CN114874334A - Chimeric fibrosome and application thereof - Google Patents

Chimeric fibrosome and application thereof Download PDF

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CN114874334A
CN114874334A CN202210459391.8A CN202210459391A CN114874334A CN 114874334 A CN114874334 A CN 114874334A CN 202210459391 A CN202210459391 A CN 202210459391A CN 114874334 A CN114874334 A CN 114874334A
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chimeric
peroxidase
laccase
lignin
scaffold protein
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CN114874334B (en
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田沈
杨秀山
杜济良
孔冬冬
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Capital Normal University
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Abstract

The invention relates to the technical field of complex enzymes, in particular to a chimeric fibrosome and application thereof. The chimeric cellulosome is assembled by laccase, peroxidase and soluble polysaccharide monooxygenase. According to the invention, the laccase, the peroxidase and the soluble polysaccharide monooxygenase are assembled into the composite enzyme in the form of the chimeric cellulose body, so that the defect that the activity of the peroxidase is reduced when the laccase and the peroxidase are compounded is overcome, the defect that the range of a lignin substrate degraded when the peroxidase and the soluble polysaccharide monooxygenase are compounded is limited is overcome, and the composite enzyme with higher stability of a lignin degrading enzyme system and lignin substrate degrading activity is obtained, so that harmless degradation of lignin and effective conversion of cellulosic ethanol in a biomass energy conversion system are realized.

Description

Chimeric fibrosome and application thereof
Technical Field
The invention relates to the technical field of complex enzymes, in particular to a chimeric fibrosome and application thereof.
Background
Lignin is a major component constituting plant cell walls, and is an aromatic polymer having the highest natural content and being difficult to degrade. When lignocellulose is used as a raw material to produce the cellulose ethanol which is a clean renewable energy source, lignin in the pretreated raw material can interfere the hydrolysis efficiency of cellulase on a substrate, and can generate irreversible adsorption with the cellulase through hydrophobic effect, electrostatic effect and hydrogen bond effect, so that the enzyme activity in a synchronous saccharification and fermentation reaction system is reduced, and the production cost is improved. Therefore, the harmless delignification has important practical significance for improving the stress resistance of the yeast and the production efficiency of the cellulosic ethanol fermentation, reducing the process cost and even reducing the emission of toxic and harmful substances in the production process.
The basic structural unit of lignin is phenyl propane, which is connected by chemical bonds to form precursor substances such as sinapyl alcohol, pinosyl alcohol, 5-hydroxy-pinosyl alcohol and coumaryl alcohol, and then the precursor substances are polymerized into a complex phenolic polymer by the precursor substances. Currently known enzyme systems for microbial degradation of lignin mainly include two major classes of lignin-modifying enzymes (LMEs) and lignin-degrading accessory enzymes (LDAs).
It was found that fungi have a relatively more powerful lignin depolymerase system than bacteria. And the high-efficiency degradation of the lignin by the fungi is mainly completed based on the synergistic effect of lignin degrading enzyme systems. Among them, laccases (laccas, Lac) and Peroxidases (Peroxidases) play an important role in the process of degrading lignin complex phenolic polymers. Laccase can act on monophenol compounds, bisphenol compounds, aminophenol compounds and the like for low oxidationPhenols and aromatic amines with a reduction potential, while peroxidases act on phenols and non-phenols with a high redox potential in addition to phenols with a low redox potential. When laccase is catalyzed by a phenolic substrate, an electron can be extracted from the oxidized phenolic molecule to promote the generation of free radicals, which in turn cause the cleavage of covalent bonds (especially alkyl-aryl groups) resulting in the depolymerization of lignin polymers. Finally, the lignin macromolecules are degraded to generate a large amount of aromatic compounds, and the laccase can still continue to use the aromatic compounds as substrates to carry out enzymatic hydrolysis of aromatic ring demethoxylation and demethylation. Peroxidases as another important lignin oxidase mainly include lignin peroxidases (LiP), manganese peroxidases (MnP), and multifunctional peroxidases (VP). Wherein, the multifunctional peroxidase (VP) can have the biological catalytic characteristics of lignin peroxidase (LiP) and manganese peroxidase (MnP), the catalytic reaction path can generate free radicals with higher oxidation-reduction potential to further initiate free radical chain reaction, the reaction can act on lignin to generate various reactions such as C-C bond or C-O bond breakage, demethylation, hydroxylation, benzyl alcohol oxidation and the like, and the products are further degraded into CO completely through different metabolic processes 2 And H 2 O, finally achieving the purpose of degrading lignin; furthermore, VP can also oxidize hydroquinone and substituted phenols directly, both of which are difficult to oxidize efficiently by other types of peroxidases.
However, the laccase alone still has certain problems: 1. are unable to catalyze non-phenolic lignin building blocks with-O-4 and 5-5'; 2. enzymatic hydrolysis is of limited efficiency and tends to "polymerize" reactions, particularly plant laccases; 3. in order to improve the catalytic hydrolysis efficiency of single laccases, some groups tried to explore the catalytic conversion of non-phenolic lignin with the help of media (such as ABTS, NHA, TEMPO, etc.), but these media all present toxicity, poor stability of intermediates and high production cost.
