A high-temperature resistant xylanase mutant and its preparation method and application

Abstract

The present invention discloses a thermostable xylanase mutant and its preparation method and application, and relates to the field of genetic engineering and genetic engineering technology. Hortaea werneckii Using the 10th Family xylanase HwXyl10A as the parent, point mutagenesis was used to screen for mutants N318W and D372L. Both mutants exhibited significantly improved thermostability and catalytic activity. The present invention identifies two key amino acid residues that influence the thermostability of Family 10 xylanases, providing important guidance for improving the thermostability of Family 10 xylanases. It also provides a basis for the industrialized production of xylanases and their application in feed preparation and biomass degradation of xylan to produce sugars.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering and genetic engineering, and in particular to a high-temperature resistant xylanase mutant and a preparation method and application thereof. Background Art

[0002] Cellulose, hemicellulose, lignin, and pectin are the main components of plant biomass in nature (Wong et al., 1988). Xylan, the most abundant component of hemicellulose, accounts for approximately one-third of Earth's renewable organic carbon, second only to cellulose. Xylan has a complex structure. Its backbone is composed of xylanose linked by β-1,4-glycosidic bonds, with side chains primarily consisting of O-acetyl, ferulic, coumaric, α-L-arabinofuranosyl, and glucuronic acid residues (Squina et al., 2009). Xylanases cleave the β-1,4-glycosidic bonds of the xylan backbone and are the primary enzymes responsible for xylan hydrolysis.

[0003] Xylanases are widely used in food, feed, beer brewing, papermaking, and bioenergy applications (Yao Bin et al., 2011). Xylanases belong to 11 glycoside hydrolase (GH) families: GH5, GH7, GH8, GH10, GH11, GH16, GH26, GH30, GH43, GH52, and GH62. GH10 family xylanases have been the most widely studied. These xylanases have a high molecular weight, mostly exceeding 35 kDa. Due to their (β / α)8 cylindrical structure, GH10 family xylanases are highly stable and have a broad substrate spectrum (Collins et al., 2005). Most GH10 family xylanases from microorganisms exhibit maximum activity in neutral or slightly acidic environments at moderate temperatures (approximately 40 to 60°C) (Collins et al., 2005). In industrial applications, most application environments are high-temperature environments, so obtaining xylanase with excellent thermal stability and high catalytic activity is crucial for industrial production.

[0004] Protein engineering is the process of modifying or modifying genes or proteins themselves to change the protein molecular structure and thus achieve the transformation of enzyme function. Protein engineering is widely used in improving enzyme properties, and the main areas involved include enzyme catalytic efficiency, thermal stability, extreme environment tolerance and substrate specificity. The main methods involved in protein engineering are rational design, semi-rational design and directed evolution (Woodley, 2013). Among them, rational design is a fast and effective means of transformation, which has high requirements for the accuracy of protein molecular structure. At present, the crystal structures of more than 50 microbial xylanases have been resolved (http: / / www.rcsb.org / pdb / ), which provides ideal structural information for improving the thermal stability of xylanases. The commonly used methods in rational design are module replacement and site-directed mutagenesis. If this method is used, Thermoascus aurantiacus The thermal stability and pH stability of the xylanase XynA derived from β-catenin were significantly improved (Angelica et al., 2016). Summary of the Invention

[0005] To address the shortcomings of the existing technology, the present invention provides thermostable xylanase mutants, their preparation methods, and references. Two thermostable xylanase mutants were obtained through site-directed mutagenesis. These mutants were screened by mutations at the key amino acid sites Asn318 and D372L in the GH10 family xylanase HwXyl10A. Compared with blind bacterial screening or artificial (natural) mutagenesis, enzyme molecular modification shortens the time required to modify enzymatic properties. These high-specific-activity thermostable xylanase mutants have broad application prospects in feed additives and biomass degradation for sugar production.

