Alkaline pectolyase scp and use thereof

Abstract

The application provides a basic pectin lyase Scp and an application thereof, and belongs to the technical field of bioengineering; the application is characterized in that a hypothetical protein from Phytophthora nicotianae is optimized in code and is expressed on a vector by bioengineering technology, is transferred into Escherichia coli which has a clear genetic background and a fast growth cycle, is amplified and expressed, and is purified to obtain a protein product; the amino acid sequence of the protein product is shown as SEQ ID NO. 1, and the nucleotide sequence for coding the protein product is shown as SEQ ID NO. 2; the application is characterized in that the protein product has high pectin lyase activity and is recorded as the basic pectin lyase Scp; the basic pectin lyase Scp has the advantages of alkali resistance, high-temperature resistance, a wide pH action range and the like, and has good application in the fields of the textile industry, food processing, pulping processing, oil extraction and industrial wastewater treatment and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering, and particularly relates to a basic pectin lyase Scp and application thereof. BACKGROUND

[0002] Pectate lyase (PL) cuts the internal alpha-1, 4-glycosidic bond of pectin to produce oligosaccharides based on beta-trans elimination, which has great application potential in the fields of food, papermaking, textile and feed. Natural PL widely exists in animals, plants and microorganisms, but the production of PL in animals and plants is low and difficult to extract; microorganisms grow rapidly, have simple culture conditions and are relatively simple to prepare, and are an important source of PL at present. The existing PL still has some limitations, such as poor alkali resistance and heat resistance, small pH action range, etc. With the wide application of pectinase, it is urgent to excavate and characterize new PL with excellent performance. SUMMARY

[0003] In view of some deficiencies in the prior art, the application provides a basic pectin lyase Scp and application thereof. The application is characterized in that a protein product is prepared by the following steps: a protein code of a hypothetical protein derived from Phytophthora nicotianae is optimized by bioengineering technology, and then the optimized protein code is expressed on a vector by heterologous expression, and then the vector is introduced into Escherichia coli which has a clear genetic background and a fast growth cycle to amplify and express, and then the protein product is prepared by purification. The amino acid sequence of the protein product is shown as SEQ ID NO. 1, and the nucleotide sequence of the gene encoding the protein product is shown as SEQ ID NO. 2. The application characterizes that the protein product has high pectin lyase activity and is recorded as basic pectin lyase Scp. The basic pectin lyase Scp has the advantages of alkali resistance, high temperature resistance and wide pH action range, and has good application in the fields of textile industry, food processing, pulping processing, oil extraction and industrial wastewater treatment.

[0004] In order to achieve the above technical effects, the application adopts the following technical means:

[0005] The application provides a basic pectin lyase Scp. The amino acid sequence of the basic pectin lyase Scp is shown as SEQ ID NO. 1.

[0006] The application also provides a gene encoding the basic pectin lyase Scp.

[0007] Preferably, the nucleotide sequence of the gene comprises SEQ ID NO. 2 or a degenerate sequence thereof.

[0008] The application also provides a recombinant vector comprising the gene.

[0009] Preferably, the expression vector of the recombinant vector comprises pET28a.

[0010] The application further provides a recombinant bacterium comprising the gene encoding the alkaline pectolytic enzyme Scp or the recombinant vector.

[0011] Preferably, the construction method of the recombinant bacterium comprises:

[0012] (1) linearizing the pET28a plasmid to obtain a linearized pET28a plasmid, then connecting the linearized pET28a plasmid and the gene encoding the alkaline pectolytic enzyme Scp through seamless cloning, transforming into E. coli competent cells through heat shock method, and obtaining the recombinant vector through PCR and Sanger sequencing;

[0013] (2) transferring the recombinant vector into a host bacterium to obtain the recombinant bacterium.

[0014] Preferably, the host bacterium of the recombinant bacterium comprises E. coli.

[0015] The application further provides a production method of the alkaline pectolytic enzyme Scp, comprising: culturing, inducing expression, and isolating and purifying the recombinant bacterium to obtain the alkaline pectolytic enzyme Scp.