In order to improve the degradation efficiency of lignin degrading enzyme, the prior art researches a compounding mode of various enzymes, for example, the degradation of lignin is promoted by matching laccase with peroxidase, but the enzymatic activity of the peroxidase is reduced due to the relative shortage of an oxidizing agent generated in the laccase catalytic process. For example, laccase and the soluble polysaccharide monooxygenase are used simultaneously, but this, in turn, increases the strength of the cellulose, resulting in a decrease in the conversion of the cellulose. For example, both peroxidase and soluble polysaccharide monooxygenase enzymes are used, but the range of lignin substrates degraded is limited. For example, the use of soluble polysaccharide monooxygenase and cellulase promotes the cellulolytic efficiency of cellulase, but soluble polysaccharide monooxygenase does not dominate the degradation of lignin and does not optimize the cellulosic ethanol fermentation system. [ teacher, your good, generally does not suggest marking the provenance of the reference in the manuscript ]
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a chimeric fibrosome and application thereof, and the composite enzyme with higher lignin degrading enzyme system stability and lignin substrate degrading activity is obtained by assembling chimeric laccase, peroxidase and soluble polysaccharide monooxygenase.
In a first aspect, the invention provides a chimeric cellulosome assembled from laccase, peroxidase and soluble polysaccharide monooxygenase.
The chimeric fibrosome is a Mini-fibrosome structure (Mini-cellosome) artificially designed to be expressed in heterologous cells according to a natural fibrosome structure and depending on species-specific recognition and high-affinity protein molecule assembly basis of an adhesion module (Cohesin) and a docking module (Dockerin). Wherein, the scaffold protein (Scaffoldin) is composed of adhesion modules from different microorganism sources, and the zymoprotein is assembled on the scaffold protein through the specific high affinity mediation between the docking module at the C end and the adhesion modules, and finally forms a 'complex enzyme system'. The structure can ensure that the reaction product of the previous step in the multiple enzymolysis reaction processes can directly enter the active center of the enzyme protein of the next reaction without diffusion to become an enzymolysis Substrate, so that the enzymolysis reaction efficiency is improved, namely, a Substrate-channel effect (Substrate-channel effect) is formed. The substrate channel effect has the advantages of promoting the reaction, avoiding adverse reaction balance and kinetic process, protecting unstable reaction intermediates and the like, and the flexibility of the cellulosome scaffold protein effectively overcomes the steric hindrance effect between adjacent enzyme components, provides guarantee for the substrate channel effect in the enzymatic reaction, and enhances the synergistic effect between multiple enzyme components and the proximity effect between enzyme and substrate, thereby endowing the chimeric enzyme with high-efficiency catalytic characteristics.
Because Lac in the lignin degrading enzyme system mainly acts on phenolic compounds with low oxidation-reduction potential and has the characteristic of high reaction rate, and VP mainly acts on phenols and non-phenols with low/high oxidation-reduction potential but has low reaction rate, the lignin degrading enzymes Lac, VP and LPMO proteins are assembled in a chimeric cellulose body structure, substrates with low/high oxidation-reduction potential can be synchronously catalytically degraded through Lac and VP, and released products such as phenols and low molecular weight lignin derivative compounds are used as electron donors, so that LPMO activity and cellulose hydrolysis efficiency can be improved. And H produced in LPMO catalyzed process 2 O 2 And then to the VP and Lac catalytic reactions, preferably for lignin oxidation.
Based on the idea of constructing a cellosome chimeric enzyme system, the invention provides guarantee for the action of a substrate channel in an enzymatic reaction, enhances the synergistic action among multiple enzyme components and the proximity effect of enzyme and a substrate, assembles laccase, peroxidase and soluble polysaccharide monooxygenase to form a chimeric cellosome, which can degrade multiple phenolic and non-phenolic components in lignin and low/high redox potential components in the chimeric cellosome, and realizes the full pollution-free degradation of lignin.
Further, assembling the laccase, peroxidase and soluble polysaccharide monooxygenase with a scaffold protein; the scaffold protein comprises at least three adhesion modules.
Further, the assembling is to assemble the laccase, the peroxidase and the soluble polysaccharide monooxygenase onto the scaffold protein through the adhesion module and the docking module; the adhesion module is derived from one or more of Clostridium thermocellum, Clostridium cellulolyticum, or Ruminococcus flavus.
Further, the adhesion module is one or more of CipA, ScaB or CipC, and the docking module is Doc-CipA, Doc-ScaB and Doc-CipC or more.
Further, the mass ratio of the laccase to the peroxidase to the soluble polysaccharide monooxygenase is 1: (0.5-2): (0.5-2).
In a second aspect, the present invention provides a method for producing chimeric fibrosomes, comprising:
the method comprises the steps of expressing a scaffold protein by adopting a saccharomyces cerevisiae a lectin display system, anchoring the scaffold protein by saccharomyces cerevisiae cell surface anchoring protein, and assembling laccase, peroxidase and soluble polysaccharide monooxygenase on the scaffold protein by an adhesion module and a docking module.
Further, comprising: the scaffold protein ScafI is expressed by adopting a saccharomyces cerevisiae alpha lectin display system, the N-terminal Aga2 signal peptide of the scaffold protein ScafI is combined with saccharomyces cerevisiae cell surface anchoring protein AGA1 to anchor the scaffold protein ScafI, and then laccase, peroxidase and soluble polysaccharide monooxygenase are assembled on the scaffold protein ScafI through an adhesion module and a docking module.