[0006] A thermostable xylanase mutant is obtained by subjecting xylanase HwXyl10A as a parent to site mutation; the site is Asn318 or Asp372, and the obtained mutants are respectively recorded as high specific activity thermostable xylanase mutant D318W or high specific activity thermostable xylanase mutant N372L.

[0007] As an improvement, the amino acid sequence of the xylanase HwXyl10A is shown as SEQ ID NO.1; the amino acid sequence of D318W is shown as SEQ ID NO.2; and the amino acid sequence of N372L is shown as SEQ ID NO.3.

[0008] Wherein, SEQ ID NO.1 is as follows:

[0009] tktetsvqmmtetstvtqsascsglsgstmtsggngstmsttmssssrtrpsttltatatatgsglndaaedagklyfgtaadipgtgeaqdpyymrefnnthdfgqatpanimkfvytepeqgvfnftggdyflnitdptknyirchnlvwgsqlptwitnpstnwtnatlsaalhnhvyttvsyfgdkcfawdvvneglsdnpagsymeniwynvigpeyipmafaaaqqaiedndlsvklyyndynieylgnkslaaqdivtelkgrgiqidgvgleshfiagstpsqsaqednmrafvnlgvevavtelNvrlnlppnatteaqqkldyyntvaacvavdgcvgitvwdfvdtyswipgtfpgqgygdlflqpdgadtpllkkaaydgclealege

[0010] SEQ ID NO. 2 is as follows:

[0011] tktetsvqmmtetstvtqsascsglsgstmtsggngstmsttmssssrtrpsttltatatatgsglndaaedagklyfgtaadipgtgeaqdpyymrefnnthdfgqatpanimkfvytepeqgvfnftggdyflnitdptknyirchnlvwgsqlptwitnpstnwtnatlsaalhnhvyttvsyfgdkcfawdvvneglsdnpagsymeniwynvigpeyipmafaaaqqaiedndlsvklyyndynieylgnkslaaqdivtelkgrgiqidgvgleshfiagstpsqsaqednmrafvnlgvevavtel W vrlnlppnatteaqqkldyyntvaacvavdgcvgitvwdfvdtyswipgtfpgqgygdlflqpdgadtpllkkaaydgclealege

[0012] SEQ ID NO. 3 is as follows:

[0013] tktetsvqmmtetstvtqsascsglsgstmtsggngstmsttmssssrtrpsttltatatatgsglndaaedagklyfgtaadipgtgeaqd pyymrefnnthdfgqatpanimkfvytepeqgvfnftggdyflnitdptknyirchnlvwgsqlptwitnpstnwtnatlsaalhnhvyttvs yfgdkcfawdvvneglsdnpagsymeniwynvigpeyipmafaaaqqaiedndlsvklyyndynieylgnkslaaqdivtelkgrgiqidgvg leshfiagstpsqsaqednmrafvnlgvevavteldvrlnlppnatteaqqkldyyntvaacvavdgcvgitvwdfvdtyswipgtfpgqgyg L lflqpdgadtpllkkaaydgclealege

[0014] As an improvement, the nucleotide sequence encoding the xylanase HwXyl10A is shown in SEQ ID NO. 4, the nucleotide sequence encoding the high specific activity thermostable xylanase mutant D318W is shown in SEQ ID NO. 5, and the nucleotide sequence encoding the high specific activity thermostable xylanase mutant N372L is shown in SEQ ID NO. 6.

