Pectinase mutant, method for producing same, and use thereof

Abstract

The application discloses a pectinase mutant and a production method and application thereof, and belongs to the technical field of bioengineering.The amino acid sequence of the pectinase mutant is that negative-charged aspartic acid at positions 162, 164 and 203 of a wild-type pectinase amino acid sequence is replaced by neutral asparagine; the amino acid sequence is shown as SEQ ID NO:2; and the pectinase mutant can be obtained by constructing a recombinant vector containing a coding gene of the pectinase mutant and expressing the pectinase mutant by a recombinant cell.The pectinase mutant provided by the application can reach more than 80% of the enzyme activity under a conventional calcium ion (1-2 mM) concentration (determined at 50 DEG C) without adding calcium ions, which is significantly higher than that of a wild-type pectinase which is almost completely inactivated under the same treatment condition.The pectinase mutant provided by the application can be widely applied to the elimination of anti-nutritional factors in feed processing and cotton and hemp degumming and refining in the textile industry, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to an enzyme engineering technology, specifically to a pectinase mutant and its production method and application. Background Technology

[0002] Pectinase is a general term for a class of enzymes that break down pectin; it is a complex enzyme with multiple components. Based on their substrate and mode of action, they are divided into three main categories: pectin lyases, pectin esterases, and pectin hydrolases. Their optimal pH is generally in the alkaline range, and their catalytic activity depends on calcium. 2+ It is currently widely used in the papermaking, food processing, and textile industries.

[0003] Pectinase is a novel feed additive that reduces feed stickiness in animal intestines, eliminates anti-nutritional factors, improves nutrient utilization, and promotes nutritional balance in animals. Examples include publicly available technical solutions such as CN119769617A, CN119234920A, and CN119073464B. Pectinase can also be used as a refining aid for cotton and linen, degrading the pectin components of cotton and linen fibers (fabrics) and improving their softness, moisture absorption, and other wearability properties (see CN118957769A and CN118704229A). However, in pectinase applications, calcium ions are usually required to activate enzyme activity, leading to unfavorable factors in the application system. For example, in feed processing, adding too much calcium and magnesium ions can result in excessively high ash content, affecting feed quality; in cotton and linen processing, adding too much calcium ions can cause calcium ions to precipitate with polyvalent anions in the additives, reducing the overall effectiveness of the additives. Therefore, developing pectinases that do not rely on calcium ions is essential.

[0004] In summary, how to obtain a pectinase that does not depend on calcium ions, thereby eliminating the need for additional exogenous calcium ions and reducing costs while increasing efficiency, is a technical problem that urgently needs to be solved. Summary of the Invention

[0005] The first technical problem to be solved by the present invention is to provide a pectinase mutant that can have high enzyme activity without relying on a calcium ion environment.

[0006] The second technical problem to be solved by the present invention is to provide a DNA molecule.

[0007] The third technical problem to be solved by the present invention is to provide a recombinant carrier.

[0008] The fourth technical problem to be solved by the present invention is to provide a recombinant cell.

[0009] The fifth technical problem to be solved by the present invention is to provide a method for producing a pectinase mutant.

[0010] The sixth technical problem to be solved by the present invention is to provide an application of a pectinase mutant in feed additives or cotton and linen refining auxiliaries.

[0011] The seventh technical problem to be solved by the present invention is to provide a feed additive containing a pectinase mutant, which can reduce the ash content of the finished feed and improve the utilization rate of nutrients in the feed.

[0012] The eighth technical problem to be solved by the present invention is to provide a cotton and linen refining auxiliary agent containing a pectinase mutant, which can simplify the process, reduce costs, and reduce inorganic pollutants.

[0013] The technical solution adopted by the present invention to solve its first technical problem is a pectinase mutant, wherein the pectinase is a wild-type pectinase, and the amino acid sequence of the pectinase mutant is such that the negatively charged aspartic acid at positions 162, 164 and 203 of the amino acid sequence of the wild-type pectinase is replaced with neutral asparagine.

[0014] Furthermore, its amino acid sequence is shown in SEQ ID NO:2.