Further, the assembly is carried out for 10-14 hours at the temperature of 0-4 ℃.
As a preferred embodiment, the present invention provides a method for producing chimeric fibrosomes, comprising:
1. amplifying to obtain gene segments of laccase, peroxidase and soluble polysaccharide monooxygenase, constructing the gene segments on a carrier, and then transforming saccharomyces cerevisiae cells to express to obtain the laccase, the peroxidase and the soluble polysaccharide monooxygenase;
2. after laccase, peroxidase and soluble polysaccharide monooxygenase are mixed, the recombinant saccharomyces cerevisiae strains with the hybrid scaffold protein ScafI displayed on the surfaces are further mixed for assembly at the temperature of 0-4 ℃.
The invention further provides application of the chimeric cellulosome in improving stress resistance of saccharomyces cerevisiae.
The invention further provides application of the chimeric cellulosome in improving the ethanol fermentation performance of cellulose.
Saccharomyces cerevisiae has the advantages of clear genetic background, mature genetic engineering operation technology, fast growth and reproduction, higher tolerance to inhibitors and toxic substances, high-efficiency expression of foreign proteins and the like, and is also a traditional strain for industrially producing ethanol. The method can effectively improve the tolerance of the saccharomyces cerevisiae to harmful substances generated by lignin decomposition and can also improve the ethanol production performance of the saccharomyces cerevisiae.
The invention has the following beneficial effects:
the chimeric cellosome reaction system capable of synergistically degrading multiple types of lignin components is constructed by the composite laccase, the peroxidase and the soluble polysaccharide monooxygenase, so that the tolerance of saccharomyces cerevisiae to toxic and harmful compounds generated by lignin degradation, such as phenol, guaiacol, vanillin and syringaldehyde, is remarkably improved; meanwhile, the method also has higher lignin degradation capability and improves the ethanol fermentation performance of the yeast.
Drawings
FIG. 1 is a schematic diagram showing the cloning results of LPMO, VP and LAC genes and fusion genes provided in example 1 of the present invention; wherein A is the result of amplification of LPMO gene, Lane M is BL2000Plus,1 is LPMO; b is the amplification result of VP gene, lane M is BL2000Plus,1 is VP; c is the amplification result of the LAC gene, lane M is BL2000Plus,1 is LAC; d is the amplification result of the docking module, lane M is BL2000Plus,1 is Doc-CipA, 2 is Doc-ScaB, 3 is Doc-CipC; e is the amplification result of the fusion gene, 1 is LPMO-CipA,2.VP-ScaB,3. LAC-CipC.
FIG. 2 shows the result of amplification of pRS423 α -MCS vector fragment provided in example 1 of the present invention; wherein A is the amplification result of a secretion signal peptide, a promoter and a terminator, a lane M is BL2000Plus,1 is alpha MF, 2 is PGK, and 3 is MATT; b is the amplification result of promoter, cell surface display signal peptide and terminator, Lane M is BL2000Plus,1 is PGK, 2 is Aga2, 3 is MATT; c is the amplification result of the large segment of promoter, secretion signal peptide and terminator gene used for secretory expression, the Lane M is BL2000Plus,1 is PGK-alpha MF-MATT; d is the amplification result of the large fragment of the promoter, surface display signal peptide and terminator gene used for cell surface display expression, and lane M is BL2000Plus, and 1 is PGK-Aga 2-MATT.
FIG. 3 shows the result of screening positive clones of recombinant Saccharomyces cerevisiae strains according to example 1 of the present invention; wherein A is a screening result of the recombinant saccharomyces cerevisiae strain W303/LPMO positive clone, a Lane M is BL2000Plus, a Lane 1 is a positive control, and Lanes 2-6 are W303/LPMO positive monoclonals; b is a screening result of the recombinant saccharomyces cerevisiae strain W303/VP positive clone, a lane M is BL2000Plus, a lane 1 is a positive control, and a lane 2 is a W303/VP monoclonal; c is the screening result of the recombinant Saccharomyces cerevisiae strain W303/LAC positive clone, lane M is BL2000Plus, lane 1 is the positive control, and lanes 2-3 are W303/Lac monoclonals.
Fig. 4 shows the results of western blot detection of LPMO protein provided in example 1 of the present invention.
FIG. 5 shows the result of immunoblot detection of VP protein provided in example 1 of the present invention.
FIG. 6 shows the result of immunoblotting detection of Lac protein provided in example 1 of the present invention.
FIG. 7 shows the result of detection of the assembly of chimeric cellulosome enzyme by confocal laser immunofluorescence microscopy imaging verification as provided in example 2 of the present invention.
FIG. 8 shows the analysis results of the stress resistance of the recombinant Saccharomyces cerevisiae strain provided in example 3 of the present invention; wherein A is a detection result for phenol, B is a detection result for guaiacol, C is a detection result for vanillin, and D is a detection result for syringaldehyde.
FIG. 9 shows the results of performance analysis of the Saccharomyces cerevisiae cells displaying chimeric cellulosome degraded alkaline lignin provided in example 4 of the present invention.
