A kind of high temperature resistant xylanase and its preparation method and application

Abstract

The present invention belongs to the field of genetic engineering technology, and relates to a thermostable xylanase, a preparation method thereof, and an application thereof. The amino acid sequence of the thermostable xylanase Xyn‑G30D / Y59T is shown in SEQ ID NO: 1. After redesign and transformation, it has significantly improved thermostable properties and activity. The present invention obtains a thermostable xylanase Xyn‑G30D / Y59T by transforming the xylanase wild type Xyn. The xylanase Xyn‑G30D / Y59T obtained by the present invention lays a foundation for the application of xylanase in the fields of fruit juice clarification, efficient utilization of lignocellulose, etc.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a high temperature resistant xylanase and a preparation method and application thereof. Background Art

[0002] Lignocellulose is mainly composed of 40-50% cellulose, 25-30% hemicellulose, 15-20% lignin, and trace amounts of pectin, nitrogen compounds, and inorganic components. Hemicellulose is the most abundant polysaccharide in the world besides cellulose. It is generally classified according to the main sugar residues in the trunk, such as xylan, mannan, and glucan; the most abundant type of hemicellulose is xylan, so xylan is also one of the most abundant renewable resources in the biological world. Xylanase produced by microorganisms can degrade xylan. Various microorganisms such as fungi, bacteria, yeast, and seaweed can produce xylanase to degrade xylan.

[0003] In recent years, more and more proteases have been used in various industrial applications, and the demand for commercial proteases is also growing; while xylanase is widely used in food, energy, papermaking, feed and other industries, and its activity research has received widespread attention. In the process of industrial production, enzyme preparations are usually used in high-temperature environments, but the optimal temperature of natural xylanase is between 40 and 60°C, and its thermal stability is poor, which makes it difficult to meet the high-temperature requirements of industry. In order to meet the requirements of industrial production, biological enzymes can be modified through protein engineering to meet the requirements of industrial production.

[0004] Protein engineering through rational design or molecular evolution can systematically dissect the structure-function relationship of proteins and generate new proteins with modified activity or new properties. In order to gain a deeper understanding of the catalytic process of xylanase and the biochemical function of amino acids that interact with substrates, the amino acids in the catalytic center of the enzyme can be modified to effectively regulate the catalytic performance of the enzyme. At present, the research on the modification of the catalytic center of xylanase is usually based on the comparison of homologous enzymes, and the xylanase with the best catalytic performance is used as a template to introduce mutation sites. For example, in the "Effect of Loop Structure A79Y and T81Q Site-Directed Mutations on the Thermal Stability of Xylanase XynASP" in the Journal of Chinese Biological Products, after site-directed mutation of wild-type XynASP, heat treatment at 60℃ for 30min, the enzyme activity of wild-type XynASP was 7.1%, while the mutated XynLQ still retained 26.4% of the enzyme activity. Although the thermal stability of the mutant has been improved, the degree of improvement has not yet reached industrial requirements.

[0005] The industrial application of xylanase requires it to be resistant to high temperatures. By modifying it using protein engineering, its thermal stability can be improved, thereby being better applied to industrial production and reducing production costs. Summary of the invention

[0006] In order to overcome the above problems, the purpose of the present invention is to provide a thermostable xylanase and its preparation method and application. The present invention uses xylanase xyn as the starting gene, and uses site-directed mutagenesis technology to mutate it, thereby overcoming the defect of poor thermal stability of wild-type xylanase Xyn; finally, xylanase and its mutants are used to hydrolyze corn straw, thereby improving the hydrolysis efficiency of xylanase. The xylanase Xyn-G30D / Y59T obtained by the present invention has better application prospects, can reduce the production cost of xylanase in industrial applications, lays a foundation for the enzyme in industry, and provides a new strategy for the molecular modification of xylanase.

[0007] To achieve the above purpose, the technical solution designed by the present invention is as follows:

[0008] The invention provides a thermostable xylanase mutant. Compared with the amino acid sequence of the wild-type xylanase shown in SEQ ID NO: 2, the glycine at position 30 of the thermostable xylanase mutant is mutated to aspartic acid, and the tyrosine at position 59 is mutated to threonine.

[0009] Furthermore, the amino acid sequence of the wild-type xylanase is derived from Streptomyces rochei L10904.

[0010] Furthermore, the amino acid sequence of the thermostable xylanase mutant is shown in SEQ ID No.1.

[0011] The present invention also provides a gene encoding the thermostable xylanase mutant. The base sequence of the thermostable xylanase mutant gene is shown in SEQ ID NO:3.

