Protein mutants having pectolytic activity and use thereof

Abstract

The present application relates to the technical field of biotechnology, and discloses a protein mutant with pectinase activity and application thereof.The protein mutant has at least one mutation in the positions of R150, R216 and A238 compared with the protein with the amino acid sequence shown in SEQ ID NO:1.The protein mutant with pectinase activity provided by the present application has higher reaction activity stability under high temperature and higher pH conditions, and has great industrial application potential.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a protein mutant with pectinase activity and its applications. Background Technology

[0002] Pectin is an acidic polysaccharide widely found in the plant kingdom, primarily in the primary cell walls and middle lamellae of plants, serving as a supporting substance for internal cells. Pectin has a complex chemical structure; its backbone is a partially methylated galactan, while the side chains vary depending on the pectin's origin. Deesterified pectin is called pectic acid or polygalacturonic acid. Enzymes that hydrolyze this type of pectin are widely known as pectinases. Pectic acid lyase (EC.4.2.2.2) degrades pectic acid by randomly cleaving the α-1,4-glycosidic bonds through β-elimination. Alkaline, heat-resistant pectinases can be applied to some traditional industrial processes, such as textile and plant fiber processing, coffee and tea fermentation, oil extraction, and the treatment of industrial wastewater containing pectin. Because microbial enzymatic methods offer significant advantages over traditional chemical treatments in biorefining and plant fiber degumming—reducing the damage to plant fibers caused by high-concentration strong alkaline solutions and the subsequent wastewater treatment—they have attracted increasing research attention.

[0003] Enzymatic properties and stability are key factors influencing enzyme applications. Since degumming processes are mostly carried out under alkaline and high-temperature conditions, the enzymes involved must be able to withstand these conditions. Thermo-alkaline stability of enzymes is particularly important for industrial applications. Furthermore, under high-temperature and alkaline conditions, enzymes react faster, shortening reaction cycles, saving costs, and also helping to prevent contamination by other microorganisms during the reaction process.

[0004] However, although there are some pectinases that have good catalytic activity under alkaline or high-temperature conditions, there is still considerable room for improvement in their thermal and alkaline stability. Summary of the Invention

[0005] The purpose of this invention is to overcome the problem of insufficient stability of existing pectinases under extreme conditions of high temperature and alkalinity, and to provide a protein mutant with pectinase activity and its application. This protein with pectinase activity has high reactivity and stability under high temperature and high pH conditions, and has great potential for industrial application.

[0006] To achieve the above objectives, the present invention provides a protein mutant with pectinase activity, which, compared with the protein with the amino acid sequence shown in SEQ ID NO:1, contains a mutation at at least one of the sites R150, R216 and A238.

[0007] The second aspect of the present invention provides the coding gene for the protein mutant as described in the first aspect above.

[0008] A third aspect of the present invention provides a recombinant vector containing the coding gene as described in the second aspect above.

[0009] A fourth aspect of the present invention provides a recombinant cell containing the coding gene as described in the second aspect above, or the recombinant cell containing the recombinant vector as described in the third aspect above.

[0010] The fifth aspect of the present invention provides a composition for degrading pectin, the composition comprising the protein mutant as described in the first aspect above.

[0011] The sixth aspect of the present invention provides a method for hydrolyzing pectin, the method comprising contacting a protein mutant as described in the first aspect, a recombinant cell as described in the fourth aspect, or a composition as described in the fifth aspect with a pectin-containing substrate, and hydrolyzing the pectin under hydrolysis conditions.

[0012] Through the above technical solution, the present invention can achieve the following beneficial effects:

[0013] (1) The protein mutant provided by the present invention has high pectinase activity and high thermal-alkali stability. Compared with its corresponding wild-type protein, the residual enzyme activity of the protein mutant is increased by more than 4 times under thermal-alkali conditions of 60°C and pH10 for 4 hours, which is more conducive to industrial application.

