A pectate lyase mutant derived from a marine bacterium and its application

Abstract

The present invention belongs to the field of enzymes, and particularly relates to a pectin lyase mutant derived from marine bacteria and its application. The present invention discovered a PNL from a halotolerant Bacillus sp. ( Halotolerant Bacillus ), named PNLHA, whose amino acid sequence is shown in SEQ ID NO:1, and its enzyme activity is as high as 229.4 U / mg. Based on the pectin lyase PNLHA, the S38G mutant with improved relative enzyme activity and thermal stability was obtained through site-directed mutagenesis. Its amino acid sequence is shown in SEQ ID NO:3, and it can effectively improve the quality of honey pomelo tea when used to enzymatically hydrolyze pomelo pulp.

Description

Technical Field

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

[0002] Pectin is an acidic heteropolysaccharide widely present 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 have a branched structure rather than a linear structure. The main components of pectin include three polysaccharides with different structural characteristics: homogalacturonan, rhamnogalacturonan I, rhamnogalacturonan II, and a small amount of xylogalacturonan. Due to this complex structure, the degradation of complex pectin usually requires the combined action of various pectinases, including pectin methyl esterase, 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, forming C4-C5 unsaturated bonds at the non-reducing ends of polysaccharide fragments. PNL acts on the substrate only through endolytic cleavage, while PEL has both endolytic and exolytic cleavage activities. The substrate cleavage of PNL does not require Ca 2+ ions, while the activity of PEL depends on Ca 2+ ions. PNL performs better in processing methylated pectin because many pectins contain methylated parts, while PEL lacks the ability to degrade this methylated pectin.

[0004] The application fields of PNL are very extensive. First, in the food industry, the use of pectin lyase greatly promotes the production efficiency of juice, jam, and honey beverage processing. The degradation of pectin can not only reduce the turbidity in juice, making it clearer, but also adding pectin lyase to honey beverages can decompose the colloidal substances in them, significantly improving the transparency and fluidity of honey, while reducing the crystallization phenomenon, making it easier to fill and store. In addition, pectin lyase can also improve the texture of jam, making it more uniform. Through the treatment of pectin lyase, the clarity of juice can be significantly improved, precipitation can be reduced, and the sensory quality of the product can be improved.

[0005] Although pectin lyase has shown great application potential in multiple fields, the pectin lyase products currently on the market are often of a mixed type 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. Such mixed enzymes often exhibit problems such as low efficiency, raw material waste, and environmental pollution in practical applications. Therefore, developing highly efficient and specific pectin lyase preparations to meet the application requirements of different fields has become an important direction of current research. Improving the performance of pectin lyase (such as heat resistance and pH adaptability) through enzyme engineering technology and developing special enzyme preparations for the characteristics of different substrates such as honey and fruit juice will help improve production efficiency, reduce environmental impact, and promote the sustainable development of related industries. Summary of the Invention

[0006] To solve the above technical problems, the present invention discovered a PNL from a halotolerant Bacillus ( Bacillus halotolerans ), and named it PNLHA. PNLHA exhibits good activity within a wide range of pH values and temperatures. To improve its thermal stability, a pectin lyase mutant was obtained through site-directed mutagenesis, further enhancing its thermal stability and pH stability, and promoting its wide application in the food and brewing fields.

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

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

[0009] The present invention also provides a recombinant vector containing the encoding gene of the above pectin lyase mutant, and the nucleotide sequence of the encoding gene is as 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 containing 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 above-mentioned pectin lyase mutant, which includes: inoculating the recombinant strain into a fermentation medium, centrifuging the fermentation broth to collect the supernatant, and obtaining the product after purifying the supernatant.

[0014] Further, it includes:

[0015] (1) Inoculate the recombinant strain (such as Pichia pastoris GS115 containing the expression cassette of the coding gene of the pectin lyase mutant) into a 5 mL YPD test tube, culture at 28 °C and 200 rpm for 18 h, and then transfer it to a liquid shake flask containing 50 mL of BMGY at an inoculation amount of 10%, and culture on a shaker at 28 °C and 200 rpm until the OD600 reaches about 5;

[0016] (2) Centrifuge at 4000×g for 10 min to collect the cells and discard the supernatant. Transfer all the precipitate to 50 mL of BMMY liquid medium, culture on a shaker at 28 °C and 200 rpm, and add 1% methanol every day;

[0017] (3) Centrifuge the obtained bacterial liquid and collect the supernatant. The supernatant is the crude enzyme solution of the pectin lyase mutant;

[0018] (4) Purify the crude enzyme solution through a Ni NTA affinity chromatography column to obtain the purified pectin lyase mutant.

