CN110592121A

CN110592121A - High-expression cellulose endonuclease gene and recombinant vector and protein thereof

Info

Publication number: CN110592121A
Application number: CN201911006736.9A
Authority: CN
Inventors: 胡兴; 李洪波
Original assignee: Huaihua University
Current assignee: HUNAN BUSKY PHARMACEUTICAL Co.,Ltd.
Priority date: 2019-10-22
Filing date: 2019-10-22
Publication date: 2019-12-20
Anticipated expiration: 2039-10-22
Also published as: CN110592121B

Abstract

The invention relates to a high-activity cellulose endonuclease artificial synthesis gene, which is shown by a nucleotide sequence of SEQ ID NO.1 in a sequence table; or a 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. The nucleotide sequence shown as SEQ ID NO.1 in the sequence table can utilize a pichia pastoris constitutive expression plasmid pGAPZ alpha A as a vector, and can realize high-level recombination secretory expression of target protein in pichia pastoris host bacteria X33. Meanwhile, the screened high-level expression transformant can realize high-level secretion of the recombinant protein by utilizing inorganic salt high-density fermentation, and a high-activity novel recombinant cellulose endonuclease protein pure product can be obtained by simple nickel affinity chromatography purification. The recombinant protein has strong capacity of hydrolyzing cellulose substances and generating glucose, and has important application value in the aspects of biological energy, feed, food industry and the like.

Description

High-expression cellulose endonuclease gene and recombinant vector and protein thereof

Technical Field

The invention belongs to the technical field of biological gene engineering, and relates to a novel high-expression cellulose endonuclease gene from poria cocos, and a recombinant vector and a protein thereof.

Background

The annual yield of only crop straws in China can reach 6 multiplied by 10⁸～7×10⁸Ton, but these valuable cellulose resources are not being utilized efficiently. The use of cellulase to decompose cellulose is an efficient and environmentally friendly method. The cellulase is used for degrading cellulose and converting the cellulose into fuels, foods and chemical products, such as sugar, ethanol, feed protein and the like, has great economic significance for relieving energy crisis and the shortage of food and feed resources, and plays an important role in the fields of food, feed, environmental protection, energy and resource development and the like. Cellulases can be classified into two classes according to their structure: cellulase complex and non-complex cellulases. The non-complex cellulase consists of endoglycosidase, exoglycosidase and beta-glucanase, wherein the cellulose endoglycosidase is a key enzyme determining the biological activity of the cellulase.

Cellulases are widely present in natural organisms, and many organisms such as bacteria, fungi, plants, animals, etc. can produce cellulases. Since cellulase produced by organisms can be added to a cellulose raw material to utilize the raw material, how to produce cellulase in large quantities becomes a major bottleneck restricting the use of cellulose. Along with the development of biotechnology, the development of high-activity and high-yield cellulase resources by using genetic engineering means is more and more emphasized by scholars at home and abroad. At present, the cloning and expression of cellulase genes have been greatly developed, but reports on successful acquisition of a large amount of recombinant cellulase are few. As the cellulase has wide requirements in the fields of industry, agriculture, livestock, medicine and the like, the demand is increasing day by day, and the cellulase preparation has short supply and short demand and has very wide 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. Since commercially available cellulase is often a mixture of many enzymes, it is difficult to buy an enzyme preparation consisting entirely of cellulases, and thus it is one of the feasible ways to produce pure cellulase by producing the monomeric enzyme components constituting cellulase one by means of genetic engineering and mixing them in a suitable ratio.

The genetic background of the escherichia coli is clear, and the escherichia coli becomes a preferred expression system of the exogenous gene due to the characteristics of short period, high efficiency, easy operation, safe use and the like. Coli is expressed in BL21(DE3) after transformation, but all obtained inclusion bodies are inactive, and soluble protein which can only be obtained by dozens to hundreds of micrograms per liter of culture medium can be obtained by dissolving, denaturing, renaturing and purifying in vitro under proper conditions. In application nos. 201811240264.9 and 201811281667.8, the endocellulase is expressed and purified by an escherichia coli system using pET28 and pET32 as vectors to obtain a certain amount of recombinant protein with good activity, but the proteins expressed by the two vectors are intracellular proteins, and the cells need to be crushed first during purification, which is troublesome in purification steps and low in recovery rate. Furthermore, since the expression in E.coli may cause toxicity due to the presence of LPS, it is often necessary to analyze and measure the toxicity of the expressed purified product. Finally, to obtain a high purity protein, it is often necessary to perform multiple purification operations, and the more purification steps, the lower the yield of the protein and the more likely the inactivation of the target product will be. Yeast is a high-efficiency exogenous gene expression system, and the exogenous gene expression system taking Pichia pastoris as a host develops most rapidly in recent years and is most widely applied. However, whether each protein can achieve high-level secretory expression in yeast requires an appropriate gene sequence, screening of transformants for high-level secretory expression, optimization of expression conditions, and establishment of an efficient protein purification method.

Disclosure of Invention

In view of the above, the present invention aims to provide a recombinant gene capable of highly expressing cellulase, a recombinant vector thereof, a method for preparing protein, and applications of the recombinant gene and the recombinant vector.

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

1. the invention provides a gene, which is a gene for coding a cellulose endonuclease protein and is a DNA molecule of any one of the following (1) to (2):

(1) a DNA molecule shown by SEQ ID No.1 in a 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;

further, the nucleotide sequence 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;

further, the nucleotide sequence has more than 96 percent of homology with the nucleotide sequence shown in SEQ ID NO.1 and codes the protein with the same biological function;

further, the nucleotide sequence has more than 97 percent of homology with the nucleotide sequence shown in SEQ ID NO.1 and codes the protein with the same biological function;

wherein, SEQ ID No.1 in the sequence table is composed of 1221 deoxynucleotides, the sequence comprises a mature protein full-length reading frame of the cellulose endonuclease gene and an expression label, a stop codon and a restriction enzyme cutting site which are composed of 6 histidine residues, and the protein with the amino acid residue sequence of SEQ ID No.2 in the sequence table is coded.

The protein coded by SEQ ID No.1 in the sequence table belongs to the protection scope of the invention.

