CN114874334B - Chimeric fiber corpuscle and application thereof - Google Patents

Chimeric fiber corpuscle and application thereof Download PDF

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CN114874334B
CN114874334B CN202210459391.8A CN202210459391A CN114874334B CN 114874334 B CN114874334 B CN 114874334B CN 202210459391 A CN202210459391 A CN 202210459391A CN 114874334 B CN114874334 B CN 114874334B
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peroxidase
chimeric fiber
saccharomyces cerevisiae
lignin
laccase
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CN114874334A (en
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田沈
杨秀山
白子上
杜济良
孔冬冬
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Capital Normal University
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    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
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    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
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    • C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
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    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Abstract

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

Description

Chimeric fiber corpuscle and application thereof
Technical Field
The invention relates to the technical field of complex enzymes, in particular to a chimeric fiber body and application thereof.
Background
Lignin is a main component constituting plant cell walls, and is an aromatic polymer with highest natural content and difficult degradation. When lignocellulose is used as a raw material to produce clean renewable energy, namely cellulosic ethanol, lignin in the pretreated raw material not only can interfere the hydrolysis efficiency of cellulase on a substrate, but also can generate irreversible adsorption action with the cellulase through hydrophobic action, electrostatic action, hydrogen bond action, so that the enzyme activity in a synchronous saccharification and fermentation reaction system is reduced, and the production cost is increased. Therefore, the harmless delignification has important practical significance for improving the stress resistance of yeast and the production efficiency of cellulose 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 phenylpropane, which is linked by chemical bonds to form precursor substances such as sinapyl alcohol, pinitol, 5-hydroxy-pinitol, coumarol and the like, which are then polymerized into complex phenolic polymers. The currently known enzyme systems for microbial degradation of lignin mainly comprise two major classes, lignin-modifying enzymes (LME) and lignin-degrading auxiliary enzymes (lignin-degrading auxiliary enzymes, LDA).
It was found that fungi have a relatively more powerful lignin depolymerase system than bacteria. And, the efficient degradation of lignin by fungi is mainly completed based on the synergistic effect of lignin degrading enzyme systems. Among them, laccase (lacase, lac) and peroxidase (Peroxidases) play an important role in the degradation of lignin complex phenolic polymers. Laccase can act on monophenolizationPhenols and aromatic amines having low redox potential such as compounds, bisphenols and aminophenols, while peroxidases act on phenols and non-phenols having high redox potential other than phenols having low redox potential. When laccase is catalyzed by a phenolic substrate, an electron may be extracted from the oxidized phenolic molecule to trigger free radical generation, which in turn causes cleavage of covalent bonds (especially alkyl-aromatic groups), resulting in depolymerization of lignin polymers. Finally, lignin macromolecules are degraded to generate a large amount of aromatic compounds, and laccase can still be used as a substrate to carry out enzymatic hydrolysis of aromatic ring demethoxy groups and demethylation. Peroxidase is another important lignin oxidase, and mainly includes lignin peroxidase (lignin peroxidases, liP), manganese peroxidase (manganese peroxidases, mnP), multifunctional peroxidase (versatile peroxidases, VP), and the like. Wherein the multifunctional peroxidase (VP) has biological catalytic properties of lignin peroxidase (LiP) and manganese peroxidase (MnP), and the catalytic reaction path can generate free radicals with higher oxidation-reduction potential so as to trigger free radical chain reaction, and the reaction also acts on lignin to generate various reactions such as C-C bond or C-O bond cleavage, demethylation, hydroxylation, benzyl alcohol oxidation and the like, and the products are further thoroughly degraded into CO through different metabolic processes 2 And H 2 O, finally achieving the aim of degrading lignin; furthermore, VP can oxidize hydroquinone and substituted phenol directly, and both substrates are difficult to oxidize effectively by other types of peroxidases.
However, there are still problems associated with laccase alone: 1. non-phenolic lignin structural units with-O-4 and 5-5' cannot be catalyzed; 2. enzymolysis has limited efficiency and tends to "polymerize" reactions, especially plant laccases; 3. in order to improve the catalytic hydrolysis efficiency of single laccase, some groups have tried to explore catalytic conversion of non-phenolic lignin with the help of medium (such as ABTS, NHA, TEMPO, etc.), but these mediums are toxic, and the intermediate has poor stability and high production cost.
In order to improve the degradation efficiency of lignin degrading enzyme, the prior art researches a compound mode of various enzymes, such as promoting the degradation of lignin through the combination of laccase and peroxidase, but the enzyme activity of peroxidase is reduced due to relatively insufficient oxidant generated in the laccase catalysis process. For example, laccase and soluble polysaccharide monooxygenase are used simultaneously, but this increases the strength of cellulose instead, resulting in a decrease in the conversion of cellulose. For example, the use of both peroxidases and soluble polysaccharide monooxygenases, but the range of degraded lignin substrates is limited. For example, the use of a soluble polysaccharide monooxygenase and a cellulase enzyme promotes the cellulolytic efficiency of the cellulase enzyme, but the soluble polysaccharide monooxygenase does not dominate the degradation of lignin and does not optimize the fermentation system of cellulosic ethanol.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a chimeric fiber body and application thereof, and a composite enzyme with high 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 present invention provides a chimeric fiber-body assembled from laccase, peroxidase and a soluble polysaccharide monooxygenase.
