CN101845427B - A kind of halophilic cellulase and its coding gene and application - Google Patents

Abstract

The invention discloses halophilic cellulase and a coding gene and application thereof. The enzyme is a protein represented by the following or (b): a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table; (b) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1 in the sequence table, has halophilic cellulase activity and is derived from the sequence 1. The enzyme has the characteristics of wide pH tolerance, wide application temperature range and halophilic property, has wide application range, is particularly suitable for being applied in high-salt environment, and has good application prospect in the field of cellulose degradation under the conditions of hypertonicity and high salt.

Description

A kind of halophilic cellulase and its coding gene and application

technical field

The invention relates to a halophilic cellulase, its coding gene and application.

Background technique

Cellulose is a linear polymer formed by linking glucosides through β-1,4 glucosidic bonds, and is the main component of plant cell walls, accounting for 35%-50% of plant dry weight. As the most abundant renewable resource on earth, its hydrolysis into usable glucose by cellulase system has important application value.

A variety of microorganisms can produce cellulase, such as bacteria, yeast, fungi, and actinomycetes. The cellulase system produced by microorganisms is a multi-component enzyme system, mainly including endoglucanase, exoglucanase and β-glucosidase. First, the endoglucanase acts on the inside of the cellulose molecule to randomly hydrolyze the β-1,4-glucosidic bond, cut off the long-chain fiber molecule, and produce a large amount of small molecule cellulose. Then exoglucanase acts on the end of the cellulose linear molecule to hydrolyze the β-1,4-glycosidic bond, and cut off a cellobiose molecule from the non-reducing end of the cellulose chain each time. Finally, the cellobiose molecule is hydrolyzed to available glucose by β-glucosidase.

Cellulase produced by microorganisms has a wide range of applications in industry and agriculture. It is used to extract fruit and vegetable juices to increase the extraction rate, shorten the extraction time, improve the nutritional value, and reduce the viscosity of the mixture after extraction. Adding cellulase in the brewing process can make up for the lack of enzyme activity in wort, and can reduce agglomeration in fermentation, improve filtration efficiency, and increase beer production. In the animal feeding industry, the application of cellulase can improve the utilization rate of feed, balance the individual differences among animals, and increase the body weight or milk yield. In the textile industry, the application of cellulase for biological grinding can reduce the breakage rate of manual grinding, shorten the processing time, reduce the work intensity, and is conducive to the safety of the working environment. Cellulase can also improve the effect of washing clothes and reduce the cost of washing.

Contents of the invention

One object of the present invention is to provide a halophilic cellulase and its coding gene.

The halophilic cellulase provided by the present invention belongs to the glycosyl hydrolase 5 family, and is a protein as shown in (a), (b) or (c) below:

(a) a protein consisting of the amino acid sequence shown in amino acid residues 34-569 from the N-terminus in Sequence 1 in the sequence listing;

(b) a protein consisting of the amino acid sequence shown in Sequence 1 in the Sequence Listing;

(c) The amino acid sequence shown in (a) or (b) undergoes substitution and/or deletion and/or addition of one or several amino acid residues and has cellulase activity derived from (a) or (b) of protein.

The CelB protein shown in Sequence 1 consists of 569 amino acid residues.

The coding gene is the following 1) or 2) or 3) or 4) DNA molecule:

1) The nucleotide sequence is the DNA molecule shown in the 100th to 1710th nucleotides from the 5' end of Sequence 2 in the sequence listing;

2) The nucleotide sequence is the DNA molecule shown in sequence 2 in the sequence listing;

3) a DNA molecule that hybridizes to the DNA sequence defined in 1) or 2) under stringent conditions and encodes a protein with halophilic cellulase activity;

4) A DNA molecule having more than 90% homology with the DNA sequence defined in 1) or 2) or 3) and encoding a protein with halophilic cellulase activity.

The coding gene shown in Sequence 2 consists of 1710 nucleotides.

The above-mentioned stringent conditions can be hybridization at 65° C. in a solution of 6×SSC, 0.5% SDS, and then wash the membrane once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS respectively.

In order to make the protein in (a) easy to purify, the amino-terminal or carboxy-terminal of the protein consisting of the amino acid sequence shown in Sequence 1 in the Sequence Listing can be linked with the tags shown in Table 1.

Sequence of Table 1 Tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

The protein in (a) or (b) above can be synthesized artificially, or its coding gene can be synthesized first, and then biologically expressed. The gene encoding the protein in the above (b) can be deleted by deleting one or several amino acid residue codons in the DNA sequence shown in the sequence 2 from the 1st to 1710th nucleotides of the 5' end in the sequence listing, and/or Or carry out a missense mutation of one or several base pairs, and/or connect the coding sequence of the tag shown in Table 1 to its 5' end and/or 3' end.

Recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing any of the above-mentioned coding genes also belong to the protection scope of the present invention.

