Marine bacterium-derived pectate lyase mutant and application thereof

Abstract

The invention belongs to the field of enzymes, and particularly relates to a pectate lyase mutant derived from marine bacteria and application of the pectate lyase mutant. According to the present invention, a PNL is found from Bacillus halotolans, and is named as PNLHA, the amino acid sequence of the PNL is represented by SEQ ID NO: 1, and the enzyme activity of the PNL is as high as 229.4 U / mg; the S38G mutant with improved relative enzyme activity and thermal stability is obtained through site-specific mutagenesis on the basis of pectate lyase PNLHA, the amino acid sequence of the S38G mutant is shown as SEQ ID NO: 3, and when the S38G mutant is used for enzymolysis of grapefruit pulp, the quality of honey grapefruit tea can be effectively improved.

Description

Technical Field

[0001] The invention belongs to the field of enzymes, and in particular relates to a pectin lyase mutant derived from marine bacteria and application thereof. Background Art

[0002] Pectin is an acidic heteropolysaccharide widely found in the cell walls of higher plants. Its main chain is composed of D-galacturonic acid, connected by α-1,4-glycosidic bonds, and the side chains include units such as rhamnose, arabinose, galactose and xylose. Most natural pectin molecules are branched rather than linear. The main components of pectin include three polysaccharides with different structural characteristics: homogalacturonic acid, rhamnogalacturonic acid I, rhamnogalacturonic acid II and a small amount of xylosylgalacturonic acid. Due to this complex structure, the degradation of complex pectin usually requires the combined action of various pectinases, including pectin methylesterase, polygalacturonase, pectate lyase and pectin lyase.

[0003] Pectin lyase (PNL) and pectate lyase (PEL) act on pectin chains, but they target different substrates. PNL acts on methylated pectin, while PEL acts on unmethylated pectin. These enzymes catalyze β-elimination reactions to form C4-C5 unsaturated bonds at the non-reducing ends of polysaccharide fragments. PNL acts on substrates only by endocleavage, while PEL has both endo- and exo-cleavage activity. PNL does not require Ca for substrate cleavage. 2+ ions, while the activity of PEL depends on Ca 2+ ions. PNL performs better when processing methylated pectins, since many pectins contain methylated moieties and PEL lacks the ability to degrade such methylated pectins.

[0004] PNL has a wide range of applications. First, in the food industry, the use of pectin lyase has greatly promoted the production efficiency of juice, jam and honey beverage processing. The degradation of pectin can not only reduce the turbidity in the juice and make it clearer, but also add pectin lyase to the honey beverage to decompose the colloidal substances, significantly improve the transparency and fluidity of the honey, and reduce the crystallization phenomenon, making it easier to fill and store. In addition, pectin lyase can also improve the texture of jam and make it more uniform. Through the treatment of pectin lyase, the clarity of the juice can be significantly improved, the precipitation can be reduced, and the sensory quality of the product can be improved.

[0005] Although pectin lyase has shown great application potential in many fields, the pectin lyase products currently on the market are often mixed and lack targeted specificity. For example, in honey processing, the use of non-specific pectin lyase may over-decompose other components, affecting the natural flavor and nutritional value of honey. This mixed enzyme often exhibits problems such as low efficiency, waste of raw materials and environmental pollution in practical applications. Therefore, the development of efficient and specific pectin lyase preparations to meet the application needs of different fields has become an important direction of current research. Improving the performance of pectin lyase (such as temperature resistance and pH adaptability) through enzyme engineering technology and developing special enzyme preparations for the characteristics of different matrices such as honey and juice will help improve production efficiency, reduce environmental impact, and promote the sustainable development of related industries. Summary of the invention

[0006] In order to solve the above technical problems, the present invention is to obtain a salt-tolerant Bacillus ( Bacillus halotolerans ) and named it PNLHA. PNLHA showed good activity in a wide range of pH values ​​and temperatures. In order to improve its thermal stability, a pectin lyase mutant was obtained by site-directed mutagenesis, which further improved its thermal stability and pH stability, promoting its wide application in the fields of food and brewing.

[0007] The present invention first provides a pectin lyase derived from marine bacteria, the amino acid sequence of which is shown in SEQ ID NO:1, and the nucleotide sequence of the encoding gene thereof is shown in SEQ ID NO:2.

[0008] Based on the pectin lyase derived from marine bacteria, the present invention provides a pectin lyase mutant derived from marine bacteria through site-directed mutagenesis, whose amino acid sequence is shown in SEQ ID NO:3, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:4.

