CN109280672B

CN109280672B - Recombinant cellulose endonuclease gene and its protein and protein preparing process

Info

Publication number: CN109280672B
Application number: CN201811240711.0A
Authority: CN
Inventors: 李洪波; 胡兴; 江素珍; 米丹; 张赛名
Original assignee: Huaihua University
Current assignee: HUNAN BUSKY PHARMACEUTICAL Co.,Ltd.
Priority date: 2018-10-23
Filing date: 2018-10-23
Publication date: 2020-10-30
Anticipated expiration: 2038-10-23
Also published as: CN109280672A

Abstract

The invention relates to a recombinant cellulose endonuclease gene, wherein the nucleotide sequence of the gene is shown as SEQ ID NO.1, and the encoded protein is shown as SEQ ID NO. 2. The invention also provides a method for efficiently expressing and purifying the recombinant protein, the purity of the purified protein is as high as 95%, the protein concentration is higher, and the protein has enzyme activity for hydrolyzing cellulose to generate glucose, so that the protein has important industrial value for the industries of production of biofuel ethanol, food, feed and the like.

Description

Recombinant cellulose endonuclease gene and its protein and protein preparing process

Technical Field

The invention belongs to the field of biological gene engineering, and particularly relates to a recombinant cellulose endonuclease gene, and also relates to a protein encoded by the gene and a preparation method of the protein.

Background

Cellulose is a polysaccharide composed of glucose with beta-1, 4 glycosidic bonds, is an important component for constituting plant cell walls, and is the most abundant renewable resource on the earth at present. At present, the utilization rate of cellulose is very low, and how to improve the utilization rate of cellulose is still a world-level topic. Efficient use of cellulose is not required to degrade cellulose-related enzymes. Cellulase belongs to glycoside hydrolase, is a general name of enzymes specially used for catalyzing and hydrolyzing beta-1, 4-glycosidic bond in cellulose chain, is a high-activity biocatalyst, and can decompose cellulose to produce glucose. Cellulases can be classified into two classes according to their structure: cellulase complex and non-complex cellulases. The cellulase complex is a multienzyme protein complex with a supramolecular structure and is composed of a plurality of subunits. The non-complex cellulase consists of endoglucanase, exoglucanase and beta-glucanase. Non-complex cellulases are produced primarily by aerobic filamentous fungi, which are the most important sources of enzymes for the breakdown of cellulose.

With the gradual depletion of petroleum and coal resources, how to more effectively convert and utilize cellulose, a renewable organic resource and polysaccharide substance which are distributed most widely, most abundantly and cheapest in nature, is an important research field concerned by China. Therefore, the cellulose is degraded by the cellulase and is converted into fuels, foods and chemical products, such as sugar, ethanol, feed protein and the like, so that the cellulase has great economic significance for relieving energy crisis and shortage of food and feed resources, and the cellulase plays an important role in the fields of food, feed, environmental protection, energy and resource development and the like. Although a great variety of organisms producing cellulase, such as bacteria, fungi, actinomycetes, insects, mollusks and the like, are found at present, the demand of the cellulase is increasing day by day as the cellulase has wide requirements in the fields of industry, agriculture, livestock, medicine and the like, and the cellulase preparation is short of supply and has a very broad prospect. However, the industrial preparation of cellulase in China is still in the research and development stage, and the application of cellulase is limited due to the problems of low cellulase activity, high production cost, long production period and the like in the production of cellulase, so the bottleneck of mass production of cellulase needs to be overcome. The action mechanism of the cellulase is deeply researched, the molecular biology research on the cellulase is strengthened, and particularly, the application of a DNA gene recombination technology is fully utilized to produce the recombinant protein with high enzyme activity. The cellulase system comprises 3 enzymes of endonuclease, exonuclease and glycosidase. Among them, endocellulase plays an important role in the decomposition of cellulose. The cellulase sold in the market at present is a mixture of various enzymes, and related products and technologies for obtaining the cellulose endonuclease with high maximum purity and high activity by using genetic engineering means are few.

Disclosure of Invention

In view of the above, the present invention aims to provide a recombinant cellulase gene and a protein encoded by the gene, and also provides a method for constructing a recombinant vector containing a gene sequence of SEQ ID No.1, and further provides a method for efficiently expressing and purifying the recombinant protein.

