A thermostable xylanase mutant and its application

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a xylanase mutant and its application. The amino acid sequence of the xylanase mutant is shown in SEQ ID No. 4. A vector containing a mutant gene is introduced into a host by means of genetic engineering to obtain a genetically engineered bacterium. The xylanase activity can reach more than 100,000 U / mL through fermentation performance verification. The optimal pH of the xylanase mutant is 3.5, which is 0.5 lower than that before the mutation. The optimal temperature is 45°C. After being treated for 24 hours under the conditions of pH 3.5 and temperature 70°C, the relative activity of the enzyme still remains about 80%. Compared with before the mutation, the heat resistance is improved, which is more conducive to application in the cellulose bioconversion industry and feed enzyme preparations.

Description

Technical field:

[0001] The invention relates to the field of biotechnology, and in particular to a thermostable xylanase mutant and application thereof. Background technology:

[0002] Xylanase (EC 3.2.1.8) is a complex enzyme system including multiple endo- and exo-enzymes, which can degrade xylan into oligo-xylose or xylo-oligosaccharides and belongs to the class of hydrolases. Xylanase is widely distributed in nature, which plays a significant role in degrading hemicellulose in nature. Microorganisms that have been reported to produce xylanase include bacteria, Streptomyces, Aspergillus, Penicillium, Trichoderma, etc. In addition, xylanase is also present in some organisms such as snails, marine algae, terrestrial plant tissues, various invertebrates and rumen of ruminants. Molds and yeasts are widely used in the fermentation industry. The main bacteria producing xylanase in industrial fermentation are: Aspergillus, Penicillium, Bacillus, Streptomyces, yeast, Trichoderma, etc.

[0003] Xylanase is widely used in practical production as an efficient, green and safe plant cell wall hydrolase. Microbial xylanase has a wide range of sources and varieties, which can meet the needs of industrial production to a certain extent. It has important application value in food, feed, pulp and paper, medicine, chemical industry, textile, energy, environmental protection and other industries.

[0004] As an important feed enzyme preparation, xylanase can effectively degrade macromolecular substances, improve the activity of animal digestive enzymes, and promote the digestion and absorption of nutrients after adding xylanase to feed in livestock breeding. In addition, after xylan is degraded, since oligoxylose can proliferate bifidobacteria, it can effectively regulate the intestinal microecological environment of animals, thereby improving the disease resistance and production performance of animals. In order to adapt to the needs of different feed processing technologies and applications, it is often necessary to screen high-temperature resistant strains, or use genetic engineering and protein engineering methods to transform the properties of xylanase to meet the requirements of various factors for xylanase.

[0005] my country has abundant and cheap wood fiber raw materials. Most of the agricultural wood fiber raw materials produced each year, such as corn cobs, wheat bran, rice bran, rice straw, corn stalks and sugarcane bagasse, are increasing in quantity. Some of them will cause pollution and harm the ecological environment. Xylanase, as a cellulose conversion enzyme, can convert these abundant hemicellulose resources into useful products and realize the large-scale feed and energy utilization of straw. Therefore, further strengthening the research and development of xylanase as a cellulose bioconversion enzyme will have great social and economic benefits for solving the problems of resource shortage and unconventional resource waste in my country.

[0006] There are many disadvantages in the process of fermentation and production of xylanase by wild strains, such as low yield, high raw material cost, difficulty in large-scale fermentation production, and low activity. In addition, some enzymatic properties of natural xylanase itself cannot fully meet the increasingly stringent production environment and are subject to some restrictions in the application process. Therefore, the limitation of xylanase in practical application can be solved by studying the heterologous expression of xylanase in engineered bacteria.

[0007] The present application selects a strain of Trichoderma that produces xylanase as the experimental material, induces mutagenesis and screens and clones one of the xylanase genes, obtains a heat-resistant xylanase mutant, and finally obtains an engineered bacterium that expresses higher xylanase activity in Pichia pastoris by constructing an expression vector for Pichia pastoris, thereby meeting the needs of industrial production of cellulose bioconversion and application of feed enzyme preparations, reducing production costs, and laying the foundation for further research and development. Summary of the invention:

[0008] The purpose of the present invention is to provide a xylanase mutant with improved enzyme activity and heat resistance and a Pichia pastoris engineered strain thereof, aiming to solve the problems of low enzyme activity of xylanase in industrial production, unsatisfactory application of cellulose bioconversion and feed enzyme preparation.

