An acidic pectinase of fungal origin, its encoding gene and heterologous expression

Abstract

The application discloses a fungal-derived acidic pectinase, a coding gene thereof and heterologous expression, and belongs to the field of genetic engineering. A pectinase gene is obtained by performing whole genome sequencing and gene cloning on Aspergillus sp. CM96 (preservation number: CGMCC No. 40340), and is successfully expressed in Escherichia coli Rosetta-gami B (DE3) pLysS. The amino acid sequence is shown as SEQ ID NO. 1. The application provides a new pectinase gene, and the pectinase coded by the pectinase gene has good properties, and can be applied to the industries of feed, food, medicine and the like.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and protein engineering technology, and specifically relates to a fungal-derived acidic pectinase, its encoding gene, and heterologous expression, and its function has been verified. Background Technology

[0002] Pectin is mainly found in the primary cell wall and intermediate layer tissues. It is an anionic acidic heteropolysaccharide composed of D-galacturonic acid residues linked by α-1,4 glycosidic bonds. Pectinase refers to a class of enzymes that degrade pectin, mainly including polygalacturonase (PG), pectin lyase (PL), and pectin esterase (PE).

[0003] Polygalacturonase (PG) hydrolyzes the α-1,4 glycosidic bonds of pectin acid molecules, belonging to the GH28 family of glycoside hydrolases, and is associated with fungal pathogenicity and toxicity. Based on different hydrolytic mechanisms, it can be divided into endo-galacturonase (endo-PG) and exo-galacturonase (exo-PG). Exo-PG can be further divided into two types: fungal exo-galacturonase (exo-PG1), whose end product is monomeric galacturonic acid; and bacterial exo-galacturonase (exo-PG2), whose end product is dimer galacturonic acid. The optimal substrate for endo-galacturonic acid is pectin acid, which is cleaved randomly, and the enzymatic hydrolysis products are mainly galacturonic acid trisaccharides and tetrasaccharides.

[0004] Pectinase has a wide range of applications. In fruit wine production, adding pectinase can improve the color and sensory appeal of the product; it can clarify the wine, making it more stable; and it can enhance the aroma and fuller flavor. In the textile industry, pectinase is combined with amylase, lipase, cellulase, and hemicellulase to remove gum-like substances from cotton. This method replaces the previous method using toxic and corrosive baking soda, making it safer and more environmentally friendly. However, because pectinase in the Chinese market is an extremely complex mixed enzyme, lacking highly efficient and specific commercially available enzymes, its application lacks specificity and cannot meet the requirements of various fields, resulting in low application efficiency, waste of raw materials, and environmental pollution. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a fungal-derived acidic pectinase, its encoding gene, and heterologous expression, and to study its enzymatic properties, thereby improving its application efficiency and specificity and promoting its widespread application in the food industry.

[0006] The acidic pectinase of this invention is derived from Aspergillus sp. CM96, which was deposited on October 21, 2022, at the China General Microbiological Culture Collection Center (No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China) with accession number CGMCC No. 40340, and has been published in Chinese Patent Publication No. CN116926044A.

[0007] The amino acid sequence of the fungal-derived acidic pectinase described in this invention is shown in SEQ ID NO:1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:2.

[0008] The present invention also provides a recombinant plasmid carrying the above-mentioned pectinase encoding gene.

[0009] The present invention also provides a recombinant bacterial strain carrying the above-mentioned recombinant plasmid, wherein the host cell of the recombinant bacterial strain is Escherichia coli, preferably Escherichia coli Rosetta-gami B(DE3)pLysS.

[0010] The recombinant expression was constructed using pColdⅠ as the expression vector and E. coli Rosetta-gami B(DE3)pLysS as the expression host.

[0011] This invention provides a method for producing the acidic pectinase using genetic engineering fermentation. The method involves activating and culturing a recombinant bacterium containing a gene encoding acidic pectinase (e.g., Rosetta-gami B(DE3)pLysS containing a gene encoding acidic pectinase), then inoculating it into 100 ml of LB fermentation medium containing 1 mM kanamycin, 1 mM chloramphenicol, and 1 mM ampicillin. The culture is carried out at 37°C and 180 rpm until the cells reach a certain growth stage (OD). 600 =0.6-0.8), add IPTG to a final concentration of 0.05 mM for induction, and adjust the temperature to 16℃ for 16 h of induction fermentation. Centrifuge the obtained bacterial culture, collect the bacterial cells, and sonicate to disrupt them. The supernatant after disruption is the crude enzyme solution of acidic pectinase. Purify the crude enzyme solution using a Ni-NTA affinity chromatography column to obtain purified acidic pectinase.

