Xylanase Mutants and Their Applications

Abstract

The xylanase mutant and its application are obtained by mutation of Ser21 and Thr324 sites of the parent HwXyl10A. The xylanase mutant provided by the present invention has greatly improved thermal stability. The half-life of mutants S21Y and T324V at 75°C is ( t 1 / 2 ) were 88 min and 34 min, which were 14.7 times and 5.7 times that of the wild type (6 min), respectively; the mutants S21Y and T324V T 50 The optimal pH and temperature were 80.0°C and 76.5°C, respectively, 12.0°C and 8.5°C higher than the wild-type (68.0°C). 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. This high-specific-activity thermostable xylanase mutant has broad application prospects in feed additives and biomass degradation for sugar production.

Description

Technical Field

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

[0002] Over 50% of biomass in nature is composed of components such as cellulose, hemicellulose, lignin, and pectin (Wong et al., 1988). Xylan, a major component of hemicellulose, is a renewable resource second only to cellulose in abundance in nature, accounting for approximately one-third of Earth's renewable organic carbon. Xylan has a complex structure, with a backbone composed of xylopyranose sugars linked by β-D-1,4-xylosidic bonds. Side chains are diverse, primarily including ferulic acid, α-L-arabinofuranosyl residues, O-acetyl groups, coumaric acid, and glucuronic acid residues (Squina et al., 2009). Therefore, the hydrolysis of xylan requires the involvement of multiple enzymes.

[0003] Xylanases are key enzymes in the hydrolysis of xylan, cleaving the β-1,4-glycosides of the xylan backbone to produce oligosaccharides or monosaccharides. Xylanases are widely used, primarily in animal feed, food, papermaking, beer brewing, and green energy (Zhang Honglian et al., 2002; Jiang Zhengqiang, 2005). Xylanases are found in 11 glycoside hydrolase families (GHs): 5, 7, 8, 10, 11, 16, 26, 30, 43, 52, and 62. GH10 and GH11 family xylanases have been the most studied. GH10 family xylanases have a (β / α)8 cylindrical structure, a higher molecular weight than GH11 family xylanases, and are more stable and have a broader substrate spectrum (Collins et al., 2005). Most GH10 family xylanases derived from microorganisms exhibit maximum activity in neutral or slightly acidic environments at moderate temperatures (approximately 40 to 60°C) (Collins et al., 2005). Industrial applications often involve high temperatures, making the development of xylanases with excellent thermostability and high catalytic activity crucial. Currently, the crystal structures of over 50 fungal xylanases have been determined (http: / / www.rcsb.org / pdb / ), laying the foundation for improved xylanase properties.

[0004] Protein engineering is widely used in enzyme modification. This involves altering the protein's molecular structure by modifying or modifying genes or proteins themselves, thereby altering enzyme function. Protein engineering is primarily used to design and modify enzyme properties such as thermostability, catalytic efficiency, substrate specificity, and tolerance to extreme environments. Key methods include directed evolution, rational design, and semi-rational design (Woodley, 2013). Rational design is a rapid and effective modification method that requires high accuracy in protein molecular structure. Commonly used methods in rational design include module replacement and site-directed mutagenesis. For example, this approach significantly improved the thermostability and pH stability of the xylanase XynA from Thermoascus aurantiacus (Angelica et al., 2016). The applicant previously used the high-specific-activity thermostable xylanase XYL10C as the material, and through rational design and site-directed saturation mutagenesis, identified the key amino acid site affecting the catalytic efficiency of GH10 family xylanases - Glu175, and used this as a guide to improve the catalytic efficiency and pH stability of the GH10 family xylanase XylE (You et al., 2018). Summary of the Invention

[0005] Technical problem to be solved: The present invention provides a thermostable xylanase mutant, which is a mutant obtained by screening after the key amino acid sites Ser21 and Thr324 in the GH10 family xylanase HwXyl10A are mutated respectively.

[0006] Technical solution: The xylanase mutant is obtained by mutating the Ser21 or Thr324 site of the parent HwXyl10A.

[0007] The nucleotide sequence of the parent HwXyl10A is shown in SEQ ID NO.1.

[0008] The amino acid sequence of the parent HwXyl10A is shown in SEQ ID NO.2.

[0009] The nucleic acid sequence after the Ser21 site mutation is shown in SEQ ID NO.3.