2. The invention provides a cellulose endonuclease protein, which is a protein of any one of the following (1) or (2):

(1) a protein consisting of an amino acid sequence shown by SEQ ID No.2 in a sequence table;

(2) and (b) a protein derived from the SEQ ID No.2, wherein the protein is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown by the SEQ ID No.2 in the sequence table.

Wherein, SEQ ID No.2 in the sequence table is composed of 400 amino acid residues, wherein the first 396 amino acid residues are mature cellulose endonuclease protein amino acid sequences, the last 6 amino acid residues are expression labels composed of histidine, and the reading frame of the coding gene comprises 1221 nucleotides.

The substitution and/or deletion and/or addition of one or several amino acid residues means the substitution and/or deletion and/or addition of not more than ten amino acid residues.

3. The recombinant vector, expression cassette or recombinant bacterium containing the gene also belongs to the protection scope of the invention.

Further, the recombinant vector consists of an empty vector and a target gene inserted into the empty vector, and the target gene is specifically synthesized and then directly inserted into an expression empty vector to obtain the recombinant vector for expressing the protein.

Further, the recombinant vector is specifically a recombinant vector for expressing the protein, which is obtained by inserting the gene between Xho I and Xba I enzyme cutting sites of the expression empty vector pGAPZ alpha A.

4. It is a fourth object of the present invention to provide a method for preparing a recombinant endo-cellulose protein, comprising the steps of:

s1: the artificially synthesized gene and the expression vector pGAPZ alpha A in the technical scheme 1 are respectively subjected to double enzyme digestion by Xho I and Xba I, purified and recovered, and then are connected by ligase at 16 ℃ to obtain a recombinant vector pGAPZ alpha A-cellulose endonuclease;

s2: the recombinant vector pGAPZ alpha A-cellulose endonuclease is linearized by Sac I single enzyme digestion, transformed into a pichia pastoris host strain by a lithium chloride transformation method, and screened by Zeocin to obtain positive clone;

s3: the positive clones were transferred to YPD plates containing higher Zeocin resistance, and transformants having higher Zeocin resistance were selected to finally obtain transformants which grew normally on YPD plates containing 2mg/ml Zeocin. The high Zeocin resistance transformant was selected and cultured in a 100ml Erlenmeyer flask containing 10ml YPG medium at 28 ℃ and 250rpm to OD₆₀₀The supernatant was centrifuged at about 10 ℃ and 20. mu.l of the supernatant was added to 80. mu.l of 1% CMC-Na-containing sodium dihydrogen phosphate and citric acid buffer (pH 4) and reacted at 40 ℃ for 1 hour; taking 50 mul and 2 mul of the mixture of NAD and ATP with the concentration of 10mmol/L of the reacted sample, and adding water to the total volume of95 μ L, reacted at room temperature for 5min, then 5 μ L of glucose-6-phosphate dehydrogenase at a concentration of 2 μmol/L was added, reacted at room temperature for 3min, and then the absorbance was measured at 340 nm. Meanwhile, a standard curve is prepared by using a glucose standard product. The transformant with the largest light absorption value is the transformant which expresses the cellulose endonuclease at the highest level.

S4: and (3) culturing the transformant of the high-expression poria cocos cellulose endonuclease to an OD600 of 10-15 by using an YPG culture medium as a seed bacterium, and culturing the seed bacterium according to the ratio of 1: 10 in proportion to the culture medium containing inorganic salt; continuously culturing for 72-96 hours at 28-30 ℃, supplementing 50% of glycerol as a carbon source, wherein the glycerol supplementation rate is connected with dissolved oxygen in series, and the dissolved oxygen of the fermentation tank is set to be 25%; the pH was adjusted with concentrated ammonia, setting the pH of the fermentor to 4.0.

Preferably, after step S4, the following steps of purifying the protein are also included:

s5: centrifuging the culture solution after S4 fermentation, taking supernate, adjusting the pH value of the supernate to 7.5-8.0 by using Tris alkali, centrifuging the supernate for 10-20 minutes at a rotating speed of more than or equal to 15000g, adding the obtained supernate into a nickel affinity chromatography column balanced by a pH 8Tris-HCl buffer solution, and rinsing the nickel affinity chromatography column by using a buffer solution which is 2-4 times of the volume of the chromatography column and contains 10mM Tris-HCl and 30mM imidazole and has pH 8;

s6: the nickel affinity column was eluted with a buffer containing 10mM Tris-HCl and 200mM imidazole, and the resulting eluate was dialyzed against 10mM Tris-HCl buffer using a dialysis bag having a molecular weight of 10kDa, followed by concentration by ultrafiltration.

Preferably, after step S5, the following steps of preserving the protein are also included:

s7: the product obtained by ultrafiltration concentration is quickly frozen at-80 ℃ and then freeze-dried.

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

The yeast transformant which is obtained by screening in the step S3 and is stable and can secrete and express the poria cocos cellulose endonuclease at a high level also belongs to the protection scope of the invention.

The application of the protein, the gene or the recombinant vector and the expression cassette is also within the protection scope of the invention.

Further, the use is the use of decomposing cellulose in the field of feed, textile, food and/or bioenergy.

The technical scheme provided by the invention has the following advantages: firstly, the expression method according to the technical scheme can secrete, express and purify to obtain the recombinant cellulose endonuclease with biological activity, and simultaneously can effectively prevent host bacteria from degrading expression products, and reduce the metabolic load of host cells and the toxic effect of the expression products on the hosts; secondly, the secretion signal alpha-factor signal peptide on the yeast vector pGAPZ alpha A-cellulose incision enzyme is utilized to guide the gene secretion expression of the target protein, the target protein can be greatly secreted into the culture solution, and an accurate space structure can be formed, so that the natural activity of the cellulose incision enzyme is maintained; thirdly, obtaining a stable yeast transformant capable of secreting and expressing the cellulose endonuclease at a high level through screening, and establishing a proper high-density fermentation culture system, wherein the key point is that the highest total protein expression amount is 4600mg/L through the fermentation system provided by the invention, the content of the target protein can reach 3700mg/L through electrophoretic gray scanning, and the enzyme activity of the cellulose endonuclease in the supernatant reaches 488.2 IU/ml. Fourthly, a method for expressing the cellulose endonuclease by using a eukaryotic host pichia pastoris and a method for quickly and efficiently purifying the cellulose endonuclease are explored, so that the cost can be reduced and mass production can be realized; fifthly, the expressed recombinant protein can be rapidly purified by nickel affinity chromatography, and the purified protein has strong biological activity of hydrolyzing cellulose. Sixthly, a constitutive expression vector is adopted, the carbon source added in the thallus culture process is glycerol which is a non-toxic and harmless substance to a human body, the biological safety of the product is ensured, and the fermented supernatant can be directly used without purification.

