A pectate lyase and its mutants, recombinant expression vectors and applications

Abstract

A pectate lyase of the present invention, its mutants, recombinant expression vectors and applications; the nucleotide sequence of the pectate lyase is shown as SEQ ID NO.1, the full length of the gene is 1056 bp, encoding 352 amino acids in total, and the amino acid sequence is shown as SEQ ID NO.2; the pectate lyase mutant is obtained by site-directed mutagenesis based on the amino acid sequence of the pectate lyase, the mutated nucleotide sequence is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO.4; compared with the existing pectate lyases, the pectate lyase and its mutants provided by the present invention have the characteristics of high activity and temperature stability, and can be widely applied to juice extraction, juice clarification and the processing of fruit and vegetable juice beverages.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly to a pectin lyase and its mutants, recombinant expression vectors and applications. Background Art

[0002] Natural pectin has various biological activities such as anti-inflammatory, anticoagulant, anti-tumor, antioxidant, antibacterial, etc., and has the ability to regulate the intestinal environment. However, natural pectin has defects in terms of being absorbed by the human body, and the high molecular weight and complexity of natural pectin will limit their bioavailability.

[0003] Pectin oligosaccharides (POSs) are degradation products of pectin and are also oligomeric products with various good biological activities. POSs can be selectively utilized by intestinal microorganisms, so they are considered to be the best choice for the second-generation prebiotic factors and have biological activities such as antibacterial, anti-cancer, antioxidant properties, etc.

[0004] The methods for degrading pectin into pectin oligosaccharides usually include physical degradation method, chemical hydrolysis method and enzymatic preparation method. Among them, enzymatic preparation is the most effective method at present, with obvious advantages such as mild reaction conditions, strong specificity, high degradation efficiency, etc.

[0005] Pectinase refers to a multi-enzyme complex system that can degrade pectin substances and can degrade various structures of pectin. According to the action mode of pectinase on the galacturonic acid backbone in pectin substances, pectinase can be divided into three categories: polygalacturonase, pectin lyase and pectin methylesterase. Different pectinases cleave different types of α-1,4-glycosidic bonds in pectin. According to the optimal enzyme activity pH, it can be divided into two major categories: alkaline and acidic. Alkaline pectinase is mainly applied in the food production process to help decompose pectin, improve the texture and taste of products, and improve digestion and absorption; acidic pectinase is mainly used for extracting and clarifying fruit and vegetable juices and fruit wines to reduce the turbidity and viscosity of products.

[0006] Among the natural sources of pectinase, microorganisms such as fungi, bacteria, actinomycetes and yeasts have become the main sources of commercial pectinase due to their advantages such as short growth cycle, easy control of culture conditions, wide distribution, etc. Among them, pectinase from fungi is the most common. Fungal sources include Rhizopus, Aspergillus niger, Aspergillus japonicus, etc. Most fungal-derived pectinases have an optimal reaction pH of 4.0 - 5.5 and an optimal reaction temperature of 25°C. Currently, most of the pectinases that can be applied in industry are derived from fungi, and most fungal pectinases have problems such as being acidic, low temperature adaptability, low enzyme activity, high extraction energy consumption, etc. And due to technical barriers, the price is expensive.

[0007] Therefore, exploring novel pectinases with high activity and high adaptability, and using genetic engineering methods to introduce pectinase-encoding genes into suitable recipient cells, followed by efficient heterologous expression and purification, are the key technologies for achieving large-scale production and industrial application of pectinases. SUMMARY OF THE INVENTION

[0008] The object of the present invention is to provide a pectin lyase and its mutants, a recombinant expression vector and applications thereof; the nucleotide sequence of the pectin lyase is as shown in SEQ ID NO.1, and the encoded amino acid is as shown in SEQ ID NO.2; the nucleotide sequence of the pectin lyase mutant is as shown in SEQ ID NO.3, and the encoded amino acid is as shown in SEQ ID NO.4; both the pectin lyase and the mutant can be applied to juice clarification. Compared with the commercial pectinase derived from Aspergillus niger, the pectin lyase and its mutants provided by the present invention have higher specific enzyme activity and better thermal stability.

[0009] In the first aspect, the present invention provides a pectin lyase gene, and the nucleotide sequence is as shown in SEQ ID NO.1.

[0010] In the second aspect, the present invention provides a pectin lyase encoded by the above pectin lyase gene, and the amino acid sequence of the pectin lyase is as shown in SEQ ID NO.2.

