Pectate lyase as well as mutant, recombinant expression vector and application thereof

Abstract

The invention relates to pectin lyase as well as a mutant, a recombinant expression vector and application thereof. The nucleotide sequence of the pectin lyase is as shown in SEQ ID NO.1, the full length of the gene is 1056 bp, 352 amino acids are coded in total, and the amino acid sequence is as shown in SEQ ID NO.2; the pectin lyase mutant is formed by site-directed mutagenesis on the basis of an amino acid sequence of pectin lyase, a mutated nucleotide sequence is as shown in SEQ ID NO.3, and the amino acid sequence is as shown in SEQ ID NO.4; compared with the existing pectin lyase, the pectin lyase and the mutant thereof provided by the invention have the characteristics of high activity and temperature stability, and can be widely applied to fruit juice extraction, fruit juice clarification and fruit and vegetable juice beverage processing.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a pectin lyase and a mutant thereof, a recombinant expression vector and application thereof. Background Art

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

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

[0004] Methods for degrading pectin into pectin oligosaccharides generally include physical degradation, chemical hydrolysis and enzymatic preparation, among which enzymatic preparation is currently the most effective method, with obvious advantages such as mild reaction conditions, strong specificity and high degradation efficiency.

[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 skeleton in pectin substances, pectinase can be divided into three categories: polygalacturonase, pectin lyase and pectin methylesterase. Different pectinases cut different types of α-1,4-glycosidic bonds in pectin. According to the optimal enzyme activity pH, it can be divided into two categories: alkaline and acidic. Alkaline pectinase is mainly used in the food production process to help decompose pectin, improve the texture and taste of the product, and improve digestion and absorption; acidic pectinase is mainly used to extract and clarify fruit and vegetable juice and fruit wine, and reduce the turbidity and viscosity of the product.

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

[0007] Therefore, the key technologies to achieve mass production and industrial application of pectinase are to explore new types of highly active and highly adaptable pectinases, introduce pectinase encoding genes into suitable receptor cells through genetic engineering, and express and purify them through efficient heterologous expression. Summary of the invention

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

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

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

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

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

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

[0014] In a sixth aspect, the present invention provides a recombinant engineered bacterium, wherein the recombinant engineered bacterium is a gene containing any one of the pectin lyase gene and the pectin lyase mutant gene. Escherichia coli BL21DE3.

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

[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, wherein 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: (1) The present invention provides a novel pectin lyase gene derived from Chitinophaga, the gene and its mutant gene are shown in SEQ ID NO.1 and SEQ ID NO.3 respectively, after optimization, the total length is 1056 bp, the base (G+C) content is 49.15% and 49.34% respectively, corresponding to encoding 351 amino acids, the amino acid sequences of pectin lyase and its mutant are shown in SEQ ID NO.2 and SEQ ID NO.4, and the thermal stability of the pectin lyase mutant is greatly improved, and the enzyme activity of pectin is effectively improved; (2) The pectin lyase and its mutant provided by the present invention effectively improve the problem of reduced enzyme activity of existing commercial pectin lyase under high temperature; (3) The pectin lyase and its mutants expressed by the present invention can maintain an enzyme activity of more than 80% when incubated at a temperature of 35-40°C and a pH of 7.0-9.0 for 6 h and 24 h, respectively, and have excellent heat resistance and pH tolerance; (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 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 commercial pectin lyase; (5) The pectin lyase and its mutants of the present invention are widely used and have good clarification effects on a variety of fruit juices. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The electrophoresis diagrams of PCR amplification of the pectin lyase prepared in Example 1 and the pectin lyase mutant gene prepared in Example 2 of the present invention are shown; Figure 2The SDS-PAGE protein electrophoresis diagram of the pectin lyase prepared in Example 1 of the present invention and the pectin lyase mutant protein expression prepared in Example 2; Figure 3 The optimum temperature determination results of the pectin lyase prepared in Example 1 and the pectin lyase mutant prepared in Example 2 of the present invention; Figure 4 The optimum pH measurement results of the pectin lyase prepared in Example 1 and the pectin lyase mutant prepared in Example 2 of the present invention; Figure 5 The results of the temperature stability test of the pectin lyase prepared in Example 1 of the present invention are as follows; Figure 6 The results of measuring the temperature stability of the pectin lyase mutant prepared in Example 2 of the present invention; Figure 7 The pH stability test results of the pectin lyase prepared in Example 1 of the present invention and the pectin lyase mutant prepared in Example 2; Figure 8 The optimum reaction temperature determination results of the pectin lyase prepared in Example 1 and the pectin lyase mutant prepared in Example 2 in the application of fruit juice clarification; Fig. 9 The comparison results of the clarification effects of the pectin lyase prepared in Example 1 of the present invention and the pectin lyase mutant prepared in Example 2 in the clarification application of citrus juice; Fig.10 The results are a comparison of the clarification effects 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 DESCRIPTION

