An acidic pectinase and its mutants

Abstract

The present invention belongs to the field of gene cloning and protein modification technology, and specifically relates to an acid pectinase and its mutant. Aspergillus sp.CM96 Random mutations were performed on the wild-type acid pectinase of CGMCC (Accession No. CGMCC No. 40340), and a mutant strain of acid pectinase with significantly improved specific activity was screened. The mutant was a pectinase mutant obtained by mutating the 10th amino acid of the wild-type acid pectinase amino acid sequence (SEQ ID NO: 1) from serine to alanine.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering and protein engineering, and particularly relates to an acid pectinase and a mutant thereof. Background Art

[0002] Pectin, primarily found in the primary cell wall and interlamellar tissue, is an anionic, acidic heteropolysaccharide composed of D-galacturonic acid residues linked by α-1,4-glycosidic bonds. Galacturonic acid exists as covalent domains, including homogalacturonic acid (HGA), xylosegalacturonic acid (XGA), rhamnogalacturonic acid I (RG-I), and rhamnogalacturonic acid II (RG-II). Homogalacturonic acid is the most abundant component of pectin, comprising up to 65% of the pectin structure. It consists of approximately 100 galacturonic acid residues linked by α-1,4-glycosidic bonds. The backbone of xylosylgalacturonic acid is homogalacturonic acid, with 25–75% of the galacturonic acid residues on HGA replaced by a single xylose residue at the C-3 position or a second xylose residue at the C-4 position. Rhamnogalacturonic acid I, comprising 20–35% of pectin, is a more complex polymer composed of repeating units of the α-D-gala-1,2-α-L-rhaa-1,4-disaccharide. RG-II is the most complex domain in pectin, consisting of a single GalA residue backbone linked by α-D-1,4-linkages consisting of 12 different sugars (arabinose, galactose, xylose, caramel, acetic acid, etc.). The diverse structure of pectin has led to a wide variety of pectinases that degrade it, resulting in a wide range of applications.

[0003] Pectinase is a complex enzyme. Generally speaking, it refers to a class of enzymes that degrade pectin. It is widely present in animals, plants and microorganisms and belongs to the polysaccharide family.

[0004] The basic mechanisms of action of pectinase include hydrolysis and trans-elimination. Hydrolysis requires water molecules. Polygalacturonase (PG) hydrolyzes the α-1,4 glycosidic bonds of pectin molecules. Based on their hydrolysis mechanisms, polygalacturonase can be divided into endo-PG and exo-PG. Exo-PG can be further divided into two types: fungal exo-PG1, whose end product is monomeric galacturonic acid; and bacterial exo-PG2, whose end product is dimeric galacturonic acid.

[0005] Pectinase was first used domestically in food processing in 1930. It wasn't until the 1960s that these enzymes were officially used for wine and juice clarification. In today's biotechnology era, pectinase has garnered global attention as an environmentally friendly biocatalyst, accounting for 25% of the global food and beverage enzyme market. Pectin also has numerous positive effects on human health, including lowering blood cholesterol and blood sugar levels, inhibiting cancer cell growth, and stimulating immune responses.

[0006] Although numerous pectinases have been obtained from nature and their properties have been studied, like many other industrial enzymes, pectinases face challenges in increasing yield, application efficiency, and specificity. The demand for pectinases with high specific activity, good thermal stability, and acid stability has driven the development of new enzyme resources, as well as research into enzyme catalytic mechanisms and protein engineering to discover new genes or pectinases with superior properties. Key areas of pectinase research include the use of genetic engineering techniques to construct genetically engineered bacteria that express high-activity pectinases, and the use of error-prone PCR to mutate pectinase genes, thereby altering pectinase properties such as heat resistance and specific activity, thereby improving their effectiveness. Summary of the Invention

[0007] In view of this, the object of the present invention is to provide an acid pectinase mutant to improve its specific activity, thereby helping to reduce the production cost of acid pectinase and promote its wide application in the food field.

[0008] The wild type acid pectinase of the present invention is derived from Aspergillus Aspergillus sp.CM96, The bacterium was deposited on October 21, 2022 at the General Microbiology Center of the China Culture Collection Administration (Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, 100101), and its deposit number is: CGMCC No.40340.

[0009] The amino acid sequence of the wild-type acid pectinase is shown in SEQ ID NO: 1, and the nucleotide sequence of the gene encoding the wild-type acid pectinase is shown in SEQ ID NO: 2.

[0010] The present invention provides an acid pectinase mutant, which is obtained by mutating the 10th amino acid in the amino acid sequence of the wild-type acid pectinase from serine to alanine, and the amino acid sequence is shown in SEQ ID NO: 3.

