A xylanase mutant with improved enzymatic properties and its application

Abstract

A xylanase mutant with improved enzymatic properties and its application, wherein the D116 and E119 sites of the xylanase XynASP are mutated, and the amino acid sequence of the resulting mutant is shown in SEQ ID NO.1-SEQ ID NO.3, or an amino acid sequence with at least 90% homology to the above amino acid sequence, and retains the function of clarifying juice. In the juice clarification application, the wild type increased the clarification rate of mango juice and pitaya juice by a maximum of 53.34% and 60.16% at 90min and 105min, respectively, while XynASP-SV reached the maximum values ​​of 65.73% and 75.61% at 75min. The xylanase mutant obtained by introducing two mutation sites in the cord region of the present invention has significantly improved enzymatic properties compared to the wild type, and has shown high application prospects in the clarification experiments of mango juice and pitaya juice.

Description

Technical Field

[0001] The invention belongs to the technical field of xylanase, and particularly relates to a xylanase mutant with improved enzymatic properties and application thereof in fruit juice clarification. Background Art

[0002] Endo-β-1,4-xylanases, often referred to as xylanases (EC 3.2.1.8), are a class of glycoside hydrolases that specialize in xylan hydrolysis. Their unique ability lies in their ability to precisely cleave β-1,4-glycosidic bonds within the xylan backbone, generating functional oligosaccharides such as xylobiose, xylotriose, and higher-order xylo-oligosaccharides. Based on differences in structural characteristics, catalytic mechanisms, and physicochemical properties, xylanases are subdivided into multiple families, including 5, 7, 8, 10, 11, and 43. The GH10 and GH11 families are particularly prominent, with the GH11 family being designated as a representative class of xylanases in the CAZy database. GH11 family xylanases are renowned for their exceptional catalytic activity and substrate specificity, effectively converting xylan into oligosaccharide products such as xylobiose and xylotriose.

[0003] However, despite the excellent catalytic efficiency of GH11 family xylanases, most natural members are mesophilic enzymes. They are not stable enough in high temperature environments and have difficulty adapting to extreme conditions such as high temperature, strong acid or strong base often encountered in industrial production. Therefore, the optimization and improvement of their performance is particularly urgent. Studies have shown that although enhancing the rigidity of the enzyme structure can effectively improve its thermal stability, it is often accompanied by a significant decrease in enzyme activity. How to improve thermal stability while maintaining high catalytic activity has become a major challenge in current research. In particular, the cord region in xylanase, as a highly flexible peptide chain, can adjust its conformation according to the substrate morphology during the catalytic process to promote substrate binding. This characteristic is crucial for the efficient catalytic effect of the enzyme. Qin et al., using protein engineering techniques, introduced three mutants (SrxynF, SrxynM, and SrxynFM) into the cord region of the xylanase Srxyn from Streptomyces rochei L10904, successfully increasing the enzyme activity to 2.1-, 3.2-, and 5.3-fold, respectively, compared to the wild-type. This finding highlights the potential impact of cord region modifications on the catalytic performance of xylanases. Despite this, a comprehensive and systematic exploration of how cord region mutations specifically affect the thermostability and enzymatic activity of xylanases remains lacking.

[0004] Due to its broad application prospects, xylanases hold a significant position in the textile industry, bioenergy development, and food processing. Freshly squeezed juice, in particular, contains large amounts of polysaccharides such as cellulose and hemicellulose. These components not only increase juice viscosity but also hinder the sedimentation of solid particles, resulting in turbidity. Xylanases specifically break down hemicellulose, converting it into small molecules such as oligosaccharides and xylose, effectively reducing juice viscosity and promoting the settling of solid particles, thereby improving juice clarity. Existing studies have shown that the optimal application conditions for xylanases from different sources in juice clarification are a temperature of 40–60°C, a treatment time of 60–150 minutes, and a pH of 3.0–5.0. Given the application limitations of mesophilic xylanases, developing xylanases with increased heat and acid resistance and catalytic efficiency through rational or semi-rational design strategies is crucial for improving juice clarification processes. Summary of the Invention

