CN110577959A

CN110577959A - Cellulose exonuclease gene, protein thereof and recombinant vector

Info

Publication number: CN110577959A
Application number: CN201911014486.3A
Authority: CN
Inventors: 胡兴; 李洪波; 李露露; 董海丽
Original assignee: Huaihua University
Current assignee: Huaihua University
Priority date: 2019-10-22
Filing date: 2019-10-22
Publication date: 2019-12-17
Anticipated expiration: 2039-10-22
Also published as: CN110577959B

Abstract

The invention relates to a cellulose exonuclease gene of tuckahoe, a recombinant vector containing the gene and a preparation method of recombinant protein expressed by the recombinant vector, wherein the gene at least contains a DNA sheet 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 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 constitutive secretory expression vector is further constructed according to the gene sequence of the invention and yeast is transformed, the screened high-level secretory expression transformant can high-level secretory expression recombinant poria cocos cellulose exonuclease, and the active recombinant protein with the purity higher than 95 percent can be obtained through nickel affinity purification.

Description

Cellulose exonuclease gene, protein thereof and recombinant vector

Technical Field

The invention belongs to the technical field of biological gene engineering, and relates to a novel poria-derived cellulose exonuclease gene, and a protein and a recombinant vector thereof.

Background

Cellulose is the most abundant natural organic matter in nature, and can be regarded as the largest total renewable resource in the world. 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 microbially produced cellulases to break down cellulose is an efficient and environmentally friendly method. 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. Exoglucanase, which can randomly cut cellulose chains to generate oligosaccharides with different lengths and new ends; exoglucanase acting on the ends of polysaccharide chains formed by exoglucanase cleavage to produce glucose and cellobiose; beta-glucosidase, hydrolyzes cellobiose to produce 2 molecules of glucose. 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.

cellulose is a very abundant resource in nature, but the industrial production of cellulase is limited by a plurality of factors such as low enzyme efficiency, poor thermal stability, high cost and the like. 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 made great progress, but there is a great space to be worth of research, and more cellulases with high activity and high yield are developed. 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 Escherichia coli, toxicity may occur due to the presence of LPS, and therefore, 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. In application No. CN201811238622.2, the expression of cellulose exonuclease using pET32 as an expression vector and BL21 as an expression strain were performed, and a certain amount of recombinant protein with good activity was obtained by purification, but the proteins expressed by the above vectors were intracellular proteins, and the cells were required to be crushed first during purification, which is troublesome in purification steps and low in recovery rate. Yeast is a high-efficiency exogenous gene expression system, and an 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

Aiming at the defects in the prior art, the invention aims to provide a novel poria-derived cellulose exonuclease gene, a protein thereof and a recombinant vector. Also provides a high-expression preparation method of the cellulose exonuclease protein.

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 exonuclease protein and is a DNA molecule of any one of the following (1) to (3):

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

(2) a DNA molecule having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the DNA sequence defined in (1) and encoding a protein having cellulolytic activity.

Wherein, SEQ ID No.1 in the sequence table consists of 1545 deoxynucleotides, the sequence comprises a mature protein full-length reading frame of the cellulose exonuclease gene, an expression label consisting of 6 histidine residues, a stop codon and a protein with an amino acid residue sequence of SEQ ID No.2 in the sequence table.

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 exonuclease protein, which is the protein described in 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) The protein is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No.2 in the sequence table.

wherein, SEQ ID No.2 in the sequence table is composed of 514 amino acid residues, wherein the first 508 amino acid residues are mature cellulose exonuclease protein amino acid sequences, the last 6 amino acid residues are expression tags composed of histidine, and the reading frame of the coding gene comprises 1545 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 double 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 cellulose exonuclease protein, comprising the steps of:

s1: the artificially synthesized gene and the expression vector pGAPZ alpha A of claim 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-tuckahoe cellulose exonuclease;

S2: the recombinant vector pGAPZ alpha A-poria cellulose exonuclease 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 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 exoenzyme at the highest level.