[0019] Compared with the prior art, the beneficial effects of the present invention at least include:

[0020] The pectin lyase PNLHA derived from marine bacteria provided by the present invention has an enzyme activity as high as 229.4 U / mg; based on the pectin lyase PNLHA, a S38G mutant with improved relative enzyme activity and thermal stability is obtained. When used for enzymolysis of pomelo pulp, it can effectively improve the quality of honey pomelo tea. Description of the Drawings

[0021] Figure 1 It is the SDS PAGE electrophoresis result of the pectin lyase PNLHA. Lane M is the molecular weight standard, lane 1 is the protein supernatant, and lane 2 is the purified protein.

[0022] Figure 2 It is the relative enzyme activity change diagram of the pectin lyase PNLHA; Figure 2 In it, A is the relative enzyme activity under different temperature conditions, Figure 2 In it, 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 In it, C is the relative enzyme activity at 50 °C and different pH values, Figure 2In D, it is the relative enzyme activity after incubation at pH values of 7, 8, 9 and 4 °C for 96 h.

[0023] Figure 3 It is the effect of different concentrations of small molecules on the enzyme activity of pectin lyase PNLHA. Figure 3 In A, it is the effect of metal ions at 1 mM and 10 mM on the enzyme activity of pectin lyase PNLHA. Figure 3 In B, it is the effect of small molecules such as 1 mM and 10 mM EDTA on the enzyme activity of pectin lyase PNLHA.

[0024] Figure 4 It is the analysis of the flexible region of pectin lyase PNLHA. Figure 4 In A, it is the ΔΔG heat map of PNLHA. Figure 4 In B, it is the visualization analysis of PNLHA and the ligand tetragalacturonic acid.

[0025] Figure 5 It is the enzymatic properties of pectin lyase mutants. Figure 5 In A, it is the relative enzyme activity of PNLHA and mutants under different temperature conditions. Figure 5 In B, it is the relative enzyme activity of PNLHA and mutants after incubation at 50 °C for 4 h. Figure 5 In C, it is the relative enzyme activity of PNLHA and mutants after incubation at 60 °C for 4 h. Figure 5 In D, it is the residual enzyme activity of PNLHA and mutants after incubation at 4 °C and pH 8 for 96 h. Detailed implementation manners

[0026] In order to make the objectives, technical solutions and beneficial effects of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation manners. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following implementation manners of the present invention are only illustrative descriptions of the specific implementation manners of the present invention, and are intended to explain the present invention, rather than limiting the present invention.

[0027] The endpoints and any values within the ranges disclosed herein are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges.

[0028] Example 1: Pectin lyase PNLHA

[0029] Isolate the marine bacterium Halobacillus halophilus from the coastal marine sediments in Aoshanwei Town, Jimo District, Qingdao City, Shandong Province ( Bacillus halotolerans), through whole-genome sequencing, followed by screening, a pectin lyase named PNLHA was finally obtained. Its 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 Huaxun Biotechnology Co., Ltd. was commissioned to synthesize the gene using pPIC9K as a vector to obtain a recombinant plasmid pPIC9K containing the pectin lyase PNLHA gene.

