Mutant high-stability catalase and construction method thereof
By obtaining the catalase sequence of thermophilic bacterial source and codon optimization, combining PCR amplification and seamless cloning technology, the target gene was cloned into the pET-28a vector, and the mutant highly stable catalase was obtained by expressing it. , solves the problem that it is difficult to obtain high stability and suitable for large-scale preparation in the prior art, and achieves the effects of high expression, heat resistance, acid and alkali resistance.
Patent Information
- Application Number
- CN202411132464.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-19
- Publication Date
- 2025-02-18
- Estimated Expiration
- 2044-08-19
AI Technical Summary
It is difficult to obtain a highly stable catalase in the prior art, which is suitable for large-scale preparation and multi-field use.
By obtaining the catalase sequence of thermophilic bacteria source in the NCBI database, codon optimization is performed to synthesize the mutational amino acid sequence, and the target gene is cloned into the pET-28a vector using PCR amplification and seamless cloning technology. Finally, Expression of mutant highly stable catalase was obtained in BL21 (DE3).
It has achieved a mutant catalase with high expression and good stability, which is suitable for large-scale preparation, with the characteristics of heat resistance and acid and alkali resistance, and is simple to operate and good soluble.
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Figure CN119464240A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a catalase and a construction method thereof, and more specifically to a mutant highly stable catalase and a construction method thereof, belonging to the technical field of biological genetic engineering. Background Art
[0002] Catalase (CAT) is widely present in humans, animals, plants, and microorganisms. For example, the enzyme is found in plant organelles, animal livers, and most bacteria, actinomycetes, and other microorganisms in cells. It is an important antioxidant enzyme that produces toxic byproducts during cell metabolism. Catalase decomposes hydrogen peroxide (H2O2) and converts it into water and oxygen, thereby protecting cells from oxidative damage. Catalase is mainly composed of a single subunit or multiple identical or different subunits. It is mainly divided into catalase, manganese-containing catalase, copper-containing catalase, and zinc-containing catalase. It can also be divided into tetrameric catalase, which is the most common type, usually composed of four identical subunits, each of which contains a heme prosthetic group as the active center, non-heme catalase: does not contain a heme prosthetic group, but still has catalytic activity; special catalase: for example, some microorganisms can produce very heat-resistant catalase, or enzymes that can remain active under extreme conditions. In current research, expressing in microorganisms to obtain large amounts of fermentation products is the trend in biological fermentation. Summary of the invention
[0003] The present invention aims to provide a mutant highly stable catalase and a construction method thereof, which has the technical characteristics of high prokaryotic expression, direct synthesis, suitability for large-scale preparation, simple operation, good solubility, etc.
[0004] In order to achieve the above object, the present invention is implemented by the following technical solutions:
[0005] The present invention provides a mutant highly stable catalase, wherein the protein sequence of the catalase is:
[0006] MEARKAPRLTADGRPIGDNQNALTAGPRGPLLIQDVQLLEQIQHFNRERIPERVVHAKGSGAYGTFTVTNDVTRYTKAAFLSEVGKQTEVFVRFSTVAGERGAADAERDVRGFAVKFYTEEGN FDLVGNNTPVFFVRDPYKFQMFIHSQKRHPKTNLRDPDMQWDFWSLCPESLHQVTILFSDRGIPASYRHMNGYGSHTYSMYNDRGELFWVKFHFKTQQGIKCLTDEAARLIGEDRETHQRDLYE AIERGDYPRWTLYIQVMTPEQAENFRWNPFDLTKVWPHAEFPLIEVGVLELNRNPENYFAEVEQAAFKPSAFVPGIGPSPDKMLQARLMSYADAHRYRLGVNYQQLKVNRPRCPVHHYQRDGFMA QIEEGSGHPNYFPNSIPGSPQDDPIYKEPAWHLGEVIVDRYDSRKDHDDYTQAGNLYRLFDEGQKDRLARAIAASLGQARLEVQKRQLGHFYRADVDYGRRVARALGFDPAAAEAELGIDASVAG.