[0021] In the examples of the present invention, the reagents and raw materials without special instructions are all commercially available; the commercial pectinase described in the present invention is from Shanghai Yuanye, CAS#9032-75-1; the vector pET-29a is from Invitrogen; Escherichia coli Escherichia coli BL21 (DE3) was from Invitrogen; Cosmosil Sugar-D chromatographic column was from Nacalai Tesuque, Kyoto, Japan; restriction endonucleases were from Takara; and seamless cloning kit was from Nanjing Novezan.

[0022] The enzyme activity described in the examples of the present invention is defined as: at 50° C., catalyzing the hydrolysis of substrate to generate 1 μmol of reducing sugar per minute is one enzyme activity unit (U).

[0023] Example 1 Example 1 of the present invention provides a method for cloning a pectin lyase gene and constructing an expression vector, comprising the following steps: S1. PCR amplification of pectin lyase gene S11. According to Chitinophaga sp. DLHW Genome sequencing orf Prediction; design primers according to the restriction sites of the nucleic acid sequence, the upstream primer F1 sequence is shown in SEQ ID NO.5, and the downstream primer R1 sequence is shown in SEQ ID NO.6; S12, performing full-length PCR amplification according to the primers designed in S11; The PCR reaction system is as follows: Chitinophaga sp. DLHW Total DNA 1.0 μL, Primers F1 and R1 1.0 μL each, PrimeSTAR ® MaxDNA Polymerase 25.0 μL, add ddH2O to make the total volume of the system 50.0 μL; PCR amplification conditions were: 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, and a final extension at 72°C for 10 min; the full-length sequence of pectin lyase (CfPelB (wild type)) was obtained, and the electrophoresis results are shown in Figure 1 As shown, Figure 1 Lane M in the figure is the DL5000 nucleic acid marker, and lane 1 is the PCR amplification product of the CfPelB (wild type) gene; S2. Construction and verification of the recombinant vector pET-29a-CfPelB (wild type) S21, performing double enzyme digestion on pET-29a to obtain double enzyme digestion products; The double enzyme digestion system is: N I 1.0 μL, XOt I 1.0 μL, 10×H Buffer 5.0 μL, pET-29a30.0 μL, ddH2O 13.0 μL; S22, recovering the double enzyme digestion product using a DNA agarose gel recovery kit, and cloning the recovered product; The cloning system is: 6.0 μL of CfPelB (wild type) PCR product obtained in S1.2, 2×Hieff Clone ® Enzyme Premix 10 μL; The cloning reaction conditions were as follows: the system reacted at 50 °C for 30 min, and then cooled on ice for 2 min to obtain the cloning product; S23, the cloned product was transformed into E. coli DH5α competent cells: Add the cloned product to 100 μL of melted E. coli In DH5α competent cells, incubate on ice for 30 min; After the ice bath, transfer to a 42 °C water bath for heat shock for 90 s, and cool on ice for 5 min after heat shock; After cooling, add 800 μL of liquid LB medium and incubate at 37 °C, 180 rpm, on a shaking table for 60 min; S24. After incubation, centrifuge at 600 rpm for 3 min, take 100 μL of competent cells and spread them on LB agar plates containing 50 mg / L kanamycin, and invert the plates in a 37 ℃ incubator for 13 h; S25. Extract multiple single colonies from the cultured plate and inoculate them into a test tube containing 4 mL of liquid LB medium. Incubate on a shaker at 37 °C and 180 rpm for 12 h. After incubation, use a plasmid miniprep kit to extract the plasmid. S26, double enzyme digestion of the plasmid extracted in step S25; The double enzyme digestion system is: N I 1.0 μL, XOt I 1.0 μL, 10×H Buffer 5.0 μL, pET-29a-CfPelB 30.0 μL, ddH2O 13.0 μL; S27, verify and send the obtained recombinant plasmid to Hunan Qingke Biotechnology Co., Ltd. for sequencing; the sequencing results showed that the cloned target fragment was inserted into the pET-29a vector, with a nucleotide length of 1056 bp, and then the recombinant expression vector pET-29a- CfP (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 expressed thereby is shown in SEQ ID NO.2; S3. Expression and purification of recombinant pectin lyase S31, the plasmid pET-29a- CfP (Wild type) heat shock transformation into host bacteria E. coli BL21(DE3) to obtain recombinant engineered bacteria containing the recombinant plasmid; S32, inoculate a single colony of the positive clone into 4 mL LB medium (50 μg / mL kanamycin), culture at 37 °C, 180 rpm shaking for 12 h. After the culture is complete, use a plasmid mini-prep kit to extract the plasmid and verify it; S33. After verification, the cultured bacteria in S32 were inoculated into 50 mL LB medium and cultured at 37°C and 180 rpm for 8 h. After the culture was completed, the culture was transferred to 400 mL LB medium at a 1% inoculum and cultured at 37°C and 180 rpm for 3.5 h. S34. After the culture is completed, measure OD 600 When it reached 0.4-0.6, IPTG (final concentration was 2 mmol / L) was added and the expression was induced at 16°C and 180 rpm for 24 h; S35. After the induction expression was completed, the culture solution was centrifuged at 5000 rpm for 0.5 h and the bacteria were collected; S36, resuspend and wash the cells with PBS buffer at a concentration of 20 mmol / L and a pH of 7.0, and then centrifuge at 5000 rpm for 0.5 h to collect the cells; S37, add 20 mmol / L PBS buffer at pH 7.0 in an ice bath, and use an ultrasonic disruptor to disrupt the cells; S38. After the fragmentation is completed, centrifuge at 4 °C and 12,000 rpm for 1 h to obtain a crude biological extract of CfPelB (wild type); S39, using Ni-NTA affinity chromatography column to purify the biological crude extract of CfPelB (wild type) to obtain purified CfPelB (wild type), and the purified product was purified by Amicon ® Ultra (3 kDa MWCO) filter, 5000 rpm, centrifugation for 0.5 h, to increase the concentration of the purified product, and finally obtain the purified and concentrated pectin lyase CfPelB (wild type).