[0015] Furthermore, the amino acid sequence of the wild-type pectinase is shown in SEQ ID NO.1, which is derived from the gene bank (GenBank accession number: JX964998). This invention involves sequence design and combined mutation screening of the wild-type pectinase, ultimately obtaining a pectinase mutant with the best overall performance. The two amino acid sequences described above contain three different sites (D162N, D164N, D203N), meaning that the mutant pectinase amino acid sequence replaces the negatively charged aspartic acid at positions 162, 164, and 203 of the wild-type pectinase amino acid sequence with neutral asparagine.

[0016] The technical solution adopted by the present invention to solve its second technical problem is a DNA molecule used to encode the pectinase mutant.

[0017] Furthermore, the DNA molecule is the coding gene of the pectinase mutant, or a DNA sequence containing the coding gene, which can express the amino acid sequence shown in SEQ ID NO.2.

[0018] Since the calcium-independent pectinase mutant provided by this invention is actually obtained by expressing a recombinant vector containing the above-mentioned DNA molecule through engineered bacteria, the DNA molecule can be adjusted according to the preferences of the engineered bacteria.

[0019] As an example, in one specific embodiment of the present invention, the DNA molecule is expressed by Escherichia coli, and the nucleotide sequence of the DNA molecule is shown in SEQ ID NO:4.

[0020] The technical solution adopted by the present invention to solve its third technical problem is a recombinant vector containing the DNA molecule, wherein the recombinant vector is a prokaryotic or eukaryotic expression vector.

[0021] The technical solution adopted by the present invention to solve its fourth technical problem is a recombinant cell containing the recombinant vector, wherein the recombinant cell includes, but is not limited to, bacteria or fungi.

[0022] The recombinant cells, namely chassis cells, are typically, but not limited to, selected from at least one of Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Candida albicans, Rhodotorula rubrum, Bacillus, Escherichia coli, Salmonella, Clostridium, Streptomyces, Staphylococcus aureus, Neisseria, and Shigella.

[0023] The technical solution adopted by the present invention to solve its fifth technical problem is a method for producing the pectinase mutant, which involves constructing a recombinant vector, introducing the recombinant vector into cells to construct recombinant cells, and obtaining the mutant through expression in the recombinant cells.

[0024] The technical solution adopted by the present invention to solve its sixth technical problem is the application of the pectinase mutant or the DNA molecule in the preparation of pectinase-containing feed additives or cotton and linen refining auxiliaries.

[0025] The technical solution adopted by this invention to solve its seventh technical problem is a feed additive, including the aforementioned pectinase mutant. The feed additive can degrade anti-nutritional factors such as pectin in feed, reduce the ash content of the finished feed by eliminating the need for adding exogenous calcium, magnesium, and other mineral ions, and improve the utilization rate of nutrients in the feed.

[0026] The technical solution adopted by this invention to solve its eighth technical problem is a cotton and linen refining auxiliary agent, comprising the aforementioned pectinase mutant. This cotton and linen refining auxiliary agent can degrade the pectin components of cotton and linen fibers (fabrics), simplifying the process, reducing costs, and decreasing inorganic pollutants by eliminating the need for additional exogenous calcium ions.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] The pectinase mutant provided by this invention exhibits enzyme activity exceeding 80% of that at conventional calcium ion (1-2 mM) concentrations (measured at 50°C) without the addition of calcium ions, significantly higher than that of wild-type pectinase, which is almost completely inactivated under the same treatment conditions. Therefore, the pectinase mutant provided by this invention is suitable for eliminating anti-nutritional factors in feed processing and for degumming and refining cotton and linen in the textile industry, demonstrating promising industrial application prospects. Attached Figure Description

[0029] Figure 1 Gel electrophoresis images of the mutant pectinase and wild-type pectinase provided in this invention. Detailed Implementation

[0030] Many specific details of the invention are set forth in the following description to enable those skilled in the art to fully understand the invention. However, the invention can be practiced in many other ways different from those described herein, and those skilled in the art can make various modifications without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0032] The term "recombinant gene" refers to DNA capable of expressing the pectinase of the present invention. Typically, the recombinant gene is initially synthesized in vitro via solid-phase phosphoramidite synthesis, TdT biosynthesis, or other suitable techniques known in the art. Once a template sequence is available, it can be amplified by PCR or other suitable techniques known in the art. With a recombinant bacterial strain, further large-scale amplification can be achieved by culturing the strain. In some embodiments, the recombinant gene may also include residual restriction enzyme sites, other accessory elements such as control elements (e.g., promoters), labeling substances (e.g., fluorescent labels), and other sequences that do not affect the expression of the target gene.