[0015] Wherein, SEQ ID NO. 4 is as follows:

[0016]

[0017] SEQ ID NO. 5 is shown below:

[0018] actaagaccgaaacctccgttcaaatgatgaccgaaacctctaccgttacccaatctgcctcttgctctggattgtctggatccaccatgacctccggaggaaacggatctaccatgtctaccaccatgtcttcttcttccagaactagaccatctactaccttgaccgccactgctactgctaccggttccggtttgaacgatgccgccgaagatgctggaaagctttactttggtactgccgccgatattccaggtactggagaagctcaagacccatactacatgagagaatttaataacactcacgatttcggacaagctactccagctaatattatgaagtttgtttacaccgaaccagaacaaggtgtttttaattttactggtggagactactttttgaacattactgacccaaccaagaactacatcagatgtcataaccttgtttggggttcccaattgccaacttggattactaacccatccactaactggactaacgctactttgtctgctgctttgcacaatcatgtttacactaccgtttcatatttcggtgataagtgttttgcctgggatgttgttaacgagggtttgtctgataacccagctggttcttatatggagaacatttggtacaacgtcattggtcctgaatacattcctatggcttttgctgctgctcaacaggctattgaagataatgatttgtctgttaagttgtactacaacgattacaacattgagtacttgggtaacaagtctttggctgctcaggacattgttactgagttgaaaggtagaggtattcaaattgatggagttggtttggagagtcatttcattgccggttctactccatctcaatctgctcaagaagataacatgagagcatttgttaatttgggtgttgaagttgccgttaccgaattggatgttagattg TGGttgccaccaaacgccactactgaggcccaacaaaaattggattactacaacaccgttgcagcttgtgttgctgttgatggttgtgttggaattaccgtttgggattttgttgatacttactcttggatcccaggaacctttccaggtcaaggatatggtgatttgtttttgcagcctgatggtgctgatactcctttgttgaagaaggctgcttatgatggttgtttggaagctttggaaggtgaa

[0019] SEQ ID NO. 6 is as follows:

[0020] TTG ttgtttttgcagcctgatggtgctgatactcctttgttgaagaaggctgcttatgatggttgtttggaagctttggaaggtgaa

[0021] A recombinant vector contains a nucleotide sequence encoding the mutant.

[0022] A recombinant strain expressing the above-mentioned recombinant vector.

[0023] The method for preparing the above-mentioned thermostable xylanase mutant comprises the following steps:

[0024] Step 1: Recombinant expression plasmid with wild-type xylanase pic9r_ HwXyl10a As template, the linear recombinant plasmids pic9r_N318W and pic9r_D372L of the wood mutant were amplified by site-directed mutagenesis method using specific primers;

[0025] Step 2: Transform the mutant recombinant vector into Pichia pastoris Gs115, induce expression with methanol, and obtain mutant engineered strains, preferably Gs115 / N318W and Gs115 / D372L;

[0026] Step 3: Fermenting and culturing the recombinant Pichia pastoris strain to induce the expression of the recombinant xylanase;

[0027] Step 4: Recover and purify the expressed high specific activity thermostable xylanase mutants N318W and D372L.

[0028] As an improvement, the Pichia pastoris is Pichia pastoris GS115.

[0029] Application of the above-mentioned thermostable xylanase mutant in preparing feed or utilizing biomass to degrade xylan and produce sugar

[0030] Beneficial effects:

[0031] Compared with the prior art, the present invention provides a thermostable xylanase mutant and its preparation method and application. The obtained xylanase mutant has greatly improved thermal stability. In terms of thermal stability, the half-life of mutants N318W and D372L at 75°C is ( t 1 / 2 ) were 40 min and 25 min, which were 6.7 times and 4.2 times that of the wild type (6 min), respectively; the mutants N318W and D372L T 50The values were 76.5°C and 74.0°C, respectively, representing increases of 8.5°C and 6.0°C compared to the wild-type (68.0°C). In terms of catalytic activity, the mutants N318W and D372L exhibited specific activities of 3100 U / mg and 2800 U / mg, respectively, which were not significantly different from the wild-type enzyme (3200 U / mg). However, the catalytic efficiencies of the mutants N318W and D372L were 4600 mL / s·mg and 3700 mL / s·mg, respectively, representing increases of 1.2-fold and 76.2% compared to the wild-type (2100 mL / s·mg). The optimal pH and temperature were essentially the same as those of the wild-type, fully meeting the requirements for feed production and biomass degradation.