[0024] Example 2 Example 2 of the present invention provides a method for cloning a pectin lyase mutant gene and constructing an expression vector, which is different from Example 1 in that: In step S11, after molecular docking and alignment, an amino acid site is selected in the substrate channel for mutation, a primer for the mutation site is designed, and PCR amplification of the pectin lyase mutation site is performed; the sequence of the upstream primer F2 is shown in SEQ ID NO.7, and the sequence of the downstream primer R2 is shown in SEQ ID NO.8; In step S12, three PCR reactions are performed; 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 condition in step S12 is 55°C, 5 s; The second PCR reaction is different from step S12 in that upstream primer F2 and downstream primer R1 are added to the PCR reaction system; the annealing condition in the PCR amplification condition in step S12 is 55°C, 5 s; After the first and second PCR amplifications are completed, two short sequences containing mutation sites are obtained, and the two short sequences are used as PCR templates for the third amplification; The third PCR amplification system is: 1.0 μL of short sequence DNA containing the mutation site, 1.0 μL of primers F1 and R1, PrimeSTAR ® Max DNA Polymerase 25.0 μL, add ddH2O to make the total volume of the system 50.0 μL; The second PCR amplification conditions were: 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, and finally extension at 72 °C for 10 min; the full-length sequence of the pectin lyase mutant (CfPelB (mutant)) was obtained; the electrophoresis results are shown in Figure 1 As shown, Figure 1 Lane M in the figure is the DL5000 nucleic acid marker, and lane 2 is the PCR amplification product of the CfPelB (mutant) gene; In step S36, the mutant cells are resuspended and washed using a Tris-HCl buffer having a concentration of 20 mmol / L and a pH of 8.0; In step S37, a Tris-HCl buffer having a concentration of 20 mmol / L and a pH of 8.0 is added; Other conditions and steps were consistent with those in Example 1, and finally a purified and concentrated pectin lyase mutant CfPelB (mutant) was obtained; the DNA sequence of CfPelB (mutant) is shown in SEQ ID NO.3, and the amino acid sequence of the pectin lyase expressed by it is shown in SEQ ID NO.4.

[0025] Performance Testing After the purity and molecular weight of the pectin lyase and its mutants prepared in Example 1 and Example 2 were detected, the performance of the pectin lyase and its mutants provided by the present invention was tested by detecting the enzyme activity (DNS method).

[0026] The purity and molecular weight of pectin lyase and its mutants were determined by SDS-PAGE. The purity identification results were as follows: Figure 2 As shown, the molecular weight is approximately 41.6 kDa; Figure 2 Lane 1 is a protein molecular weight standard, and lane 2 is a protein containing the empty vector pET-29a. E. coli BL21(DE3) supernatant, lane 3 is the one containing CfPelB (wild type) gene E. coli BL21(DE3) supernatant enzyme solution, lane 4 contains CfPelB (wild type) E. coli Lane 5 is the purified pectin lyase CfPelB (wild type) eluted with 100 mmol / L imidazole, and lane 6 is the purified pectin lyase CfPelB (mutant) gene E. coli BL21(DE3) supernatant enzyme solution, lane 7 is the one containing CfPelB (mutant) gene E. coli Lane 8 is the purified pectin lyase mutant CfPelB (mutant) eluted with 100 mmol / L imidazole.

[0027] 1. Determination of optimum temperature 1.1 Determination of the optimum temperature of the pectin lyase prepared in Example 1 Prepare a 500 μL reaction system: add 10 μL of CfPelB (wild type) prepared in Example 1 to a buffer system (pH 8.0, 20 mmol / L Tris-HCl) with an apple pectin concentration of 0.2%; The reaction system was reacted in a water bath at different temperatures (35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃) for 10 min; After the reaction is completed, add DNS (dinitrosalicylic acid) with an equal volume to the reaction system to terminate the reaction; The absorbance of the reaction system at 540 nm after reaction at different temperatures was measured. Three parallel tests were designed for each temperature, and the average value of the measured results was taken. The results are shown in Figure 3 As shown; 1.2 Determination of the optimum temperature of the pectin lyase mutant prepared in Example 2 The difference from 1.1 is that 10 μL of CfPelB (mutant) prepared in Example 2 was added to the reaction system, and other conditions and steps remained the same; the results are shown in Figure 3 As shown; See also Figure 3At 50°C, both CfPelB (wild type) prepared in Example 1 and CfPelB (mutant) prepared in Example 2 had the highest enzyme activity. Therefore, the enzyme activity of the enzyme activity reaction system at this temperature was set to the relative activity as 100%, and the relative activities of the CfPelB (wild type) and CfPelB (mutant) enzyme activity reaction systems at other temperatures were calculated respectively.