In order to achieve the purpose, the invention provides the following technical scheme:

1. a recombinant endonuclease encoding gene comprising a DNA segment of at least one of the following nucleotide sequences:

1) the nucleotide sequence of SEQ ID NO.1 in the sequence table;

2) a nucleotide sequence which has more than 95 percent of homology with the nucleotide sequence shown in SEQ ID NO.1 and codes the protein with the same biological function;

3) a nucleotide sequence which is hybridized with the nucleotide sequence shown in SEQ ID NO.1 and encodes the protein with the same biological function.

Furthermore, the conditions for hybridizing with the nucleotide sequence shown in SEQ ID NO.1 are as follows: hybridization at 50 ℃ in a mixed solution of 7% Sodium Dodecyl Sulfate (SDS), 0.5M Na3PO4, and 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃;

hybridization conditions were also 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS;

hybridization conditions were also 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃;

hybridization conditions were also 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃;

hybridization conditions were also 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃;

hybridization conditions may also be such that hybridization is carried out in a solution of 6 XSSC, 0.5% SDS at 65 ℃ and then the membranes are washed once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

2. Wherein the nucleotide sequence shown in SEQ ID NO.1 consists of 1020 deoxynucleotides, the sequence is a full-length cDNA reading frame of cellulose Endonuclease (EG), and the protein of the amino acid sequence shown in SEQ ID NO.2 in a coding sequence table comprises 340 amino acid residues.

1) The nucleotide sequence of SEQ ID NO.1 in the sequence table;

2) a nucleotide sequence which has more than 90 percent of homology with the nucleotide sequence shown in SEQ ID NO.1 and codes the protein with the same biological function;

or a nucleotide sequence which has more than 95 percent of homology with the nucleotide sequence shown in SEQ ID NO.1 and codes the protein with the same biological function;

or a nucleotide sequence which has more than 98 percent of homology with the nucleotide sequence shown in SEQ ID NO.1 and codes the protein with the same biological function;

The cellulose endonuclease obtained by coding any one of the three genes belongs to the protection scope of the invention.

3. A recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the gene sequence of SEQ ID NO. 1.

Further, the recombinant vector is composed of an empty vector and a target gene inserted into the empty vector, and the target gene is the gene according to claim 1.

Further, the empty vector was pET28 vector.

4. A method for preparing an endo-cellulose, characterized by comprising the steps of:

1) recombining the gene described in the technical scheme 1 into a pET28 vector; then transforming the strain into an escherichia coli strain to obtain an expression strain;

2) culturing the expression strain obtained in the step 1) in an LB liquid culture medium, adding 0.1-0.5mM IPTG for induction, performing ultrasonic crushing after fermentation is finished, and centrifuging to obtain a supernatant so as to obtain the soluble recombinant cellulose endonuclease.

Further, the method also comprises the following protein purification steps: purifying the supernatant obtained in step 2) with a DEAE chromatography column, equilibrating the column with equilibration buffer, passing the supernatant through the column, and purifying with a solution containing 20mM Na₂HPO₄20-30 mM NaCl, pH7.0 buffer solutionThe column was pre-washed, then washed with a solution containing 100 mM-200 mM NaCl, 20mM Na₂HPO₄And eluting the protein by using a buffer solution with pH 7.0.

The purified protein prepared by the method for preparing the protein also belongs to the protection scope of the invention.

5. The gene in the technical scheme 1, the protein obtained in the technical scheme 2 or 4, and the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacterium in the technical scheme 3 are also applied to the fields of production of biofuel ethanol, food, feeding and printing and dyeing, and belong to the protection range of the invention.

The invention has the beneficial effects that: the invention provides a new artificially modified cellulose endonuclease gene which is constructed to a pET28 vector and then connected with an escherichia coli expression strain, thereby realizing the soluble expression and the high-efficiency expression of an expression product with the activity of the cellulose endonuclease; meanwhile, a simple and effective method for purifying the active recombinant cellulose endonuclease is also provided, so that the purity of the purified protein is as high as 95 percent, a simple and efficient production and preparation method is provided for the preparation of a cellulase preparation, and a technical basis is laid for the wide application of the cellulase in the fields of industry, agriculture, livestock, medicine and the like.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 is a schematic diagram of pET28/EG vector construction in an example of the present invention.

FIG. 2 is a SDS-PAGE graph showing the expression of the target protein by pET28/EG recombinant vector in the examples of the present invention.

FIG. 3 is an elution profile of the recombinant endo-cellulose purified by a DEAE chromatography column in the example of the present invention.

