Heat-resistant, saline-alkaline-tolerant and ionic liquid-tolerant cellulase and application thereof

Abstract

The invention discloses heat-resistant, saline-alkaline-resistant and ionic liquid-tolerant cellulase and application thereof. Belongs to the technical field of cellulase. According to the invention, a new cellulase gene c5-cel4 is obtained from a metagenome of Xinjiang Abian salt lake sediment, is cloned and is subjected to heterologous expression in Escherichia coli. The C5-CEL4 has the characteristics of heat resistance, alkali resistance, salt resistance and high tolerance of ionic liquid. Besides, the enzyme can be secreted and expressed in escherichia coli, the protein C end is cut off from amino acid residues 315-448, secretion of the enzyme is related to sequences of the amino acid residues 315-448 at the protein C end, and the enzyme can tolerate most of metal ions, inhibitors and alcohols at the same time. It is indicated that C5-CEL4 is an ideal candidate enzyme for biomass conversion, washing and textile industry production, and has huge potential application prospects in feed, food and biological energy.

Description

Technical Field

[0001] The invention relates to the technical field of cellulase, and more particularly to a cellulase which is heat-resistant, salt-resistant, alkali-resistant and ionic liquid-resistant and an application thereof. Background Art

[0002] Cellulose is a renewable organic macromolecular polysaccharide, which is the most abundant in renewable plant biomass. The effective degradation of cellulose by cellulase is a key step in the conversion of lignocellulose. The complete hydrolysis of cellulose into glucose requires the combined action of endoglucanase (endo-1,4-D-glueanase, EC3.2.1.4), exoglucanase (exo-1,4-D-glucanase, EC3.2.1.91) and β-glucosidase (1,4-D-glueosidase, EC3.2.1.21). Among them, endoglucanase acts on the non-crystalline region inside the cellulose molecule, randomly attacks, and makes the polymer more susceptible to hydrolysis by other cellulose hydrolases. It is the main enzyme group for internal bond hydrolysis.

[0003] Currently, cellulase is widely used in various industries and products such as pulp and paper, biofuel, food, brewing, textiles, animal feed, bio-nanomaterials, phenolic formaldehyde, etc. However, the enzyme is easily inactivated in high temperature and high salt and alkali industrial environment. Therefore, the thermal stability and salt and alkali resistance of cellulase are particularly important.

[0004] Although many thermostable cellulases are salt-tolerant and multipolar, few can effectively catalyze conversion reactions in high-salt environments. Therefore, screening salt-alkali tolerant cellulases is beneficial to reducing costs and improving industrial efficiency.

[0005] In summary, how to provide a salt-alkali resistant cellulase is a problem that those skilled in the art need to solve urgently. Summary of the invention

[0006] In view of this, the present invention provides a cellulase that is heat-resistant, salt-resistant, alkali-resistant, and ionic liquid-resistant and its application.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A cellulase that is resistant to heat, salt, alkali and ionic liquids, characterized in that its amino acid sequence is shown in SEQ ID NO.6.

[0009] A gene encoding the above-mentioned cellulase.

[0010] Furthermore, the nucleotide sequence is shown in SEQ ID NO.5.

[0011] A recombinant vector comprises the above-mentioned coding gene.

[0012] A recombinant bacterium comprises the above-mentioned recombinant vector.

[0013] The above-mentioned cellulase is used in the degradation of CMC-Na, bagasse xylan, beech xylan and corncob xylan.

[0014] The above-mentioned cellulase is used in food, feed, textile and industrial production.

[0015] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects achieved by the present invention are:

[0016] The present invention isolated a new cellulase gene from the metagenomic data of the sediment of Xinjiang Aibi Salt Lake, named c5-cel4, cloned the gene sequence, constructed a recombinant vector, introduced it into Escherichia coli, and determined the activity of the recombinant cellulase by heterologous expression and protein purification. The results showed that the enzyme is a heat-resistant, alkali-resistant, and salt-loving ionic liquid-tolerant cellulase, and can be secreted outside the Escherichia coli, which indicates that the enzyme can provide an enzyme source reserve for the use of cellulase in the fields of papermaking, textiles, food, feed, and biofuels. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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 or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0018] Figure 1 The extracellular fermentation broth and activity monitoring of the recombinant cellulase C5-CEL4 in Example 1 of the present invention, wherein A represents the extracellular fermentation situation; B represents the activity result;

[0019] Figure 2 The SDS-PAGE gel image of the recombinant cellulase C5-CEL4 in Example 1 of the present invention, wherein A represents the gel image of the extracellular purified enzyme; B represents the gel image of the intracellular purified enzyme; Lane 1 represents the protein molecular weight marker; Lane 2 represents the bacteria of Escherichia coli DH5α / pSHY211-c5-cel4; Lane 3 represents the purified enzyme C5-CEL4; Lane 4 represents the zymogram analysis of the purified enzyme;

[0020] Figure 3 This is the protein C-terminal sequencing analysis based on LC-MS / MS of the recombinant cellulase C5-CEL4 in Example 1 of the present invention, wherein A represents the total ion current chromatogram, Chymotrypsin TIC map; B represents the total ion current chromatogram, Pepsin-TIC map; C represents the C-terminal secondary mass spectrum;

[0021] Figure 4 The phylogenetic tree diagram obtained by the maximum likelihood analysis based on the amino acid sequence in Example 1 of the present invention shows the phylogenetic position of C5-CEL4 and related cellulases, and the bootstrap values ​​(expressed as a percentage of 1000 replications) are given at the nodes;