[0010] The amino acid sequence after the Ser21 site mutation is shown in SEQ ID NO.4.

[0011] The nucleic acid sequence after the above-mentioned Thr324 site mutation is shown in SEQ ID NO.5.

[0012] The amino acid sequence after the above-mentioned Thr324 site mutation is shown in SEQ ID NO.6.

[0013] The recombinant plasmid pic9r_S21Y comprises the above-mentioned thermostable xylanase mutant gene.

[0014] The recombinant plasmid pic9r_T324V comprises the above-mentioned thermostable xylanase mutant gene.

[0015] Recombinant bacteria containing the above recombinant plasmid.

[0016] Application of the above recombinant bacteria in the preparation of feed additives.

[0017] The present invention also provides a method for preparing the high specific activity thermostable xylanase mutant, and the technical scheme thereof is as follows:

[0018] 1) Using the wild-type xylanase recombinant expression plasmid pic9r_HwXyl10a as a template, specific primers were used to amplify the xylanase mutant linear recombinant plasmids pic9r_S21Y and pic9r_T324V by site-directed mutagenesis;

[0019] 2) Transforming the mutant recombinant vector into Pichia pastoris Gs115 and inducing expression with methanol to obtain mutant engineered strains, preferably Gs115 / S21Y and Gs115 / T324V;

[0020] 3) Cultivating the recombinant strain and inducing the expression of the recombinant xylanase;

[0021] 4) Recovering and purifying the expressed high specific activity thermostable xylanase mutants S21Y and T324V.

[0022] Beneficial effects: The thermostability of the xylanase mutants provided by the present invention is greatly improved, and the half-life (t 1 / 2 ) were 88 min and 34 min, which were 14.7 times and 5.7 times that of the wild type (6 min), respectively; the T 50 The values were 80.0°C and 76.5°C, respectively, 12.0°C and 8.5°C higher than the wild-type (68.0°C). In terms of catalytic activity, the specific activities of mutants S21Y and T324V were 3400 U / mg and 4000 U / mg, respectively, showing no significant difference compared to the wild-type enzyme (3260 U / 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. Compared with 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

[0023] Figure 1 is the optimal pH of the wild type and the two mutants;

[0024] Figure 2 pH stability of the wild type and two mutants;

[0025] Figure 3 is the optimum temperature of the wild type and the two mutants;

[0026] Figure 4 is the half-life of the wild type and two mutants at 80°C 1 / 2 ;

[0027] Figure 5 T of the wild type and two mutants 50 value. DETAILED DESCRIPTION

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0029] 1. Strains and vectors: The expression host Pichia pastoris GS115 was purchased from Invitrogen.

[0030] 2. Enzymes and other biochemical reagents: High-fidelity polymerase was purchased from Fermentas, beechwood xylan was purchased from Sigma, and all other reagents were domestic analytical grade (all available from common biochemical reagent companies).

[0031] 3. Culture medium:

[0032] 1) YPD medium: 2% glucose, 2% peptone, 1% yeast extract;

[0033] 2) LB medium: 1% peptone, 0.5% yeast extract, 1% NaCl, 1% agar powder (solid);

[0034] 3) MD medium: 1.5% agarose, 2% glucose, 0.00004% Biotin, 1.34% YNB;

[0035] 4) BMGY medium: 2% peptone, 1% yeast extract, 1% glycerol (v / v), 0.00004% Biotin, 1.34% YNB;

[0036] 5) BMMY medium: 2% peptone, 1% yeast extract, 1.34% YNB, 0.5% methanol (V / V), 0.00004% Biotin.

[0037] Example 1 Obtaining a gene encoding a high specific activity thermostable xylanase mutant

[0038] Using the recombinant expression vector pic9r_HwXyl10a of the xylanase gene HwXyl10a from Hortaea werneckii as a template, site-directed mutagenesis was performed at sites S21 and T324, respectively. The primer designs are shown in Table 1. The mutagenesis and cloning methods are described in the literature (You et al., 2016).

[0039] Table 1 Site-directed mutagenesis primers for xylanase HwXyl10A

[0040]

[0041] Example 2 Preparation of thermotolerant xylanase mutants

[0042] 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 / S21Y and Gs115 / T324V.