[0028] 2. Determination of optimal pH 2.1 Determination of the optimum pH of the pectin lyase prepared in Example 1 Prepare 500 μL of 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%; The buffer system was divided into four groups, each of which was configured with a concentration of 50 mmol / L and a reaction system of different pH values, and reacted in a 50 ℃ water bath for 10 min; the first group, a citric acid-sodium citrate aqueous solution with a pH of 4.0-6.0; the second group, a Na2HPO4-NaH2PO4 aqueous solution with a pH of 6.0-8.0; the third group, a Tris-HCl aqueous solution with a pH of 8.0-9.0; the fourth group, a Glycine-NaOH aqueous solution with a pH of 9.0-10.0; After the reaction is completed, add DNS (dinitrosalicylic acid) with an equal volume to the reaction system to terminate the reaction; The absorbance of the reaction system at 540 nm after reaction at pH was determined. Three parallel tests were designed for each pH group, and the average value of the results was taken. Figure 4 As shown; 2.2 Determination of the optimal pH of the pectin lyase mutant prepared in Example 2 The difference from 2.1 is that 10 μL of CfPelB (mutant) prepared in Example 2 was added to the buffer system, and other conditions and steps remained the same; the results are shown in Figure 4 As shown; See also Figure 4 In the reaction system with a pH of 8, both CfPelB (wild type) prepared in Example 1 and CfPelB (mutant) prepared in Example 2 have the highest enzyme activity. Therefore, the enzyme activity of the enzyme activity reaction system under this pH condition is set as the relative activity 100%, and the relative activities of CfPelB (wild type) and CfPelB (mutant) enzyme activity reaction systems under other pH reaction systems are calculated respectively.

[0029] 3. Thermal stability determination 3.1 Determination of thermal stability of pectin lyase prepared in Example 1 The CfPelB (wild type) prepared in Example 1 was incubated at different temperatures (35°C, 40°C, 45°C, 50°C) for 1 h, 2 h, 4 h, 6 h, and 8 h, respectively; Prepare a 500 μL reaction system: add 10 μL of CfPelB (wild type) prepared in Example 1 after incubation at different temperatures into a buffer system with an apple pectin concentration of 0.2% (pH 8.0, 50 mmol / L Tris-HCl); The reaction system was reacted at 50 °C in a water bath for 10 min; After the reaction is completed, add DNS (dinitrosalicylic acid) with an equal volume to the reaction system to terminate the reaction; The absorbance of the reaction system at 540 nm after reaction at different temperatures was measured. Three parallel tests were designed for each temperature. CfPelB (wild type) without incubation treatment was used as the control group, and the enzyme activity was 100%. The relative enzyme activity of CfPelB (wild type) after incubation treatment was calculated. The average value of the measurement results was taken. The results are shown in Figure 5 As shown; 3.2 Determination of thermal stability of the pectin lyase mutant prepared in Example 2 The difference from 3.1 is that 10 μL of CfPelB (mutant) after incubation was added to the reaction system, and other conditions and steps remained the same; the results are shown in Figure 6 As shown; See also Figures 5 and 6 When incubated at 35 ℃, CfPelB (wild type) still had more than 50% residual enzyme activity after 8 h, but after incubation at 40 ℃ for 2 h, the residual enzyme activity of CfPelB (wild type) dropped to less than 50%. After incubation at 45 ℃ and 50 ℃ for 2 h, the enzyme activity dropped faster, and CfPelB (wild type) had less than 20% residual enzyme activity. However, CfPelB (mutant) still had higher residual enzyme activity than CfPelB wild type after incubation at 40-45 ℃. After incubation at 35 ℃ for 8 h, it could still maintain more than 90% residual enzyme activity. After incubation at 40 ℃ for 6 h, it still had more than 90% residual enzyme activity. After incubation at 45 ℃ for 2 h, the residual enzyme activity was more than 20%. When incubated at 50 ℃, the enzyme activity also dropped rapidly, and lost enzyme activity after incubation for 2 h. Since after incubation at 40°C, CfPelB (mutant) has obvious enzyme activity difference compared with CfPelB (wild type), therefore, by comparing the residual enzyme activities of CfPelB (wild type) and CfPelB (mutant) incubated at 40°C for different lengths, at 40°C, the CfPelB (mutant) prepared in Example 2 can maintain better enzyme activity than the 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 is less than 50%, while the CfPelB (mutant) prepared in Example 2 still maintains more than 90% of the residual enzyme activity after being kept at 40°C for 6 h; therefore, the CfPelB (mutant) prepared in Example 2 of the present invention has better thermal stability at 40°C.