[0012] The present invention also provides a method for preparing a thermostable xylanase mutant, comprising the following steps:

[0013] (1) obtaining the sfGFP-sumo fusion-promoting gene sequence as shown in SEQ ID NO: 4, loading the sfGFP-sumo fusion-promoting gene sequence into the pET23a vector to obtain the pET23a-sfGFP-sumo plasmid. Then, by PCR amplification technology, primers sfgfp-sumo-F and sfgfp-sumo-R, and the pET23a-sfGFP-sumo plasmid as a template, the sfGFP-sumo-Sxyn gene fragment is obtained, and the C-terminus of the sfGFP-sumo-Sxyn gene fragment contains the wild-type xylanase gene homologous sequence as shown in SEQ ID NO: 13;

[0014] (2) obtaining a wild-type xylanase xyn gene as shown in SEQ ID NO: 13, loading the wild-type xylanase xyn gene into a pET23a vector to obtain a pET23a-xyn plasmid, and obtaining a xyn-SsfGFP-sumo gene fragment by PCR amplification technology, primers xyn-F and xyn-R, and a template of the pET23a-xyn plasmid, wherein the N-terminus of the xyn-SsfGFP-sumo gene fragment contains a homologous sequence of the sfGFP-sumo fusion-promoting gene as shown in SEQ ID NO: 4;

[0015] (3) The sfGFP-sumo-Sxyn gene and the xyn-SsfGFP-sumo gene were fused together by fusion PCR technology and primers sfgfp-sumo-F and xyn-R to obtain the sfGFP-sumo-xyn fragment;

[0016] (4) The sfGFP-sumo-xyn fragment was recombined with the pET23a vector using seamless cloning enzyme to obtain the pET23a-sfGFP-sumo-xyn recombinant plasmid.

[0017] (5) performing a first site-directed mutagenesis on the pET23a-sfGFP-sumo-xyn recombinant plasmid: first, mutating the amino acid at position 30 of the wild-type xylanase amino acid sequence as shown in SEQ ID NO: 2, mutating the glycine at position 30 into aspartic acid, the first site-directed mutagenesis is achieved by PCR technology, primers G30D-F and G30D-R, and the template is pET23a-sfGFP-sumo-xyn, to obtain the pET23a-sfGFP-sumo-xyn-g30d plasmid;

[0018] (6) performing a second site-directed mutagenesis on the pET23a-sfGFP-sumo-xyn-g30d recombinant plasmid: mutating the amino acid at position 59 of the wild-type xylanase amino acid sequence, mutating the tyrosine at position 59 to threonine. The second site-directed mutagenesis was achieved by PCR technology, primers Y59T-F and Y59T-R, and template pET23a-sfGFP-sumo-xyn-g30d to obtain pET23a-sfGFP-sumo-xyn-g30d / y59t plasmid;

[0019] (7) The pET23a-sfGFP-sumo-xyn-g30d / y59t plasmid was transferred into the host bacterium Escherichia coli, cultured in shake flasks for protein expression, and the cells were broken to obtain a crude enzyme solution;

[0020] (8) The crude enzyme solution is purified by nickel column and ultrafiltration to obtain a thermostable xylanase mutant.

[0021] Furthermore, the preparation method of the thermostable xylanase mutant is:

[0022] (1) In the step 1), the amino acid sequences of the primers sfgfp-sumo-F and sfgfp-sumo-R used in the PCR amplification process are shown in SEQ ID No. 5 and SEQ ID No. 6;

[0023] (2) In step 2), the amino acid sequences of the primers xyn-F and xyn-R used in the PCR amplification process are shown in SEQ ID No. 7 and SEQ ID No. 8;

[0024] (3) In step 5), the amino acid sequences of primers G30D-F and G30D-R used in the PCR amplification process are shown in SEQ ID No. 9 and SEQ ID No. 10;

[0025] (4) In step 6), the amino acid sequences of primers Y59T-F and Y59T-R used in the PCR amplification process are shown in SEQ ID No. 11 and SEQ ID No. 12.

[0026] The present invention also provides a site-directed mutagenesis recombinant expression plasmid, comprising a thermostable xylanase mutant gene with a sequence as shown in SEQ ID No. 3, and an expression vector is pET23a.

[0027] The present invention also provides a site-directed mutagenesis recombinant expression strain, comprising the recombinant expression plasmid, wherein the host cell of the recombinant expression strain is Escherichia coli.

[0028] The present invention also provides an application of the thermostable xylanase mutant, wherein the thermostable xylanase mutant is applied to hydrolyzing corn stalks.

[0029] Furthermore, the application of the thermostable xylanase mutant, the hydrolysis environment is: pH 5-7, temperature 45-60 ° C, 1-1.5% powdered corn stalks, the concentration of the thermostable xylanase mutant added to the hydrolysis system is 8-10 mg / g 底物质量 , rotation speed is 120-160rpm, and hydrolysis time is 22-28h.