[0014] (2) The protein mutant provided by the present invention is derived from a mutant of natural pectinase. It is not easy to cause environmental pollution when used, and it can be induced to be expressed and produced by genetic engineering, which is conducive to large-scale industrial application and promotion. Attached Figure Description

[0015] Figure 1 This is a graph showing the results of thermostability testing for different mutant enzymes in Test Example 2. Detailed Implementation

[0016] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0017] In this invention, unless otherwise specified, "wild-type pectinase" ("wild-type protein") refers to BacPelA expressed by Bacillus clausii S10, abbreviated as WT. "Pectinase protein mutant 1A" is a mutant of BacPelA with a single-point R150G mutation, abbreviated as 1A. "Pectinase protein mutant 2B" is a mutant of BacPelA with a single-point R216H mutation, abbreviated as 2B. "Pectinase protein mutant 3C" is a mutant of BacPelA with a single-point A238C mutation, abbreviated as 3C. "Pectinase protein mutant 4AB" is a mutant of BacPelA with a double-point R150G / R216H mutation, abbreviated as 4AB. "5AC" is a mutant of BacPelA pectinase with a double-point mutation of R150G / A238C, abbreviated as 5AC. "6BC" is a mutant of BacPelA pectinase with a double-point mutation of R216H / A238C, abbreviated as 6BC. "FCM" is a mutant of BacPelA pectinase with a triple-point mutation of R150G / R216H / A238C, abbreviated as FCM.

[0018] The inventors of this invention discovered an alkaliphilic Bacillus clausii S10 strain that expresses a pectinase, BacPelA (amino acid sequence shown in SEQ ID NO:1), which exhibits high catalytic activity against different methylated pectins, especially hypermethylated pectins, under alkaline conditions. This pectinase also demonstrates good heat resistance and promising industrial application potential. However, industrial applications require not only heat and alkali resistance but also good stability under high-temperature alkaline conditions. Based on this, the inventors further discovered that by incorporating specific mutations at specific sites (R150, R216, and A238) into BacPelA, the resulting mutant not only exhibits good heat and alkali resistance but also high thermal-alkali stability, making it even more suitable for industrial degumming applications.

[0019] ANVNFSMQGFATLNGGTTGGAGGDVVTVSTGDQLIAALKNKKANTPLTIYIDGTITPANTSASKIDIKDVNDVSLLGVGTNGELNGIGIKVWRANNVIIRNLKIHHVNTGDKDAISIEGPSKNIWVDHNELYNSLDVHKDYYDGLFDVKRDADY ITFSWNYVHDSWKSMLMGSSDSDSYGRKITFHNNYFENLNSRVPSVRFGEAHIFSNYYADIRETGINSRMGAQVRIEENYFERANNPIVSRDSKEIGYWHLVNNRYVSSTGEQPTVSTTTYNPPYSYQATPVNQVKDVVRANAGVGVISP(SEQ ID NO:1)

[0020] Based on the above findings, the present invention provides a protein mutant with pectinase activity, which, compared with the protein with the amino acid sequence shown in SEQ ID NO:1 (i.e., wild-type pectinase BacPelA), contains a mutation at at least one of the sites R150, R216 and A238.

[0021] According to a preferred embodiment of the present invention, the protein mutant contains at least one mutation of R150G, R216H and A238C compared to the protein with the amino acid sequence shown in SEQ ID NO:1.

[0022] Preferably, the amino acid sequence of the protein mutant is as shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

[0023] ANVNFSMQGFATLNGGTTGGAGGDVVTVSTGDQLIAALKNKKANTPLTIYIDGTITPANTSASKIDIKDVNDVSLLGVGTNGELNGIGIKVWRANNVIIRNLKIHHVNTGDKDAISIEGPSKNIWVDHNELYNSLDVHKDYYDGLFDVKGDADYITFSWNYVHDSWASMLGSSSDSYGRKITFHNNYFENLNSRVPSVAFGEAHIFSNYYADIRETGINSRMGAQVRIEENYFERANNPIVSRDSKEIGYWHLVNNRYVSSTGEQPTVSTTTYNPPYSYQATPVNQVKDVVVRANAGVGVISP(SEQ ID NO:2)

[0024] ANVNFSMQGFATLNGGTTGGAGGDVVTVSTGDQLIAALKNKKANTPLTIYIDGTITPANTSASKIDIKDVNDVSLLGVGTNGELNGIGIKVWRANNVIIRNLKIHHVNTGDKDAISIEGPSKNIWVDHNELYNSLDVHKDYYDGLFDVKRDADYITFSWNYVHDSWASMLGSSSDSYGRKITFHNNYFENLNSRVPSVAFGEAHIFSNYYADIHETGINSRMGAQVRIEENYFERANNPIVSRDSKEIGYWHLVNNRYVSSTGEQPTVSTTTYNPPYSYQATPVNQVKDVVRANAGVGVISP(SEQ ID NO:3)