[0009] The present invention also provides a recombinant vector comprising a gene encoding the pectin lyase mutant, wherein the nucleotide sequence of the gene encoding is shown in SEQ ID NO:4.

[0010] Furthermore, the recombinant vector is a pPIC9K expression vector.

[0011] The present invention also provides a recombinant strain comprising the above recombinant vector.

[0012] Furthermore, the host cell of the recombinant strain is Pichia pastoris GS115.

[0013] The present invention also provides a method for producing the pectin lyase mutant, comprising: inoculating the recombinant strain into a fermentation medium, collecting the supernatant by centrifugation after fermentation, and obtaining the pectin lyase mutant after purifying the supernatant.

[0014] Further, including: (1) inoculating the recombinant strain (e.g., Pichia pastoris GS115 containing the expression frame of the gene encoding the pectin lyase mutant) into a 5 mL YPD test tube, culturing at 28° C., 200 rpm for 18 h, then transferring the inoculation volume to a liquid shake flask containing 50 mL BMGY at a 10% inoculation rate, and culturing at 28° C., 200 rpm in a shaker until the OD600 reaches about 5; (2) Centrifuge at 4000 × g for 10 min to collect the cells and discard the supernatant. Transfer all the precipitates to 50 mL of BMMY liquid culture medium and culture at 28°C, 200 rpm, and add 1% methanol every day. (3) After centrifuging the obtained bacterial solution, the supernatant is collected. The supernatant is the crude enzyme solution of the pectin lyase mutant; (4) Purify the crude enzyme solution using a Ni NTA affinity chromatography column to obtain a purified pectin lyase mutant.

[0015] Compared with the prior art, the beneficial effects of the present invention include at least: The pectin lyase PNLHA derived from marine bacteria provided by the present invention has an enzyme activity of up to 229.4 U / mg; based on the pectin lyase PNLHA, a S38G mutant with improved relative enzyme activity and thermal stability is obtained through site-directed mutagenesis, which can effectively improve the quality of honey pomelo tea when used for enzymatic hydrolysis of pomelo pulp. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The SDS PAGE electrophoresis results of pectin lyase PNLHA, lane M is the molecular weight standard, lane 1 is the protein supernatant, and lane 2 is the purified protein.

[0017] Figure 2 This is the relative enzyme activity change diagram of pectin lyase PNLHA; Figure 2 A in the figure is the relative enzyme activity under different temperature conditions. Figure 2 B is the relative enzyme activity measured every 1 h after incubation at 30°C, 40°C, 50°C, and 60°C for 4 h. Figure 2 C is the relative enzyme activity at 50℃ and different pH values. Figure 2 D in the figure is the relative enzyme activity after incubation at pH values ​​of 7, 8, 9 and 4°C for 96 h.

[0018] Figure 3The effect of different concentrations of small molecules on the activity of pectin lyase PNLHA. Figure 3 Effects of metal ions at 1mM and 10mM on the activity of pectin lyase PNLHA. Figure 3 Figure B shows the effect of small molecules such as 1mM and 10mM EDTA on the enzymatic activity of pectin lyase PNLHA.

[0019] Figure 4 Analysis of the flexible region of pectin lyase PNLHA, Figure 4 A is the ΔΔG heat map of PNLHA. Figure 4 Figure B shows the visualization analysis of PNLHA and its ligand tetragalacturonic acid.

[0020] Figure 5 The enzymatic properties of pectin lyase mutants are shown in Figure 2. Figure 5 A in the figure is the relative enzyme activity of PNLHA and mutants under different temperature conditions. Figure 5 B is the relative enzyme activity of PNLHA and mutants incubated at 50℃ for 4h. Figure 5 C is the relative enzyme activity of PNLHA and mutants after incubation at 60℃ for 4h. Figure 5 D in the figure is the residual enzyme activity of PNLHA and mutants after incubation at 4°C and pH 8 for 96 h. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and beneficial effects of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the present invention are only exemplary descriptions of the specific embodiments of the present invention, and are intended to be used to explain the present invention, but do not constitute a limitation of the present invention.

[0022] The endpoints of ranges and any values ​​disclosed herein are not limited to the exact range or value, and these ranges or values ​​should be understood to include approximations to these ranges.