[0030] Beneficial effects of the present invention:

[0031] The invention obtains a xylanase mutant by molecularly modifying a wild-type xylanase Xyn, and the xylanase mutant has significantly improved thermal stability compared with the wild-type xylanase. In addition, the xylanase mutant has a wider suitable reaction temperature range. The xylanase mutant is used to hydrolyze corn stalks, and the results show that when hydrolyzed to 24 hours, the reducing sugar content of the xylanase mutant Xyn-G30D / Y59T is increased by 38.12% compared with the reducing sugar content of the wild-type xylanase Xyn; it can be seen that the xylanase mutant has a better hydrolysis effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the electrophoresis diagram of sfGFP-sumo fragment;

[0033] Figure 2 This is the electrophoresis diagram of pET23a vector and xyn fragment;

[0034] Figure 3 This is the electrophoresis diagram of sfGFP-sumo-xyn fragment;

[0035] Figure 4 This is a comparison of protein expression before and after the insertion of the sfGFP-sumo fusion-promoting tag;

[0036] Figure 5 Diagram of the construction process of pET23a-sfGFP-sumo-xyn-g30d plasmid;

[0037] Figure 6 is the three-dimensional structure diagram of xylanase;

[0038] Figure 7 constructing procedures for site-directed mutagenesis;

[0039] Figure 8 To obtain the electrophoresis of the PCR amplification product of pET23a-sfGFP-sumo-xyn-g30d plasmid;

[0040] In the figure, M: 10 kb Marker; 1: pET23a-sfGFP-sumo-xyn-g30d;

[0041] Fig. 9 To obtain the electrophoresis of the PCR amplification product of pET23a-sfGFP-sumo-xyn-g30d / y59t plasmid;

[0042] In the figure, M: 10 kb Marker; 1: pET23a-sfGFP-sumo-xyn-g30d / y59t;

[0043] Fig.10 is the standard curve of xylose concentration;

[0044] Fig.11 This is the result diagram of the optimum reaction temperature of the thermostable xylanase;

[0045] Fig.12 This is the result diagram of the thermostability of the thermostable xylanase;

[0046] In the figure, Fig.12 A: Thermal stability at 50°C; Fig.12 B: Thermal stability at 55°C; Fig.12 C: thermal stability at 60°C; Fig.12 D: Thermal stability at 65°C; Fig.12 E: thermal stability at 70°C;

[0047] Fig.13 The results of protein hydrolysis of corn stover that inhibited the growth of the strain;

[0048] Fig.14 The figure shows the results of protein hydrolysis of corn stover which does not inhibit the growth of the strain. DETAILED DESCRIPTION

[0049] In order to better understand the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific examples.

[0050] In the following examples, various molecular biological operations not specifically described were performed according to the instructions of the purchased enzymes or kits, or according to the Molecular Cloning Experiment Guide.

[0051] Example 1: Construction of the gene of the thermostable xylanase mutant xyn-g30d / y59t

[0052] 1. Materials

[0053] Strains and plasmids:

[0054] Escherichia coli expression competent cells BL21 (DE3) were purchased from Nanjing Novazon Biotech Co., Ltd., and the expression plasmid pET23a and the instruments and reagents required for constructing recombinant plasmids and recombinant strains were all purchased from conventional commercial sources.

[0055] Culture medium:

[0056] Preparation of LB liquid medium (1L): Prepare 1L of pure water, weigh 5g of yeast extract powder, 10g of peptone and 10g of sodium chloride, add the weighed items to 1L of pure water, stir and mix thoroughly until dissolved. Sterilize at 121℃ for 30min to allow strain growth.

[0057] Preparation method of LA liquid culture medium (1L): prepare 1L of pure water, weigh 5g of yeast extract powder, 10g of peptone and 10g of sodium chloride, add the weighed items to 1L of pure water, stir and mix thoroughly until dissolved, then sterilize at 121°C for 30min to allow strain growth, and add 0.1% of 0.1M ampicillin when used.

[0058] Preparation method of LA solid medium (1 L): Add 15 g of agar powder to the formula of LA liquid medium, then sterilize at 121°C for 30 min to allow strain growth, and add 0.1% of 0.1 M ampicillin when used.

[0059] (II) Construction of plasmid pET23a-sfGFP-sumo-xyn

[0060] 2.1. The base sequence of the gene sfGFP-sumo is shown in SEQ ID NO: 3. The gene was synthesized by Shanghai Sangon Biotechnology Co., Ltd. and loaded into the pET23a vector to obtain the pET23a-sfGFP-sumo plasmid. Using the pET23a-sfGFP-sumo plasmid as a template, the sfGFP-sumo-Sxyn gene fragment containing the wild-type xylanase gene homologous sequence shown in SEQ ID NO: 13 at the C-terminus was amplified by PCR using Phanta high-fidelity enzyme, primers sfgfp-sumo-F and sfgfp-sumo-R. The primers are shown in Table 1. The PCR amplification system is 2×Phanta MaxBuffera: 25μL; dNTPMix (10mM each): 1μL; Primer-F: 2μL; Primer-R: 2μL; Phanta enzyme: 1μL; template: 1μL; ddH2O: 18μL. The PCR amplification procedure was as follows: pre-denaturation at 95°C for 30 seconds; denaturation at 95°C for 15 seconds, annealing at 65°C for 15 seconds, extension at 72°C for 1.5 minutes, and the cycle was repeated 33 times; the final extension was at 72°C for 5 minutes. The correctness of the PCR product was verified using 0.7% agarose gel electrophoresis ( Figure 1 ), the target band size should be 1011 bp. The PCR product was subjected to 1% agarose gel electrophoresis, and the product was recovered using a product recovery kit to obtain the sfGFP-sumo-Sxyn gene fragment.