[0030] Example 2: Transformation of Recombinant Plasmid pPIC9K and Expression of Pectin Lyase PNLHA

[0031] 1. Preparation of Competent Cells of Pichia pastoris GS115

[0032] Pick a single colony of Pichia pastoris GS115 strain and inoculate it into 50 mL of YPD liquid medium. Incubate with shaking at 28 °C and 180 rpm for 12 - 18 h. Take 3 mL of the culture broth and inoculate it into fresh YPD liquid medium, and continue to incubate at 28 °C and 180 rpm for 6 - 8 h. Transfer 35 mL of the bacterial solution to a 50 mL centrifuge tube, centrifuge at 3000 × g for 5 min, and discard the supernatant. Add 20 mL of 1 M sorbitol solution to resuspend the cells, centrifuge at 4 °C and 3000 × g for 5 min, and discard the supernatant to collect the cell pellet. Add 5 mL of transformation treatment solution to resuspend the cell pellet, and add 50 μL of 1 M DTT solution to a final concentration of 10 mM. Mix well and let stand at room temperature for 30 min, then centrifuge at 3000 × g for 5 min and discard the supernatant. Add 20 mL of ice-cold 1 M sorbitol solution to resuspend the cells, centrifuge at 4 °C and 3000×g for 5 min and discard the supernatant. Repeat the addition of sorbitol solution to resuspend the cells and centrifuge twice. After the last wash, centrifuge the cell suspension at 4 °C and 3000 × g for 5 min, and discard the supernatant to collect the cell pellet. 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 them on ice for later use.

[0033] 2. Transformation of Linearized Plasmid

[0034] The recombinant plasmid pPIC9K of Example 1 was used with SacⅠ Perform single enzyme digestion to obtain linear DNA fragments. Add 10 μL of the recovered linear DNA fragments to the 80 μL of Pichia pastoris GS115 competent cells prepared above. After mixing, transfer the mixture into an electroporation cuvette and place it on ice for 10 - 15 min. Connect the electroporation chamber to the electroporator, put the electroporation cuvette into the electroporation chamber for electroporation treatment under the conditions of: voltage 1500 V, resistance 200 Ω, capacitance 25 μF, and pulse time 5 ms. Immediately after electroporation, add 1 mL of ice-precooled 1 M sorbitol solution to the electroporation cuvette. Mix the cells and transfer them into a 1.5 mL sterilized EP tube. After standing at 30 °C for 1 h, take an appropriate amount of the cell suspension and evenly spread it on the YNB medium (3% glucose, 1% YNB, 0.5% ammonium sulfate). Invert the plate and culture it in an incubator at 28 °C for 3 - 4 d, and observe the growth situation.

[0035] 3. Induction expression and purification of yeast containing recombinant plasmid pPIC9K

[0036] After the electrotransformation, pick a single colony of recombinant Pichia pastoris GS115 on the YNB screening plate and transfer it to a 5 mL YPD test tube. Culture it at 28 °C and 200 rpm for 18 h, then transfer it to a liquid shaking flask containing 50 mL of BMGY at an inoculation amount of 10% and culture it on a shaker at 28 °C and 200 rpm until the OD 600 reaches about 5. Centrifuge at 4000 × g for 10 min to collect the cells and discard the supernatant. Transfer all the precipitate to 50 mL of BMMY liquid medium and culture it on a shaker at 28 °C and 200 rpm. Induce with 1% methanol added every day. After inducing the recombinant bacteria for 7 d, centrifuge the bacterial solution at 4 °C and 8000 × g for 15 min to obtain the supernatant containing crude pectin lyase solution. Use a Ni-IDA chromatography column to purify the recombinant protein by imidazole gradient elution on an AKTA protein purifier. First, equilibrate the chromatography column with 20 mM phosphate buffer solution (pH 7.4). Filter the fermentation supernatant through a 0.22 μm microporous filter membrane and then load the sample. Gradient elute the target protein with 20 mM phosphate buffer solution (pH 7.4) containing 0.5 M imidazole to obtain the purified pectin lyase solution. SDS-PAGE electrophoresis shows that the molecular weight of pectin lyase PNLHA is about 34.7 kDa (as Figure 1 shown).

[0037] 4. Enzyme activity assay of pectin lyase PNLHA

[0038] Take the above crude enzyme solution or purified alkaline protease. After incubating 30 μL of 10 g / L pectin solution at 50 °C for 5 min, add 2 μL of the enzyme solution to be tested, react at a constant temperature of 50 °C for 15 min, add 30 μL of 1 M NaOH solution to terminate the reaction, incubate at 80 °C for 10 min, cool to room temperature, add 40 μL of 1 M HCl to acidify the reaction system, mix well, and then add 30 μL of 0.04 M TBA solution. React at 80 °C for 10 min for color development, and measure the absorbance value at a wavelength of 550 nm. The enzyme activity unit is defined as: the amount of enzyme required to increase the ultraviolet absorbance value at 550 nm by 0.01 in 1 min. The measured enzyme activity of PNLHA is 229.4 U / mg.