The chimeric fiber corpuscles are protein molecule assembly bases with high affinity and specific recognition between species depending on a Cohesin module (Cohesin) and a Dockerin module (Dockerin) according to a natural fiber corpuscle structure, and are manually designed into a micro fiber corpuscle structure (Mini-cellulose) expressed in heterologous cells. Wherein the scaffold protein (Scaffolden) is composed of adhesion modules derived from different microorganisms, and the enzyme protein is assembled on the scaffold protein through the specific high affinity mediation between the docking module at the C end and the adhesion modules, thus finally forming a complex enzyme system. The structure can ensure that the enzyme protein active center of the last reaction product in the process of a plurality of enzymolysis reactions can directly enter the next reaction without diffusion to become an enzymolysis Substrate, so that the enzymolysis reaction efficiency is improved, namely a Substrate channel effect (Substrate-channeling effect) is formed. The substrate channel effect has the advantages of promoting the reaction, avoiding unfavorable reaction balance and dynamics process, protecting unstable reaction intermediates and the like, and the flexibility of the fiber small scaffold protein effectively overcomes the steric hindrance effect between adjacent various enzyme components, provides guarantee for the substrate channel effect in the enzymatic reaction, enhances the synergistic effect between various enzyme components and the proximity effect of the enzyme and the substrate, thereby endowing the chimeric enzyme with high-efficiency catalytic characteristics.
Since Lac in the lignin degrading enzyme system mainly acts on phenolic compounds with low oxidation-reduction potential and has the characteristic of quick reaction rate, and VP mainly acts on phenolic and non-phenolic substances with low/high oxidation-reduction potential but has slower reaction rate, the lignin degrading enzyme Lac, VP and LPMO proteins are assembled in a chimeric fiber small body structure, the Lac and VP can synchronously catalyze and degrade substrates with low/high oxidation-reduction potential, and released products such as phenols and low molecular weight lignin derivative compounds are used as electron donors, so that the LPMO activity and the hydrolysis efficiency of cellulose can be improved. While H is produced in LPMO catalytic process 2 O 2 And VP and Lac catalytic reaction are supplied, and lignin oxidation is preferentially performed.
The invention is based on the concept of constructing a fiber small chimeric enzyme system, provides guarantee for substrate channel action in enzymatic reaction, enhances the synergistic effect between various enzyme components and the proximity effect of enzyme and substrate, assembles laccase, peroxidase and soluble polysaccharide monooxygenase into a chimeric fiber small body which can degrade various phenols and non-phenols in lignin and low/high oxidation-reduction potential components therein, realizes fully pollution-free degradation of lignin, and simultaneously can not only improve the stress resistance and fermentation performance of strains in a synchronous saccharification and fermentation reaction system, but also can be used as an auxiliary enzyme system of commercial cellulase, and improves the substrate utilization rate and ethanol yield under the condition of not adding additional cellulase.
Further, assembling the laccase, peroxidase and soluble polysaccharide monooxygenase with scaffold proteins; the scaffold protein comprises at least three adhesion modules.
Further, the assembling is to assemble the laccase, peroxidase and soluble polysaccharide monooxygenase onto the scaffold protein through the adhesion module and docking module; the adhesion module is derived from one or more of clostridium thermocellum, clostridium defibricum, or ruminococcus flavum.
Further, 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.
Further, the mass ratio of laccase, peroxidase and soluble polysaccharide monooxygenase is 1: (0.5-2): (0.5-2).
In a second aspect, the present invention provides a method for producing a chimeric fiber body, comprising:
expressing a scaffold protein by using a Saccharomyces cerevisiae a lectin display system, anchoring the scaffold protein by using a Saccharomyces cerevisiae cell surface anchoring protein, and assembling laccase, peroxidase and soluble polysaccharide monooxygenase on the scaffold protein by using an adhesion module and a docking module.
Further, the method comprises the steps of: expressing a scaffold protein ScafI by using a Saccharomyces cerevisiae a lectin display system, anchoring the scaffold protein ScafI by combining an N-terminal Aga2 signal peptide of the scaffold protein ScafI with an anchoring protein AGA1 on the surface of Saccharomyces cerevisiae cells, and assembling laccase, peroxidase and soluble polysaccharide monooxygenase on the scaffold protein ScafI by using an adhesion module and a docking module.
Further, the assembly is carried out for 10 to 14 hours under the environment of 0 to 4 ℃.