[0061] Table 1 PCR amplification primer sequence list

[0062]

[0063] 2.2. Using pET23a-xyn plasmid as template, PCR amplified the gene fragment xyn-SsfGFP-sumo containing the homologous part of sfGFP-sumo at the N-terminus by Phanta high-fidelity enzyme, primers xyn-F and xyn-R. The primers are shown in Table 1. The PCR amplification system and procedure are as shown above. The correctness of the PCR product was verified by 0.7% agarose gel electrophoresis technology ( Figure 2 ), the target band size should be 717 bp. The PCR product was subjected to 1% agarose gel electrophoresis, and the product was recovered using a product recovery kit to obtain the xyn-SsfGFP-sumo gene fragment.

[0064] 2.3. Using sfgfp-sumo-F and xyn-R as primers, fusion PCR was used to fuse the sfGFP-sumo-Sxyn gene and the xyn-SsfGFP-sumo gene fragments. The PCR amplification system and procedure are as described above. The correctness of the PCR product was verified using 0.7% agarose gel electrophoresis technology ( Figure 3 ), the target band size should be 1752 bp. The PCR product was subjected to 1% agarose gel electrophoresis, and the product was recovered using a product recovery kit to obtain the sfGFP-sumo-xyn fragment.

[0065] 2.4. Since the xyn gene alone cannot be successfully expressed in a soluble form during protein expression, in order to promote the soluble expression of xylanase Xyn, a fusion tag sfGFP-sumo was inserted at the N-terminus of xyn. After the fusion tag sfGFP-sumo was inserted at the N-terminus of xyn, soluble expression was successfully performed. The protein band size of xyn is 26kDa. After sfGFP-sumo is inserted at the N-terminus, xyn and sfGFP-sumo become a whole protein with a protein band size of 68kDa. The protein expression before and after the insertion of the fusion tag sfGFP-sumo is shown in Figure 2. Figure 4 Before the insertion of the fusion tag sfGFP-sumo, most of the protein appeared in the precipitate, forming inclusion bodies. After the insertion of the fusion tag sfGFP-sumo, most of the protein appeared in the supernatant, forming soluble expression.

[0066] 2.5. Use seamless cloning enzyme to efficiently recombine the sfGFP-sumo-xyn fragment and pET23a vector to obtain the recombinant plasmid pET23a-sfGFP-sumo-xyn. The recombinant reaction system is shown in Table 2. The required content of DNA fragment = 0.04 × the size of DNA fragment; the required content of vector = 0.02 × the size of vector. The corresponding volume can be calculated according to the concentration of the fragment and the vector. The total volume of the fragment and the vector cannot be greater than 4μL. After adding according to the corresponding system, react in a 37℃ water bath for 30min. The recombinant product can be stored in a -20℃ refrigerator for one week. Transfer the recombinant product into the host bacteria (Escherichia coli), add 5 μL of the product to 50 μL of Escherichia coli BL21 competent cells, and let it stand on ice for 30 minutes. When the time is up, heat shock the mixture in a 42°C water bath for 45 seconds, take it out and immediately put it on ice, then add 1 mL of pre-cooled LB liquid culture medium to the mixture, place the centrifuge tube in a shaker at 37°C and 220 rpm and culture for 40 to 60 minutes, take out the centrifuge tube, centrifuge at 12000 rpm for 1 minute, and pour out the supernatant. Use the remaining trace amount of liquid to resuspend the bacteria and spread it on the LA solid plate. Place the coated plate upside down in a 37°C constant temperature incubator and culture overnight until transformants grow. Obtain the plasmid pET23a-sfGFP-sumo-xyn. The plasmid construction process is as follows Figure 5 shown.

[0067] Table 2 Recombination reaction system

[0068]

[0069] (III) Construction of the thermostable xylanase gene xyn-g30d / y59t

[0070] Combination Figure 6 As shown, by analyzing the three-dimensional structure of xylanase Xyn, site-directed mutagenesis of wild-type xylanase Xyn was performed, and a total of 2 mutation sites (G30D, Y59T) were formed, and the mutation sites were analyzed using PyMol. The Loop region of the protein can affect the rigidity of the protein. The Y59T (tyrosine mutation to threonine) site is located in the Loop region of the protein. The introduction of this mutation site will enhance the rigidity of the protein Loop region, thereby increasing the thermal stability of the protein. Disulfide bonds are related to the thermal stability of proteins. The introduction of disulfide bonds in proteins can enhance the thermal stability of proteins. The introduction of the G30D (glycine mutation to aspartic acid) site can increase the disulfide bonds of the protein, thereby increasing the thermal stability of the protein.

[0071] To construct the thermostable xylanase xyn-g30d / y59 gene, the xyn-g30d gene was first constructed, and then the xyn-g30d / y59 gene was constructed based on the xyn-g30d gene. Figure 7 shown.