FIG. 4 is a SDS-PAGE result of the purified recombinant endo-cellulose in the examples of the present invention.

FIG. 5 is a diagram showing the results of SDS-PAGE detection of the concentrated recombinant endonuclease in the example of the present invention.

FIG. 6 is a graph showing the results of SDS-PAGE detection of the expression of the target protein in the comparative example.

FIG. 7 is a HPLC result chart of enzyme activity detection in the example of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or under conditions recommended by the manufacturers. The materials, reagents and the like used in the examples are commercially available unless otherwise specified. In the examples,% is by mass unless otherwise specified. In the quantitative tests in the following examples, three replicates were set, and the data are the mean or the mean ± standard deviation of the three replicates.

The Escherichia coli expression strain, the vector amplification strain TOP10 and the expression vector pET28 are purchased from Invitrogen corporation of America.

The formula of the culture medium is as follows:

1) LB liquid medium: 10g of NaCl, 10g of peptone, 5g of yeast extract and 1L of distilled water, and carrying out autoclaving and room-temperature storage;

2) LB/Kan plates: 10g of NaCl, 10g of peptone, 5g of yeast extract, 1L of distilled water and 15g of agar powder, sterilizing at high pressure, cooling to below 70 ℃, adding 1mL of kanamycin (Kan) with the concentration of 100mg/mL, fully mixing, pouring out, and storing at 4 ℃ in a dark place;

3) LB/Kan Medium: 10g of NaCl, 10g of peptone, 5g of yeast extract and 1L of distilled water, sterilizing at high pressure, cooling to below 70 ℃, adding 1mLKan (100mg/ml), mixing completely, and storing at 4 ℃; LB liquid medium: NaCl10g, peptone 10g, yeast extract 5g, distilled water 1L, autoclaving, and storing at room temperature.

4)50 × TAE agarose gel electrophoresis buffer: 121g of Tris alkali, 28.6mL of glacial acetic acid, 50mL of 0.5mol/L EDTA (pH8.0), adding distilled water to a constant volume of 500mL, and storing at room temperature;

5)50mg/mL kanamycin stock solution: 0.5g of kanamycin, adding distilled water to dissolve and fix the volume to 10mL, subpackaging and storing at-20 ℃;

6)5 XSDS-PAGE Loading buffer: 1.25mL of 1M Tris-HCl (pH 6.8), 0.5g of SDS, 25mg of BPB and 2.5mL of glycerol, adding deionized water for dissolving, then fixing the volume to 5mL, subpackaging (about 500 mu L of each part), then storing at room temperature, adding 25 mu L of beta-mercaptoethanol into each part, and uniformly mixing;

7)5 XSDS-PAGE running buffer: 15.1g of Tris, 94g of glycine and 5.0g of SDS, adding about 800mL of deionized water, fully stirring and dissolving, then fixing the volume to 1L, and storing at room temperature;

8) coomassie brilliant blue R-250 staining solution: adding 225mL of methanol, 46mL of glacial acetic acid and 225mL of deionized water into the Coomassie brilliant blue R-2500.25g, uniformly stirring, removing particulate matters by using filter paper, and storing at room temperature;

9) coomassie brilliant blue destaining solution: 50mL of glacial acetic acid, 150mL of methanol and 300mL of deionized water are fully mixed and stored at room temperature.

Example 1

The embodiment provides an optimized artificially and chemically synthesized recombinant cellulose endonuclease gene EG, wherein the specific sequence is shown as SEQ ID NO.1 in a sequence table, and the protein sequence corresponding to the gene is shown as SEQ ID NO.2 in the sequence table. The synthesized DNA sequence has no obvious similarity in NCBI database, is a DNA molecule optimized and synthesized according to the characteristics of Escherichia coli and expression vector pET28, and is one of the DNA molecules obtained by utilizing the data optimization of the preference of Escherichia coli codon, the influence of gene higher structure on transcription, GC content and the like.

Connecting the optimized gene to an escherichia coli expression vector pET28 to obtain a recombinant vector, carrying out heat shock transformation on the recombinant vector verified by sequencing to obtain competent cells of an escherichia coli expression strain, coating a corresponding resistant LB (Langmuir-Blodgett) plate, culturing for 12 hours in a constant-temperature incubator at 37 ℃, and screening transformants, wherein pET28/EG vector construction is shown in figure 1, and figure 1 is a schematic diagram of pET28/EG vector construction in the embodiment of the invention.