The recombinant vector is a recombinant expression vector obtained by inserting any of the above-mentioned coding genes into the multiple cloning site of the departure vector pET28a; The obtained recombinant expression vector was obtained by transforming Escherichia coli Rosetta (DE3).

An existing expression vector can be used to construct a recombinant expression vector containing the gene. When using the gene to construct a recombinant expression vector, any enhanced promoter or constitutive promoter can be added before its transcription initiation nucleotide, and they can be used alone or in combination with other promoters; in addition, When using the gene of the present invention to construct a recombinant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be consistent with the coding The reading frames of the sequences are identical to ensure correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene.

Another object of the present invention is to provide a method for preparing halophilic cellulase.

The method for preparing halophilic cellulase provided by the present invention is to cultivate any of the above-mentioned recombinant bacteria to obtain the protein.

The method for preparing halophilic cellulase provided by the present invention can specifically be: culture the recombinant bacteria at 37°C for 3 hours, when OD ₆₀₀ =0.7-1.0, add IPTG to a final concentration of 0.1-0.8mM, and transfer to 18°C Continue to cultivate for 4-20h.

A pair of primers for amplifying the full length of any of the above-mentioned coding genes and any fragment thereof also falls within the protection scope of the present invention.

One primer sequence in the primer pair is shown as sequence 3 in the sequence listing, and the other primer sequence is shown as sequence 4 in the sequence listing.

The application of any of the above-mentioned proteins, any of the above-mentioned coding genes, any of the above-mentioned recombinant expression vectors, the above-mentioned expression cassettes, transgenic cell lines or recombinant bacteria in degrading cellulose also belongs to this invention. protection scope of the invention. Wherein, the cellulose may specifically be carboxymethyl cellulose.

In the above application, the carboxymethyl cellulose can be a soluble salt of carboxymethyl cellulose, specifically sodium carboxymethyl cellulose; the protein degrades carboxymethyl cellulose under high-salt conditions (0.5-4M) In the case of plain sodium, the temperature of the reaction system is 20-70° C., preferably 55° C., and the pH value of the reaction system is 4-10, preferably 5.5; the salt concentration in the reaction system is shown in 1) or 2) below: 1) The salt is NaCl, and the salt concentration in the reaction system is 0.25-4M, preferably 2.5M; 2) the salt is KCl, and the salt concentration in the reaction system is 0.25-3.5M, preferably 3M.

The CelB protein of the present invention has halophilic cellulase activity and belongs to halophilic cellulase. Compared with the amino acid sequences of other characterized cellulases, the CelB protein has a similarity of less than 55%, and belongs to a member of the cellulase family. Cellulase of the present invention still has higher enzymatic activity under the condition of high salt (2.5M sodium chloride), and is 33U/mg protein dry powder, and general cellulase has already been obtained under the condition of 2.5M sodium chloride Complete or partial denaturation, complete or partial loss of enzyme activity. Under the condition of 2.5M sodium chloride, in the temperature range of 40 ℃-70 ℃, the enzyme of the present invention has higher activity, and the optimum temperature is 55 ℃; Within the pH value range of pH 10, the enzyme of the present invention has relatively high activity, and the optimum pH value is 5.5. Therefore, the enzyme of the present invention has wide pH tolerance, wide applicable temperature range and salt-tolerant characteristics. The enzyme of the present invention has a wide range of applications, and is particularly suitable for applications in high-salt environments. Fibers under hypertonic and high-salt conditions It has a good application prospect in the field of degradation of toxins.

Description of drawings

Figure 1 is the SDS-PAGE electrophoresis of CelB protein.

Figure 2 is the change of CelB protein activity with salt concentration

Figure 3 is the change of halophilic cellulase activity with temperature.

Figure 4 shows the change of halophilic cellulase activity with pH.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Genetic resource name: Bacillus sp.BG-CS10. Direct source: Acquisition time in 2004; Collection method: Collection place (country, province (city)) Barjian Lake, Inner Mongolia, name (name) of the collector Xue Yanfen, contact information of the collector e-mail: xueyfim.ac.cn.

Embodiment 1, the discovery of halophilic cellulase

1. Extraction of total DNA from Bacillus sp.BG-CS10

Using Bacillus sp.BG-CS10, get 20 grams of fresh wet cells, suspend in 10 ml of 50mM Tris buffer (pH8.0), add a small amount of lysozyme and 8 ml of 0.25mM EDTA (pH8.0), mix After homogenization, place at 37°C for 20 minutes; then add 2 ml of 10% SDS, place at 55°C for 5 minutes, and extract once with equal volumes of phenol and chloroform respectively; take the last supernatant solution, add 2 times the volume of ethanol, recover DNA, and separate Wash with 70% and absolute ethanol; dissolve the precipitate in 0.5ml TE buffer (pH8.0, 10mM Tris, 1mMEDTA), add 10mg/ml RNase 3μl, keep warm at 37°C for 1 hour, and extract with equal volumes of phenol and chloroform respectively Extract once; add 2 times the volume of ethanol to the supernatant solution, recover DNA, wash with 70% and absolute ethanol respectively, dry in vacuum, and dissolve with deionized water.