[0025] ANVNFSMQGFATLNGGTTGGAGGDVVTVSTGDQLIAALKNKKANTPLTIYIDGTITPANTSASKIDIKDVNDVSLLGVGTNGELNGIGIKVWRANNVIIRNLKIHHVNTGDKDAISIEGPSKNIWVDHNELYNSLDVHKDYYDGLFDVKRDADYITFSWNYVHDSWASMLGSSSDSYGRKITFHNNYFENLNSRVPSVAFGEAHIFSNYYADIRETGINSRMGAQVRIEENYFERCNNPIVSRDSKEIGYWHLVNNRYVSSTGEQPTVSTTTYNPPYSYQATPVNQVKDVVRANAGVGVISP(SEQ ID NO:4)

[0026] ANVNFSMQGFATLNGGTTGGAGGDVVTVSTGDQLIAALKNKKANTPLTIYIDGTITPANTSASKIDIKDVNDVSLLGVGTNGELNGIGIKVWRANNVIIRNLKIHHVNTGDKDAISIEGPSKNIWVDHNELYNSLDVHKDYYDGLFDVKGDADYITFSWNYVHDSWKSMLMGSSDSSYGRKITFHNNYFENLNSRVPSVRFGEAHIFSNYYADIHETGINSRMGAQVRIEENYFERANNPIVSRDSKEIGYWHLVNNRYVSSTGEQPTVSTTTYNPPYSYQATPVNQVKDVVRANAGVGVISP(SEQ ID NO:5)

[0027] ANVNFSMQGFATLNGGTTGGAGGDVVTVSTGDQLIAALKNKKANTPLTIYIDGTITPANTSASKIDIKDVNDVSLLGVGTNGELNGIGIKVWRANNVIIRNLKIHHVNTGDKDAISIEGPSKNIWVDHNELYNSLDVHKDYYDGLFDVKGDADYITFSWNYVHDSWKSMLMGSSDSSYGRKITFHNNYFENLNSRVPSVRFGEAHIFSNYYADIRETGINSRMGAQVRIEENYFERCNNPIVSRDSKEIGYWHLVNNRYVSSTGEQPTVSTTTYNPPYSYQATPVNQVKDVVRANAGVGVISP(SEQ ID NO:6)

[0028] ANVNFSMQGFATLNGGTTGGAGGDVVTVSTGDQLIAALKNKKANTPLTIYIDGTITPANTSASKIDIKDVNDVSLLGVGTNGELNGIGIKVWRANNVIIRNLKIHHVNTGDKDAISIEGPSKNIWVDHNELYNSLDVHKDYYDGLFDVKRDADYITFSWNYVHDSWKSMLMGSSSDSYGRKITFHNNYFENLNSRVPSVRFGEAHIFSNYYADIHETGINSRMGAQVRIEENYFERCNNPIVSRDSKEIGYWHLVNNRYVSSTGEQPTVSTTTYNPPYSYQATPVNQVKDVVRANAGVGVISP(SEQ ID NO:7)

[0029] ANVNFSMQGFATLNGGTTGGAGGDVVTVSTGDQLIAALKNKKANTPLTIYIDGTITPANTSASKIDIKDVNDVSLLGVGTNGELNGIGIKVWRANNVIIRNLKIHHVNTGDKDAISIEGPSKNIWVDHNELYNSLDVHKDYYDGLFDVKGDADY ITFSWNYVHDSWASMLMGSSDSDSYGRKITFHNNYFENLNSRVPSVAFGEAHIFSNYYADIHETGINSRMGAQVRIENYFERCNNPIVSRDSKEIGYWHLVNNRYVSSTGEQPTVSTTTYNPPYSYQATPVNQVKDVVRANAGVGVISP(SEQ ID NO:8)

[0030] According to the present invention, there are no particular limitations on the method of obtaining the protein mutant, as long as a pectinase protein mutant with the above-described amino acid sequence can be obtained. For example, the protein mutant can be obtained artificially, or its encoding gene can be obtained from the amino acid sequence of the protein mutant, and then obtained through corresponding biological expression.

[0031] The second aspect of the present invention provides the coding gene for the protein mutant as described in the first aspect above.

