A cellulase mutant and its gene, application and product

Abstract

The present invention belongs to the field of genetic engineering and enzyme engineering, and specifically relates to a cellulose enzyme mutant and its gene, application and product. Specifically, the present invention relates to a cellulose enzyme preparation XYN04 with improved stability and its gene and application. By modifying the amino acid sequence as shown in SEQ ID NO.1 xyn04 The sequence was mutated to N179L, G204A and E243I to obtain a mutant XYN04M. Compared with XYN04, the thermal stability of the mutant was improved by 10°C, and the enzyme activity could still be maintained at more than 60% at 65°C for 3 minutes, while XYN04 could only maintain 30% of the enzyme activity at 65°C for 3 minutes. After being treated at pH 4.0 for 60 minutes, the stability was improved from about 3% to more than 40%. The cellulase mutant of the invention has an optimum pH of 7.0 and an optimum temperature of 40°C, and still has high enzyme activity under low temperature conditions and good stability.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and enzyme engineering, and specifically relates to a cellulose enzyme mutant and its gene, application and product. Background Art

[0002] In the field of biomass conversion, cellulase preparations play a vital role. They are a class of biocatalysts that can decompose cellulose, hemicellulose, etc. in plant cell walls into fermentable sugars. In the traditional sense, cellulase is a complex enzyme composed of endoglucanase, exoglucanase, and β-glucosidase, which only have a degrading effect on cellulase. Plant cell walls are mainly composed of cellulose, hemicellulose, and lignin, so several enzymes are required to participate in the degradation, especially enzymes such as xylanases, which show significant activity in decomposing hemicellulose. Hemicellulose, like cellulose, is one of the key polysaccharides that constitute plant cell walls. As an extension of cellulase, xylanases are gradually being explored in industrial applications. They act on xylan, a polysaccharide that is closely related to cellulose in structure and also exists in plant cell walls. Through its catalytic action, xylanases can effectively decompose xylan into smaller sugar units, providing raw materials for the production of biofuels and other industrial processes. Given the functional similarities between xylanase and cellulase, as well as their synergistic effects in applications, xylanase is also classified as a cellulase enzyme preparation.

[0003] Cellulase is a type of enzyme that can degrade cellulose and has a wide range of applications. In the food industry, when performing alcohol fermentation, adding cellulase can significantly improve the utilization rate of raw materials, increase the yield of alcohol and liquor, reduce the viscosity of the fermentation liquid, and shorten the fermentation time. At the same time, cellulase can also improve the taste of the wine and reduce the content of fusel oil. In the brewing process of soy sauce, cellulase can destroy the cell membrane of soybean raw materials, release the protein and carbohydrates hidden in the cells, thereby improving the concentration and quality of soy sauce and shortening the production cycle. In the processing of fruits and vegetables, cellulase can promote the softening of plant tissues, improve the taste, and simplify the processing technology. In agriculture and animal husbandry, cellulase can be used as a feed additive to supplement the deficiency of homologous enzymes in animals, promote the digestion and absorption of plant cellulose by animals, and improve the utilization rate of feed. At the same time, cellulase can also destroy plant cell walls, release mineral elements and other trace elements, and promote the healthy growth of animals. In the textile industry, cellulase can act on the surface of cotton fibers to break the cellulose on the surface of cotton fibers, thereby reducing the hairiness, blurring, pilling and other phenomena on the surface of cotton fibers, and improving the smoothness and color of cotton fabrics. At the same time, cellulase can also relax the surface of cotton fibers, which is convenient for subsequent printing and dyeing treatments, and improves the absorption rate of dyes and the uniformity of dyeing. In the environmental protection industry, cellulase can decompose organic matter such as cellulose in waste, promote the degradation of organic matter, and reduce environmental pollution and waste treatment costs. In the pharmaceutical industry, cellulase can not only be directly used to treat digestive system diseases and improve the extraction efficiency of Chinese herbal medicines, but can also be used as a drug carrier and to prepare biodegradable materials.

[0004] With the continuous progress of biotechnology, in-depth research and improvement of cellulase will help improve the stability and adaptability of cellulase, which will not only promote the optimization of existing industrial processes, but also provide possibilities for the development of new bioconversion technologies. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a cellulose enzyme mutant, which has the advantages of higher enzyme activity under low temperature conditions and good stability.

[0006] In one aspect, the present invention provides a cellulase mutant, the amino acid sequence of the cellulase mutant is shown in SEQ ID NO.2.

[0007] In yet another aspect, the present invention provides a nucleic acid encoding the aforementioned cellulase mutant.

[0008] Specifically, the sequence of the nucleic acid is SEQ ID NO.3 or a sequence having 70% sequence identity with SEQ ID NO.3.