[0011] The present invention also provides a gene encoding an acid pectinase mutant, the nucleotide sequence of which is shown in SEQ ID NO: 4.

[0012] The present invention also provides a recombinant plasmid carrying the pectinase mutant encoding gene.

[0013] The present invention also provides a recombinant strain carrying the above recombinant plasmid, wherein the host cell of the recombinant strain is Escherichia coli, preferably Escherichia coli Rosetta-gami B (DE3) pLysS.

[0014] The present invention also provides a method for obtaining the acid pectinase mutant, specifically, the method comprises the following steps: Aspergillus sp.CM96 The wild-type acid pectinase gene (SEQ ID NO: 2) of the strain CGMCC (Accession No. 40340) was used as a template for random mutagenesis using error-prone PCR. The PCR products were recovered from agarose gel and ligated with the vector pCold I treated with Nde I and Xba I. The cells were then chemically transformed into E. coli Rosetta-gami B (DE3) pLysS competent cells, plated on LB plates (containing 1 mM Kanamycin, 1 mM Ampicillin, and 1 mM Chloramphenicol), and cultured overnight at 37°C to obtain transformants. Some of the transformant colonies were spotted on LB pectin plates (containing 1% pectin, 1 mM Kanamycin, 1 mM Ampicillin, 1 mM Chloramphenicol, and 0.05 mM IPTG). After incubation at 37°C for a period of time, 20 ml of the substrate plate was added. After 10 minutes of 1% CTAB incubation, observe the formation of a hydrolysis zone. Single colonies corresponding to the formation of a hydrolysis zone indicate pectinase activity. Transformants with hydrolysis zones are selected for expansion, induction of expression, ultrasonic disruption, and enzyme activity assay. Using the wild-type as a control, transformants with improved enzyme activity are screened.

[0015] The recombinant expression uses pColdI as an expression vector and E. coli Rosetta-gami B (DE3) pLysS as an expression host to construct a recombinant bacterium.

[0016] The primers used for error-prone PCR are:

[0017] FP:5 ’ -ggctgaagcttacgtagaattcTCCGCCGCTGCCGTCGGCGT-3 ’ ;

[0018] RP: 5 ’ -gtgatgatgattcgcggccgcCAGGTTGCCCTGACCGGCGT-3 ’ ;

[0019] The present invention provides a method for producing the acid pectinase mutant by using genetic engineering fermentation, which comprises activating a recombinant bacterium containing a gene encoding a mutant acid pectinase (for example, Rosetta-gamiB(DE3)pLysS containing a gene encoding a mutant acid pectinase) and then inoculating it into 100 ml of LB fermentation medium containing 1 mM kanamycin, culturing it at 37°C and 180 rpm, and growing the bacteria to a certain stage (OD 600 =0.6-0.8), IPTG was added to a final concentration of 0.05 mM for induction, and the temperature was adjusted to 16°C for 14 hours of fermentation. The resulting bacterial liquid was centrifuged and the cells were ultrasonically disrupted. The supernatant was the crude acid pectinase enzyme solution, which contained the mutant. The crude enzyme solution was purified using a Ni-NTA affinity chromatography column to obtain the purified acid pectinase mutant.

[0020] The acid pectinase and the acid pectinase mutant provided by the present invention are applied in the field of food industry production.

[0021] The acid pectinase mutant provided by the present invention has significantly increased specific enzyme activity compared to the wild-type acid pectinase, thereby helping to reduce the production cost of pectinase. The specific activity of the mutant W-8 is 3781.56 U / mg, a 15% increase over the wild-type, achieving excellent technical results. The pectin lyase mutant can be widely used in the food industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is an error-prone PCR electrophoresis diagram;

[0023] Figure 2 The SDS-PAGE electrophoresis diagram of wild-type and mutant pectinase;

[0024] Figure 3 is the relative enzyme activity of wild type and mutant pectinase;

[0025] Figure 4 The SDS-PAGE electrophoresis diagram of the purified wild-type and mutant pectinase;

[0026] Figure 5 The effect of pH on enzyme activity;

[0027] Figure 6 The effect of temperature on enzyme activity;

[0028] Figure 7 is the temperature stability curve;

[0029] Figure 8 The protein standard curve. Implementation Method

[0030] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0031] The wild type acid pectinase of the present invention is derived from Aspergillus Aspergillus sp.CM96, The bacterium was deposited on October 21, 2022 at the General Microbiology Center of the China Culture Collection Administration (Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, 100101), and its deposit number is: CGMCC No.40340.

[0032] The present invention discloses an acid pectinase mutant with improved enzyme activity, belonging to the field of genetic engineering and enzyme engineering. Aspergillus sp.CM96 The acid pectinase encoding gene pelA of the plant (CGMCC No. 40340) was randomly mutagenized by error-prone PCR, and the single mutant W-8 was obtained by screening.