2. Discovery of halophilic cellulase

1. Take 10 μl of the total DNA solution (about 50 μg DNA), partially digest it with the restriction endonuclease Sau3AI, and recover the 3-9kb DNA fragment by agarose gel electrophoresis.

2. Ligation reaction for 16 hours

Ligation system (20μl): 2μl (5μg) Sau3AI digested DNA fragment;

1 μl (1 μg) of EarI-digested plasmid pHBM803DNA;

2 μl 10× ligation buffer;

1 μl T4 DNA ligase;

14 μl of water.

3. Transform competent Escherichia coli XL-Gold with the product of the ligation reaction, and then spread it on a solid medium containing 50 μg/ml Amp (ampicillin), culture it at 37°C for 16-18 hours, and copy the obtained colony to a medium containing 50 μg/ml Amp (Ampicillin), 0.5mM IPTG, 0.025% Triben blue and 1% konjac flour on the solid medium, the colony with a transparent circle around it is a positive clone.

4. Positive clones were cultured in Amp-LB medium at 37°C for 16-18 hours, and had cellulase activity after the activity test.

Recombinant plasmids in positive clones were sequenced. Sequencing results showed that in the recombinant plasmid, a DNA fragment was inserted into the pHBM803 DNA backbone, and the DNA fragment contained an open reading frame (ORF) with a length of 1710 bp. The 1st to 99th nucleotides at the 'end are signal peptides. See references for plasmid pHBM803 (Cloning and enzymatic characterization of a xylanase gene from a soil-derived metagenomic library with an efficient approach. Applied Microbial Biotechnology, 2008, 80(5): 823-30).

The protein encoded by the nucleotides shown in Sequence 2 is shown in Sequence 1 in the Sequence Listing (referred to as CelB protein), and the protein consists of 569 amino acid residues. The CelB protein belongs to the cellulase superfamily, and is 80% identical to the cellulase B derived from Bacillus agaradhaerens, and is 68% identical to the cellulase B of the whole genome sequenced Bacillus halodurans C-125 strain; and Geobacillu sp.Y412MC10 The identity of cellulase is 55%, but the enzymatic properties of these proteins have not been analyzed; the gene encoding CelB protein is named as celB gene.

Embodiment 2, expression of halophilic cellulase

1. Preparation of celB gene

The following method can be used to prepare the celB gene; the celB gene shown in the 100th to 1710th nucleotides from the 5' end of the sequence 2 in the sequence listing can also be obtained by artificial synthesis, and the two ends of the celB gene are respectively equipped with BamHI Restriction site and SalI restriction site.

According to the nucleotide sequence of celB gene (as shown in the sequence 2 of sequence listing), design primer pair as follows:

Forward primer: 5′-cgcggatccgatgaaggtgctaagcaaacagatattc-3′;

Reverse primer: 5′-agcgtcgacttatcgtctaccttgaacatggtttcc-3′;

The underlined part of the forward primer is the restriction site of BamHI, and the underlined part of the reverse primer is the restriction site of SalI.

Using the total DNA of Bacillus sp.BG-CS10 as a template, PCR amplification was performed with the designed primer pair.

PCR reaction system:

10× buffer 5 μl

dNTP 4μl

exTaq DNA polymerase 0.5μl

Forward primer 1 μl

Reverse primer 1μl

Template 0.5μl

Water 38 μl.

PCR reaction conditions: pre-denaturation at 94°C for 5 minutes, then denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 2 minutes, 30 cycles, and finally extension at 72°C for 10 minutes.

The yield and specificity of the PCR product were detected by 1% agarose gel electrophoresis, and purified with a DNA purification kit (ultra-thin spin column type, produced by Tiangen Company). The purified PCR product was sequenced, and the result showed that the sequence of the PCR product was as shown in the 100th to 1710th nucleotides from the 5' end of sequence 2 in the sequence listing.

2. Construction of recombinant expression vector

1. Digest the correctly sequenced PCR products or artificially synthesized fragments with BamHI and SalI, and recover the digested products by agarose electrophoresis.

2. Digest the plasmid pET28a (Cat.N0 69864-3, Novogen) with BamHI and SalI, and recover the digested product by agarose electrophoresis.

3. Ligate the digested product of step 1 and the digested product of step 2, transform the ligated product into Escherichia coli DH5α by electric shock, spread it on an LB plate containing 50 μg/ml kanamycin, and culture it overnight at 37°C. The transformants were colony PCRed with forward and reverse primers, and the recombinant bacteria containing the celB gene were screened, and the plasmids of the recombinant bacteria were extracted for sequencing verification. The results showed that the BamHI and SalI restriction sites of pET28a were inserted between The insertion direction of the celB gene shown in the 100th to 1710th nucleotides from the 5' end of Sequence 2 in the sequence listing is correct, and the recombinant plasmid is named pET28a-celB.