[0072] The plasmid pET23a-sfGFP-sumo-xyn was PCR amplified using Phanta enzyme and primers G30D-F and G30D-R (Table 1) to obtain the pET23a-sfGFP-sumo-xyn-g30d plasmid. The PCR amplification system and procedure are as described above. The correctness of the PCR product was verified using 0.7% agarose gel electrophoresis technology ( Figure 8 ), the target band size should be 5415bp. The PCR product was digested with DNA template to remove the excess plasmid template, and DpnⅠ enzyme was added to the PCR product, and reacted in a 37℃ water bath for 1.5h to obtain the digestion product.

[0073] The digestion product was transferred into the host bacteria (E. coli) and the transformation process was as described above. The coated plate was placed upside down in a 37°C constant temperature incubator and cultured overnight until transformants grew. The mutation site was sent for sequencing to verify whether it was correct. The mutation site was correct.

[0074] The plasmid of the transformant with the correct mutation site was extracted using the plasmid extraction kit to obtain the pET23a-sfGFP-sumo-xyn-g30d plasmid. The plasmid pET23a-sfGFP-sumo-xyn-g30d was PCR amplified using Phanta enzyme, primers Y59T-F and Y59T-R to obtain the pET23a-sfGFP-sumo-xyn-g30d / y59t plasmid (Table 1). The PCR amplification system and procedure were the same as above. The correctness of the PCR product was verified using 0.7% agarose gel electrophoresis technology ( Fig. 9 ), the target band size should be 5415bp. The PCR product was digested with DNA template to remove the excess plasmid template, and DpnⅠ enzyme was added to the PCR product, and reacted in a water bath at 37℃ for 1.5h to obtain the digestion product. According to the above steps, the digestion product was transferred into Escherichia coli, and after the transformant was grown, it was sent for sequencing to verify the correct mutation site, and the xylanase mutant xyn-g30d / y59t gene was obtained.

[0075] Example 2: Obtaining the Thermoresistant Xylanase Xyn-G30D / Y59T Protein Solution

[0076] (I) Expression of thermostable xylanase Xyn-G30D / Y59T

[0077] The xylanase Xyn-G30D / Y59T Escherichia coli BL21 expression strain was selected and placed in LA liquid medium. The culture temperature was adjusted to 37°C and the culture speed was adjusted to 220 rpm until the OD 600 Reach 0.8-1.2. Add 0.4% 0.1M IPTG, and before low-temperature induction expression, ensure that the shaker has been pre-cooled to 20°C and cultured at 200 rpm for 8 hours.

[0078] After protein expression, centrifuge at 8000rpm for 2min to collect the cells, discard the supernatant, add an appropriate volume of 20mM Tris-HCl solution with pH 7.0, resuspend the cells, centrifuge at 8000rpm for 2min, and repeat the above steps once. Add protein lysis buffer containing PMSF and lysozyme, resuspend the cells, and place in a 4℃ refrigerator for 2h. Use an ultrasonic disruptor to break the bacteria, set the power to 45%, set the time to 30min, start 1.5s, and stop 3.0s. After the end, centrifuge at 8500rpm for 15min to precipitate the cell debris, take the supernatant, discard the precipitate, and the supernatant is the crude enzyme solution.

[0079] (II) Purification of protein solution by ultrafiltration

[0080] First, regenerate the Ni column, add Stripping Buffer evenly to the Ni column, ensure that the filler is completely infiltrated, rinse sufficiently until the filler turns completely white, and rinse the Ni column twice with pure water to ensure that the Stripping Buffer is completely removed. Add 0.1M nickel sulfate to replenish nickel ions. Then rinse the column twice with 20mM Tris-HCl to complete the regeneration process. Pour the crude enzyme solution into the Ni column and place it in a 4℃ refrigerator for 40-60min for loading. Connect the column to the pipeline of the UV detector, select medium speed column flow, let the bacterial solution flow out, add low concentration imidazole eluent to elute the impurities at a low speed until the absorbance remains unchanged. Add 400mM imidazole eluent to elute the target protein, collect the sample, add buffer to rinse the Ni column, add pure water to rinse, and seal the column with 20% ethanol.

[0081] Add 0.1M sodium hydroxide solution to the protein ultrafiltration tube and soak for 30 minutes. After soaking, centrifuge at 4°C and 3000g for 30 minutes. The upper and lower centrifugal forces are 3. The centrifugation conditions during the protein ultrafiltration process are all the same. Pour out the sodium hydroxide, rinse with pure water, add pure water, centrifuge for 30 minutes, and repeat this step once. After pouring out the pure water, add the purified protein, centrifuge for 30 minutes, and centrifuge several times until the enzyme solution is added. Add the buffer used for purification and centrifuge for 30 minutes. This step needs to be repeated 2-3 times. Collect the protein concentrate (i.e., protein solution), add 0.1M sodium hydroxide to the ultrafiltration tube, soak for 30 minutes, pour out the sodium hydroxide, and add 20% ethanol to seal the tube.