[0012] The acidic pectinase provided by this invention can be applied in the food industry.

[0013] The acidic pectinase provided by this invention has a specific enzyme activity as high as 513.24 U / mg, achieving excellent technical results. This pectinase can be widely used in the food and other fields. Attached image description:

[0014] Figure 1 This is a PCR electrophoresis image of the target gene;

[0015] Figure 2 Electrophoresis image of the expression vector PCoIdⅠ digested with enzymes;

[0016] Figure 3 This is an SDS-PAGE electrophoresis image of pectinase.

[0017] Figure 4 The effect of pH on enzyme activity;

[0018] Figure 5 The effect of temperature on enzyme activity;

[0019] Figure 6 The effects of different metal ions on enzyme activity;

[0020] Figure 7 Temperature stability curve;

[0021] Figure 8 For pH stability;

[0022] Figure 9 This is a TLC image of the acidic pectinase hydrolysis products of the present invention;

[0023] Figure 10 This is the protein standard curve. Detailed Implementation

[0024] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0025] The acidic pectinase of this invention is derived from Aspergillus sp. CM96, which was deposited on October 21, 2022, at the China General Microbiological Culture Collection Center (No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China) with accession number CGMCC No. 40340, and has been published in Chinese Patent Publication No. CN116926044A.

[0026] This invention discloses an acidic pectinase mutant, belonging to the fields of genetic engineering and enzyme engineering. The invention involves cloning and expressing the acidic pectinase encoding gene pgaA from Aspergillus sp. CM96 (accession number: CGMCC No. 40340).

[0027] The following is a detailed description through specific examples.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0029] The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0030] Example 1: Obtaining acidic pectinase

[0031] I. Obtaining total RNA from Aspergillus sp. CM96

[0032] Aspergillus sp. CM96 was inoculated into liquid PDA medium and cultured at 28°C and 160 rpm for 3 days. The bacterial culture was then transferred to centrifuge tubes and centrifuged at 10,000 rpm for 10 minutes to collect bacterial pellets. The pellets were then ground in liquid nitrogen, dissolved in an RNA isolater, and 1 / 5 volume of chloroform was added. The mixture was vigorously mixed, incubated at 4°C for 5 minutes, and centrifuged at 12,000 rpm for 4 minutes. The supernatant was collected, and an equal volume of isopropanol was added. The mixture was incubated at 4°C for 10 minutes and centrifuged at 12,000 rpm for 4 minutes. The mixture was washed with 75% ethanol and centrifuged at 12,000 rpm for 5 minutes. The supernatant was discarded, the mixture was air-dried, and ddH2O was added. The total RNA of Aspergillus sp. CM96 was measured using NanoDrop to be 2945 ng / μL. Finally, the RNA was analyzed using Novizan (HiScript III 1). st The cDNA of this strain was obtained by reverse transcription using the Strand cDNA Synthesis Kit.

[0033] II. Obtaining the target gene pgaA

[0034] Primer pairs were designed based on the nucleotide sequence of the acidic pectinase gene pgaA (GenBank: name: Aspergillus tubingensis putativeendopolygalacturonaseA (AtWU_09859), partial mRNA accession number: XM_035504965) as follows:

[0035] FP:5 ’ -CATATCGAAGGTAGG CATATG GCGCAGGGTGTCACTGGCTCC-3 ’ ;

[0036] RP:5 ’ -AGCAGAGATTACCTA TCTAGA ATAGCCACGGCCGTGACCCTT-3 ’ ;

[0037] The primer pair was synthesized by Suzhou Genewiz Biotechnology Co., Ltd.

[0038] Using cDNA from Aspergillus sp. CM96 as a template, PCR amplification was performed using designed primers.