[0005] Technical Problems to be Solved: In order to solve the above problems, the present invention provides a xylanase mutant with improved enzymatic properties and its application, involving two potentially beneficial mutation sites, and combining the mutations to form a xylanase mutant XynASP-SV with improved enzymatic properties, wherein both thermal stability and catalytic efficiency are improved. The optimum temperature of the mutant XynASP-SV is 5°C higher than that of the wild type, reaching 50°C; T m The value increased by 6℃ to 52℃, and the half-life at 50℃ increased by 4.62 times to 24.51min; the conversion efficiency (kcat / Km) increased by 27.7 times to 1765.80mL mg -1 s -1 The specific enzyme activity was 156 U / mg, a 3.04-fold increase compared to the wild-type enzyme. Even in a low-acid environment (pH 3.0), XynASP-SV maintained high catalytic activity, with a 30% increase in residual enzyme activity compared to the wild-type enzyme. In juice clarification applications, XynASP-SV achieved a maximum improvement in mango juice clarification of 65.73% and pitaya juice clarification of 75 minutes, respectively, reaching 75.61%.

[0006] Technical solution: A xylanase mutant, wherein the D116 and E119 sites of the xylanase XynASP are mutated, and the amino acid sequence of the obtained mutant is shown in SEQ ID NO.1-SEQ ID NO.3, or an amino acid sequence with at least 90% homology to the above amino acid sequence, and retains the function of clarifying juice.

[0007] Among them, the amino acid sequence of the xylanase mutant D116S is shown in SEQ ID NO.1; the amino acid sequence of the xylanase mutant E119V is shown in SEQ ID NO.2; and the amino acid sequence of the xylanase mutant XynASP-SV is shown in SEQ ID NO.3.

[0008] The gene encoding the xylanase mutant has a nucleotide sequence as shown in SEQ ID NO.4-SEQ ID NO.6, or a nucleic acid sequence having at least 90% homology with the above nucleic acid sequence, and can encode a protein with the function of clarifying juice.

[0009] Among them, the nucleotide sequence of the xylanase mutant D116S is shown in SEQ ID NO.4; the nucleotide sequence of the xylanase mutant E119V is shown in SEQ ID NO.5; and the nucleotide sequence of the xylanase mutant XynASP-SV is shown in SEQ ID NO.6.

[0010] An expression vector containing the above nucleic acid sequence.

[0011] Preferably, the above expression vector is a plasmid.

[0012] A host cell containing the above expression vector.

[0013] Preferably, the host cell is Escherichia coli or yeast.

[0014] A method for producing xylanase using the host cell comprises culturing the host cell and collecting the xylanase mutant expressed by the cell.

[0015] Application of the above xylanase mutants in the juice clarification process.

[0016] Preferably, the fruit juice is apple juice, orange juice, peach juice, mango juice or dragon fruit juice.

[0017] Beneficial effects: The present invention found through determination that the optimal pH of the mutant XynASP-SV is 5.0, which is slightly lower than that of the wild type (pH 6.0). When incubated at 40°C for 1 hour under pH 3.0-9.0 conditions, the residual enzyme activity of XynASP-SV under various pH conditions is greater than that of the wild type; the optimal temperature of the mutant XynASP-SV is 50°C higher than that of the wild type; when treated at 50°C and pH 5.0 for 60 minutes, the wild type only retains 16.84% of the enzyme activity, while the mutant XynASP-SV has an enzyme activity of 55.16%, which is 3.27 times higher than that of the wild type; mThe value increased by 6℃ to 52℃; the half-life at 50℃ increased by 4.62 times to 24.51min; the conversion efficiency (kcat / Km) increased by 27.7 times to 1765.80mL mg -1 s -1 ; The specific enzyme activity was increased by 3.04 times compared with the wild type, reaching 156U / mg. XynASP-SV showed good acid tolerance, with residual enzyme activities of 75.86%, 89.01%, and 83.92% at pH 3.0-5.0, which were improved compared with the residual enzyme activities of 45.86%, 65.69%, and 60.10% of the wild type. In the application of juice clarification, the maximum improvement in the clarification rate of mango juice and pitaya juice by the wild type was 53.34% and 60.16% at 90min and 105min, respectively, while XynASP-SV reached the maximum at 75min, which was 65.73% and 75.61%. The xylanase mutant obtained by introducing two mutation sites in the cord region of the present invention has significantly improved enzymatic properties compared with the wild type, and has shown high application prospects in the clarification experiments of mango juice and pitaya juice. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the three-dimensional model of xylanase XynASP.

