Cellulase CelL7 as well as preparation, modification and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to cellulase CelL7 as well as preparation, modification and application thereof. The amino acid sequence of the cellulase CelL7 is as shown in SEQ ID NO.2, and the cellulase CelL7 shows degradation capacity on sodium carboxymethyl cellulose, cellobiose and xylan and can promote the dispersion of methicillin-resistant staphylococcus aureus and pseudomonas aeruginosa biological membranes. The cellulase CelL7 and a CBM3 binding structural domain are fused, and modified cellulase CelL7-CBM3 is obtained; compared with cellulase CelL7, the modified cellulase CelL7-CBM3 has the advantages that the specific activity and the pH stability are improved. The method has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a cellulase CelL7 and the preparation, transformation and application thereof. Background Art

[0002] Cellulase is a complex enzyme that can catalyze the hydrolysis of cellulose to produce glucose, cellobiose and cellulose oligosaccharides, and belongs to the glycoside hydrolase (GH) family. According to the mode of action and substrate specificity of the enzyme, it is divided into endoglucanase, exoglucanase, β-glucosidase and polysaccharide cleavage monooxidase, and plays an important role in the food industry, papermaking and pulping, biofuels and biomedicine. Cellulose in wood fiber biomass can be converted into fermentable reducing sugars by hydrolysis, and then converted into green energy such as bioethanol or other chemical products through fermentation. Among them, enzymatic degradation of cellulose is an important method and means of biomass cellulose degradation.

[0003] Cellulolytic enzymes come from a wide range of sources, including various fungi, bacteria, plants, and ruminant herbivores. Fungi, as the most studied plant biomass-degrading microorganisms, have a strong ability to secrete enzymes outside the cell. Cellulolytic fungi mainly include Ascomycota and Basidiomycota. In contrast, bacterial-derived cellulolytic enzymes are less studied. Most bacterial cellulolytic enzymes come from Bacillus, Acinetobacter, Clostridium, and Cellulomonas. Most fungi can degrade crystalline and amorphous cellulose at the same time, while the vast majority of bacteria can only effectively degrade amorphous structures (such as CMC). At present, cellulase is widely used in important industries such as food and feed, papermaking and pulping, and biofuels. In particular, in the biopharmaceutical industry, cellulase exhibits good antibacterial properties and inhibits the biofilm formed by common pathogenic bacteria at the site of medical implants, which can reduce the infection rate during surgery. Microorganisms are the main producers of cellulase, but most cellulose-degrading bacteria have low enzyme production, low enzyme catalytic efficiency, and poor thermal stability, which limits large-scale industrial applications.

[0004] The CBMs domain has a binding site that specifically interacts with cellulose, usually present at the N-terminus or C-terminus, and is connected to the CD region through a highly glycosylated flexible linker peptide, which helps to form a relatively stable aggregate between different enzyme molecules. The core function of the CBM domain is to recognize and specifically bind to insoluble cellulose. This binding guides the catalytic domain CD of cellulase to the cellulose substrate, thereby enhancing the nucleophilicity and hydrolysis activity of cellulase to the substrate. Based on the functional characteristics of CBM, fusing CBMs from different sources with the catalytic domain of cellulase to develop chimeric enzymes is an effective means to improve the hydrolysis efficiency of enzymes.

[0005] In order to solve the limitations of the above factors on the industrial application of cellulase, the heterologous expression of a cellulase is achieved by constructing genetically engineered bacteria, which is of great significance for improving the catalytic activity and stability of cellulase, exploring enzyme-substrate interactions and the application potential of enzymes, thereby meeting the needs of industrial production. Summary of the invention

[0006] The purpose of the present invention is to provide a cellulase CelL7 and its preparation, modification and application.

[0007] To achieve the above object, the present invention adopts the following technical solutions: A cellulase CelL7, the amino acid sequence of the cellulase CelL7 is shown as SEQ ID NO.2.