[0009] The present invention is specifically implemented through the following technical solutions:

[0010] First, a mutagenized strain was obtained by screening Trichoderma sp. M014 preserved in the applicant's laboratory through the ARTP mutagenesis method, and the gene encoding the xylanase mutant was cloned from the mutagenized strain and a recombinant plasmid was constructed, which was then expressed in Pichia pastoris GS115 to obtain a Pichia pastoris engineered strain, and the xylanase mutant with improved enzyme activity and heat resistance was produced therefrom.

[0011] One of the technical solutions provided by the present invention is a xylanase mutant, which is obtained by causing at least one of the following mutations on the basis of the M014 wild-type xylanase shown in SEQ ID No. 2: mutating the serine at position 61 to threonine, mutating the glutamine at position 64 to histidine, mutating the aspartic acid at position 82 to histidine, mutating the lysine at position 238 to arginine, or mutating the isoleucine at position 240 to asparagine;

[0012] Preferably, the xylanase mutant is a mutant having mutations at the above five sites simultaneously: Ser61Thr / Gln64His / Asp82His / Lys238Arg / Ile240Asn, and the amino acid sequence is shown in SEQ ID No. 4 in the sequence listing;

[0013] The present invention also provides a gene encoding the above-mentioned xylanase mutant;

[0014] Furthermore, the nucleotide sequence of the gene encoding the xylanase mutant Ser61Thr / Gln64His / Asp82His / Lys238Arg / Ile240Asn is shown in the sequence listing SEQ ID No.3.

[0015] The enzymatic properties of the xylanase mutant Ser61Thr / Gln64His / Asp82His / Lys238Arg / Ile240Asn are as follows:

[0016] (1) Optimum pH: The enzyme activity is stable at pH 3.0-4.5, and the optimal action pH is 3.5;

[0017] (2) Optimum temperature: Enzyme activity is stable at 40°C-65°C, and the optimal temperature is 45°C;

[0018] (3) Heat resistance: Under the conditions of pH 3.5 and temperature 70°C, the enzyme activity still retains more than 80% after 24 hours.

[0019] The second technical solution provided by the present invention is: a recombinant vector or a recombinant strain comprising the above-mentioned xylanase mutant encoding gene, reconstructing the above-mentioned xylanase mutant encoding gene into a recombinant vector, and efficiently expressing it in Pichia pastoris to obtain a recombinant strain producing high-activity xylanase, and obtaining high-activity xylanase by fermentation, extraction and other technologies;

[0020] Furthermore, the host cell used to express the xylanase mutant is Pichia pastoris GS115;

[0021] Furthermore, the expression vector used to express the xylanase mutant is a pPIC9K plasmid;

[0022] Preferably, the recombinant strain is obtained by connecting the gene encoding the xylanase mutant shown in SEQ ID No. 3 to the expression vector pPIC9K and expressing it in Pichia pastoris GS115.

[0023] The third technical solution provided by the present invention is the application of the recombinant vector or recombinant strain described in the second technical solution, especially in the fermentation production of the xylanase mutant shown in SEQ ID No.4.

[0024] The fourth technical solution provided by the present invention is the application of the xylanase mutant shown in SEQ ID No. 4, especially in the degradation of xylan;

[0025] Furthermore, it is applied in the cellulose bioconversion industry, such as degrading xylan in wood fiber raw materials to convert raw materials into feed and energy;

[0026] Furthermore, the invention can be used in feed enzyme preparations. For example, after adding the xylanase to feed, it can effectively degrade macromolecular substances, improve the activity of animal digestive enzymes, and promote the digestion and absorption of nutrients.

[0027] The following definitions are used in the present invention:

[0028] 1. Nomenclature of amino acid and DNA sequences

[0029] The generally accepted IUPAC nomenclature for amino acid residues is used, either in the form of three-letter or one-letter codes. The generally accepted IUPAC nomenclature for DNA nucleic acid sequences is used.

[0030] 2. Identification of xylanase mutants

[0031] "Original amino acid + position + replaced amino acid" is used to represent the mutated amino acid in the mutant. For example, Ser61Thr means that the amino acid at position 61 is replaced by Thr from wild-type Ser, and the position number corresponds to the amino acid sequence number of the wild-type xylanase in SEQ ID No.2. In the present invention, MT-1 represents wild-type xylanase, and AMT-2 represents the xylanase mutant Ser61Thr / Gln64His / Asp82His / Lys238Arg / Ile240Asn, and the information is as follows:

[0032]

[0033] Beneficial effects:

[0034] The invention discloses a new xylanase mutant, which has the characteristics of high enzyme activity and high heat resistance. The enzyme activity of the mutant fermentation liquid can reach more than 100000 U / mL.