[0082] Example 3: Determination of enzymatic properties of thermostable xylanase Xyn-G30D / Y59T

[0083] (I) Drawing of xylose concentration standard curve

[0084] Weigh 10 mg of xylose into 10 mL of pure water, mix well, add appropriate amount of pure water to dilute, and dilute to concentrations of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mg / mL respectively. Pipette 400 μL of xylose solution of each concentration gradient into a new centrifuge tube, and then pipette 400 μL of DNS solution and mix it evenly in a ratio of 1:1. React in a metal bath at 100°C for 10 minutes. After the reaction, cool the sample to room temperature. When adding the cooled reaction solution to a 96-well plate, ensure that it is added evenly to avoid bubbles. Use an enzyme reader for measurement, set the wavelength to 540 nm, use the data for statistical analysis, calculate the linear relationship between xylose concentration and absorbance, and obtain the equation of the standard curve ( Fig.10 ).

[0085] (II) Determination of the optimal reaction temperature of thermostable xylanase Xyn-G30D / Y59T

[0086] Prepare an appropriate volume of 1% beech xylan solution, draw 990μL of xylan solution and 10μL of xylanase protein solution to react, set up 8 experimental groups, react at 50, 55, 60, 65, 70, 75, 80, 85℃, react for 5min, take out immediately after the time is up, centrifuge briefly to sink xylan to the bottom of the tube, draw 400μL of reaction solution into a new centrifuge tube, add 400μL of DNS reaction and mix it in a ratio of 1:1, react in a metal bath at 100℃ for 10min to terminate the reaction. At the same time, set up 2 blank control groups: ①: 990μL of 1% beech solution + 10μL of pure water (to remove the influence of substrate); ②: 990μL of pure water + 10μL of protein solution (to remove the influence of protein solution itself); these 2 blank control groups react at the same time, and 3 groups of experimental and control groups are set in parallel. Prepare a 96-well plate, take 200 μL of the stop reaction solution and place it in the small well, use a microplate reader to measure the absorbance value, and set the wavelength at 540nm. Calculate the xylose concentration according to the xylose concentration standard curve and calculate the enzyme activity. The enzyme activity at the optimal reaction temperature is defined as 100%. The enzyme activity of xylanase is defined as: under the optimal conditions, the amount of enzyme required to generate 1 μg of reducing sugar per minute is defined as 1U.

[0087] The results are as follows Fig.11 As shown in the figure, the optimum reaction temperature of Xyn is 70°C, and the optimum reaction temperature of Xyn-G30D / Y59T is 65°C. The enzyme activity of Xyn-G30D / Y59T can be maintained above 90% at 50, 55, 60, 65, 70, 75, 80, and 85°C. The reaction temperature range of Xyn-G30D / Y59T becomes wider.

[0088] (III) Determination of thermal stability of thermostable xylanase Xyn-G30D / Y59T

[0089] Prepare a 1% beech xylan solution with a pH of 6. Dilute the protein solution to an appropriate multiple and incubate it in a water bath at 50, 55, 60, 65, and 70°C, take samples at 0, 5, 15, 30, and 60 minutes, and react the incubated protein solution with an appropriate substrate solution at 60°C for 5 minutes. Take it out immediately after the time is up, centrifuge it briefly to sink the xylan to the bottom of the tube, draw 400μL of the reaction solution into a new centrifuge tube, add 400μL of DNS reaction and mix it evenly in a ratio of 1:1, and react in a metal bath at 100°C for 10 minutes to terminate the reaction. At the same time, set up two blank control groups: ①: 990μL of 1% beech solution + 10μL of pure water (to remove the influence of the substrate); ②: 990μL of pure water + 10μL of protein solution (to remove the influence of the protein solution itself); these two blank control groups react at the same time, and three parallel groups are set for both the experimental group and the control group. Prepare a 96-well plate, take 200 μL of the stop reaction solution and place it in a small well, use a microplate reader to measure the absorbance value, and set the wavelength to 540 nm. Calculate the xylose concentration according to the xylose concentration standard curve and calculate the enzyme activity. The enzyme activity of the unincubated protein is defined as 100%.

[0090] The results are as follows Fig.12 As shown. Xyn-G30D / Y59T showed higher thermal stability. At 50°C, after incubation for 1 hour, the residual enzyme activities of Xyn and Xyn-G30D / Y59T were 64.38% and 99.72%, respectively. At 55°C, after incubation for 1 hour, the residual enzyme activities of Xyn and Xyn-G30D / Y59T were 45.24% and 95.74%, respectively. Under the conditions of 60°C, after incubation for 1 hour, the residual enzyme activity of Xyn-G30D / Y59T was 87.34%, while the enzyme activity of Xyn was 23.45%. Under the conditions of 65°C, after incubation for 1 hour, the residual enzyme activity of Xyn-G30D / Y59T was 79.73%, while the enzyme activity of Xyn was 0 at 30 minutes of incubation. Under the condition of 70℃, after incubation for 5 minutes, the enzyme activity of Xyn was only 9.68%, while the residual enzyme activity of Xyn-G30D / Y59T was 90.39%. The results showed that the thermal stability of Xyn-G30D / Y59T was greatly improved.

