Recombinant pectate lyase from bacillus pumilus B.15-1, construction method and application of recombinant pectate lyase and low-temperature ramie degumming method

Abstract

The invention relates to the technical field of biology, in particular to recombinant pectate lyase from bacillus pumilus B.15-1, a construction method and application of the recombinant pectate lyase and a low-temperature ramie degumming method. The invention provides bacillus pumilus B.15-1, which is preserved in the China Center for Type Culture Collection, the address is Wuhan University, Wuhan, China, the preservation date is December 23, 2024, and the preservation number is CCTCC NO: M20242888. The strain is amplified to obtain a gene with pectate lyase activity, and the recombinant pectate lyase rBpumPel-Ec obtained by coding of the gene can be used for degumming ramie under a low-temperature condition. A novel method is provided for green degumming of ramie.

Description

Technical Field

[0001] The invention relates to the field of biological technology, and in particular to a recombinant pectate lyase from Bacillus pumilus B.15-1, a construction method and application thereof, and a low-temperature ramie degumming method. Background Art

[0002] Ramie is an important economic crop. Ramie fiber exists in the bast layer of ramie plants. As one of the longest and strongest natural hemp fibers in the world, it enjoys the reputation of "king of natural fibers" and is widely used in textiles, clothing, industrial materials and other industries.

[0003] Ramie fiber is mainly distributed in the bast part of ramie stems. After "peeling" and "scraping", it still contains pectin, hemicellulose, protein, fat and ash, which leads to poor separation performance and low softness, and cannot be used for high-quality spinning. Appropriate degumming methods are needed to remove colloids from ramie bast fibers to separate fibers and ensure that the fibers meet textile requirements. Ramie contains 4% to 5% pectin. Pectin is one of the main components of bonding fibers. It is very important for achieving separation between fibers and is the focus of ramie degumming. Pectinase is a general term for a class of enzymes that can degrade pectin substances. Pectinase degrades pectin into oligomers with various physiological activities through depolymerization or degreasing reactions. According to its active site, pectinase can be divided into hydrolases, esterases and lyases. Pectate lyase (Pels), also known as polygalacturonase (PGL), mainly acts on low-degree methyl-esterified pectin to generate oligogalacturonic acid or digalacturonic acid.

[0004] Traditional textile technology uses chemical methods to treat impurities. The degumming process requires the use of strong alkali and boiling, which not only destroys the structure of cellulose, but also produces toxic waste and pollutes the environment. Compared with chemical degumming, enzymatic degumming has many advantages such as mild processing conditions, flexible and environmentally friendly operation, little damage to fibers, and easy quality control. Therefore, green and environmentally friendly pectate lyase has great application potential in textile technology.

[0005] Due to the complex composition of ramie phloem, biological degumming cannot completely remove the impurities of ramie fiber. Ramie after degumming still has a high residual gum rate, so chemical post-treatment is needed to completely remove the gum. It has been reported that the effect of treating ramie phloem with pectate lyase first and then with NaOH is better than the effect of either treatment alone. This method includes two processes: pectate lyase degumming in the first step and NaOH heat treatment degumming in the second step. Among them, in the enzymatic degumming process, the current related research focuses on the screening of thermophilic or alkaliphilic pectate lyases, aiming to use high temperature and NaOH conditions to achieve the removal of pectin in ramie fiber.

[0006] Ramie can be degummed at room temperature or low temperature with good degumming effect, which can save energy consumption for maintaining the temperature used for enzymatic degumming, and a low-pollution solution has not been found. Based on this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a recombinant pectate lyase from Bacillus pumilus B.15-1 and a construction method and application thereof and a low-temperature ramie degumming method.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a strain having pectate lyase activity, wherein the strain is Bacillus pumilus B.15-1, which is deposited in China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, with a deposit date of December 23, 2024 and a deposit number of CCTCC NO: M20242888.

[0010] The invention also provides application of the strain in preparing a product for hydrolyzing polygalacturonic acid.

[0011] The invention also provides application of the strain in preparing a product with low-temperature ramie degumming effect.

[0012] The present invention also provides a recombinant pectate lyase, wherein the recombinant pectate lyase is a recombinant pectate lyase rBpumPel-Ec, and the recombinant pectate lyase is constructed by the strain, and the amino acid sequence of the constructed recombinant pectate lyase rBpumPel-Ec is shown in SEQ ID NO: 1;

[0013] The gene encoding the amino acid shown in SEQ ID NO:1 is shown in SEQ ID NO:2.