[0035] The optimum reaction pH of the xylanase mutant obtained by the present invention is 3.5. Compared with the wild-type M014 xylanase, the optimum reaction pH is reduced by 0.5, indicating that the acid resistance is improved.

[0036] The xylanase obtained by the invention retains more than 80% of its enzyme activity after 24 hours under the conditions of pH 3.5 and 70°C, and has significantly improved heat resistance compared with the wild-type M014 xylanase. Description of the drawings:

[0037] Figure 1 Optimum pH curve;

[0038] Figure 2 Optimum temperature curve;

[0039] Figure 3 Heat resistance curve. Specific implementation method:

[0040] The present invention is described in more detail by specific embodiments, which are only used as examples and are not intended to limit the scope of the present invention. For those skilled in the art, improvements that can be made based on the principles of the present invention should also be considered as the scope of protection of the present invention. The molecular biology experimental methods that are not specifically described in this embodiment can be referred to in the "Molecular Cloning Experiment Guide".

[0041] The present invention provides a xylanase mutant with improved enzyme activity and heat resistance. The mutant is selected from a mutagenized strain obtained by ARTP mutagenesis of a Trichoderma sp. M014 preserved in the applicant's laboratory. The mutation sites obtained by gene sequencing are that the 61st serine is mutated to threonine, the 64th glutamine is mutated to histidine, the 82nd aspartic acid is mutated to histidine, the 238th lysine is mutated to arginine, and the 240th isoleucine is mutated to asparagine. The present invention first obtains the encoding gene of the xylanase mutant, connects the mutant encoding gene with a pPIC9K vector to construct a recombinant plasmid, transfers it into a corresponding host bacterium GS115 for heterologous expression, and obtains the corresponding xylanase of the mutant by fermentation. The xylanase has high enzyme activity and is suitable for industrial production, cellulose bioconversion industry and feed enzyme preparations.

[0042] Some of the materials and methods involved in the present invention are as follows:

[0043] 1. Experimental materials and reagents:

[0044] Experimental strains and vectors: The wild-type strain is the Trichoderma strain M014 preserved in the applicant's laboratory; the expression host bacteria and vectors: GS115 and pPIC9K were purchased from Novagen; host bacteria: DH5α.

[0045] Main reagents: DNA polymerase, T4 DNA ligase, DNA gel recovery kit, plasmid extraction kit, RNA extraction kit, pNPG, DNA marker, agarose, ampicillin, birchwood xylan, IPTG, X-gal: all purchased from Shanghai Bioengineering; various restriction endonucleases: purchased from NEB.

[0046] Experimental instruments: gel imager (Bio-Rad), protein electrophoresis instrument (Bio-Rad), nucleic acid electrophoresis instrument (Bio-Rad), PCR amplification instrument (Bio-Rad), high-speed centrifuge (Eppendorf).

[0047] 2. The method for determining xylanase activity used in the present invention:

[0048] (1) Definition of enzyme activity:

[0049] Under certain conditions (unless otherwise specified, the conditions are: 37° C., pH 5.5), the amount of enzyme required to degrade and release 1 μmol of reducing sugar from a 5 mg / mL xylan solution per minute is one enzyme activity unit U.

[0050] (2) Drawing of standard curve:

[0051] Weigh 23.14 g of sodium acetate trihydrate, add 1.70 mL of glacial acetic acid, add water to dissolve, and dilute to 2000 mL. Measure the pH of the solution, adjust to pH 5.5 with 0.1 mol / L acetic acid solution or 0.1 mol / L sodium acetate solution, and set aside.

[0052] Weigh 1.0000 g of anhydrous xylose, add the above acetic acid-sodium acetate buffer solution to dissolve it, and make up to 100 mL.

[0053] Pipette 4.0 mL of acetic acid-sodium acetate buffer, add 5 mL of DNS reagent, heat in a boiling water bath for 5 min, cool to room temperature with tap water, add water to make up to 25 mL to prepare a standard blank sample.