As a preferred embodiment, the present invention provides a method for producing a chimeric fiber body, comprising:
1. amplifying to obtain gene fragments of laccase, peroxidase and soluble polysaccharide monooxygenase, constructing the gene fragments on a carrier, and then converting saccharomyces cerevisiae cells to express to obtain laccase, peroxidase and soluble polysaccharide monooxygenase;
2. after mixing laccase, peroxidase and soluble polysaccharide monooxygenase, further mixing recombinant Saccharomyces cerevisiae strain with surface displaying heterozygous scaffold protein ScafI, and assembling at 0-4deg.C.
The invention further provides application of the chimeric fiber body in improving stress resistance of saccharomyces cerevisiae.
The invention further provides application of the chimeric fiber body in improving ethanol fermentation performance of cellulose.
Saccharomyces cerevisiae (Saccharomyces cerevisiae) has the advantages of clear genetic background, mature genetic engineering operation technology, rapid growth and propagation, higher tolerance to inhibitors and toxic substances, high-efficiency expression of exogenous proteins and the like, and is also a traditional strain for industrially producing ethanol. The chimeric fiber corpuscles obtained by the method are transformed into the saccharomyces cerevisiae, so that the tolerance of the saccharomyces cerevisiae to harmful substances generated by lignin decomposition can be effectively improved, and the ethanol production performance of the saccharomyces cerevisiae can be improved.
The invention has the following beneficial effects:
the chimeric fiber body reaction system with synergistic degradation of various lignin components is constructed by compounding laccase, peroxidase and 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 saccharomycetes.
Drawings
FIG. 1 is a schematic diagram showing cloning results of LPMO, VP and LAC genes and fusion genes provided in example 1 of the present invention; wherein A is the amplification result of the LPMO gene, lane M is BL2000 Plus, and 1 is LPMO; b is the amplification result of VP gene, lane M is BL2000 Plus, and 1 is VP; c is the result of amplification of LAC gene, lane M is BL2000 Plus,1 is LAC; d is the result of amplification of the docking module, lane M is BL2000 Plus,1 is Doc-CipA,2 is Doc-ScaB, and 3 is Doc-CipC; e is the result of amplification of the fusion gene, 1 is LPMO-CipA,2.VP-ScaB,3.LAC-CipC.
FIG. 2 shows the amplification results of pRS423 alpha-MCS vector fragment provided in example 1 of the present invention; wherein A is the result of amplification of secretion signal peptide, promoter and terminator, lane M is BL2000 Plus,1 is αMF,2 is PGK, and 3 is MATT; b is the amplification result of a promoter, a cell surface display signal peptide and a terminator, lane M is BL2000 Plus,1 is PGK, 2 is Aga2, and 3 is MATT; c is the amplification result of large fragments of a promoter, a secretion signal peptide and a terminator gene used for secretory expression, lane M is BL2000 Plus, and 1 is PGK-alpha MF-MATT; d is the result of amplification of large fragments of the promoter, surface display signal peptide and terminator gene used for cell surface display expression, lane M is BL2000 Plus, and 1 is PGK-Aga2-MATT.
FIG. 3 shows the results of screening positive clones of recombinant Saccharomyces cerevisiae strains provided in example 1 of the present invention; wherein A is the result of screening positive clones of recombinant Saccharomyces cerevisiae strain W303/LPMO, lane M is BL2000 Plus, lane 1 is positive control, lanes 2-6 are W303/LPMO positive monoclonal; b is the result of screening recombinant Saccharomyces cerevisiae strain W303/VP positive clone, lane M is BL2000 Plus, lane 1 is positive control, and lane 2 is W303/VP monoclonal; c is a result of screening for recombinant Saccharomyces cerevisiae strain W303/LAC positive clones, lane M is BL2000 Plus, lane 1 is positive control, and lanes 2-3 are W303/LAC monoclonal.
FIG. 4 shows the Western blotting detection result of LPMO protein provided in example 1 of the present invention.
FIG. 5 shows the result of immunoblotting 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 is a test result of the assembly of chimeric fiber-like enzyme using fluorescence microscopy imaging of laser copolymerization Jiao Mianyi provided in example 2 of the present invention.
FIG. 8 is a graph showing the results of stress resistance analysis of the recombinant Saccharomyces cerevisiae strain provided in example 3 of the present invention; wherein A is the detection result for phenol, B is the detection result for guaiacol, C is the detection result for vanillin, and D is the detection result for syringaldehyde.
FIG. 9 is a graph showing the results of a performance analysis of chimeric fiber bodies to degrade alkaline lignin by Saccharomyces cerevisiae cells provided in example 4 of the present invention.