[0022] Figure 5 is a multiple alignment diagram of the amino acid sequence of C5-CEL4 in Example 1 of the present invention, 8BQA, 8BQC, 8C10, 4MIR, and 1EGZ are PBD IDS of the amino acid sequence, and residues with a homology level of 100% are highlighted in red, wherein 8BQA and 8BQC are from Cellvibriojaponicus, 8C10 is from Teredinibacter waterburyi, 4MIR is from soil metagenome, and 1EGZ is from Dickeya chrysanthemi;

[0023] Figure 6 The protein structure analysis of C5-CEL4 in Example 1 of the present invention, wherein A is the domain prediction of the protein sequence of C5-CEL4 using InterPro, and the structure describes a cellulase catalytic domain (CD); B is the GH5 family cellulase C5-CEL4, and homology modeling was performed on the SWISS-MODEL server using the GH5 family endoglucanase from Microbulbifer rhizosphaerae as a template (UniProt accession number: A0A7W4WCK3);

[0024] Figure 7 The effect of temperature and pH on the activity and stability of the recombinant cellulase C5-CEL4 in Example 1 of the present invention, wherein A represents the effect of temperature on the activity of C5-CEL4; B represents the effect of pH on the activity of C5-CEL4; C represents the effect of temperature on the stability of the intracellular enzyme C5-CEL4; D represents the effect of pH on the stability of the intracellular enzyme C5-CEL4; the initial activity is 100%, each value in the figure represents the mean ± SEM (n = 3), extracellular enzyme 100% = 24.36 + 0.63 U / mg, intracellular enzyme 100% = 19.6 ± 3.47 U / mg;

[0025] Figure 8The effects of NaCl and ionic liquid on the intracellular enzyme C5-CEL4 in Example 1 of the present invention, wherein A represents the effect of NaCl on the activity of C5-CEL4; B represents the effect of NaCl concentration on the stability of C5-CEL4 after incubation for 9 months; C represents the effect of ionic liquid on the activity of the intracellular enzyme C5-CEL4; D represents the effect of ionic liquid on the stability of the intracellular enzyme C5-CEL4 after incubation at 4°C for 24h; the initial activity is 100%, and each value in the figure represents the mean ± SEM, 100% = 19.6 ± 3.47 U / mg;

[0026] Fig. 9 is the predicted surface electrostatic potential of C5-CEL4 in Example 1 of the present invention, wherein A represents the surface electrostatic potential of C5-CEL4; B represents the surface electrostatic potential obtained by flipping Figure A by 180° in Pymol 2.6; negative electrostatic potential and positive electrostatic potential are represented by red and blue, respectively;

[0027] Fig.10 This is a thin layer chromatography analysis of the hydrolysis of CMC-Na by C5-CEL4 in Example 1 of the present invention, wherein channel 1, standard substances: glucose (G1), cellobiose (G2), cellotriose (G3), cellotetraose (G4); channel 2, CMC-Na without enzyme solution; channel 3, C5-CEL4 purified by hydrolysis of CMC-Na. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The drugs required for the present invention are conventional experimental drugs, which are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0030] Example 1

[0031] 1 Materials and methods

[0032] 1.1 Sample collection and metagenomic sequencing

[0033] The sampling point is located at Lake Aibi in Xinjiang (45°09′35″N, 83°53′21″E). The surface temperature of Lake Aibi is around 7.8℃ and the pH value is 8.49. The sediment samples were quickly frozen on dry ice for DNA isolation and metagenomic sequencing. DNA was isolated using the powersoil Kit (MOBIO dnasy PowerSoil Kit, USA). Metagenomic sequencing was performed by Suzhou GENWIZ using the HiSeq 2500 instrument, and sequence analysis was performed using the IMG server (https: / / img.jgi.doe.gov / cgi-bin / mer / main.cgi).

[0034] 1.2 Cellulase sequence prediction and sequence analysis

[0035] Based on functional prediction, a cellulase gene sequence was predicted from the metagenomic database and named c5-cel4. The amino acid sequence was translated using the Expasy-Translate tool. The protein sequence of c5-cel4 was aligned using the BLASTp program (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Amino acid multiple sequence alignment and homology modeling were performed using RCSB PDB, SWISS-MODEL, AlphaFold Protein Structure Database (ebi.ac.uk), MEGA 7, and ESPript3.0. Signal peptides were predicted using SignalP (http: / / www.cbs.dtu.dk / services / SignalP / ) and protein domains were predicted using Search-InterPro (ebi.ac.uk). Phylogenetic analysis was performed using MEGA 7, and a phylogenetic tree was constructed using the maximum likelihood method and Poisson correction model.

[0036] 1.3 Cloning, expression, purification and identification of C5-CEL4

[0037] Primer 5.0 was used to design degenerate primers: c5-cel4-F (CATCATCATCATCATCATGAAATGATATTCGGCGCAGGTGCCCAG, SEQ ID NO.1) and c5-cel4-R (GTGCTCGAGTGCGGCCGCAAGTCAGTTGGCGCTGCATTCCGGC, SEQ ID NO.2) to amplify the full-length c5-cel4 gene. The underlined sequence represents the homologous recombination fragment of the pSHY211 vector (Characterization of a GH10 extremelythermophilic xylanase from the metagenome of hot spring for prebiotic production.) after digestion with BamH I and Hind III. PCR was performed by TransStarFastPfu Fly DNA polymerase (TransGenBiotech, China). The PCR amplification program was: 95°C pre-denaturation for 3 min, 98°C for 20 s, 55°C for 30 s, 72°C for 2 min, 29 cycles, and incubation at 72°C for 10 min for final extension. The PCR product was inserted into the pSHY211 vector using the pEASY-Uni Seamless Cloning and Assembly Kit (TransGenBiot ech, China) to obtain the expression plasmid pSHY211-c5-cel4. Escherichia coli DH5α was used for cloning the c5-cel4 gene. Escherichia coli was grown in LB medium containing 100 μg / mL kanamycin, and the DNA isolation and purification kit was purchased from Sangon (China).