[0014] Preferably, the enzymatic properties of the recombinant pectate lyase are as follows: the optimum pH is 8.5, and it remains stable at pH 7.5-9.0; the optimum enzymatic hydrolysis temperature is 55° C.; and the recombinant pectate lyase is an endo-pectate lyase.

[0015] The present invention also provides a method for constructing a recombinant pectate lyase, comprising the following steps:

[0016] (1) using the genomic DNA of the strain as a template and using a specific primer pair to perform PCR amplification to obtain a gene encoding a recombinant pectate lyase;

[0017] (2) Connecting the gene encoding recombinant pectate lyase to the pET30a(+) vector to construct the pectate lyase recombinant expression plasmid pET30a(+)-BpumPel;

[0018] (3) transforming the pectate lyase recombinant expression plasmid pET30a(+)-BpumPel into Escherichia coli Rosetta (DE3) to obtain recombinant Escherichia coli pET30a(+)-BpumPel / E. coli Rosetta (DE3);

[0019] (4) Spread the recombinant Escherichia coli pET30a(+)-BpumPel / E. coli Rosetta (DE3) on LB solid medium and culture for 15 to 17 hours, pick a single colony and culture it in LB liquid medium for 15 to 17 hours to obtain a bacterial solution;

[0020] (5) After inducing the bacterial solution, the bacterial cells are collected, the cells are broken, centrifuged, and the supernatant is taken to obtain a crude enzyme solution;

[0021] (6) purifying the crude enzyme solution by nickel-NTA column affinity chromatography to obtain recombinant pectate lyase;

[0022] The specific primer pair in step (1) is shown in SEQ ID NO: 3-4;

[0023] The culture temperature in step (4) is 35-39° C.

[0024] The induction culture method in step (5) is: first pre-culture in LB liquid culture medium containing kanamycin for 2 to 3 hours, and then add isopropyl-β-D-thiogalactoside and culture for 15 to 17 hours.

[0025] The present invention also provides the use of the recombinant pectate lyase or the recombinant pectate lyase constructed by the construction method in hydrolyzing polygalacturonic acid or in low-temperature ramie degumming.

[0026] The present invention also provides a low-temperature ramie degumming method, comprising the following steps: mixing ramie fiber with a buffer solution containing recombinant pectate lyase, and incubating at 20-30° C. for 3.5-4.5 hours to obtain an enzymatic hydrolysis mixture; mixing the enzymatic hydrolysis mixture with a sodium hydroxide solution, and incubating at 20-30° C. for 1.5-2.5 hours;

[0027] The recombinant pectate lyase is the recombinant pectate lyase or the recombinant pectate lyase constructed according to the construction method;

[0028] The preparation method of the buffer containing recombinant pectate lyase is as follows: the recombinant pectate lyase is mixed with the buffer; the final concentration of the recombinant pectate lyase in the buffer containing the recombinant pectate lyase is 1-2.5 U / mL;

[0029] The mass volume ratio of the ramie fiber and the buffer containing recombinant pectate lyase is 1 g: 18-22 mL;

[0030] The initial concentration of the sodium hydroxide solution is 0.05-0.1 wt %.

[0031] The present invention provides an expression plasmid, comprising a gene encoding a recombinant pectate lyase and an empty vector;

[0032] The recombinant pectate lyase is the recombinant pectate lyase;

[0033] The gene encoding the recombinant pectate lyase is shown in SEQ ID NO: 2;

[0034] The empty vector is a pET30a(+) vector.

[0035] The present invention also provides a recombinant expression strain, comprising a gene encoding a recombinant pectate lyase and a host bacterium;

[0036] The recombinant pectate lyase is the recombinant pectate lyase;

[0037] The gene encoding the recombinant pectate lyase is shown in SEQ ID NO: 2;

[0038] The host bacteria is Escherichia coli Rosetta (DE3).

[0039] The present invention provides a recombinant pectate lyase from Bacillus pumilus B.15-1, a construction method thereof, and an application thereof, and a method for low-temperature ramie degumming. The present invention provides a strain of Bacillus pumilus B.15-1, which is deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China, with a deposit date of December 23, 2024, and a deposit number of CCTCC NO: M20242888. A gene with pectate lyase activity was amplified from the strain, and the gene was connected to pET30a(+) to construct an expression plasmid pET30a(+)-BpumPel, and the expression plasmid pET30a(+)-BpumPel was connected to Escherichia coli Rosetta(DE3) to obtain recombinant Escherichia coli pET30a(+)-BpumPel / E.coli Rosetta(DE3), and the recombinant Escherichia coli pET30a(+)-BpumPel / E.coli Rosetta(DE3) was induced to produce recombinant pectate lyase, and the obtained recombinant pectate lyase can hydrolyze polygalacturonic acid, and can be combined with sodium hydroxide solution to degumming ramie under low temperature conditions, providing a new method for green degumming of ramie with low pollution and mild conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is the phylogenetic tree;