[0032] Compared to blind screening or artificial (natural) mutagenesis, enzyme molecular modification shortens the time required to modify enzymatic properties. This high-specific-activity, thermostable xylanase mutant has broad application prospects in feed additives and biomass degradation for sugar production. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The optimum pH values for the wild-type xylanase and two xylanase mutants N318W and D372L;

[0034] Figure 2 The pH stability of wild-type xylanase and two xylanase mutants N318W and D372L;

[0035] Figure 3 The optimum temperatures for the wild-type xylanase and two mutants, N318W and D372L;

[0036] Figure 4 The half-lives of wild-type xylanase and two mutants N318W and D372L at 75°C t 1 / 2 The measurement results;

[0037] Figure 5 The wild-type xylanase and two mutants N318W and D372L T 50 The measurement results of the value. DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0039] 1. Strains and vectors: expression host Pichiapastoris GS115 was purchased from Invitrogen.

[0040] 2. Enzymes and other biochemical reagents: High-fidelity polymerase was purchased from Fermentas, beechwood xylan was purchased from Sigma, and the others were domestic analytical grade reagents (all available from common biochemical reagent companies).

[0041] 3. Culture medium:

[0042] 1) YPD medium: 2% glucose, 2% peptone, 1% yeast extract;

[0043] 2) LB medium: 1% peptone, 0.5% yeast extract, 1% NaCl, 1% agar powder (solid);

[0044] 3) MD medium: 1.5% agarose, 2% glucose, 0.00004% Biotin, 1.34% YNB;

[0045] 5) BMGY medium: 2% peptone, 1% yeast extract, 1% glycerol (v / v), 0.00004% Biotin, 1.34% YNB;

[0046] BMMY medium: 2% peptone, 1% yeast extract, 1.34% YNB, 0.5% methanol (v / v), 0.00004% Biotin.

[0047] It should be noted that the technical solutions not mentioned in the following embodiments are commonly used in the art and do not require special explanation.

[0048] Example 1 Obtaining a gene encoding a high specific activity thermostable xylanase mutant

[0049] Based on Hortaea werneckii Xylanase genes HwXyl10a (GenBank: RMY90018.1) recombinant expression vector pic9r_HwXyl10a As a template, site-directed mutagenesis was performed on the N318 and D372 sites respectively. The primer design is shown in Table 1. The mutation and cloning methods are referred to the literature (You, et al., 2022).

[0050] Table 1 Site-directed mutagenesis primers for xylanase HwXyl10A

[0051]

[0052] Example 2 Preparation of thermotolerant xylanase mutants

[0053] The linear recombinant expression vector obtained by PCR was directly transformed into DMT competent cells and verified by colony PCR to obtain the nucleic acid sequences of 19 other mutants at this site. The recombinant plasmid was linearized and transformed into Pichia pastoris GS115 to obtain recombinant yeast strains Gs115 / N318W and Gs115 / D372L.

[0054] The Gs115 strain containing the recombinant plasmid was inoculated into 2 mL of BMGY medium in a 10 mL test tube and incubated at 30°C, 220 rpm, and shaken for 48 hours. The culture was then centrifuged at 3000 g for 5 minutes, the supernatant discarded, and the pellet resuspended in 2 mL of BMMGY medium containing 0.5% methanol. The tube was then incubated again at 30°C, 220 rpm, for 48 hours. The supernatant was used for enzyme activity assays, and mutants Gs115 / N318W and Gs115 / D372L were identified, demonstrating both improved thermostability and catalytic activity compared to the wild-type enzyme.