FIG. 10 shows the production of ethanol by steam exploded corn stalk fermentation with different enzymes provided in example 4 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1 construction of recombinant Saccharomyces cerevisiae strains expressing chimeric enzyme proteins and determination of enzyme protein expression levels and enzyme activities
1. Cloning of the chimeric enzyme Gene:
respectively extracting total RNA from Neurospora crassa, Pleurotus eryngii and trametes hirsuta by using RNA extraction kit (RNAioso, Takara Bio Inc.), and respectively reverse transcribing with total RNA to obtain cDNA template: (
Figure BDA0003619961010000071
First-Strand cDNA Synthesis SuperMix, Kyoto Total gold Biotechnology, Inc.).
Using Neurospora crassa cDNA as a template, the LPMO gene sequence (GI: XM-960505.2) was amplified by the following primer pair:
LPMO-F：5’-CGGAATTCCACACCATCTTCCAGAAGGTGTCC-3’，
LPMO-R：5’-TCACCGCGGTTAATGGTGATGGTGATGATGAGGGAGGCACTGGCTG-3’；
the VP gene sequence (GI: AF007221.1) was amplified by the following primer pair using Pleurotus eryngii cDNA as template:
VP-F：5’-CGGAATTCGCAACTTGCGACGACGGACGCACC-3’，
VP-R：5’-TCACCGCGGTTAATGGTGATGGTGATGATGCGATCCAGGGACGGG-3’；
the LAC gene sequence (GI: KU055621.1) was amplified using trametes furiosa cDNA as a template by the following primer set:
Lac-F：5’-CGGAATTCGCCATCGGGCCAGTCGCAGACCTC-3’，
Lac-R：5’-TCACCGCGGTTAATGGTGATGGTGATGATGCTGGTCGTCAGGCGAG-3’。
PCR conditions were as follows: 30s at 98 ℃; 10s at 98 ℃; at 58 ℃ for 35 s; 72 ℃ for 30 s; 72 ℃ for 10 min; 30 cycles. After agarose gel electrophoresis, the target bands are LPMO (1037bp), VP (993bp) and LAC (1488bp), and the target gene bands are recovered and purified as shown in A, B and C.
Cloning of butt-joint module genes: the sequence of the Doc-CipA gene (CipA is GI:125972525) was amplified using the cDNA of Clostridium thermocellum as a template by the following primers:
Doc-CipA-F：5’-CAGCCAGTGCCTCCCTCGAAACAGTGCTTTC-3’，
Doc-CipA-R：5’-TCACCGCGGTTAATGGTGATGGTGATGATGTAATATATACCTCTTC-3’；
the sequence of the docking module Doc-ScaB gene (ScaB is GI:13277318)) was amplified using Ruminococcus xanthus cDNA as a template by the following primer set:
Doc-ScaB-F：5’-CCCGTCCCTGGATCGACAAAGCTCGTTCCTAC-3’，
Doc-ScaB-R：5’-TCACCGCGGTTAATGGTGATGGTGATGATGCTGAGGAAGTGTGATG-3’。
the sequence of the Doc-CipC gene (CipC is GI:11056042) of the docking module was amplified using Clostridium cellulolyticum cDNA as template by the following primer pairs:
Doc-CipC-F：5’-CTCGCCTGACGACCAGTACCTTGATGAAAAG-3’，
Doc-CipC-R：5’-TCACCGCGGTTAATGGTGATGGTGATGATGTAACAAGAATGATTTG-3’。
PCR conditions were as follows: 30s at 98 ℃; 10s at 98 ℃; at 58 ℃ for 35 s; 72 ℃ for 10 s; 72 ℃ for 10 min; 30 cycles. The result is shown as D in FIG. 1.
LPMO and Doc-CipA fragments are used as templates, and an LPMO chimeric enzyme gene fragment LPMO-CipA is obtained by using an Over-lap PCR method and primers LPMO-F and Doc-CipA-R.
VP and Doc-ScaB are taken as templates, VP chimeric enzyme gene fragment VP-ScaB is obtained by using an Over-lap PCR method and primers VP-F and Doc-ScaB-R,
using LAC and Doc-CipC segments as templates, and obtaining the LAC chimeric enzyme gene segment LAC-CipC by using an Over-lap PCR method and primers LAC-F and Doc-CipC-R.
PCR conditions were as follows: 30s at 98 ℃; 10s at 98 ℃; at 58 ℃ for 35 s; 60s at 72 ℃; 72 ℃ for 10 min; 30 cycles. The result is shown as E in FIG. 1.