[0019] Figure 2 These are the optimum temperatures and pH values ​​for xylanases XynASP, D116S, E116V, and XynASP-SV, where A represents the optimum temperature and B represents the optimum pH value.

[0020] Figure 3 The pH thermostability of xylanases XynASP, D116S, E116V and XynASP-SV.

[0021] Figure 4 The thermostability of xylanases XynASP, D116S, E116V and XynASP-SV at 50°C.

[0022] Figure 5 The clarification rates of mango juice and pitaya juice treated with xylanase XynASP and XynASP-SV. DETAILED DESCRIPTION

[0023] The present invention discloses a xylanase mutant, its preparation method and application, a DNA molecule encoding the xylanase mutant, a vector, and a host cell. Those skilled in the art can refer to the contents herein and appropriately modify the process parameters to achieve the desired results. The methods and applications of the present invention have been described through preferred embodiments. It is apparent that those skilled in the art can modify, alter, and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0024] Materials required for the experiment in Example 1:

[0025] The xylanase gene XynASP was derived from Aspergillus saccharolyticus JOP1030-1. Escherichia coli DH5α, Escherichia coli BL21(DE3), and pET-28a(+) were purchased from Novagen. The recombinant plasmid pET-28a-xynASP was fully synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. High-fidelity DNA polymerase, deoxyribonucleoside triphosphates (dNTPs), and the restriction endonuclease DpnI were purchased from New England Biolabs. Isopropyl-β-D-thiogalactopyranoside (IPTG) and kanamycin (Kan) were purchased from Takara. Beechwood xylan used in this study was purchased from Megazyme. SYPRO dye was purchased from Sigma-Aldrich. All other reagents were domestically produced and of analytical grade.

[0026] Preparation of main reagents:

[0027] LB medium:

[0028] Weigh 0.5 g of yeast powder, 1 g of peptone, and 1 g of NaCl, dissolve them fully in 70 mL of deionized water, make up to 100 mL, and sterilize at 121°C for 20 min.

[0029] Solid LB medium: Add 2% agar powder to the liquid medium.

[0030] TB medium recipe

[0031] 6 g of tryptone, 12 g of yeast extract, 2 mL of glycerol, add distilled water to 450 mL, and divide into 500 mL Erlenmeyer flasks, 90 mL per bottle, for a total of 5 bottles.

[0032] 3,5-Dinitrosalicylic acid (DNS reagent)

[0033] First, weigh 91g of potassium sodium tartrate and dissolve it in 300mL of hot water. Then, add 10.48g of NaOH, 3.15g of 3,5-dinitrosalicylic acid, 2.5g of phenol, and 2.5g of anhydrous sodium sulfite in sequence. Stir and dissolve while heating. After cooling, make up to 500mL. Protect from light and store at room temperature. Use after about one week.

[0034] Sodium hydrogen phosphate-citrate buffer (pH 5.0)

[0035] Weigh 33.5 g of disodium hydrogen phosphate and 11.2 g of citric acid, dissolve them in 800 mL of ddH2O, dilute to 1000 mL, and adjust the pH to 5.0.

[0036] Preparation of 0.5% beechwood xylan solution

[0037] Take 100 mL of disodium hydrogen phosphate-citric acid buffer (pH 4.6), weigh 0.5 g of beech wood xylan, put it into a conical flask, heat to boiling, boil for 5 minutes, cool, dilute to 100 mL with distilled water, and store at 4°C.

[0038] Construction of mutant model and determination of mutation sites

[0039] The amino acid sequence of the recombinant xylanase XynASP was submitted to Alphafold2 (https: / / alphafold2.biodesign.ac.cn / ) for homology modeling. Five resulting models were evaluated using the Predicted Local Distance Difference Test (PLDDT) and a model quality distribution plot was plotted to determine the most plausible model structure. The specific steps were performed using the software's default settings. After obtaining a high-quality model, protein sequence analysis and model structure delineation revealed that the cord region consists of the 12 amino acids connecting B5 and B6 in β-sheet B (Asp116, Tyr117, Gly118, Glu119, Tyr120, Asn121, Pro122, Gly123, Ser124, Ala125, Gly126, and Thr127).