[0007] Preferably, the mutant highly stable catalase has a molecular weight of 57.07 kDa and a theoretical isoelectric point of 6.584.
[0008] Preferably, the mutant highly stable catalase has no signal peptide and transmembrane domain.
[0009] The present invention provides a method for constructing a mutant highly stable catalase, the method comprising the following steps:
[0010] Step 1) obtaining a catalase sequence from a thermophilic bacteria in the NCBI database;
[0011] Step 2) synthesizing the mutated amino acid sequence using codon optimization;
[0012] Step 3) amplifying the target gene and pET-28a vector using PCR;
[0013] Step 4) Clone the target gene into the pET-28a vector using seamless cloning;
[0014] Step 5) Transform into BL21(DE3) for expression.
[0015] Preferably, step 1)-step 2) specifically includes: obtaining the catalase sequence WP_012845258.1 of thermophilic bacteria from the NCBI database, mutating the amino acids, and then optimizing the codons using Escherichia coli as the host, placing the gene synthesis sequence on pGEX-4T-1, using TOP10 as the host bacteria, and obtaining a plasmid containing the optimized target gene.
[0016] Preferably, the plasmid is constructed as follows: using the synthesized plasmid containing pGEX-4T-1-CAT as a template, using primers Cat-f and Cat-r to amplify the target gene CAT by PCR, using XF and XR to amplify the linearized vector fragment of pET28a containing homology arms by PCR, and the product is electrophoresed through 1% agarose gel, and the size of the electrophoresis band of the product is compared with the expected size using ultraviolet detection. If they are consistent, a gel recovery kit is used for recovery.
[0017] Preferably, Cat-f primer sequence (5'-3')
[0018] GAGATATACCATGGAAGCGCGCAAAGCGCCGCG; Cat-r primer sequence (5'-3')TTCTTAGCCCGCCACGCTCGCATCAATGCCCA; XF primer sequence (5'-3')GCGTGGCGGGCTAAGAATTCGAGCTCCGTC; XR primer sequence (5'-3')
[0019] GCGCTTCCATGGTATATCTCCTTCTTAAAGTTAAAC.
[0020] Preferably, seamless cloning: the target gene CAT obtained by PCR recovery product and the linearized vector of pET28a containing homology arms are connected by seamless cloning, and the reaction conditions are incubated at 50° C. for 30 minutes.
[0021] Preferably, step 5) specifically includes heat shock transformation of DH5α, plasmid extraction, heat shock transformation of BL21 (DE3), and strain induction expression; heat shock transformation of DH5α: transforming the seamless cloning product into DH5α competent cells, and the specific steps are:
[0022] 1) Take out the DH5α competent cells from the -80℃ freezer and thaw them on ice;
[0023] 2) Add the seamless cloning product to the thawed DH5α competent cells, tap the wall of the centrifuge tube to fully mix the seamless cloning product and DH5α competent cells, place on ice for 30 minutes, and turn on the 42°C water bath;
[0024] 3) Heat shock in a 42°C water bath for 45 seconds, place on ice for 3-5 minutes, add 700uL culture medium, and culture at 37°C, 220rpm for 60 minutes;
[0025] 4) Centrifuge at 12000rpm, 4℃ for 1min, discard the supernatant, keep 200uL for resuspending, apply on LB plate of corresponding resistance, and culture at 37℃ for 12h;
[0026] 4) Plaque verification: colonies were picked from the above LB plate and placed in 5 mL LB medium. They were cultured at 37°C and 220 rpm for 12 h for identification. If the sequence was correct, the bacteria and plasmid were returned.
[0027] Preferably, plasmid extraction: transfer the returned plasmid, culture it at 37°C, 220rpm for 12h, then use a plasmid small-scale extraction kit to extract the plasmid, measure the plasmid concentration by ultraviolet spectrophotometer, and store it at -20°C.