S4: culturing the transformant with high expression of poria cocos cellulose exoenzyme to OD by using YPG culture medium₆₀₀Is 10 ℃15 as seed bacteria, and the seed bacteria are mixed according to the proportion of 1: 10 into a fermenter containing a medium with inorganic salts. Continuously culturing for 72-96 hours at 28-30 ℃, supplementing 60% of glycerol as a carbon source, wherein the glycerol supplementation rate is in series connection with dissolved oxygen, and the dissolved oxygen of the fermentation tank is set to be 40%; the pH was adjusted with concentrated ammonia, setting the pH of the fermentor to 4.5.

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 of the supernate to 7.5-8.5 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 or S6, the following steps of preserving the protein are further 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, is stable and can secrete and express the poria cocos cellulose exonuclease at a high level, and 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, a new gene capable of efficiently expressing cellulose exonuclease is provided, and the recombinant cellulose exonuclease with biological activity, which is obtained by secretory expression and purification according to the expression method of the technical scheme, can effectively prevent host bacteria from degrading expression products, and reduce the load of host cell metabolism and the toxic effect of the expression products on hosts; secondly, the secretion signal alpha-factor signal peptide on the yeast vector pGAPZ alpha A-cellulose exonuclease 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 spatial structure can be formed, so that the natural activity of the cellulose exonuclease is maintained; thirdly, obtaining a stable yeast transformant capable of secreting and expressing cellulose exonuclease 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 950mg/L through the fermentation system provided by the invention, the content of target protein can reach 400mg/L through electrophoresis gray scale scanning, and after purification, each microgram of cellulose exonuclease can be hydrolyzed to generate 7.2nmol of glucose after reacting for 1 hour. Fourthly, a method for expressing the cellulose exonuclease by using eukaryotic host pichia pastoris and a method for quickly and efficiently purifying the cellulose exonuclease 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. The fermentation supernatant fluid of the secretory expression target protein does not contain methanol, has high protein concentration, and can be directly applied by using the protein 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 construction of an expression vector pGAPZ alpha A-cellulose exonuclease in an embodiment of the 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 chart of cellulose exonuclease proteins eluted and purified from imidazole with different concentrations in the embodiment of the present invention.

FIG. 5 is a SDS-PAGE detection result of the cellulose exonuclease protein purified in the example of the present invention.

FIG. 6 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. 7 is a photograph showing the results of SDS-PAGE detection of the target protein in the supernatant of the transformant of the comparative example yeast.

FIG. 8 is a graph showing the results of thin layer chromatography staining of glucose produced by hydrolysis of filter paper by exocellulose in example 4 of the present invention.

FIG. 9 is a graph showing the results of thin layer chromatography staining of glucose produced by hydrolysis of filter paper by exocellulose in example 5 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, autoclaving at 121 deg.C for 15-20min, cooling to about 70 deg.C, and adding 100ml 20% sterilized glucose solution. YPD solid medium can be prepared by adding 1.5-1.8% 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 cellulose exonuclease gene with a 6 XHis tag at the C-terminal, the specific sequence of the gene 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.

under the condition of artificial planting of tuckahoe, the tuckahoe can be formed in three months, and the tuckahoe can be dug in six months. Poria cocos is generally harvested for no more than two years, otherwise it dies and decays because the nutrition of pine is completely absorbed. The main components of the wood are cellulose, hemicellulose and lignin, wherein the content of the cellulose in the wood is 40-50%. Therefore, there is a high possibility that enzymes having high activity, which degrade polysaccharides such as cellulose, lignin, hemicellulose, etc., are present in the mycelia of Poria cocos. Through screening, a novel cellulose exonuclease with high activity is obtained from the tuckahoe. Therefore, the high-efficiency production of the enzyme protein by using a genetic engineering means is helpful for researching the action mechanism of the enzyme and is applied to actual industrial production.

The DNA sequence synthesized according to the sequence characteristics of the poria cellulose exonuclease gene and the preference of yeast codons, the natural DNA of the poria cellulose exonuclease before optimization and other artificial DNA sequences synthesized only according to the preference of the yeast codons are respectively connected to a 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 exoenzyme at the highest level.