[0054] 1.00mL, 2.00mL, 3.00mL, 4.00mL, 5.00mL, 6.00mL and 7.00mL of xylose solution were respectively taken and made up to 100mL with the above-mentioned acetic acid-sodium acetate buffer solution to prepare xylose standard solutions with concentrations of 0.10mg / mL, 0.20mg / mL, 0.30mg / mL, 0.40mg / mL, 0.50mg / mL, 0.60mg / mL and 0.70mg / mL.

[0055] Take 2.00mL of the above concentration series of xylose standard solutions and add them to the graduated test tubes, then add 2.00mL of acetic acid-sodium acetate buffer and 5.0mL of DNS reagent. Electromagnetic oscillation for 3s to 5s, and heat in a boiling water bath for 5min. Then cool to room temperature with tap water, and add distilled water to 25mL. Use the standard blank sample as a reference to adjust to zero, and measure the absorbance A value at 540nm.

[0056] Draw a standard curve with xylose concentration as the Y axis and absorbance A value as the X axis. The standard curve needs to be redrawn each time a new DNS reagent is prepared.

[0057] (3) Enzyme activity determination method:

[0058] 10.0 mL of xylan solution (5 mg / mL) was pipetted and equilibrated at 37°C for 20 min.

[0059] Pipette 10.0 mL of appropriately diluted enzyme solution and equilibrate at 37°C for 10 min.

[0060] Pipette 2.00mL of appropriately diluted enzyme solution and add it to a graduated test tube, then add 5mL of DNS reagent and oscillate electromagnetically for 3s to 5s. Then add 2.0mL of xylan solution, keep warm at 37°C for 30min, and heat in a boiling water bath for 5min. Cool to room temperature with tap water, add water to make up to 25mL, and oscillate electromagnetically for 3s to 5s. Use the standard blank sample as a blank control and measure the absorbance A at 540nm B .

[0061] Pipette 2.00mL of appropriately diluted enzyme solution (equilibrated at 37℃) and add it to a graduated test tube. Then add 2.0mL of xylan solution (equilibrated at 37℃), electromagnetically oscillate for 3s to 5s, and keep the temperature at 37℃ for 30min. Add 5mL of DNS reagent and electromagnetically oscillate for 3s to 5s to terminate the enzymatic reaction. Heat in a boiling water bath for 5min, cool to room temperature with tap water, add water to make up to 25mL, electromagnetically oscillate for 3s to 5s, use the standard blank sample as the blank control, and measure the absorbance A at 540nm. E .

[0062] (4) Calculation formula for xylanase activity:

[0063]

[0064] Where:

[0065] X------xylanase activity of the sample, U / mL;

[0066] A E ------Absorbance of enzyme reaction solution;

[0067] A B ------Absorbance of enzyme blank sample;

[0068] K-------The slope of the standard curve;

[0069] Co------intercept of the standard curve;

[0070] M-------molar mass of xylose, M(C5H 10 O5) = 150.2 g / mol;

[0071] t-------enzyme reaction time, min;

[0072] n------the dilution multiple of the sample;

[0073] 1000----Conversion factor, 1mmol=1000μmol.

[0074] In the present invention, the amino acid sequence of the wild-type xylanase is shown in SEQ ID No. 2:

[0075] MKFSALLFTASLVAAMPASVYPTKVPVDDDSDDDCAETDARKEKDGLLFNAQYQPKAAYTSLAQAAGLKYFGSAVDNGYLSDAPYSKLADDVEEFGQLVPQGKFNFGGADQV VNFAAQNGFQGHLIVGETPSRSELAATFKRFTDLNVEVAITELDIRHELKVRGHALVWHSQLPQWVHNFQGHLIVGETPSRSELAATFFRAARAADPNAKLYINDYSIDDPN AAKLKAGMVANVKKWIYPTKVPVDDDSDDDCAETDANGVQAALQQMASTGVKEVAITELDIRSAPAADYATVTKACEPRQAQDSINKLIKNKGKTYSLRKDSPFTQVLGEEF VGIAFRAARAADPNAKLYINDYSIDDPNAAKLKAGMVANVKKWIYGTITDPNLLQSQQNNAIIKADFGQVTPENSMKWDATEPQQGGNPFQQGGAQPQQEQAAGESCQSN*.