[0040] Enzyme activity determination method

[0041] The DNS method is used. 1.5 mL of 0.5% beechwood xylan is reacted with 1 mL of appropriately diluted enzyme solution at an appropriate temperature for 15 minutes. The reaction is terminated by adding 2.5 mL of DNS and then boiled in a water bath for 7 minutes. After cooling, 5 mL of ddH2O is added, the mixture is shaken and mixed, and the absorbance is measured at 540 nm. A unit of xylanase activity (U) is defined as the amount of enzyme required to hydrolyze xylan to produce 1 μmol of xylose per minute under the above assay conditions.

[0042] Construction of saturation library of mutation sites and library screening

[0043] Using pET-28a-xynASP as the template, D116X-F, Y117X-F, G118X-F, E119X-F, Y120X-F, N121X-F, P122X-F, G123X-F, S124X-F, A125X-F, G126X-F, and T127X-F as upstream primers (Table 1), and T7-R as downstream primer, saturation mutagenesis was performed on the Asp116, Tyr117, Gly118, Glu119, Tyr120, Asn121, Pro122, Gly123, Ser124, Ala125, Gly126, and Thr127 codons by whole-plasmid PCR. The PCR products were digested with DpnI and transformed into E. coli BL21(DE3) to construct a saturation mutagenesis library. For each mutation site, 97 transformants (E. coli / xynD116X, E. coli / xyn Y117X, E. coli / xyn G118X, E. coli / xyn E119X, E. coli / xyn Y120X, E. coli / xyn N121X, E. coli / xynP122X, E. coli / xyn G123X, E. coli / S124X, E. coli / xynA125X, E. coli / xynG126X, and E. coli / xyn T127X, where X represents any amino acid) were selected from LB solid medium, inoculated into 1 mL of LB medium containing Kan (final concentration of 50 μg / mL), cultured at 37°C and 220 rpm for 12 h, and then transferred to 1 mL of the same medium at a 2% inoculum volume fraction and cultured until the OD 600When the concentration is about 0.6-0.8, add 0.5mmol / L IPTG and induce expression at 28℃ for 5h. Centrifuge at 4℃ and 8000r / min for 5min to collect the bacteria, add 200μL, 1mg / mL lysozyme solution, place in an incubator at 37℃ for lysis for 2h, and collect the supernatant after centrifugation as the crude enzyme solution. After the enzyme solution is appropriately diluted, take 5μL and add it to 95μL 0.5g / mL beech wood xylan solution (pH 5.5, 50mmol / L citric acid-sodium dihydrogen phosphate buffer), use a PCR instrument to carry out catalytic and colorimetric reaction (75℃ for 10min, add 50μL DNS reagent, 95℃ for 5min, cool to 10℃), add it to a 96-well plate, and measure the OD with a microplate reader. 540 Mutants corresponding to xylanases with enzyme activity greater than 30% of the original enzyme were defined as positive mutants and subjected to DNA sequencing. Combinatorial mutations were performed on sites with significantly improved enzyme activity, and the optimal mutants were selected using the DNS method.

[0044] Table 1 Mutation primer design

[0045]

[0046] Mutant expression and purification

[0047] The recombinant plasmid was transformed into E.coli BL21 (DE3) cells. Positive colonies were selected and inoculated into 2 mL of LB liquid culture medium containing Kan+ (final concentration of 50 μg / mL). The culture medium was then cultured overnight at 37°C and 180 rpm. Subsequently, 1 mL of the bacterial solution was transferred to TB culture medium, cultured at 37°C and 180 rpm for 3 hours, and then 200 μL of IPTG (final concentration of 2 mmol / L) was added to induce at 30°C and 180 rpm for 2 hours. After completion, the cells were broken and the supernatant was extracted as a crude enzyme solution. The recombinant xylanase was purified using a 10 cm nickel agarose gel (Ni Sepharose HP) column. The column was filled with 50 mmol / L phosphate buffer (pH 7.8, containing 300 mmol / L NaCl and imidazole at a concentration of 40.00-500.00 mM). Use Purification was performed using a fast protein liquid chromatography (FPLC) system. Purification of the purified recombinant xylanase was assessed by SDS-PAGE using a 10% separating gel and a 5% stacking gel. Protein concentration was quantified by Coomassie Brilliant Blue staining using bovine serum albumin (BSA) as a standard.