Example 2

The embodiment provides a method for preparing cellulose exonuclease 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 bias of yeast codons, namely the DNA in SEQ ID No.1, in the embodiment 1 is connected to a Pichia pastoris constitutive secretory expression vector pGAPZ alpha A to obtain a recombinant vector pGAPZ alpha A-cellulose exonuclease, 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 exonuclease construction in the embodiment of the invention. The main vector construction steps are preferably as follows:

(1) The plasmid containing the synthetic Poria cocos cellulose exonuclease gene was digested with Xho I and Xba I to obtain the desired fragment, and the reaction system was as follows (both the enzyme and the buffer were purchased from the company "TAKARA":

(2) pGAPZ. alpha.A was double-digested with Xho I and Xba I to obtain vector fragments in the following reaction system (both the enzyme 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 exonuclease is linearized by Sac I single enzyme digestion, and the recombinant vector is transformed into a Pichia pastoris host bacterium according to a lithium chloride transformation method provided by an Invitrogen operation manual, wherein X-33 is selected in the embodiment. 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. The Pichia pastoris transformant growing on YPD plate of Zeocin antibiotic 100 microgram/ml is washed with sterile water, and then is coated and inoculated on YPD plate with Zeocin antibiotic final concentration of 250 microgram/ml, the transformant growing on the resistant plate is further coated on YPD plate with Zeocin antibiotic final concentration of 500 microgram/ml, and the Pichia pastoris transformant capable of growing normally on 2mg/ml Zeocin YPD plate is finally obtained according to the Zeocin concentration doubling method. 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 exoenzyme at the highest level. The light absorption value has a certain positive correlation with the transformant expression level of the cellulose exonuclease.

S4: culturing the transformant with high expression of poria cocos cellulose exoenzyme to OD by using YPG culture medium₆₀₀10-15 as seed bacteria, and the seed bacteria are mixed according to the proportion of 1: 10 into a fermenter containing a medium with inorganic salts. Continuously culturing for 96 hours at 28 ℃ and automatically supplementing 60% of glycerol as a carbon source, wherein the glycerol supplementing rate is in series connection with dissolved oxygen, and the dissolved oxygen of the fermentation tank is set to be 40%; the pH was adjusted with concentrated ammonia, setting the pH of the fermentor to 4.5.

It should be noted that, when a transformant with high Zeocin resistance is expressed in a small amount by using YPG medium, 4 yeast transformants which stably and highly secretly express Pachymase exoenzyme are selected from the transformants with high resistance (the resistance level is 2mg/ml Zeocin) through SDS-PAGE analysis, and the secretion and expression ability of the target protein of the transformant with 50 strains of Zeocin YPD plates with the resistance level lower than 500 mug/ml is obviously lower than that of the transformant with high resistance and the enzyme activity detection has the same result, the SDS-PAGE result of the target protein secreted by a part of the transformants with high Zeocin resistance is shown in FIG. 2, and the target protein of the transformant with 70kDa is obviously expressed.

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 1-4 days, the total amount of the target protein obtained by culturing is higher by continuously supplementing glycerol, and the result of the specific target protein accounting for the total amount of the supernatant protein is shown in the following table 1.

TABLE 1 Total protein content

Incubation time	1 day	2 days	3 days	4 days
					Total protein concentration (mg/L)	250	360	880	950

Taking the supernatant of the fermentation liquid cultured for 1-4 days, performing SDS-PAGE, wherein the supernatant has obvious target protein expression at about 70kDa, 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 positively correlated 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	1 day	2 days	3 days	4 days
					Percentage content (%)	12	18	39	42

It was calculated that although the ratio of the target protein was decreasing, the total amount of the target protein was still significantly increased due to the faster increase of total protein, and the results of the target protein content are shown in table 3 below.

TABLE 3 Total amount of target protein

Incubation time	1 day	2 days	3 days	4 days
					Cellulose exonuclease (mg/L)	30	65	340	400

Taking supernatant of fermentation liquor cultured for 1-4 days, adding 10 mu l of the supernatant into 80 mu l of sodium dihydrogen phosphate and citric acid buffer solution (pH is 4) containing 1% CMC-Na, and reacting for 1 hour at 40 ℃; 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. As can be seen from the results, the enzyme activity was close to the maximum value after the culture to day 3.

TABLE 4 variation results of absorbance values

Incubation time	1 day	2 days	3 days	4 days
					Absorbance at 340nm	0.22	0.45	1.89	1.95

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 exonuclease gene in pichia pastoris and establish a high-density fermentation culture system.