[0009] More specifically, the sequence of the nucleic acid is SEQ ID NO.3 or a sequence having 80% sequence identity with SEQ ID NO.3.

[0010] More specifically, the sequence of the nucleic acid is SEQ ID NO.3 or a sequence having 85% sequence identity with SEQ ID NO.3.

[0011] Preferably, the sequence of the nucleic acid is SEQ ID NO.3 or a sequence having 90% sequence identity with SEQ ID NO.3.

[0012] Further preferably, the sequence of the nucleic acid is SEQ ID NO.3 or a sequence having 95% sequence identity with SEQ ID NO.3.

[0013] More preferably, the sequence of the nucleic acid is SEQ ID NO.3 or a sequence having 98% sequence identity with SEQ ID NO.3.

[0014] In yet another aspect, the present invention provides a recombinant vector comprising the aforementioned nucleic acid.

[0015] Specifically, the vector is one of a plasmid, a phage or a virus.

[0016] Preferably, the vector is a plasmid.

[0017] More preferably, the vector is pPIC9K Plasmid.

[0018] In yet another aspect, the present invention provides a host cell comprising the aforementioned nucleic acid or recombinant vector.

[0019] Specifically, the cell can be a eukaryotic cell or a prokaryotic cell.

[0020] Preferably, the cell can be a Pichia pastoris, a Saccharomyces cerevisiae cell or a Pseudomonas polymorpha cell.

[0021] More preferably, the cell is Pichia pastoris.

[0022] More preferably, the cell is Pichia pastoris GS115 .

[0023] In another aspect, the present invention provides a method for preparing the aforementioned cellulase mutant, comprising the following steps:

[0024] S1. transforming host cells with the aforementioned recombinant vector to obtain a recombinant strain;

[0025] S2, culturing the recombinant strain to induce the expression of cellulase mutants;

[0026] S3. Recover and purify the expressed cellulase mutant.

[0027] In yet another aspect, the present invention provides an enzyme preparation comprising the aforementioned cellulase mutant.

[0028] In yet another aspect, the present invention provides a medicine comprising the aforementioned cellulase mutant.

[0029] In another aspect, the present invention provides the use of the aforementioned cellulose enzyme mutants in agriculture, animal husbandry, textile industry, environmental protection industry and pharmaceutical industry.

[0030] Specifically, the agriculture and animal husbandry include but are not limited to: feed additives.

[0031] Specifically, the textile industry includes but is not limited to: textile processing.

[0032] Specifically, the environmental protection industry includes but is not limited to: waste disposal.

[0033] The technical effects achieved by the present invention are:

[0034] (1) The optimum pH value of the cellulase mutant of the present invention is 7.0, and the catalytic activity is maintained at more than 50% in the pH range of 5.0-8.0.

[0035] (2) The cellulase mutant of the present invention is very stable between pH 4.0 and 10.0, and the residual enzyme activity is above 40% after treatment for 60 minutes within this pH range.

[0036] (2) The optimum temperature of the cellulase mutant of the present invention is 40°C, and the enzyme activity remains high at 20-60°C.

[0037] (3) The cellulase mutant of the present invention has good thermal stability and can maintain more than 60% of the enzyme activity when incubated at 65°C for 3 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the optimum pH for XYN04, XYN04M and XYN04M2.

[0039] Figure 2 pH stability of XYN04, XYN04M, and XYN04M2.

[0040] Figure 3 It is the optimum temperature for XYN04, XYN04M and XYN04M2.

[0041] Figure 4 Temperature resistance of XYN04, XYN04M, and XYN04M2. DETAILED DESCRIPTION

[0042] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0043] Main test materials and reagents of the present invention

[0044] 1. Strains and vectors: Cellulase genes of the present invention xyn04M Synthesized by Beijing Ruibo Xingke Biotechnology Co., Ltd., Pichia pastoris expression vector pPIC9K and strains GS115 Purchased from Invitrogen.

[0045] 2. Enzymes and other biochemical reagents: Endonucleases were purchased from TaKaRa, ligases were purchased from Invitrogen, mannan was purchased from Sigma, and the others were domestic reagents (all available from common biochemical reagent companies).

[0046] 3. Culture medium:

[0047] (1) Yeast medium YPD: 1%wt peptone, 0.5%wt yeast extract, 1%wt glucose, 2%wt agar, pH 7.0.

[0048] (2) Escherichia coli culture medium LB: 1% wt peptone, 0.5% wt yeast extract, 1% wt NaCl, pH 7.0.

[0049] (3) BMGY medium: 1% wt yeast extract, 2% wt peptone, 1.34% wt YNB, 0.00004% biotin, 1% glycerol (V / V).

[0050] (4) BMMY medium: except that 0.5% methanol is used instead of glycerol, the rest of the ingredients are the same as BMGY.