[0023] Example 1: Pectin lyase PNLHA The marine bacteria halotolerant Bacillus ( Bacillus halotolerans ), through whole genome sequencing and screening, a pectin lyase was finally obtained, named PNLHA, whose amino acid sequence is shown in SEQ ID NO: 1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 2. Suzhou Hongxun Biotechnology Co., Ltd. was commissioned to perform gene synthesis using pPIC9K as a vector to obtain a recombinant plasmid pPIC9K containing the pectin lyase PNLHA gene.

[0024] Example 2: Transformation of recombinant plasmid pPIC9K and expression of pectin lyase PNLHA 1. Preparation of Pichia pastoris GS115 competent cells Pick the Pichia GS115 strain and inoculate it into 50mL YPD liquid medium, shake and culture it at 28℃ and 180 rpm for 12-18 h, take 3mL of the culture solution and inoculate it into fresh YPD liquid medium, and continue to culture it at 28℃ and 180 rpm for 6-8h. Take 35mL of the bacterial solution to a 50mL centrifuge tube, centrifuge it at 3000×g for 5 min, and discard the supernatant. Add 20mL of 1M sorbitol solution to resuspend the cells, centrifuge it at 4℃ and 3000×g for 5 min, and discard the supernatant to collect the bacteria. Add 5mL of transformation treatment solution to resuspend the bacteria, and add 50μL of 1M DTT solution to a final concentration of 10 mM. After mixing, let it stand at room temperature for 30 min, centrifuge it at 3000×g for 5 min, and discard the supernatant. Add 20mL of ice-cold 1M sorbitol solution to resuspend the cells, centrifuge it at 4℃ and 3000×g for 5min, and discard the supernatant. Repeat the process twice to add sorbitol solution to resuspend the cells and centrifuge them. After the last wash, centrifuge the bacterial suspension at 3000 × g for 5 min at 4°C, discard the supernatant and collect the bacteria. Resuspend the cells with 5 mL of ice-cold 1 M sorbitol solution, and dispense the cell suspension into sterilized 1.5 mL EP tubes (80 μL per tube) and place on ice for later use.

[0025] 2. Transformation of Linearized Plasmid The recombinant plasmid pPIC9K of Example 1 is used Sac Ⅰ Perform single enzyme digestion to obtain linear DNA fragments. Add 10 μL of the recovered linear DNA fragments to the 80 μL Pichia GS115 competent cells prepared above, mix well and add to the electroporation cup, and place on ice for 10-15 min. Connect the electroporation chamber to the electroporator, and place the electroporation cup in the electroporation chamber for electroporation treatment. The conditions are: voltage 1500 V, resistance 200 Ω, capacitance 25 μF, pulse time 5 ms. Immediately after the electroporation, add 1 mL of ice-cold 1 M sorbitol solution to the electroporation cup, mix the cells and transfer them to a 1.5 mL sterilized EP tube, stand at 30℃ for 1 h, then take an appropriate amount of cell suspension and evenly spread it on YNB medium (glucose 3%, YNB 1%, ammonium sulfate 0.5%). Incubate the plate upside down in a 28℃ incubator for 3-4 d and observe the growth.

[0026] 3. Induced expression and purification of yeast containing recombinant plasmid pPIC9K After electroporation, a single colony of recombinant Pichia pastoris GS115 was picked from the YNB screening plate and placed in a 5 mL YPD tube. After culturing at 28°C and 200 rpm for 18 h, the colony was transferred to a liquid shake flask containing 50 mL BMGY at a 10% inoculation rate and cultured at 28°C and 200 rpm until OD 600 to about 5. The cells were collected by centrifugation at 4000 × g for 10 min, and the supernatant was discarded. The precipitate was transferred to 50 mL BMMY liquid medium and cultured at 28°C and 200 rpm in a shaking table. 1% methanol was added every day for induction. After the recombinant bacteria were induced and cultured for 7 days, the bacterial solution was centrifuged at 4°C and 8000 × g for 15 min to obtain the supernatant containing the crude pectin lyase solution. The recombinant protein was purified by imidazole gradient elution using a Ni-IDA chromatography column on an AKTA protein purifier. The chromatographic column was pre-equilibrated with 20 mM phosphate buffer solution (pH 7.4), and the fermentation supernatant was filtered with a 0.22 μm microporous filter membrane and then loaded. The target protein was gradient eluted with 20 mM phosphate buffer solution (pH 7.4) with an imidazole concentration of 0.5 M to obtain a purified pectin lyase solution. SDS-PAGE electrophoresis showed that the molecular weight of pectin lyase PNLHA was approximately 34.7 kDa (such as Figure 1 shown).