[0038] Recombinant E. coli DH5α was expressed and purified using pSHY211-c5-cel4. In 15 mL LB liquid medium containing 100 μg / mL kanamycin, cultured overnight at 37°C, 180 r / min as seed solution. The seed solution was inoculated with 5% inoculum into a mixed liquid medium containing 200 mL basal salt and 100 mL LB, the mixed liquid medium contained 100 μg / mL kanamycin, and cultured at 37°C, 180 r / min, and shaken for 36 hours. Centrifuged at 4000 r / min for 30 minutes, and the supernatant was collected as the extracellular crude enzyme solution. The E. coli biomass was collected, and the E. coli biomass was resuspended with 25-30 mL PBS buffer (pH 7.6) containing 10 mM imidazole, placed in an ice water bath for ultrasonic disruption, and the lysate was centrifuged at 4°C, 12,000 r / min for 20 minutes, and the supernatant was taken as the intracellular crude enzyme solution. The filtered intracellular and extracellular crude enzyme solutions were added to the balanced Ni-NTA nickel column (Histrap, TransGenBiotech, China), the nickel column filler was resuspended, and placed in a 500 mL conical flask, and shaken at 150 r / min for 1.5 h at ≤15 ° C to bind and increase the amount of bound protein. Intracellular and extracellular proteins were purified according to the method previously reported by Yin et al. (2017) (The Hybrid Strategy of Thermoactinospora rubra YIM 77501T for Utilizing Cellulose as a Carbon Source at Different Temperatures) for characterization of recombinant cellulase. The protein concentration was determined using a Bradford protein assay kit (Order NO. C5-CEL403031, Sangon Biotech, China), with bovine serum albumin as the standard. The purified C5-CEL4 enzyme was electrophoresed using 12% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and the protein bands were stained with Coomassie Brilliant Blue R-250. The cellulase activity within the gel was assessed using zymography. Briefly, the samples were resolved on a 12% SDS-acrylamide gel containing 0.5% (w / v) CMC-Na. After electrophoresis, the gels were washed with 2.5% (v / v) Triton X-100 (pH 7.0) for 30 min, soaked in 200 mM phosphate buffer for 15 min, and incubated in phosphate buffer (pH 7.0) at 45 °C for 1 hour. Finally, the gels were stained with 0.2% (w / v) Congo red at 25 °C for 10 min, and Congo red was removed using 1 mol / L sodium chloride until a clear active band was visible against the background of the gel.

[0039] Cut the protein target band into 1mm pieces 3The micelles were placed in a 1.5mL EP tube. Decolorize with 50% ACN (acetonitrile)-50% 50mM NH4HCO3 (ammonium bicarbonate) solution, place for 10-30 minutes, aspirate and discard, repeat this operation until the micelles are colorless. Add 1000μL of 100% ACN, place for 30 minutes, wait until the micelles turn white and shrink to a ball, discard the ACN, and place at room temperature to dry. Reductive alkylation: Take 100μL of 10mM DTT (dithiothreitol) solution and add it to the sample, reduce it in a 56℃ water bath for 1h, aspirate and discard the supernatant; take 100μL of 20mM IAM (iodoacetamide) solution and add it to the sample, react at room temperature in the dark for 1h, aspirate and discard the supernatant. Take 500μL of decolorizing solution and add it to the sample, vortex and then aspirate and discard; take 1000μL of 100% ACN and add it to the sample. When the colloid particles shrink to a white mass, discard the ACN and place it to dry at room temperature. Add 50μL of trypsin with a concentration of 25ng / μL (diluted with 50mMNH4HCO3), seal and place in a 37℃ water bath for enzyme digestion for 16h. Peptide extraction: add 100μL / tube of extraction solution (5% trifluoroacetic acid TFA-50% ACN-45% water), after 1h in a 37℃ water bath, ultrasonicate for 5min, centrifuge for 5min, transfer the extraction solution to another new EP tube, repeat the extraction once, combine the extraction solutions, and vacuum centrifuge and dry. After enzyme digestion, the peptides are desalted using a desalting column and the solvent is evaporated in a 45℃ vacuum centrifugal concentrator.

[0040] Using Easy-nLC 1200 / Q Exactive TM Hybrid Quadrupole-Orbitrap TMMassSpectrometer high-resolution liquid-mass spectrometry system was used for data acquisition. Chromatographic separation: mobile phase A is an aqueous solution containing 0.1% formic acid, mobile phase B is 0.1% FA (formic acid) and 80% ACN; 0-2min, 4-8% B; 2-35min, 8-28% B; 35-55min, 28-40% B; 55-56min, 95%-95% B, 56-66min, 95%-95% B, flow rate 0.6μL / min. Chromatographic column: 150μm id×170mm, packing: Reprosil-Pur 120C18-AQ 1.9μm. Mass spectrometry acquisition: Full MS: Resolution (70000), AGC target (4e8), Maximum IT (20ms), Number of scan ranges (1), Scan range Spectrum (300-1800m / z), Datatype (Profile); dd-MS2: Resolution (17500), AGC target (1e5), Maximum IT (50ms); TopN: (20); NCE stepped NCE (30). Byonic software was used for analysis.