[0011] In the third aspect, the present invention provides a pectin lyase mutant gene, which is obtained by mutating the nucleotide sequence shown in SEQ ID NO.1 at positions 349 - 351, and the nucleotides at positions 349 - 351 are mutated from TAC to GCC; the nucleotide sequence of the pectin lyase mutant gene is as shown in SEQ ID NO.3.

[0012] In the fourth aspect, the present invention provides a pectin lyase mutant encoded by the above pectin lyase mutant gene, and the amino acid sequence of the pectin lyase mutant is as shown in SEQ ID NO.4.

[0013] In the fifth aspect, the present invention provides a recombinant vector, including an empty vector, the above pectin lyase gene and / or pectin lyase mutant gene.

[0014] In the sixth aspect, the present invention provides a recombinant engineering bacterium, and the recombinant engineering bacterium is BL21 DE3 containing any one of the above pectin lyase gene and pectin lyase mutant gene. Escherichia coli BL21 DE3

[0015] In the seventh aspect, the present invention provides an application of the pectin lyase, and the application includes juice extraction, juice clarification, and processing of fruit and vegetable juice beverages.

[0016] Optionally, the conditions for the hydrolysis reaction of the pectin lyase in juice clarification are: 40 - 50 °C and pH 8.0 - 9.0.

[0017] In an eighth aspect, the present invention provides an application of a pectin lyase mutant, and the application includes juice extraction, juice clarification, and fruit and vegetable juice beverage processing.

[0018] Optionally, the conditions for the hydrolysis reaction of the pectin lyase mutant in juice clarification are: 40 - 50 °C and pH 8.0 - 9.0.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The present invention provides a novel pectin lyase gene derived from the genus Chitinophaga. The gene and its mutant gene are shown in SEQ ID NO.1 and SEQ ID NO.3 respectively. After optimization, the full length is 1056 bp, and the base (G + C) contents are 49.15% and 49.34% respectively, encoding 351 amino acids correspondingly. The amino acid sequences of the pectin lyase and its mutant are shown in SEQ ID NO.2 and SEQ ID NO.4 respectively. Moreover, the thermal stability of the pectin lyase mutant is significantly improved, and the enzyme activity of pectin is effectively enhanced;

[0021] (2) The pectin lyase and its mutant provided by the present invention effectively improve the problem that the enzyme activity of the existing commercial pectin lyase decreases at higher temperatures;

[0022] (3) The pectin lyase and its mutant expressed by the present invention are incubated at 35 - 40 °C and pH 7.0 - 9.0 for 6 h and 24 h respectively, and the enzyme activity can still maintain above 80% level, having excellent heat resistance and pH tolerance;

[0023] (4) The pectin lyase of the present invention has the highest enzyme activity under the conditions of 40 - 50 °C and pH 8.0, and the specific activity of hydrolyzing apple pectin reaches 517 U / mg, which is 1.03 times higher than the activity of the commercial pectin lyase; the pectin lyase mutant has the highest enzyme activity under the conditions of 40 - 50 °C and pH 8.0, and the specific activity of hydrolyzing apple pectin reaches 621 U / mg, which is 1.24 times higher than the activity of the commercial pectin lyase;

[0024] (5) The pectin lyase and its mutant of the present invention have a wide range of applications and have good clarification effects on a variety of fruit juices. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1PCR amplification electrophoresis diagram of the pectin lyase prepared in Example 1 of the present invention and the pectin lyase mutant gene prepared in Example 2;

[0026] Figure 2 SDS-PAGE protein electrophoresis diagram of the protein expression of the pectin lyase prepared in Example 1 of the present invention and the pectin lyase mutant prepared in Example 2;

[0027] Figure 3 Optimum temperature determination results of the pectin lyase prepared in Example 1 of the present invention and the pectin lyase mutant prepared in Example 2;

[0028] Figure 4 Optimum pH determination results of the pectin lyase prepared in Example 1 of the present invention and the pectin lyase mutant prepared in Example 2;

[0029] Figure 5 Temperature stability determination results of the pectin lyase prepared in Example 1 of the present invention;

[0030] Figure 6 Temperature stability determination results of the pectin lyase mutant prepared in Example 2 of the present invention;

[0031] Figure 7 pH stability determination results of the pectin lyase prepared in Example 1 of the present invention and the pectin lyase mutant prepared in Example 2;

[0032] Figure 8 Optimum reaction temperature determination results of the pectin lyase prepared in Example 1 of the present invention and the pectin lyase mutant prepared in Example 2 in the application of fruit juice clarification;