3. Preparation of Engineering Bacteria

The plasmid pET28a-celB was transformed into Escherichia coli Rosetta (DE3) (Cat.NOCD801, Quanshijin Company) by electroporation, and spread on an LB plate containing 50 μg/ml kanamycin, and cultured overnight at 37°C to obtain the plasmid containing pET28a-celB The engineered bacterium was denoted as Rosetta/pET28a-celB.

Replace pET28a-celB with pET28a, transform Escherichia coli Rosetta (DE3), and use the same procedure as above to obtain a recombinant bacterium containing pET28a as a control bacterium. The positive recombinant bacteria transformed into pET28a were recorded as Rosetta/pET28a.

4. Preparation and purification of halophilic cellulase

Ni-NTA His·Bind Superflow purification column was purchased from Novagen, the product catalog number is 70971-3.

The positive recombinant Rosetta/pET28a-celB prepared in step 3 was cultured in LB medium containing 50 μg/ml kanamycin, and cultured at 37°C for 3 hours; when OD ₆₀₀ =0.7, IPTG was added to its concentration in LB medium The final concentration was 0.8mM, and the culture was continued at 18°C for 16h.

Collect the bacteria by centrifugation at 5000rpm for 10min, suspend in solution A (20mM Tris-HCl, pH7.9, 0.5MNaCl, 10mM imidazole), ultrasonically break in an ice bath (60w, 10min; ultrasonic 2s, stop 2s), then 15000rpm Centrifuge for 10min to remove cell debris, take the supernatant; pass the supernatant through the Ni-IDA His BindSuperflow purification column, wash with 5ml solution A, and then wash with 10ml solution B (20mM Tris-HCl, pH7.9, 0.5M NaCl, 60mM imidazole), and finally eluted with 5ml solution C (20mM Tris-HCl, pH7.9, 0.5MNaCl, 500mM imidazole), and the eluate was collected. Then, the eluate was desalted by FPLC (fast protein liquid chromatography) to obtain a solution of purified CelB protein, which was freeze-dried in vacuum to obtain CelB protein dry powder.

The control bacteria prepared in step 3 were cultured and purified in the same steps, and the obtained solution was used as the control enzyme solution.

SDS-PAGE electrophoresis showed that the molecular weight of the purified CelB protein was about 62kDa, which was in line with the theoretical deduction of 62kDa. The results are as shown in Figure 1. In Figure 1, swimming lane 1 represents the CelB protein after Ni-NTA column purification, and swimming lane 2 represents the supernatant after Escherichia coli Rosetta/pET28a-celB breaks bacteria, and swimming lane 3 represents protein molecular weight standard (100 , 75, 50, 35, 25kDa).

The control bacteria did not obtain the target protein.

Example 3, functional verification of protein

The enzyme activity unit is defined as the amount of enzyme required to catalyze the production of 1 μmol of glucose in 1 min.

(1) Effect of salt on enzyme activity

Experimental group 1: the activity measurement reaction system is 0.5ml, is made up of the dilution of the CelB protein dry powder that 0.48ml solution A and 0.02ml embodiment 2 obtain; The pH value of activity measurement reaction system is 8.8 (this pH buffer system is affected by NaCl concentration little impact). After incubating the reaction system at 40° C. for 30 minutes, 0.5 ml of dinitrosalicylic acid solution (DNS) was added to terminate the reaction, and then the absorbance value at 520 nm was measured after 5 minutes in a boiling water bath. The same operation was carried out by using Tris-HCl buffer solution with a pH value of 8.8 instead of the CelB protein dilution solution as control 1.

The composition of solution A: by the Tris-HCl buffer solution of 50mM pH8.8, NaCl and sodium carboxymethyl cellulose (CMC); The concentration of NaCl in solution A is 0.05-4M, and sodium carboxymethyl cellulose is in The concentration in solution A is 0.5g/100ml.

Experimental group 2: the activity measurement reaction system is 0.5ml, is made up of the dilution of the CelB protein dry powder that 0.48ml solution A and 0.02ml embodiment 2 obtain; The pH value of activity measurement reaction system is 8.8 (this pH buffer system is affected by KCl concentration little impact). After incubating the reaction system at 40° C. for 30 minutes, 0.5 ml of dinitrosalicylic acid solution (DNS) was added to terminate the reaction, and then the absorbance value at 520 nm was measured after 5 minutes in a boiling water bath. The same operation was performed by using Tris-HCl buffer solution with a pH value of 8.8 instead of the CelB protein dilution solution as control 2.

The composition of solution A: by the Tris-HCl buffer solution of 50mM pH8.8, KCl and sodium carboxymethylcellulose (CMC); The concentration of KCl in solution A is 0.5-3.5M, sodium carboxymethylcellulose The concentration in solution A is 0.5g/100ml.