[0041] Primer5.0 was used to design degenerate primers: c5-cel4-gh5-F2 (CATCATCATCATCATCATGAAATGATATTCGGCGCAGGTGC, SEQ ID NO.3) and c5-cel4-gh5-R2 (GTGCTCGAGTGCGGCCGCAAGTCAGCTGGAGCTCGAAGAGGAA, SEQ ID NO.4) to amplify the c5-cel4-gh5 enzyme gene. The cloning, expression and purification methods of c5-cel4-gh5 were consistent with those of c5-cel4. C5-CEL4 and C5-CEL4-GH5 monoclonal bacteria were inoculated into a mixed liquid medium containing 6 mL of basal salt and 3 mL of LB, respectively, and the mixed liquid medium contained 100 μg / mL of kanamycin. C5-CEL4 and C5-CEL4-GH5 were inoculated into three test tubes, respectively, and three replicates were set up, and the shaking culture was carried out at 37°C, 180 r / min, and 36 h. Centrifuge at 4°C, 12,000 r / min for 20 min, and collect the supernatant as the extracellular crude enzyme solution. Collect the E. coli biomass, resuspend it in 5 mL PBS buffer (pH 7.6), place it in an ice-water bath for ultrasonic disruption, centrifuge the lysate at 4°C, 12,000 r / min for 20 min, and take the supernatant as the intracellular crude enzyme solution. Compare the differences in the intracellular and extracellular activities of C5-CEL4 and C5-CEL4-GH5.

[0042] 1.4 Enzyme activity assay

[0043] Using 1% (w / v) sodium carboxymethyl cellulose (CMC-Na) as the substrate, the 3,5-dinitrosalicylic acid (DNS) method was used to determine the amount of reducing sugar released, and the activity against CMC-Na was determined. Each unit of CMCase activity was defined as the amount of enzyme that releases 1 μmol of glucose equivalent reducing sugar per minute. Determination method: Take 10 μL of purified enzyme and add it to 90 μL of the optimal pH buffer containing 1% CMC-Na, react at the optimal temperature for 30 minutes, take it out and quickly add 150 μL of DNS to terminate the reaction, and heat shock at 90°C for 10 minutes to develop color. After cooling, take 150 μL of the reaction mixture and add it to a 96-well culture plate, and use an enzyme reader to measure its absorbance at 540 nm. Set up 3 parallels and 1 control for each group of experiments.

[0044] 1.5 Enzyme properties

[0045] The optimum pH of the purified enzyme C5-CEL4 was determined at pH 3.0-10.0 (citrate-sodium hydrogen phosphate buffer, pH 3.0-8.0; glycine-sodium hydroxide buffer, pH 8.0-10.0). The optimum temperature was determined by measuring the activity of C5-CEL4 at different temperatures (20-75°C) at the optimum pH. To evaluate the thermal stability and pH stability, the purified C5-CEL4 was incubated at different temperatures (40, 45, 50, 55 and 60°C) for different times (0, 20, 40, 60, 80, 100 and 120 min) and gradient pH (3.0-13.0) and the residual enzyme activity was determined after incubation for different times (4°C, 12 and 24 h).

[0046] 1.6 Effects of salts, ionic liquids, metal ions and chemical reagents on enzyme activity

[0047] The optimal salt concentration (0, 0.1, 0.3, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 M NaCl) was determined at 50°C and pH 7. After the purified enzyme solution was incubated (4°C) in a pH 7.0 salt solution (0-5 M) for 9 months, the residual enzyme activity was determined (determined without desalting). The surface electrostatic potential of C5-CEL4 was predicted using the AlphaFold Protein Structure Database and Pymol 2.6 software.

[0048] The relative enzyme activity was determined by adding different ionic liquids (i.e., 1-butyl-3-methylimidazolium tetrachloroborate BMIM-BF4, 1-butyl-3-methylimidazolium acetate BMIM-Ac, and 1-ethyl-3-methylimidazolium chloride EMIM-Cl) at different concentrations (w / v, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%) at 50°C and pH 7. After the purified enzyme solution was incubated (4°C) in the above three ionic liquid solutions (w / v, 1%, 5%, 10%, 20%, 30%, 40%, 45%, 50%) at pH 7.0 for 24 hours, the residual enzyme activity was determined.

[0049] Effects of metal ions, chemical reagents and ionic liquids on the activity of C5-CEL4. 1mM and 10mM metal ions (Na + ,K + Mg 2+ ,Fe 3+ ,Ca 2+ ,Zn 2+ ,Co 2+ ,Cu 2+ ,Ag + ,Mn 2+ ,Pb 2+ and Ni 2+ ), 0.1% and 1% of different chemical reagents (EDTA, PMSF, DTT, Tween80, SDS, CTAB, the corresponding full names are ethylenediaminetetraacetic acid, phenylmethylsulfonyl fluoride, dithiothreitol, polysorbate-80, sodium dodecyl sulfate, hexadecyltrimethylammonium bromide), 1% and 10% of different alcohols (Methanol, Ethanol, IPA, β-ME, the corresponding full names are methanol, ethanol, isopropanol, β-mercaptoethanol) were added to the reaction system of the enzyme solution, and the enzyme activity measured by the untreated enzyme solution was used as the control (100%) to determine the relative enzyme activity.