[0091] (IV) Determination of kinetic parameters of thermostable xylanase Xyn-G30D / Y59T

[0092] Use pH 6 buffer to prepare beech xylan solutions with concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0%. Dilute the protein solution by a large multiple to avoid the situation where the absorbance values ​​corresponding to some larger concentrations remain unchanged. Take the protein solution and react with substrate solutions of different concentrations at 60°C for 5 minutes, and take the supernatant to react with DNS. Each experimental group has 2 blank control groups, and 3 parallel groups are set up for the experimental group and the control group, and the absorbance value at 540nm is measured. Calculate the xylose concentration according to the xylose concentration standard curve, and use the double reciprocal plot method to calculate K m and K max value.

[0093] The results are shown in Table 3. m The K value can reflect the affinity of the protein to the substrate. m The values ​​are 1.16 and 3.05 respectively, indicating that the former has a better affinity for the substrate than the latter. cat The K value can reflect the catalytic efficiency of the protein. cat The values ​​were 8.75 and 5.05, respectively, indicating that the enzyme catalytic efficiency of the former was higher than that of the latter. The results showed that the xylanase Xyn-G30D / Y59T showed higher affinity and catalytic efficiency for the substrate.

[0094] Table 3 Kinetic parameters of thermostable xylanase Xyn-G30D / Y59T

[0095]

[0096] Example 4: Hydrolysis of corn stover by wild-type xylanase and its mutants

[0097] (I) Protein hydrolysis of corn stover to inhibit bacterial growth

[0098] Five experimental groups were designed: ① blank (no protein added); ② Xyn (wild-type xylanase); ③ Xyn-G30D / Y59T (thermoresistant xylanase mutant). The solution system was 30 mL, to which 0.3 g corn stalk (powdered), 0.03 mL of 2.5 mg / mL hydrochloric acid-tetracycline solution (to inhibit bacterial growth), 0.06 mL of Kathon solution (to inhibit fungal growth; it needs to be placed at room temperature for 1 hour before use), 12 mL of 0.25 mg / mL Xyn or 10 mL of 0.30 mg / mL Xyn-G30D / Y59T were added. Under the conditions of pH 6, temperature 50°C, and rotation speed 150 rpm, the reaction was carried out for 24 hours, and samples were taken every 3 hours in the first 9 hours until 24 hours. The taken samples were placed in a 100°C metal bath for 5 minutes to remove the enzymatic activity of the xylanase, and stored in a -20°C refrigerator for later use. Take an appropriate volume of sample solution and react with DNS to determine the reducing sugar content therein, the reaction condition is 100℃ for 10min. Set up 3 parallel experiments for each experimental group, measure the absorbance value at 540nm, and calculate the corresponding reducing sugar content using the xylose concentration standard curve.

[0099] The results are as follows Fig.13 As shown, after 24 hours of hydrolysis, the reaction no longer occurs. The hydrolysis effect of Xyn-G30D / Y59T is improved compared to Xyn. After 24 hours of hydrolysis, the reducing sugar content of Xyn-G30D / Y59T reached 0.67 mg / mL, and the reducing sugar content of Xyn was 0.49 mg / mL, which was 38.12% higher than that of Xyn. The results show that the hydrolysis rate of the thermostable xylanase Xyn-G30D / Y59T is improved compared to the control protein Xyn.

[0100] (II) Protein hydrolysis of corn stover that does not inhibit bacterial growth

[0101] Five experimental groups were designed: ① blank (without any protein); ② Xyn (wild-type xylanase); ③ Xyn-G30D / Y59T (thermoresistant xylanase mutant). The solution system was 30 mL, to which 0.3 g corn straw (powder) and 12 mL Xyn with a concentration of 0.25 mg / mL or 10 mL Xyn-G30D / Y59T with a concentration of 0.30 mg / mL were added. Under the conditions of pH 6, temperature 50°C, and rotation speed 150 rpm, the reaction was carried out for 24 hours, and samples were taken every 3 hours in the first 9 hours until 24 hours. The taken samples were placed in a 100°C metal bath for 5 minutes to remove the enzymatic activity of the xylanase, and stored in a -20°C refrigerator for later use. An appropriate volume of sample solution was taken to react with DNS to determine the content of reducing sugars therein, and the conditions were 100°C for 10 minutes. Three parallel experiments were set up for each experimental group, the absorbance value at 540 nm was measured, and the corresponding reducing sugar content was calculated using the xylose concentration standard curve.

[0102] The results are as follows Fig.14 As shown, after 24 hours of hydrolysis, the reducing sugar content of Xyn-G30D / Y59T reached 0.90 mg / mL, and the reducing sugar content of Xyn was 0.71 mg / mL, which was 26.76% higher than that of Xyn. No hydrochloric acid-tetracycline and casone solution were added to the hydrolysis environment, and reducing sugars would be produced even without adding protein. This is because microorganisms will grow during the hydrolysis process, thereby using corn stalks to produce reducing sugars.

[0103] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A thermostable xylanase mutant, characterized in that: Compared with the amino acid sequence of the wild-type xylanase shown in SEQ ID NO: 2, the glycine at position 30 of the thermostable xylanase mutant is mutated to aspartic acid, and the tyrosine at position 59 is mutated to threonine.