FIG. 10 shows the ethanol production by steam exploded corn stalk fermentation of different enzymes according to example 4 of the present invention.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
EXAMPLE 1 construction of recombinant strain of Saccharomyces cerevisiae expressing chimeric enzyme protein and measurement of enzyme protein expression amount and enzyme Activity
1. Cloning of chimeric enzyme gene:
extracting total RNA from Neurospora crassa, pleurotus eryngii and trametes bristle by using RNA extraction kit (RNAiso, takara Bio-engineering (Dalian) limited company), and respectively performing reverse transcription to obtain cDNA templateFirst-Strand cDNA Synthesis SuperMix, beijing full gold biotechnology Co., ltd.).
The LPMO gene sequence (GI: XM_ 960505.2) was amplified using Neurospora crassa cDNA as a template by the following primer pair:
LPMO-F：5’-CGGAATTCCACACCATCTTCCAGAAGGTGTCC-3’，
LPMO-R：5’-TCACCGCGGTTAATGGTGATGGTGATGATGAGGGAGGCACTGGCTG-3’；
the VP gene sequence (GI: AF 007221.1) was amplified by the following primer pair using the Pleurotus Citrinopileatus Sing cDNA as a template:
VP-F：5’-CGGAATTCGCAACTTGCGACGACGGACGCACC-3’，
VP-R：5’-TCACCGCGGTTAATGGTGATGGTGATGATGCGATCCAGGGACGGG-3’；
the LAC gene sequence (GI: KU 055621.1) was amplified using the. Bristle. Key. CDNA as a template by the following primer pair:
Lac-F：5’-CGGAATTCGCCATCGGGCCAGTCGCAGACCTC-3’，
Lac-R：5’-TCACCGCGGTTAATGGTGATGGTGATGATGCTGGTCGTCAGGCGAG-3’。
PCR conditions: 98 ℃ for 30s;98 ℃ for 10s;58 ℃ for 35s;72 ℃,30s;72 ℃ for 10min;30 cycles. After agarose gel electrophoresis, the target bands were LPMO (1037 bp), VP (993 bp) and LAC (1488 bp), and the results were shown as A, B and C, and the target gene bands were recovered and purified.
Gene cloning of the docking module: the sequence of the Doc-CipA gene (CipA is GI: 125972525) was amplified using the Clostridium thermocellum cDNA as a template with 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: GI: 13277318)) was amplified using the ruminococcus flavus cDNA as a template by the following primer pair:
Doc-ScaB-F：5’-CCCGTCCCTGGATCGACAAAGCTCGTTCCTAC-3’，
Doc-ScaB-R：5’-TCACCGCGGTTAATGGTGATGGTGATGATGCTGAGGAAGTGTGATG-3’。
the sequence of the docking module Doc-CipC gene (CipC is GI: 11056042) was amplified using clostridium cellulolyticum cDNA as a template by the following primer pair:
Doc-CipC-F：5’-CTCGCCTGACGACCAGTACCTTGATGAAAA G-3’，
Doc-CipC-R：5’-TCACCGCGGTTAATGGTGATGGTGATGATGT AACAAGAATGATTTG-3’。
PCR conditions: 98 ℃ for 30s;98 ℃ for 10s;58 ℃ for 35s;72 ℃,10s;72 ℃ for 10min;30 cycles. The result is shown as D in fig. 1.
LPMO and Doc-CipA fragments are used as templates, and the LPMO chimeric enzyme gene fragments LPMO-CipA are obtained by using an Over-lap PCR method and primers LPMO-F and Doc-CipA-R.
VP and Doc-ScaB are used 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,
LAC-CipC gene fragments are obtained by using LAC and Doc-CipC fragments as templates and using an Over-lap PCR method and primers LAC-F and Doc-CipC-R.
PCR conditions: 98 ℃ for 30s;98 ℃ for 10s;58 ℃ for 35s;72 ℃,60s;72 ℃ for 10min;30 cycles. The results are shown as E in FIG. 1.
2. Promoter PGK, signal peptides αMF and AGA2, and terminator MATT sequence amplification
The Saccharomyces cerevisiae S288c genomic DNA was used as a template to amplify the glycerol triphosphate kinase (Phosphaglycerate kinase, PGK) promoter PGK sequence by the following primer pair:
PGK-F：5’-GAGGAAGCTGAAACGCAATATTTTAGATTCCTGA CTTC-3’，
PGK-R：5’-GTAAAAATTGAAGGAAATCTCATCGTTTTGTTTT ATATTTGTTG-3’。
AGA2 and MATT sequences were amplified using the sequence of the commercial vector pYD1 as template by the following primer pair:
AGA2-F：5’-CAACAAATATAAAACAGTAATAAAAGTATCAAC-3’，
AGA2-R：5’-CCGCGGGGATCCACTAGTGTCGACCTCGAGGA TATCGAATTCAGAACCACCACCACCAG-3’；
MATT-F：5’-GAATTCGATATCCTCGAGGTCGACACTAGTGGA TCCCCGCGGGTTTAAACCCGCTGATC-3’，
MATT-R：5’-ATTATTATCATCATTTTTTATTACTGAGTAGTATT TATTTAAG-3’；
the αmf sequence was amplified using the commercial vector pPIC9K as a template by the following primer pair:
αMF-F：5’-CAACAAATATAAAACAAAACGATGAGATTTCCTT CAATTTTTAC-3’，
αMF-R：5’-CCGCGGGGATCCACTAGTGTCGACCTCGAGGAT ATCGAATTCAGCTTCAGCCTCTCTTT-3’。
amplification procedure: 98℃30s,98℃10s,56℃25s,72℃30s,72℃10min,30 cycles.