[0028] Preferably, the specific steps of heat shock transformation of BL21 (DE3) are:
[0029] 1) Take out the BL21(DE3) competent cells from the -80℃ freezer and thaw them on ice;
[0030] 2) Add 10uL of the above plasmid to 100uL of thawed BL21(DE3) competent cells, flick the wall of the centrifuge tube to mix the plasmid and BL21(DE3) competent cells thoroughly, and place on ice for 30 minutes;
[0031] 3) Turn on the 42°C water bath, heat shock for 45 seconds at 42°C, place on ice for 3-5 minutes, add 700uL culture medium, and culture at 37°C, 220rpm for 60 minutes;
[0032] 4) Centrifuge at 12000 rpm and 4°C for 1 min, discard the supernatant, keep 200uL for resuspending, spread on LB plates with corresponding resistance, and then culture at 37°C for 12h.
[0033] Preferably, the strain is induced to express: 1) pick a single colony into 5 mL LB medium, add 5 uL 50 mg / mL Kan solution, and culture at 37°C, 220 rpm until OD600 is 0.6-0.8; 2) add 5 uL 1M IPTG, and culture at 37°C, 220 rpm for 12 h to obtain catalase protein.
[0034] Beneficial effects: The mutant catalase of the present invention has a short peptide segment and a molecular weight of only 57.07 kDa, can be directly synthesized, and is suitable for large-scale preparation; a large amount of protein is obtained by eukaryotic expression, and the relative enzyme activity of the protein is higher than that of the catalase products on the market; the mutant catalase of the present invention has the effects of heat resistance, acid and alkali resistance, can maintain stable activity during transportation and storage, and has high stability; it has the technical characteristics of simple operation, good solubility, and suitability for use in multiple fields. BRIEF DESCRIPTION OF THE DRAWINGS
[0035] Figure 1 It is the gel electrophoresis diagram of the target gene of the pET-28a vector of the present invention.
[0036] Figure 2 It is the gel electrophoresis diagram of the CAT target gene of the present invention.
[0037] Figure 3 It is the pET28a-CAT plasmid map of the present invention.
[0038] Figure 4 It is a sequence comparison diagram of the sequencing results of the present invention.
[0039] Figure 5 This is a protein gel image of the CAT protein of the present invention expressed in BL21 (DE3).
[0040] Figure 6 It is the protein expression diagram of the standard control and the CAT crude enzyme solution of the present invention.
[0041] Figure 7 It is a comparison chart of the enzyme activities of the standard product of the present invention and the crude CAT enzyme solution.
[0042] Figure 8 It is a test chart of the CAT acidic and alkaline environment tolerance of the present invention.
[0043] Fig. 9 This is a CAT 10min temperature environment tolerance test chart of the present invention.
[0044] Fig.10 This is a graph showing changes in CAT enzyme activity within 100 minutes of heating at 50°C according to the present invention.
[0045] Fig.11 This is a graph showing changes in CAT enzyme activity within 100 minutes of heating at 60°C according to the present invention. DETAILED DESCRIPTION
[0046] The present invention will be further described below in conjunction with the accompanying drawings, but the present invention is not limited to the following embodiments.
[0047] The invention can construct the mutant catalase gene into a plasmid for soluble expression, and the expression amount reaches the standard of industrial production.
[0048] like Figure 1-11 The following is a specific example of a mutant highly stable catalase and a method for constructing the same. To implement the mutant highly stable catalase and a method for constructing the same in this example, the following test was used.
[0049] 1. Materials, instruments and reagents for the experiment
[0050] 1.1 Main Materials
[0051] Escherichia coli DH5α, Escherichia coli BL21 (DE3), and pET-28a (+) plasmid were purchased from Suzhou Jinweizhi Biotechnology Co., Ltd. Catalase standard (22092200510) was from Shandong Longkote Enzyme Preparation Co., Ltd.
[0052] 1.2 Main instruments
[0053] Table 1 Main instruments
[0054]
[0055]
[0056] 1.3 Main Reagents
[0057] Table 2 Main reagents
[0058]
[0059] 1.4 Preparation of main reagents
[0060] IPTG(1M):
[0061] 1) Weigh 2.4 g IPTG powder and place it in a 15 mL centrifuge tube.
[0062] 2) Add 8 mL of sterile water, mix thoroughly to dissolve, and then add double distilled water to 10 mL.