Control 3: The activity measurement reaction system is 0.5ml, which is composed of 0.48ml solution A and 0.02ml dilution of CelB protein dry powder obtained in Example 2; the pH value of the activity measurement reaction system is 8.8. After incubating the reaction system at 40° C. for 30 minutes, 0.5 ml of dinitrosalicylic acid solution (DNS) was added to terminate the reaction, and the absorbance value at 520 nm was measured after 5 minutes in a boiling water bath.

The composition of solution A: consists of 50mM Tris-HCl buffer solution with pH 8.8 and sodium carboxymethylcellulose (CMC); the concentration of sodium carboxymethylcellulose in solution A is 0.5g/100ml. The solution does not contain NaCl or KCl.

The absorbance value measured without NaCl or KCl (ie, control 3) was regarded as 100% of relative activity, and the ratio of the absorbance value of other groups to this absorbance value was regarded as their respective relative activities.

The experiment was repeated 3 times. The change of CelB protein solution activity with salt concentration is shown in Figure 2. In an environment with a pH value of 8.8, the CelB protein solution has the highest enzyme activity when it contains 2.5M NaCl or 3M KCl, which is more than ten times higher than that without salt. Under the condition of salt concentration of 0.5-4M, the protein has relatively high activity, indicating that the protein has halophilic properties.

No enzyme activity was detected in both Control 1 and Control 2.

The above experiment was carried out with the protein obtained from the control bacteria Rosetta/pET28a in Example 2 (referred to as the control enzyme solution). As a result, no matter what the salt concentration was, the control enzyme solution had no activity.

The above experiments prove that the CelB protein of the present invention has cellulase activity, is a cellulase, and has halophilic properties, and is a halophilic cellulase.

(2) Optimum temperature

Experimental group: The activity measurement reaction system is 0.5ml, which is composed of 0.48ml solution A and 0.02ml dilution of CelB protein dry powder obtained in Example 2; the pH value of the reaction system is 8.8; after incubating the reaction system at a specific temperature for 30min , adding 0.5ml of dinitrosalicylic acid solution (DNS) to terminate the reaction, and then measuring the absorbance at 520nm after 5min in a boiling water bath.

The composition of solution A: by the Tris-HCl buffer solution of 50mM pH8.8, NaCl and sodium carboxymethylcellulose (CMC); The concentration of NaCl in solution A is 2.5M, sodium carboxymethylcellulose in solution The concentration in A is 0.5g/100ml.

Control group: the activity measurement reaction system is 0.5ml, which is composed of 0.48ml solution A and 0.02ml dilution of CelB protein dry powder obtained in Example 2; the pH value of the reaction system is 8.8; after incubating the reaction system at a specific temperature for 30min , adding 0.5ml of dinitrosalicylic acid solution (DNS) to terminate the reaction, and then measuring the absorbance at 520nm after 5min in a boiling water bath.

The composition of solution A: consists of 50mM Tris-HCl buffer solution with pH 8.8 and sodium carboxymethylcellulose (CMC); the concentration of sodium carboxymethylcellulose in solution A is 0.5g/100ml. The solution does not contain sodium chloride.

The experiment was repeated 3 times.

When containing 2.5M NaCl, at 55°C, the halophilic cellulosic enzyme solution has the highest enzyme activity, which is 20U/mg protein dry powder. The absorbance of the system with the highest enzyme activity was taken as 100% of the relative activity, and the ratio of the absorbance of the other reaction systems to the absorbance of the system with the highest enzyme activity was taken as their respective relative activities.

The change of halophilic cellulase activity with temperature is shown in Figure 3. In an environment with a pH value of 8.8, under salt-free conditions, the cellulase solution has the highest enzyme activity at 35°C; under the condition of 2.5M NaCl, at 40°C-70°C, CelB protein (halophilic fiber CelB protein (halophilic cellulase solution) has the best enzymatic activity at 55°C.

The above experiment was carried out with the protein obtained from the control bacteria Rosetta/pET28a in Example 2 (referred to as the control enzyme solution). As a result, no matter under which temperature conditions, the control enzyme solution had no activity.

(3) Optimum pH

Experimental group: the activity assay system is 0.5ml, consisting of 0.48ml solution B (B1, B2, B3, B4, B5, B6 or B7) and 0.02ml dilution of CelB protein dry powder obtained in Example 2;

The composition of solution B1: consists of 50mM Na ₂ HPO ₄ -citrate buffer, NaCl and sodium carboxymethylcellulose (CMC); the concentration of NaCl in solution B1 is 2.5M, sodium carboxymethylcellulose in solution The concentration in B1 is 0.5 g/100 ml; the pH of solution B1 is 4.0.

Composition of solution B2: the same composition as solution B1, the difference is that the pH value of solution B2 is 5.0.