[0008] The preparation method of the cellulase CelL7 is as follows: BamHⅠ and XhoⅠ restriction sites are introduced at both ends of the cellulase gene celL7, and then connected to the pMD18-T vector to obtain the recombinant vector pMD18-T-celL7; the recombinant vector pMD18-T-celL7 and the pET28a vector are double-digested with BamHⅠ and XhoⅠ, and then connected by T4 DNA ligase to obtain the recombinant vector pET28a-celL7; the recombinant vector pET28a-celL7 is transformed into the host bacteria E.coil BL21 (DE3) to obtain the recombinant bacteria E.coil BL21 (DE3)-pET28a-celL7; the recombinant bacteria E.coil BL21 (DE3)-pET28a-celL7 is fermented and induced to express and purified to obtain the cellulase CelL7; Wherein, the nucleotide sequence of the cellulase gene celL7 is shown as SEQ ID NO.1.

[0009] The above-mentioned cellulase CelL7 is used in any of the following applications: 1) in the degradation of sodium carboxymethyl cellulose; 2) in the degradation of cellobiose; 3) in the degradation of xylan; 4) in the preparation of a drug for promoting the dispersion of bacterial biofilms; Furthermore, the bacteria include methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa.

[0010] A method for modifying the cellulase CelL7, wherein the cellulase CelL7 is fused with the CBM3 binding domain to obtain a modified cellulase CelL7-CBM3; the modified cellulase CelL7-CBM3 has improved specific activity and pH stability compared to the cellulase CelL7; Furthermore, the amino acid sequence of the modified cellulase CelL7-CBM3 is shown in SEQ ID NO.4; Furthermore, the method specifically comprises: using Overlap PCR technology to fuse the cellulase gene celL7 with the CBM3 binding domain encoding gene to obtain the fusion gene celL7-cbm3; the fusion gene celL7-cbm3 and the pET-28a vector are double-digested with BamHⅠ and XhoⅠ, and then connected by T4 DNA ligase to obtain the recombinant vector pET28a-celL7-cbm3; the recombinant vector pET28a-celL7-cbm3 is transformed into the host bacteria E.coil BL21 (DE3) to obtain the recombinant bacteria E.coil BL21 (DE3)-pET28a-celL7-cbm3; the recombinant bacteria E.coil BL21 (DE3)-pET28a-celL7-cbm3 is fermented and induced to express and purified to obtain the modified cellulase CelL7-CBM3; wherein the nucleotide sequence of the fusion gene celL7-cbm3 is shown in SEQ ID NO.3.

[0011] A modified cellulase CelL7-CBM3 is obtained by modification using the above method.

[0012] The above-mentioned modified cellulase CelL7-CBM3 is used in any of the following: 1) in the degradation of sodium carboxymethyl cellulose; 2) in the degradation of cellobiose; 3) in the degradation of xylan; 4) in the preparation of a drug for promoting the dispersion of bacterial biofilms; Furthermore, the bacteria include methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa.

[0013] The significant advantages of the present invention are: (1) The cellulase CelL7 provided by the present invention is a new member of the glycoside hydrolase GH5 family, and its base sequence does not match a similar cellulase gene fragment.

[0014] (2) The present invention successfully achieved heterologous expression of cellulase CelL7 through genetic engineering means. After separation and purification of CelL7 by Ni-NTA agarose purification resin, the specific enzyme activity of recombinant cellulase CelL7 was measured to be 2249.81 U / mg. At the same time, cellulase CelL7 can degrade sodium carboxymethyl cellulose (CMC-Na), cellobiose, and xylan, mainly acting on β-1,4-glycosidic bonds, and is an endoglucanase.

[0015] (3) The cellulase CelL7 provided by the present invention has an optimum reaction temperature of 50°C and an optimum reaction pH of 5.0, and is a medium-temperature acidic enzyme. Existing literature shows that cellulase, as a glycoside hydrolase, can degrade polysaccharide substances in the extracellular polymers of pathogenic bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa, and target degradation of β-1,4-glycosidic bonds in bacterial EPS, thereby inducing the spread of biofilms, which is an effective method for solving biofilm infections clinically. Therefore, cellulase CelL7 has good application prospects for degrading biofilms.