[0032] Any gene capable of encoding the above-mentioned protein mutant falls within the scope of protection of this invention. It is well known to those skilled in the art that genetic codons are degenerate; therefore, given the amino acid sequence of the above-mentioned pectinase protein, those skilled in the art can obtain coding genes with different nucleotide sequences that can encode the above-mentioned pectinase protein using conventional techniques. For example, based on the coding gene of wild-type pectinase BacPelA (e.g., the coding gene with the nucleotide sequence shown in SEQ ID NO:9), the codon encoding the amino acid at the mutation site can be adjusted accordingly. Furthermore, the coding gene can be optimized based on the characteristics of the expression host to improve the expression level and / or expression efficiency of the protein mutant.

[0033] (SEQ ID NO:9)

[0034] According to a preferred embodiment of the present invention, the nucleotide sequence of the encoding gene is as shown in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16.

[0035] (SEQ ID NO:10)

[0036] (SEQ ID NO:11)

[0037] (SEQ ID NO:12)

[0038] (SEQ ID NO:13)

[0039] (SEQ ID NO:14)

[0040] (SEQ ID NO:15)

[0041] gccaatgtgaatttctcaatgcaagggtttgccactcttaatggagggactacagggggtgctggaggagatgttgtaaccgtttctaccggtgaccaattgatagccgcgctaaaaaataaaaaggcgaacacgcctttaaccatttatatagacggaacgatcacgcctgcaaacacatcggcgagcaagattgacattaaagatgtcaatgatgtgtctttgttaggagtaggaacaaatggggagcttaacggaattggcattaaagtatggcgggccaataacgtgatcatccgcaatttgaaaatccatcatgtgaatacaggcgataaagatgcaatcagcattgaaggcccgtccaaaaacatttgggtggaccataacgaactctataatagccttgatgtgcataaggactattatgatggattgtttgatgtgaaacgggatgctgattacattactttttcatggaactatgtacacgacagctgggcgtcgatgctgatgggttcgtctgacagcgattcatacggacgaaaaatcacgttccacaataattactttgaaaacttgaattctcgtgtgccttctgttgcttttggcgaagcccatattttcagcaattactacgcagatattagagagacagggatcaattctcggatgggggctcaagtacgaatcgaggaaaactattttgaaaggtgcaacaatccaattgtaagccgagacagcaaggaaatcgggtattggcatctggtcaataatcgctatgtttcttcgactggcgagcagccaactgtctcgacaacgacttataatccaccatacagctatcaagcaacgcctgtaaaccaagtgaaggatgtagtgcgggcgaacgccggagtcggggttatttctccttaa(SEQ ID NO:16)

[0042] In a third aspect, the present invention provides a recombinant vector, wherein the recombinant vector comprises the coding gene according to the second aspect described above.

[0043] Any starting vector that can be used in the art to construct recombinant vectors for expressing exogenous genes using engineered bacteria or other host cells to obtain target proteins can be used for constructing the recombinant vectors provided in this invention. For example, commonly used plasmid vectors in the art, such as pET28a and pMA5, can be selected.

[0044] A fourth aspect of the present invention provides a recombinant cell containing the coding gene as described in the second aspect above, or the recombinant cell containing the recombinant vector as described in the third aspect above.

[0045] Any engineered bacteria or other host cells capable of expressing exogenous genes in the art can be used for the recombinant cell construction provided by this invention, as long as they can express the protein mutants described in the first aspect of this invention. According to a preferred embodiment of this invention, the host cells are selected from host cells of prokaryotic expression systems, such as *Escherichia coli* and *Bacillus subtilis*.

[0046] When using different host cells, the methods / conditions for expressing the protein mutant with pectinase activity provided in this invention using the obtained recombinant cells will also differ.

[0047] For example, when using *E. coli* as the host cell, the recombinant cells can be cultured at 35-38°C. When the OD value reaches 0.5-0.8, an inducer is added and cultured for another 4-6 hours. The inducer is typically isopropyl thiogalactoside (IPTG). The final concentration of the inducer only needs to be sufficient to induce the recombinant cells to express the target protein (i.e., the protein mutant with pectinase activity provided by this invention), for example, 0.8-1 mM.

[0048] The fifth aspect of the present invention provides a composition for degrading pectin, the composition comprising the protein mutant as described in the first aspect above.

[0049] The composition provided by the present invention may contain only the protein mutant as the (sole) active component, or it may contain other substances with pectin hydrolysis ability as active components in addition to the protein mutant.

[0050] Considering that the protein mutant provided by the present invention has better heat resistance, alkali resistance and thermal-alkali stability than existing pectinases in the art, according to a preferred embodiment of the present invention, the composition contains only the protein mutant as described in the first aspect above as an active component.