[0041] Figure 2 The electrophoretic pattern of the gene encoding the recombinant pectate lyase of the PCR product is detected by agarose gel electrophoresis;

[0042] Figure 3 The figure is the SDS-gel electrophoresis spectrum of the purified recombinant pectate lyase BpumPel-Ec (lane 1 represents the protein molecule marker; lane 2 represents the total protein obtained by ultrasonically disrupting Escherichia coli Rosetta (DE3) cells containing the blank vector pET30a (+) after IPTG induction; lane 3 represents the total protein obtained by ultrasonically disrupting Escherichia coli Rosetta (DE3) cells containing the plasmid pET30a (+)-BpumPel after IPTG induction, and then the total soluble protein obtained by centrifugation to remove the precipitate; lane 4 represents the purified recombinant pectate lyase BpumPel-Ec);

[0043] Figure 4The results of the effects of pH and temperature on the enzyme activity and stability of the recombinant pectate lyase BpumPel-Ec are shown in FIG. (a represents the effect of different pH on the enzyme activity of the recombinant pectate lyase BpumPel-Ec, b represents the effect of different pH on the stability of the recombinant pectate lyase BpumPel-Ec, c represents the effect of different temperatures on the enzyme activity of the recombinant pectate lyase BpumPel-Ec, and d represents the effect of different temperatures on the stability of the recombinant pectate lyase BpumPel-Ec);

[0044] Figure 5 This is a high performance liquid chromatography analysis diagram of the hydrolysis of polygalacturonic acid PGA by recombinant pectate lyase rBpumPel-Ec.

[0045] Collection Instructions

[0046] Bacillus pumilus B.15-1 is deposited in the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China. The deposit date is December 23, 2024, and the deposit number is CCTCC NO: M20242888. DETAILED DESCRIPTION

[0047] In the present invention, Bacillus pumilus B.15-1 was screened from soil samples of cassava fields in the Guangxi University Farm.

[0048]

[0049] In the present invention, the amino acid sequence of the recombinant pectate lyase rBpumPel-Ec is shown in SEQ ID NO: 1;

[0050] SEQ ID NO: 1: EAKAAVKYPESPSIMANFNQQGFSTLNGGTTGGEGGKTVTVKTGNELLAALKSKGTNEKLKIVVDGTITPSNTSANKIDVKDTNNVSIVGKGTKGEFNGIGIKVWRGNNIIIRNLKIHHSKIGDKDAIGIEGASKNVWVDHNELYNTLNSGKDDYD GLFDVKNDSDYITFSWNYVHDSWKTMLMGSSDNDNYNRKITFHNNRFENLNSRVPSMRFGEGHVYNNYYKGILTTAINSRMGAKMRIEHNVFENTKNAIGSWDSRQVGTWHVINNSFINSTGSLPTSSTGTYNPPYNYSLLNVNNVKSEVVSNAGVGKVNP.

[0051] The gene encoding the amino acid shown in SEQ ID NO: 1 is as shown in SEQ ID NO: 2, SEQ ID NO: 2: GAGGCAAAAGCGGCAGTAAAGTATCCCGAAAGCCCTTCTATTATGGCCAATTTTAATCAACAAGGATTCTCCACATTAAATGGCGGCACAACAGGCGGTGAAGGCGGTAAAACCGTCACAGTCAAAACTGGAAATGAGCTATTGGCTGCCCTCAAAAGTAAAGGAACAAACGAAAAATTAAAAATTGTTGTCGACGGGACGATTACCCCTTCTAATACATCTGCCAATAAAATCGATGTGAAAGATACAAACAATGTCTCAATCGTCGGCAAAGGCACAAAAGGTGAATTTAATGGAATTGGCATTAAAGTATGGCGTGGCAATAACATCATCATCCGCAACCTGAAAATCCATCACAGCAAAATCGGTGATAAGGATGCCATCGGAATCGAAGGCGCTTCTAAAAACGTATGGGTCGATCATAATGAACTGTACAATACATTAAACTCTGGTAAAGATGATTATGATGGATTATTCGATGTGAAAAATGACTCCGATTATATTACCTTCTCATGGAACTATGTTCATGACAGCTGGAAAACGATGCTCATGGGCAGCTCTGATAACGACAACTACAACCGAAAAATCACATTCCACAACAACCGTTTCGAAAACCTGAATTCTCGTGTACCATCTATGCGCTTTGGGGAAGGACATGTCTATAACAACTATTACAAAGGCATCCTGACAACTGCAATTAACTCTCGTATGGGCGCAAAAATGAGAATTGAGCACAACGTATTTGAAAATACAAAAAATGCGATCGGCAGCTGGGACAGCCGTCAAGTCGGTACATGGCATGTCATCAACAACTCATTTATTAACAGCACAGGCAGTCTGCCAACGTCTTCTACAGGTACGTACAACCCTCCTTATAACTACTCCTTGCTTAACGTGAACAATGTCAAATCAGAAGTTGTATCAAATGCTGGTGTTGGAAAGGTAAACCCT。