[0016] The application further provides application of the alkaline pectolytic enzyme Scp, the alkaline pectolytic enzyme Scp encoded by the gene, or the alkaline pectolytic enzyme Scp produced by the recombinant bacterium in juice clarification, hemp degumming, industrial wastewater, extraction of perfume oil and carotenoid medical raw materials, fermentation of coffee and tea, treatment of pectin-containing wastewater, extraction of crude oil, purification of plant viruses, pulp bleaching, and biological refining of textiles.

[0017] Compared with the prior art, the application has the beneficial effects that:

[0018] The application obtains a protein product by bioengineering technology, wherein a hypothetical protein from Phytophthora nicotianae is codon-optimized and expressed on a vector, and then is transferred into E. coli with a clear genetic background and a fast growth cycle for amplification and expression; the amino acid sequence of the protein product is shown as SEQ ID NO. 1, and the nucleotide sequence of the protein product is shown as SEQ ID NO. 2; the application finds that the protein product has high pectolytic enzyme activity and is recorded as alkaline pectolytic enzyme Scp. The alkaline pectolytic enzyme Scp has high pectolytic enzyme activity under high alkalinity, and has the advantages of simple operation, rapid preparation, high purity and the like.

[0019] The alkaline recombinant pectolytic enzyme has an enzyme activity of 51.6±0.6 U / mg at pH=10 and 55℃; the relative enzyme activity of the alkaline recombinant pectolytic enzyme can reach more than 60% in the temperature range of 50-65℃, and the relative enzyme activity is still more than 10% at 75℃; the relative enzyme activity of the alkaline recombinant pectolytic enzyme can reach more than 90% in the pH range of 9.0-10.0, and the relative enzyme activity is still more than 20% in the pH range of 8.0-12.0.

[0020] Compared with other pectolytic enzymes, the hypothetical protein of the application is verified to have excellent performance of pectolytic enzyme; the alkaline pectolytic enzyme Scp has never been characterized, has high pectolytic enzyme activity, alkali resistance, high temperature resistance, wide pH action range and other outstanding advantages, has good application in the fields of textile industry, food processing, pulp processing, oil extraction and industrial wastewater treatment, and can be used in the fields of fruit juice clarification, degumming of hemp, extraction of industrial wastewater, perfume oil and carotenoid medical raw materials, fermentation of coffee and tea, treatment of pectin-containing wastewater, extraction of crude oil, purification of plant virus, pulp bleaching and textile biofinishing. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is an electropherogram of the target gene amplified by PCR in Example 1.

[0022] Figure 2 It is a map of the recombinant vector pET28a-Scp.

[0023] Figure 3 It is an SDS-PAGE map of the expression product after shake flask fermentation of the recombinant Escherichia coli JJ3; in the figure, M: standard protein Marker; 1: supernatant of cell disruption after induction of BL21(DE3) / pET28a; 2: supernatant of cell disruption after induction of JJ3; 3: precipitate of cell disruption after induction of JJ3; 4: pectolytic enzyme Scp protein purified by Ni-NTA.

[0024] Figure 4 It is the activity of the alkaline pectolytic enzyme Scp under different pH (A) and temperature (B). DETAILED DESCRIPTION

[0025] The application will be further described below in conjunction with the drawings and specific examples, but the protection scope of the application is not limited thereto. In the following examples, various processes and methods not described in detail are conventional methods known in the art. The source of the reagents used, trade name, and necessary composition components are indicated at the first occurrence, and the same reagents used thereafter are not specifically indicated, and are the same as the source indicated at the first time; the reagents, materials and the like involved are commercially available without special indication.

[0026] In the following examples, pET28a plasmid, JM109 competent cells, E. coli BL21(DE3) are general materials in the art, so they will not be described again; the LB medium components in the following examples include: 1% tryptone + 0.5% yeast powder + 1% NaCl + 8 g / L glucose + 50 μg / mL kanamycin sulfate.

[0027] Example 1: Obtaining of the target gene

[0028] The present application codon optimizes the hypothetical protein (Genbank accession no. XM_008899953.1) in Phytophthora nicotianae according to E. coli BL21(DE3), and the codon-optimized sequence is shown as SEQ ID NO. 2, and the encoded protein is shown as SEQ ID NO. 1. The target gene (SEQ ID NO. 2, same below) fragment is synthesized by Suzhou Jiwizhi Biotechnology Co., Ltd.