[0039] Example 3: Enzymatic properties of pectin lyase PNLHA

[0040] Prepare the substrate using glycine and sodium hydroxide buffer solution with pH 10.0, carry out the reaction in the temperature range of 30 - 80 °C, and determine the optimal reaction temperature of PNLHA by detecting the activity. The experimental results are as shown in Figure 2 Figure A. The experimental results show that the pectin lyase has a significant effect at a temperature of 45 °C, and the effect decreases when the temperature is higher or lower than 45 °C. At 37.5 °C and 65 °C, the relative enzyme activity is still 70%, indicating that the enzyme has a wide temperature range of action and can adapt to different temperatures.

[0041] To evaluate the thermal stability of PNLHA, incubate PNLHA at four temperatures of 30 °C, 40 °C, 50 °C, and 60 °C for 4 h, and measure the activity every 1 h. The experimental results are as shown in Figure 2 Figure B. The pectin lyase can still maintain 90% of its enzyme activity after being incubated at 50 °C for 2 h, and still retains more than 60% of its activity at 60 °C. The pectin lyase provided by the present invention has relatively excellent thermal stability.

[0042] To determine the optimal reaction pH of PNLHA, use the substrate dissolved in different pH buffer solutions at 50 °C, carry out the reaction in the range of pH 3.0 - 12.0, and determine the optimal reaction pH by detecting the activity. The experimental results are as shown in Figure 2 Figure C. The experimental results show that the pectin lyase has a significant effect at pH 8.0. When the pH is 6.0 and 10.0, the enzyme activity can be maintained above 70%, indicating that the pectin lyase produced by this bacterium can maintain its enzyme effect in a relatively wide pH range.

[0043] PNLHA was incubated 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), and its pH stability was detected by estimating the remaining enzyme activity. The experimental results are as Figure 2 shown in D. Pectin lyase showed excellent stability in the alkaline pH range of 7 - 9. In this pH range, the enzyme still retained more than 80% of its original activity after 24 h at 25 °C and 70% of its original activity after 48 h.

[0044] Take different metal ions Cu at 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+ , as well as EDTA, SDS, DTT, β - mercaptoethanol, and Tween 80 were added to a 100 μL reaction system, and the enzyme activity of the purified pectin lyase was measured under the optimal pH and temperature conditions. Taking the enzyme activity of pectin lyase measured without adding metal ions as 100%, the experimental results are as Figure 3 shown, Figure 3 in which A shows the effect of metal ions at 1 mM and 10 mM on the enzyme activity of pectin lyase PNLHA, Figure 3 and B in which shows the effect of small molecules such as EDTA at 1 mM and 10 mM on the enzyme activity of pectin lyase PNLHA. The results show that higher concentrations of 10 mM Cu 2+ , Fe 3+ , Al 3+ , SDS, DTT, β - mercaptoethanol significantly reduced the enzyme activity. Higher concentration of 1 mM Zn + significantly increased the enzyme activity by 22.2%, and the effects of other ions and compounds on the enzyme activity were relatively small.

[0045] Example 4: Pectin lyase mutants

[0046] 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 4 In 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.

[0047] Example 5: Enzymatic properties of pectin lyase mutants

[0048] 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 5Figure A shows that S38G has better thermal stability than PNLHA and higher enzyme activity at 30 - 70 °C. The relative enzyme activities of S38G are 0.9%, 2.0%, 9.0%, 13.3% and 2.4% higher than those of PNLHA respectively. T37Y and S310R also show slight improvements at 50 °C and 60 °C, while there is no significant difference between S310Q and PNLHA. Figure 5 Figure B and Figure 5 Figure C shows the remaining enzyme activities of PNLHA and each mutant after incubation at 50 °C and 60 °C. Within 2 h of incubation, there is little difference in the remaining enzyme activities of PNLHA and the mutants. After incubation at 50 °C for 3 h and 4 h, the relative enzyme activities of the S38G mutant are 86.89% and 68.73% respectively, which are significantly higher than 77.8% and 50.3% of PNLHA. Figure 5 Figure D shows that the pH stabilities of the four mutants are basically the same as that of PNLHA. In addition, after incubation at 60 °C for 1 h, the relative enzyme activity of the mutants is 84.2%, while that of PNLHA is only 57.1%, which proves that the thermal stability of PNLHA is 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.