[0052] In the present invention, the specific primer pair for amplifying the gene encoding the recombinant pectate lyase is shown in SEQ ID NOs: 3-4;

[0053] Upstream primer SEQ ID NO: 3: 5′-CCCAAGCTTGG GAGGCAAAAGCGGCAG-3′;

[0054] Downstream primer SEQ ID NO: 4: 5'-CCGCTCGAG AGGGTTTACCTTTCCAACACC-3'.

[0055] In the present invention, the nucleotide sequence of the constructed expression plasmid pET30a(+)-BpumPel is shown in SEQ ID NO: 6. SEQ ID NO: 6:

[0056]

[0057]

[0058] (The single underlined bases are the parts brought into the vector pET30a(+), the bold GG is the base added to avoid changing the reading frame, the double underlined bases represent the start codon and the stop codon, and the remaining bases are the pectate lyase gene).

[0059] In the present invention, the primers used to verify whether the pectate lyase gene has been transferred into Escherichia coli DH5α are T7 universal primers, as shown in SEQ ID NOs: 7 to 8:

[0060] Upstream primer SEQ ID NO: 7: 5′-TAATACGACTCACTATAGGG-3′;

[0061] Downstream primer SEQ ID NO: 8: 5'-GCTAGTTATTGCTCAGCGG-3'.

[0062] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0063] The LB liquid culture medium involved in the embodiment of the present invention uses water as a solvent and also includes the following components in concentrations: 5 g / L yeast extract, 10 g / L peptone, and 10 g / L NaCl.

[0064] The LB solid culture medium involved in the embodiment of the present invention uses water as a solvent and also includes the following components in the following concentrations: 5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl, and 15 g / L agar.

[0065] The separation plate culture medium described in the embodiment of the present invention uses water as a solvent and further includes the following components in concentrations: 10 g / L orange peel powder, 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 20 g / L agar powder, pH 7.0.

[0066] The screening culture medium described in the embodiment of the present invention uses water as a solvent and further includes the following components in concentrations: 10 g / L orange peel powder, 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0.

[0067] The soil samples in the embodiments of the present invention were taken from a cassava field in the Guangxi University Farm.

[0068] Example 1

[0069] (1) Isolation of Bacillus pumilus B.15-1

[0070] Isolation of strains: Place 1g of soil sample in a finger-shaped bottle filled with 10mL of sterile water and place it in a shaker set at 200rpm for 10min. Dilute the suspension with sterile water in a 10-fold gradient, then spread the dilution on the isolation plate culture medium and culture at 37℃ for 2 days.

[0071] Screening of pectate lyase-producing microorganisms: Inoculate the colonies grown on the separation plate culture medium into the screening culture medium (50 mL of culture medium in a 250 mL Erlenmeyer flask), culture in a shaker at 37°C and 200 rpm for 2 days, centrifuge the cultured bacterial solution at 10,000 rpm for 1 min, take the supernatant, and determine the pectate lyase activity in different bacterial solutions.

[0072] Determination of pectate lyase activity: 40 μL of appropriately diluted enzyme solution was mixed with 280 μL of Tris-HCl (0.2 M, pH 8.5) solution containing 0.15% polygalacturonic acid (PGA), incubated at 50°C for 10 min, and then 640 μL of HCl was added. 3 PO 4 The reaction was terminated by adding 30 mM solution. The activity of pectate lyase was reflected by measuring the increase of unsaturated bonds at 235 nm. One unit of enzyme activity was defined as the amount of enzyme required to produce 1 μmol of unsaturated bonds per minute, where the molecular extinction coefficient at 235 nm was 4600 M -1 cm -1 The enzyme solution was placed in a boiling water bath for 5 min and the rest of the operation was the same as above as a control.