S6: the results of SDS-PAGE of the eluted proteins are shown in FIG. 4, wherein the proteins eluted by the 50mM and 100mM imidazole buffers also contain a lot of impurity proteins, and when a sample eluted by the 200mM imidazole buffer is collected, the target protein has high purity. Dialyzing the obtained eluate 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 to more than 5 mg/ml. The efficiency and recovery of protein purification in each step per 100ml of the supernatant of the fermentation broth are shown in Table 5.

TABLE 5 results of protein purification in the respective steps

In application No. CN201811238622.2, pET32 was used as an expression vector, BL21 was used as an expression strain (hereinafter, expressed in escherichia coli expression system), and after disruption by ultrasonication under the induction of IPTG, soluble target protein was purified by nickel affinity chromatography, and only 1.1mg of target protein was purified per 100ml of medium. The SEQ ID No.1 of the invention takes pGAPZ alpha A as an expression vector and X33 as an expression strain, and the target protein which can be purified by high-density fermentation every 100ml of fermentation liquid is 25mg, and finally the recovery rate of the target protein can reach 63 percent and the purity is more than 95 percent, and both the recovery rate and the purity are higher than those of an escherichia coli expression system. Compared with an escherichia coli expression system, pGAPZ alpha A is used as an expression vector, X33 is used as an expression strain to realize high-level secretory expression and efficient purification of the recombinant exonuclease under a high-density fermentation condition, and the recombinase obtained by the method provided by the invention is 25 times that of a comparative patent finally under the condition of the same culture volume.

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 the supernatant, and carrying out SDS-PAGE analysis, wherein the result is shown in figure 5, the target protein is detected and is extremely single band, which indicates that the protein treatment method does not denature and degrade a large amount of protein. Mass spectrum identification is carried out on the purified recombinant exonuclease protein by using Nano-LC-MS/MS, and 10 sequences are obtained in total, and the sequences and one section of the sequences belong to SEQ ID No.2 provided by the invention, wherein the detected secondary mass spectrum result and the matched sequence of one section are shown in FIG. 6, and the detected peptide CSGSDCGQGSDR is just one section of the full-length sequence. This result indicates that the purified protein is the target protein.

Comparative example

The inventor utilizes transcriptome technology to analyze the expression profile of poria cellulose exonuclease 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 secretion type expression vector pGAPZ alpha A, wherein one artificial sequence synthesized only according to the yeast codon preference optimization is shown as SEQ ID NO.4 in the sequence table (represented by the sequence). The poria cellulose exonuclease gene sequence was double-digested with Xho I and Xba I and ligated to the pGAPZ. alpha.A expression vector, which was also double-digested with Xho I and Xba I. The recombinant vector pGAPZ alpha A-Poria cellulose exonuclease is linearized by Sac I 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. 7, in which the target protein band was not detected at the target position. The results show that only the DNA sequences provided by the invention and synthesized according to the sequence characteristics of the poria cellulose exonuclease gene and the preference of yeast codonsThe sequence shown in the SEQ ID NO.1 in the list can be transformed into pichia pastoris to realize high-level secretory expression of the target protein.

Example 3

In this embodiment, the enzyme activity of the purified soluble poria cocos cellulose exonuclease is detected, and the specific steps and results are as follows:

The recombinant poria cocos cellulose exonuclease freeze-dried powder purified and concentrated by 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: 1g/L 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 purified Poria cellulose exonuclease 5mg/ml and 20mM citrate buffer solution (pH 4.0) 10 μ l into 90 μ l sodium dihydrogen phosphate and citric acid buffer solution (pH 4) containing 1% CMC-Na, 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 after 1 hour of reaction per microgram of cellulose exonuclease, it was hydrolyzed to yield 7.2nmol of glucose.

(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. The protease activity can be maintained above 73% at the pH of 3-6.

TABLE 7 relative enzyme activity at different pH values

pH	3	4	5	6	7
						Relative enzyme activity (%)	84	100	87	73	51

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

example 4

in this example, the cellulose hydrolysis activity of purified soluble poria cocos cellulose exonuclease is detected by thin layer chromatography, and the specific steps and results are as follows:

100. mu.l of Poria cocos cellulose exonuclease at a final concentration of 1mg/ml was added to 100. mu.l of a 10mg/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 color development results are shown in FIG. 8, which shows that the enzyme has hydrolysis of the filter paper and glucose generation by adding the pachyman cellulose exonuclease and no enzyme (0 hour), so that spots appear at the same positions as the glucose standard, and the color depth of the spots is positively correlated with the time of the enzyme action.