[0033] Figure 9 Clarification effect comparison results of the pectin lyase prepared in Example 1 of the present invention and the pectin lyase mutant prepared in Example 2 in the application of citrus fruit juice clarification;

[0034] Figure 10 Clarification effect comparison results of the pectin lyase prepared in Example 1 of the present invention and the pectin lyase mutant prepared in Example 2 in the application of navel orange juice clarification. Detailed implementation manners

[0035] In the examples of the present invention, reagents and raw materials without special instructions are all commercially available; the commercial pectinase described in the present invention is from Yuanye Biotechnology Co., Ltd., Shanghai, CAS#9032-75-1; the vector pET-29a is from Invitrogen Corporation; Escherichia coli Escherichia coliBL21(DE3) was purchased from Invitrogen; Cosmosil Sugar-D column was from Nacalai Tesuque, Kyoto, Japan; restriction endonucleases were from Takara; seamless cloning kit was from Nanjing Novoprotein.

[0036] In the examples of the present invention, the definition of enzyme activity is as follows: at 50 °C, the hydrolysis of the substrate to generate 1 μmol of reducing sugar per minute is defined as one unit of enzyme activity (U).

[0037] Example 1

[0038] Example 1 of the present invention provides a method for cloning a pectate lyase gene and constructing an expression vector, comprising the following steps:

[0039] S1. PCR amplification of the pectate lyase gene

[0040] S11. Prediction was performed according to Chitinophaga sp. DLHW the genomic sequence; primers were designed according to the restriction enzyme sites of the nucleic acid sequence. The sequence of the upstream primer F1 was as shown in SEQ ID NO.5, and the sequence of the downstream primer R1 was as shown in SEQ ID NO.6; orf

[0041] S12. According to the primers designed in S11, full-length PCR amplification was performed;

[0042] The PCR reaction system was as follows:

[0043] Chitinophaga sp. DLHW Total DNA 1.0 μL, each of primers F1 and R1 1.0 μL, PrimeSTAR Max DNA Polymerase 25.0 μL, supplemented with ddH2O to make the total volume of the system 50.0 μL;

[0044] The PCR amplification conditions were:

[0045] Figure 1 Pre-denaturation at 98 °C for 5 min, denaturation at 98 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min, a total of 30 cycles were performed, and finally extension at 72 °C for 10 min; the full-length sequence of pectate lyase (CfPelB (wild type)) was obtained. The electrophoresis results were as shown in Figure 1 wherein lane M in was DL5000 nucleic acid Marker, and lane 1 was the PCR amplification product of the CfPelB (wild type) gene;

[0046] S2. Construction and verification of the recombinant vector pET-29a-CfPelB (wild type)

[0047] ​

[0047] S21. Double digest pET-29a to obtain a double-digested product;

[0048] The double-digest system is as follows: Nde I 1.0 μL, Xho I 1.0 μL, 10×H Buffer 5.0 μL, pET-29a 30.0 μL, ddH2O 13.0 μL;

[0049] S22. Recover the double-digested product using a DNA agarose gel recovery kit, and clone the recovered product;

[0050] The cloning system is as follows: 6.0 μL of the CfPelB (wild type) PCR product obtained in S1.2, 2×Hieff Clone ® Enzyme Premix 10 μL;

[0051] The cloning reaction conditions are as follows: The system reacts at 50 °C for 30 min, and after the reaction, it is cooled on ice for 2 min to obtain a cloning product;

[0052] S23. Transform the cloning product into E.coli DH5α competent cells by heat shock:

[0053] Add the cloning product to 100 μL of melted E.coli DH5α competent cells and incubate on ice for 30 min;

[0054] After the ice bath, transfer to a 42 °C water bath for heat shock for 90 s, and after heat shock, cool on ice for 5 min;

[0055] After cooling, add 800 μL of liquid LB medium, incubate at 37 °C, 180 rpm on a shaker for 60 min;

[0056] S24. After incubation, centrifuge at 600 rpm for 3 min, take 100 μL of the competent cells and spread them on an LB agar plate containing 50 mg / L kanamycin, and invert the plate and culture it in a 37 °C incubator for 13 h;

[0057] S25. Pick multiple single colonies from the cultured plate, inoculate them into a test tube containing 4 mL of liquid LB medium, incubate at 37 °C, 180 rpm on a shaker for 12 h, and after incubation, extract the plasmid using a plasmid miniprep kit;

[0058] S26. Double digest the plasmid extracted in step S25;