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

FIG. 1 is a schematic diagram of the construction of an expression vector pGAPZ alpha A-endocellulase in the embodiment of the present invention.

FIG. 2 is a photograph showing SDS-PAGE of the supernatant of the culture of the yeast transformant having high Zeocin resistance (2mg/ml) in the examples of the present invention.

FIG. 3 is a SDS-PAGE graph showing the expression of the target protein at different time points under the high-density fermentation culture conditions in the example of the present invention.

FIG. 4 is a SDS-PAGE detection result of endocellulase proteins eluted and purified from imidazole of different concentrations in the examples of the present invention.

FIG. 5 shows the second mass spectrum result and the retrieved sequence of one of the polypeptides obtained by mass spectrometric identification of the recombinant protein.

FIG. 6 is a SDS-PAGE result of the supernatant of the transformant culture medium showing the comparative example gene sequence in the examples of the present invention.

FIG. 7 is a graph showing the results of detecting glucose produced by hydrolyzing filter paper with recombinant endo-cellulose.

FIG. 8 is a graph showing the results of thin layer chromatography staining of glucose produced by the hydrolysis of filter paper with endo-cellulose 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 used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the following 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 Pichia pastoris strain and the integrative expression plasmid pGAPZ alpha A are both purchased from Invitrogen corporation of America.

The formula of the culture medium is as follows:

1) YPD medium

Completely dissolving 10g yeast extract and 20g peptone, diluting to 900ml, steam autoclaving at 121 deg.C for 15-20min, cooling to about 70 deg.C, and adding 100ml 20% sterilized glucose solution. YPD solid medium was prepared by adding 1.8% of agar thereto.

2) YPG medium

Completely dissolving 10g yeast extract, 20g peptone and 20g glycerol, diluting to 1000ml, and steam autoclaving at 121 deg.C for 15-20 min.

3) Inorganic salt culture medium

Each liter of culture solution contains 6 g of potassium sulfate, 5g of magnesium sulfate, 1g of potassium hydroxide, 7 ml of concentrated phosphoric acid, 30 g of glycerol, a proper amount of antifoaming agent and 0.3 g of calcium sulfate, ammonia water is used for adjusting the pH to 4.5, and after moist heat sterilization and cooling to a room, 5 ml of trace element solution is added according to each liter of culture medium during inoculation.

4) Solution of trace elements

Each liter of the trace element solution comprises: 65g of FeSO₄·7H₂O,24g MoNa₂O₄·2H₂O,20g ZnCl₂,6g CuSO₄·5H₂O,3g MnSO₄·H₂O,0.5g CoCl₂0.2g of biotin, 0.09g of KI,0.02g H₃BO₃And 5.0ml of concentrated H₂SO₄After filtration through a 0.22 μm bacterial filter, the cells were stored in a refrigerator at 4 ℃ until use.

Example 1

The embodiment provides an optimized artificially synthesized poria cocos cellulose endonuclease gene with a 6 XHis tag at the C-terminal, 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. And the optimized DNA sequence is compared by NCBI, and has no obvious similarity to the natural sequence.

The invention is based on the sequence characteristics of tuckahoe cellulose endonuclease gene andthe DNA sequence synthesized by yeast codon preference, the natural DNA of tuckahoe cellulose endonuclease before optimization and other artificial DNA sequences synthesized only after yeast codon preference optimization are respectively connected to pichia pastoris secretion type expression vector pGAPZ alpha A to obtain recombinant vectors, then the recombinant vectors are respectively transformed into pichia pastoris host bacteria X-33 by a lithium chloride transformation method provided by an Invitrogen company operation manual, and YPD plates containing 100 mu g/ml Zeocin antibiotics are used for screening after transformation. The Pichia pastoris transformant growing on YPD plate of Zeocin antibiotic 100 microgram/ml is washed with sterile water, and then is spread on YPD plate with final concentration of 250 microgram/ml Zeocin antibiotic, the transformant growing on the resistant plate is spread on YPD plate of Zeocin antibiotic 500 microgram/ml, and high-resistance Pichia pastoris transformant capable of growing normally on 2 microgram/ml Zeocin YPD plate is finally obtained according to the Zeocin concentration doubling method. The high Zeocin resistance transformant was selected and cultured in a 100ml Erlenmeyer flask containing 10ml YPG medium at 28 ℃ and 250rpm to OD₆₀₀The supernatant was centrifuged at about 10 ℃ and 20. mu.l of the supernatant was added to 80. mu.l of 1% CMC-Na-containing sodium dihydrogen phosphate and citric acid buffer (pH 4) and reacted at 40 ℃ for 1 hour; taking 50 mu L and 2 mu L of the mixture of NAD and ATP with the concentration of 10mmol/L of the reacted samples, adding water until the total volume is 95 mu L, reacting for 5min at room temperature, adding 5 mu L of glucose-6-phosphate dehydrogenase with the concentration of 2 mu mol/L, reacting for 3min at room temperature, and measuring the light absorption value at 340 nm. Meanwhile, a standard curve is prepared by using a glucose standard product. The transformant with the largest light absorption value is the transformant which expresses the poria cocos cellulose endonuclease at the highest level.