[0029] SEQ ID NO. 1:

[0030] MGSSHHHHHHSSGLVPRGSSLPNGSWPASKGTVQYSQAYIVKAGEVFDGKMKTFERSDVSCEGQTESGSDTAVFKVEAGGHLKNVIIGKNQMEGVHCDKHDCIIENVWWDDVCEDALSVKGGTASSVTKVIGGGARYADDKVIQHNGFGPEGHAVFLVEAGGTLKNAIIGKNQKEGVHCDDHDCTIENVWWDDVCEDALSIKGGSASSVTTVTNCGARYAEDKVVQHNGYGTVKIKGFFAQEFGKLYRSCGTCGNIPRKVTVENVYAIDPLVSVVTVNKNNNDQATFKNIYVKTTDGKKNVKVCQWSQASKTPSNLGDGPSGKLCQYSSSDVHINED

[0031] SEQ ID NO. 2:

[0032] AGCCTGCCGAATGGCAGCTGGCCGGCAAGTAAAGGCACCGTTCAGTATAGCCAGGCATATATTGTGAAAGCAGGCGAAGTTTTTGATGGCAAAATGAAAACCTTTGAACGCAGCGATGTGAGCTGTGAAGGCCAGACCGAAAGTGGTAGTGATACCGCCGTTTTTAAAGTGGAAGCCGGTGGTCATCTGAAAAATGTGATTATTGGCAAAAACCAGATGGAAGGTGTTCATTGCGATAAACATGATTGTATTATCGAAAACGTGTGGTGGGATGATGTGTGTGAAGATGCACTGAGCGTTAAAGGCGGCACCGCAAGCAGTGTGACCAAAGTTATTGGTGGCGGTGCACG CTATGCCGATGATAAAGTGATTCAGCATAATGGTTTTGGTCCGGAAGGCCATGCCGTTTTTCTGGTTGAAGCAGGTGGTACCCTGAAAAATGCCATTATTGGCAAGAATCAGAAAGAAGGTGTGCATTGTGATGATCATGATTGTACCATTGAAAATGTTTGGTGGGATGACGTTTGCGAAGATGCCCTGAGCATTAAAGGTGGTAGTGCAAGTAGCGTTACCACCGTTACCAATTGTGGCGCCCGCTATGCAGAAGATAAAGTTGTTCAGCATAATGGCTATGGTACCGTGAAAATTAAAGGTTTTTTCGCCCAGGAATTTGGCAAACTGTATCGTAGTTGTGGTACCTGTGGCAATATTCCGCGTAAAGTGACCGTTGAAAATGTTTATGCAATTGATCCGCTGGTTAGCGTGGTGACCGTGAATAAAAATAATAATGATCAGGCAACCTTCAAGAATATTTATGTTAAAACCACCGACGGCAAAAAAAATGTGAAAGTGTGTCAGTGGAGTCAGGCCAGTAAAACCCCGAGTAATCTGGGTGATGGTCCGAGCGGCAAACTGTGCCAGTATAGCAGTAGTGATGTGCATATTAATGAAGATtaactcgag.

[0033] The above synthesized gene fragment was used as a template, and PCR amplification was performed by primers F1 and R1. The product obtained by PCR amplification was verified by agarose gel electrophoresis, and the verification result is shown in Figure 1 .

[0034] F1: ctggtgccgc gcggcagc AGCCTGCCGAATGGCAGCTGGC (SEQ ID NO. 3, the underlined part is a homologous arm);

[0035] R1: ggtggtggt ggtggtgct cgagtta ATCTTCATTAATATGCAC (SEQ ID NO. 4, the underlined part is a homologous arm).

[0036] Figure 1 The electrophoretogram of the product obtained by PCR amplification is shown in the figure. As can be seen from the figure, the length of the product obtained by PCR amplification is 963 bp. This is because the primer for seamless cloning is relatively long and contains a homologous arm fragment. Finally, the band after PCR is larger than the band of the target gene fragment only, and the size of the band is consistent with the sum of the size of the target gene and the size of the primer. This indicates that the target gene has been successfully obtained.