The target gene bands obtained by the PCR amplification method are as follows: PGK (778 bp), AGA2 (296 bp), alpha MF (267 bp) and MATT (367 bp) to recover and purify the target gene. PGK, AGA2, alphaMF, MATT sequences are ligated by Over-lap RCR to form PGK-alphaMF-MCS-MATT and PGK-AGA2-MCS-MATT fragments.
Amplification procedure: 98℃30s,98℃10s,56℃60s,72℃55s,72℃10min,30 cycles.
The band length of the target gene fragment is 1784bp and 1755bp respectively, the fragment is recovered and purified, and the implementation result is shown in figure 2. After double enzyme digestion, the vector is connected with pRS423 commercial vector, and after sequencing verification, high copy expression vectors pRS 423-PGK-alpha MF and pRS423-PGK-Aga2 are constructed.
LAC-CipC, VP-ScaB and LPMO-CipA are linked with pRS 423-PGK-alpha MF vector, scafI is linked with pRS423-PGK-Aga2, then E.coli DH5 alpha competent cells are transformed by adopting a chemical transformation method, single colonies are selected and cultured, and high copy expression vectors pRS423-LPMO, pRS423-VP, pRS423-Lac and pRS423-ScafI plasmid are obtained through bacterial liquid PCR screening and sequencing verification, wherein 3 'ends of LPMO and LAC contain 6 XHis tag sequences and 3' ends of ScafI contain Xpress tag sequences.
3. Saccharomyces cerevisiae transformation and positive clone screening
pRS423-LPMO, pRS423-VP, pRS423-Lac and pRS423-ScafI plasmids were transferred into Saccharomyces cerevisiae W303 cells and pRS423-ScafI plasmid was transferred into Saccharomyces cerevisiae EBY100 cells by a lithium acetate transfer method. Then extracting yeast genome by alkali thermal cracking method, carrying out genome PCR verification, and screening the result to obtain positive monoclonal W303/LPMO, W303/VP, W303/Lac and EBY100/ScafI, wherein the result is shown in figure 3.
4. Enzyme Western blot analysis
And culturing the positive recombinant saccharomyces cerevisiae strain obtained by screening for 48 hours, measuring the extracted protein by an ultra-micro spectrophotometer, adjusting the protein concentration to be consistent, and analyzing by using a western immunoblotting method.
SDS-PAGE proteins were electrophoresed as follows: the recombinant Saccharomyces cerevisiae cells were first cultured at 150rpm at 30℃for 48 hours in 5mL and centrifuged at 3000rpm at 4℃for 5min. The supernatant was aspirated, transferred to a ultrafilter tube and centrifuged at 4000rpm for 10min at 4 ℃.
The ultrafiltered protein concentrate was aspirated and placed in a 2mL centrifuge tube for further use. mu.L of the protein concentrate was taken and added to 5. Mu.L of loading buffer (5X) and mixed well.
The sample was applied to a 10% SDS-PAGE separating gel, the voltage was 80V, and the gel was electrophoresed for about 20min, and then the gel was electrophoresed at 120V for 60min.
Protein transfer was then performed: and (3) carrying out ice water bath, setting the current to 250mA, and transferring the film for 1.5h.
Nitrocellulose membrane blocking: the mixture was blocked with a 5% nonfat dry milk solution at room temperature for 1h.
Incubation resistance: the enzyme protein contains His tag and is incubated for 1.5h by adopting 1:10000ProteinFind Anti-His Mouse Monoclonal Antibody;
secondary antibody incubation: the incubation was performed for 1H using a ProteinFind Goat Anti-Mouse IgG (H+L), HRP Conjugate, of 1:2000.
The cells were washed 3 times with TBST buffer for 5min each. Finally, mixing the solution A and the solution B in EasySee Western Blot Kit (Beijing full gold biotechnology Co., ltd.) according to the ratio of 50:1, and adding 1%o of solution C for uniform mixing. Then uniformly spread on a nitrocellulose membrane, and developed by exposure to light in GE ImageQuant LAS4000 mini.
The color band is analyzed for the protein of interest based on a protein molecular weight standard. As shown in FIG. 4 (LPMO), FIG. 5 (VP) and FIG. 6 (Lac), western blot analysis demonstrated that the developed protein bands were molecular weights consistent with the target protein molecular weights LPMO (39.35 kDa), VP (44.5 kDa) and Lac (61.95 kDa), which demonstrated that the recombinant strains W303/LPMO, W303/VP and W303/Lac, respectively, could correctly express the enzyme proteins.