The composition of solution B3: the same composition as solution B1, the difference is that the pH value of solution B2 is 6.0, and the 50mM Na ₂ HPO ₄ -citric acid buffer is replaced by 50mM NaH ₂ PO ₄ -Na ₂ HPO ₄ buffer .

The composition of solution B4: the same composition as solution B1, the difference is that the pH value of solution B4 is 7.0, 50mM Na ₂ HPO ₄ -citric acid buffer is replaced by 50mM NaH ₂ PO ₄ -Na ₂ HPO ₄ buffer .

Composition of solution B5: the same composition as solution B1, except that the pH of solution B5 is 8.0, and 50 mM Na ₂ HPO ₄ -citric acid buffer is replaced by 50 mM Tris-HCl buffer.

Composition of solution B6: the same composition as solution B1, except that the pH of solution B6 is 9.0, and 50 mM Na ₂ HPO ₄ -citric acid buffer is replaced by 50 mM Glycine-NaOH buffer.

Composition of solution B7: the same composition as solution B1, except that the pH of solution B7 is 10.0, and 50 mM Na ₂ HPO ₄ -citric acid buffer is replaced by 50 mM Glycine-NaOH buffer.

After incubating the reaction system at 55° C. for 30 minutes, 0.5 ml of dinitrosalicylic acid solution (DNS) was added to terminate the reaction, and the absorbance at 520 nm was measured after 5 minutes in a boiling water bath.

Control group: The difference between the reaction system and the experimental group is that the control group does not contain NaCl, and the reaction is carried out at 35°C.

The experiment was repeated 3 times.

In the experimental group, when the pH value of the reaction system was 5.5, the halophilic cellulosic enzyme liquid had the highest enzyme activity, which was 33U/mg protein dry powder. The absorbance of the system with the highest enzyme activity was taken as 100% of the relative activity, and the ratio of the absorbance of the other reaction systems to the absorbance of the system with the highest enzyme activity was taken as their respective relative activities.

In the control group, when the pH value of the reaction system was 5.5, the halophilic cellulosic enzyme solution had the highest enzyme activity, which was 3U/mg protein dry powder. The absorbance of the system with the highest enzyme activity was taken as 100% of the relative activity, and the ratio of the absorbance of the other reaction systems to the absorbance of the system with the highest enzyme activity was taken as their respective relative activities.

The change of halophilic cellulase activity with pH is shown in Figure 4 (A represents the experimental group, B represents the control group). Results: Under the condition of high salt (2.5M NaCl) or no salt, CelB protein (halophilic cellulosic enzyme solution) was active at 55°C, pH4-pH10, and CelB protein (halophilic cellulosic enzyme solution) was active at pH5.5. Suzyme solution) has the best enzyme activity. This shows that salt has little effect on the optimum pH of the enzyme.

The above experiment was carried out with the protein obtained from the control bacteria Rosetta/pET28a in Example 2 (referred to as the control enzyme solution). As a result, the control enzyme solution had no activity regardless of the pH conditions.

(4) Enzyme thermostability

After diluting the enzyme with buffer containing 2.5M NaCl and without NaCl, respectively, place it in a water bath at 30, 40, 50, and 60°C for 15 and 30 minutes, and measure the residual activity of the enzyme. The results show that the enzyme has good thermal stability in the presence of 2.5M NaCl, and there is no decrease in enzyme activity when treated at 60°C for 30 minutes; the enzyme is stable at 30°C and 40°C without NaCl, but it is completely stable at 50°C for 15 minutes. Lost activity. This suggests that salt can increase the thermostability of the enzyme.

Sequence Listing

<110>Institute of Microbiology, Chinese Academy of Sciences

<120>A kind of halophilic cellulase and its coding gene and application

<160>4

<210>1

<211>569

<212>PRT

<213> Bacillus sp.