2. The thermostable xylanase mutant according to claim 1, characterized in that: The amino acid sequence of the wild-type xylanase is derived from Streptomyces roqueforti.

3. The thermostable xylanase mutant according to claim 1, characterized in that: The amino acid sequence of the thermostable xylanase mutant is shown in SEQ ID No.

1.

4. A gene encoding the mutant of the thermostable xylanase according to claim 1, characterized in that: The base sequence of the mutant gene of the thermostable xylanase is shown in SEQ ID NO:

3.

5. A method for preparing a thermostable xylanase mutant, characterized in that: The steps include: (1) obtaining a sfGFP-sumo fusion-promoting gene sequence as shown in SEQ ID NO: 4, loading the sfGFP-sumo fusion-promoting gene sequence into a pET23a vector to obtain a pET23a-sfGFP-sumo plasmid; then, by PCR amplification technology, primers sfgfp-sumo-F and sfgfp-sumo-R, and a template of the pET23a-sfGFP-sumo plasmid, obtaining a sfGFP-sumo-Sxyn gene fragment, wherein the C-terminus of the sfGFP-sumo-Sxyn gene fragment contains a wild-type xylanase gene homologous sequence as shown in SEQ ID NO: 13; (2) obtaining a wild-type xylanase xyn gene as shown in SEQ ID NO: 13, loading the wild-type xylanase xyn gene into a pET23a vector to obtain a pET23a-xyn plasmid, and obtaining a xyn-SsfGFP-sumo gene fragment by PCR amplification technology, primers xyn-F and xyn-R, and a template of the pET23a-xyn plasmid, wherein the N-terminus of the xyn-SsfGFP-sumo gene fragment contains a homologous sequence of the sfGFP-sumo fusion-promoting gene as shown in SEQ ID NO: 4; (3) The sfGFP-sumo-Sxyn gene and the xyn-SsfGFP-sumo gene were fused together by fusion PCR technology and primers sfgfp-sumo-F and xyn-R to obtain the sfGFP-sumo-xyn fragment; (4) Using seamless cloning enzyme, the sfGFP-sumo-xyn fragment was recombined with the pET23a vector to obtain the pET23a-sfGFP-sumo-xyn recombinant plasmid; (5) performing a first site-directed mutagenesis on the pET23a-sfGFP-sumo-xyn recombinant plasmid: first, mutating the amino acid at position 30 of the wild-type xylanase amino acid sequence as shown in SEQ ID NO: 2, mutating the glycine at position 30 into aspartic acid, the first site-directed mutagenesis is achieved by PCR technology, primers G30D-F and G30D-R, and the template is pET23a-sfGFP-sumo-xyn, to obtain the pET23a-sfGFP-sumo-xyn-g30d plasmid; (6) performing a second site-directed mutagenesis on the pET23a-sfGFP-sumo-xyn-g30d recombinant plasmid: mutating the amino acid at position 59 of the wild-type xylanase amino acid sequence, mutating the tyrosine at position 59 to threonine. The second site-directed mutagenesis was achieved by PCR technology, primers Y59T-F and Y59T-R, and template pET23a-sfGFP-sumo-xyn-g30d to obtain pET23a-sfGFP-sumo-xyn-g30d / y59t plasmid; (7) The pET23a-sfGFP-sumo-xyn-g30d / y59t plasmid was transferred into the host bacterium Escherichia coli, cultured in shake flasks for protein expression, and the cells were broken to obtain a crude enzyme solution; (8) Purifying the crude enzyme solution by nickel column and ultrafiltration to obtain a thermostable xylanase mutant; In the step 1), the sequences of the primers sfgfp-sumo-F and sfgfp-sumo-R in the PCR amplification process are shown in SEQ ID No. 5 and SEQ ID No. 6; In the step 2), the sequences of the primers xyn-F and xyn-R used in the PCR amplification process are shown in SEQ ID No. 7 and SEQ ID No. 8; In the step 5), the sequences of primers G30D-F and G30D-R used in the PCR amplification process are shown in SEQ ID No. 9 and SEQ ID No. 10; In the step 6), the sequences of primers Y59T-F and Y59T-R used in the PCR amplification process are shown in SEQ ID No.11 and SEQ ID No.

12.

6. A site-directed mutagenesis recombinant expression plasmid, characterized in that: The invention comprises a thermostable xylanase mutant gene with a sequence as shown in SEQ ID No. 3, and the expression vector is pET23a.

7. A site-directed mutagenesis recombinant expression strain, characterized in that: Comprising the recombinant expression plasmid as claimed in claim 6, the host cell of the recombinant expression strain is Escherichia coli.

8. Use of the thermostable xylanase mutant according to claim 1, characterized in that: The thermostable xylanase mutant is applied to the hydrolysis of corn straw.

9. The use of the thermostable xylanase mutant according to claim 8, characterized in that: The hydrolysis environment is: pH 5-7, temperature 45-60°C, 1-1.5% powdered corn stalks, and the concentration of the thermostable xylanase mutant added to the hydrolysis system is 8-10 mg / g 底物质量 , rotation speed is 120-160rpm, and hydrolysis time is 22-28h.

Images