[0039] Table 1: PCR reaction system

[0040] Reverse primer (concentration 10 μM) 1μL cDNA 1μL Gold Medal MiX 47μL

[0041] PCR reaction conditions: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, 55℃ annealing for 1 min, 71℃ extension for 70 s, 35 cycles, and a final extension at 72℃ for 10 min. PCR product yield and specificity were detected by 0.8% agarose gel electrophoresis. Figure 1 The DNA was purified using a DNA purification kit (purchased from Novizan).

[0042] The purified PCR product was ligated into the vector pColdⅠ. The successfully ligated plasmid was transformed into *E. coli* DH5α competent cells and sent to Genewiz for sequencing. The correctly sequenced plasmid was then chemically transformed into *E. coli* Rosetta-gamiB(DE3)pLysS competent cells for expression. The gene with high expression levels of soluble protein and high pectinase activity was sequenced, and the nucleotide sequence of the target gene is shown in SEQ ID NO: 2. This target gene plasmid was then used for further experiments.

[0043] Example 2: Expression and purification of acidic pectinase

[0044] The plasmid pColdⅠ-pgaA was transformed into E. coli Rosetta-gami B(DE3)pLysS competent cells to obtain the recombinant strain Rosetta-gami B(DE3)pLysS-pColdⅠ-pgaA. The recombinant strain Rosetta-gami B(DE3)pLysS-pColdⅠ-pgaA was cultured in LB medium containing 1 mM kanamycin, 1 mM chloramphenicol, and 1 mM ampicillin at 37°C and 180 rpm for 3 h; OD 600When the concentration is 0.6-0.8, add IPTG to a final concentration of 0.05 mM in LB medium, and continue culturing at 16°C for 16 h. The bacterial cells were collected by centrifugation at 8000 rpm for 10 min and resuspended in solution A (500 mM NaCl, 20 mM Tris-HCl, pH 8.0). The cells were then sonicated in an ice-water bath (160 W, 25 min; sonication for 1 s, pause for 3 s), followed by centrifugation at 10000 rpm for 15 min to remove cell fragments. The supernatant was collected and purified using a Ni-NTA column. The supernatant was washed with 100 ml of solution B (50 mM Tris-HCl; 500 mM NaCl; 10 mM imidazole; 5% glycerol, pH 8.0), then rinsed with 15 ml of 10% solution C (50 mM Tris-HCl; 500 mM NaCl; 500 mM imidazole; 5% glycerol, pH 8.5), and finally eluted with 5 ml of 40% solution C. The eluent was collected to obtain the purified acidic pectinase solution. SDS-PAGE electrophoresis showed that the molecular weight of pCold-pgaA protein was approximately 46 kDa (e.g., ...). Figure 3 ). Figure 2 Electrophoresis image of the expression vector PCoIdⅠ digested with enzymes.

[0045] Example 3: Determination of specific enzyme activity and protein content of acidic pectinase

[0046] Using pectin solution as a substrate, the crude enzyme solution from Example 2 or the acidic pectinase solution purified by Ni-NTA affinity chromatography column was added. After reacting accurately for 30 min, 1 mL of the reaction solution was added to 3 mL of DNS and shaken well. The mixture was then boiled in a water bath for 10 min, cooled, and brought to a final volume of 25 mL. The absorbance was measured at a wavelength of 540 nm. The absorbance of the blank control group was recorded as A1, and the absorbance of the experimental group was recorded as A2.

[0047] Enzyme activity calculation:

[0048] UI=(A2-A1)×1000×N / (0.1*K*30)

[0049] UI: Pectinase activity (U / mL); N: Crude enzyme solution dilution factor; A2: Absorbance of the experimental group; A1: Absorbance of the control group; 1000: 1mg = 1000μg; 0.1: Enzyme solution addition amount / mL; K: Slope of the galacturonic acid standard curve; 30: Reaction time min.

[0050] II. Protein Concentration Measurement Methods

[0051] Using BSA as the protein standard solution, Bradford Reagent was added, and the mixture was vortexed and incubated at room temperature for 5-10 minutes. The absorbance at 595 nm was measured using a microplate reader, with the absorbance without BSA as the blank control. A standard curve was plotted with protein concentration (μg) on ​​the x-axis and absorbance on the y-axis (e.g., ...). Figure 10 The crude enzyme solution from Example 2 or the acidic pectinase solution purified by Ni-NTA affinity chromatography was diluted with deionized water to an appropriate concentration, and the absorbance of the sample at A595 nm was measured. The protein concentration was calculated according to the standard curve equation.