[0016] (4) The present invention constructs a recombinant plasmid pET28a-celL7-cbm3 by adding the CBM3 binding domain gene to the C-terminus of the cellulase gene celL7, and successfully constructs the fusion protein CelL7-CBM3 genetic engineering bacteria E.coilBL21 (DE3)-pET28a-celL7-cbm3. The specific activity of CelL7-CBM3 to CMC-Na is 2915.76U / mg, which is 1.3 times higher than the original enzyme's 2249.81U / mg. Its optimal reaction temperature is 50°C and the optimal reaction pH is 5.5. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : SDS-PAGE image of cellulase CelL7 after separation and purification. Lane M is the marker; Lanes 3-4 are the purified cellulase CelL7.

[0018] Figure 2 :The effect of temperature on the cellulase CelL7. A shows the effect of temperature on the activity of the cellulase CelL7; B shows the effect of temperature on the stability of the recombinant cellulase CelL7.

[0019] Figure 3 :The effect of pH on the cellulase CelL7. A shows the effect of pH on the activity of cellulase CelL7; B shows the effect of pH on the stability of cellulase CelL7.

[0020] Figure 4 : Effects of metal ions and chelating agents on cellulase CelL7.

[0021] Figure 5 : Substrate specificity of cellulase CelL7.

[0022] Figure 6 : Dispersing effect of cellulase CelL7 on bacterial biofilm. A shows the dispersing effect of cellulase CelL7 on methicillin-resistant Staphylococcus aureus biofilm; B shows the dispersing effect of cellulase CelL7 on Pseudomonas aeruginosa biofilm.

[0023] Figure 7: SDS-PAGE image of the modified cellulase CelL7-CBM3 after separation and purification. Lane M is the marker; Lane 3 is the purified modified cellulase CelL7-CBM3.

[0024] Figure 8 :The effect of temperature on the modified cellulase CelL7-CBM3. A shows the effect of temperature on the activity of the modified cellulase CelL7-CBM3; B shows the effect of temperature on the stability of the modified cellulase CelL7-CBM3.

[0025] Fig. 9 :The effect of pH on the modified cellulase CelL7-CBM3. A shows the effect of pH on the activity of the modified cellulase CelL7-CBM3; B shows the effect of pH on the stability of the modified cellulase CelL7-CBM3. DETAILED DESCRIPTION

[0026] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0027] The Zobellia sp. in the present invention is provided by Fuzhou University.

[0028] The cellulase gene celL7 in the present invention is amplified from the genome DNA of Zobellia sp. by PCR. The nucleotide sequence of the cellulase gene celL7 is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it (i.e., cellulase celL7) is shown in SEQ ID NO.2. The homology comparison results show that no cellulase gene fragment similar to the nucleotide sequence of the cellulase gene celL7 is matched, and the cellulase CelL7 is a new member of the glycoside hydrolase GH5 family.

[0029] The formula of the BR buffer in the present invention is: to a mixture of three acids (phosphoric acid, acetic acid, boric acid, all with a concentration of 0.04 mol / L), add a 0.2 mol / L NaOH solution to adjust the pH to a corresponding value.

[0030] The formula of NTA-0 Buffer in the present invention is: 20 mM Tris-HCl (pH=7.9), 0.5 M NaCl, and 10% glycerol.

[0031] The formula of NTA-10 Buffer to NTA-500 Buffer in the present invention is: adding imidazole with a final concentration of 10 to 500 mM to NTA-0 Buffer. For example, the formula of NTA-10 Buffer is: adding imidazole with a final concentration of 10 mM to NTA-0 Buffer; the formula of NTA-20 Buffer is: adding imidazole with a final concentration of 20 mM to NTA-0 Buffer.

[0032] The formula of the 2× protein treatment solution in the present invention is: 1.25mL 5mol / L Tris-HCl (pH=6.8), 2mL glycerol, 2mL 10wt% SDS, 1mL β-mercaptoethanol, 0.5mL 0.1wt% bromophenol blue are mixed evenly, and distilled water is added to make up to 10mL.