[0059] The double-digest system is as follows: NdeI 1.0 μL, Xho I 1.0 μL, 10×H Buffer 5.0 μL, pET-29a-CfPelB 30.0 μL, ddH2O 13.0 μL;

[0060] S27. Verify and send the obtained recombinant plasmid to Hunan Qingke Biotechnology Co., Ltd. for sequencing; the sequencing results show that the cloned target fragment is inserted into the pET-29a vector, with a nucleotide length of 1056 bp, and then the recombinant expression vector pET-29a- CfPelB (wild type), the DNA sequence of CfPelB (wild type) is shown in SEQ ID NO.1, and the amino acid sequence of the pectin lyase it expresses is shown in SEQ ID NO.2;

[0061] S3. Expression and purification of recombinant pectin lyase

[0062] S31. Heat shock transform the plasmid pET-29a- CfPelB (wild type) obtained in step S2.5 into the host bacterium E.coli BL21(DE3) to obtain a recombinant engineering bacterium containing the recombinant plasmid;

[0063] S32. Inoculate a single colony of the positive clone into 4 mL of LB medium (50 μg / mL kanamycin), culture it at 37 °C with shaking at 180 rpm for 12 h. After the culture is completed, extract the plasmid using a plasmid miniprep kit and verify it;

[0064] S33. After verification, inoculate the bacterial liquid obtained in S32 into 50 mL of LB medium, culture it at 37 °C with shaking at 180 rpm for 8 h. After the culture is completed, transfer it to 400 mL of LB medium at an inoculation amount of 1%, and culture it at 37 °C with shaking at 180 rpm for 3.5 h;

[0065] S34. After the culture is completed, measure OD 600 When it reaches 0.4 - 0.6, add IPTG (final concentration 2 mmol / L), and induce expression at 16 °C with shaking at 180 rpm for 24 h;

[0066] S35. After the induction of expression is completed, centrifuge the culture solution at 5000 rpm for 0.5 h to collect the bacteria;

[0067] S36. Resuspend and wash the bacteria with PBS buffer at a concentration of 20 mmol / L and pH 7.0, then centrifuge at 5000 rpm for 0.5 h to collect the bacteria;

[0068] S37. In an ice bath, add PBS buffer with a concentration of 20 mmol / L and a pH of 7.0, and use an ultrasonic cell disruptor to lyse the cells;

[0069] S38. After the disruption is completed, centrifuge at 4 °C and 12,000 rpm for 1 h to obtain a crude extract of CfPelB (wild type);

[0070] S39. Use a Ni-NTA affinity chromatography column to purify the crude extract of CfPelB (wild type) to obtain purified CfPelB (wild type). The purified product is passed through an Amicon ® Ultra (3 kDa MWCO) filter and centrifuged at 5,000 rpm for 0.5 h to increase the concentration of the purified product, and finally obtain purified and concentrated pectate lyase CfPelB (wild type).

[0071] Example 2

[0072] Example 2 of the present invention provides a method for cloning a pectate lyase mutant gene and constructing an expression vector. The difference from Example 1 is as follows:

[0073] In step S11, after molecular docking and comparison, amino acid sites are selected for mutation in the substrate channel, primers for the mutation sites are designed, and PCR amplification of the pectate lyase mutation sites is carried out. The sequence of the upstream primer F2 is as shown in SEQ ID NO.7, and the sequence of the downstream primer R2 is as shown in SEQ ID NO.8;

[0074] In step S12, a total of three PCR reactions are carried out;

[0075] The difference between the first PCR reaction and step S12 is that the upstream primer F1 and the downstream primer R2 are added to the PCR reaction system. The annealing condition in the PCR amplification conditions in step S12 is 55 °C for 5 s;

[0076] The difference between the second PCR reaction and step S12 is that the upstream primer F2 and the downstream primer R1 are added to the PCR reaction system. The annealing condition in the PCR amplification conditions in step S12 is 55 °C for 5 s;

[0077] After the first and second PCR amplifications are completed, two short sequences containing the mutation sites are obtained, and the two short sequences are used as PCR templates for the third amplification;

[0078] The system for the third PCR amplification is as follows:

[0079] 1.0 μL of the short sequence DNA containing the mutation site, 1.0 μL each of primers F1 and R1, PrimeSTAR ®Add ddH2O to make the total volume of the system up to 50.0 μL with 25.0 μL of Max DNAPolymerase;

[0080] The conditions for the second PCR amplification were as follows:

[0081] Pre-denaturation at 98 °C for 5 min, denaturation at 98 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min, for a total of 30 cycles, and finally extension at 72 °C for 10 min; to obtain the full-length sequence of the pectate lyase mutant (CfPelB (mutant)); the electrophoresis results were as Figure 1 shown, Figure 1 In the lane M was DL5000 nucleic acid Marker, and the lane 2 was the PCR amplification product of the CfPelB (mutant) gene;

[0082] In step S36, the mutant cell pellet was resuspended and washed with Tris-HCl buffer at a concentration of 20 mmol / L and pH 8.0;

[0083] In step S37, Tris-HCl buffer at a concentration of 20 mmol / L and pH 8.0 was added;

[0084] Other conditions and steps were the same as those in Example 1, and finally, the purified and concentrated pectate lyase mutant CfPelB (mutant) was obtained; the DNA sequence of CfPelB (mutant) was as shown in SEQ ID NO.3, and the amino acid sequence of the expressed pectate lyase was as shown in SEQ ID NO.4.

[0085] Performance test

[0086] After detecting the purity and molecular weight of the pectate lyase and its mutant prepared in Example 1 and Example 2, the performance of the pectate lyase and its mutant provided by the present invention was tested by detecting the enzyme activity (DNS method).

[0087] The purity and molecular weight of the pectate lyase and its mutant were determined by SDS-PAGE, and the purity identification results were as Figure 2 shown, with a molecular weight of approximately 41.6 kDa; Figure 2 In the lane 1 was the protein molecular weight standard, and the lane 2 was the supernatant enzyme solution of BL21(DE3) containing the empty vector pET-29a E.coli BL21(DE3) supernatant enzyme solution, the lane 3 was the supernatant enzyme solution of BL21(DE3) containing the CfPelB (wild type) gene E.coli BL21(DE3) supernatant enzyme solution, the lane 4 was the supernatant enzyme solution of BL21(DE3) containing the CfPelB (wild type) E.coliThe precipitate of BL21(DE3), lane 5 is the purified pectin lyase CfPelB (wild type) eluted with 100 mmol / L imidazole, and lane 6 is the one containing the CfPelB (mutant) gene E.coli The supernatant enzyme solution of BL21(DE3), lane 7 is the one containing the CfPelB (mutant) gene E.coli The precipitate of BL21(DE3); lane 8 is the purified pectin lyase mutant CfPelB (mutant) eluted with 100 mmol / L imidazole.

[0088] 1. Determination of the optimum temperature

[0089] 1.1 Determination of the optimum temperature of the pectin lyase prepared in Example 1

[0090] Prepare a 500 μL reaction system: Add 10 μL of CfPelB (wild type) prepared in Example 1 to a buffer system with an apple pectin concentration of 0.2% (pH 8.0, 20 mmol / L Tris-HCl);

[0091] Incubate the reaction system in a water bath at different temperatures (35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C) for 10 min;

[0092] After the reaction, add an equal volume of DNS (dinitrosalicylic acid) to the reaction system to terminate the reaction;

[0093] Measure the absorbance value of the reaction system at 540 nm after the reaction at different temperatures. Design 3 parallel tests for each temperature, and take the average of the measurement results; The results are as Figure 3 shown;

[0094] 1.2 Determination of the optimum temperature of the pectin lyase mutant prepared in Example 2

[0095] The difference from 1.1 is that 10 μL of CfPelB (mutant) prepared in Example 2 is added to the reaction system, and other conditions and steps remain the same; The results are as Figure 3 shown;

[0096] See Figure 3 that at 50 °C, both CfPelB (wild type) prepared in Example 1 and CfPelB (mutant) prepared in Example 2 have the highest enzyme activity. Therefore, set the enzyme activity of the enzyme activity reaction system at this temperature as the relative activity of 100%, and calculate the relative activities of the CfPelB (wild type) and CfPelB (mutant) enzyme activity reaction systems at other temperatures respectively.