[0051] Note: The molecular biology experimental methods not specifically described in the following examples are all carried out with reference to the specific methods listed in the book Molecular Cloning Experiment Guide (3rd Edition) by J. Sambrook, or according to the kits and product instructions.

[0052] Example 1 Flavobacterium ( Flavobacterium ) Cellulase encoding genes xyn04M Synthesis

[0053] The present invention uses a cellulase from Flavobacterium xyn04 The gene (SEQ ID NO.1) was used as a reference, and the following mutations were made to its sequence (N179L, G204A, E243I), and the 5' and 3' ends of the mutated sequence were added EcoR I and Not I The restriction enzyme cutting site was removed and the sequence was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for artificial gene synthesis. The amino acid sequence of the artificially synthesized cellulase mutant is shown in SEQ ID NO.2, and its nucleotide sequence is shown in SEQ ID NO.3.

[0054] SEQ ID NO.1:

[0055] CNAKKQEVETISLKDSYKDDFYIGTALSADQIEEKDAKVDSLVKKEFNAITAENSMKSMFVHPFPNKYDFALTDKFVAYGEKNKMFIHGHTLIWHSQLAPWMEKIKDSTEMKAFMKDHITTIVSKYKGRINSWDVVNEALNEDGTLRKSVFLSTLGERYLVDAFKLAAAADPKVDLY YNDYNIEEPAKREGAIKLIKKIKAAGGKIDGVGIQGHWRLASPSLDEIEKSILAYSALGIKVAITELDITVLPNPWDLKGADVNQNFEGSAKMNPYPEKLPDSIQVKLADRYASIFKIFLKHKDKISRVTFWGVHDGQSWLNDWPIKGRTNYPLLFDTKLQPKKAYNSVLELKETKK;

[0056] SEQ ID NO.2:

[0057] CNAKKQEVETISLKDSYKDDFYIGTALSADQIEEKDAKVDSLVKKEFNAITAENSMKSMFVHPFPNKYDFALTDKFVAYGEKNKMFIHGHTLIWHSQLAPWMEKIKDSTEMKAFMKDHITTIVSKYKGRINSWDVVVNEALNEDGTLRKSVFLSTLGERYLVDAFKLAAAADPKVDLYYLDYNIEEPAKREGAIKLIKKIKAAGAKIDGVGIQGHWRLASPSLDEIEKSILAYSALGIKVAITILDITVLPNPWDLKGADVNQNFEGSAKMNPYPEKLPDSIQVKLADRYASIFKIFLKHKDKISRVTFWGVHDGQSWLNDWPIKGRTNYPLLFDTKLQPKKAYNSVLELKETKK;

[0058] SEQ ID NO.3:

[0059]

[0060] Example 2 Cellulase Genes xyn04M Clone

[0061] The synthesized gene vector was stored in the form of puncture bacteria. The puncture bacteria were picked up with a sterile toothpick in the clean bench and placed in an LB shaking tube containing Amp antibiotic (working concentration: 100 μg / mL). The tube was cultured overnight at 37°C and 220 rpm. The next day, the gene-containing vector was extracted according to the instructions of the PurePlasmid Mini Kit (CW0500).

[0062] According to the cellulase gene sequence, the following primers were designed and synthesized:

[0063] P1 (SEQ ID NO.4):

[0064] 5'-CCGGAATTCCGGTGTAATGCGAAAAAGCAAGAAGTAG-3';

[0065] P2 (SEQ ID NO.5):

[0066] 5'-TTGCGGCCGCTTTTTTTGTTTCTTTTTAATTCCAGAAC-3'.

[0067] The extracted vector was used as a template for PCR amplification. The PCR reaction parameters were: 94℃ denaturation for 5 min; then 94℃ denaturation for 30 sec, 55℃ annealing for 30 sec, 72℃ extension for 1 min, and 72℃ incubation for 10 min after 30 cycles. A fragment of about 1084 bp was obtained, which was recovered and connected to the pMD19 vector and sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. The predicted protein molecular weight was 40.3 kDa.

[0068] According to the nucleotide sequence obtained by sequencing, the obtained nucleotide sequence was compared with the xyn04M sequence using DNA Man software and confirmed to be correct.

[0069] Example 3 Preparation of recombinant cellulase XYN04M

[0070] The expression vector pPIC9K Double enzyme digestion ( EcoR I+ Not I), while the genes encoding cellulase xyn04M Double enzyme digestion ( EcoR I+ Not I), enzymatically cut out the gene fragment encoding mature cellulase and the expression vector pPIC9K Connect and obtain the cellulase gene xyn04M Recombinant plasmid pPIC9K - XYN04M And transformed Pichia pastoris GS115 to obtain the recombinant Pichia pastoris strain GS115 / XYN04M .

