measured at 30°C, 50°C, and 60°C. Figure 8 As shown in Figure B, after 9 hours of incubation at 50°C, the StGH11-5 recombinant protein can maintain more than 65% activity, while at higher or lower temperatures, the enzyme activity drops significantly after only 3 hours. The relative enzyme activity of the above recombinant protein was further measured at a reaction temperature of 50°C and a reaction system pH of 4.0-11.0. The results are shown in Figure 2. Figure 8 As shown in Figure C, the enzyme activity of the recombinant protein StGH11-5 was the highest at pH 5. The stability of the recombinant protein StGH6-1 at pH = 5 and pH = 7 was tested, and the results showed that after treatment at pH = 5.5 and pH = 7 for 3 hours, the enzyme activity decreased significantly ( Figure 8 (D) The enzyme activity of StGH11-5 recombinant protein under the optimal conditions was 18.54 U / mL.

[0124] 2.4 Heterologous expression of polysaccharide monooxygenase and analysis of its enzymatic properties

[0125] 2.4.1 Construction of prokaryotic expression vector of polysaccharide monooxygenase gene

[0126] The polysaccharide monooxygenase gene StAA9-11 was heterologously expressed. The cDNA synthesized by reverse transcription of RNA extracted from corn leaf blight pathogen was used as a template, and the cDNA of the above gene was amplified using specific primers with upstream and downstream homologous arms. The results are as follows: Fig. 9 As shown in A, the size of the PCR product is consistent with the theoretical prediction of the target fragment size, indicating that the above gene cDNA fragment was successfully amplified. The heterologous expression vector pet32a was double-digested with restriction endonucleases, and the corresponding restriction product fragment of about 5950 bp was recovered from the gel to obtain the linearized vector pet32a. The above gene cDNA fragment was seamlessly cloned and fused with its corresponding linearized vector and transformed into competent Escherichia coli cells. The positive transformants of the pet32a-StAA9-11 recombinant vector were verified by ampicillin resistance screening and bacterial liquid PCR amplification. The results are shown in Fig. 9As shown in B. Transformants with corresponding fragment sizes were amplified respectively, and sequencing of the bacterial liquid of the positive transformants confirmed that the sequence was consistent with the cDNA fragment sequence of the above gene, indicating that the prokaryotic expression vector was successfully constructed.

[0127] 2.4.2 Transformation and screening of recombinant vectors

[0128] The recombinant vectors obtained above were transformed into competent E. coli BL21, and single colonies that grew well on LB solid medium with ampicillin resistance were selected for bacterial liquid PCR verification. The positive clones corresponding to the correct and bright bands detected by agarose gel electrophoresis in the bacterial liquid PCR were selected, and a bacteria with the ability to degrade cellulose and hemicellulose was obtained after further screening using the Congo red method. The target substrate CMC-Na was used to detect whether the positive clone strain had the ability to degrade polysaccharide monooxygenase. The above positive clones had hydrolysis circles and the protein size was correct ( Fig.10 ).

[0129] 2.4.3 Analysis of enzymatic properties of recombinant protein StAA9-11

[0130] The StAA9-11 recombinant protein was induced to express at different times, and the enzymatic activity of the crude enzyme solution was determined. It was found that the enzymatic activity of the crude enzyme solution was higher under the induction time of 9h. The StAA9-1 and StAA9-11 recombinant proteins were induced to express with different final concentrations of IPTG (0.2, 0.4, 0.6, 0.8, 1.0mM), and the enzymatic activity of the crude enzyme solution was determined. Under different IPTG concentrations, the enzymatic activity of the StAA9-1 crude enzyme solution was higher when induced under the condition of a final IPTG concentration of 0.2mM. The enzymatic activity of the StAA9-1 crude enzyme solution was higher when induced under the condition of a final IPTG concentration of 1mM.

[0131] The selected enzymatic reaction temperature range is 30-60℃, and the reaction pH is between 4.0-12.0. Using CMC-Na as substrate, the optimal conditions for the StAA9-11 recombinant enzyme catalytic reaction were determined. The results are shown in Fig.11 .like Fig.11 As shown in Figure A, the enzyme activity of StAA9-11 is highest at 50°C. The temperature stability of StAA9-11 was measured at 30°C, 50°C, and 60°C. Fig.11 As shown in B, the temperature stability is the highest at 50°C. The relative enzyme activity of StAA9-11 recombinant protein was further measured at a reaction temperature of 50°C when the reaction system pH was 4.0-12.0. The results are as follows: Fig.11 As shown in C, the enzyme activity of the recombinant protein StAA9-11 is the highest at pH 5, and the enzyme activity of StAA9-11 is stable at pH = 5.0. The enzyme activity of the StAA9-11 recombinant protein under the optimal conditions is 26.82 U / mL.