[0097] 2. Determination of the optimum pH

[0098] 2.1 Determination of the Optimal pH of the Pectate Lyase Prepared in Example 1

[0099] Prepare a 500 μL buffer system: Add 10 μL of CfPelB (wild type) prepared in Example 1 to a buffer system with an apple pectin concentration of 0.2%;

[0100] Divide the buffer system into four groups and configure reaction systems with different pH values of 50 mmol / L respectively. React in a water bath at 50 °C for 10 min; The first group is a citric acid - sodium citrate aqueous solution with a pH of 4.0 - 6.0; The second group is a Na2HPO4 - NaH2PO4 aqueous solution with a pH of 6.0 - 8.0; The third group is a Tris - HCl aqueous solution with a pH of 8.0 - 9.0; The fourth group is a Glycine - NaOH aqueous solution with a pH of 9.0 - 10.0;

[0101] After the reaction is completed, add an equal volume of DNS (dinitrosalicylic acid) to the reaction system to terminate the reaction;

[0102] Measure the absorbance value of the reaction system at 540 nm after the reaction at the corresponding pH. Design 3 parallel tests for each pH value of each group, and take the average of the measurement results; The results are as Figure 4 shown;

[0103] 2.2 Determination of the Optimal pH of the Pectate Lyase Mutant Prepared in Example 2

[0104] The difference from 2.1 is that 10 μL of CfPelB (mutant type) prepared in Example 2 is added to the buffer system, and other conditions and steps remain the same; The results are as Figure 4 shown;

[0105] See Figure 4 , in the reaction system with a pH of 8, both CfPelB (wild type) prepared in Example 1 and CfPelB (mutant type) prepared in Example 2 have the highest enzyme activity. Therefore, set the enzyme activity of the enzyme activity reaction system under this pH condition as the relative activity of 100%, and calculate the relative activities of the CfPelB (wild type) and CfPelB (mutant type) enzyme activity reaction systems under other pH reaction systems respectively.

[0106] 3. Thermal Stability Determination

[0107] 3.1 Determination of the Thermal Stability of the Pectate Lyase Prepared in Example 1

[0108] Incubate CfPelB (wild type) prepared in Example 1 at different temperatures (35 °C, 40 °C, 45 °C, 50 °C) for 1 h, 2 h, 4 h, 6 h, 8 h respectively;

[0109] Prepare a 500 μL reaction system: Add 10 μL of CfPelB (wild type) prepared in Example 1 after incubation at different temperatures to a buffer system with an apple pectin concentration of 0.2% (pH 8.0, 50 mmol / L Tris-HCl);

[0110] React the reaction system in a water bath at 50 °C for 10 min;

[0111] After the reaction is completed, add an equal volume of DNS (dinitrosalicylic acid) to the reaction system to terminate the reaction;

[0112] Measure the absorbance value of the reaction system at 540 nm after reaction at different temperatures. Design 3 parallel experiments at each temperature. CfPelB (wild type) without incubation treatment is used as a control group, and the enzyme activity is 100%. Calculate the relative enzyme activity of CfPelB (wild type) after incubation treatment; The measurement results are averaged; The results are as Figure 5 shown;

[0113] 3.2 Determination of the thermal stability of the pectin lyase mutant prepared in Example 2

[0114] The difference from 3.1 is that 10 μL of CfPelB (mutant) after incubation treatment is added to the reaction system, and other conditions and steps remain the same; The results are as Figure 6 shown;

[0115] See Figures 5 to 6 , when incubated at 35 °C, after 8 h, CfPelB (wild type) still has more than 50% residual enzyme activity. However, after incubation at 40 °C for 2 h, the residual enzyme activity of CfPelB (wild type) drops below 50%. After incubation at 45 °C and 50 °C for 2 h, the enzyme activity decreases even faster, and CfPelB (wild type) has only less than 20% residual enzyme activity; While CfPelB (mutant) still has higher residual enzyme activity than CfPelB wild type after incubation at 40 - 45 °C. After incubation at 35 °C for 8 h, it can still maintain more than 90% residual enzyme activity. After incubation at 40 °C for 6 h, it still has more than 90% residual enzyme activity. After incubation at 45 °C for 2 h, the residual enzyme activity is above 20%. When incubated at 50 °C, the enzyme activity also decreases rapidly and loses its enzyme activity after incubation for 2 h;

[0116] Since there are significant differences in enzyme activity between CfPelB (mutant) and CfPelB (wild type) after incubation at 40 °C, the residual enzyme activities of CfPelB (wild type) and CfPelB (mutant) incubated at 40 °C for different durations were compared. At 40 °C, CfPelB (mutant) prepared in Example 2 could maintain better enzyme activity than CfPelB (wild type) prepared in Example 1. After incubation at 40 °C for 2 h, the residual enzyme activity of CfPelB (wild type) prepared in Example 1 was less than 50%, while CfPelB (mutant) prepared in Example 2 still maintained more than 90% residual enzyme activity after incubation at 40 °C for 6 h. Therefore, CfPelB (mutant) prepared in Example 2 of the present invention has better thermal stability at 40 °C.