[0076] In the present invention, the amino acid sequence of the xylanase mutant Ser61Thr / Gln64His / Asp82His / Lys238Arg / Ile240Asn is shown in SEQ ID No.4:

[0077] MKFSALLFTASLVAAMPASVYPTKVPVDDDSDDDCAETDARKEKDGLLFNAQYQPKAAYTTLAHAAGLKYFGSAVDNGYLSHAPYSKLADDVEEFGQLVPQGKFNFGGADQV VNFAAQNGFQGHLIVGETPSRSELAATFKRFTDLNVEVAITELDIRHELKVRGHALVWHSQLPQWVHNFQGHLIVGETPSRSELAATFFRAARAADPNAKLYINDYSIDDPN AAKLKAGMVANVKRWNYPTKVPVDDDSDDDCAETDANGVQAALQQMASTGVKEVAITELDIRSAPAADYATVTKACEPRQAQDSINKLIKNKGKTYSLRKDSPFTQVLGEEF VGIAFRAARAADPNAKLYINDYSIDDPNAAKLKAGMVANVKKWIYGTITDPNLLQSQQNNAIIKADFGQVTPENSMKWDATEPQQGGNPFQQGGAQPQQEQAAGESCQSN*.

[0078] The present invention is further explained below through specific implementation modes.

[0079] Example 1 Obtaining the gene encoding the xylanase mutant AMT-2

[0080] 1. Starting strain: a xylanase-producing Trichoderma strain M014 preserved in the applicant's laboratory.

[0081] 2. Culture medium:

[0082] (1) Seed culture medium: 1.5% glucose, 0.4% peptone, 0.12% potassium dihydrogen phosphate, 0.5% magnesium sulfate, pH 4.5, sterilized at 121°C for 20 min;

[0083] (2) YPD solid medium: 1% yeast extract, 2% peptone, 2% glucose, 1.5% agar powder, pH 4.5, sterilized at 121°C for 20 min;

[0084] (3) Primary screening medium: YPD solid medium supplemented with 1% birchwood xylan, sterilized at 115°C for 20 min;

[0085] (4) Shake flask rescreening culture medium: 2% glucose, 1.5% peptone, 1% corn steep liquor, 1% ammonium sulfate, pH 4.5, sterilized at 121°C for 20 min.

[0086] 3.ARTP mutagenesis:

[0087] (1) Take a fresh slant of strain M014, wash the cells with sterile water, shake in a beaded flask to disperse the cells, shake on a shaker for 50 min, filter through 4 layers of lens paper, perform gradient dilutions, count on a hemocytometer and adjust the spore concentration to 10. 7 -10 8 About 1000 g / mL was used as the mutagenic starting bacterial suspension.

[0088] (2) Turn on the normal temperature and pressure plasma system, wipe the inside and outside of the operating room with alcohol cotton, and turn on the ultraviolet lamp for sterilization for 30 minutes. After sterilization, take 10 μL of bacterial suspension and apply it to the rough surface of the slide, and transfer the slide to the operating room table with tweezers under sterile conditions. Open the helium valve, set the gas flow rate and mutagenesis time for mutagenesis. The mutagenesis time was set to 30s, 60s, 90s, 120s, and 150s respectively.

[0089] (3) After each mutagenesis, the slide was placed in an EP tube containing 990 μL of sterile saline and vortexed for 1 min. The diluted slide was spread on the primary screening culture plate and then cultured in a 30°C incubator for 5 days. After the culture was completed, the strains with good growth were randomly selected from the plate for primary screening.

[0090] 4. Screening of mutant strains

[0091] (1) Primary screening: The primary screening culture medium plate was stained with 1% Congo red for 20 min, and then decolorized with 1 mol / L NaCl for 20 min. The strains were screened according to the size of the transparent spots. The seven strains with the largest transparent circles were selected and inoculated onto YPD solid culture medium plates and continued to be cultivated until a pure culture was obtained. The seven strains were named MA-01, MA-02, MA-03, MA-04, MA-05, MA-06, and MA-07, respectively.