The optimized pET28 recombinant vector of the gene sequence of the natural cellulose endonuclease is used as an expression vector, the corresponding transformants of the expression strain are induced by IPTG with the concentration of 0.1-0.5mM at the temperature of 18 ℃ to detect the expression of the target protein, the SDS-PAGE result of the total bacterial protein is shown in figure 2, the molecular weight of the recombinant cellulose endonuclease is about 40kDa, and the expressed target protein is shown by an arrow.

Example 2

This example provides a method for preparing a protein, comprising the steps of:

s1: optimizing genes, constructing a prokaryotic expression vector and transforming: artificially chemically synthesizing an optimized mature cellulose endonuclease gene, connecting the gene to a pUC universal vector to obtain pUC/EG, carrying out double digestion of pUC/EG by using BamHI and HindIII, and subcloning the obtained EG fragment into an expression vector pET28 to obtain a recombinant expression vector pET28/EG, wherein the vector construction is shown in figure 1. The main steps of pET28/EG vector construction are as follows:

(1) the target fragment EG is obtained by double digestion of the recombinant vector pUC/EG with BamH I and Hind III, and the reaction system is as follows (both the endonuclease and the buffer are purchased from the company TaKARA):

(2) the vector fragment was obtained by double digestion of pET28 with BamH I and Hind III in the following reaction scheme (both the endonuclease and the buffer were purchased from Dalian TAKARA):

(3) the target fragment and the vector fragment obtained in steps (1) and (2) were recovered by using a DNA gel retrieval kit purchased from Dalian TAKARA, and the detailed procedures were carried out according to the kit instructions.

(4) The target fragment and the vector recovered in the step (3) are connected by T4DNA ligase (purchased from TaKARA company of Dalian province) to ensure that the target gene is accurately inserted into the reading frame of the expression vector, and the reaction system is as follows:

transforming the recombinant vector pET28/EG into an Escherichia coli TOP10 strain, and extracting the recombinant vector pET28/EG from TOP 10; the recombinant vector pET28/EG is transferred into a host cell escherichia coli expression strain by a heat shock method, and an LB plate containing Kan resistance is used for screening to obtain an escherichia coli expression strain transformant containing the recombinant vector pET 28/EG.

S2: expression and extraction of soluble recombinant cellulose endonuclease: the E.coli recombinant transformant containing pET28/EG vector of the optimized artificially synthesized gene is cultured in a liquid LB culture medium at 37 ℃ until OD600 is 0.5-0.7, IPTG with the concentration of 0mM, 0.1mM, 0.2mM and 0.5mM is respectively added, induction is carried out for 24 hours at 18 ℃, the collected thallus after induction is subjected to ultrasonication, the crushing power is 300W, the thallus is crushed for 2s and is separated for 6s, and after circulation is carried out for 90 times, the supernatant is centrifuged to obtain the recombinant cellulose endonuclease, and the SDS-PAGE result is shown in figure 2.

S3: purification of recombinant endo-cellulose: after amplification culture and induction with 0.1mM IPTG at 18 deg.C for 20-24 hr, the thallus of the expression bacteria after IPTG induction expression is collected and resuspended in 40ml of buffer solution A (containing 20mM Na)₂HPO₄And 1mM protease inhibitor PMSF, pH7.0), then crushing by using an ultrasonic crusher, wherein the crushing power is 300W, the crushing time is 2s, the gap is 6s, and the cycle is 90 times; centrifuging the crushed bacterial liquid at the temperature of 4 ℃ at 30000g for 15 min; adding the supernatant obtained by centrifugation into a DEAE chromatographic column pre-balanced by a buffer solution A; with 100ml of buffer B (containing 20mM Na)₂HPO₄20-30 mM NaCl, pH7.0) rinsing the protein purification column, addingAdding buffer solution C (containing 20mM Na) with 100-200 mM NaCl₂HPO₄pH7.0), the elution curve is shown in figure 3, the protein sample of the target arrow peak in the figure is collected, the SDS-PAGE analysis of the sample protein by SDS-PAGE is shown in figure 4, the protein eluted by 100-200 mM NaCl is the recombinant endo-cellulose enzyme with the purity of more than 95%.