5. Enzyme activity assay
LPMO enzyme activity assay: the LPMO activity was determined using locust bean gum as substrate and 3, 5-dinitrosalicylic acid colorimetric method.
The reaction system comprises: enzyme solution 15. Mu.L, 0.5% (w/v) locust bean gum 60. Mu.L.
The reaction flow is 60 ℃ for 12 hours. Then 75. Mu.L of DNS reagent was added and incubated at 100℃immediately for 10min. Naturally cooling to room temperature, taking 130 mu L, and measuring OD by using an enzyme-labeled instrument 540nm Is used for the light absorption value of (a). The enzyme protein deactivated by heating at 100 ℃ is used as a blank control, the enzyme amount required for degrading locust bean gum per 1mg/L reducing sugar is defined as one enzyme activity unit (U), and the concentration of the enzyme protein is measured by using an ultra-micro spectrophotometer.
VP enzyme activity assay: the guaiacol is used as a substrate, and the enzymatic reaction system is as follows: HAc-NaAc Buffer (pH 4.5) 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 spectrophotometryMeasurement of OD at room temperature 465nm The VP enzyme activity was calculated according to the following formula at the change of absorbance within 5min.
Wherein the extinction coefficient epsilon 465nm ＝12100L/(mol·cm)，V Total (S) : total volume of reaction solution (mL), V Enzymes : crude enzyme solution volume (mL), delta A : difference in absorbance, delta t : enzymatic reaction time
VP enzyme activity definition: the amount of enzyme required to catalyze a reaction of 1. Mu. Mol of substrate per minute is one enzyme activity unit (U).
And Lac enzyme activity determination, wherein ABTS is used as a substrate, and the reaction system is as follows: HAc-NaAc Buffer (pH 4.5) 125. Mu.L, 0.6mM ABT 125. Mu.L, and enzyme solution 50. Mu.L. Determination of OD Using an enzyme-labeled Instrument 420nm Change in absorbance within 5min.
Wherein the extinction coefficient epsilon 420nm ＝36000L/(mol·cm)，V Total (S) : total volume of reaction solution (mL), V Enzymes : crude enzyme solution volume (mL), delta A : difference in absorbance, delta t : enzymatic reaction time
Lac enzyme activity definition: the amount of enzyme required to catalyze a reaction of 1. Mu. Mol of substrate per minute is one enzyme activity unit (U).
TABLE 1 measurement of enzyme Activity of secretion expressed LPMO, VP and Lac
Note that: * Expressed as the average of three measured values
The results of the enzyme activity measurement are shown in Table 1, the enzyme activities of the secretion expression LPMO, VP and Lac 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 results demonstrate that the recombinant strain of Saccharomyces cerevisiae exocytosis Lac, VP and LPMO enzyme proteins have enzymatic activity.
EXAMPLE 2 Saccharomyces cerevisiae EBY100 cell surface self-assembled chimeric fiber corpuscles with Lac, VP, and LPMO catalytic modules
1. Taking recombinant Saccharomyces cerevisiae cells cultured for 20-48 hours, and centrifuging for 10min at 4 ℃ and 3000 rpm. Wherein the EBY100/ScafI strain was centrifuged to discard the supernatant, the cell pellet was retained, and the supernatant was washed with a resuspension buffer (50 mM Tris-HCl,100mM NaCl,10mM CaCl 2 ) Resuspending the EBY100/ScafI cells; the culture supernatant after centrifugation was kept as a crude enzyme solution for each of the enzyme protein secretion expression strains W303/Lac, W303/VP and W303/LPMO.
2. 1mL of W303/ScafI was added to 1mL of crude enzyme solution, the mixture was incubated at 4℃for 12 hours to complete the assembly of chimeric fiber bodies, and finally, the supernatant was discarded by centrifugation at 3000rpm at 4℃for 5 minutes to collect cell pellets.
3. With 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) the chimeric fibroblast pellet was resuspended and centrifuged at 3000rpm for 10min at 4 ℃. The supernatant was then discarded and 250. Mu.L of 1% BSA (in PBS) was added to the pellet for resuspension.
4. Adding a primary antibody (Mouse anti-Xpress tag; rabbit anti-6 XHis tag) according to a ratio (1:1000 v/v), uniformly mixing, incubating for 1h at room temperature, and reversing and uniformly mixing every 15min to make cells in a suspension state. The cells were centrifuged at 3000rpm for 10min at 4℃and the supernatant was discarded, and the cell pellet was washed twice with 1mL of PBS.