[0030] 4. pH stability determination 4.1 Determination of pH stability of pectin lyase prepared in Example 1 Prepare 500 μL buffer system: incubate 10 μL of CfPelB (wild type) prepared in Example 1 in buffer systems of different pH for 12 h; set up 4 buffer systems of different pH, all with a concentration of 50 mmol / L; Group 1: citric acid-sodium citrate aqueous solution with a pH of 4.0-6.0; Group 2: Na2HPO4-NaH2PO4 aqueous solution with a pH of 6.0-8.0; Group 3: Tris-HCl aqueous solution with a pH of 8.0-9.0; Group 4: Glycine-NaOH aqueous solution with a pH of 9.0-10.0; The incubated CfPelB (wild type) was added to a buffer system with a 0.2% apple pectin concentration (pH 8.0, 50 mmol / L Tris-HCl) and reacted in a water bath at 50°C for 10 min. After the reaction is completed, add DNS (dinitrosalicylic acid) with an equal volume to the reaction system to terminate the reaction; The absorbance at 540 nm of the reaction system after reaction at pH was determined. Three parallel experiments were designed for each pH group. CfPelB (wild type) without incubation treatment was used as the control group, and the enzyme activity was 100%. The relative enzyme activity of CfPelB (wild type) after incubation treatment in different pH systems was calculated; the average value of the measurement results was taken. The results are shown in Figure 7 As shown; 4.2 pH stability determination of the pectin lyase mutant prepared in Example 2 The difference from 4.1 is that 10 μL of CfPelB (mutant) prepared in Example 2 after incubation with different pH systems was added to the apple pectin buffer system with a concentration of 0.2%, and other conditions and steps remained the same. The results are as follows Figure 7 As shown; See also Figure 7 In the buffer system of pH 8.0-9.0, the residual enzyme activity of CfPelB (wild type) and CfPelB (mutant) provided by the present invention is still maintained at more than 90%, proving that the pectin lyase and its mutants of the present invention have excellent stability at pH 8.0-9.0.

[0031] 5. Determination of the optimum temperature for the pectin lyase prepared in Example 1 when used in fruit juice clarification Citrus juice preparation: Scrub the citrus fruits repeatedly in water to remove impurities such as sand and dust on the surface; The citrus fruits are peeled and deseeded, and then crushed using a blade crusher; after the processing, they are added to a hydraulic juicer for juicing; After the juicing is completed, filter using a 100-120 mesh filter cloth to obtain citrus juice; Prepare a 5 mL reaction system: add 50 μL of CfPelB (wild type) prepared in Example 1 to 5 mL of citrus juice; The reaction system was reacted in a water bath at 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, and 60 ℃ for 2 h. After the reaction was completed, the reaction was terminated by boiling in a water bath for 10 min. After termination, let it stand for 30 min and measure the OD using a UV spectrophotometer. 660 Transmittance, measurement results such as Figure 8 As shown; See also Figure 8 At a temperature of 40°C, the clarity of the juice has reached 80%, and at 50°C, the clarity of the juice 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.