[0073] The bacterial cells were collected from the enzyme-active bacterial liquid, and the genome was extracted and amplified to obtain the partial coding region sequence of its 16S RNA. The obtained amplified product was sequenced as shown in SEQ ID NO: 5. The 16S RNA sequence was sequenced, compared with NCBI, and physiological and biochemical identification was performed. Finally, a strain belonging to Bacillus pumilus was obtained and named B.15-1. The strain was deposited in the China Center for Type Culture Collection with the deposit number: CCTCC NO: M20242888. The phylogenetic tree after NCBI comparison is shown in Figure 1 shown.

[0074] (2) Construction of recombinant E. coli pET30a(+)-BpumPel / E. coli Rosetta(DE3)

[0075] The hypothetical protein predicted as pectate lyase was searched in the genome of Bacillus pumilus strain published in NCBI (accession number QKN79634). The signal peptide and catalytic domain of the hypothetical protein were predicted using SMART online software (http: / / smart.embl-heidelberg.de / ) to determine the cloning region and design specific primers. The genomic DNA of Bacillus pumilus B.15-1 was used as a template, and PCR amplification was performed using specific primers BpumPel-F (SEQ ID NO.3: 5'-CCCAAGCTTGGGAGGCAAAAGCGGCAG-3') and BpumPel-R (SEQ ID NO.4: 5'-CCGCTCGAGAGGGTTTACCTTTCCAACACC-3'). The amplification program was: 95℃ pre-denaturation for 3min, followed by 30 cycles, each cycle including 95℃ denaturation for 15s, 61℃ annealing for 15s, 72℃ extension for 40s, and finally 72℃ extension for 10min. After the PCR reaction, the purity of the PCR product (gene encoding recombinant pectate lyase) was detected by agarose gel electrophoresis. Figure 2 shown.

[0076] The gene encoding the recombinant pectate lyase is shown in SEQ ID NO:2.

[0077] The pET30a(+) vector and PCR product were double-digested with HindШ-XhoⅠ (37°C, 4h), and then the digested product was purified and ligated with T4 DNA ligase at 16°C for 30min to obtain the ligation product. The ligation product was transferred into Escherichia coli DH5α, spread on LB (50μg / mL Kan) solid culture medium plate, and cultured at 37°C for 16h. A single colony was picked in LB (50μg / mL Kan) liquid culture medium, cultured at 37°C and 220rpm for 16h, and the bacterial solution was taken for PCR verification with T7 universal primers (SEQ ID NO: 7-8), and the successfully verified bacterial solution was sent for sequencing. After sequence identification, the nucleotide sequence of the expression plasmid pET30a(+)-BpumPel obtained is shown in SEQ ID NO: 6. The nucleotide sequence shown in SEQ ID NO: 6 is 1155bp.

[0078] The recombinant expression plasmid was transferred into E. coli Rosetta (DE3) to obtain a recombinant expression strain producing pectate lyase, namely, recombinant E. coli pET30a (+) -BpumPel / E. coli Rosetta (DE3).

[0079] (3) Preparation and detection of recombinant pectate lyase rBpumPel-Ec

[0080] The recombinant E. coli pET30a(+)-BpumPel / E. coli Rosetta(DE3) was spread on a LB solid (50μg / mL Kan) medium plate and cultured at 37°C for 16h. A single colony was picked and placed in a LB (50μg / mL Kan) liquid medium. After culturing at 37°C and 220rpm for 16h, 1mL of the bacterial solution was added to 100mL of LB liquid medium with kanamycin, pre-cultured at 37°C and 220rpm for 2.5h, and then 100μL of 1M isopropyl-β-D-thiogalactoside (IPTG) was added and induced at 16°C and 100rpm for 16h. At the same time, the blank vector pET30a(+) was transferred into E. coli Rosetta(DE3) as a blank control group, and cultured and induced in the same manner as the recombinant E. coli pET30a(+)-BpumPel / E. coli Rosetta(DE3).

[0081] After induction, the cells were collected and 18 mL of cell lysis buffer (300 mM NaCl, 50 mM NaH 2 PO 4 , 10 mM imidazole, pH 8.5) was used to disrupt the cells with ultrasound, and centrifuged at 12000 rpm for 30 min to obtain the supernatant as the crude enzyme solution. The crude enzyme solution was purified using nickel-NTA column affinity chromatography to obtain recombinant pectate lyase.

[0082] The amino acid sequence of the obtained recombinant pectate lyase is shown in SEQ ID NO:1.