[0033] The following is an introduction through specific embodiments.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0035] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Example 1

[0036] 1. Acquisition of the original target gene pelA

[0037] Will Aspergillus sp.CM96 The cells were ground with liquid nitrogen to extract RNA, which was then reverse transcribed to obtain the cDNA of the strain. Each gene was cloned using primers synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and ligated to the pCold vector. The successfully ligated plasmids were chemically transformed into the competent cell culture medium of E. coli Rosetta-gami B(DE3)pLysS for expression. Genes with high protein expression and high pectinase activity were sequenced. The target gene obtained was named pelA, and its nucleotide sequence is shown in SEQ ID NO: 2. This target gene plasmid was used for the next experiment.

[0038] 2. Random mutation to obtain W-8

[0039] The primer pairs were designed based on the nucleotide sequence of the acid pectinase gene pelA as follows:

[0040] FP:5 ’ -ggctgaagcttacgtagaattcTCCGCCGCTGCCGTCGGCGT-3 ’ ;

[0041] RP: 5 ’ -gtgatgatgattcgcggccgcCAGGTTGCCCTGACCGGCGT-3 ’ ;

[0042] The original gene pelA was used as a template (sequence information is shown in SEQ ID NO: 2), and error-prone PCR amplification was performed using the designed primers.

[0043] PCR reaction system:

[0044] Forward primer (concentration 10 μM) 5μL Reverse primer (concentration 10 μM) 5μL <![CDATA[MnCl2(5Mm)]]> 10 μL <![CDATA[MgCl2(25Mm)]]> 13.30μL dATP (10 mM) 2.20 μL dCTP (10 mM) 2.00μL dGTP (10 mM) 3.40 μL dTTP (100 mM) 2.35 μL BSA (0.5 μg) 1.00μL Template pelA (50 ng / μL) 1.00μL Taq DNA polymerase 1.00μL 10×buffer 10.00μL water 43.75μL

[0045] PCR reaction conditions: 95℃ pre-denaturation for 5 min, then 91℃ denaturation for 30 min, 55℃ annealing for 1 min, 71℃ extension for 70 s, 30 cycles, and finally 72℃ extension for 10 min. The yield and specificity of the PCR products were detected by 0.8% agarose gel electrophoresis (such as Figure 1 ) and purified using a DNA purification kit (purchased from Novozymes).

[0046] The PCR product fragment was enzymatically ligated with the vector pCold I treated with Nde I and Xba I. After enzymatic ligation, the product was chemically transformed into E. coli Rosetta-gami B(DE3)pLysS (purchased from Shanghai Weidi). The strain was plated on LB plates containing 1 mM kanamycin and cultured overnight at 37°C. The strain isolated on the plate was the acid pectinase gene mutant. Transformants were spotted on LB pectin plates (containing 1% pectin, 1 mM kanamycin, 1 mM ampicillin, 1 mM chlorophenicol, and 0.05 mM IPTG). After incubation at 37°C, 20 ml of 1% CTAB was added to the substrate plate and allowed to stand for 10 minutes. The plate was then observed for the formation of hydrolysis zones. Single colonies corresponding to hydrolysis zones indicated pectinase activity. The transformants with hydrolysis circles were selected for expansion culture, induced expression, ultrasonically disrupted, and enzyme activity detected. The wild type was used as the control, and the strain with the highest enzyme activity was selected as the acidic pectinase-producing mutant strain, named W-8 (e.g. Figure 2 、 Figure 3 The plasmid pCold-W-8 obtained from the mutant strain was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing, and the plasmid was found to carry the sequence shown in SEQ ID NO: 4. Example 2

[0047] The plasmid pCold-W-8 was transformed into E. coli Rosetta-gami B (DE3) pLysS competent cells to obtain the recombinant bacteria Rosetta-gami B (DE3) pLysS-pCold-W-8.

[0048] The recombinant strain Rosetta-gami B (DE3) pLysS-pCold-W-8 was cultured in LB medium containing 1 mM kanamycin at 37°C and 180 rpm for 3 h; OD 600 =0.6-0.8, add IPTG to a final concentration of 0.5 mM in LB medium, transfer to 16°C and continue culturing for 14 h. The cells were collected by centrifugation at 8000 rpm for 10 min, suspended in solution A (500 mM NaCl, 20 mM Tris-HCl, pH 8.0), and ultrasonically disrupted in an ice-water bath (160 W, 10 min; ultrasonication for 1 s, pause for 3 s). The cell fragments were then removed by centrifugation at 10000 rpm for 15 min, and the supernatant was collected. The supernatant was passed through a Ni-NTA purification column, rinsed with 100 ml of solution B (50 mM Tris-HCl; 500 mM NaCl; 10 mM imidazole; 5% glycerol, pH 8.0), and then rinsed with 15 ml of 10% solution C (50 mM Tris-HCl; 500 mM NaCl; 500 mM imidazole; 5% glycerol, pH 8.5). Finally, the column was eluted with 5 ml of 40% solution C. The eluate was collected to obtain a solution of purified acid pectinase. SDS-PAGE electrophoresis showed that the molecular weight of pCold-W-8 protein was approximately 45 kDa (e.g. Figure 4 ).