[0092] (2) Rescreening: The above 7 strains were then rescreened by fermentation shake flasks for 3 batches. After shake flask fermentation tests, a strain MA-03 with a higher xylanase production was screened based on the average xylanase activity of the 3 batches. The rescreening results are shown in the following table:

[0093]

[0094] 5. Genetic stability experiment of xylanase high-yielding strain MA-03

[0095] The xylanase high-yielding mutant strain MA-03 was streaked and cultured on the screening and separation plate, and the single colony with good growth was picked and placed on the slant medium and cultured at 30°C for 5 days. Then it was cultured in seed bottles and shake flasks respectively, and the xylanase activity was measured at the end of the culture. The strain was subcultured continuously, and the results of the shake flask after 10 subcultures are shown in the following table:

[0096]

[0097] The mutant strain was subcultured for 10 generations, and the experimental results showed that the mutant strain had good genetic stability.

[0098] MA-03 obtained by mutagenesis screening was used as the starting strain, and PCR primers (Forward / Rerverse) were designed according to the nucleotide sequence of the wild-type xylanase MT-1 encoding gene. The restriction site Xho I was added to the 5' end and the restriction site Not I was added to the 3' end. The encoding gene of the mutant AMT-2 was obtained by PCR. The nucleotide sequence obtained by sequencing was SEQ ID No.3, and the corresponding amino acid sequence was SEQ ID No.4.

[0099] Primer sequences:

[0100] Forward: 5'-GAC CTCGAG TTAGTTGCTCTGGCA-3' (the underline indicates the restriction endonuclease Xho I recognition site);

[0101] Reverse: 5'-CAG GCGGCCGC ATGAAGTTCTCTGCC-3' (the underline indicates the recognition site of restriction endonuclease Not I).

[0102] In the present invention, by comparing the nucleotide sequences of the wild type and the mutant, the mutation site information is obtained as shown in the following table:

[0103]

[0104] Example 2 Construction of recombinant vector pPIC9K-AMT-2

[0105] The mutant AMT-2 encoding gene (SEQ ID No. 3) and plasmid pPIC9K were digested with Xho I and Not I, respectively, and the products were recovered. The recovered AMT-2 encoding gene and pPIC9K were mixed in proportion, ligated with T4 ligase overnight at 16° C., and the ligation product was transformed into Escherichia coli DH5α competent cells. The transformation product was spread on an LB (containing ampicillin) solid plate, inverted and cultured at 37° C. overnight, a single colony was picked to LB liquid culture medium, cultured at 37° C., and the target sequence was obtained by colony PCR. Sequencing comparison showed the nucleotide sequence shown in SEQ ID NO. 3, that is, the obtained recombinant vector contained the correct mutant gene, and the obtained recombinant vector was named pPIC9K-AMT-2.

[0106] Example 3 Recombinant plasmid transformation of Pichia pastoris

[0107] 1. Preparation of Pichia pastoris GS115 competent cells

[0108] (1) Pick a single colony from a Pichia pastoris plate and inoculate it into 5 mL of YPD medium at 30°C and 220 rpm overnight;

[0109] (2) Take 0.5 mL of overnight culture solution and inoculate it into 50 mL of freshly prepared YPD medium. Cultivate at 30°C and 220 rpm with shaking until the OD 600 The value reaches 1.3-1.5;

[0110] (3) Centrifuge the culture medium at 4°C, 3000 rpm for 5 min;

[0111] (4) Discard the supernatant, add 50 mL of ice-cold sterile water, and vortex to resuspend the cells;

[0112] (5) Centrifuge at 4°C, 3000 rpm for 5 min, discard the supernatant, drain the residual liquid on the tube wall, add 25 mL of ice-cold sterile water, and vortex to resuspend the cells;

[0113] (6) Centrifuge at 4°C, 3000 rpm for 5 min, discard the supernatant, drain the residual liquid on the tube wall, add 10 mL of ice-cold 1 mol / L sterile sorbitol solution, and resuspend the cells;

[0114] (7) Centrifuge at 4°C, 3000 rpm for 5 min, discard the supernatant, drain the residual liquid on the tube wall, add 1 mL of ice-cold 1 mol / L sterile sorbitol solution (pre-added with glycerol to a final concentration of 15%), and vortex to mix;

[0115] (8) Aliquot 100 μL / tube into sterile EP jars and store in a -70°C refrigerator (freshly prepared competent cells have better results).

[0116] 2. Transformation of Linearized Plasmid

[0117] The positive clones obtained in Example 2 were extracted to obtain the recombinant plasmid pPIC9K-AMT-2, which was digested with Sal I to obtain a linearized plasmid. Freshly prepared (or frozen at -70°C) GS115 competent cells were placed in an ice bath to completely thaw.