[0050] 1.7 Determination of substrate specificity

[0051] The control conditions were tested using the same procedure as above, and no additives were added to the reaction mixture. CMC-Na, sugarcane bagasse xylan, beechwood xylan, corncob xylan, microcrystalline cellulose, and cellobiose were used as substrates (1%, w / v) to investigate the substrate specificity of intracellular and extracellular C5-CEL4 enzymes. The kinetic constants of intracellular and extracellular C5-CEL4 enzymes were determined with different concentrations of CMC-Na and sugarcane bagasse xylan from 2 to 20 mg / mL, and incubated at 50 °C and pH 7 for 5 / 10 min. The maximum velocity Km (Michaelis-Menten constant) and Vmax (maximum velocity of the reaction) constants of the reaction were calculated using the Lineweaver-Burk plot.

[0052] 1.8 Thin layer chromatography analysis of hydrolysis products

[0053] The reaction mixture composed of 1% CMC-Na and 10 μg of purified enzyme was incubated at the optimal pH and temperature for 2 h. The hydrolysis products of CMC were characterized by thin layer chromatography (TLC) using a silica gel 60 plate (Merck, Darmstadt, Germany). The solvent was 1-butanol / acetic acid / water (2:1:1, v / v / v). Sugars were detected after spraying with freshly prepared 5% (v / v) H2SO4 ethanol and treating at 120 °C for 10 min. Sugar standards were glucose (G1), cellobiose (G2), cellotriose (G3), and cellotetraose (G4).

[0054] 1.9 Statistical analysis

[0055] Unless otherwise stated, all experiments were set up in triplicate, and all analyses were performed using the mean values. The results were statistically analyzed using SPSS 20.0 and expressed as mean ± SEM. Statistical analysis was performed using one-way analysis of variance, and Tukey's test was used for comparison of multiple experimental groups (0.01 < p value < 0.05).

[0056] 2 Results

[0057] 2.1 Cloning, heterologous expression, and purification of the C5-CEL4 gene

[0058] DNA samples were extracted from the sediment of Lake Aibi in Xinjiang for sequencing. A candidate cellulase gene sequence was obtained by similarity search of the cellulase sequence, named c5-cel4. The full-length nucleotide sequence of c5-cel4 was 1347 bp, encoding 448 amino acids. No obvious signal peptide sequence was found in c5-cel4, suggesting that the enzyme might be localized in the cytoplasm. The deduced signal peptide-free protein consisted of 448 amino acids, with a theoretical molecular size of 47.54 KDa and a theoretical pI of 4.51.

[0059] The nucleotide sequence of the cellulase gene is as follows:

[0060]

[0061] The amino acid sequence of the cellulase is shown below:

[0062] MIFGAGAQAVEPLTVNGNRILAGGEVRSLAGPSFFWSNTGWGAERFYNESAVRWVKNDWNATIVRASLGVDGEGGYLEDPAGNKSRVVELVEAAIANDLYVIIDWHSHHAEDH PAEAVAFFEEMARNYGHHDNVIYEIYNEPLQISWSNTIKPYAETVISAIRAIDPDNLIVVGTPTWSQDVDTASWDPIQGHANIAYTLHFYAGTHTQYLRDKARTALNNGIALF VTEWGTVNANGDGGVAQDETQRWMDFLEANHISHANWALNDKDEGASALVPGVSATGGWGQSELTESGRLVRDIVRGWDGGGSSSSSSSSSSSSSSSSSSSSSSS SSSSSGGSGALSCDPGNADVWNSGFVLSNVSVHNDGGADVDGWQVSLQFPQPIQLTNGWNGDFSLSGDGKTLTVANIGWNGYLQPGQSTSFGLQGSYSGNFLMPECSAN, SEQ ID NO.6.

[0063] The cellulase gene was successfully cloned into pSHY211 C-His (pSHY211 vector with 6 His tags) as a His-tag fusion protein and further confirmed by sequencing. The recombinant cellulase C5-CEL4 can be secreted into the extracellular space. The extracellular activity monitoring results show that the optimal fermentation time for C5-CEL4 is 37°C, 36h, and the specific activity of the enzyme is 0.886U / mL ( Figure 1 A). The intracellular crude enzyme solution and the extracellular crude enzyme solution were purified by Ni-NTA nickel column. The purified intracellular and extracellular proteins showed a 33KDa band for protein marker on 12% SDS-PAGE. Figure 2 As shown. The actual protein molecular size is quite different from the theoretical molecular size (47.54KDa). By analyzing the sequenced sequence, the N-terminus of the protein sequence has a His tag, while the C-terminus has no His tag and no stop codon. Therefore, the C-terminus was selected for protein sequencing analysis.

[0064] The protein C-terminal sequencing analysis based on LC-MS / MS showed that the C-terminal sequence of C5-CEL4 protein was IVRGWDGGGSSSSSS, SEQ ID NO.7, and the C-terminus terminated at the 314th amino acid of the protein sequence ( Figure 3 ). Therefore, the theoretical size of the expressed target protein is 34.12KDa, and the theoretical pI is 4.69. The premature termination of the amino acid sequence causes the expressed protein to become smaller, and the actual protein molecular weight is consistent with the theoretical protein molecular weight (34.12KDa), indicating that the target protein has been successfully expressed and purified. Cellulase activity was detected by zymography, where positive activity was manifested as a clear band on a red background ( Figure 2 ). Due to the cleavage of the amino acid sequence or premature termination during expression, C5-CEL4 lacked C-terminal amino acid residues 315 to 448, including the CBM domain. Primers were designed to amplify the catalytic domain of C5-CEL4 (C5-CEL4-GH5) and compare the activity differences between the two. The results showed that the intracellular activities of C5-CEL4 and C5-CEL4-GH5 were not much different, but the extracellular activities were quite different. The extracellular activity of C5-CEL4-GH5 was extremely low, which may be due to the missing part of the amino acid sequence of C5-CEL4 related to the extracellular secretion in Escherichia coli ( Figure 1 B).