[0043] 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 BMMY medium containing 0.5% methanol. The culture was then incubated again at 30°C, 220 rpm, for 48 hours. The supernatant was used for enzyme activity testing, and mutants S21Y and T324V were identified, showing improved thermostability and catalytic activity compared to the wild-type enzyme.

[0044] A scaled-up fermentation system was established for the wild-type strain GS115 / HwXyl10A and two mutant strains, Gs115 / S21Y and Gs115 / T324V. 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 hours. The culture was then centrifuged at 3000 g for 5 minutes, the supernatant discarded, and the pellet resuspended in 100 mL of BMMY medium containing 0.5% methanol. The culture was then incubated again at 30°C and 220 rpm for induction. 0.5 mL of methanol was added every 12 hours 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.

[0045] Example 3 Comparative analysis of enzymatic properties of recombinant high specific activity thermostable xylanase mutant and wild type

[0046] 1. DNS method: The specific method is as follows: At the respective optimal pH and temperature conditions, a 1 mL reaction system consists of 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 sample is 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.

[0047] 2. Determination of properties of recombinant high-specific-activity thermostable xylanase mutants and wild-type

[0048] 1. The optimal pH and pH stability of the recombinant high specific activity thermostable xylanase mutant and wild type were determined as follows:

[0049] The purified xylanase mutants and wild type were subjected to enzymatic reactions at different pH values (1.0-6.5) to determine their optimal pH. The xylanase activity was determined at 75°C using 0.1 mol / L citric acid-sodium hydrogen phosphate buffer at different pH values. Figure 2 As shown in Figure 2, the optimal reaction pH of the wild type and mutants is similar between 4.0-4.5; the residual enzyme activity of the wild type xylanase and mutants was measured after being treated at 37℃ for 1 hour in an environment with a pH of 1.0-12.0. The results are shown in Figure 2. Figure 3 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.

[0050] 2. The optimal temperature determination method for wild-type xylanase and mutants is as follows:

[0051] The optimal temperature of the recombinant high specific activity thermostable xylanase mutant and the wild type was determined by performing the enzymatic reaction in a 0.1 mol / L citric acid-sodium hydrogen phosphate buffer (pH 4.5) at different temperatures (35-90°C). Figure 3 The results showed that the optimum temperature of the recombinant high specific activity thermostable xylanase mutant and the wild type (70℃) were between 70-75℃, and the relative enzyme activity of the two mutants at high temperature (80-90℃) was significantly improved compared with the wild enzyme.

[0052] 3. The thermostability of wild-type xylanase and mutants was determined as follows:

[0053] Half-life at 75℃ (t 1 / 2 ): The mutant and wild type were treated at 75℃ for different time periods, up to 120 min, and the residual enzyme activity of each was detected. The half-life determination results at 75℃ were as follows: Figure 5 It showed that the mutants S21Y and T324V had 1 / 2The thermal stability of mutant S21Y was 88 min and 34 min, respectively, which were 14.7 times and 5.7 times that of the wild type (6 min).

[0054] T 50 The mutant and wild-type enzymes were treated at 60-85°C for half an hour. The temperature at which the enzyme activity remained half of its original value was the T of the enzyme. 50 The result is as follows Figure 5 It showed that the T 50 The values were 80.0℃ and 76.5℃, respectively, which were 12.0℃ and 8.5℃ higher than the wild type (68.0℃), and the results were consistent with the half-life determination results. That is, the order of thermal stability was: S21Y>T324V>HwXyl10A.

[0055] 4. The kinetic parameter determination method of the recombinant high specific activity thermostable xylanase mutant and wild type is as follows:

[0056] The detection method was referred to the literature (Luo, et al., 2009) to determine the first-order reaction time of the reaction. 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. K was calculated using GraFit7 software. m Value and V max .

[0057] The catalytic efficiency (k cat / K m ) were both 2400 mL / s·mg, which was not much different from the wild type (2100 mL / s·mg); the specific activities of S21Y and T324V were 3400 U / mg and 4000 U / mg, respectively, of which the mutant T324V was 25% higher than that of the wild type (3200 U / mg) (Table 2).

[0058] Table 2 Specific activities and kinetic parameters of xylanase mutants and wild type

[0059]

Claims

1. A xylanase mutant, characterized in that The amino acid sequence of the Ser21 mutation of the parent gene HwXyl10A is shown in SEQ ID NO. 4.

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