[0118] (1) Transfer 100 μL of competent cells to a new sterile EP tube, add 10 μL of linearized plasmid, blow gently to mix, and transfer to a 0.2 cm electroporation cuvette;

[0119] (2) Place the conversion cup in an ice bath for 5-10 minutes to keep it low temperature;

[0120] (3) Electroporation transformation conditions: 1500 V, 200 Ω, 25 μF, discharge time of about 5 ms, one shock;

[0121] (4) After electroporation, immediately add 1 mL of 1 mol / L sorbitol solution precooled at 4°C to the electroporation transformation cup, pipette and mix evenly, and place in an ice bath;

[0122] (5) Aseptically apply MD medium (1.34% YNB; 4×10 -5 % biotin; 2% glucose plate), 150 μL / plate, the coated plate was inverted and cultured at 30°C for 3-4 days;

[0123] (6) Three recombinant strains were screened on MD plates, and the target sequences were obtained by colony PCR. Sequencing comparison showed the nucleotide sequence shown in SEQ ID NO.3, that is, the obtained recombinant strains contained the correct mutant gene. The three recombinant strains were named MD-01, MD-02, and MD-03, respectively.

[0124] Example 4 Induced expression of yeast containing recombinant plasmid pPIC9K-AMT-2

[0125] BMGY medium formula: 1.4% yeast extract, 2.0% peptone, 0.1 mol / L pH 5.5 phosphate buffer, 1.5% YNB, 3.5×10 -5 % biotin, 1.5% glycerol, and the rest water.

[0126] BMMY medium formula: 1.4% yeast extract, 2.0% peptone, 0.1 mol / L pH 5.5 phosphate buffer, 1.5% YNB, 3.5×10 -5 % biotin, 1.2% methanol, and the remainder water.

[0127] MD-01, MD-02, MD-03, and the recombinant strain MD constructed by the same method as in Example 3 with the wild-type xylanase encoding gene (nucleotide sequence shown in SEQ ID NO.1) were inoculated into flasks containing 30 mL of BMGY medium and cultured at 30°C and 200 r / min until OD 600 The fermentation temperature was about 10, and the cells were collected by centrifugation. The cells were resuspended in 35 mL of BMMY induction medium and cultured for 60 h at 30 °C and 200 r / min. The xylanase activity in the supernatant was determined after centrifugation of the fermentation broth. The results are shown in the following table.

[0128] strain Xylanase activity (U / mL) MD 2782 MD-01 6512 MD-02 6425 MD-03 6620

[0129] Example 5 MD-03 Fermentation Performance Verification

[0130] The MD-03 obtained in Example 3 was used as the production strain for fermentation tank culture to produce xylanase.

[0131] Seed tank medium formula: 4.5% glycerol, 2.1% ammonium dihydrogen phosphate, 1.5% potassium dihydrogen phosphate, 0.7% magnesium sulfate, 1.0% potassium sulfate, 0.08% calcium sulfate, 0.5% potassium hydroxide, the rest is water, pH 5.5;

[0132] Fermentation tank medium formula: 4.5% glycerol, 2.1% ammonium dihydrogen phosphate, 1.5% potassium dihydrogen phosphate, 0.7% magnesium sulfate, 1.0% potassium sulfate, 0.08% calcium sulfate, 0.5% potassium hydroxide, the rest is water, pH 5.5;

[0133] Carbon source: 55% glycerol;

[0134] Methanol: pure methanol;

[0135] Seed tank culture: culture temperature 30℃, initial speed 200r / min, initial air volume 2.0m 3 / h, aeration and stirring culture, pH 5.0, dissolved oxygen maintained at 30-40%, when dissolved oxygen is lower than 30%, control it by increasing the rotation speed and air volume, and transplant when the wet weight increases to 85g / L;

[0136] Fermentation tank culture: culture temperature 30℃, initial speed 200r / min, initial air volume 2.0m 3 / h, culture with ventilation and stirring, inoculation amount of 10%, pH 5.0, in the 0-15h period, 55% glycerol as a carbon source is added at a flow rate of 600g / h, the dissolved oxygen is maintained at 30-40%, and when it is lower than 30%, it is controlled by increasing the rotation speed and air volume, and the bacteria are cultured to a wet weight of 220g / L; after the 16th hour period, the carbon source is stopped, and the dissolved oxygen rebounds to more than 80% and is maintained for 0.5h; then methanol is added at an initial rate of 190g / h, and the dissolved oxygen is controlled to be maintained at 30-40% by adjusting the feeding rate during the methanol addition process, and the culture is cultured until the total fermentation period is 170h and the fermentation is completed.