[0033] The enzyme activity of the crude enzyme solution in the present invention is determined by the 3,5-dinitrosalicylic acid (DNS) method, and the specific operation is as follows: take 120 μL of 1.0% (W / V) CMC-Na and mix it evenly with an equal volume of enzyme solution, incubate at 50°C for 30 minutes (three parallels are set for each group), add 100 μL of DNS reagent after the incubation, heat and color develop for 10 minutes, add UP water to 1 mL after cooling and mix evenly, centrifuge at 10000rpm for 1 minute, and take the supernatant. The blank control group is a crude enzyme solution that has been heated and inactivated for 10 minutes, and the absorbance value of the supernatant at a wavelength of 540nm is finally determined. The reducing sugar content is calculated according to the glucose standard curve equation. Definition of enzyme activity unit: Under the above experimental conditions, the amount of enzyme required to catalyze the hydrolysis of the substrate to produce 1 μg of reducing sugar per 1 minute is one enzyme activity unit (U / mL). The enzyme activity calculation formula is: Wherein: A1——absorbance of the experimental group at 540nm; A0——absorbance of the control group at 540nm; n——dilution multiple of enzyme solution; T——reaction time (min); V——volume of enzyme solution (mL).

[0034] Example 1: Preparation of Cellulase CelL7 Primers celL7F1 and celL7R1 were used to add BamHⅠ and XhoⅠ restriction sites at both ends of the cellulase gene celL7 sequence, and the PCR reaction was carried out under the following conditions: pre-denaturation at 95℃ for 10 min; denaturation at 95℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 2 min, 30 cycles; extension at 72℃ for 8 min. The PCR product was subjected to 1% agarose nucleic acid electrophoresis and then gel-cut and recovered to obtain the cellulase gene celL7 with restriction site. The cellulase gene celL7 with restriction site was connected to the pMD18-T vector using the TA cloning kit purchased from TAKATA. The connection system and conditions were as follows: 5 μL of the target band was recovered by gel; 4.3 μL of Solution I; 0.7 μL of pMD18-T. The connection system was placed at 16°C for overnight connection. The next day, the connection product was transformed into E.coil (DH5α) competent cells, and the positive clones were picked for DNA sequencing. The recombinant vector pMD18-T-celL7 was extracted from the positive clones that were successfully sequenced. The recombinant vector pMD18-T-celL7 and pET28a vector were double-digested with restriction endonucleases BamHⅠ and XhoⅠ. The digestion products were recovered by gel cutting after 1% agarose nucleic acid electrophoresis. The fragments recovered by gel were ligated with T4 DNA ligase. After overnight ligation at 16°C, the ligation products were transformed into E.coil (DH5α) competent cells, and positive clones were selected for DNA sequencing. The recombinant vector pET28a-celL7 was extracted from the positive clones with successful sequencing; the recombinant vector pET28a-celL7 was transformed into the host bacteria E.coilBL21 (DE3) to obtain the recombinant bacteria E.coil BL21 (DE3)-pET28a-celL7.

[0035] celL7F1:5'-CG GGATCC ATGAAAATGATTAAATTAATTTGTGCTGTGGTATGTATG-3' (the underline is the BamHⅠ restriction site) celL7R1:5'-CCG GAATTC AAATTTAGGGTTTTTCGCCAATATTTCGG-3' (the underline is the XhoⅠ restriction site) Fermentation induction of cellulase celL7: The recombinant bacteria E.coil BL21 (DE3)-pET28a-celL7 was streaked in three zones on a LB solid plate containing 50 μg / mL kanamycin, and a single colony was picked and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured in a shaking incubator at 37°C overnight to obtain a seed solution; the seed solution was inoculated into 1 L of LB liquid medium containing 50 μg / mL kanamycin at an inoculum volume of 1%, and cultured until OD 600When the pH value was 0.6-0.8, add isopropylthiogalactoside (IPTG) with a final concentration of 0.8mM, induce at 20℃ for 16h; collect the cells by centrifugation at 4℃ and 10000rpm for 15min, resuspend the cells in pre-cooled 20mL BR buffer (pH 7.4), ultrasonically disrupt for 20min, centrifuge at 4℃ and 10000rpm for 15min to collect the supernatant, which is the crude enzyme solution of cellulase celL7. Using CMC-Na as substrate, the enzyme activity of crude enzyme solution of cellulase celL7 was determined by 3,5-dinitrosalicylic acid (DNS) method.