[0117] 4. pH Stability Determination

[0118] 4.1 pH Stability Determination of Pectate Lyase Prepared in Example 1

[0119] Prepare a 500 μL buffer system: Incubate 10 μL of CfPelB (wild type) prepared in Example 1 in buffer systems with different pH values for 12 h. A total of 4 different pH buffer systems with a concentration of 50 mmol / L were set up. The first group: Citric acid-sodium citrate aqueous solution with a pH of 4.0 - 6.0; the second group: Na2HPO4-NaH2PO4 aqueous solution with a pH of 6.0 - 8.0; the third group: Tris-HCl aqueous solution with a pH of 8.0 - 9.0; the fourth group: Glycine-NaOH aqueous solution with a pH of 9.0 - 10.0.

[0120] Add the incubated CfPelB (wild type) to a buffer system with an apple pectin concentration of 0.2% (pH 8.0, 50 mmol / L Tris-HCl), and react in a water bath at 50 °C for 10 min.

[0121] After the reaction, add an equal volume of DNS (dinitrosalicylic acid) to the reaction system to terminate the reaction.

[0122] Measure the absorbance value of the reaction system at 540 nm after the reaction at the pH. Three parallel tests were designed for each pH group. CfPelB (wild type) without incubation treatment was used as the control group with an enzyme activity of 100%, and calculate the relative enzyme activity of CfPelB (wild type) after incubation treatment in different pH systems. The measurement results were averaged, and the results are as Figure 7 shown;

[0123] 4.2 pH Stability Determination of the Pectate Lyase Mutant Prepared in Example 2

[0124] The difference from 4.1 is that 10 μL of CfPelB (mutant) prepared in Example 2 after incubation in different pH systems was added to an apple pectin buffer system with a concentration of 0.2%, and other conditions and steps were kept the same. The results are as Figure 7 shown;

[0125] See Figure 7 , in the buffer system with pH 8.0 - 9.0, the residual enzyme activity of CfPelB (wild type) and CfPelB (mutant) provided by the present invention remains above 90%, proving that the pectin lyase and its mutants of the present invention have excellent stability at pH 8.0 - 9.0.

[0126] 5. Determination of the optimal temperature of the pectin lyase prepared in Example 1 when applied to juice clarification

[0127] Preparation of citrus juice:

[0128] The citrus was repeatedly rubbed in water to remove impurities such as surface sediment and dust;

[0129] The citrus was peeled and pitted, and then crushed using a blade crusher; after the treatment was completed, it was added to a hydraulic juicer for juicing;

[0130] After juicing, it was filtered using a 100 - 120 - mesh filter cloth to obtain citrus juice;

[0131] Prepare a 5 - mL reaction system: Add 50 μL of CfPelB (wild type) prepared in Example 1 to 5 mL of citrus juice;

[0132] The reaction system was subjected to a water bath reaction at 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, and 60 °C for 2 h respectively; after the reaction was completed, the reaction was terminated by a boiling water bath for 10 min;

[0133] After termination, it was left standing for 30 min, and the OD 660 transmittance was measured using an ultraviolet spectrophotometer. The measurement results are as Figure 8 shown;

[0134] See Figure 8 , at a temperature of 40 °C, the juice clarity has reached 80%, and at 50 °C, the juice clarity reaches the highest. Therefore, 40 - 50 °C is the optimal enzyme reaction temperature of CfPelB (wild type) provided in Example 1 of the present invention.

[0135] 6. Verification of the effect of juice clarification in citrus juice

[0136] 6.1 Verification of the effect of the pectin lyase prepared in Example 1

[0137] Prepare a 5 mL reaction system: Add 50 μL of CfPelB (wild type) prepared in Example 1 to 5 mL of citrus juice;

[0138] At 40 °C, subject the reaction system to water bath reactions for 0.5 h, 1 h, 2 h, 3 h, 5 h, and 8 h respectively; after the reaction is completed, terminate the reaction by boiling water bath for 10 min;

[0139] After termination, let it stand for 30 min, and use an ultraviolet spectrophotometer to measure the OD 660 transmittance. The measurement results are as shown in Figure 9 A in; the change in juice clarity is as shown in Figure 9 B in;

[0140] 6.2 Verification of the effect of the pectin lyase mutant prepared in Example 2

[0141] The difference from 6.1 is that CfPelB (mutant type) is added to the reaction system, and other conditions and steps remain unchanged. The transmittance measurement results are as shown in Figure 9 A in; the change in juice clarity is as shown in Figure 9 C in;

[0142] See Figure 9 , during the water bath reaction for 0.5 - 3 h, the juice clarity of both CfPelB (wild type) and CfPelB (mutant type) reaches 60%; after 5 h, CfPelB (mutant type) makes the juice clarity reach 90% and above. However, the juice clarity of CfPelB (wild type) does not increase, which is consistent with the previous thermal stability measurement results. CfPelB (mutant type) has better thermal stability than CfPelB (wild type). Therefore, in the application of juice clarification, CfPelB (mutant type) prepared in Example 2 of the present invention shows better effects.