Example 2

The embodiment provides a method for preparing a cellulose endonuclease protein, which specifically comprises the following steps:

s1: constructing an expression vector and transforming: the DNA sequence synthesized by the sequence characteristics of the gene itself and the yeast codon preference in the embodiment 1, namely the DNA in SEQ ID No.1, is connected to a Pichia pastoris constitutive secretory expression vector pGAPZ alpha A to obtain a recombinant vector pGAPZ alpha A-cellulose endonuclease, the vector construction is shown as a figure 1, and the figure 1 is a schematic diagram of the eukaryotic expression vector pGAPZ alpha A-cellulose endonuclease construction in the embodiment of the invention. The main vector construction steps are preferably as follows:

(1) the plasmid containing the synthesized cellulase gene was digested with Xho I and Xba I to obtain the desired fragment in the following reaction system (both the endonuclease and the buffer were purchased from Takara GmbH):

(2) pGAPZ alpha A was double-digested with Xho I and Xba I to obtain vector fragments in the following reaction system (both the endonuclease and the buffer were purchased from Dalian TAKARA Co.):

(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), the target gene is accurately inserted into the reading frame of the secretory vector containing a secretory signal alpha-factor, and the reaction system is as follows:

s2: transformation of recombinant plasmid: the recombinant vector pGAPZ alpha A-Poria cellulose endonuclease was linearized by Sac I single digestion, and transformed into Pichia pastoris host bacteria according to the lithium chloride transformation method provided by the Invitrogen operating manual, X-33 being selected in this example. After transformation, YPD plates containing 100. mu.g/ml Zeocin antibiotic were used for selection.

S3: screening of high-level secretion expression yeast transformants and expression of proteins: after transformation, YPD plates containing 100. mu.g/ml Zeocin antibiotic were used for selection. Growing YPD plates of 100. mu.g/ml Zeocin antibioticThe Pichia pastoris transformants were washed with sterile water, plated on YPD plates inoculated to a final concentration of 250. mu.g/ml Zeocin antibiotic, and transformants grown on the resistant plates were plated on YPD plates of 500. mu.g/ml Zeocin antibiotic, and Pichia transformants capable of growing normally on 2mg/ml Zeocin YPD plates were finally obtained by Zeocin concentration doubling. The transformants with high Zeocin resistance were selected and selected, and cultured at 28 ℃ and 250rpm in a 100ml Erlenmeyer flask containing 10ml YPG medium to OD₆₀₀The supernatant was centrifuged at about 10 ℃ and 20. mu.l of the supernatant was added to 80. mu.l of 1% CMC-Na-containing sodium dihydrogen phosphate and citric acid buffer (pH 4) and reacted at 40 ℃ for 1 hour; taking 50 mu L and 2 mu L of the mixture of NAD and ATP with the concentration of 10mmol/L of the reacted samples, adding water until the total volume is 95 mu L, reacting for 5min at room temperature, adding 5 mu L of glucose-6-phosphate dehydrogenase with the concentration of 2 mu mol/L, reacting for 3min at room temperature, and measuring the light absorption value at 340 nm. Meanwhile, a standard curve is prepared by using a glucose standard product. The transformant with the largest light absorption value is the transformant which expresses the poria cocos cellulose endonuclease at the highest level. The light absorption value has a certain positive correlation with the transformant expression level of the cellulose endonuclease.

S4: and (3) culturing the transformant of the high-expression poria cocos cellulose endonuclease to an OD600 of 10-15 by using an YPG culture medium as a seed bacterium, wherein the seed bacterium is cultured in a volume ratio of 1: 10 is inoculated into a fermentation tank containing inorganic salt culture medium for fermentation; continuously culturing for 96 hours at 28 ℃ and automatically supplementing 50 percent by mass of glycerol as a carbon source, wherein the glycerol supplementation rate is in series connection with dissolved oxygen, and the dissolved oxygen of a fermentation tank is set to be 25 percent; the pH was adjusted with concentrated ammonia, setting the pH of the fermentor to 4.0.

It should be noted that, when transformants with high Zeocin resistance are expressed in a small amount in YPG medium, and analyzed by SDS-PAGE, 3 yeast transformants with stable and high secretory expression of Poria cocos cellulose endonuclease were selected from the transformants with high resistance (resistance level is 2mg/ml Zeocin), and the secretion of target protein from 20 transformants with resistance level lower than 200. mu.g/ml Zeocin YPD plate was significantly lower than that of the transformants with high resistance and the same results were obtained by enzyme activity detection, and the SDS-PAGE results of the target protein secretion of some of the transformants with high Zeocin resistance are shown in FIG. 2.

It should also be noted that SDS-PAGE is used to detect the expression of the target protein at different times in the feeding culture conditions in the fermentor, and the electrophoresis results are shown in FIG. 3, and FIG. 3 is a graph of the SDS-PAGE detection of the expression of the target protein at different time points in the examples of the present invention. After culturing for 4 days, the total amount of the target protein obtained by culturing is higher by continuously supplementing glycerol, and the results of the total protein concentration of each supernatant are shown in the following table 1.

TABLE 1 Total protein content

Incubation time (hours)	12	24	36	48	60	72	96
								Total protein concentration (mg/L)	300	840	1400	2200	3400	4300	4600

Taking the supernatant of the fermentation liquor cultured for 1-4 days, performing SDS-PAGE, wherein the supernatant has obvious target protein expression at about 45kDa, and continuously adding glycerol to obtain a higher total amount of the target protein by culture, wherein the grayscale scanning result of SDS-PAG gel shows that the proportion of the target protein is in positive correlation with the culture time, and the proportion result of the target protein in the total protein of the supernatant is shown in the following table 2.

TABLE 2 results of percentage of target protein in supernatant protein obtained at different induction times

Incubation time (hours)	12	24	36	48	60	72	96
								Proportion of target protein (%)	53	61	68	74	77	79	80

The results of calculating the content of the target protein are shown in the following table 3.

TABLE 3 Total amount of target protein

Incubation time (hours)	12	24	36	48	60	72	96
								Target protein content (mg/L)	160	510	950	1630	2600	3400	3700

Taking supernatant of fermentation liquor cultured for 1-4 days, adding 1 mu l of supernatant into 99 mu l of sodium dihydrogen phosphate and citric acid buffer solution (pH is 4) containing 1% CMC-Na, and reacting at 40 ℃ for 1 hour; taking 10 mu L and 2 mu L of the mixed solution of NAD and ATP with the concentration of 10mmol/L of the reacted samples, adding water until the total volume is 95 mu L, reacting for 5min at room temperature, adding 5 mu L of glucose-6-phosphate dehydrogenase with the concentration of 2 mu mol/L, reacting for 3min at room temperature, and measuring the light absorption value at 340 nm. . The resulting absorbance changes are shown in Table 4.