S4: concentration of recombinant endocellulase: protein samples were incubated at pH4.0 with 20mM NaH₂PO₄And dialyzing with citric acid buffer solution, after dialysis, performing ultrafiltration concentration by using an ultrafiltration tube with the molecular weight cutoff of 15kDa to obtain the high-concentration recombinant cellulose endonuclease with the purity of more than 95%, and detecting the purity of the concentrated protein by using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), wherein the obtained protein is the high-purity recombinant cellulose endonuclease protein as shown in figure 5. The concentration of the target protein was determined by gel scanning combined with the Bradford method, and Table 1 shows the yield and purity of the soluble recombinant cellulolytic enzyme protein in 100ml of IPTG-induced bacteria after each purification step.

TABLE 1 protein purification results

In addition, SDS-PAGE sample buffer was added to the supernatant obtained in step S2, and the soluble protein was analyzed. Soluble recombinant endocellulolytic enzymes were obtained at IPTG concentrations of 0.1, 0.2 and 0.5mM at a temperature of 18 ℃. In order to save cost and shorten production period, the invention preferably adopts IPTG with induction temperature of 18 ℃ and 0.1mM for induction expression.

Comparative example

The tuckahoe is formed by that tuckahoe mycelium parasitizes on dead pine wood under proper conditions to continuously decompose the nutrition in the pine wood and accumulate and rapidly expand the residual substance after the bacteria transformation, and the formed nutrition storage organ and dormant organ are sclerotia, which is commonly called as tuckahoe. The main component of wood is cellulose. Therefore, highly active secretory cellulases are highly likely to be present in the mycelia of Poria cocos. Invention of the inventionThe expression spectrum of the cellulase of tuckahoe is analyzed by a human by using the transcriptome technology to find the high-abundance cellulase genes. The data obtained by the tuckahoe transcriptome is utilized to design a primer, a target gene is amplified by RT-PCR and is connected to a cloning vector, the sequence of the amplified target gene is shown as SEQ ID NO.3 in a sequence table, the tuckahoe recombinant cellulose endonuclease gene is subjected to double enzyme digestion by BamH I and Hind III and is connected to a pET28 expression vector which is also subjected to double enzyme digestion by BamH I and Hind III. The recombinant vector is transformed into competent cells of an escherichia coli expression strain through heat shock, a corresponding resistant LB plate is coated, the culture is carried out in a constant temperature incubator at 37 ℃ for 12 hours, and transformants are screened. Coli recombinant transformants containing pET28/EG vector for Pre-optimization Gene were cultured to OD in liquid LB medium at 37 ℃₆₀₀0.4, then adding IPTG with the concentration of 0, 0.1, 0.2 and 0.5mM respectively, inducing for 24 hours at 18 ℃, carrying out ultrasonic disruption on the collected thalli after induction, breaking power of 300W, breaking for 2s and spacing for 6s, circulating for 90 times, centrifuging and taking supernatant fluid, obtaining no soluble recombinant cellulose endonuclease, and showing SDS-PAGE results in figure 6. The comparative example results show that only the artificially optimized tuckahoe recombinant cellulose endonuclease gene can realize soluble expression in escherichia coli.

Example 3

The invention determines the enzyme activity by detecting the capability of the sodium carboxymethylcellulose (CMC-Na) hydrolyzed by the endonuclease to generate a small amount of glucose by using the high performance liquid chromatography. The specific method comprises the following steps: adding 200 μ g of purified recombinant cellulose endonuclease into 1% CMC-Na solution with pH4, and reacting at 40 deg.C for 8 hr; after the reaction is finished, the sample is filtered to a sample bottle by a 0.22 mu m microporous filter membrane and is subjected to liquid chromatography analysis. The liquid phase method is as follows: a chromatographic column: an agent amino column, 250X 4.6mm, 5 μm; mobile phase: acetonitrile: 70 parts of water: 30 (volume ratio), flow rate: 1.0mL/min, sample size: 10uL, column temperature: 35 ℃, detector: a differential refractive detector. The HPLC results are shown in FIG. 7, in which the upper panel a shows that no glucose peak is detected in the absence of the addition of the recombinant endo-cellulose, and the lower panel b shows that a significant glucose peak is detected after 8 hours of the reaction of adding CMC-Na to the endo-cellulose prepared according to the present invention, the retention time of the peak is 5.581 minutes, and the glucose concentration is about 56 ng/. mu.L, indicating that the endo-cellulose does have the ability to hydrolyze cellulose to produce glucose.