[0036] Separation and purification of cellulase celL7: Use Ni-NTA pre-packed column for purification. Before purification, filter the crude enzyme solution of cellulase celL7 with a 0.22μm filter membrane, and use all solutions used for purification after filtration. Drain the 20% ethanol in the column, wash the column material with 3 to 5 column volumes of UP water, balance the column material with 3 to 5 column volumes of NTA-0Buffer, take 1 column volume of crude enzyme solution and adsorb for 3 to 5 minutes, wash the column with 4 to 6 column volumes of NTA-0Buffer to remove unbound proteins, and elute with 1 to 2 column volumes of NTA-10Buffer to NTA-500Buffer in sequence to collect the eluate, and obtain the purified cellulase celL7 (hereinafter referred to as "pure enzyme celL7").

[0037] Take the pure enzyme celL7 and add an equal volume of 2× protein treatment solution, boil it in a water bath for 10 minutes, cool it to room temperature, centrifuge it at 10000rpm for 5 minutes, and take the supernatant for SDS-PAGE protein electrophoresis analysis. The gel concentration is 12%, the voltage of the concentrated gel is 100V, and the voltage of the separation gel is 120V. The protein gel is stained with Coomassie Brilliant Blue R-250 for 3 hours, destained with a decolorizing solution, and photographed for analysis. Figure 1 As shown, the molecular weight of cellulase CelL7 is about 40 kDa, which is consistent with the predicted results.

[0038] BR buffer (pH = 5.0) was used as solvent to prepare 1wt% CMC-Na as the substrate for enzymatic reaction. The enzyme activity (unit: U / mL) of pure enzyme celL7 on the substrate was determined by DNS method, the concentration of pure enzyme celL7 was determined by Coomassie Brilliant Blue G-25 (unit: mg / mL), and the specific activity (unit: U / mg) was calculated. The results showed that cellulase celL7 had a significant degradation effect on 1% CMC-Na, and the specific activity on CMC-Na was 2249.81U / mg.

[0039] Example 2: Enzymatic properties of cellulase CelL7 (1) Effect of temperature on the activity of cellulase CelL7 120 μL of pure enzyme CelL7 and an equal volume of 1 wt% CMC-Na substrate prepared with BR buffer (pH = 5.0) were uniformly mixed, and the reaction system was placed at different temperatures (20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C and 90°C) for 30 min of enzymatic reaction. After the incubation, 100 μL of DNS reagent was quickly added and the color reaction was completed by boiling water bath treatment for 10 min. After cooling to room temperature, UP water was added to make the volume 1 mL and fully mixed, centrifuged at 10000 rpm for 1 min, and the supernatant was taken to determine its absorbance value at a wavelength of 540 nm. At the same time, the inactivated enzyme treated with boiling water bath for 10 min was used as the blank control group, and the highest enzyme activity obtained was set as 100%, and the relative enzyme activity at different temperatures was calculated as follows: Figure 2 As shown, the optimal reaction temperature of cellulase CelL7 is 50°C.

[0040] (2) Effect of temperature on the stability of cellulase CelL7 The pure enzyme CelL7 was incubated at 40°C, 45°C, 50°C, 55°C, and 60°C, and different incubation times (10min, 20min, 30min, 40min, 50min, and 60min) were set. After the incubation, an equal volume of 1wt% CMC-Na substrate prepared with BR buffer (pH=5.0) under the optimal reaction conditions (50°C) was taken and mixed evenly. At the same time, the inactivated enzyme treated in a boiling water bath for 10min was used as a blank control group. The highest enzyme activity obtained by the measurement was set as 100%, and the relative enzyme activity under different incubation times and different temperature conditions was calculated. Figure 2 As shown, cellulase CelL7 can remain relatively stable in the range of 40-55°C and can maintain more than 80% relative enzyme activity, indicating that cellulase CelL7 has good thermal stability.