[0052] Table 2. Detection of pectinase activity before and after purification.

[0053]

[0054]

[0055] Example 4: Study on the enzymatic properties of pectinase

[0056] I. Optimal pH of pectinase

[0057] Following the method for pectinase activity determination in Example 3, citrate buffer solutions with pH values ​​of 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 5.0, and 6.0, and phosphate buffer solutions with pH values ​​of 7.0 and 8.0 were prepared. The purified pectinase activity was measured after reacting at 50°C for 30 minutes, with the highest pectinase activity considered as 100%. The experimental results are as follows: Figure 4 .

[0058] II. Optimal temperature for pectinase

[0059] Enzyme activity was measured under optimal pH conditions and at reaction temperatures ranging from 40 to 70°C, with increments of 5°C. The experimental results are as follows: Figure 5 .

[0060] III. Effects of Metal Ions on Pectinase Activity

[0061] Different metal ions K were selected with a final concentration of 1 mM. + Fe 2+ Ba 2+ Ni 2+ Cu 2+ Mn 2+ Mg 2+ Zn 2+ Ca 2+ The purified pectinase activity was added to a 10 mL reaction system and measured under optimal pH and temperature conditions. The pectinase activity measured without the addition of metal ions was taken as 100%. The experimental results are as follows: Figure 6 .

[0062] IV. pH and temperature stability of pectinase

[0063] Pectinase was appropriately diluted with buffer (optimal pH) and incubated at room temperature for 8 hours. Samples were taken at 0h and 8h to measure pectinase activity under optimal reaction conditions, with the activity measured at 0h considered 100%. The experimental results are as follows: Figure 7 .

[0064] The purified pectinase was placed in an environment of 55℃, and enzyme activity was measured at 0, 10, 30, 60, and 90 min, with the enzyme activity measured at 0 h considered as 100%. The experimental results are as follows: Figure 8 .

[0065] Enzymatic property tests of pCOId-pgaA show that the optimal pH for this enzyme is 3.6 (citrate buffer), and it exhibits good stability and acid resistance under acidic conditions. The optimal reaction temperature is 55℃. Different metal ions have significantly different effects on the enzyme activity of pCOId-pgaA, with Cu... 2+ Ca 2+ Mn 2+ It has a significant inhibitory effect on enzyme activity, Mg 2+ Fe 2+ Ba 2+ It significantly improves enzyme activity.

[0066] V. Pectinase Degradation of Pectin Solution

[0067] The specific steps are as follows: Weigh 0.04g of pectin into 10mL of buffer solution to prepare a pectin solution for later use. Mix the purified pectinase solution and the pectin solution in a 1:1 ratio and incubate at 50℃ for 2, 4, 6, and 8 hours. Use ethyl acetate-glacial acetic acid-water (2:1:1) as the developing solvent, and spray with anisaldehyde solution (0.5ml anisaldehyde, 10ml glacial acetic acid, 85ml methanol, and 5ml sulfuric acid) for color development. Analyze the product components using thin-layer chromatography (TLC). Figure 9 As shown, this proves that the hydrolysis product is D-galacturonic acid.

Claims

1. An acidic pectinase derived from fungi, characterized in that, The acidic pectinase is derived from Aspergillus ( Aspergillus sp.) CM96, the biological preservation number of Aspergillus CM96 is CGMCC No.40340; the amino acid sequence of the acid pectinase is shown in SEQ ID NO:

1.

2. A gene encoding an acidic pectinase derived from a fungus as described in claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

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

4. The recombinant vector according to claim 3, characterized in that, The recombinant vector used was pColdⅠ as the expression vector.

5. A recombinant bacterium, characterized in that, The recombinant bacteria comprises the recombinant vector as described in claim 3.

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

7. The application of an acidic pectinase derived from fungi as described in claim 1, characterized in that, The application of the fungal-derived acidic pectinase in catalyzing the hydrolysis of pectin to produce D-galacturonic acid.

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