[0083] The purity of the purified recombinant pectate lyase was detected by SDS-PAGE gel electrophoresis and compared with the crude enzyme solution of the induced cells of E. coli Rosetta (DE3) containing the blank vector pET30a(+) and the induced cells of E. coli Rosetta (DE3) containing the plasmid pET30a(+)-BpumPel. Figure 3 shown.

[0084] The purified recombinant pectate lyase was desalted (the replacement fluid for desalting was sterile ultrapure water) and concentrated using a centrifugal ultrafiltration tube with a molecular weight cutoff of 10 kDa. After concentration, the concentration of the recombinant pectate lyase was determined by the Coomassie Brilliant Blue R-250 method, and the protein concentration was 3.93. The concentrated enzyme solution was used for enzymatic property analysis. After testing, the specific enzyme activity of the purified recombinant pectate lyase rBpumPel-Ec was 78.26 U / mg. The enzyme was named recombinant pectate lyase rBpumPel-Ec.

[0085] Example 2

[0086] Using 0.15% polygalacturonic acid PGA (w / v) as substrate, the effect of pH on the enzyme activity and stability of the recombinant pectate lyase rBpumPel-Ec obtained in Example 1 was studied. The specific process is as follows:

[0087] Use Na 2 HPO 4 -citrate buffer (0.1 M, pH 6.0-7.0), Tris-HCl buffer (0.1 M, pH 7.0-9.0), and glycine-NaOH buffer (0.1 M, pH 9.0-12.0) were used for the experiments.

[0088] (1) Effect of pH on enzyme activity

[0089] 0.15% PGA (w / v) substrate was prepared using buffer solutions with pH values ​​of 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, and 11.5, respectively, and the enzyme activity of the recombinant pectate lyase rBpumPel-Ec obtained in Example 1 on the substrate at different pH values ​​was measured at 50°C for 10 min. The optimal enzyme activity was defined as 100%, and the other enzyme activities were calculated as relative values. The test results are shown in FIG. Figure 4As shown in (a), it can be seen that the enzyme activity of the recombinant pectate lyase rBpumPel-Ec is low in an acidic environment. As the pH value increases, its enzyme activity increases and reaches the highest at pH 8.5.

[0090] (2) Effect of pH on stability

[0091] The recombinant pectate lyase rBpumPel-Ec was placed in 0.1 mol / L, pH 5.0 to 11.0 buffer, incubated at 30°C for 24 h, and then its enzyme activity was measured at pH 8.5 and 50°C. The activity of the enzyme not incubated in the buffer was defined as 100%, and the activity of the enzyme after incubation was calculated as a relative value. The test results are shown in Figure 4 As shown in (b), it can be seen that the enzyme activity of the recombinant pectate lyase rBpumPel-Ec is not lost at pH 8.5, and the enzyme activity of rBpumPel-Ec placed at other pH values ​​is lost to varying degrees, and is relatively stable at pH 7.5-9.0.

[0092] Example 3

[0093] Using 0.15% PGA (w / v) and pH 8.5 buffer as substrate, the effect of temperature on the enzyme activity and stability of the recombinant pectate lyase rBpumPel-Ec obtained in Example 1 was studied and tested. The specific process is as follows:

[0094] (1) Effect of temperature on enzyme activity

[0095] The enzyme activity of the recombinant pectate lyase rBpumPel-Ec was measured at 30, 35, 40, 45, 50, 55, 60, 65, and 70°C, and the enzyme activity at the optimal enzymatic hydrolysis temperature was defined as 100%. Figure 4 As shown in (c), it can be seen that the optimal enzymatic hydrolysis temperature of the recombinant pectate lyase rBpumPel-Ec is 55°C.

[0096] (2) Effect of temperature on stability

[0097] The enzyme activity of the recombinant pectate lyase rBpumPel-Ec was measured after incubation at 30, 35, 40, 45, 50, 55 and 60°C for 15, 30, 45 and 60 min, and the enzyme activity was measured at pH 8.5 and 55°C. The activity of the unincubated enzyme was set as 100%. The test results are shown in Figure 4 As shown in (d), it can be seen that rBpumPel-Ec is stable at lower temperatures, and the enzyme activity is basically unchanged after incubation at 30°C for 1 hour; it becomes unstable as the temperature rises, and the enzyme activity remains at 22.05±0.83% after incubation at 60°C for 1 hour, and the enzyme activity is basically lost after the incubation time is extended to 1 hour.