[0027] 4. Pectin lyase PNLHA activity assay Take the above crude enzyme solution or purified alkaline protease, incubate 30 μL 10 g / L pectin solution at 50℃ for 5 minutes, add 2 μL of the enzyme solution to be tested, react at 50℃ for 15 minutes, add 30 μL 1M NaOH solution to terminate the reaction, incubate at 80℃ for 10 minutes, add 40 μL 1M HCI to acidify the reaction system after cooling to room temperature, mix well, and add 30uL 0.04M TBA solution. React at 80℃ for 10 minutes to develop color, and measure its absorbance at 550 nm. The enzyme activity unit is defined as: the amount of enzyme required to increase the 550 nm ultraviolet absorbance by 0.01 in 1 minute. The PNLHA enzyme activity was measured to be 229.4U / mg.

[0028] Example 3: Enzymatic properties of pectin lyase PNLHA The substrate was prepared using a pH 10.0 glycine and sodium hydroxide buffer solution, and the reaction was carried out in the temperature range of 30-80°C. The optimal reaction temperature of PNLHA was determined by detecting the activity. The experimental results are shown in Figure 2 A. The experimental results show that pectin lyase has a significant effect at a temperature of 45°C, and the effect is reduced at temperatures above or below 45°C. At 37.5°C and 65°C, the relative enzyme activity is still 70%, which shows that the enzyme has a wide temperature range and can adapt to different temperatures.

[0029] In order to evaluate the thermal stability of PNLHA, PNLHA was incubated at four temperatures of 30°C, 40°C, 50°C, and 60°C for 4 h, and the activity was measured every 1 h. Figure 2 Middle B. Pectin lyase can still maintain 90% of its enzyme activity when kept at 50° C. for 2 h, and still retain more than 60% of its activity at 60° C. The pectin lyase provided by the present invention has relatively excellent thermal stability.

[0030] In order to determine the optimal reaction pH of PNLHA, the substrate dissolved in different pH buffer solutions was used at 50°C and the reaction was carried out in the pH range of 3.0-12.0. The optimal reaction pH was determined by detecting the activity. The experimental results are shown in Figure 2 C. The experimental results show that the pectin lyase has a significant effect at pH 8.0, and the enzyme activity can be maintained above 70% at pH 6.0 and 10.0, indicating that the pectin lyase produced by the bacteria can maintain its enzymatic effect in a wider pH range.

[0031] The pH stability of PNLHA was tested by estimating the residual enzyme activity after incubation of PNLHA at 4°C for 12 h in different pH buffer solutions (citric acid-sodium citrate 3.0-6.0, disodium hydrogen phosphate-sodium dihydrogen phosphate 7.0-8.0, glycine-sodium hydroxide 9.0-12.0). The experimental results are shown in Figure 2 D. Pectin lyase showed excellent stability in the alkaline pH range of 7-9. In this pH range, the enzyme retained more than 80% of its original activity after 24 h at 25°C and 70% of its original activity after 48 h.

[0032] Take different metal ions Cu with final concentrations of 1 mM and 10 mM 2+ Mg 2+ , Ba 2+ , Ca 2+ 、Co 2+ , Fe 3+ , K + 、Zn + 、Na + , Mn 2+ 、Al 3+ , EDTA, SDS, DTT, β-mercaptoethanol, and Tween 80 were added to a 100 μL reaction system, and the activity of the purified pectin lyase was determined under the optimal pH and temperature conditions. The pectin lyase activity measured without adding metal ions was taken as 100%. The experimental results are shown in Figure 3 As shown, Figure 3 Effects of metal ions at 1mM and 10mM on the activity of pectin lyase PNLHA. Figure 3The results show that the higher concentration of Cu 2+ , Fe 3+ 、Al 3+ , SDS, DTT, β-mercaptoethanol, significantly reduced the enzyme activity, and a higher concentration of 1 mM Zn + The enzyme activity was significantly improved by 22.2%, while other ions and compounds had little effect on the enzyme activity.