[0132] 2.5 Multi-enzyme complex degradation of lignocellulose

[0133] In order to detect the degradation and promotion effect of multi-enzyme compounding on the degradation of lignocellulose, two or three of the recombinant endoglucanase StGH5-9, xylanase StGH11-5 and polysaccharide monooxygenase StAA9-11 were compounded to detect the degradation effect on corn straw powder (the total amount of enzymes added in each group remained unchanged), and the results are shown in Table 4. The results showed that compared with the treatment of each recombinant enzyme alone and the compounding of two by two, the saccharification rate of corn straw powder after the endoglucanase StGH5-9, xylanase StGH11-5 and polysaccharide monooxygenase StAA9-11 were compounded in a ratio of 1:1:1, and the saccharification rate reached 46.05% after 3 days of treatment of corn straw powder, which was 37.41% higher than that of the recombinant protein StGH5-9 alone. The three enzymes StGH5-9, StGH11-5 and StAA9-11 were further compounded in different proportions. It was found that after being compounded in a ratio of 1:1:3, the saccharification rate could reach 55.32% and the synergy could reach 2.36, which also showed that StAA9-11 could significantly enhance the hydrolysis effect of cellulase on corn straw.

[0134] Table 4 Saccharification rate and synergy coefficient of single and mixed enzymes in corn straw

[0135]

[0136] Note: Synergistic coefficient = actual saccharification rate / theoretical saccharification rate; taking the StGH5-9 + StGH11-5 + StAA9-11 group as an example, the theoretical saccharification rate = StGH5-9 saccharification rate × StGH5-9 compound proportion + StGH11-5 saccharification rate × StGH11-5 compound proportion + StAA9-11 saccharification rate × StAA9-11 compound proportion. A synergistic coefficient > 1 indicates a synergistic effect.

[0137] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An enzyme combination for degrading lignocellulose, characterized in that: Including at least two of StGH5-9 protein, StGH11-5 protein and StAA9-11 protein; The amino acid sequence of the StGH5-9 protein is shown in SEQ ID NO.1; The amino acid sequence of the StGH11-5 protein is shown in SEQ ID NO.3; The amino acid sequence of the StAA9-11 protein is shown in SEQ ID NO.

5.

2. The enzyme combination according to claim 1, characterized in that When the enzyme combination includes two proteins, the mass ratio of the two proteins in the enzyme combination is 1:

1.

3. The enzyme combination according to claim 1, characterized in that When the enzyme combination includes the StGH5-9 protein, the StGH11-5 protein and the StAA9-11 protein; the mass ratio of the StGH5-9 protein, the StGH11-5 protein and the StAA9-11 protein is (1-3): (1-3): (1-3).

4. The enzyme combination according to claim 3, characterized in that The mass ratio of the StGH5-9 protein, the StGH11-5 protein and the StAA9-11 protein is 1:1:(2-3).

5. The enzyme combination according to claim 1, characterized in that The mass ratio of the StGH5-9 protein, the StGH11-5 protein and the StAA9-11 protein is 1:1:

3.

6. Use of the enzyme combination according to any one of claims 1 to 5 in the preparation of a saccharifying enzyme preparation for lignocellulose.

7. A saccharifying enzyme preparation for lignocellulose, characterized in that: The active ingredient comprises the enzyme combination according to any one of claims 1-5.

8. Use of the enzyme combination according to any one of claims 1 to 5 or the saccharifying enzyme preparation according to claim 7 in degrading lignocellulose.

9. A method for degrading lignocellulose, characterized in that: The method comprises the step of enzymatically degrading lignocellulose using the enzyme combination according to any one of claims 1 to 5 or the saccharifying enzyme preparation according to claim 7.

10. The method according to claim 9, characterized in that The temperature for the enzyme-catalyzed degradation is 50° C. and the pH is 5.0.

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