[0051] In addition to the active ingredient, the composition may also contain auxiliary components, such as excipients, preservatives, and protective agents. Any of the above-mentioned auxiliary components commonly used in the art can be used in this invention, as long as they do not have a significant adverse effect on the pectinase activity of the protein mutant provided by this invention (for example, after adding the auxiliary component, the pectinase activity when using the composition for pectin degradation is not less than 80% of the pectinase activity when using the protein mutant provided by this invention alone, preferably not less than 90%).

[0052] The sixth aspect of the present invention provides a method for hydrolyzing pectin, the method comprising contacting a protein mutant as described in the first aspect, a recombinant cell as described in the fourth aspect, or a composition as described in the fifth aspect with a pectin-containing substrate, and hydrolyzing the pectin under hydrolysis conditions.

[0053] Because the pectinase-active protein provided by this invention has good thermal and alkaline stability, it can undergo pectin hydrolysis under high temperature and alkalinity conditions. Based on this, according to a preferred embodiment of the invention, the hydrolysis conditions include: a temperature not exceeding 70°C and a pH not exceeding 10.

[0054] Preferably, the hydrolysis conditions include: temperature 60-70℃ and pH 8.5-10.

[0055] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to further explain and illustrate the content of the present invention by way of example, and are not intended to limit the present invention.

[0056] Unless otherwise specified, the experimental methods used in the following examples were performed under standard conditions, such as those described in *Molecular Cloning: A Laboratory Manual* or those recommended by the respective reagent manufacturers (e.g., refer to the methods and conditions in the kit instructions). All reagents and materials used were purchased from reputable biological or chemical reagent / material suppliers unless otherwise stated, and all reagents were of analytical purity.

[0057] Example 1

[0058] This example illustrates the construction of a site-directed saturation mutant library of pectinase BacPelA expressed by Bacillus clausii S10.

[0059] The inventors discovered that mutations at three sites—R150, R216, and A238—in the pectinase BacPelA protein are most likely to increase its thermo-alkali stability. Based on this, a site-directed saturation mutant library was constructed using the primers listed in Table 1 targeting these three sites, employing the following method.

[0060] First, the pectinase BacPelA gene pelA was amplified from the genome of Bacillus clausii S10 using PCR, and the recombinant plasmid pET28a-pelA was constructed using pET28a plasmid.

[0061] Then, using the recombinant plasmid pET28a-pelA as a template, the MEGAWHOP (Megaprimer PCR for wholeplasmid) whole plasmid PCR amplification method was adopted. Nucleotide mutations were introduced into three different sites of pelA in vitro using degenerate primers and the amplification system and conditions in Table 2.

[0062] Table 1 Primers used to construct site-directed saturation mutant libraries.

[0063]

[0064] Table 2 PCR System and Conditions

[0065]

[0066] The obtained product was digested with DpnI overnight and then electroporated into E. coli BL21(DE3) electrocompetent cells (Shanghai Weidi Biotechnology Co., Ltd.). It was then plated on LB agar plates (containing 50 ng / μL kanamycin). After overnight culture, single clones were picked up with sterile toothpicks and placed into 96-well plates containing 160 μL LB liquid medium (containing 50 ng / μL kanamycin). After overnight culture, 50 μL of 60% glycerol was added, mixed, and stored at -80℃ to construct a site-directed saturation mutant library. Each site of the mutant library contained approximately 500 clones.

[0067] Example 2

[0068] This example illustrates the screening of protein mutants with improved thermal alkali stability.

[0069] The mutant and wild-type strains were replicated using a sterile 96-well plate needle replicator in fresh LB liquid medium (150 μL per well, containing 50 ng / μL kanamycin and 0.1 mM IPTG) and induced overnight. The resulting bacterial cultures were used for further screening as follows.

[0070] 1. Initial screening in 96-well plates: Add 10 μL of RIPA lysis buffer to the induced bacterial culture and incubate at 37°C for 30 min. Dilute the lysed bacterial culture 10-fold in glycine-NaOH buffer (pH 10), incubate at 80°C for 15 min, and then cool on ice. Mix 10 μL of the diluted bacterial culture with 70 μL of glycine-NaOH buffer (pH 10.5, 0.1 mM CaCl2) containing 0.2% polygalacturonic acid (PGA) (w / v), incubate at 70°C for 10 min, cool on ice, add 80 μL of DNS (3,5-dinitrosalicylic acid) solution, and incubate at 98°C for 8 min. Measure the OD. 540 .