[0143] 7. Verification of the effect of juice clarification in navel orange juice

[0144] 7.1 Verification of the effect of the pectin lyase prepared in Example 1

[0145] Prepare navel orange juice using the preparation method of citrus juice;

[0146] Prepare a 5 mL reaction system: Add 50 μL of CfPelB (wild type) prepared in Example 1 to 5 mL of navel orange juice;

[0147] At 40 °C, subject the reaction system to water bath reactions for 0.5 h, 1 h, 2 h, 3 h, 5 h, and 8 h respectively; after the reaction is completed, terminate the reaction by boiling water bath for 10 min;

[0148] After termination, let it stand for 30 min, and use an ultraviolet spectrophotometer to measure the OD 660 transmittance. The measurement results are as shown in Figure 10 A in it. The change in the clarity of navel orange juice is as shown in Figure 10 B in it;

[0149] 7.2 Effect verification of the pectate lyase mutant prepared in Example 2

[0150] The difference from 7.1 is that CfPelB (mutant) is added to the reaction system, and other conditions and steps remain unchanged. The measurement results of the transmittance are as shown in Figure 10 A in it. The change in the clarity of navel orange juice is as shown in Figure 10 C in it;

[0151] See Figure 10 , after reacting for 4 h, CfPelB (mutant) provided in Example 2 can decompose most of the juice, reduce the viscosity, significantly improve the transmittance of the juice, improve the clarity of the juice, and the transmittance of the juice reaches more than 90%; while CfPelB (wild type) provided in Example 1, after reacting for 4 h, the clarity of the juice does not increase significantly, because the thermal stability of CfPelB (wild type) after reacting for 4 h is lower than that of CfPelB (mutant), and as the reaction time becomes longer, CfPelB (wild type) is inactivated, resulting in a decrease in the ability to decompose pectin and a slow increase in clarity.

[0152] Although the embodiments of the present invention have been described in detail above, it is obvious to those skilled in the art that various modifications and changes can be made to these embodiments. However, it should be understood that such modifications and changes are all within the scope and spirit of the present invention described in the claims. Moreover, the present invention described herein can have other embodiments and can be implemented or realized in various ways.

Claims

1. A pectin lyase gene, characterized in that, The nucleotide sequence is as shown in SEQ ID NO.

1.

2. A pectin lyase, characterized in that, Encoded by the pectate lyase gene described in claim 1, the amino acid sequence of the pectate lyase is as shown in SEQ ID NO.

2.

3. A pectin lyase mutant gene, characterized in that, Obtained by mutating the nucleotide sequence shown in SEQ ID NO.1 at positions 349 - 351, where the nucleotides at positions 349 - 351 are mutated from TAC to GCC; the nucleotide sequence of the pectate lyase mutant gene is as shown in SEQ ID NO.

3.

4. A pectin lyase mutant, characterized in that, Encoded by the pectate lyase mutant gene described in claim 3, the amino acid sequence of the pectate lyase mutant is as shown in SEQ ID NO.

4.

5. A recombinant vector, characterized in that, The recombinant vector includes an empty vector, the pectate lyase gene described in claim 1 and / or the pectate lyase mutant gene described in claim 3.

6. A recombinant engineering bacterium, characterized in that, The recombinant engineering bacteria contain any one of the pectin lyase genes described in claim 1 and the pectin lyase mutant genes described in claim 3 Escherichia coli BL21DE3.

7. The use of the pectin lyase according to claim 2, characterized in that, The application is for the processing of fruit and vegetable juice beverages.

8. The application according to claim 7, wherein The conditions for the hydrolysis reaction of the pectate lyase in juice clarification are: 40 - 50 °C and pH 8.0 - 9.

0.

9. Use of the pectate lyase mutant according to claim 4, characterized in that, The application is for the processing of fruit and vegetable juice beverages.

10. The application according to claim 9, wherein The conditions for the hydrolysis reaction of the pectate lyase mutant in juice clarification are: 40 - 50 °C and pH 8.0 - 9.0.

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