[0037] Example 2: Construction of recombinant vector containing target gene and recombinant bacteria

[0038] The pET28a plasmid was used as a template, and primers F2 and R2 were used for PCR linearization to obtain a linearized plasmid fragment. Then the linearized plasmid fragment and the target gene obtained in Example 1 were connected by seamless cloning to obtain the recombinant vector pET28a-Scp.

[0039] The obtained recombinant vector pET28a-Scp was transformed into JM109 competent cells by heat shock method, and PCR and Sanger sequencing verification were performed. The recombinant vector pET28a-Scp was successfully constructed. The map of the recombinant vector pET28a-Scp is shown in Figure 2 .

[0040] The sequences of primers F2 and R2 are as follows:

[0041] F2: caccaccaccaccaccac (SEQ ID NO. 5);

[0042] R2: gctgccgcgcggcaccag (SEQ ID NO. 6).

[0043] (2) Construction of recombinant bacteria

[0044] The recombinant vector pET28a-Scp was transformed into E. coli BL21 (DE3) to obtain a recombinant strain, which was designated as recombinant E. coli JJ3 and was used for standby.

[0045] In this step, the recombinant E. coli JJ3 after shake flask fermentation was also investigated by taking the empty vector strain as a control. The empty vector strain pET28a / BL21 (DE3) was obtained by directly transforming the pET28a plasmid into E. coli BL21 (DE3). The investigation step was specifically as follows:

[0046] S1. Seed liquid preparation:

[0047] The empty vector strain pET28a / BL21 (DE3) and the recombinant E. coli JJ3 preserved by glycerol were respectively streaked on a plate, and a single colony was inoculated into a 250 ml conical flask containing 50 mL of LB liquid medium, which was then incubated at 37°C and 220 r / min overnight to obtain the overnight culture of the empty vector strain pET28a / BL21 (DE3) and the recombinant E. coli JJ3.

[0048] S2. Fermentation:

[0049] The overnight culture of the empty vector strain pET28a / BL21 (DE3) and the recombinant E. coli JJ3 was centrifuged, and the bacterial cells were resuspended with LB medium and inoculated into a shake flask fermentation medium, so that the initial OD 600 of the bacterial cells was 0.1. Then, when the OD 600 of the bacterial cells was about 0.6, 1 mM IPTG was added to induce the empty vector strain pET28a / BL21 (DE3) and the recombinant E. coli JJ3. After induction, the culture conditions were adjusted to 16°C and 100 r / min, and the culture was incubated for 24 h to obtain the induced culture of the empty vector strain pET28a / BL21 (DE3) and the recombinant E. coli JJ3.

[0050] S3. Sample treatment:

[0051] 1 mL of the induced culture of the empty vector strain pET28a / BL21 (DE3) and the recombinant E. coli JJ3 was taken into a 1.5 mL centrifuge tube, and the bacterial cells were collected by centrifugation at room temperature, 10000 r / min and 5 min. Then, 100 μL of PBS buffer (pH 7.0) was added, and the mixture was boiled in a water bath for 10 min. After centrifugation (room temperature, 6000 r / min, 5 min), the supernatant and the precipitate of the lysate were obtained, and the supernatant and the precipitate of the lysate were subjected to SDS-PAGE electrophoresis. The electrophoresis results are shown in Figure 3 .

[0052] Figure 3The SDS-PAGE map of the expression product of the recombinant E. coli JJ3 after shake flask fermentation shows that there is a specific protein band at 36 kDa in the supernatant and the precipitate of the lysate of the recombinant E. coli JJ3 after IPTG induction, while there is no such band in the control of the empty vector strain, which indicates that the Scp gene has been effectively expressed in the E. coli BL21 (DE3), and thus the target protein Scp can be obtained by culturing, inducing expression and isolating and purifying the recombinant E. coli JJ3.

[0053] Example 3: Purification and characterization of the target protein Scp

[0054] In this example, the recombinant E. coli JJ3 in Example 2 is cultured, induced for expression, isolated and purified to obtain the target protein Scp, and the function of the target protein Scp is characterized, and the specific steps are as follows:

[0055] (1) Obtain the crude enzyme solution:

[0056] The recombinant E. coli JJ3 is treated and fermented by the steps S1 and S2 in Example 3, and then the recombinant E. coli JJ3 induction culture solution is collected in a 50 mL centrifuge tube by a refrigerated centrifuge (4°C, 6000 r / min, 5 min), and washed twice with PBS buffer (pH 7.0). After washing, 2 mL of equilibration buffer (pH 7.0) is used to resuspend the bacterial body, and the bacterial body is placed on ice for ultrasonic disruption for 15-20 min (power 550 W, working time 3 s, interval time 35 s). Whether the bacterial solution is clear and transparent is used to determine whether the disruption is complete.