TABLE 4 variation results of absorbance values

Incubation time (hours)	12	24	36	48	60	72	96
								Absorbance at 340nm	0.16	0.51	0.96	1.57	2.28	2.65	2.674

The enzyme activity of the cellulase in the supernatant reaches 488.2IU/ml after 96 hours of induction culture. Preferably, after step S4, the following steps of purifying the protein are also included:

s6: the SDS-PAGE results of the eluted proteins, which were sequentially eluted from the nickel affinity column with 10mM Tris-HCl buffers containing 50 mM, 100mM, 200mM and 400mM of imidazole in the order of imidazole concentration from low to high, are shown in FIG. 4, and it can be seen that the target protein had a high purity only when samples eluted with 100mM and 200mM of imidazole buffers were collected. Therefore, the pH8 buffer containing 200mM imidazole can be used for elution as required, or the pH8 buffer containing 50-200mM imidazole from low to high concentration can be used for elution in several times. Dialyzing the obtained eluate containing the target protein in 10mM Tris-HCl buffer solution by using a dialysis bag with the molecular weight of 10kDa, and then performing ultrafiltration concentration on the recombinant protein, wherein the concentration can be detected to 10 mg/ml.

The SEQ ID No.1 sequence provided by the invention takes pGAPZ alpha A as an expression vector, X33 as an expression strain, the target protein which can be purified by fermenting every 100mL of the fermentation solution is about 300 mg, the recovery rate of the target protein can reach nearly 80% and the purity is more than 95%, and the sequence SEQ ID No.1 provided by the invention takes pGAPZ alpha A as the expression vector and X33 as a pichia pastoris expression system of the expression strain, the expressed target protein has high expression level, less foreign protein and easy purification, and the related purification results are shown in Table 5.

TABLE 5 fermentation supernatant protein purification results

Preferably, after step S6, the following steps of preserving the protein are also included:

s7: and (3) quickly freezing the product obtained by ultrafiltration and concentration at-80 ℃, and then freeze-drying to obtain the freeze-dried powder protein. Dissolving the freeze-dried powder in physiological saline, centrifuging at the rotating speed of 15000g for 20 minutes at 4 ℃, taking supernate, and carrying out SDS-PAGE analysis to detect target protein which is extremely single strip, which indicates that the protein treatment method does not lead a large amount of protein denaturation and degradation. The purified recombinant endonuclease protein is subjected to mass spectrum identification by using Nano-LC-MS/MS, 10 sequences are obtained in total, the sequences and one segment of the sequence 2 provided by the invention belong to, the detected secondary mass spectrum result and the matched sequence of one segment are shown in figure 5, and the detected peptide segment sequence is as follows:

WGGEIIGQGGPTNEQFAATWGAI, the sequence is just one segment of the full-length sequence. This result indicates that the purified protein is the target protein.

As the constitutive transformant does not need to use methanol and other flammable and harmful substances in the thallus growth process, the key points are to realize the constitutive secretion expression of the novel poria cellulose endonuclease gene in pichia pastoris and establish a high-density fermentation culture system.

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 inventor utilizes transcriptome technology to analyze the expression profile of poria cellulose endonuclease gene of poria cocos and finds several high-abundance cellulolytic enzyme genes. Using the data obtained by the transcriptome, designing a primer, amplifying a target gene by RT-PCR and connecting the target gene to a cloning vector, wherein the natural sequence of the amplified target gene is shown as SEQ ID NO.3 in a sequence table; similarly, other artificial DNA sequences synthesized only according to the yeast codon preference optimization are respectively connected to the pichia pastoris secretory expression vector pGAPZ alpha A, and one of the artificial DNA sequences is optimized only according to the yeast codon preferenceThe artificial sequence synthesized later is shown as SEQ ID NO.4 in the sequence list (represented by the sequence), and DNA synthesis is also carried out according to codon preference of Escherichia coli. The above-mentioned Poria cocos cellulose endonuclease gene sequence was double-digested with Xho I and Xba I and ligated to pGAPZ. alpha.A expression vector which was also double-digested with Xho I and Xba I. The recombinant vector pGAPZ alpha A-Poria cellulose endonuclease is linearized by SacI single enzyme digestion, transformed into a pichia host strain by a lithium chloride transformation method, and screened by Zeocin to obtain a transformant which can normally grow on a YPD plate of 2mg/ml Zeocin. The transformants with high Zeocin resistance were selected and selected, and cultured at 28 ℃ and 250rpm in a 100ml Erlenmeyer flask containing 10ml YPG medium to OD₆₀₀The supernatant was centrifuged at about 10 ℃ and 20. mu.l of the supernatant was added to 80. mu.l of 1% CMC-Na-containing sodium dihydrogen phosphate and citric acid buffer (pH 4) and reacted at 40 ℃ for 1 hour; taking 50 mu L and 2 mu L of the mixture of NAD and ATP with the concentration of 10mmol/L of the reacted samples, adding water until the total volume is 95 mu L, reacting for 5min at room temperature, adding 5 mu L of glucose-6-phosphate dehydrogenase with the concentration of 2 mu mol/L, reacting for 3min at room temperature, and measuring the light absorption value at 340 nm. Meanwhile, a standard curve is prepared by using a glucose standard product. As a result, the difference in absorbance of the above transformants was hardly detected and was almost all 0. Meanwhile, the expression of the target protein in the supernatant was detected by SDS-PAGE, and the result of SDS-PAGE is shown in FIG. 6, and no target protein band was detected at the target position. The result shows that the high-level secretory expression of the target protein can be realized only by transforming the sequence shown by SEQ ID NO.1 in the DNA sequence table synthesized according to the sequence characteristics of the poria cellulose endonuclease gene and the preference of the yeast codon into pichia pastoris.

Example 3

In this embodiment, the enzyme activity of the purified soluble tuckahoe cellulose endonuclease is detected by the following specific steps and results:

the recombinant poria cocos cellulose endonuclease freeze-dried powder purified, concentrated and freeze-dried in the step S7 in the embodiment 2 is dissolved in physiological saline again by taking p-nitrophenyl-beta-D-glucopyranoside as a substrate and p-nitrophenol as a standard substance, and enzyme activity detection is carried out after the protein concentration is adjusted to 1 mg/ml.