[0063] 3) Then filter and sterilize using a 0.22 μm filter membrane in a clean bench.
[0064] 4) After aliquoting (1 mL / aliquot), store at -20°C.
[0065] Kanamycin sulfate solution (Kan, 50 mg / mL):
[0066] 1) Weigh 0.5 g Kan powder and place it in a 15 mL centrifuge tube.
[0067] 2) Add 8 mL of sterile water, mix thoroughly to dissolve, and then add double distilled water to 10 mL.
[0068] 3) Then filter and sterilize using a 0.22 μm filter membrane in a clean bench.
[0069] 4) After aliquoting (1 mL / aliquot), store at -20°C.
[0070] Coomassie Brilliant Blue R-250 staining solution (1L):
[0071] 1) Weigh 1 g of Coomassie Brilliant Blue R-250 powder and place it in a 1 L beaker.
[0072] 2) Measure 250 mL of isopropanol and add it to a beaker, stirring to dissolve.
[0073] 3) Add 100 mL of glacial acetic acid and stir well, then add 650 mL of double distilled water and stir well.
[0074] 4) After removing particulate matter with filter paper, store at room temperature.
[0075] Coomassie Brilliant Blue Destaining Solution (1L):
[0076] 1) Measure the following solutions and place them in a 1L beaker.
[0077] 50mL of anhydrous ethanol 100mL of glacial acetic acid
[0078] 2) Add double distilled water to make up to 1 L and mix thoroughly before use.
[0079] 10×PBS(1L):
[0080] 1) Weigh the following reagents and place them in a 1L beaker.
[0081] Sodium chloride 80.0g Disodium hydrogen phosphate dodecahydrate 35.8g Potassium dihydrogen phosphate 2.4g Potassium chloride 2.0g
[0082] 2) Add about 800 mL of double distilled water to the beaker and stir thoroughly to dissolve.
[0083] 3) Add concentrated hydrochloric acid to adjust the pH value to 7.2-7.4, and then add double distilled water to make the solution volume to 1L.
[0084] 4) After sterilization at 121℃ for 30min, store at room temperature and dilute to 1× PBS before use.
[0085] LB liquid medium (1L):
[0086] 1) Weigh the following reagents and place them in a 1L beaker.
[0087] Peptone 10g Sodium chloride 10g Yeast powder 5g
[0088] 2) Add about 800 mL of double distilled water and stir thoroughly to dissolve.
[0089] 3) Add about 0.2 mL of NaOH to adjust the pH to 7.0.
[0090] 4) Add double distilled water to make the volume of the culture medium to 1 L.
[0091] 5) After high temperature and high pressure sterilization at 121℃ for 20min, store at 4℃.
[0092] SDS-PAGE electrophoresis buffer:
[0093] 1) Weigh the following reagents and place them in a 1L beaker.
[0094]
[0095] 2) Add about 800 mL of double distilled water to the beaker and stir to dissolve.
[0096] 3) Add double distilled water to make the solution volume 1L, store at room temperature, and dilute to 1× SDS-PAGE electrophoresis buffer before use.
[0097] 5×SDS-PAGE Loading Buffer:
[0098] 1) Take the following reagents and place them in a 10 mL plastic centrifuge tube.
[0099] 1M Tris-HCl (pH 6.8) 1.25 mL
[0100] SDS 0.5g
[0101] Bromophenol blue (BPB) 25 mg
[0102] Glycerol 2.5mL
[0103] 2) Add double distilled water to dissolve and make up to 5 mL.
[0104] 3) Aliquot the solution into small portions (500 μl / portion) and store at room temperature.
[0105] 4) Add 25 μl of β-mercaptoethanol to each aliquot before use, and the aliquot can be stored at room temperature for about one month.
[0106] 2. Construction method of mutant highly stable catalase:
[0107] 2.1 CAT gene synthesis
[0108] The catalase sequence of thermophilic bacteria (WP_012845258.1) was obtained from the NCBI database, the penultimate amino acid I was mutated to A, and then codon optimization was performed using Escherichia coli as the host. The gene synthesis sequence was placed on pGEX-4T-1, and TOP10 was used as the host bacteria. Gene synthesis obtained a plasmid containing the optimized target gene.