Example 5

Respectively adding 100 mu g of purified cellulose exonuclease and equivalent cellulose exonuclease expressed by an escherichia coli expression system in CN201811238622.2 into a sodium carboxymethylcellulose solution with pH of 4 and containing 1 percent, and reacting for 1 hour at 40 ℃; after the reaction is finished, reacting for 1 hour at 40 ℃; 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 sample, 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 concentration of glucose produced by the action of the purified cellulose exonuclease of the present invention was calculated to be about 274 ng/. mu.L, while the concentration of glucose produced by the action of the recombinase expressed by E.coli was calculated to be 95 ng/. mu.L. Therefore, the activity of the cellulose exonuclease expressed by pGAPZ alpha A serving as an expression vector and X33 serving as an expression strain is about 2.85 times that of the same cellulose exonuclease expressed by pET32 serving as the expression vector and BL21 serving as the expression strain.

In addition, purified Escherichia coli-expressed and Pichia pastoris-expressed recombinant Poria cocos cellulose exonucleases were dissolved in 20mM citrate buffer solution pH4.0, respectively, to a final concentration of 1mg/ml, and an equal mass of filter paper ground into a slurry was added to the above enzyme solution, followed by reaction at 40 ℃ for 1 hour. Activating a silica gel G plate in an oven at 100 ℃, cooling to room temperature, respectively taking 2 mu L of filter paper liquid supernatant without enzyme, recombinant exonuclease and filter paper reaction liquid expressed by an escherichia coli expression system and recombinant exonuclease and filter paper reaction liquid expressed by a yeast expression system by using a capillary, and spotting to a thin silica gel plate, wherein the spotting position is 1.5cm from the lower end of the silica gel plate, 2cm from both sides, and the distance between each sample spot is 1 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. When the spreading agent moves to a position 2cm away from the upper end, the silica gel plate is taken out for drying, a color developing agent (25% sulfuric acid) is uniformly sprayed on the silica gel plate, and the silica gel plate is taken out from the oven after the color developing agent is developed for 20min at 100 ℃. The color development results are shown in FIG. 9, from which it can be seen that there are significant color development spots at the positions where the 20. mu.g of glucose standard was added; in the supernatant of the filter paper liquid without the recombinant exonuclease, no chromogenic spot of glucose is detected; in a recombinant exonuclease sample added with escherichia coli for expression, a weak color development spot also appears at the position of a glucose standard product through color development, which indicates that the recombinant exonuclease expressed by the escherichia coli has the capability of hydrolyzing filter paper to generate glucose, but the color development of the spot is weak, which indicates that the generated glucose amount is less; in a recombinant cellulose exonuclease sample added with pichia pastoris for expression, obvious color spots appear at the position of a glucose standard product through color development, which shows that the recombinant exonuclease expressed by the yeast has stronger capability of hydrolyzing filter paper to generate glucose, and the generation amount of the glucose is obviously higher than that of an expression product of escherichia coli. It can be seen that the method provided by the invention provides more efficient cellulose exonuclease with stronger enzyme activity than an expression method of escherichia coli.

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 cellulose exonuclease gene, and protein and recombinant vector thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1545

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cagcaagctg gtactcagac cgctgagaac catccacagt tgtcctctca aaagtgtact 60