Therefore, according to the above results, the new endonuclease generated by the recombinant vector constructed according to the gene sequence shown in SEQ ID NO.1 provided by the present invention is a novel recombinant endonuclease capable of decomposing sodium carboxymethylcellulose to generate glucose.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Sequence listing

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<120> recombinant cellulose endonuclease gene, protein thereof and method for preparing protein

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<213> Artificial Sequence (Artificial Sequence)

<400>1

atgttgaaca tcaaggcagc ctctctcgtg ctactggcag cggcagcgcc ggtggtactt 60

ggtcatggtc aagtccactc tgtcatcatc gcttcccctt ctgcgacgtt cccagctgca 120

gacgcctacg ctgctgctga tcccactagc cctctgcgca aactaaacac ctacggacct 180

gctgccaact ttactggccc tgacattacg tgtggaccag gagggaatat ccccatcacg 240

ccattggccc ctgtagatgc tggaagtttg gtcacatttg attggcagag ttggaactcc 300

gttcaccctg ggcctgtcat gacgtatatcgccaaatgtc cggatggctg cgctaacttt 360

aagggcgacg aaggcaacgt ttgggttaaa attgaccagg accaatacaa tcccgatcgt 420

ggttctgatc tcgcttgggg tgaagagttg ctgcgtttga agacaaactc aaagtatagc 480

gtcaacgttc ctgcaggttt agaaaacggg gagtatatcc ttcgtcacga aattcttggt 540

ctccatgttg ctggcaccgt catgggtgcc caattctacc cgaactgctt gcaaatcaag 600

gttcaaaacg gtggaagtgt tactctaccc gaaggtatcc ctcttccagg gtcctatgac 660

ccatatgatc cgggtatcct tgttcagctc tggcaaatta ctcttcagaa ccccaactac 720

accgctcctg gtggccctgt gcttcttcct ggtggttcag gcgactgggc cgaggctaac 780

tacggatcga aggccgtcag ttcacctgca gcccctccaa caacgacagc atctcagtcg 840

gcgcacccga gcaccacaac ggctgctcct actaccatca actcatcacc cgcgtcgtcc 900

cctactggcg ttacggtcca gaagtacggg caatgcggag gtcagggata ttcgggagcg 960

acactttgtg catcgggaac gacttgcacg gctttaaacg attactactc tcaatgcctc 1020

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Met Leu Asn Ile Lys Ala Ala Ser Leu Val Leu Leu Ala Ala Ala Ala