[0041] (3) Effect of pH on the activity of cellulase CelL7 At the optimal reaction temperature (50°C), BR buffers with different pH values ​​(3.0-11.0) were added to the reaction system to determine the enzyme activity of the pure enzyme CelL7. The group with the highest enzyme activity was taken as 100% to calculate the relative enzyme activity of each group. Figure 3 As shown, the optimum pH of cellulase CelL7 is 5.0.

[0042] (4) Effect of pH on the stability of cellulase CelL7 After the pure enzyme CelL7 was mixed with the above-mentioned BR buffer of different pH in equal volumes, it was incubated at 4°C for 4 hours, and then the pH stability of the cellulase was tested under the optimum temperature and optimum pH conditions, and the enzyme activity without incubation treatment was taken as 100%. As shown in the figure, the cellulase CelL7 has good stability under the condition of pH = 4.0-6.5.

[0043] (5) Effects of metal ions and chelating agents on cellulase CelL7 Pure enzyme CelL7 was thoroughly mixed with an equal volume of 1 wt% CMC-Na substrate, and metal ions (Na + , Fe 3+ 、Co 2+ 、Zn 2+ , Cu 2+ 、Ni 2+ , Fe 2+ , Mn 2+ Mg 2+ , Li + , Ba 2+ , K + , Ca 2 + ), chelating agent EDTA and surfactant SDS were added to the samples for enzyme activity determination, and the enzyme activity without adding any metal ions and chelating agents was taken as 100%. Figure 4 As shown, 1-10 mM Mn + and 10 mM Fe 2+ The promoting effect on cellulase CelL7 was obvious. 1mM Li + It can also improve the enzymatic activity of cellulase CelL7; other metal ions and chelating agents have an inhibitory effect on enzyme activity, among which Cu 2+ The inhibitory effect was the most obvious.

[0044] (6) Substrate specificity of cellulase CelL7 CMC-Na, cellobiose, Avicel (α-cellulose), xylan, laminarin, glucan, chitosan and maltose were prepared with BR buffer at pH 5.0 at a concentration of 1 wt%. Appropriate amounts of pure enzyme CelL7 were mixed with equal volumes of the above different substrates. After reacting under optimal conditions, the relative enzyme activity of cellulase CelL7 on different substrates was determined. Figure 5As shown in the results, cellulase CelL7 showed degradation ability on CMC-Na, cellobiose and xylan, with the greatest catalytic effect on CMC-Na, while it had no obvious degradation effect on crystalline cellulose Avicel, laminarin, glucan, chitosan and maltose, indicating that cellulase CelL7 mainly hydrolyzes β-1,4-glycosidic bonds. It can be seen that cellulase CelL7 has endoglucanase and β-glucanase activities.

[0045] Example 3: Dispersion of bacterial biofilm by cellulase CelL7 Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (P. aeruginosa) were streaked on LB solid medium, and single colonies were selected and transferred to LB liquid medium. The culture was carried out at 37°C and 180 rpm for 12 h. After counting the bacteria, the bacterial suspension was diluted to a concentration of 1×10 6 CFU / mL; pure enzyme CelL7 was prepared with PBS (pH 7.4) at a concentration of 5.0-60 μg / mL.