gccggtggat cttgtacttc tgcttccact tcggttgtcc tggactctaa ttggagatgg 120

gttcacacca cttccggtta caccaactgc tacactggta acacttggga cgcctccatc 180

tgttctgacc cagttacttg tgctcagaac tgcgctttgg atggtgctga ttacgccggt 240

acttacggaa ttactacttc tggtgacgcc ctgaccttga agttcgttac tggttctaac 300

gtcggattca gagtctacct gatggaagat gagactaact accagctgtt caagctgatg 360

aatcaagagt tcaccttcga cgtggacgtt tccaacttgc catgtggttt gaacggtgcc 420

gtttacttcg ttcaaatgga ccaagatggt ggttcctcta agttcccaaa caacaaggct 480

ggtgccaagt tcggaactgg ttactgtgat tcccaatgtc cacaggacat caagttcatc 540

aacggtgagg ccaacatcgt taactggacc gcttctgcag gtgatgctaa ctctggaact 600

ggttccttcg gtacttgttg ccaagagatg gatatctggg aagccaactc tatctccgct 660

gcctacactc cacatccatg taccgttact gagcagacca gatgttctgg ttctgattgt 720

ggacaaggtt ccgacagata caacggtatt tgcgacccag acggttgcga cttcaactcc 780

tttagaatgg gaaacaccga gttctacggt aagggattga ctgttgacac ttctcagaag 840

ttcaccatcg tcacacagtt catttccgac gatggcactg ctgacggtaa cttggctgag 900

atcagaagat tctacgtcca gaacggtaag gtcatcccaa actctgttgt tcagatcact 960

ggtatcgacc cagtcaactc tatcaccgag gatttctgta cacagcaaaa gaccgtgttc 1020

ggcgacaaca acaacttcgc tgcaaaaggt ggtctgcagc aaatgggaga agctgtcaag 1080

aacggtatgg ttctggcttt gtctctgtgg gatgactacg ctgcacaaat gttgtggctg 1140

gactctgact acccaactac cgctgatcca tctaagccag gagttgccag aggcacttgt 1200

ccaaccactt caggtgttcc atcgcaggtt gaaggtcaag aaggatcttc ctctgttatc 1260

tactccaaca ttaagttcgg cgacctgaac tccactttca ccggtacttt gactaaccca 1320

tcctctccag cttctccacc agttacatct tcaccatctc aaccatctca gtccactcag 1380

ccttctcagc ctgctcaacc ttcacaacca gctggaactg ctgctcaatg ggcccagtgt 1440

ggaggtatgg gttttactgg tccaaccgtt tgtgcttctc cattcacttg tcacgtcttg 1500

aacccttact actcccaatg ctaccatcac caccaccatc attaa 1545

<210> 2

<211> 514

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Gln Gln Ala Gly Thr Gln Thr Ala Glu Asn His Pro Gln Leu Ser Ser