(1) Drawing a standard curve: 1mg/mL of glucose was taken and diluted to 800, 400, 200, 100, 50, 25 and 0. mu.g/mL with 20mM citrate buffer solution pH4.0, respectively. 10. mu.l of each of the above dilutions was added to 90. mu.l of 1% CMC-Na-containing sodium dihydrogen phosphate and citric acid buffer (pH 4) and reacted at 40 ℃ for 1 hour; taking 50 mu L and 2 mu L of the mixture of NAD and ATP with the concentration of 10mmol/L of the reacted samples, adding water until the total volume is 95 mu L, reacting for 5min at room temperature, adding 5 mu L of glucose-6-phosphate dehydrogenase with the concentration of 2 mu mol/L, reacting for 3min at room temperature, and measuring the light absorption value at 340 nm. And drawing a standard curve, wherein the obtained standard curve equation is as follows: y is 0.0023X-0.0073, R²Table 6 shows the results of the test on the standard samples as 0.9999.

TABLE 6 test results for the standards

Standard substance concentration (μ g/ml)	0	25	50	100	200	400	800
								OD₃₄₀	0	0.05	0.11	0.23	0.45	0.93	1.87

(2) And (3) measuring the enzyme activity of the sample: adding 1 μ l of purified Poria Endocellulose endonuclease with concentration of 1mg/ml and 10 μ l of 20mM citrate buffer solution with pH4.0 into 90 μ l of 1% CMC-Na-containing sodium dihydrogen phosphate and citric acid buffer solution (pH 4), and reacting at 40 deg.C for 1 hr; taking 50 mu L and 2 mu L of the mixture of NAD and ATP with the concentration of 10mmol/L of the reacted samples, adding water until the total volume is 95 mu L, reacting for 5min at room temperature, adding 5 mu L of glucose-6-phosphate dehydrogenase with the concentration of 2 mu mol/L, reacting for 3min at room temperature, and measuring the light absorption value at 340 nm. According to the standard curve, the amount of glucose produced after the reaction was calculated. It was calculated that 211. mu.g of glucose could be produced per microgram of protein in a 1 hour reaction.

(3) According to the method (2), the enzyme activity was detected using citric acid and phosphate buffer solutions having a pH of 3-7, respectively. The relative activities at different pH values are shown in Table 7, and the optimum pH of the enzyme is about 4. Table 7 shows the results of the detection of the relative enzyme activities at different pH values.

TABLE 7 relative enzyme activity at different pH values

pH	3	4	5	6	7
						Relative enzyme activity (%)	86	100	91	82	51

Therefore, according to the above results, the novel poria cocos cellulose endonuclease prepared by the present invention is an endonuclease capable of efficiently hydrolyzing cellulose, and the optimum pH is about 4.

Example 4

In this example, the cellulose hydrolysis activity of purified soluble tuckahoe cellulose endonuclease was measured by glucose production and thin layer chromatography, and the specific steps and results are as follows:

determination of glucose production: mu.l of purified Poria cocos Endocellulose endonuclease with a concentration of 100. mu.g/ml and 10. mu.l of 20mM citrate buffer solution with pH4.0 were added to 90. mu.l of a 1% filter paper fiber and citric acid buffer solution (pH 4) and reacted at 40 ℃ for 1 hour; taking 50 mu L and 2 mu L of the mixture of NAD and ATP with the concentration of 10mmol/L of the reacted samples, adding water until the total volume is 95 mu L, reacting for 5min at room temperature, adding 5 mu L of glucose-6-phosphate dehydrogenase with the concentration of 2 mu mol/L, reacting for 3min at room temperature, and measuring the light absorption value at 340 nm. According to the standard curve, the amount of glucose produced after the reaction was calculated. The results are shown in FIG. 7, from which it can be seen that 50. mu.g/ml glucose was produced by hydrolysis over 2 hours, indicating that the enzyme has the ability to hydrolyze fibers to produce glucose.

Thin layer chromatography: 100. mu.l of Poria cocos Endocellulose endonuclease at a final concentration of 1mg/ml was added to an equal amount of 0.01g/ml filter paper solution and reacted at 40 ℃ for 0, 1, 2 and 4 hours, respectively. And (3) activating the silica gel G plate in a drying oven at 100 ℃, cooling to room temperature, and spotting 1 mu L of the silica gel G plate by using a capillary tube, wherein the spotting position is 1.5cm from the lower end of the silica gel plate, the two sides of the silica gel G plate are 2-3 cm, and the distance between every two sample points is 1-1.5 cm. The spreading agent (ethyl acetate: acetic acid: water 2: 1) was equilibrated in a chromatography cylinder, and after a few minutes, a silica gel plate was placed. And when the spreading agent moves to a position 2-3 cm away from the upper end, taking out the silica gel plate for drying, uniformly spraying a color developing agent (25% sulfuric acid), developing at 100 ℃ for 5-30 min, and taking out from the oven. The coloration results are shown in FIG. 8, which shows that the addition of the enzyme with and without (0 hour) indicates that hydrolysis of the filter paper and glucose production occur due to the addition of the enzyme with the aid of the enzyme, resulting in spots at the same locations as in the glucose standards, and the coloration of the spots is positively correlated with the duration of the action of the enzyme.

Although the embodiments of the present invention have been shown and described above, it is understood that the above preferred embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail through the above preferred embodiments, those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing the essence of the corresponding technical solutions from the technical solutions of the embodiments of the present invention, which should be covered in the scope of the claims and the specification of the present invention.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although the embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and those skilled in the art can make changes, modifications, substitutions and alterations to the above embodiments within the scope of the present invention without departing the essence of the corresponding embodiments from the scope of the embodiments of the present invention, which should be covered in the claims and the specification of the present invention.