2. Amplification of promoter PGK, Signal peptides α MF and AGA2, and terminator MATT sequence
The genomic DNA of Saccharomyces cerevisiae S288c is used as a template, and the sequence of the phosphoglycerate kinase (PGK) promoter PGK is amplified through the following primer pairs:
PGK-F：5’-GAGGAAGCTGAAACGCAATATTTTAGATTCCTGACTTC-3’，
PGK-R：5’-GTAAAAATTGAAGGAAATCTCATCGTTTTGTTTTATATTTGTTG-3’。
the sequences of the commercial vector pYD1 were used as templates to amplify AGA2 and MATT sequences by the following primer pairs:
AGA2-F：5’-CAACAAATATAAAACAGTAATAAAAGTATCAAC-3’，
AGA2-R：5’-CCGCGGGGATCCACTAGTGTCGACCTCGAGGATATCGAATTCAGAACCACCACCACCAG-3’；
MATT-F：5’-GAATTCGATATCCTCGAGGTCGACACTAGTGGATCCCCGCGGGTTTAAACCCGCTGATC-3’，
MATT-R：5’-ATTATTATCATCATTTTTTATTACTGAGTAGTATTTATTTAAG-3’；
the α MF sequence was amplified by the following primer pair using the commercial vector pPIC9K as template:
αMF-F：5’-CAACAAATATAAAACAAAACGATGAGATTTCCTTCAATTTTTAC-3’，
αMF-R：5’-CCGCGGGGATCCACTAGTGTCGACCTCGAGGATATCGAATTCAGCTTCAGCCTCTCTTT-3’。
and (3) amplification procedure: 30 cycles of 98 ℃ for 30s, 98 ℃ for 10s, 56 ℃ for 25s, 72 ℃ for 30s, 72 ℃ for 10 min.
The target gene band obtained by the PCR amplification method is as follows: PGK (778bp), AGA2(296bp), alpha MF (267bp) and MATT (367bp), and recovering and purifying the target gene. PGK, AGA2, alpha MF and MATT sequences are connected through an Over-lap RCR to form PGK-alpha MF-MCS-MATT and PGK-AGA2-MCS-MATT fragments.
And (3) amplification procedure: 30 cycles of 98 ℃ 30s, 98 ℃ 10s, 56 ℃ 60s, 72 ℃ 55s, 72 ℃ 10 min.
The lengths of the target gene fragment bands are 1784bp and 1755bp respectively, the fragments are recovered and purified, and the implementation result is shown in figure 2. After double enzyme digestion, the vector is connected with a pRS423 commercial vector, and the high-copy expression vectors pRS 423-PGK-alpha MF and pRS423-PGK-Aga2 are constructed through sequence verification without errors.
LAC-CipC, VP-ScaB and LPMO-CipA are linked with pRS 423-PGK-alpha MF vector, Scafi and pRS423-PGK-Aga2 are linked, then Escherichia coli DH5 alpha competent cells are transformed by a chemical transformation method, single colonies are picked and cultured, bacterial liquid PCR screening and sequencing verification are carried out, and high copy expression vectors pRS423-LPMO, pRS423-VP, pRS423-Lac and pRS423-Scafi plasmid are obtained, wherein the 3 'ends of LPMO and LAC contain 6 XHis tag sequences, and the 3' end of Scafi contains Xpress tag sequences.
3. Saccharomyces cerevisiae transformation and positive clone screening
pRS423-LPMO, pRS423-VP, pRS423-Lac and pRS423-Scafi plasmids are respectively transformed into Saccharomyces cerevisiae W303 cells and pRS423-Scafi plasmids are transformed into Saccharomyces cerevisiae EBY100 cells by using a lithium acetate transformation method. Then, yeast genomes were extracted by an alkaline heat lysis method, and genome PCR was verified, and positive monoclonals W303/LPMO, W303/VP, W303/Lac and EBY100/ScafI were obtained by screening the results, as shown in FIG. 3.
4. Enzyme western blot analysis
And culturing the screened positive recombinant saccharomyces cerevisiae strain for 48 hours, determining the extracted protein by using an ultramicro spectrophotometer, adjusting the protein concentration to be consistent, and analyzing by using a protein immunoblotting method.
The SDS-PAGE protein electrophoresis process is as follows: 5mL of recombinant Saccharomyces cerevisiae cells cultured at 30 ℃ for 48h at 150rpm are taken and centrifuged at 3000rpm at 4 ℃ for 5 min. The supernatant was aspirated, transferred to an ultrafiltration tube, and centrifuged at 4000rpm for 10min at 4 ℃.
And sucking the protein concentrated solution after ultrafiltration, and placing the protein concentrated solution in a 2mL centrifuge tube for later use. mu.L of the protein concentrate was added to 5. mu.L of the loading buffer (5X) and mixed well.
The gel was applied to a 10% SDS-PAGE gel at 80V for about 20min and then at 120V for 60 min.
Then, protein membrane transfer is carried out: and (5) performing ice water bath, setting the current to be 250mA, and rotating the membrane for 1.5 h.
Sealing a nitrocellulose membrane: blocking with 5% skimmed milk powder solution at room temperature for 1 h.
Primary antibody incubation: the enzyme protein contains His label, and is incubated for 1.5h by adopting 1:10000protein Find Anti-His Mouse Monoclonal Antibody;
and (3) secondary antibody incubation: incubation was performed for 1H with 1:2000 ProteinFind Goat Anti-Mouse IgG (H + L), HRP Conjugate.
Wash 3 times with TBST buffer for 5min each time. Finally, the solution A and the solution B in the easy See Western Blot Kit (Beijing all-type gold biotechnology limited) are mixed according to the proportion of 50:1, and then 1 per thousand of the solution C is added and mixed evenly. Then spread evenly onto nitrocellulose membrane and developed by exposure in GE ImageQuant LAS4000 mini.