[0065] The nucleotide sequence (1347 bp) of the cellulase gene c5-cel4 expressed in Escherichia coli is shown in SEQ ID NO.5.

[0066] The nucleotide sequence (945 bp) of the cellulase gene c5-cel4-gh5 expressed in E. coli is as follows:

[0067] ATGATATTCGGCGCAGGTGCCCAGGCGGTGGAGCCGTTGACCGTCAATGGCAACCGGATACTGGCCGGTGGTGAAGTGCGCAGCCTGGCCGGGCCCAGCTTCTTCTGGAGCAACACCGGCTGGGGCGCCGAGCGTTTCTACAACGAAAGCGCGGTGCGCTGGGTCAAGAACGACTGGAACGCCACCATCGTGCGCGCCTCCCTGGGTGTGGACGGAGAGGGCGGCTACCTGGAAGACCCGGCCGGCAACAAGAGCCGGGTGGTCGAACTGGTGGAGGCAGCCATTGCCAACGACCTCTACGTGATCATCGACTGGCACTCCCACCACGCCGAGGACCACCCCGCCGAAGCGGTAGCGTTTTTTGAGGAAATGGCCCGCAACTACGGTCATCACGACAACGTCATCTACGAAATCTACAACGAGCCATTACAGATTTCCTGGAGCAACACCATCAAGCCTTATGCGGAGACGGTCATTTCCGCCATCCGCGCCATCGATCCGGACAACCTGATCGTGGTGGGCACACCCACCTGGTCCCAGGACGTGGACACCGCCTCCTGGGACCCGATCCAGGGACACGCCAATATCGCCTACACCCTGCACTTCTACGCCGGCACCCATACGCAATACCTGCGCGACAAGGCGCGGACCGCATTGAACAACGGCATCGCGCTGTTCGTCACCGAGTGGGGTACCGTCAACGCCAACGGCGACGGCGGTGTCGCCCAGGATGAAACCCAGCGCTGGATGGACTTCCTCGAAGCCAACCATATCAGCCACGCAAACTGGGCACTGAACGACAAGGACGAGGGCGCCTCCGCCCTGGTGCCTGGCGTCAGCGCCACCGGCGGCTGGGGGCAGAGCGAACTGACCGAATCCGGCCGGCTGGTGCGGGATATAGTGCGGGGCTGGGATGGAGGCGGTTCCTCTTCGAGCTCCAGCTGA,SEQ ID NO.8。

[0068] The amino acid sequence of cellulase C5-CEL4-GH5 (314 residues) expressed in E. coli is as follows:

[0069] MIFGAGAQAVEPLTVNGNRILAGGEVRSLAGPSFFWSNTGWGAERFYNESAVRWVKNDWNATIVRASLGVDGEGGYLEDPAGNKSRVVELVEAAIANDLYVIIDWHSHHAEDHPAEAVAFFEEMARNYGHHDNVIYEIYNEPLQISWSNTIKPYAETVI SAIRAIDPDNLIVVGTPTWSQDVDTASWDPIQGHANIAYTLHFYAGTHTQYLRDKARTALNNGIALFVTEWGTVNANGDGGVAQDETQRWMDFLEANHISHANWALNDKDEGASALVPGVSATGGWGQSELTESGRLVRDIVRGWDGGGSSSSSS, SEQ ID NO.9.

[0070] By comparing in NCBI, the amino acid sequence of C5-CEL4 is 90.97% similar to the cellulase from Microbulbifer litoralis (all sequence analyses used the actual expressed amino acid sequence). Figure 4 As shown, phylogenetic analysis of the amino acid sequence showed that C5-CEL4 belonged to the cellulase family glycosyl hydrolase, and was clustered with the cellulase from Microbulbifer halophilus (GenBank: WP 265722981.1) with a support rate of 85%. Microbulbifer halophilus is a moderately halophilic bacterium.

[0071] In the PDB database, the protein has a similarity of 70% with two GH5 family endoglucanases from Cellvibrio japonicus (PDB: 8BQA, 8BQC). Multiple sequence alignment results showed that C5-CEL4 has a high homology with GH5 family cellulases 8BQA, 8BQC, 8C10, 4M1R, and endoglucanase 1EGZ ( Figure 5 ).

[0072] Further protein sequence analysis showed that C5-CEL4 contained a cellulase catalytic domain (CD) and belonged to the GH5 family of cellulases ( Figure 6A). By SWISS-MODEL homology modeling, the highest GMQE value of this protein with GH5 family endoglucanases from Microbulbiferrhizosphaerae was 0.94. C5-CEL4 has a highly conserved overall structure, namely a typical (β / α)8TIM barrel fold with 8 loops around the catalytic cleft ( Figure 6 B) This structural folding provides the loop with abundant degrees of variation, which endows the protein with catalytic diversity, including substrate specificity, thermal stability, and pH stability.