[0055] A scaled-up fermentation system was developed for the wild-type strain GS115 / HwXyl10A and two mutant strains, Gs115 / N318W and Gs115 / D372L. A seed culture was first inoculated into YPD medium. This culture was then inoculated at 1% inoculum into 300 mL of BMGY medium in a 1-L Erlenmeyer flask and incubated at 30°C and 220 rpm for 48 h. The culture was then centrifuged at 3000 g for 5 min, the supernatant discarded, and the pellet resuspended in 100 mL of BMMY medium containing 0.5% methanol. The culture was then inducible again at 30°C and 220 rpm. Every 12 h, 0.5 mL of methanol was added to maintain a methanol concentration of 0.5%. The supernatant was collected for enzyme activity assays. Finally, the supernatant was concentrated to 20 mL, and the protein was purified by anion exchange for enzymatic property analysis and comparison.

[0056] Example 3 Comparative analysis of enzymatic properties of recombinant high specific activity thermostable xylanase mutant and wild type

[0057] 1. DNS method: The specific method is as follows: Under the respective optimal pH and temperature conditions, a 1 mL reaction system includes 100 μL of diluted enzyme solution and 900 μL of substrate. The reaction is allowed to proceed for 10 minutes. 1.5 mL of DNS is added to terminate the reaction, and the reaction is then boiled in water for 5 minutes. After cooling, the OD value is measured at 540 nm. One unit (U) of enzyme activity is defined as the amount of enzyme required to degrade xylan to produce 1 μmol of reducing sugars per minute under the given conditions.

[0058] 2. Determination of properties of recombinant high-specific-activity thermostable xylanase mutants and wild-type

[0059] 1. The optimal pH and pH stability of the recombinant high specific activity thermostable xylanase mutant and wild type were determined as follows:

[0060] Purified mutant and wild-type xylanase were subjected to enzymatic reactions at different pH levels (1.0-6.5) to determine their optimal pH. The xylanase activity was determined at 75°C using the substrate xylan in 0.1 mol / L citric acid-sodium hydrogen phosphate buffer at different pH values.

[0061] The results are as follows Figure 1 As shown in Figure 2, the optimal reaction pH of the wild type and mutants is similar, at 3.5-4.5; the residual enzyme activity of the wild type xylanase and mutants was measured after being treated at 37°C for 1 hour in an environment with a pH of 1.0-12.0. The results are shown in Figure 2. Figure 2 As shown, there was no significant difference in pH stability between the wild type and the two mutants, and both were stable between pH 2.0 and pH 10.0.

[0062] 2. The optimal temperature determination method for wild-type xylanase and mutants is as follows:

[0063] The optimal temperatures of the recombinant high specific activity thermostable xylanase mutant and the wild type were determined by performing enzymatic reactions in a 0.1 mol / L citric acid-disodium hydrogen phosphate buffer (pH 4.5) system at different temperatures (35-90°C).

[0064] The results of determination of the optimum temperature of enzyme reaction are as follows Figure 3 The results showed that the thermotolerant xylanase mutants N318W and D372L were 5℃ and 10℃ higher than those of the wild type (70℃), respectively. The relative enzyme activities of the two mutants at high temperatures (80-90℃) were significantly higher than those of the wild type.

[0065] 3. The thermostability of wild-type xylanase and mutants was determined as follows:

[0066] Half-life at 75℃ ( t 1 / 2 ): The mutant and wild type were treated at 75°C for different time periods, up to 120 min, and their respective residual enzyme activities were detected.

[0067] The half-life determination results at 75℃ are as follows Figure 4 It showed that the half-lives of mutants N318W and D372L at 75°C ( t 1 / 2 ) were 40 min and 25 min, which were 6.7 times and 4.2 times that of the wild type (6 min), respectively.

[0068] T 50: The mutant and wild enzyme were treated at 60-85℃ for half an hour. The temperature corresponding to when the enzyme activity remained half of the original was the enzyme activity. T 50 value.

[0069] The results are as follows Figure 5 It showed that the mutants N318W and D372L T 50 The values were 76.5°C and 74.0°C, respectively, 8.5°C and 6.0°C higher than the wild type (68.0°C). The N318W mutant showed the highest thermal stability, a trend consistent with the half-life measurements. The order of thermal stability was: N318W > D372L > HwXyl10A.