1 5 10 15

Pro Val Val Leu Gly His Gly Gln Val His Ser Val Ile Ile Ala Ser

20 25 30

Pro Ser Ala Thr Phe Pro Ala Ala Asp Ala Tyr Ala Ala Ala Asp Pro

35 40 45

Thr Ser Pro Leu Arg Lys Leu Asn Thr Tyr Gly Pro Ala Ala Asn Phe

50 55 60

Thr Gly Pro Asp Ile Thr Cys Gly Pro Gly Gly Asn Ile Pro Ile Thr

65 70 75 80

Pro Leu Ala Pro Val Asp Ala Gly Ser Leu Val Thr Phe Asp Trp Gln

85 90 95

Ser Trp Asn Ser Val His Pro Gly Pro Val Met Thr Tyr Ile Ala Lys

100 105 110

Cys Pro Asp Gly Cys Ala Asn Phe Lys Gly Asp Glu Gly Asn Val Trp

115 120 125

Val Lys Ile Asp Gln Asp Gln Tyr Asn Pro Asp Arg Gly Ser Asp Leu

130 135 140

Ala Trp Gly Glu Glu Leu Leu Arg Leu Lys Thr Asn Ser Lys Tyr Ser

145 150 155 160

Val Asn Val Pro Ala Gly Leu Glu Asn Gly Glu Tyr Ile Leu Arg His

165 170 175

Glu Ile Leu Gly Leu His Val Ala Gly Thr Val Met Gly Ala Gln Phe

180 185 190

Tyr Pro Asn Cys Leu Gln Ile Lys Val Gln Asn Gly Gly Ser Val Thr

195 200 205

Leu Pro Glu Gly Ile Pro Leu Pro Gly Ser Tyr Asp Pro Tyr Asp Pro

210 215 220

Gly Ile Leu Val Gln Leu Trp Gln Ile Thr Leu Gln Asn Pro Asn Tyr

225 230 235 240

Thr Ala Pro Gly Gly Pro Val Leu Leu Pro Gly Gly Ser Gly Asp Trp

245 250 255

Ala Glu Ala Asn Tyr Gly Ser Lys Ala Val Ser Ser Pro Ala Ala Pro

260 265 270

Pro Thr Thr Thr Ala Ser Gln Ser Ala His Pro Ser Thr Thr Thr Ala

275 280 285

Ala Pro Thr Thr Ile Asn Ser Ser Pro Ala Ser Ser Pro Thr Gly Val

290 295 300

Thr Val Gln Lys Tyr Gly Gln Cys Gly Gly Gln Gly Tyr Ser Gly Ala

305 310 315 320

Thr Leu Cys Ala Ser Gly Thr Thr Cys Thr Ala Leu Asn Asp Tyr Tyr

325 330 335

Ser Gln Cys Leu

340

<210>3

<211>1020

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<213> Artificial Sequence (Artificial Sequence)

<400>3

atgttaaata ttaaggcagc ctcgctggtg ctactcgcag cggcagcgcc cgtcgtactg 60

ggacacgggc aggtgcattc ggtcatcatc gcttcccctt cggcgacgtt cccggctgca 120

gacgcctacg ctgctgctga tccgactagc cctttgcgca aactaaacac ctacggacct 180

gctgccaact ttactggacc ggacattacg tgtggaccag gcggcaatat cccgatcacg 240

ccattggccc cagtagatgc tggaagtttg gtcacatttg attggcagag ttggaactca 300

gttcaccctg gacctgtcat gacgtatatc gccaaatgtc cggatggctg cgctaacttt 360

aagggcgacg aagggaacgt ttgggttaaa attgaccagg accaatacaa tccagatcgt 420

ggctcggatc tcgcttgggg cgaagagttg ctgcgtttga agacaaactc caagtatagc 480

gtcaacgttc cggcaggctt agaaaacggc gagtatatcc ttcgtcacga aattcttggc 540

ttgcatgttg ctggaaccgt catgggcgcc caattctacc ccaactgctt gcaaatcaag 600

gttcaaaacg gcggcagtgt tactctacct gaaggcatcc ctcttccagg ctcatatgac 660

ccatatgatc cgggcatcct ggttcagctc tggcaaatta ctcttcagaa ccccaactac 720

accgctccgg ggggccctgt gcttcttcct ggaggttcag gcgactgggc cgaggctaac 780

tacggctcca aggccgtcag ttcgccagca gccccgccca caacgacagc atcccagtcg 840

gcgcacccaa gcaccacaac ggctgctccc actaccatca actcgtcgcc ggcgtcctca 900

ccgactgggg ttacggtcca gaagtacggc caatgcggcg gacaggggta ttccggggcg 960

acactttgtg catcgggcac gacttgcacg gctttaaacg attactactc acaatgcctc 1020

Claims

1. The recombinant cellulose endonuclease gene is characterized in that the sequence of the gene is a nucleotide sequence of SEQ ID NO.1 in a sequence table.

2. The gene of claim 1 encodes the obtained endo-cellulose.

3. An endo-cellulose according to claim 2, characterized in that: the amino acid sequence of the cellulose endonuclease is shown as SEQ ID NO.2 in the sequence table.

4. A recombinant vector, expression cassette, transgenic cell line or recombinant bacterium comprising the gene of claim 1.

5. The recombinant vector according to claim 4, which comprises an empty vector and a target gene inserted into the empty vector, wherein the target gene is the gene according to claim 1.

6. The recombinant vector according to claim 5, wherein the empty vector is the pET28 vector.

7. A method for producing the recombinant endo-cellulose according to claim 2 or 3, characterized by comprising the steps of:

1) the gene of claim 1 is recombined and constructed into a pET28 vector; then transforming the strain into an escherichia coli strain to obtain an expression strain;

8. The method of claim 7, further comprising a protein purification step: purifying the supernatant obtained in step 2) with a DEAE chromatography column, equilibrating the column with equilibration buffer, passing the supernatant through the column, and purifying with a column containing 20mM Na₂HPO₄20 to 30mM NaCl, pH7.0 buffer solution, and then using 100mM to 200mM NaCl, 20mM Na₂HPO₄And eluting the protein by using a buffer solution with pH 7.0.

9. A protein obtained by the production method according to claim 7 or 8.

10. Use of the gene of claim 1, the protein of claim 2, 3 or 9, the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacterium of claim 4 in the fields of biofuel ethanol production, food, feeding and/or printing.