[0048] Characterization of mutant enzymatic properties

[0049] The optimum pH of xylanase was determined at 37°C in the pH range of 3.0-9.0. The pH stability of xylanase was determined by incubating the enzyme at 45°C in a pH 3.0-9.0 buffer for 15 min and measuring the residual enzyme activity. The optimum temperature of xylanase was determined by measuring the activity in 50 mM citric acid buffer at intervals of 5°C for 15 min in the range of 35°C-60°C. The pH stability of xylanase was determined by incubating the enzyme at 40°C at pH 3.0-9.0 for 60 min. The thermostability of xylanase was evaluated by incubating the enzyme at different times and temperatures. The protein concentration of pure enzyme was appropriately diluted with pH 5.0 citric acid buffer. The enzyme was kept at 35°C-50°C for 60 min and then rapidly cooled in an ice-water bath. The untreated xylanase activity was defined as 100%. The residual enzyme activity of the mutants and XynASP was calculated. The half-life t 1 / 2 Equal to ln2 / k, the formula is y=Ae -kt (A is the initial enzyme activity, “t” is the time, and “k” is the decay constant). The half-lives of the mutant and XynASP at 45°C and 50°C were calculated according to the formula.

[0050] Enzyme conformational stability was determined by differential scanning fluorimetry (DSF) using the CFX Touch 96-well system. Melting temperature (Tm) was measured. Based on DSF analysis, enzyme concentrations of 0.8-1.0 mg / mL were used, along with SYPRO dye. Samples were heated from 10°C to 95°C in a gradient of 0.5°C / s. Fluorescence was monitored at 480 / 580 nm (excitation / emission).

[0051] The enzyme was incubated with different concentrations of metal ions and chemical reagents at 40°C for 1 hour to detect the residual enzyme activity. The initial enzyme activity without any treatment was set as 100% to calculate the enzyme's tolerance to these substances. The metal ions (including K+, Ca2+, and Mg2+) at a concentration of 5 mM were detected. 2 +, Na+, Mg2+, Al3+, Mn2+, Fe2+, Fe3+, Ni 2 +, Cu2+, Zn2+ and Ba 2 +). And the resistance to the chemical reagents sodium dodecyl sulfate (SDS) and ethylenediaminetetraacetic acid (EDTA) at a concentration of 5 mM. In addition, the resistance to methanol, ethanol and isopropanol at a final concentration of 5% (vol / vol) was also studied.

[0052] Kinetic parameters were measured using different concentrations of beechwood xylan under the optimal reaction conditions for each enzyme. Km and Vmax values ​​were calculated using the Michaelis-Menten equation using GraphPad Prism software. All experiments were repeated three times.

[0053] Application of mutants in juice clarification

[0054] Fresh fruits (apples, mangoes, oranges, peaches, and pitaya) were thoroughly washed and peeled, then blended in a high-speed blender to a homogenous pulp. The pulp was then mixed with xylanase at a 1:1 volume / volume ratio (v / v) and incubated at 40°C for 150 minutes. Samples were collected every 15 minutes during this period. Each sample was boiled in boiling water for 5 minutes to inactivate the enzyme and then centrifuged at 4000g for 10 minutes. The resulting supernatant was analyzed for transmittance (Tt) at 640 nm. A control group not treated with xylanase was also analyzed for transmittance (Tc). The percent increase in clarity was calculated as follows: Percent increase in clarity = (Tt - Tc) / Tc × 100, where Tt = transmittance of the test sample and Tc = transmittance of the control sample.