[0071] After incubation at the selected temperature and alkaline conditions for 15 minutes, the residual enzyme activity of the wild type was basically 0, resulting in a lighter color. If the mutant was darker in color after incubation at the selected temperature and alkaline conditions for 15 minutes, it indicated that the residual enzyme activity of the mutant was significantly higher than that of the wild type. It was preliminarily identified as a mutant protein with improved thermal and alkaline stability and proceeded to the next step of rescreening.

[0072] 2. Mutant rescreening: In order to further determine the thermo-alkali stability of the mutants, the mutant proteins with improved thermo-alkali stability obtained from the initial screening and wild-type BacPelA were purified and their thermo-alkali stability, thermo-alkali stability and the improvement of thermo-alkali stability were tested.

[0073] The enzyme protein purification process is as follows:

[0074] E. coli expressing the mutant protein and wild-type BacPelA were activated overnight, and the resulting culture was transferred to 100 mL of fresh LB liquid medium (containing 50 μg / mL kanamycin) at a 1% (v / v) inoculation rate and incubated at 37°C until OD500. 600 When the concentration reaches approximately 0.6, add IPTG to a final concentration of 1 mM and continue induction culture for 5 hours. Centrifuge the culture medium at 6000g for 10 min to collect the bacteria, then resuspend it in 10 mL of binding buffer (20 mM Tris-HCl, 500 mM NaCl, 10 mM imidazole, pH 7.9). Sonicate the cells (16V, 20 min), centrifuge at 15000g for 10 min at 4℃, and collect the supernatant, which is the crude enzyme solution.

[0075] Add the crude enzyme solution to His Bind Column (Novagen) equilibrated with binding buffer, wash with 10 bed volumes of binding buffer to remove unbound contaminants; wash with 5 bed volumes of rinsing buffer (20 mM Tris-Cl, 0.5 M NaCl, 60 mM imidazole, pH 7.9) to remove weakly bound contaminants; elute with 5 bed volumes of elution buffer (20 mM Tris-Cl, 0.5 M NaCl, 500 mM imidazole, pH 7.9), and collect approximately 5 mL of the target protein eluent.

[0076] The target protein eluent was desalted on an AKTA FPLC system to obtain a purified enzyme protein solution. The desalting buffer was 20 mM Tris-HCl, pH 8.0.

[0077] T 50 15 The detection process for the value (i.e., the temperature at which 50% of the enzyme activity remains after 15 minutes of heat treatment) is as follows:

[0078] The purified enzyme protein solution was diluted to 1 μg / mL and incubated at different temperatures for 15 min under neutral conditions (pH 7.5) and alkaline conditions (pH 10.0). The residual enzyme activity was then measured. The enzyme activity measured after incubation at room temperature (25±3℃) for 15 min was taken as the initial enzyme activity. The incubation temperature corresponding to a residual enzyme activity of half the initial enzyme activity was defined as the Tenzyme at that pH value. 50 15 Value. Among them, the T value of pectinase protein under neutral conditions at pH 7.5 is taken as an example. 50 15 The value indicates its thermal stability, expressed as T0 under alkaline conditions at pH 10. 50 15 The value indicates its thermal alkali stability.

[0079] Enzyme activity testing methods:

[0080] 10 μL of the diluted purified enzyme protein solution was mixed with 190 μL of glycine-NaOH buffer (pH 10.5, 0.1 mM CaCl2) containing 0.2% (w / v) polygalacturonic acid (PGA), and the mixture was incubated at 70 °C for 10 min. The specific activity and residual enzyme activity of the purified enzyme protein were determined using A... 235 The method involves terminating the reaction by adding 300 μL of 0.03 M phosphoric acid, and then measuring its OD. 235 Value. One unit of enzyme activity is defined as the amount of enzyme required to produce 1 μmol of unsaturated galacturonic acid per minute under the above conditions. The formula for calculating enzyme activity (U / mL) is as follows:

[0081]

[0082] In the formula, ΔOD 235 = Absorbance value of experimental group at 235nm - Absorbance value of control group at 235nm;

[0083] 4600 is the molar absorptivity of unsaturated polygalacturonic acid at 235 nm, expressed in L·mol⁻¹. -1 ·cm -1 ;

[0084] t represents the enzyme-catalyzed reaction time, in minutes.