And analyzing whether the color development strip is the target protein according to the protein molecular weight standard. As shown in FIG. 4(LPMO), FIG. 5(VP) and FIG. 6(Lac), Western blot analysis demonstrated that the molecular weights of the developed protein bands match those of the target proteins LPMO (39.35kDa), VP (44.5kDa) and Lac (61.95kDa), and that the recombinant strains W303/LPMO, W303/VP and W303/Lac, respectively, were able to correctly express the enzyme proteins.
5. Enzyme activity assay
And (3) LPMO enzyme activity determination: the activity of LPMO is determined by using locust bean gum as a substrate and adopting a 3, 5-dinitrosalicylic acid colorimetric method.
The reaction system comprises: enzyme solution 15 μ L, 0.5% (w/v) locust bean gum 60 μ L.
The reaction flow is that the reaction is carried out for 12 hours at 60 ℃. Then 75. mu.L DNS reagent was added and immediately incubated at 100 ℃ for 10 min. Naturally cooling to room temperature, collecting 130 μ L, and measuring OD with enzyme labeling instrument 540nm The absorbance of (a). Enzyme protein inactivated by heating at 100 ℃ is used as blank control, the enzyme amount required for degrading locust bean gum to generate 1mg/L reducing sugar is defined as one enzyme activity unit (U), and the concentration of the enzyme protein is measured by using a ultramicro spectrophotometer.
And (3) measuring the activity of the VP enzyme: the guaiacol is used as a substrate, and an enzymatic reaction system is as follows: HAc-NaAc Buffer (pH4.5)1.5mL, 2.4mM guaiacol 0.5mL, 3mM MnSO 4 0.5mL of enzyme solution 0.4mL, 3mM H 2 O 2 0.1mL。
Using ultraviolet raysDetermination of OD by Room temperature measurement with a Photometer 465nm The change of the absorbance value within 5min, and the activity of the VP enzyme is calculated according to the following formula.
Figure BDA0003619961010000121
Wherein the extinction coefficient epsilon 465nm ＝12100L/(mol·cm)，V General assembly : total volume of reaction solution (mL), V Enzyme : crude enzyme solution volume (mL), Δ A : difference in absorbance, Δ t : time of enzymatic reaction
VP enzyme activity is defined as follows: the amount of enzyme required to catalyze a reaction of 1. mu. mol of the substrate per minute is one enzyme activity unit (U).
And (3) detecting the Lac enzyme activity, namely taking ABTS as a substrate and adopting the following reaction system: 125. mu.L of HAc-NaAc Buffer (pH4.5), 125. mu.L of 0.6mM ABTS, and 50. mu.L of enzyme solution. Determination of OD Using microplate reader 420nm Change in absorbance within 5 min.
Figure BDA0003619961010000131
Wherein the extinction coefficient ε 420nm ＝36000L/(mol·cm)，V General assembly : total volume of reaction solution (mL), V Enzyme : crude enzyme solution volume (mL), Δ A : difference in absorbance, Δ t : time of enzymatic reaction
Lac enzyme activity is defined as follows: the amount of enzyme required to catalyze a reaction of 1. mu. mol of the substrate per minute is one enzyme activity unit (U).
TABLE 1 measurement of enzymatic Activity for secretion of LPMO, VP and Lac
Figure BDA0003619961010000132
Note: expressed as the mean of three determinations
The enzyme activity determination results are shown in Table 1, the enzyme activities of LPMO, VP and Lac secreted and expressed are 5.620U/mL,6.298U/mL and 6.831U/mL respectively, and the corresponding specific enzyme activities are 16.788U/mg 18.782U/mg and 20.078U/mg. The result proves that the recombinant strain of saccharomyces cerevisiae extracellularly secretes Lac, and the enzyme proteins of VP and LPMO have enzyme activities.
Example 2 Saccharomyces cerevisiae EBY100 cell surface self-assembly of chimeric cellulosomes with Lac, VP, and LPMO catalytic modules
1. And (3) centrifuging the recombinant saccharomyces cerevisiae cells cultured for 20-48 h at 4 ℃ and 3000rpm for 10 min. Wherein, the EBY100/ScafI strain was centrifuged and the supernatant was discarded, the cell pellet was retained, and the suspension buffer (50mM Tris-HCl, 100mM NaCl, 10mM CaCl) was used 2 ) Resuspending EBY100/Scafi cells; the enzyme protein secretion expression strains W303/Lac, W303/VP and W303/LPMO retain culture supernatant after centrifugation as crude enzyme solution.
2. 1mL of W303/ScafI was added to 1mL of the crude enzyme solution, incubated at 4 ℃ for 12 hours to complete the assembly of chimeric fibrosomes, and finally centrifuged at 4 ℃ and 3000rpm for 5min to discard the supernatant and collect the cell pellet.
3. 1mL of Phosphate buffer (Phosphate Buffered Saline, PBS; containing 137mM NaCl, 2.7mM KCl, 10mM Na) 2 HPO 4 ，1.8mM NaH 2 PO 4 pH 7.4) and the chimeric fibrocyte pellet was centrifuged at 3000rpm at 4 ℃ for 10 min. The supernatant was then discarded and 250. mu.L of 1% BSA (in PBS) was added to the pellet and resuspended.
4. Adding primary antibody (Mouse anti-Xpress tag; Rabbit anti-6 XHis tag) according to the proportion (1:1000v/v), mixing uniformly, incubating for 1h at room temperature, and mixing uniformly by reversing every 15min to enable the cells to be in a suspension state. After centrifugation at 3000rpm for 10min at 4 ℃ again, the supernatant was discarded, and the cell pellet was washed twice with 1mL of PBS.