[0109] 2.2 Plasmid construction
[0110] (1) PCR: The target gene CAT was amplified by PCR using the synthesized plasmid containing pGEX-4T-1-CAT as a template using primers Cat-f and Cat-r, and the linearized vector fragment of pET28a containing homology arms was amplified by PCR using XF and XR. The specific primer sequences are shown in Table 3. The PCR reaction system is shown in Table 4, and the PCR reaction procedure is shown in Table 5.
[0111] Table 3 Primer sequences
[0112]
[0113] Table 4 PCR system
[0114]
[0115]
[0116] Table 5 PCR reaction program
[0117]
[0118] After the PCR is completed, the product is electrophoresed through 1% agarose gel. The size of the electrophoresis band of the product detected by ultraviolet light is the same as the expected size, and then it is recovered using a gel recovery kit.
[0119] (2) Seamless cloning: The target gene CAT obtained by PCR recovery and the linearized vector of pET28a containing homology arms were connected by seamless cloning in an appropriate ratio. The reaction conditions were incubated at 50°C for 30 min.
[0120] (3) Heat shock transformation of DH5α: Transform the seamless clone product into DH5α competent cells. Specific steps: 1) Take out the DH5α competent cells from the -80℃ refrigerator and thaw them on ice. 2) Add the seamless clone product to the thawed DH5α competent cells, flick the wall of the centrifuge tube to fully mix the seamless clone product and DH5α competent cells, and place it on ice for 30 minutes. Turn on the 42℃ water bath. 3) Heat shock in a 42℃ water bath for 45 seconds, and immediately place it on ice for 3-5 minutes after heat shock. Add 700uL of culture medium and culture at 37℃, 220rpm for 60min. 4) Centrifuge at 12000rpm, 4℃ for 1min, and discard the supernatant. Keep 200uL for resuspending and apply it on the LB plate of the corresponding resistance. Culture at 37℃ for 12h. 4) Plaque verification: colonies were picked from the above LB plates and cultured in 5 mL LB medium at 37°C, 220 rpm for 12 h. If the identification and sequence were correct, the bacteria and plasmids were returned.
[0121] 2.3 Plasmid extraction
[0122] Transfer the returned plasmid and culture at 37°C and 220 rpm for 12 hours. Then use a plasmid small-scale extraction kit to extract the plasmid. Measure the plasmid concentration using an ultraviolet spectrophotometer and store at -20°C.
[0123] 2.4 Heat shock transformation of BL21(DE3)
[0124] (1) Take out the BL21(DE3) competent cells from the -80℃ freezer and thaw them on ice.
[0125] (2) Add 10uL of the above plasmid to 100uL of thawed BL21(DE3) competent cells, flick the wall of the centrifuge tube to mix the plasmid and BL21(DE3) competent cells thoroughly, and place on ice for 30 minutes. Turn on the 42℃ water bath.
[0126] (3) Heat shock in a 42°C water bath for 45 seconds, then place on ice for 3-5 minutes. Add 700uL of culture medium and incubate at 37°C, 220rpm for 60 minutes.
[0127] (4) Centrifuge at 12000 rpm, 4°C for 1 min, discard the supernatant, keep 200uL for resuspending, apply on LB plate of corresponding resistance, and incubate at 37°C for 12h.
[0128] 2.5 Induced expression of strains
[0129] (1) Pick a single colony and transfer it to 5 mL LB medium. Add 5 μL 50 mg / mL Kan solution and culture at 37°C and 220 rpm until the OD600 is 0.6-0.8.
[0130] (2) Add 5uL 1M IPTG and culture at 37°C, 220rpm for 12h.
[0131] 3. Testing Catalase Protein
[0132] 3.1 Ultrasonic fragmentation
[0133] (1) Collection: Take the cultured bacterial solution and centrifuge it at 4℃ 12000rpm for 3min. Take 500uL 1*PBS to resuspend the bacterial solution and centrifuge it at 4℃ 12000rpm for 3min. Repeat this process.