1 5 10 15

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

20 25 30

Val Leu Asp Ser Asn Trp Arg Trp Val His Thr Thr Ser Gly Tyr Thr

35 40 45

Asn Cys Tyr Thr Gly Asn Thr Trp Asp Ala Ser Ile Cys Ser Asp Pro

50 55 60

Val Thr Cys Ala Gln Asn Cys Ala Leu Asp Gly Ala Asp Tyr Ala Gly

65 70 75 80

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

85 90 95

Thr Gly Ser Asn Val Gly Phe Arg Val Tyr Leu Met Glu Asp Glu Thr

100 105 110

Asn Tyr Gln Leu Phe Lys Leu Met Asn Gln Glu Phe Thr Phe Asp Val

115 120 125

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

130 135 140

Gln Met Asp Gln Asp Gly Gly Ser Ser Lys Phe Pro Asn Asn Lys Ala

145 150 155 160

Gly Ala Lys Phe Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Gln Asp

165 170 175

Ile Lys Phe Ile Asn Gly Glu Ala Asn Ile Val Asn Trp Thr Ala Ser

180 185 190

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

195 200 205

Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Ala Ala Tyr Thr Pro

210 215 220

His Pro Cys Thr Val Thr Glu Gln Thr Arg Cys Ser Gly Ser Asp Cys

225 230 235 240

Gly Gln Gly Ser Asp Arg Tyr Asn Gly Ile Cys Asp Pro Asp Gly Cys

245 250 255

Asp Phe Asn Ser Phe Arg Met Gly Asn Thr Glu Phe Tyr Gly Lys Gly

260 265 270

Leu Thr Val Asp Thr Ser Gln Lys Phe Thr Ile Val Thr Gln Phe Ile

275 280 285

Ser Asp Asp Gly Thr Ala Asp Gly Asn Leu Ala Glu Ile Arg Arg Phe

290 295 300

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

305 310 315 320

Gly Ile Asp Pro Val Asn Ser Ile Thr Glu Asp Phe Cys Thr Gln Gln

325 330 335

Lys Thr Val Phe Gly Asp Asn Asn Asn Phe Ala Ala Lys Gly Gly Leu

340 345 350

Gln Gln Met Gly Glu Ala Val Lys Asn Gly Met Val Leu Ala Leu Ser

355 360 365

Leu Trp Asp Asp Tyr Ala Ala Gln Met Leu Trp Leu Asp Ser Asp Tyr

370 375 380

Pro Thr Thr Ala Asp Pro Ser Lys Pro Gly Val Ala Arg Gly Thr Cys

385 390 395 400

Pro Thr Thr Ser Gly Val Pro Ser Gln Val Glu Gly Gln Glu Gly Ser

405 410 415

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

420 425 430

Phe Thr Gly Thr Leu Thr Asn Pro Ser Ser Pro Ala Ser Pro Pro Val

435 440 445

Thr Ser Ser Pro Ser Gln Pro Ser Gln Ser Thr Gln Pro Ser Gln Pro

450 455 460

Ala Gln Pro Ser Gln Pro Ala Gly Thr Ala Ala Gln Trp Ala Gln Cys

465 470 475 480

Gly Gly Met Gly Phe Thr Gly Pro Thr Val Cys Ala Ser Pro Phe Thr

485 490 495

Cys His Val Leu Asn Pro Tyr Tyr Ser Gln Cys Tyr His His His His

500 505 510

His His

<210> 3

<211> 1524

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cagcaggcag gtacacagac cgcagaaaat catccgcagc tgagcagcca gaaatgtacc 60