[0085] b represents the thickness of the cuvette, in cm;

[0086] V0 is the total volume of the system, in mL;

[0087] V1 represents the volume of the enzyme solution, in mL.

[0088] The enzyme activity (U / mL) calculated using the above formula is converted to the specific activity (U / mg) of the enzyme protein according to the enzyme concentration (mg / mL) in the diluted purified enzyme protein solution.

[0089] After screening, mutants of pectinase protein 1A, 2B, 3C, 4AB, 5AC, 6BC, and FCM were found to have significantly improved thermal alkali stability and activity. Sequencing (contracted to Beijing Qingke Biotechnology Co., Ltd.) confirmed their amino acid mutations. The results are shown in Table 3.

[0090] Table 3. Characteristics of pectinase protein mutants obtained through screening.

[0091]

[0092] Note: R150G, R216H, and A238C represent mutations based on the amino acid sequence of wild-type pectinase protein (WT), with arginine at position 150 replaced by glycine, arginine at position 216 replaced by histidine, and alanine at position 238 replaced by cysteine; R150G / R216H, R150G / R216H, and R216H / A238C represent the presence of two of the above three mutations; R150G / R216H / A238C represents the presence of all three mutations.

[0093] As shown in Table 3, mutant proteins 1A, 2B, 3C, 5AC, 6BC, and FCM exhibited improved enzyme activity, thermostability, and thermo-alkali stability compared to wild-type pectinase BacPelA (WT), while 4AB showed a slight decrease in activity but significantly improved thermostability and thermo-alkali stability. Specifically, the activities of mutant proteins 1A, 2B, 3C, 5AC, 6BC, and FCM were approximately 6.7%, 17.1%, 35.6%, 31.5%, 31.4%, and 25.1% higher than WT, respectively. Under alkaline conditions (pH 10), the thermostability of mutant proteins 1A, 2B, 3C, 4AB, 5AC, 6BC, and FCM was significantly higher than that of WT. 50 15 The values ​​increased by 6.5℃, 6℃, 3℃, 11.0℃, 4.5℃, 5.5℃ and 8.5℃ respectively.

[0094] To further determine the improved thermo-alkali stability of the mutant proteins, wild-type pectinase BacPelA and mutant proteins 1A, 2B, 3C, 4AB, 5AC, 6BC, and FCM were incubated at 60℃ and pH 10 for 4 hours (the ramie degumming reaction conditions for pectinase BacPelA). The residual enzyme activity was then measured, and the results are as follows: Figure 1 As shown in the figure, the residual enzyme activity of wild-type pectinase BacPelA (WT) after incubation under the same hot-alkali conditions for 4 hours was only 3.4%, while the residual enzyme activities of mutant proteins 1A, 2B, 3C, 4AB, 5AC, 6BC, and FCM were 24.6%, 17.6%, 33.4%, 50.9%, 75.6%, 63.1%, and 86.2%, respectively, which are approximately 7.2 times, 5.2 times, 9.8 times, 15.0 times, 22.2 times, 18.6 times, and 25.4 times that of WT. Clearly, these mutant proteins have better hot-alkali stability and are more suitable for industrial applications.

[0095] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A protein mutant having pectolytic activity, characterized in that, This protein mutant is modified by at least one of the following mutations: R150G, R216H, and A238C, compared to the protein with the amino acid sequence shown in SEQ ID NO:

1.

2. The encoding gene of the protein mutant according to claim 1.

3. A recombinant vector, characterized in that, The recombinant vector contains the encoding gene as described in claim 2.

4. A recombinant cell, characterized in that, The recombinant cell contains the encoding gene as described in claim 2, or the recombinant cell contains the recombinant vector as described in claim 3.

5. A composition for degrading pectin, characterized in that, The composition contains the protein mutant of claim 1.

6. The composition according to claim 5, wherein, The composition contains only the protein mutant of claim 1 as the active component.

7. A method for hydrolyzing pectin, characterized in that, The method includes contacting the protein mutant of claim 1, the recombinant cell of claim 4, or the composition of claim 5 or 6 with a pectin-containing substrate and hydrolyzing the pectin under hydrolytic conditions.

8. The method according to claim 7, wherein, The hydrolysis conditions include: temperature not exceeding 70°C and pH not exceeding 10.

9. The method according to claim 8, wherein, The hydrolysis conditions include: temperature 60-70℃, pH 8.5-10.

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