[0098] Example 4

[0099] The product analysis of the hydrolysis of polygalacturonic acid PGA by the recombinant pectate lyase rBpumPel-Ec obtained in Example 1 is as follows:

[0100] The recombinant pectate lyase rBpumPel-Ec was incubated with PGA at 35°C for 10 min. After 24 h, the hydrolyzate was analyzed by HPLC at a wavelength of 235 nm. Figure 5 It can be seen that rBpumPel-Ec hydrolyzes PGA to produce 4,5-unsaturated oligogalacturonic acid including unsaturated trigalacturonic acid (uG3) and unsaturated digalacturonic acid (uG2), but does not produce unsaturated galacturonic acid (uG1), which shows that rBpumPel-Ec is an endo-pectate lyase.

[0101] Example 5

[0102] The effects of the recombinant pectate lyase rBpumPel-Ec, NaOH and their combination on ramie degumming obtained in Example 1 were tested. The specific process is as follows:

[0103] Effect of enzyme dosage on ramie degumming

[0104] 1g of ramie fiber was wound into a small circle (ramie circle) with a diameter of 5.0±0.1cm, and immersed in 20mL of Tris-HCl buffer (pH 8.5) containing different amounts of recombinant pectate lyase rBpumPel-Ec (1U / mL and 2.5U / mL) at a solid-liquid ratio of 1:20, incubated at 30°C for 6h, and then fully rinsed with ultrapure water, drained, and dried at 100°C to constant weight. Weight loss rate (%) = (weight before degumming - weight after degumming) × 100 / weight before degumming.

[0105] Effect of NaOH concentration on ramie degumming

[0106] The ramie ring was immersed in 20 mL of NaOH solution of different concentrations (0.05 wt%, 0.1 wt%), incubated at 30°C for 6 h, then fully rinsed with ultrapure water, drained and dried at 100°C to constant weight, and the weight loss rate was calculated.

[0107] Effect of recombinant pectate lyase rBpumPel-Ec combined with NaOH on ramie degumming

[0108] 1 g of ramie fiber was immersed in 20 mL Tris-HCl buffer (pH 8.5) containing different amounts of rBpumPel-Ec enzyme at a solid-liquid ratio of 1:20 and incubated at 30 ° C for 4 h. After the enzymatic degumming was completed, the enzyme-treated ramie fiber was immersed in 20 mL of NaOH solution with different concentrations (w / v), incubated at 30 ° C for 2 h, drained and dried at 100 ° C to constant weight, and the weight loss rate was calculated.

[0109] The results are shown in Table 1. When rBpumPel-Ec or NaOH was used alone, the weight loss rates of 1U / mL and 2.5U / mL rBpumPel-Ec were 11.85±0.50% and 13.47±0.60%, respectively, and the weight loss rates of 0.05% and 0.1% NaOH were 15.45±0.50% and 17.53±0.58%, respectively. However, the effect was greatly improved when rBpumPel-Ec was used in combination with NaOH.

[0110] Table 1 Effect of rBpumPel-Ec and NaOH on ramie degumming

[0111] deal with Weight loss rate / % pH 8.5 Tris-HCl buffer (negative control) 8.60±0.41 1U / mLrBpumPel-Ec 11.85±0.50 2.5U / mLrBpumPel-Ec 13.47±0.60 0.05% NaOH 15.45±0.50 0.1% NaOH 17.53±0.58 1U / mL rBpumPel-Ec+0.05%NaOH 18.73±0.55 1U / mL rBpumPel-Ec+0.1%NaOH 21.65±0.39 2.5U / mLrBpumPel-Ec+0.05%NaOH 20.13±0.20 2.5U / mL rBpumPel-Ec+0.1%NaOH 22.40±0.33

[0112] It can be seen from the above embodiments that the present invention provides a recombinant pectate lyase from Bacillus pumilus B.15-1, a construction method and application thereof, and a method for low-temperature ramie degumming. The present invention provides a strain of Bacillus pumilus B.15-1, which is deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with a deposit date of December 23, 2024, and a deposit number of CCTCC NO: M20242888. A gene with pectate lyase activity was amplified from the strain, and the gene was connected to pET30a(+) to construct an expression plasmid pET30a(+)-BpumPel, and the expression plasmid pET30a(+)-BpumPel was connected to Escherichia coli Rosetta(DE3) to obtain recombinant Escherichia coli pET30a(+)-BpumPel / E.coli Rosetta(DE3), and the recombinant Escherichia coli pET30a(+)-BpumPel / E.coli Rosetta(DE3) was induced to produce pectate lyase, and the obtained pectate lyase can hydrolyze polygalacturonic acid, and can be combined with sodium hydroxide solution to degumming ramie under low temperature conditions. A new method for green degumming of ramie with low pollution and mild conditions is provided.