[0137] The above fermentation method was used to carry out a 50L fermentation tank scale-up verification experiment with a fermentation cycle of 170h. The fermentation enzyme production of the three batches is shown in the following table. The average enzyme production level is 105392U / mL, indicating that the strain MD-03 not only has a high production of xylanase, but also has a certain stability in its fermentation performance and the enzyme activity of the xylanase it produces.

[0138] Fermentation enzyme production of 3 batches of genetically engineered bacteria

[0139] batch Fermentation cycle (h) Fermentation activity (U / mL) 1 170 105420 2 170 104556 3 170 106200

[0140] Example 6 Enzymatic properties of xylanase

[0141] (1) Optimal pH

[0142] The supernatant of the fermentation broth of the recombinant bacteria MD-03 obtained in Example 4 was used as the mutant xylanase sample, and the supernatant of the MD fermentation broth was used as the wild-type control sample. The xylanase activity determination method of the present invention was used to determine the relative enzyme activities under different pH conditions of 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 at 37°C, and the highest xylanase activity of the two enzymes was taken as the benchmark 100%. Figure 1 It can be seen that the enzyme activity of the xylanase mutant of the present invention is stable in the pH range of 3.0-4.5, and the optimal pH is 3.5, and its optimal pH is 0.5 lower than that of the control. The above results show that compared with before mutation, the enzyme activity of the xylanase after mutation of the present invention is higher under more acidic conditions, and its pH range of action is wider, which is more suitable for cellulose bioconversion industry and feed enzyme preparations.

[0143] (2) Optimum working temperature

[0144] The supernatant of the fermentation broth of the recombinant bacteria MD-03 obtained in Example 4 was used as the mutant xylanase sample, and the supernatant of the MD fermentation broth was used as the wild-type control sample. The xylanase activity determination method of the present invention was used to determine the enzyme activity at different temperatures of 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C under the condition of pH 3.5. The relative enzyme activity was calculated with the maximum enzyme activity of the xylanase measured by each of the two enzymes as 100%. The results are shown in the figure. Figure 2 As shown, the xylanase mutant of the present invention has stable enzyme activity at 40-65°C, and the optimum action temperature is 45°C. Compared with the wild-type xylanase, the optimum reaction temperature does not change, but the optimum temperature range is improved.

[0145] (3) Heat resistance

[0146] The supernatant of the fermentation broth of the recombinant bacteria MD-03 obtained in Example 4 was used as the mutant xylanase sample, and the supernatant of the fermentation broth of MD was used as the wild-type control sample. The xylanase activity of each untreated sample was taken as 100% benchmark. The two samples were heat-treated at 70°C under pH 3.5, and the enzyme activity was measured at 37°C and buffer pH 5.5 every 2 hours. The residual enzyme activity was calculated. Figure 3It can be seen that after 24 hours, the relative activity of the xylanase mutant of the present invention still remains more than 80%, and the heat resistance of the mutant xylanase is greatly improved compared with the wild-type xylanase, indicating that the xylanase mutant of the present invention has good heat resistance. The above results show that compared with before mutation, the improvement of heat resistance makes the mutated xylanase more suitable for application in the cellulose bioconversion industry and feed enzyme preparations.

[0147] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A xylanase mutant, characterized in that: The nucleotide sequence of the gene encoding the xylanase mutant is shown in SEQ ID NO.

3.

2. A recombinant vector or recombinant strain containing a gene encoding the xylanase mutant according to claim 1.

3. The recombinant vector according to claim 2, characterized in that The expression vector is pPIC9K plasmid.

4. The recombinant strain according to claim 2, characterized in that The host cell used was Pichia pastoris GS115.

5. The recombinant strain according to claim 2, characterized in that The recombinant strain is obtained by connecting the gene encoding the xylanase mutant shown in SEQ ID No. 3 to the expression vector pPIC9K and expressing it in Pichia pastoris GS115.

6. Use of the recombinant vector or recombinant strain according to claim 2 in producing the xylanase mutant according to claim 1.

7. Use of the xylanase mutant according to claim 1, characterized in that: It is used in the decomposition of xylan, or in the bioconversion of cellulose and the preparation of feed enzyme preparations.

Images