5. Resuspend the cell pellet in 250. mu.L of 1% Bovine Serum (BSA), then add 1. mu.L of secondary antibody (Goat anti-Mouse IgG (H + L), Alexa) at a ratio (1:250v/v)
Figure BDA0003619961010000141
488；Goat anti-Rabbit IgG(H+L),Alexa
Figure BDA0003619961010000144
647) Mixing, standing in dark for 1.5 hr, and continuously mixing to make it always in suspension state。
6. Centrifuge at 5000rpm for 5min at 4 ℃, wash the cell pellet twice with PBS, and resuspend the cells in 200 μ L PBS. A1. mu.L cell suspension was dropped onto a slide glass and observed in a laser scanning confocal microscope imaging system ZEISS LSM LIVE 780. Wherein, Alexa
Figure BDA0003619961010000142
Figure BDA0003619961010000142
488 exhibits green fluorescence, Alexa, under excitation of laser with wavelength of 488nm
Figure BDA0003619961010000143
The fluorescent material emits far infrared fluorescence under the excitation of laser with the wavelength of 633 nm. Photographs were taken with the Carl Zeiss Zen 2011 version of the software and analyzed.
The results are shown in FIG. 7, where EBY100/Scafi exhibits green fluorescence under 488nm laser excitation and no fluorescence reaction under 633nm laser excitation, at the left of line 1, demonstrating that the Scafi protein can be anchored on the EBY100 cell surface through the a lectin system. When the Lac, VP and LPMO proteins are respectively assembled with EBY100/ScafI, green fluorescence appears under 488nm laser excitation, and red fluorescence reaction appears under 633nm laser excitation, as shown by results in lines 2 to 4 in FIG. 7, which proves that the chimeric enzyme proteins Lac, VP and LPMO are combined and assembled with ScafI through the docking module, and the invention innovatively constructs the chimeric fibrosome with the function of lignin synergistic degradation enzyme system.
Example 3 analysis of the stress resistance of recombinant Saccharomyces cerevisiae strains
Preparation of a culture medium: YPD solid media containing different concentrations of inhibitors (e.g., phenol, guaiacol, vanillin, and syringaldehyde) were prepared on 24-well plates, respectively.
The blank Saccharomyces cerevisiae strain EBY100 is used as a negative control, the recombinant Saccharomyces cerevisiae strains EBY100/Lac, EBY100/VP, EBY100/LPMO and EBY100/Lac-VP-LPMO are used as experimental groups, and the low-temperature induction culture is carried out in YPG culture medium at the temperature of 150rpm and the total inoculum size of 2%. The induced control strain and experimental strain were uniformly spread on YPD solid medium containing inhibitor in 24-well plate, and cultured at 30 ℃ for 72 hours.
As shown in FIG. 8, the maximum tolerated concentrations of chimeric cellulosome (EBY100/Lac-VP-LPMO) assembled on the cell surface of Saccharomyces cerevisiae to toxic harmful compounds produced by lignin degradation were: 12mmol/L phenol, 1g/L guaiacol, 0.75g/L vanillin, 2.4g/L syringaldehyde. The concentrations of vanillin and phenol in willow hydrolysate are respectively 0.43g/L and 4mmol/L, and the concentration of syringaldehyde in spruce hydrolysate is 0.107 g/L.
The recombinant saccharomyces cerevisiae strain displaying the chimeric enzyme provided by the invention has certain universality because the maximum tolerance concentration of vanillin, phenol and syringaldehyde is higher than the concentration of an inhibitor generated after pretreatment in an actual reaction system, which indicates that the recombinant saccharomyces cerevisiae strain has application value in developing a technology for producing cellulosic ethanol by using a lignocellulose raw material.
Example 4
The chimeric enzyme system provided in this example as provided in example 2 above has specific properties for degrading lignin:
1. performance analysis of degraded alkaline lignin substrates
YPD liquid medium containing 0.5g/L alkali lignin was prepared, pH was adjusted to about 4.5, and the mixture was dispensed into 250mL Erlenmeyer flasks at 100mL per flask. Recombinant Saccharomyces cerevisiae strains were added in combination according to Table 2, with an inoculum size of 0.5g/L enzyme protein, three in each group being parallel.
TABLE 2 combination of recombinant Saccharomyces cerevisiae degraded alkali lignin
Figure BDA0003619961010000161
Note: 1 copper sulfate was added to a final concentration of 0.5 mM; 2 manganese sulfate was added to a final concentration of 0.5 mM; 3 150 μ M hydrogen peroxide was added and replenished every 24 hours; 4 ascorbic acid was added at 0.5mM and supplemented every 24 hours.
Determination of alkali lignin degradation rate: and centrifuging the alkali lignin enzymolysis sample at 10000rpm for 5min, measuring the light absorption value of the alkali lignin in the enzymolysis liquid at 280nm, and drawing a degradation curve of each combined alkali lignin by taking the time as an abscissa and the light absorption value as an ordinate. Obtaining the alkali lignin content according to the light absorption value and the alkali lignin standard curve, and calculating the alkali lignin degradation rate according to the following formula:
Figure BDA0003619961010000162
wherein: c 0 : alkali lignin content before degradation; c t : alkali lignin content after degradation.