[0073] 2.2 Effects of temperature and pH on C5-CEL4

[0074] The optimal reaction temperature for the activity of C5-CEL4 pure enzyme inside and outside the cell was 50℃, and the activity was kept above 80% at 35~60℃. Figure 7 A). The optimal pH for the activity of C5-CEL4 pure enzyme both intracellularly and extracellularly was 7.0, and more than 70% of the maximum activity was maintained between pH 6.0 and 8.0 ( Figure 7 B). Thermal stability analysis showed that C5-CEL4 maintained 100% activity after heat treatment at 40°C for 2 h and 74% activity after heat treatment at 50°C for 2 h. Its half-life at 55°C and 60°C was 70 min ( Figure 7 C). pH stability analysis showed that within the pH range of 3.0 to 13.0, its activity remained above 50% of the initial activity, and after incubation of the pure enzyme solution at 4°C for 12h and 24h, its activity remained above 80% within the pH range of 4.0 to 12.0 ( Figure 7 D).

[0075] 2.3 Effects of salt and ionic liquid on C5-CEL4

[0076] like Figure 8 As shown in A, the optimal NaCl concentration for C5-CEL4 activity is 2.5-3.0 M, and the activity remains above 100% at a near-saturated salt concentration of 5 M. Salinity tolerance analysis showed that after C5-CEL4 was treated at different salt concentrations at 4°C for 9 months, it still maintained about 100% activity in 0.5M to 5.0M NaCl concentrations ( Figure 8 B). When 1mM / 10mM NaCl, NaNO3 and Na2SO3 were added to the reaction mixture, the activity of C5-CEL4 remained close to 100%, indicating that the addition of different sodium salt anions had little effect on the activity of C5-CEL4 (Table 1). In the 1% to 10% EMIM-Cl ionic liquid, the activity of C5-CEL4 was significantly increased to more than 110% of the initial activity ( Figure 8C), it retained more than 60% relative activity in three 20% ionic liquids. In addition, after incubation in three 40% ionic liquids for 24 h, the residual activity of C5-CEL4 was as high as more than 90%, and even slightly activated ( Figure 8 D) These results indicate that C5-CEL4 is a salt- and ionic liquid-tolerant cellulase.

[0077] like Fig. 9 As shown, the distribution of acidic amino acids on the protein surface is relatively high, making the overall electrostatic potential negative, which is beneficial to weakening the hydrophobicity or enhancing the hydrophilicity of the enzyme surface, improving the water binding ability of the enzyme, and preventing the aggregation of the enzyme in high salt solutions.

[0078] 2.4 Effects of metal ions and chemical reagents on C5-CEL4

[0079] As shown in Table 1, C5-CEL4 activity can be inhibited by 1 mM Co 2+ (114.4±1.1%), 1 mM Mn 2+ (135.6±8.7%), 10 mM Mn 2+ (212.4±0.4%) activation at 1mM and 10mM K + Mg 2+ , Fe 3+ , Ca 2+ 、Zn 2+ , Cu 2+ 、Ag + 、Ni 2+ Moderate to mild inactivation, in 10 mM Pb 2+ The relative activity of Co 2+ The relative activity of Mn increased by about 14%. 2+ Strongly activates enzyme activity (increased by about 2.1 times). 0.1% inhibitors EDTA, PMSF, and DTT have no obvious effect on C5-CEL4, but when the concentration increases to 1.0%, the enzyme is slightly inactivated in EDTA and PMSF. 0.1% Tween80 increases the relative activity of the enzyme by about 37%, and it is still slightly activated when the concentration increases to 1%. 0.1% / 1.0% SDS and CTAB slightly inhibit its activity. 1% and 10% methanol, ethanol, and isopropanol do not affect enzyme activity. In 1% β-ME, it still maintains 76% relative activity, but when the concentration increases to 10%, the enzyme is directly inactivated.

[0080] Table 1 Effects of different ions and chemical reagents on the activity of intracellular enzyme C5-CEL4

[0081]

[0082]

[0083] Note: The experimental data were analyzed for significance (p>0.05 was not significant, 0.05>p>0.01 was significant and marked with *, p<0.01 was extremely significant and marked with **), 100% = 19.6±3.47 U / mg.

[0084] 2.5C5-CEL4 substrate specificity and kinetic analysis

[0085] The substrate specificity of C5-CEL4 is shown in Table 2. The extracellular enzyme C5-CEL4 was active on CMC-Na (24.36 ± 0.23 U / mg), bagasse xylan (13.24 ± 0.61 U / mg), beech xylan (14.06 ± 0.53 U / mg), but not on corncob xylan, microcrystalline cellulose and cellobiose. Similarly, the intracellular enzyme C5-CEL4 was active on CMC-Na (19.60 ± 3.47 U / mg), bagasse xylan (2.50 ± 0.77 U / mg), beech xylan (1.48 ± 2.01 U / mg), but not on corncob xylan, microcrystalline cellulose and cellobiose.

[0086] The Km, Vmax and Kcat of the extracellular enzyme C5-CEL4 for CMC-Na were 45.03 mg / mL, 158.73 μmol / min / mg and 98.20 S -1 The Km, Vmax and Kcat of bagasse xylan were 16.05 mg / mL, 15.55 μmol / min / mg and 12.32 S -1 The Km, Vmax and Kcat of the intracellular recombinant enzyme C5-CEL4 for CMC-Na were 101.71 mg / mL, 357.14 μmol / min / mg and 220.95 S -1 (See Table 3).

[0087] Table 2 Substrate specificity of C5-CEL4

[0088]

[0089] Table 3 Kinetic parameters of C5-CEL4

[0090]

[0091]

[0092] 2.6 Thin layer chromatography (TLC) analysis of CMC-Na hydrolysis by C5-CEL4

[0093] like Fig.10As shown, the hydrolysis product of CMC-Na was analyzed by TLC using C5-CEL4. The results showed that C5-CEL4 randomly cleaved the cellulose oligosaccharide chains and hydrolyzed them to produce cellotriose (G3).