5. The resuspended cells were pelleted in 250. Mu.L of 1% calf serum solution (Albumin from bovine serum, BSA) and 1. Mu.L of secondary antibody (Goat anti-Mouse IgG (H+L), alexa was then added in a ratio (1:250 v/v)488；Goat anti-Rabbit IgG(H+L),Alexa/>647 Uniformly mixing, placing in a dark place for 1.5h, and continuously reversing and uniformly mixing to ensure that the mixture always presents a suspension state.
6. The cell pellet was washed twice with PBS and resuspended in 200. Mu.L of PBS after centrifugation at 5000rpm for 5min at 4 ℃. mu.L of the cell suspension was taken, dropped onto a slide glass, and observed in a ZEISS LSM LIVE 780 of a confocal laser microscope imaging system. Wherein Alexa488 exhibits green fluorescence under excitation of laser light having wavelength 488nm, alexa +.>And emits far infrared fluorescence under the excitation of laser with wavelength of 633 nm. Photographs were taken and analyzed with Carl Zeiss Zen 2011 software.
The results are shown in FIG. 7, where the EBY100/ScafI on the left of row 1 exhibited green fluorescence under 488nm laser excitation, while no fluorescence reaction occurred under 633nm laser excitation, demonstrating that the ScafI protein was able to anchor to the EBY100 cell surface via the a lectin system. When Lac, VP and LPMO proteins are assembled with EBY100/ScafI respectively, green fluorescence appears under 488nm laser excitation, and red fluorescence reaction appears under 633nm laser excitation, as shown in the results of lines 2 to 4 in FIG. 7, which proves that chimeric enzyme proteins Lac, VP and LPMO are combined and assembled with ScafI through a docking module, and the invention innovatively constructs chimeric fiber minibodies with lignin synergistic degradation enzyme system functions.
EXAMPLE 3 stress resistance analysis of recombinant Saccharomyces cerevisiae strains
Configuration of the culture medium: YPD solid media containing different concentrations of inhibitors (e.g., phenol, guaiacol, vanillin, and syringaldehyde) were placed on 24-well plates, respectively.
The blank Saccharomyces cerevisiae strain EBY100 was used as a negative control, and recombinant Saccharomyces cerevisiae strains EBY100/Lac, EBY100/VP, EBY100/LPMO and EBY100/Lac-VP-LPMO were used as experimental groups, and low temperature induction culture was performed in YPG medium at 20deg.C and 150rpm according to a total inoculum size of 2%. The control strain and the experimental strain after the induction were uniformly spread on YPD solid medium containing the inhibitor in a 24-well plate, respectively, and were subjected to stationary culture at 30℃for 72 hours.
As a result, as shown in FIG. 8, the maximum tolerance concentrations of chimeric fiber bodies (EBY 100/Lac-VP-LPMO) assembled with Lac, VP and LPMO on the cell surface of Saccharomyces cerevisiae against toxic and harmful compounds generated by lignin degradation were respectively: 12mmol/L phenol, 1g/L guaiacol, 0.75g/L vanillin, 2.4g/L syringaldehyde. In the prior art, the concentrations of vanillin and phenol in the willow hydrolysate are respectively 0.43g/L and 4mmol/L, and the concentration of the vanillin in the spruce hydrolysate is 0.107g/L.
The maximum tolerance concentration of the recombinant saccharomyces cerevisiae strain for displaying chimeric enzyme provided by the invention to vanillin, phenol and syringaldehyde is higher than the concentration of the inhibitor generated after pretreatment in an actual reaction system, and the recombinant saccharomyces cerevisiae strain has certain universality, so that the recombinant saccharomyces cerevisiae strain has application value in developing and utilizing lignocellulose raw materials to produce cellulosic ethanol.
Example 4
Specific lignin degrading properties of the chimeric enzyme system provided in example 2 above provided in this example:
1. performance analysis of alkaline lignin substrate degradation
YPD liquid medium containing 0.5g/L alkali lignin is prepared, the pH is regulated to about 4.5, and the liquid medium is packaged into 250mL conical flasks with 100mL of liquid medium per flask. Recombinant Saccharomyces cerevisiae strains were added in combination according to Table 2 at an inoculum size of 0.5g/L enzyme protein, three replicates per group.
TABLE 2 combination of recombinant Saccharomyces cerevisiae to degrade alkali lignin
Note that: 1 copper sulphate was added at a final concentration of 0.5 mM; 2 manganese sulfate was added at a final concentration of 0.5 mM; 3 150. Mu.M hydrogen peroxide was added and replenished every 24 hours; 4 0.5mM ascorbic acid was added and added every 24 hours.
And (3) measuring the degradation rate of alkali lignin: and (3) centrifuging an 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 the degradation curve of each combined alkali lignin by taking time as an abscissa and the light absorption value as an ordinate. According to the absorbance value and the alkali lignin standard curve, the alkali lignin content is obtained, and the alkali lignin degradation rate is calculated according to the following formula:
wherein: c (C) 0 : alkali lignin content before degradation; c (C) t : alkali lignin content after degradation.