[0055] The results show

[0056] Table 2 shows some enzymatic properties. The optimal pH for D116S, E119V, and XynASP-SV is 5.0. This is slightly lower than the optimal pH of 6.0 for the wild-type (WT). When incubated at 40°C for 1 hour within the pH range of 3.0 to 9.0, the pH fluctuations for both the wild-type and mutant enzymes were relatively stable, indicating a wide pH adaptability. Specifically, within the pH range of 3.0 to 5.0, XynASP-SV exhibited residual enzyme activities of 75.86%, 89.01%, and 83.92%, respectively. In comparison, the wild-type enzyme exhibited activities of 45.86%, 65.69%, and 60.10%, respectively, at the same pH levels. This indicates that XynASP-SV is more active than the wild-type enzyme within this pH range. The optimal temperature for D116S, E119V, and XynASP is 50°C, a 5°C increase compared to the wild-type (WT) enzyme's optimal temperature of 45°C. After treatment at 50°C and pH 5.0 for 60 minutes, both the wild-type and mutant enzymes retained more than 50% of their activity after 10 minutes of heat treatment. However, after 20 minutes of heat treatment, the activity of the wild-type enzyme decreased rapidly, retaining only 16.84% of its original activity. In contrast, the mutant enzymes D116S, E119V and XynASP-SV retained 41.75%, 44.44% and 55.16% of their activity, respectively. Compared with the wild type, these increased by 2.47 times, 2.64 times and 3.27 times, respectively. The melting temperature (Tm) values ​​of the enzymes and the half-life of the enzyme activity are listed in Tables 2 and Figure 5The Tm value of the wild-type enzyme is 46°C, while that of D116S, E119V, and XynASP-SV increases to 50°C, 50°C, and 52°C, respectively. At 45°C, the half-life of the wild-type enzyme is 22.34 minutes, while all mutants have longer half-lives. At 50°C, the half-life of the wild-type enzyme is only 5.31 minutes, but that of XynASP-SV is 24.51 minutes, 4.62-fold longer than that of the wild-type enzyme. The kinetic parameters of the wild-type (WT), D116S, E119, and XynASP-SV enzymes using beechwood xylan as a substrate are shown in Table 2. Compared with the wild-type, the mutants exhibited reduced Michaelis-Menten constants (Km) and enhanced substrate affinity. Catalytic efficiency was significantly improved, with the ratios of the catalytic constant to the Michaelis-Menten constant (kcat / Km) for D116S, E119, and XynASP-SV being 6.93-fold, 15.65-fold, and 27.69-fold higher than those of the wild-type, respectively. XynASP-SV exhibited the lowest Km value and the highest catalytic efficiency (kcat / Km), resulting in a significant increase in substrate release rate. In juice clarification applications, the wild-type achieved maximum improvements in mango and pitaya juice clarification rates of 53.34% and 60.16% at 90 and 105 minutes, respectively, while XynASP-SV achieved maximum improvements of 65.73% and 75.61% at 75 minutes, respectively. Comparison with previously published data on xylanases from various sources in juice clarification (Table 4) reveals that our xylanases exhibit superior performance in juice clarification.

[0057] Table 2 Enzyme property parameters

[0058]

[0059] Table 3 Clarification effect of various juices

[0060]

[0061] Table 4 Application of xylanases from different sources in juice clarification

[0062]

[0063] In summary, the present invention screened 10 potentially beneficial mutants in the cord region of the xylanase XynASP and constructed an optimal mutant, XynASP-SV, through rational combination. This mutant significantly improved thermostability, acid tolerance, and catalytic activity, and exhibited broad application potential in juice clarification, with the best clarification effect on mango and pitaya juices.

Claims

1. A xylanase mutant, characterized in that The D116 and E119 sites of the xylanase XynASP were mutated, and the amino acid sequences of the obtained mutants are shown in SEQ ID NO.1 to SEQ ID NO.

3.

2. The gene encoding the xylanase mutant according to claim 1, characterized in that The nucleotide sequences are shown in SEQ ID NO. 4 to SEQ ID NO.

6.

3. An expression vector containing the gene according to claim 2.

4. The expression vector according to claim 3, characterized in that The expression vector is a plasmid.

5. A host cell containing the expression vector according to claim 4.

6. The host cell according to claim 5, characterized in that The host cell is Escherichia coli or yeast.

7. A method for producing xylanase using the host cell according to claim 5, characterized in that: The method comprises culturing the host cells and collecting the xylanase mutants expressed by the cells.

8. Use of the xylanase mutant according to claim 1 in a juice clarification process.

9. The use according to claim 8, characterized in that The fruit juice is apple juice, orange juice, peach juice, mango juice or dragon fruit juice.

Images