[0057] The disrupted bacterial solution is collected by low-temperature high-speed centrifugation to obtain the supernatant and the precipitate of the lysate. The supernatant is filtered by a MCE filter membrane with a pore size of 0.22 μm, and the obtained filtrate is the crude enzyme solution.

[0058] (2) Purification:

[0059] The above obtained crude enzyme solution is loaded into the equilibrated Ni 2+ -NTA affinity chromatography column, and the column is gently inverted to resuspend the resin. The column is continuously inverted for 30-60 min to allow the crude enzyme solution to fully bind to the resin. The column is vertically placed for 5-10 min to allow the resin to sediment under the action of gravity, and the liquid is discharged. 5 mL of washing solution (pH 8.0) is added, the column is gently inverted to resuspend the resin, and the column is vertically placed for 5-10 min to allow the resin to sediment under the action of gravity, and the liquid is discharged. This step is repeated 3 times. 1 mL of elution solution (pH 8.0) is added, the column is gently inverted to resuspend the resin, and the column is vertically placed for 5-10 min to allow the resin to sediment under the action of gravity. The elution solution is discharged into a new centrifuge tube, which is the soluble recombinant protein. This step is repeated 3 times.

[0060] The eluted protein was dialyzed with double distilled water to remove salt particles, and the purified alkaline recombinant pectolyase Scp was obtained and stored in a 4-degree refrigerator for standby use. The purified target protein Scp was subjected to SDS-PAGE electrophoresis, and the electrophoresis result is shown in Figure 3 As shown in Figure 3 After the pET28a-Scp / BL21(DE3) JJ3 strain was purified by Ni 2+ -NTA, the purified enzyme had a specific protein band at 36 kDa, indicating that the target protein Scp was successfully purified.

[0061] (3) Characterization of the target protein Scp:

[0062] This step characterized the pectolyase activity of the target protein Scp purified in step (2) by specific activity. The specific principle is as follows:

[0063] Since pectolyase acts on the α-1, 4 glycosidic bond in pectin to generate an unsaturated oligogalacturonide with an unsaturated bond between the C4 and C5 positions at the reducing end, there is a characteristic absorption peak at 235 nm. Therefore, the protein content was determined by BCA protein quantitative determination, and then the increase in absorbance at 235 nm was determined to represent the activity of pectolyase, and the specific activity of pectolyase was calculated.

[0064] The specific determination steps are as follows: 20 μL of 10-fold diluted enzyme solution was added to 2 mL of buffer containing 0.2% polygalacturonide to start the enzyme reaction under the set conditions for 15 min, and the reaction was terminated with 3 mL of 0.03 mol / L phosphoric acid. The absorbance value was determined at 235 nm. The blank control was the same amount of heat-inactivated diluted enzyme solution. The amount of enzyme required to produce 1 mol of unsaturated galacturonide per minute was taken as 1 enzyme activity unit (U), and the experiment was repeated three times.

[0065] According to the above method, the activity of the target protein Scp at different pH (5.0-12.0) and different temperatures (40°C-75°C) was tested, and the highest enzyme activity was taken as 100%. The enzyme activity measured at other temperatures was compared with it, and the relative enzyme activity at that temperature was obtained. The test results are shown in Figure 4 .

[0066] From Figure 4It can be seen that the enzyme activity of the target protein Scp presents a trend of first increasing and then decreasing with the change of pH at a temperature of 55℃, and the enzyme activity reaches the maximum at pH 10, which is 50±2.6 U / mg. The enzyme activity of Scp presents a trend of first increasing and then decreasing with the change of temperature at pH 10, and the enzyme activity reaches the maximum at a temperature of 55℃, which is 51.6±0.6 U / mg. Therefore, the target protein Scp has the activity of pectin lyase, the optimum reaction temperature is 55℃, and the optimum reaction pH is 10.0, which is recorded as the alkaline pectin lyase Scp.