[0032] 6. Verification of the effect of juice clarification in citrus juice 6.1 Verification of the effect of the pectin lyase prepared in Example 1 Prepare a 5 mL reaction system: add 50 μL of CfPelB (wild type) prepared in Example 1 to 5 mL of citrus juice; The reaction system was reacted in a water bath at 40 °C for 0.5 h, 1 h, 2 h, 3 h, 5 h, and 8 h respectively; after the reaction was completed, the reaction was terminated by boiling water bath for 10 min; After termination, let it stand for 30 min and measure the OD using a UV spectrophotometer. 660 Transmittance, measurement results such as Fig. 9 As shown in A, the clarity of the juice changes as Fig. 9As shown in B; 6.2 Verification of the effect of the pectin lyase mutant prepared in Example 2 The difference from 6.1 is that CfPelB (mutant) was added to the reaction system, and other conditions and steps remained unchanged. The transmittance measurement results are as follows Fig. 9 As shown in A, the clarity of the juice changes as Fig. 9 As shown in C; See also Fig. 9 , during the water bath reaction for 0.5-3 h, the juice clarity of CfPelB (wild type) and CfPelB (mutant) both reached 60%; after 5 h, CfPelB (mutant) made the juice clarity reach 90% and above, however, the juice clarity of CfPelB (wild type) did not improve, which was consistent with the previous thermal stability test results. CfPelB (mutant) had better thermal stability than CfPelB (wild type). Therefore, in the application of juice clarification, CfPelB (mutant) prepared in Example 2 of the present invention showed better effect.

[0033] 7. Verification of the effect of juice clarification in navel orange juice 7.1 Verification of the effect of the pectin lyase prepared in Example 1 The navel orange juice is prepared by using the citrus juice preparation method; Prepare 5 mL of reaction system: add 50 μL of CfPelB (wild type) prepared in Example 1 to 5 mL of navel orange juice; The reaction system was reacted in a water bath at 40 °C for 0.5 h, 1 h, 2 h, 3 h, 5 h, and 8 h respectively; after the reaction was completed, the reaction was terminated by boiling water bath for 10 min; After termination, let it stand for 30 min and measure the OD using a UV spectrophotometer. 660 Transmittance, measurement results such as Fig.10 As shown in A, the clarity of navel orange juice changes as follows Fig.10 As shown in B; 7.2 Verification of the effect of the pectin lyase mutant prepared in Example 2 The difference from 7.1 is that CfPelB (mutant) was added to the reaction system, and other conditions and steps remained unchanged. The transmittance measurement results are as follows Fig.10 As shown in A, the clarity of navel orange juice changes as follows Fig.10 As shown in C; See also Fig.10After 4 h of reaction, the CfPelB (mutant) provided in Example 2 can decompose most of the juice, reduce the viscosity, and significantly improve the transmittance of the juice, and the clarity of the juice is improved, and the transmittance of the juice reaches more than 90%; while the CfPelB (wild type) provided in Example 1 does not significantly improve the clarity of the juice after 4 h of reaction, because the thermal stability of CfPelB (wild type) after 4 h of reaction is lower than that of CfPelB (mutant), and as the reaction time increases, CfPelB (wild type) is inactivated, which reduces the ability to decompose pectin and the clarity is slowly improved.

[0034] Although the embodiments of the present invention are described in detail above, it is obvious to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as described in the claims. Moreover, the present invention described herein may have other embodiments and may be implemented or realized in a variety of ways.

Claims

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

1.

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

2.

3. A pectin lyase mutant gene, characterized in that: The nucleotide sequence shown in SEQ ID NO.1 is mutated 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 shown in SEQ ID NO.

3.

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

4.

5. A recombinant vector, characterized in that: The recombinant vector comprises an empty vector, the pectin lyase gene according to claim 1 and / or the pectin lyase mutant gene according to claim 3.

6. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria contains any one of the pectin lyase gene according to claim 1 and the pectin lyase mutant gene according to claim 3. Escherichia coli BL21DE3.

7. The use of pectin lyase according to claim 2, characterized in that: The applications include juice extraction, juice clarification, and fruit and vegetable juice beverage processing.

8. The use according to claim 7, characterized in that: The conditions for the pectin lyase to perform hydrolysis reaction in juice clarification are: 40-50°C and pH 8.0-9.

0.

9. The use of the pectin lyase mutant according to claim 4, characterized in that: The applications include juice extraction, juice clarification, and fruit and vegetable juice beverage processing.

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

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