[0049] The enzymatic properties test of WT and W-8 showed that their optimum pH did not change, both being 4.8 (phosphate buffer) (e.g. Figure 5 ), the optimal reaction temperature is 55℃, but the relative enzyme activity of the mutants at other temperatures has increased (such as Figure 6 ), its reaction temperature range is relatively wide. When measuring thermal stability, Figure 7 The results showed that the residual enzyme activity of W-8 in the first 60 min was much higher than that of WT. Example 3

[0050] 1. Determination of pectinase activity

[0051] Add 5 mL of pectin solution to test tubes A and B, preheat in a 50°C water bath for 5 minutes, then add 1 mL of diluted enzyme solution (add inactivated crude enzyme solution to tube A), 4 mL of phosphate buffer (pH 3.5), and shake well. After a 30-minute reaction, add 1 mL of the reaction solution to 3 mL of DNS, shake well, and boil in a boiling water bath for 10 minutes. Cool and dilute to 25 mL. Measure absorbance at 540 nm. The absorbance of group A (blank control) is designated A1, and the absorbance of group B (experimental group) is designated A2.

[0052] Enzyme activity calculation:

[0053] UI=(A2-A1)×1000×N / (0.1*K*30)

[0054] UI: pectinase activity (U / mL); N: dilution factor of crude enzyme solution; A2: absorbance of the experimental group; A1: absorbance of the control group; 1000: 1 mg = 1000 μg; 0, 1: enzyme solution addition amount / mL; K: slope of the galacturonic acid standard curve; 30: reaction time in min.

[0055] 2. Protein Concentration Determination Method

[0056] 1. Prepare protein standard solution: dilute 20 mg / ml BSA stock solution according to the table below.

[0057] serial number Volume of protein diluent (μL) BSA standard volume Final BSA concentration (mg / ml) A 95 5μL 1 B 20 Take 80 μL from tube A 0.8 C 20 Take 60 μL from tube B 0.6 D 20 Take 40 μL from tube C 0.4 E 30 Take 30 μL from tube D 0.2 F 30 Take 30 μL from tube E 0.1 G 50 - 0

[0058] To draw the standard curve, take a 96-well ELISA plate and add reagents according to the data in the following table:

[0059] Hole number G F E D C B A Protein standards 10 10 10 10 10 10 10 Bradford Reagent (μl) 300 300 300 300 300 300 300 Corresponding protein content (μg) 0 1 2 4 6 8 10

[0060] After vortexing and mixing, let it stand at room temperature for 5-10 min.

[0061] The absorbance at 595 nm was measured using a microplate reader, and the absorbance without BSA was used as the blank control.

[0062] With protein content (μg) as the horizontal axis and absorbance value as the vertical axis, draw a standard curve (such as Figure 8 ).

[0063] Sample Assay: Dilute the protein sample to be tested with deionized water to the appropriate concentration. Take 10 μl of sample and add 300 μl of Bradford Reagent. Mix thoroughly and let stand for 5-10 minutes. Then, using well 0 as a control, measure the sample's absorbance at A595 nm. The protein content of the sample can be determined from the standard curve using the measured absorbance. Calculate protein concentration: Use the absorbance value of the sample protein as the Y value. Substitute the value of X into the standard curve equation to calculate the sample protein concentration.

[0064] Table 1 Comparison of specific enzyme activities between wild-type enzyme and acid pectinase mutants

[0065] enzymes Specific enzyme activity (U / mg) pCold-W 3281.21 pCold-W-8 3781.56

Claims

1. An acid pectinase mutant, characterized in that: The amino acid sequence of the acid pectinase mutant is shown in SEQ ID NO:

3.

2. A gene, characterized in that Encoding the acid pectinase mutant according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence thereof is shown in SEQ ID NO:

4.

4. A recombinant vector, characterized in that Comprising the gene according to claim 2 or 3.

5. A recombinant bacterium, characterized in that Comprising the recombinant vector according to claim 4.

6. The recombinant bacterium according to claim 5, characterized in that The host cell of the recombinant bacteria is Escherichia coli Rosetta-gami B (DE3) pLysS.

7. Use of the acid pectinase mutant according to claim 1 in enzymatic hydrolysis of pectin.

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