[0033] The term "expression" refers to the process by which DNA is transcribed into messenger RNA (mRNA) and then translated into protein.

[0034] The term "expression vector" refers to the ability to incorporate and express heterologous polynucleotide fragments into host cells. Many prokaryotic and eukaryotic expression vectors are commercially available. Choosing a suitable expression vector is within the knowledge of a technician.

[0035] The term "chassis cell" refers to a suitable host vector for expressing DNA containing the DNA of the present invention. The host can be any organism capable of containing and expressing the nucleic acids or genes disclosed herein, but is not limited thereto. Chassis cells can be prokaryotes or eukaryotes, single-celled or multicellular, including mammalian cells, plant cells, fungi, etc. According to existing technology, those skilled in the art can achieve heterologous expression of the recombinant DNA of the present invention in different disclosed chassis cells by adjusting parameters through a limited number of experiments. Chassis cells can be selected from at least one of *Escherichia coli*, *Pichia pastoris*, *Saccharomyces cerevisiae*, *Hansenula polymorpha*, *Candida*, *Rhodotorula*, *Bacillus*, *Escherichia coli*, *Salmonella*, *Clostridium*, *Streptomyces*, *Staphylococcus*, *Neisseria*, and *Shigella*. This invention merely lists types of chassis cells and does not constitute a limitation on the types of chassis cells. Chassis cells are preferably *Escherichia coli*, and suitable *E. coli* strains (including many others) include BL21(DE3), C600, DH5αF′, 1113101, JM83, JM101, JM103, JM105, JM107, JM109, JM110, MC1061, MC4100, MM294, NM522, NM554, TGI, χ1776, XL1-Blue, and Y1089. + The above E. coli strains are all commercially available strains.

[0036] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The reagents used in this embodiment are all commercially available products.

[0037] Example 1

[0038] Sequence design and high-throughput screening were performed on wild-type pectinase (its amino acid sequence is shown in SEQ ID NO.1), ultimately yielding a calcium-independent pectinase mutant (its amino acid sequence is shown in SEQ ID NO.2). The two amino acid sequences contain three distinct sites (D162 N, D164 N, D203 N), meaning the amino acid sequence of the pectinase mutant is obtained by replacing the negatively charged aspartic acid at positions 162, 164, and 203 of the wild-type pectinase amino acid sequence with neutral asparagine.

[0039] 1. Preparation of pectinase mutants

[0040] 1.1 Construction of recombinant cells

[0041] The coding genes of wild-type pectinase and pectinase mutants were used as target genes. The nucleotide sequences of the target genes were synthesized by Beijing Qingke Biotechnology Co., Ltd., and these nucleotide sequences were inserted into expression vectors respectively.

[0042] The coding gene sequence for wild-type pectinase is shown in SEQ ID NO.3, and the coding gene sequence for calcium-independent pectinase mutant is shown in SEQ ID NO.4.

[0043] Specifically, the plasmid is inserted into plasmid pET28a(+) to obtain the corresponding plasmid. The synthesized plasmid is then transformed into chassis cells (E. coli BL21(DE3)), thereby constructing E. coli strains containing different plasmids. Many other plasmids and chassis cells are available in the prior art; this embodiment only provides one specific approach.

[0044] 1.2 Expression and purification of pectinase mutants

[0045] The recombinant bacteria were inoculated into liquid LB medium (5g yeast extract, 10g peptone, 10g NaCl) and cultured at 37℃ in a shaker (220 r / min) until OD. 600 The pH was set to approximately 1.0, then IPTG was added to a concentration of 200 μM, and the temperature was lowered to 16°C for overnight incubation. The overnight culture was collected by centrifugation at 4000g for 10 min, and the cells were resuspended in 10% NTA buffer (50 mM Tri-HCl, 300 mM NaCl, pH 8.0). Next, the cells were sonicated and centrifuged at 12000 rpm for 1 h. The supernatant was collected as the crude enzyme solution. Many other methods for inducing recombinant bacterial expression are available in the prior art; this embodiment only provides one specific method.