<400>1

Met Val His Ile Gln Arg Lys Phe Ile Leu Thr Lys Ser Leu Ile Val

1 5 10 15

Val Phe Thr Met Leu Leu Cys Leu Ser Leu Met Thr Pro Pro Leu Leu

20 25 30

Ala Asp Glu Gly Ala Lys Gln Thr Asp Ile Gln Ser Tyr Val Ala Asp

35 40 45

Met Gln Pro Gly Trp Asn Leu Gly Asn Thr Phe Asp Ala Val Gly Asp

50 55 60

Asp Glu Thr Ala Trp Gly Asn Pro Arg Val Thr Arg Glu LeuIle Lys

65 70 75 80

Thr Ile Ala Asp Glu Gly Tyr Lys Ser Ile Arg Ile Pro Val Thr Trp

85 90 95

Glu Asn Gln Met Gly Asn Ala Pro Asp Tyr Thr Ile Asn Glu Asp Phe

100 105 110

Phe Ser Arg Val Glu Gln Val Ile Asp Trp Ala Leu Glu Glu Asp Leu

115 120 125

Tyr Val Met Leu Asn Leu His His Asp Ser Trp Leu Trp Ile Tyr Asn

130 135 140

Met Glu His Asn Tyr Asp Glu Val Met Ala Lys Tyr Thr Ala Leu Trp

145 150 155 160

Glu Gln Leu Ser Glu Arg Phe Gln Gly His Ser His Lys Leu Met Phe

165 170 175

Glu Ser Val Asn Glu Pro Arg Phe Thr Arg Asp Trp Gly Glu Ile Gln

180 185 190

Glu Asn His His Ala Phe Leu Glu Glu Leu Asn Thr Ala Phe Tyr His

195 200 205

Ile Val Arg Glu Ser Gly Gly Ser Asn Thr Glu Arg Pro Leu Val Leu

210 215 220

Pro Thr Leu Glu Thr Ala Thr Ser Gln Asp Leu Leu Asn Arg Leu His

225 230 235 240

Gln Thr Met Lys Asp Leu Asn Asp Pro Asn Leu Ile Ala Thr Val His

245 250 255

Tyr Tyr Gly Phe Trp Pro Phe Ser Val Asn Val Ala Gly Tyr Thr Arg

260 265 270

Phe Glu Glu Glu Thr Gln Gln Asp Ile Ile Asp Thr Phe Asn Arg Val

275 280 285

His Asn Thr Phe Thr Ala Asn Gly Ile Pro Val Val Leu Gly Glu Phe

290 295 300

Gly Leu Leu Gly Phe Asp Thr Ser Thr Asp Val Ile Gln Gln Gly Glu

305 310 315 320

Lys Leu Lys Phe Phe Glu Phe Leu Ile His His Leu Asn Glu Arg Asp

325 330 335

Val Thr His Met Leu Trp Asp Asn Gly Gln His Leu Asn Arg Glu Thr

340 345 350

Tyr Ser Trp Tyr Asp Gln Glu Phe His Asn Met Leu Lys Ala Ser Trp

355 360 365

Glu Gly Arg Ser Ala Thr Ala Glu Ser Asn Leu Ile His Val Arg Asp

370 375 380

Gly Glu Pro Ile Arg Asp Gln Asp Ile Gln Leu His Leu His Gly Asn

385 390 395 400

Glu Leu Thr Gly Leu Gln Val Asp Gly Asp Ser Leu Ala Leu Gly Glu

405 410 415

Asp Tyr Glu Leu Ala Gly Asp Val Leu Thr Met Lys Ala Asp Ala Leu

420 425 430

Thr Ala Leu Met Thr Pro Gly Glu Leu Gly Thr Asn Ala Val Ile Thr

435 440 445

Ala Gln Phe Asn Ser Gly Ala Asp Trp His Phe Gln Leu Gln Asn Val

450 455 460

Asp Glu Pro Thr Leu Glu Asn Thr Glu Gly Ser Thr Ser Asn Phe Ala

465 470 475 480

Ile Pro Ala His Phe Asn Gly Asp Ser Val Ala Thr Met Glu Ala Val

485 490 495

Tyr Ala Asn Gly Glu Phe Ala Gly Pro Gln Asn Trp Thr Ser Phe Lys

500 505 510

Glu Phe Gly Tyr Thr Phe Ser Pro Val Tyr Asp Lys Gly Glu Ile Val

515 520 525

Ile Thr Asp Ala Phe Phe Asn Glu Val Arg Asp Asp Asp Ile His Leu

530 535 540

Thr Phe His Phe Trp Ser Gly Glu Met Val Glu Tyr Thr Leu Ser Lys

545 550 555 560

Asn Gly Asn His Val Gln Gly Arg Arg

565

<210>2

<211>1710

<212>DNA

<213> Bacillus sp.