[0070] 4. The kinetic parameter determination method of the recombinant high specific activity thermostable xylanase mutant and wild type is as follows:

[0071] The detection method was referred to the literature (You, et al., 2022) to determine the first-order reaction time of the reaction. K m and V max The reaction time was 5 min. Different concentrations of xylan (1.25, 1.0, 0.8, 0.4, 0.2, 0.15 and 0.1 mg / mL) were used as substrates. The enzyme activity was measured under the optimal conditions (temperature, pH), and the corresponding reaction rate was calculated using GraFit7 software. K m Value and V max .

[0072] Under their respective optimal conditions, using xylan as a substrate, the specific activities of the recombinant thermostable xylanase mutants N318W and D372L were 3100 U / mg and 2800 U / mg, respectively, which were not significantly different from the wild-type enzyme (3200 U / mg). However, the catalytic efficiencies of the mutants N318W and D372L were 4600 mL / s·mg and 3700 mL / s·mg, respectively, which were 1.2-fold and 76.2% higher than those of the wild-type enzyme (2100 mL / s·mg) (Table 2).

[0073] Table 2 Specific activities and kinetic parameters of xylanase mutants and wild type

[0074]

[0075] The thermal stability of the obtained xylanase mutants was greatly improved. In terms of thermal stability, the half-life of mutants N318W and D372L at 75℃ was ( t 1 / 2) were 40 min and 25 min, which were 6.7 times and 4.2 times that of the wild type (6 min), respectively; the mutants N318W and D372L T 50 The values were 76.°C and 74.0°C, respectively, representing increases of 8.5°C and 6.0°C compared to the wild-type (68.0°C). In terms of catalytic activity, mutants N318W and D372L exhibited specific activities of 3100 U / mg and 2800 U / mg, respectively, which were not significantly different from the wild-type enzyme (3200 U / mg). However, the catalytic efficiencies of mutants N318W and D372L were 4600 mL / s·mg and 3700 mL / s·mg, respectively, representing increases of 1.2-fold and 76.2% compared to the wild-type (2100 mL / s·mg). The optimal pH and temperature were essentially the same as those of the wild-type, fully meeting the requirements for feed production and biomass degradation.

Claims

1. A thermostable xylanase mutant, characterized in that: The mutants are obtained by performing site mutation on the parent xylanase HwXyl10A; the site is Asn318 or Asp372, and the obtained mutants are respectively recorded as high specific activity and heat-resistant xylanase mutant D318W or high specific activity and heat-resistant xylanase mutant N372L; the amino acid sequence of the xylanase HwXyl10A is shown in SEQ ID NO.1; the amino acid sequence of the D318W is shown in SEQ ID NO.2; and the amino acid sequence of the N372L is shown in SEQ ID NO.

3.

2. A recombinant vector, characterized in that: Contains a nucleotide sequence encoding the mutant according to claim 1.

3. A recombinant strain, characterized in that A strain expressing the recombinant vector according to claim 2.

4. The method for preparing the thermostable xylanase mutant according to claim 1, characterized in that: The following steps are involved: Step 1: Recombinant expression plasmid with wild-type xylanase pic9r_ HwXyl10a As template, the linear recombinant plasmids pic9r_N318W and pic9r_D372L of the wood mutant were amplified by site-directed mutagenesis method using specific primers; Step 2: Transform the mutant recombinant vector into Pichia pastoris Gs115, induce expression with methanol, and obtain mutant engineered strains, preferably Gs115 / N318W and Gs115 / D372L; Step 3: Fermenting and culturing the recombinant Pichia pastoris strain to induce the expression of the recombinant xylanase; Step 4: Recover and purify the expressed high specific activity thermostable xylanase mutants N318W and D372L.

5. Use of the thermostable xylanase mutant according to claim 1 or claim 4 in preparing feed or utilizing biomass to degrade xylan to produce sugar.

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