Sequence listing

<110> college of bosom

<120> a high expression cellulose endonuclease gene and its recombinant vector and protein

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1221

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ctcgagaaaa gacaatccca agtttggcaa cagtgtggtg gtactggttt ctctggttcc 60

actacttgtg tttccggttc ctactgttcc gagatcaacg actactactc ccagtgtgtt 120

ccaggtactg atccaaacgc tccttctcaa tcttctgctt cttccgctcc accatctcaa 180

cctactggta cttctcctcc tgctgcttct ggtccattga agttctacgg tgttaacatt 240

gccggtttcg acttcggttg taacaccgat ggtaactgtc aggcttctgc tgcttggcca 300

cctttgttga agtattacgg tcacgacggt gagggtcaaa tggaccactt tgttaaggac 360

gacggtttca acgccttcag attgcctgtt ggttggcagt tcttgaccaa cgatgttctt 420

ggtggtccaa tcaacgacgc caacttgcaa gaatacgatg acttggtcca ggcctgtatt 480

aactctggtg ctgctggttg tatcatcgac atccacaact acgctagatg gaacggtgag 540

atcattggtc aaggtggtcc taccaacgaa cagtttgctg ctacttgggg tgctatcgct 600

gctaagtacg ctaacaactc caagatcctg ttcggtgtca tgaacgaacc acacgacgtt 660

ccagatatta acgcttgggc tgactctgtt caggctgctg ttactgctat tagaaacgct 720

ggtgctactt cccagttgat cttgttgcca ggtaacaact ggacttccgc cgaaactttc 780

gtttctaacg gttctgctga cgccctgaac aaggttacta atccagacgg tactaagacc 840

ggcttgatct tcgacgttca caagtatttg gactccgaca actctggtac tcacgctgat 900

tgtgtcacca acaacattgc taatgcctgg cagccattgg ctacttggtt gagagctaat 960

ggtagacagg ctctgaacac tgaaactggt ggtggtaaca ctgactcttg tgcccaattc 1020

ttgtgtgagc agatcgcttt ccaagagcag aactccgatg ttttcctggg ttactttggt 1080

tgggctgctg gtaacttcga cccatcttac gttttgggtg aagtcccaac tcaatccggt 1140

tctacttgga ctgacacttc cttggtttcc gcttgtttgg ctcctaacaa gcaccatcac 1200

catcaccatc actaatctag a 1221

<210> 2

<211> 400

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Gln Ser Gln Val Trp Gln Gln Cys Gly Gly Thr Gly Phe Ser Gly Ser