[0046] 1 mL / well of diluted MRSA and P. aeruginosa bacterial suspensions were inoculated in a 24-well plate, and cultured in a 37°C constant temperature incubator for 48 hours to form a mature biofilm. After the culture was completed, the supernatant was aspirated, and 500 μL of pure enzyme CelL7 of different concentrations was added. PBS was added as a blank control group, and the culture was carried out for 12 hours. Then, 500 μL PBS was used to gently rinse three times to remove the floating cells, and the amount of crystal violet biofilm in vitro was determined. The specific steps are as follows: After rinsing with PBS, 300 μL of methanol solution was added, and the cells were fixed for 20 minutes and then dried. The remaining biofilm was fully stained with 300 μL of 0.1% CV reagent for 10 minutes, rinsed three times with PBS and then dried. 1.5 mL of 95% ethanol was added for decolorization at 100 rpm for 30 minutes, and the absorbance at a wavelength of 595 nm was measured with an enzyme marker to evaluate the dispersion effect of different concentrations of cellulase CelL7 on MRSA and P. aeruginosa biofilms. Figure 6 As shown, compared with the control group, 10-60 μg / mL of cellulase CelL7 could significantly disperse the biofilm formed by MRSA and P. aeruginosa (P<0.001).

[0047] Example 4: Preparation of modified cellulase CelL7-CBM3 The cellulase CelL7 was fused with the CBM3 binding domain to obtain the modified cellulase CelL7-CBM3: In the first round of PCR, primers celL7-wf-F and celL7-cbm3-R were used to amplify the cellulase gene celL7 with a partially overlapping sequence at the 3' end. In the second round of PCR, primers celL7-cbm3-F and cbm3-wf-R were used to amplify the CBM3 binding domain gene cbm3 with a partially overlapping sequence at the 5' end. The amplified products of the two rounds of PCR were recovered by gel and the DNA concentration was determined. Since the two fragments contained a homology arm sequence, the fusion gene celL7-cbm3 was amplified by the third round of Overlap PCR using primers celL7-wf-F and cbm3-wf-R. The nucleotide sequence of the fusion gene celL7-cbm3 is shown in SEQ ID NO.3, and the amino acid sequence of the protein encoded by it (i.e., the modified cellulase CelL7-CBM3) is shown in SEQ ID NO.4. The fusion gene celL7-cbm3 and the pET-28a vector were separated by restriction enzyme QuickCut TM BamHI and QuickCut TM XhoⅠ was used for double enzyme digestion at the same time, and the digestion products were recovered from the gel and ligated with T4 DNA ligase. The ligation products were transferred into E.coliDH5α competent cells, and the positive clones were picked for DNA sequencing. The recombinant vector pET28a-celL7-cbm3 was extracted from the positive clones with successful sequencing; the recombinant vector pET28a-celL7-cbm3 was transformed into the host bacteria E.coilBL21(DE3) to obtain the recombinant bacteria E.coli BL21(DE3)-pET28a-celL7-cbm3.

[0048] celL7-wf-F: 5'-TGGACAGCAAATGGGTCGCGGATCCATGAAAATGATTAAATTAAT TTGTGCTG-3' celL7-cbm3-R:5'- CCGGAACCACCACCACCA GAACCGCCACCGCCAAATTTAGGGTTTTTCGCCAATATTTCGG-3' (the underline indicates the overlapping sequence) celL7-cbm3-F:5'-GGTTC TGGTGGTGGTGGTTCCGG TGGCGGTGGTAGCGTACAATACAAAGCAGGGGATGGGG-3' (the underline indicates the overlapping sequence) cbm3-wf-R: 5'-AGTGGTGGTGGTGGTGGTGCTCGAGCAGTTCCAGATAGGTATCTGCACCT-3' The fermentation-induced expression, separation and purification of the recombinant bacteria E. coli BL21 (DE3)-pET28a-celL7-cbm3 were carried out with reference to the recombinant bacteria E. coli BL21 (DE3)-pET28a-celL7. Figure 7 As shown, the target molecular weight of the isolated and purified modified cellulase CelL7-CBM3 (hereinafter referred to as "pure enzyme CelL7-CBM3") is about 50 kDa, and the specific activity measured with CMC-Na as substrate is 2915.76 U / mg, which is 1.3 times higher than the specific activity of cellulase CelL7 of 2249.81 U / mg.