[0046] 1.3 Pass the crude enzyme solution through Ni 2+ Resin affinity chromatography was performed because the target protein has a histidine tag at its C-terminus, which can bind to the resin. The resin was washed with NTA buffer and imidazole buffer, followed by elution with NTA buffer. The resulting solution was dialyzed to remove imidazole, with a dialysis bag cutoff volume of 1 kDa. The dialyzed solution was concentrated until the protein concentration was 0.2 mg / mL.

[0047] According to existing technology, those skilled in the art can purify pectinase by adjusting parameters through a limited number of experiments, which will not be described in detail here. There are many other available methods for purifying pectinase in the prior art; this embodiment only provides one specific solution.

[0048] Wild-type pectinase (Pel) and a calcium-independent pectinase mutant (ΔPel) were heterologously expressed and purified according to the above-described pectinase preparation method. The calcium-independent pectinase mutant was validated by SDS-PAGE, and the results are shown below. Figure 1As shown, the size (37.8 kD) of the pectinase mutant and wild-type pectinase is consistent with expectations.

[0049] 2. Calcium ion dependence test

[0050] Different concentrations of CaCl2 solution were added to the crude enzyme solution in the same reaction system. 2+ The final concentration was 0-3.5 mmol / L. Under the same conditions, the enzyme activity was determined using the standard method.

[0051] 3. Methods for determining enzyme activity

[0052] 3.1 Assay of pectinase activity

[0053] The test was performed according to the method specified in QBT 4482-2013 Alkaline Pectinase Preparations.

[0054] 3.2 Solution Preparation

[0055] (1) pH 9.0 glycine-sodium hydroxide solution

[0056] Solution A: 0.8 mol / L glycine solution

[0057] Solution B: 0.8 mol / L sodium hydroxide solution

[0058] Mix solutions A and B to prepare a buffer solution with a pH of 9.0. Calibrate the solution with a pH meter to ensure that the pH is within the range of 9.0 ± 0.01. Store at room temperature. The solution is effective for one week.

[0059] (2) 0.03 mol / L phosphoric acid solution

[0060] Dilute 85wt% phosphoric acid to 0.03mol / L before use. It is effective within one week when left at room temperature.

[0061] (3) Sodium polygalacturonic acid (sodium pectate) solution

[0062] 0.2 g of sodium polygalacturonate (Sigma, No. P350655) was dissolved in glycine-sodium hydroxide solution (A.2.1) and stirred at room temperature for 30 min to ensure complete dissolution before bringing the volume to 100 mL.

[0063] The substrate solution should be prepared before use. If the amount needed is small, it can be prepared in a 50 mL volumetric flask.

[0064] 3.3 Test Procedure

[0065] (1) Sample preparation

[0066] Liquid pectinase preparation: Dilute the enzyme solution to be tested directly with glycine-sodium hydroxide solution to an appropriate ratio. The final sample OD value is between 0.2 and 0.6.

[0067] Preparation of sample blank: Take 1 mL of diluted enzyme solution, inactivate it in a boiling water bath for 20 min, and cool it to room temperature for later use.

[0068] (2) Measurement

[0069] Take a 20mL test tube with a frosted stopper and proceed with the reaction in the following order. Each sample should be prepared in triplicate. During the reaction, the time interval between adding reagents to each test tube should be consistent, starting from the addition of the substrate. React at 50℃ for 15 minutes.

[0070] The reaction steps and reagent dosages are shown in Table 1.

[0071] Table 1 Reaction steps and reagent usage

[0072]

[0073]

[0074] (3) Calculation of results

[0075] Under the conditions of a substrate sodium polygalacturonate (Sigma, No. P350655) concentration of 2 mg / mL, a temperature of 50 °C, and a pH of 9.0, the amount of enzyme required per minute to cleave sodium polygalacturonate to produce the equivalent of 1 μmol of unsaturated sodium oligogalacturonate is defined as one alkaline pectinase activity unit (u). The absorbance values ​​of the sample blank (A0) and the sample solution (A) were measured at a wavelength of 235 nm using a spectrophotometer.

[0076] The alkaline pectinase activity in the sample was calculated according to formula (A.1).