[0033] Example 4: Pectin lyase mutants The flexible region of pectin lyase PNLHA was analyzed by molecular dynamics simulation GROMACS. The simulation conditions were set at 300K and 330K, with simulation times of 50 ns and 300 ns, respectively, and the RMSD (root mean square deviation) and RMSF (root mean square fluctuation) of the protein were calculated. Subsequently, virtual saturation mutagenesis was performed on the determined flexible region using FoldX software to generate all possible amino acid mutants, and the free energy change (ΔΔG) of each mutant was calculated to determine mutations that may improve thermal stability. Based on the FoldX energy calculation results, specific mutation sites were selected to improve thermal stability. RMSD analysis showed that the overall structure of the protein remained stable at both 300K and 330K, but the fluctuations observed at 330K were slightly larger, indicating that higher temperatures may cause greater conformational changes. RMSF analysis identified highly flexible regions of the protein, especially residues 37-41, 146-152, 285-288, and 306-310. Figure 4 In Figure A, the FoldX virtual mutation results show that some mutations in these flexible regions significantly reduce free energy, which may enhance local rigidity or stabilize the protein structure, thereby improving thermal stability. Based on energy evaluation and specific site analysis, four mutation sites, 37T, 38S, and 310S, were selected for experimental verification (the mutation sites on the protein are shown in Figure 4B). The binding energy of PNLHA with tetragalacturonic acid is -5.5 kcal / mol, indicating that the two are stably bound. Generally, a binding energy below -5 kcal / mol indicates that the interaction between the ligand and the protein is stable. Figure 4In B, tetragalacturonic acid binds to PNLHA through five salt bridges and ten hydrogen bonds. The ligand forms hydrogen bonds with LYS184, LYS292, GLN262, ASN150, ARG246 and ASP290 of the receptor, with bond distances ranging from 2.8 Å to 4.1 Å. These interactions contribute to the stable binding of the ligand and protein. Based on the above analysis, four mutants (T37Y, S38G, S310R and S310Q) were designed using site-directed mutagenesis to improve thermal stability. Suzhou Hongxun Biotechnology Co., Ltd. was commissioned to synthesize mutant genes using pPIC9K as a vector, and the mutants were expressed according to the method of Example 2.

[0034] Example 5: Enzymatic properties of pectin lyase mutants According to the method of Example 3, the enzymatic properties of the pectin lyase mutant were studied and compared with the PNLHA pectin lyase. The results are as follows Figure 5 shown. Figure 5 Figure A shows that S38G has better thermal stability than PNLHA and has higher enzyme activity at 30-70°C. The relative enzyme activity of S38G is 0.9%, 2.0%, 9.0%, 13.3%, and 2.4% higher than that of PNLHA, respectively. T37Y and S310R also showed slightly improved performance at 50°C and 60°C, while S310Q showed no significant difference compared with PNLHA. Figure 5 Medium B and Figure 5 Figure C shows the residual enzyme activity of PNLHA and each mutant after incubation at 50℃ and 60℃. Within 2h of incubation, the residual enzyme activity of PNLHA and the mutants was not much different. After incubation at 50℃ for 3h and 4h, the relative enzyme activity of the S38G mutant was 86.89% and 68.73%, respectively, which was significantly higher than that of PNLHA at 77.8% and 50.3%. Figure 5 Figure D shows that the pH stability of the four mutants is basically the same as that of PNLHA. In addition, after incubation at 60°C for 1 h, the relative enzyme activity of the mutants was 84.2%, while that of PNLHA was only 57.1%, which proves that the thermal stability of PNLHA was improved by site-directed mutagenesis. The S38G mutant has the best performance, and its amino acid sequence is shown in SEQ ID NO: 3, and its encoding gene is shown in SEQ ID NO: 4.

[0035] Example 6: Improvement of the quality of honey grapefruit tea by pectin lyase mutants Take 500g of grapefruit pulp, 300g of honey, and 1000mL of purified water. Prepare pectin lyase S38G mutant, using an amount of 0.02%-0.1% of the weight of grapefruit pulp. Chop the grapefruit pulp, mix it with purified water, heat it to 45-50°C, and keep it at a constant temperature. Evenly add the pectin lyase S38G mutant to the grapefruit pulp mixture, stir it at 150rpm for 60min, and ensure that the enzyme is fully in contact with the pulp. Heat the enzymatically hydrolyzed grapefruit pulp mixture to 85°C and keep it for 15 minutes to inactivate the pectin lyase S38G mutant. Mix the treated grapefruit pulp mixture with honey and stir evenly. Use a 100-mesh filter to filter the honey grapefruit tea to remove residual pomace and colloidal substances. Fill the filtered honey grapefruit tea into a sterile container and seal it for storage.