[0067] It can be seen from Figure 4 It can also be seen from the above that the relative enzyme activity of the alkaline pectin lyase Scp can reach more than 60% in the temperature range of 50-65℃, and the relative enzyme activity is still more than 10% at 75℃, which shows that the alkaline pectin lyase Scp has good heat resistance. In addition, the relative enzyme activity of the alkaline pectin lyase Scp can reach more than 90% in the pH range of 9.0-10.0, and the relative enzyme activity is still more than 20% in the pH range of 8.0-12.0, which shows that the pH action range of the alkaline pectin lyase Scp is wide, but the enzyme activity is low under acidic conditions, and it is a typical alkaline pectinase.

[0068] It can be seen that compared with other pectin lyases, the optimum reaction temperature and pH value are 35-65℃ and pH 3.0-5.5; the alkaline recombinant pectin lyase Scp of the present application is more heat-resistant and alkali-resistant, and has a wide pH action range.

[0069] In summary, the present application realizes the expression of the alkaline pectin lyase Scp by bioengineering technology, which is codon-optimized and heterologously expressed on a vector derived from a hypothetical protein from Phytophthora nicotianae and introduced into Escherichia coli with clear genetic background and fast growth cycle. The alkaline pectin lyase Scp has high pectin lyase activity under high alkaline conditions, and has the advantages of simple operation, rapid preparation, high purity and the like. The alkaline pectin lyase Scp has not been characterized before, and compared with other pectinases, it has the advantages of high pectin lyase activity, alkali resistance, high temperature resistance, wide pH action range and the like, and has good application in the fields of textile industry, food processing, pulping processing, oil extraction and industrial wastewater treatment.

[0070] The above embodiments are preferred embodiments of the present application, but the present application is not limited to the above embodiments, and any obvious improvements, replacements or modifications made by those skilled in the art without departing from the essential content of the present application shall fall within the protection scope of the present application.

Claims

1. An alkaline pectin lyase Scp, wherein the amino acid sequence of the alkaline pectin lyase Scp is shown in SEQ ID NO.

1.

2. The gene encoding the alkaline pectin lyase Sn as described in claim 1.

3. The gene according to claim 2, characterized in that, The nucleotide sequence of the gene includes SEQ ID NO.2, or its degenerate sequence.

4. A recombinant vector comprising the gene of claim 2 or 3.

5. The recombinant vector according to claim 4, characterized in that, The expression vector for the recombinant vector includes pET28a.

6. A recombinant bacterium, characterized in that, The recombinant bacteria comprises the gene encoding alkaline pectin lyase Sn as described in claim 2 or 3, or the recombinant vector as described in claim 4 or 5.

7. The recombinant bacteria according to claim 6, characterized in that, The method for constructing the recombinant bacteria includes: (1) Linearize the pET28a plasmid to obtain a linearized pET28a plasmid, then connect the linearized pET28a plasmid with the gene encoding alkaline pectin lyase Scp as described in claim 2 through seamless cloning, transform it into competent Escherichia coli cells by heat shock, and obtain a recombinant vector after PCR and Sanger sequencing. (2) The recombinant vector is transferred into the host bacteria to obtain the recombinant bacteria.

8. The recombinant bacteria according to claim 7, characterized in that, The host bacteria of the recombinant bacteria include Escherichia coli.

9. The method for producing alkaline pectin lyase Scp according to claim 1, characterized in that, The method includes: culturing, inducing expression, separating and purifying the recombinant bacteria according to any one of claims 6 to 8 to obtain alkaline pectin lyase Scp.

10. The application of the alkaline pectin lyase Scp according to claim 1, or the gene-encoded alkaline pectin lyase Scp according to claim 2 or 3, or the alkaline pectin lyase Scp produced by the recombinant bacteria according to any one of claims 6 to 8 in fruit juice clarification, hemp degumming, industrial wastewater treatment, extraction of spice oils and carotenoid medicinal raw materials, coffee and tea fermentation, treatment of pectin-containing wastewater, purification of plant viruses, pulp bleaching, and biorefining of textiles.

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