[0049] Example 6: Improvement of the quality of honey pomelo tea by pectin lyase mutants

[0050] Take 500 g of pomelo pulp, 300 g of honey, and 1000 mL of pure water. Prepare the S38G mutant of pectin lyase, and the usage amount is 0.02% - 0.1% of the weight of pomelo pulp. Chop the pomelo pulp, mix it with pure water, heat it to 45 - 50 °C, and keep it at a constant temperature. Uniformly add the S38G mutant of pectin lyase into the pomelo pulp mixture, stir at a speed of 150 rpm for 60 min to ensure full contact between the enzyme and the pulp. Heat the enzymolyzed pomelo pulp mixture to 85 °C and keep it for 15 minutes to inactivate the S38G mutant of pectin lyase. Mix the treated pomelo pulp mixture with honey and stir evenly. Filter the honey pomelo tea through a 100 - mesh sieve to remove residual fruit dregs and colloidal substances. Fill the filtered honey pomelo tea into a sterile container and seal it for storage.

[0051] The turbidity of the processed honey pomelo tea is reduced to below 20 NTU, and the transparency is significantly improved. The texture of the pomelo pulp is finer, the taste is smoother, and there is no obvious granular feeling. The stability of the honey pomelo tea is significantly improved, and there is no obvious stratification or precipitation after 30 days of storage. The turbidity of the honey pomelo tea without pectinase treatment is 80 NTU, and obvious stratification and precipitation occur after 30 days of storage. The turbidity of the honey pomelo tea treated with pectinase is 20 NTU, and there is no obvious stratification or precipitation after 30 days of storage, the texture is uniform, and the taste is delicate. It shows that the pectin lyase S38G mutant can significantly improve the quality of honey pomelo tea.

[0052] The sequences involved in the present invention are shown as follows:

[0053] SEQ ID NO:1

[0054] VALKRFSGFPLFAVSLSSRLLLLLPEKALGAVDFPNTSTNGILGFAGNAKNEKGVSKASTTGGKNGQIVYIQSLNDLKTHLSGSTPKILVLQNDLIASSKTTVTIGSNKTLVGSYAKKTLKNIYLTTSSSSGNVIFQNLTFEHSPQINGNNDIQLYLDSGINYWIDHVTFSGHSYSAGGSDLDKLLYIGKSADYITISNSKFANHKYGLILGYPDDSQHQYDGYPHMTIANNYFENLYVRGPGLMRYGYFHVKNNYSNNFNQAITIATKAKIYSEYNYFGKGSEKGGILDDKGTGYFKDTGSYPSLNRQSSPLTTWNPGSNYSYRVQTPQYTKEFVTKYAGSQSTTLVFGY

[0055] SEQ ID NO:2

[0056]

[0057] SEQ ID NO:3

[0058] VALKRFSGFPLFAVSLSSRLLLLLPEKALGAVDFPNTGTNGILGFAGNAKNEKGVSKASTTGGKNGQIVYIQSLNDLKTHLSGSTPKILVLQNDLIASSKTTVTIGSNKTLVGSYAKKTLKNIYLTTSSSSGNVIFQNLTFEHSPQINGNNDIQLYLDSGINYWIDHVTFSGHSYSAGGSDLDKLLYIGKSADYITISNSKFANHKYGLILGYPDDSQHQYDGYPHMTIANNYFENLYVRGPGLMRYGYFHVKNNYSNNFNQAITIATKAKIYSEYNYFGKGSEKGGILDDKGTGYFKDTGSYPSLNRQSSPLTTWNPGSNYSYRVQTPQYTKEFVTKYAGSQSTTLVFGY

[0059] SEQ ID NO:4

[0060]

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation to the content of the present invention. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within 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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