As a result, the percentage of alkali lignin removed by the chimeric fiber small structures EBY100/Lac, EBY100/VP, EBY100/LPMO and EBY100/Lac-VP-LPMO was 47.96%,44.63%,7.82% and 67.08%, respectively, as shown in FIG. 9. The results illustrate that: based on a saccharomyces cerevisiae cell surface display system and the chimeric enzyme proteins Lac, VP and LPMO assembled on the scaffold protein ScafI, the synergistic effect of three enzymes in lignin substrate degradation reaction can be enhanced, and lignin degradation efficiency is improved.
2. Cellulose ethanol fermentation performance research of recombinant saccharomyces cerevisiae
(1) Synergistic fermentation of lignocellulose
Addition of commercial cellulases to lignocellulosic fermentation media10FPU/g (purchased from NoveXin Co.) was mixed with four strains of recombinant Saccharomyces cerevisiae, inoculated with 1.2g/L enzyme protein as the experimental group, and the same inoculated amount of Saccharomyces cerevisiae EBY100 host cells as the control group, and the experiment was performed in triplicate. Culturing at 150rpm at 30 ℃ for 120 hours, and sampling 2mL every 12 hours.
(2) Glucose and ethanol content determination
After centrifugation of the broth sample at 10000rpm for 5min, it was filtered with a 0.22 μm filter and the ethanol content was measured using a high performance liquid chromatograph (HPLC, mode 1260,Agilent Technologies). The detection conditions are as follows: agilent Zorbax Eclipse XDB-C18 column (250 mm. Times.4.6 mm,5 μm), column temperature 40 ℃, mobile phase methanol: water=5:95, flow rate 0.6mL/min, sample injection amount 5. Mu.L, differential detector temperature 40 ℃, run for 10min.
As shown in FIG. 10, after EBY100/Lac-VP-LPMO fermentation for 96 hours, the maximum ethanol concentration reached 4.49g/L, and the maximum ethanol concentration produced by EBY100 cell fermentation in the control group was 3.40g/L, and the chimeric fiber structure increased the ethanol concentration by 32%.
The result shows that on one hand, the recombinant yeast strain EBY100/Lac-VP-LPMO anchoring three chimeric enzymes can degrade lignin through the synergistic effect of the chimeric enzymes, so that the irreversible adsorption effect of lignin in a reaction system on cellulase is reduced, the accessibility of the cellulase and a substrate is improved, the enzymolysis saccharification level of cellulose is improved, and the enzyme consumption is reduced; on the other hand, EBY100/Lac-VP-LPMO has higher tolerance of fermentation inhibitor and ethanol production performance of simultaneous saccharification and fermentation.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
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Claims (10)

1. A chimeric fiber multimer, wherein the chimeric fiber multimer: based on a Saccharomyces cerevisiae cell surface display system and assembled laccase, peroxidase and soluble polysaccharide monooxygenase on a scaffold protein ScafI.
2. The chimeric fiber multimer according to claim 1, wherein the scaffold protein comprises at least three adhesion modules.
3. The chimeric fiber multimer according to claim 1 or 2, wherein the assembling is assembling the laccase, peroxidase and soluble polysaccharide monooxygenase onto the scaffold protein via an adhesion module and a docking module; the adhesion module is derived from one or more of clostridium thermocellum, clostridium defibricum, or ruminococcus flavum.
4. The chimeric fiber multimer according to 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 fiber multimer according to claim 1 or 2 or 4, wherein the mass ratio of laccase, peroxidase and soluble polysaccharide monooxygenase is 1: (0.5-2): (0.5-2).
6. A method for producing the chimeric fiber multimer according to any one of claims 1 to 5, comprising:
expressing a scaffold protein by using a Saccharomyces cerevisiae a lectin display system, anchoring the scaffold protein by using a Saccharomyces cerevisiae cell surface anchoring protein, and assembling laccase, peroxidase and soluble polysaccharide monooxygenase on the scaffold protein by using an adhesion module and a docking module.
7. The method of manufacturing according to claim 6, comprising:
expressing a scaffold protein ScafI by using a Saccharomyces cerevisiae a lectin display system, anchoring the scaffold protein ScafI by combining an N-terminal Aga2 signal peptide of the scaffold protein ScafI with an anchoring protein AGA1 on the surface of Saccharomyces cerevisiae cells, and assembling laccase, peroxidase and soluble polysaccharide monooxygenase on the scaffold protein ScafI by using an adhesion module and a docking module.
8. The method of manufacturing according to claim 7, comprising:
the assembly is carried out in an environment of 0-4 ℃ for 10-14 h.
9. Use of the chimeric fiber multimer of any one of claims 1-5 for increasing stress resistance in Saccharomyces cerevisiae.
10. Use of the chimeric fiber multimer of any one of claims 1-5 to improve ethanol fermentation performance of cellulose.
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