[0077]

[0078] In the formula:

[0079] X — Alkaline pectinase activity, u / mL;

[0080] A – Absorbance value of the enzyme reaction solution;

[0081] A0—Absorbance value of enzyme blank sample;

[0082] N – Dilution factor;

[0083] V1 — The total volume of the reaction system, in milliliters (mL);

[0084] V2 — The volume of diluted enzyme solution added, in milliliters (mL);

[0085] 4600 — Molar absorptivity of sodium unsaturated polygalacturonate at 235 nm, in L-mol -1 cm -1 ;

[0086] t — Enzyme-catalyzed reaction time (within the linear range of the enzyme reaction), in minutes (min);

[0087] b – Thickness of the cuvette, in centimeters (cm).

[0088] Considering that crude enzyme solution may be used in industrial production, its activity was also determined using the same method as above.

[0089] The enzyme activity unit U is defined as: at 50℃ and pH 9.0, the amount of unsaturated sodium polygalacturonate produced in 1 minute from a 0.2% sodium polygalacturonate solution is equivalent to one μmol of unsaturated sodium polygalacturonate. Specific enzyme activity U / mL: the number of enzyme activity units contained in each mL of enzyme solution.

[0090] The enzyme activity of wild-type pectinase (Pel, SEQ ID NO:1) and pectinase mutant (△Pel, SEQ ID NO:2) was determined according to the above-described enzyme activity assay method. The specific data are shown in Tables 2 and 3.

[0091] Table 2. Enzyme activity of crude enzyme solution at different calcium ion concentrations

[0092] Calcium ion concentration (mmol / L) Pel (U / mL) △Pel (U / mL) 0 0.23 4137.1 0.5 1602.1 4544.7 1.0 2247.4 4783.5 1.5 3190.9 5029.1 2.0 2612.7 4691.1 2.5 2042.5 4283.2

[0093] As shown in Table 2, for the crude enzyme solution, under the condition of 0 mmol / L calcium ions, the enzyme activities of Pel and ΔPel were 0.23 U / ml and 4137.1 U / ml, respectively; without the addition of calcium ions, Pel was almost completely inactivated, while the activity percentage of ΔPel was still 82.3%. Therefore, the pectinase mutant ΔPel of the present invention has significantly greater activity than the wild-type pectinase Pel in both the presence and absence of calcium ions, and belongs to a calcium-independent pectinase mutant.

[0094] Table 3. Enzyme activity of pure enzyme solution at different calcium ion concentrations

[0095] Calcium ion concentration (mmol / L) Pel (U / mg) △Pel(U / mg) 0 0.89 8409.4 0.5 3612.3 8764.8 1.0 5547.2 9681.6 1.5 9891.9 10020.2 2.0 7689.4 9031.7 2.5 5236.7 8553.8

[0096] As shown in Table 1, for pure enzymes, Pel is almost completely inactivated under the condition of no calcium ions, and the activity percentage of ΔPel is still 83.9%.

[0097] The above results indicate that, for both crude enzyme solution and pure enzyme, ΔPel exhibits a significantly higher percentage of activity than Pel under calcium ion-free conditions.

[0098] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A pectinase mutant, characterized in that, The amino acid sequence of the pectinase mutant is formed by replacing the negatively charged aspartic acid at positions 162, 164, and 203 of the wild-type pectinase amino acid sequence with neutral asparagine; the amino acid sequence of the pectinase mutant is shown in SEQ ID NO:

2.

2. A DNA molecule, characterized in that, The DNA molecule is the encoding gene of the pectinase mutant as described in claim 1.

3. The DNA molecule as described in claim 2, characterized in that, The nucleotide sequence of the DNA molecule is shown in SEQ ID NO:

4.

4. The method for producing the pectinase mutant according to claim 1, characterized in that, It is obtained by constructing a recombinant vector containing the DNA molecule as described in claim 2 or 3, and expressing it in recombinant cells.

5. The method for producing the pectinase mutant according to claim 4, characterized in that, The recombinant vector is a prokaryotic expression vector; the recombinant cell is Escherichia coli.

6. The use of the pectinase mutant as described in claim 1, or the DNA molecule as described in claim 2 or 3, in the preparation of pectinase-containing feed additives.

7. The use of the pectinase mutant as described in claim 1, or the DNA molecule as described in claim 2 or 3, in the preparation of pectinase cotton and linen refining adjuvants.

8. A feed additive, characterized in that, Including the pectinase mutant as described in claim 1.

9. A cotton and linen refining auxiliary agent, characterized in that, Including the pectinase mutant as described in claim 1.

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