[0113] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain having pectate lyase activity, characterized in that: The strain is Bacillus pumilus B.15-1, which is deposited in the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China. The deposit date is December 23, 2024, and the deposit number is CCTCCNO: M20242888.

2. Use of the strain according to claim 1 in preparing a product for hydrolyzing polygalacturonic acid.

3. Use of the strain according to claim 1 in preparing a product having the effect of low-temperature ramie degumming.

4. A recombinant pectate lyase, characterized in that: The recombinant pectate lyase is a recombinant pectate lyase rBpumPel-Ec, which is constructed by the strain according to claim 1, and the amino acid sequence of the constructed recombinant pectate lyase rBpumPel-Ec is shown in SEQ ID NO: 1; The gene encoding the amino acid shown in SEQ ID NO:1 is shown in SEQ ID NO:

2.

5. The recombinant pectate lyase according to claim 4, characterized in that The enzymatic properties of the recombinant pectate lyase are as follows: the optimum pH is 8.5, and it remains stable at pH 7.5-9.0; the optimum enzymolysis temperature is 55° C.; and the recombinant pectate lyase is an endo-pectate lyase.

6. A method for constructing a recombinant pectate lyase, characterized in that: The steps include: (1) using the genomic DNA of the strain described in claim 1 as a template and using a specific primer pair to perform PCR amplification to obtain a gene encoding a recombinant pectate lyase; (2) Connecting the gene encoding recombinant pectate lyase to the pET30a(+) vector to construct the pectate lyase recombinant expression plasmid pET30a(+)-BpumPel; (3) transforming the pectate lyase recombinant expression plasmid pET30a(+)-BpumPel into Escherichia coli Rosetta (DE3) to obtain recombinant Escherichia coli pET30a(+)-BpumPel / E. coli Rosetta (DE3); (4) Spread the recombinant Escherichia coli pET30a(+)-BpumPel / E. coli Rosetta (DE3) on LB solid medium and culture for 15 to 17 hours, pick a single colony and culture it in LB liquid medium for 15 to 17 hours to obtain a bacterial solution; (5) After inducing the bacterial solution, the bacterial cells are collected, the cells are broken, centrifuged, and the supernatant is taken to obtain a crude enzyme solution; (6) purifying the crude enzyme solution by nickel-NTA column affinity chromatography to obtain recombinant pectate lyase; The specific primer pair in step (1) is shown in SEQ ID NO: 3-4; The culture temperature in step (4) is 35-39° C. The induction culture method in step (5) is: first pre-culture in LB liquid culture medium containing kanamycin for 2 to 3 hours, and then add isopropyl-β-D-thiogalactoside and culture for 15 to 17 hours.

7. Use of the recombinant pectate lyase according to any one of claims 4 to 5 or the recombinant pectate lyase constructed by the construction method according to claim 6 in hydrolyzing polygalacturonic acid or in low-temperature ramie degumming.

8. A method for low-temperature ramie degumming, characterized in that: The method comprises the following steps: mixing ramie fiber with a buffer solution containing recombinant pectate lyase, incubating at 20-30°C for 3.5-4.5 hours to obtain an enzymatic hydrolysis mixture; mixing the enzymatic hydrolysis mixture with a sodium hydroxide solution, incubating at 20-30°C for 1.5-2.5 hours; The recombinant pectate lyase is the recombinant pectate lyase according to any one of claims 4 to 5 or the recombinant pectate lyase constructed according to the construction method of claim 6; The preparation method of the buffer containing recombinant pectate lyase is as follows: the recombinant pectate lyase is mixed with the buffer; the final concentration of the recombinant pectate lyase in the buffer containing the recombinant pectate lyase is 1-2.5 U / mL; The mass volume ratio of the ramie fiber and the buffer containing recombinant pectate lyase is 1 g: 18-22 mL; The initial concentration of the sodium hydroxide solution is 0.05-0.1 wt %.

9. An expression plasmid, characterized in that including a gene encoding a recombinant pectate lyase and an empty vector; The recombinant pectate lyase is the recombinant pectate lyase according to claim 4 or 5; The gene encoding the recombinant pectate lyase is shown in SEQ ID NO: 2; The empty vector is a pET30a(+) vector.

10. A recombinant expression strain, characterized in that: It includes a gene encoding a recombinant pectate lyase and a host bacteria; The recombinant pectate lyase is the recombinant pectate lyase according to claim 4 or 5; The gene encoding the recombinant pectate lyase is shown in SEQ ID NO: 2; The host bacteria is Escherichia coli Rosetta (DE3).

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