<400>2

atggtacaca tccaaagaaa gttcatactg acaaaatctc taatagtcgt gttcacaatg 60

cttttgtgtt tgtcattaat gactccccca ttgttagctg atgaaggtgc taagcaaaca 120

gatattcaat cttatgtagc tgacatgcag cctggttgga atttaggaaa cacgtttgac 180

gctgttggcg atgatgaaac agcttggggg aatcctcgtg tgacgagaga gttaataaaa 240

acgattgctg atgaagggta taaaagtatt cgtatccccag tgacatggga aaatcagatg 300

gggaatgcgc cagattatac gataaatgaa gattttttta gccgtgtgga gcaagtgatc 360

gattgggcgt tggaagaaga cttatatgtc atgttaaatc tgcatcatga ttcatggctg 420

tggatatata atatggaaca caattacgat gaagtgatgg ctaaatatac agctctttgg 480

gagcagttat cagagcgttt tcaaggccat tctcataaat taatgtttga gagtgtcaat 540

gagcctcggt ttacgcgaga ttggggagag attcaagaaa atcaccatgc tttcttagaa 600

gagttaaata cggcatttta tcatattgtc agagagtcgg gaggaagtaa tacagaacgg 660

cctctagttt taccaacatt agaaacagca acgtcacagg atttactcaa tcggttgcat 720

caaacgatga aagacttaaa tgatccaaac ttgatagcca cagttcatta ttatggcttc 780

tggccatta gtgtaaatgt agcaggttac accccgttttg aggaggagac acaacaggat 840

atcatagaca cgtttaatcg tgttcataac acatttacag cgaatggcat tcctgtcgta 900

ttgggtgaat ttggcttgtt aggctttgat acaagtacag acgtcattca gcaaggtgag 960

aaattaaaat tttttgagtt tctcatccat catctcaatg aacgtgatgt aacccatatg 1020

ttatgggaca acggtcagca tttaaatcga gaaacttatt catggtatga tcaagaattt 1080

cataacatgc taaaagcgag ctgggaagga cgttctgcta cagcagagtc taatttaatt 1140

catgtgaggg acggggagcc aattagagac caagatatac agcttcattt acacggaaat 1200

gagctaacag gtttacaggt agatggagat tcgcttgctc taggggga ttatgagcta 1260

gcgggatg tgctaacgat gaaagcggac gccctaacag cactaatgac acctggagag 1320

ttagggacca atgccgtcat aacagctcaa tttaattctg gagctgactg gcattttcaa 1380

ctacagaatg tagacgaacc aactttagaa aatacggaag gctcaacttc gaatttcgca 1440

atacctgctc attttaatgg ggatagtgtt gcgacgatgg aagctgttta tgcgaatggg 1500

gaatttgccg gaccacaaaa ttggacatcg tttaaagaat tcggttatac cttttcacct 1560

gtttacgaca agggagaaat tgtcataaca gacgcattct ttaacgaggt acgcgatgat 1620

gacattcat taacctttca tttttggagc ggggagatgg tggaatatac attaagtaaa 1680

aatggaaacc atgttcaagg tagacgataa 1710

<210>3

<211>37

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>3

cgcggatccg atgaaggtgc taagcaaaca gatattc 37

<210>4

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>4

agcgtcgact tatcgtctac cttgaacatg gtttcc 36

Claims (16)

1. A protein, which is a protein as shown in (a) or (b) below: (a) a protein consisting of the amino acid sequence shown in amino acid residues 34-569 from the N-terminus in Sequence 1 in the sequence listing; (b) A protein consisting of the amino acid sequence shown in Sequence 1 in the Sequence Listing. 2. The gene encoding the protein of claim 1. 3. The coding gene according to claim 2, characterized in that: the coding gene is the following 1) or 2) DNA molecule: 1) The nucleotide sequence is the DNA molecule shown in the 100th to 1710th nucleotides from the 5' end of Sequence 2 in the sequence listing; 2) The nucleotide sequence is the DNA molecule shown in Sequence 2 in the sequence listing. 4. A recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the coding gene of claim 2 or 3. 5. The recombinant vector according to claim 4, characterized in that: the recombinant vector is a recombinant expression vector obtained by inserting the coding gene of claim 2 or 3 into the multiple cloning site of the departure vector pET28a. 6. recombinant bacterium according to claim 4, is characterized in that: described recombinant bacterium is the recombinant expression vector transformation Escherichia coli Rosetta ( DE3) obtained. 7. A method for preparing the protein according to claim 1, comprising culturing the recombinant bacteria according to claim 6 to obtain the protein. 8. Amplify the full-length primer pair of the described coding gene of claim 2 or 3, it is characterized in that: a primer sequence in the primer pair is as shown in sequence 3 in the sequence listing, and another primer sequence is as shown in the sequence listing Sequence 4 is shown. 9. The application of the protein described in claim 1 in degrading cellulose. 10. The application of the coding gene according to claim 2 or 3 in degrading cellulose. 11. The use of the recombinant vector according to claim 4 or 5 in degrading cellulose. 12. The application of the recombinant bacteria described in claim 4 or 6 in degrading cellulose. 13. The application of the expression cassette according to claim 4 in degrading cellulose. 14. The application of the transgenic cell line according to claim 4 in degrading cellulose. 15. The application according to any one of claims 9-14, characterized in that: the cellulose is carboxymethyl cellulose; the degradation conditions include: the temperature of the reaction system is 20-70°C, the reaction system The pH value of the reaction system is 4-10; the salt concentration in the reaction system is as follows 1) or 2): 1) the salt is NaCl, and the salt concentration in the reaction system is 0.25-4M; 2) the salt is KCl, the reaction The salt concentration in the system is 0.25-3.5M. 16. The application according to claim 15, characterized in that: the degradation conditions include: the temperature of the reaction system is 55°C, the pH value of the reaction system is 5.5; the salt concentration in the reaction system is as follows 1) or 2) Shown: 1) the salt is NaCl, and the salt concentration in the reaction system is 2.5M; 2) the salt is KCl, and the salt concentration in the reaction system is 3M.

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