gcaggcggta gctgtaccag cgcaagcacc agcgttgttc tggatagcaa ttggcgttgg 120

gttcatacca ccagtggtta taccaattgt tataccggta atacctggga tgcaagcatt 180

tgtagcgatc cggttacctg tgcgcagaat tgtgcactgg atggtgcaga ttatgcaggc 240

acctatggta ttaccacctc aggtgatgca ctgaccctga aatttgttac cggtagcaat 300

gttggtagcc gtgtttatct gatggaagat gaaaccaatt accagctgtt caaactgatg 360

aatcaagagt ttaccttcga tgtggatgtt agcaatctgc cgtgtggtct gaatggtgcc 420

gtttattttg ttcagatgga tcaggatggt ggcagcagca aatttccgaa taacaaagcc 480

ggtgcaaaat ttggtacagg ttattgtgat agccagtgtc cgcaggatat caaatttatc 540

aatggcgaag ccaacattgt taattggacc gcaagtgccg gtgatgcaaa tagcggcacc 600

ggtagctttg gcacctgttg tcaagaaatg gatatttggg aagccaatag cattagcgca 660

gcatatacac cgcatccgtg taccgttacc gaacagaccc gttgtagcgg tagcgattgt 720

ggtcagggta gcgatcgtta taatggtatt tgtgatccgg atggctgtga tttcaatagc 780

tttcgtatgg gcaacaccga gttttatggt aaaggtctga ccgttgatac ctcgcagaaa 840

tttaccattg tgacccagtt tattagtgat gatggcaccg cagatggtaa tctggcagaa 900

attcgtcgtt tttatgtgca gaatggtaaa gtgattccga atagcgtggt tcagattacc 960

ggcattgatc cggtgaatag tattaccgaa gatttttgta cccagcagaa aaccgtgttt 1020

ggcgataata acaattttgc agcaaaaggt ggtctgcagc agatgggtga agcagttaaa 1080

aatggtatgg ttctggcact gagcctgtgg gatgattatg ccgcacagat gctgtggctg 1140

gatagcgatt atccgaccac agcagatccg agcaaaccgg gtgttgcacg tggtacatgt 1200

ccgacaacca gcggtgttcc gagccaggtt gaaggtcaag aaggtagcag cagcgttatc 1260

tatagcaaca tcaaatttgg cgatctgaac agcaccttta ccggtacact gaccaatccg 1320

agcagtccgg caagccctcc ggtgaccagc agtccgagtc agccgagcca gagcacccag 1380

ccgtcacagc ctgcacagcc gagtcaaccg gcaggtacgg cagcacagtg ggcacagtgc 1440

ggtggtatgg gttttaccgg tccgaccgtt tgtgcaagcc cgtttacctg tcatgttctg 1500

aatccgtatt acagccagtg ctat 1524

<210> 4

<211> 1524

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

caacaggccg gcacgcagac cgctgagaac caccctcagc tgtcatccca aaagtgtact 60

gctggtggta gctgcacctc cgcctccacc agtgttgtgc tcgactccaa ctggcgttgg 120

gtccacacca cttccggtta cactaactgc tacactggaa acacgtggga tgcctccatc 180

tgctctgacc cagttacctg tgcccagaac tgtgccctcg atggcgctga ctatgccggc 240

acttacggta tcaccaccag tggcgacgct cttactctca agtttgtgac gggctccaac 300

gtcggctctc gtgtctacct catggaggac gagaccaact accagctgtt caagctcatg 360

aaccaggagt tcactttcga cgtcgacgtc tccaacctcc cctgcggtct taacggtgct 420

gtttacttcg tccagatgga tcaggacggt ggtagctcca agttccccaa caacaaggct 480

ggagccaagt tcggtaccgg ttactgtgac tctcagtgcc ctcaggacat caagttcatc 540

aacggagagg ccaacatcgt caactggact gcctctgctg gagatgctaa ctccggtact 600

ggttctttcg gtacttgctg ccaggagatg gacatctggg aggccaactc catctccgct 660

gcctacaccc ctcacccttg cacggtcact gagcagaccc gctgctctgg ctctgactgt 720

ggacagggca gcgaccgcta caacggtatc tgcgaccccg atggctgtga cttcaactcc 780

ttccgcatgg gtaacaccga gttctacggc aagggcctta ccgttgacac tagccagaag 840

ttcaccatcg tcacccagtt catctctgac gacggcactg ctgacggaaa cctcgccgaa 900

attcgtcgct tctacgtcca aaacggcaag gttatcccta acagcgttgt tcagatcact 960

ggcatcgacc ctgtcaactc catcactgag gacttctgca ctcaacaaaa gaccgtgttc 1020

ggcgacaaca acaacttcgc cgctaagggt ggcctccagc agatgggtga ggctgtcaag 1080

aacggaatgg tcctcgctct ttcgctctgg gacgactacg ccgctcagat gctctggctc 1140

gactccgact accccaccac cgctgacccg tccaagcccg gtgttgctcg tggtacctgc 1200

cccaccacct ccggtgtccc cagccaagtc gagggtcagg aaggcagctc gtccgtcatc 1260

tactcgaaca tcaagttcgg tgacctcaac tcgactttca ccggcaccct caccaacccc 1320

agcagcccag ctagcccacc cgtcaccagc tctccctcgc agccatccca gtctactcag 1380

ccttctcagc ccgctcaacc ctcccagccg gctggtaccg ctgctcagtg ggctcagtgt 1440

ggtggtatgg gcttcaccgg cccaaccgtc tgcgcaagtc ccttcacttg ccacgtcctc 1500

aacccctact actctcagtg ctac 1524

Claims

1. A cellulose exonuclease gene, said gene comprising a DNA fragment 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 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 resultant cellulose exonuclease.

3. the exocellulose enzyme of claim 2, characterized in that: the cellulose exonuclease is protein of 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 is derived from the SEQ ID NO.2 and has the cellulose exonuclease 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 the 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 pGAPZ α A vector.

7. A method for producing the cellulose exonuclease according to claim 2 or 3, 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 high-expression cellulose exonuclease by using Zeocin;

2) Culturing the high-expression cellulose exonuclease transformant 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 following components in a volume ratio of 1: 10 to be inoculated into a culture medium containing inorganic salt for fermentation, the fermentation is continuously cultured for 72 to 96 hours at the temperature of 28 ℃, 60 percent of glycerol is supplemented as a carbon source, the glycerol supplementation rate is in series connection with dissolved oxygen, and the dissolved oxygen is set to be 40 percent; the pH value is adjusted to 4.5 by using strong ammonia water, and the fermentation supernatant contains a large amount of cellulose exonuclease protein.

8. the method according to claim 7, wherein 2mg/ml Zeocin is used for selection of a transformant having a high level of expression in step 1).

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. The protein obtained by the preparation method according to any one of claims 7 to 9 and the application of the protein in the industries of bioenergy, feed, food and the like.