[0049] The enzymatic properties of the pure enzyme CelL7-CBM3 were studied with reference to Example 1: like Figure 8 and Fig. 9 As shown, the modified cellulase CelL7-CBM3 showed the highest enzyme activity at 50°C, and its optimal temperature was basically consistent with that of cellulase CelL7; the modified cellulase CelL7-CBM3 reached the highest enzyme activity at pH = 5.5, which was close to the optimal reaction pH = 5.0 of cellulase CelL7; after incubation for 2 hours under pH = 3.0-10.0 conditions, the relative enzyme activity of the modified cellulase CelL7-CBM3 remained above 80%, and the addition of the binding domain improved the pH stability of cellulase CelL7 in alkaline environment, which had a certain effect on the enzyme's adaptation to extreme alkaline environment.

[0050] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A cellulase CelL7, characterized in that: The amino acid sequence of the cellulase CelL7 is shown in SEQ ID NO.

2.

2. The method for preparing the cellulase CelL7 according to claim 1, characterized in that: In cellulase genes celL7 Introduced at both ends Bam H Ⅰ and XOt I restriction site, and then connected to the pMD18-T vector to obtain the recombinant vector pMD18-T- celL7 ; Recombinant vector pMD18-T- celL7 Both pET28a vectors were Bm Ⅰ and XOt Ⅰ double restriction enzyme digestion, and then connected with T4 DNA ligase to obtain the recombinant vector pET28a- celL7 ; The recombinant vector pET28a- celL7 Transformed host bacteria E.coil BL21(DE3), obtained recombinant bacteria E.coil BL21(DE3)-pET28a- celL7 ; Recombinant bacteria E.coil BL21(DE3)-pET28a- celL7 Fermentation-induced expression and purification were performed to obtain cellulase CelL7; Wherein, the cellulase gene celL7 The nucleotide sequence is shown in SEQ ID NO.

1.

3. Use of the cellulase CelL7 as claimed in claim 1 in any of the following: 1) in the degradation of sodium carboxymethyl cellulose; 2) in the degradation of cellobiose; 3) in the degradation of xylan; 4) in the preparation of a drug for promoting the dispersion of bacterial biofilms.

4. The use according to claim 3, characterized in that: The bacteria include methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa.

5. A method for transforming the cellulase CelL7 according to claim 1, characterized in that: The cellulase CelL7 is fused with the CBM3 binding domain to obtain a modified cellulase CelL7-CBM3; the modified cellulase CelL7-CBM3 has improved specific activity and pH stability compared with the cellulase CelL7.

6. The method according to claim 5, characterized in that: The amino acid sequence of the modified cellulase CelL7-CBM3 is shown in SEQ ID NO.

4.

7. The method according to claim 6, characterized in that: Overlap PCR technique was used to clone the cellulase gene celL7 Fusion with the CBM3 binding domain encoding gene to obtain a fusion gene celL7-cbm3 ; Fusion gene celL7- cbm3 Both pET-28a vectors were Bm Ⅰ and XOt Ⅰ double restriction enzyme digestion, and then connected with T4 DNA ligase to obtain the recombinant vector pET28a- celL7-cbm3 ; The recombinant vector pET28a- celL7-cbm3 Transformed host bacteria E.coil BL21(DE3), obtained recombinant bacteria E.coil BL21(DE3)-pET28a- celL7-cbm3 ; Recombinant bacteria E.coil BL21(DE3)-pET28a- celL7-cbm3 Fermentation-induced expression and purification were performed to obtain the modified cellulase CelL7-CBM3; Wherein, the fusion gene celL7-cbm3 The nucleotide sequence is shown in SEQ ID NO.

3.

8. A modified cellulase CelL7-CBM3, characterized in that: The method is modified by any one of claims 5 to 7.

9. Use of the modified cellulase CelL7-CBM3 as claimed in claim 8 in any of the following: 1) in the degradation of sodium carboxymethyl cellulose; 2) in the degradation of cellobiose; 3) in the degradation of xylan; 4) in the preparation of a drug for promoting the dispersion of bacterial biofilms.

10. The use according to claim 9, characterized in that: The bacteria include methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa.

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