[0094] 3 Conclusion

[0095] The present invention obtains a new cellulase gene c5-cel4 from the Xinjiang Aibi Salt Lake sediment metagenome, clones it, and heterologously expresses it in Escherichia coli. The protein structure analysis of C5-CEL4 shows that the rich distribution of acidic amino acids on the protein surface causes the overall electrostatic potential to be negative, which helps to enhance the hydrophilicity of the enzyme surface and improve the water binding capacity of the enzyme, so that the enzyme has salt tolerance. Enzymatic properties show that C5-CEL4 has hydrolysis activity on both CMC-Na and sugarcane bagasse xylan, is a multifunctional cellulase, and has the characteristics of heat resistance, alkali resistance, salt resistance and high tolerance to ionic liquids. In addition, the enzyme can tolerate most metal ions, inhibitors and alcohols. This indicates that C5-CEL4 is an ideal candidate enzyme for biomass conversion, washing and textile industry production, and has great potential application prospects in feed, food and bioenergy.

[0096] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0097] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A cellulase that is heat-resistant, salt-alkali-resistant, and ionic liquid-resistant, It is characterized in that Its amino acid sequence is shown in SEQ IDNO.6; MIFGAGAQAVEPLTVNGNRILAGGEVRSLAGPSFFWSNTGWGAERFYNESAVRWVKNDWNATIVRASLGVDGEGGYLEDPAGNKSRVVELVEAAIANDLYVIIDWHSHHAEDHPAEAVAFFEEMARNYGHHDNVIYEIYNEPLQISWSNTIKPYAETVI SAIRAIDPDNLIVVGTPTWSQDVDTASWDPIQGHANIAYTLHFYAGTHTQYLRDKARTALNNGIALFVTEWGTVNANGDGGVAQDETQRWMDFLEANHISHANWALNDKDEGASALVPGVSATGGWGQSELTESGRLVRDIVRGWDGGGSSSSSS, SEQ ID NO.

6.

2. The encoding gene of cellulase as claimed in claim 1.

3. The encoding gene of claim 2, It is characterized in that The nucleotides are shown in SEQ ID NO.5; ATGATATTCGGCGCAGGTGCCCAGGCGGTGGAGCCGTTGACCGTCAATGGCAACCGGATACTGGCCGTGGTGAAGTGCGCAGCCTGGCCGGCCCAGCTTCTTCTGGAGCAACACCGGCTGGGGCGCCGAGCGTTTCTACAACGAAAGCGGTGCGCTGGGTCAAG AACGACTGGAACGCCACCATCGTGCGCCTCCCCTGGGTGTGGACGGAGAGGCGGCTACCTGGAAGACCCGGCCGCAACAAGAGCCGGTGGTCGAACTGGTGGAGGCAGCCATTGCCAACGACCTCTACGTGATCATCGACTGGCACTCCCACCACGCCGAGGACC ACCCCGCCGAAGCGGTAGCGTTTTTGAGGAAATGGCCGCAACTACGGTCATCACGACAACGTCATCTACGAAATCTACTACAACGAGCCATTACAGATTTCCTGGAGCAACCACCATCAAGCCTTATGCGGAGACGGTCATTTCCGCCATCCGCCATCGATCCGGACAA CCTGATCGTGGTGGGCACACCACCCACCTGGTCCCAGGACGTGGACACCGCCTCCTGGGACCCGATCCAGGGACACGCCAATATCGCCTACACCCTGCACTTCTACGCCGGCACCATACGCAATACCTGCGCGACAAGGCGGACCGCATTGAACAACGGCATCGCGCTG TTCGTCACCGAGTGGGGTACCGTCAACGCCAACGGCGGCGGTGTCGCCCAGGATGAAACCCCAGCGCTGGATGGACTTCCTCGAAGCCAACCATATCAGCCACGCAAACTGGGCACTGAACGACAAGGACGAGGGCGCCTCCGCCCTGGTGCCTGGCGTCAGCGCCA CCGGCGGCTGGGGGCAGAGCGAACTGACCGAATCCGGCCGCTGGTGCGGGATATAGTGCGGGCTGGATGGAGGCGGTTCCTCTTCGAGCTCCAGCTCGTCCAGTTCCAGTAGTTCCAGCTCAAGTTCCTCCCAGCTCCCAGCTCGTCGTCGAGTTC GAGTTCTTCGTCCAGCTCCGGTGGCAGCGCGCACTGAGCTGCGATCCCGGCAATGCGGACGTCTGGAATTCCGGCTTTGTACTGAGCAATGTCAGCGTGCACAACGATGGCGGAGCGACGTCGACGGCTGGCAGGTTTCACTGCAATTCCCCAGCCCATCCAGCTC ACCAATGGCTGGAACGGCGACTTCAGCCTCTCGGGCGATGGCAAAACCCCTGACCGTGGCGAATATCGGCTGGAACGGCTACCTCCCAGCCGGGCAATCCACCTTCGTTCGGGCTGCAGGCAGTTACAGCGGTAATTTCCTGATGCCGGAATGCAGCCAACTGA, SEQ ID NO.

5.

4. A recombinant vector, It is characterized in that Contains the encoding gene as claimed in claim 3.

5. A recombinant bacteria, It is characterized in that Contains the recombinant vector as claimed in claim 4.

6. The use of cellulase as claimed in claim 1 in degrading CMC-Na, bagasse xylan, beech xylan, and corn cob xylan.

7. The use of cellulase as claimed in claim 1 in food, feed, textile and industrial production.

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