1 5 10 15

Thr Thr Cys Val Ser Gly Ser Tyr Cys Ser Glu Ile Asn Asp Tyr Tyr

20 25 30

Ser Gln Cys Val Pro Gly Thr Asp Pro Asn Ala Pro Ser Gln Ser Ser

35 40 45

Ala Ser Ser Ala Pro Pro Ser Gln Pro Thr Gly Thr Ser Pro Pro Ala

50 55 60

Ala Ser Gly Pro Leu Lys Phe Tyr Gly Val Asn Ile Ala Gly Phe Asp

65 70 75 80

Phe Gly Cys Asn Thr Asp Gly Asn Cys Gln Ala Ser Ala Ala Trp Pro

85 90 95

Pro Leu Leu Lys Tyr Tyr Gly His Asp Gly Glu Gly Gln Met Asp His

100 105 110

Phe Val Lys Asp Asp Gly Phe Asn Ala Phe Arg Leu Pro Val Gly Trp

115 120 125

Gln Phe Leu Thr Asn Asp Val Leu Gly Gly Pro Ile Asn Asp Ala Asn

130 135 140

Leu Gln Glu Tyr Asp Asp Leu Val Gln Ala Cys Ile Asn Ser Gly Ala

145 150 155 160

Ala Gly Cys Ile Ile Asp Ile His Asn Tyr Ala Arg Trp Asn Gly Glu

165 170 175

Ile Ile Gly Gln Gly Gly Pro Thr Asn Glu Gln Phe Ala Ala Thr Trp

180 185 190

Gly Ala Ile Ala Ala Lys Tyr Ala Asn Asn Ser Lys Ile Leu Phe Gly

195 200 205

Val Met Asn Glu Pro His Asp Val Pro Asp Ile Asn Ala Trp Ala Asp

210 215 220

Ser Val Gln Ala Ala Val Thr Ala Ile Arg Asn Ala Gly Ala Thr Ser

225 230 235 240

Gln Leu Ile Leu Leu Pro Gly Asn Asn Trp Thr Ser Ala Glu Thr Phe

245 250 255

Val Ser Asn Gly Ser Ala Asp Ala Leu Asn Lys Val Thr Asn Pro Asp

260 265 270

Gly Thr Lys Thr Gly Leu Ile Phe Asp Val His Lys Tyr Leu Asp Ser

275 280 285

Asp Asn Ser Gly Thr His Ala Asp Cys Val Thr Asn Asn Ile Ala Asn

290 295 300

Ala Trp Gln Pro Leu Ala Thr Trp Leu Arg Ala Asn Gly Arg Gln Ala

305 310 315 320

Leu Asn Thr Glu Thr Gly Gly Gly Asn Thr Asp Ser Cys Ala Gln Phe

325 330 335

Leu Cys Glu Gln Ile Ala Phe Gln Glu Gln Asn Ser Asp Val Phe Leu

340 345 350

Gly Tyr Phe Gly Trp Ala Ala Gly Asn Phe Asp Pro Ser Tyr Val Leu

355 360 365

Gly Glu Val Pro Thr Gln Ser Gly Ser Thr Trp Thr Asp Thr Ser Leu

370 375 380

Val Ser Ala Cys Leu Ala Pro Asn Lys His His His His His His His

385 390 395 400

<210> 3

<211> 1182

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

caatcccaag tgtggcaaca gtgcggcggt accggtttct ctggatcaac tacttgcgtg 60

tcgggcagct actgcagcga gatcaacgac tactattcgc aatgtgtccc tggcactgac 120

cccaatgccc ctagtcaatc ctcggcttcc tcagctcccc caagccaacc cactgggact 180

agtcctccag ctgccagcgg ccctctcaag ttctacggcg tcaacatcgc tggttttgat 240

ttcggctgca acaccgacgg aaactgccaa gcctctgctg catggcctcc actccttaaa 300

tactacggcc acgatggcga aggtcagatg gaccacttcg tgaaagatga cggattcaac 360

gccttccgtc tccctgtcgg ctggcagttc ctgacgaacg acgttctcgg aggtcctatc 420

aacgacgcta acctccagga gtacgatgat ctcgttcagg cttgcatcaa ctcaggtgct 480

gccggatgta ttattgatat ccacaactat gctcgttgga acggcgagat tattggacaa 540

ggtggtccca cgaacgaaca gttcgctgct acctggggtg ctatcgcagc caagtacgcc 600

aacaactcaa agatcctctt cggagttatg aacgagcccc atgacgttcc cgacatcaat 660

gcttgggcag attcagttca ggctgctgtt actgccattc gtaacgcagg tgctacttcc 720

caactcatcc tccttcccgg gaacaactgg acctctgcag aaacattcgt ctctaatgga 780

tccgccgatg ccttgaacaa ggtcaccaac cccgacggta ccaaaaccgg attgatcttc 840

gatgttcaca agtacctcga ctccgacaac tccggcacac acgccgactg cgtcactaac 900

aatatcgcca acgcctggca acctctcgcc acctggctgc gggccaacgg ccgtcaagcc 960

ctcaacaccg agaccggtgg aggaaacacg gattcgtgcg ctcagttctt gtgcgagcag 1020

attgctttcc aggagcagaa ctctgatgtg ttccttggct acttcggttg ggctgcaggg 1080

aacttcgacc caagctacgt tcttggggaa gtacccactc agagcggcag tacttggacg 1140

gacacgtccc tcgtttcggc ttgcttggct cctaacaagc ac 1182

<210> 4

<211> 1182

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cagagccagg tttggcagca gtgtggtggc accggtttta gcggtagcac cacctgtgtg 60

agcggtagct attgtagcga aattaacgat tattacagcc agtgtgttcc gggtacagat 120

ccgaatgcac cgagccagag cagcgccagc agcgcacctc cgagtcagcc gaccggtaca 180

agccctccgg cagcaagcgg tccgctgaaa ttctatggtg ttaatattgc cggttttgac 240

tttggctgca ataccgatgg taattgtcag gcaagcgcag catggcctcc gctgctgaaa 300

tactacggtc atgatggtga aggtcagatg gatcattttg tgaaagatga tggctttaat 360

gcatttcgtc tgccggttgg ttggcagttt ctgaccaatg atgttttagg tggtccgatt 420

aatgatgcca acctgcaaga atatgatgat ctggttcagg cctgtattaa tagcggtgca 480

gcaggttgta ttatcgatat tcataactat gcccgttgga acggtgaaat tattggtcaa 540

ggcggtccga ccaatgaaca gtttgcagca acctggggtg caattgccgc aaaatatgca 600

aataacagca aaatcctgtt cggcgttatg aatgaaccgc atgatgttcc ggatattaat 660

gcatgggcag atagcgttca ggcagcagtt accgcaattc gtaatgccgg tgcaaccagc 720

cagctgattc tgctgccagg taataattgg accagcgcag aaacctttgt tagcaatggt 780

agcgcagatg cactgaataa agttaccaat ccggatggca ccaaaaccgg tctgattttt 840

gatgtgcata aatatctgga tagcgataat tcaggcaccc atgcagattg tgttaccaat 900

aacattgcaa atgcatggca gccgctggca acctggctgc gtgcaaatgg tcgtcaggcc 960

ctgaataccg aaaccggtgg tggtaatacc gatagctgtg cccagtttct gtgtgaacaa 1020

attgcatttc aagaacagaa cagcgacgtg tttctgggtt attttggttg ggcagcaggt 1080

aattttgatc cgagctatgt tctgggtgaa gttccgacac agagcggtag tacctggacc 1140

gataccagcc tggttagcgc atgtctggca ccgaataaac at 1182

Claims

1. A high-expression cellulose endonuclease gene, which is characterized in that the cellulose endonuclease gene at least contains a DNA fragment with 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 at least 90%, 95%, 96%, 97%, 98% or 99% homology with the nucleotide sequence shown in SEQ ID NO.1 and encodes the same biologically functional protein.

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

3. An endo-cellulose according to claim 2, characterized in that: the cellulose endonuclease is protein as the following (1) or (2):

(1) the amino acid sequence of the protein is shown as SEQ ID NO.2 in the sequence table;

(2) the protein which has the cellulose endonuclease activity and is obtained by substituting, deleting and/or adding one, a plurality of or dozens of amino acid residues in the amino acid sequence shown in SEQ ID NO.2 in the sequence table.

4. A recombinant vector, expression cassette 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 a constitutively secretory pGAPZ α A vector.

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

1) recombining the gene of claim 1 into a pGAPZ alpha A vector, transforming the pGAPZ alpha A vector into a pichia host cell, and screening a transformant of the high-expression cellulose endonuclease by using Zeocin;

2) culturing the transformant with high expression of the cellulose endonuclease obtained in the step 1) by using an YPG culture medium until OD600 is 10-15, and taking the transformant as a seed bacterium, wherein the seed bacterium is prepared by mixing the seed bacterium and the YPG culture medium according to a volume ratio of 1: 10 is inoculated into a culture medium containing inorganic salt for fermentation,

the resulting supernatant contained a large amount of endo-cellulose protein.

8. The preparation method of claim 7, wherein in the step 2), the fermentation is continuously cultured at 28 ℃ for 72-96 hours and supplemented with 50% of glycerol as a carbon source, the glycerol supplementation rate is connected with dissolved oxygen in series, and the dissolved oxygen of the fermentation is kept at 25%; the pH was adjusted to 4.0 with concentrated ammonia.

9. The method of claim 7, further comprising a protein purification step: purifying the supernatant obtained in step 2) by using a nickel affinity chromatography column, firstly balancing the chromatography column by using an equilibrium buffer solution, then passing the supernatant through the column, pre-washing the column by using a pH 8.0 buffer solution containing 30mM imidazole, and then eluting the fusion protein by using a pH 8.0 buffer solution containing 200mM imidazole.

10. Use of an endo-cellulose according to claim 2 or 3 in the field of feed, textile, food and/or bioenergy.