As shown in FIG. 9, the removal percentages of alkali lignin by the chimeric cellulosome structures EBY100/Lac, EBY100/VP, EBY100/LPMO and EBY100/Lac-VP-LPMO were 47.96%, 44.63%, 7.82% and 67.08%, respectively. The results show that: based on a saccharomyces cerevisiae cell surface display system and the assembly of chimeric enzyme proteins Lac, VP and LPMO on scaffold protein ScafI, the synergistic effect of the three enzymes in the lignin substrate degradation reaction can be enhanced, and the lignin degradation efficiency is improved.
2. Research on cellulose ethanol fermentation performance of recombinant saccharomyces cerevisiae
(1) Synergistic fermentation of lignocellulose
Addition of commercial cellulase to lignocellulosic fermentation media
Figure BDA0003619961010000163
CTec2 (purchased from Novixin) 10FPU/g, four recombinant Saccharomyces cerevisiae were mixed and cultured, the inoculum size of 1.2g/L enzyme protein was used as the experimental group, the same inoculum size of Saccharomyces cerevisiae EBY100 host cells was used as the control group, and the experiments were performed in triplicate. Incubate at 30 ℃ for 120h at 150rpm, and sample 2mL at 12h intervals.
(2) Determination of glucose and ethanol content
After centrifugation at 10000rpm for 5min, the fermentation broth sample was filtered through a 0.22 μm filter, and the ethanol content was measured by high performance liquid chromatography (HPLC, mode 1260, Agilent Technologies). The detection conditions are as follows: an Agilent Zorbax Eclipse XDB-C18 column (250 mm. times.4.6 mm, 5 μm), a column temperature of 40 ℃, a mobile phase of methanol and water of 5:95, a flow rate of 0.6mL/min, a sample volume of 5 μ L, a differential detector temperature of 40 ℃, and a running time of 10 min.
As shown in FIG. 10, after the EBY100/Lac-VP-LPMO containing three chimeric enzymes was fermented for 96h, the maximum ethanol concentration reached 4.49g/L, the maximum ethanol production concentration by the EBY100 cell fermentation of the control group was 3.40g/L, and the ethanol concentration was increased by 32% due to the chimeric cellulosome structure.
The results show that, on one hand, the recombinant yeast strain EBY100/Lac-VP-LPMO anchoring three chimeric enzymes can degrade lignin through the synergistic action of the chimeric enzyme system, so that the irreversible adsorption of the lignin in a reaction system on cellulase is reduced, the accessibility of the cellulase and a substrate is improved, the enzymatic saccharification level of the cellulose is improved, and the enzyme dosage is reduced; on the other hand, EBY100/Lac-VP-LPMO has higher tolerance of fermentation inhibitors and ethanol production performance of simultaneous saccharification and fermentation.
Although the invention has been described in detail with respect to the general description and the specific embodiments thereof, it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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Claims (10)

1. A chimeric cellulosome, which is assembled from laccase, peroxidase and soluble polysaccharide monooxygenase.
2. The chimeric cellulosome according to claim 1, wherein the laccase, peroxidase and soluble polysaccharide monooxygenase are assembled using a scaffold protein; the scaffold protein comprises at least three adhesion modules.
3. The chimeric fibrosome according to claim 1 or 2, wherein the assembly is of the laccase, peroxidase and soluble polysaccharide monooxygenase onto the scaffold protein via the adhesion module and docking module; the adhesion module is derived from one or more of Clostridium thermocellum, Clostridium cellulolyticum, or Ruminococcus flavus.
4. The chimeric fibrosome of claim 3, wherein the adhesion module is one or more of CipA, ScaB or CipC and the docking module is one or more of Doc-CipA, Doc-ScaB and Doc-CipC.
5. The chimeric fibrosome according to any of claims 1 to 4, wherein the laccase, peroxidase and soluble polysaccharide monooxygenase are present in a mass ratio of 1: (0.5-2): (0.5-2).
6. A method for producing a chimeric fibrosome according to any one of claims 1 to 5, comprising:
the method comprises the steps of expressing a scaffold protein by adopting a saccharomyces cerevisiae a lectin display system, anchoring the scaffold protein by saccharomyces cerevisiae cell surface anchoring protein, and assembling laccase, peroxidase and soluble polysaccharide monooxygenase on the scaffold protein by an adhesion module and a docking module.
7. The method of claim 6, comprising:
the scaffold protein ScafI is expressed by adopting a saccharomyces cerevisiae alpha lectin display system, the N-terminal Aga2 signal peptide of the scaffold protein ScafI is combined with saccharomyces cerevisiae cell surface anchoring protein AGA1 to anchor the scaffold protein ScafI, and then laccase, peroxidase and soluble polysaccharide monooxygenase are assembled on the scaffold protein ScafI through an adhesion module and a docking module.
8. The method of claim 7, comprising:
the assembly is carried out for 10-14 h in an environment of 0-4 ℃.
9. Use of the chimeric cellulosome of any one of claims 1 to 5 to improve the stress resistance of s.
10. Use of the chimeric cellulosome of any one of claims 1-5 to improve the ethanol fermentation performance of cellulose.
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