[0071] Take the GS115 strain containing the recombinant plasmid and the control strain (i.e., the unmutated strain GS115 / xyn04), inoculate them into 300 mL BMGY culture medium, shake culture at 30℃ 200 rpm for 48 hours, and collect the bacteria by centrifugation. Then resuspend them in 150 mL BMMY medium and shake culture at 30℃ 200 rpm. After 72 hours of induction, collect the supernatant by centrifugation and measure the activity of cellulase.

[0072] Example 4 Activity Analysis of Recombinant Cellulase XYN04M

[0073] DNS method: The specific method is as follows: at pH 7.0 and 37°C, 1 mL of the reaction system includes 100 μL of appropriately diluted enzyme solution and 900 μL of substrate. The reaction lasts for 10 min, 1.5 mL of DNS is added to terminate the reaction, the reaction is boiled in boiling water for 5 min, and the OD value is measured at 540 nm after cooling. One unit of enzyme activity (U) is defined as the amount of enzyme that releases 1 μmol of reducing sugar per minute under given conditions.

[0074] Example 5 Determination of properties of recombinant cellulase XYN04M

[0075] The enzymatic properties of the recombinant cellulase XYN04M and the unmutated cellulase XYN04 were measured and compared. At the same time, the mutant recombinant cellulase XYN04M2 was added for enzymatic property comparison. The sequence of XYN04M2 was mutated E243A and T327V based on SEQ ID NO.1. The specific preparation methods of the XYN04M mutant and the XYN04M2 recombinant enzyme are as described in the above examples.

[0076] 1. The optimum pH and pH stability of the recombinant cellulase XYN04M were determined as follows:

[0077] The purified cellulase XYN04, XYN04M, and XYN04M2 were subjected to enzymatic reactions at different pH values ​​to determine their optimum pH. The enzyme activity of the substrate xylan was determined at 37°C in 0.1 mol / L citric acid-sodium hydrogen phosphate buffer at different pH values. Figure 1) showed that the optimum pH of the three was 7.0, but the recombinant enzyme XYN04M had more than 50% relative enzyme activity at pH 4.0-8.0, while XYN04 and XYN04M2 had less than 40% enzyme activity at pH 4.0, indicating that the pH application range of the recombinant enzyme XYN04M was significantly improved.

[0078] The purified cellulase XYN04, XYN04M, and XYN04M2 were treated in the above-mentioned buffers of different pH values ​​at 37°C for 60 min, and then the enzyme activity was measured in a pH 7.0 buffer system at 40°C to study the pH tolerance of the enzyme. Figure 2 ) showed that the recombinant cellulase XYN04M was very stable between pH 4.0 and 10.0. After treatment for 60 minutes in this pH range, the residual enzyme activity was above 40%, and the relative enzyme activity was significantly improved compared with XYN04 and XYN04M2, indicating that the recombinant enzyme had good pH stability in a wider pH range.

[0079] 2. The optimum temperature and thermal stability determination method of recombinant cellulase XYN04M is as follows:

[0080] The optimum temperature of cellulase was determined by performing enzymatic reactions in a citric acid-sodium hydrogen phosphate buffer (pH 7.0) system at different temperatures. The temperature resistance was determined by treating cellulase at different temperatures for different times and then measuring enzyme activity at 40°C. The optimum temperature of the recombinant cellulase XYN04M reaction was determined by the following method: Figure 3 ) showed that its optimum temperature was 40°C, and the enzyme activity remained high at 20-60°C.

[0081] The enzyme thermal stability test showed that ( Figure 4 ), XYN04M has good thermal stability and can maintain more than 60% enzyme activity after incubation at 65℃ for 3 minutes. However, the remaining enzyme activity of XYN04 is only 30%, and that of XYN04M2 is only 40%. Compared with the two, XYN04M has significantly improved thermal stability and good heat resistance.

Claims

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

2.

2. A nucleic acid encoding the cellulase mutant according to claim 1.

3. The nucleic acid according to claim 2, characterized in that The sequence of the nucleic acid is SEQ ID NO.

3.

4. A recombinant vector, characterized in that: Comprising the nucleic acid of claim 2 or 3.

5. The recombinant vector according to claim 4, characterized in that The vector is one of a plasmid, a bacteriophage or a virus.

6. A host cell, characterized in that It comprises the nucleic acid according to any one of claims 2-3 or the recombinant vector according to any one of claims 4-5.

7. A method for preparing the cellulase mutant according to claim 1, characterized in that: The following steps are involved: S1. Transforming a host cell with the recombinant vector according to claim 4 or 5 to obtain a recombinant strain; S2, culturing the recombinant strain to induce the expression of cellulase mutants; S3. Recover and purify the expressed cellulase mutant.

8. An enzyme preparation, characterized in that The invention comprises the cellulase mutant according to claim 1.

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