[0036] The turbidity of the treated honey grapefruit tea was reduced to below 20NTU, and the transparency was significantly improved. The texture of the grapefruit pulp was more delicate, the taste was smoother, and there was no obvious granularity. The stability of the honey grapefruit tea was significantly improved, and there was no obvious stratification or precipitation after storage for 30 days. The honey grapefruit tea that was not treated with pectinase had a turbidity of 80NTU, and obvious stratification and precipitation occurred after storage for 30 days. The honey grapefruit tea treated with pectinase had a turbidity of 20NTU, and there was no obvious stratification or precipitation after storage for 30 days, with uniform texture and delicate taste. It shows that the pectin lyase S38G mutant can significantly improve the quality of honey grapefruit tea.

[0037] The sequence involved in the present invention is as follows: SEQ ID NO:1 VALKRFSGFPLFAVSLSSRLLLLLPEKALGAVDFPNTSTNGILGFAGNAKNEKGVSKASTTGGKNGQIVYIQSLNDLKTHLSGSTPKILVLQNDLIASSKTTVTIGSNKTLVGSYAKKTLKNIYLTTSSSSGNVIFQNLTFEHSPQINGNNDIQLYLDSGINYWIDHVTFSGHSY SAGGSDLDKLLYIGKSADYITISNSKFANHKYGLILGYPDDSQHQYDGYPHMTIANNYFENLYVRGPGLMRYGYFHVKNNYSNNFNQAITIATKAKIYSEYNYFGKGSEKGGILDDKGTGYFKDTGSYPSLNRQSSPLTTWNPGSNYSYRVQTPQYTKEFVTKYAGSQSTTLVFGY SEQ ID NO:2 SEQ ID NO:3 VALKRFSGFPLFAVSLSSRLLLLLPEKALGAVDFPNTGTNGILGFAGNAKNEKGVSKASTTGGKNGQIVYIQSLNDLKTHLSGSTPKILVLQNDLIASSKTTVTIGSNKTLVGSYAKKTLKNIYLTTSSSSGNVIFQNLTFEHSPQINGNNDIQLYLDSGINYWIDHVTFSGHSYSAGGSDLDKLLYIGKSADYITISNSKFANHKYGLILGYPDDSQHQYDGYPHMTIANNYFENLYVRGPGLMRYGYFHVKNNYSNNFNQAITIATKAKIYSEYNYFGKGSEKGGILDDKGTGYFKDTGSYPSLNRQSSPLTTWNPGSNYSYRVQTPQYTKEFVTKYAGSQSTTLVFGY SEQ ID NO:4 Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and do not constitute a limitation on the content of the present invention. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A pectin lyase mutant derived from marine bacteria, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

3.

2. A recombinant vector, characterized in that: A gene encoding the pectin lyase mutant according to claim 1.

3. The recombinant vector according to claim 2, characterized in that The nucleotide sequence of the encoding gene is shown in SEQ ID NO:

4.

4. The recombinant vector according to claim 2, characterized in that The recombinant vector is a pPIC9K expression vector.

5. A recombinant strain, characterized in that: Comprising the recombinant vector according to any one of claims 2 to 4.

6. The recombinant strain according to claim 5, characterized in that The host cell of the recombinant strain is Pichia pastoris GS115.

7. A method for producing the pectin lyase mutant according to claim 1, characterized in that: include: The recombinant strain according to claim 5 or 6 is inoculated into a fermentation medium, and after fermentation, the supernatant is collected by centrifugation, and the supernatant is purified to obtain the product.

8. The method according to claim 7, characterized in that include: (1) The recombinant strain described in claim 5 or 6 was inoculated into a 5 mL YPD test tube, cultured at 28°C and 200 rpm for 18 h, and then transferred to a liquid shake flask containing 50 mL BMGY at a 10% inoculum, and cultured in a shaker at 28°C and 200 rpm until the OD600 reached about 5; (2) Centrifuge at 4000 × g for 10 min to collect the cells and discard the supernatant. Transfer all the precipitates to 50 mL of BMMY liquid culture medium and culture at 28°C, 200 rpm, and add 1% methanol every day. (3) After centrifuging the obtained bacterial solution, the supernatant is collected. The supernatant is the crude enzyme solution of the pectin lyase mutant; (4) Purify the crude enzyme solution using a Ni NTA affinity chromatography column to obtain a purified pectin lyase mutant.

9. The use of the pectin lyase mutant according to claim 1, characterized in that: The pectin lyase mutant is used in the processing of honey grapefruit tea.

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