[0134] (2) Disruption: Use an ultrasonic disruptor on ice to disrupt the bacteria. Disrupt the bacteria for 3 seconds, pause for 5 seconds, and use 70% power for 5 minutes.
[0135] (3) Supernatant: After ultrasonic disruption, centrifuge at 12,000 rpm for 3 min at 4°C and collect the supernatant.
[0136] (4) Precipitation: Resuspend the precipitate in an equal volume of 1*PBS.
[0137] 3.2 SDS-PAGE electrophoresis
[0138] (1) Protein sample preparation: Take 80uL of the sample to be prepared and place it in a 1.5mL centrifuge tube. Add 20uL of 5* loading buffer and blow evenly. Heat at 100℃ for 10min.
[0139] (2) Protein gel preparation: Prepare 12.5% SDS-PAGE protein gel according to the manufacturer's instructions.
[0140] (3) SDS-PAGE electrophoresis: The heated sample was centrifuged at 12000 rpm for 3 min, and 10 μl of the sample was added to the protein gel channels in sequence. The electrophoresis condition was 150 V for 50 min.
[0141] (4) Protein gel staining: After electrophoresis, remove the concentrated gel and retain the separation gel, add Coomassie Brilliant Blue (R-250) and stain for 1 hour. After staining, add destaining solution to destain until clear bands are seen.
[0142] (5) Taking photos: Use a gel imager to take photos.
[0143] 3.3 CAT crude enzyme solution and standard enzyme activity detection and grayscale analysis
[0144] (1) Use Image J software to perform grayscale analysis on the obtained CAT crude enzyme solution and standard product to obtain the grayscale value.
[0145] (2) Use the total CAT detection kit to determine the activity of CAT crude enzyme solution and standard CAT.
[0146] (3) Use relative enzyme activity = CAT activity / gray value.
[0147] 3.4 Test of acidic and alkaline environment tolerance of CAT crude enzyme solution and standard products
[0148] (1) The strain is induced to express CAT to obtain a crude enzyme solution.
[0149] (2) Use the total CAT detection kit to detect the enzyme activity of CAT crude enzyme solution and standard product.
[0150] (3) Dilute the crude CAT enzyme solution and the standard 10 times with phosphate buffer of different pH (3-12), incubate at 37°C for 30 min, and measure the CAT activity. Each group is repeated three times.
[0151] (4) Enzyme activity: The CAT enzyme activity measured by diluting the sample ten-fold with double distilled water was set to 1. The relative enzyme activity at each pH was calculated accordingly.
[0152] 3.5 High temperature environment tolerance test of CAT crude enzyme solution and standard product
[0153] (1) The strain is induced to express CAT to obtain a crude enzyme solution.
[0154] (2) Use the total CAT detection kit to detect the enzyme activity of CAT crude enzyme solution and standard product.
[0155] (3) Place the crude enzyme solution and the standard in a metal bath and heat at 25°C, 35°C, 45°C, 55°C, 65°C, and 75°C for 10 min. Detect the CAT enzyme activity.
[0156] (4) Place the crude enzyme solution and the standard in a metal bath and keep them at 50°C and 60°C for 20 min, 40 min, 60 min, 80 min, 100 min, and 120 min. Then use a CAT kit to detect the CAT activity. Each group is repeated three times.
[0157] (5) The enzyme activity at 25°C was set as 1. The relative enzyme activity at each temperature and incubation time was calculated accordingly.
[0158] 4. Explanation of the results with the attached figures
[0159] 4.1 Construction of pET-28a-CAT
[0160] After PCR amplification, agarose gel electrophoresis was performed and the results were as follows Figure 1-2 Each temperature gradient can PCR out the target band.
[0161] Then use FastPure Gel DNA Extraction Mini kit to recover the DNA from the gel according to the instructions. Then use seamless cloning technology to clone the target gene into the pET-28a vector. The target plasmid pET28a-CAT is obtained. Figure 3 Then the sequencing is verified as correct. Figure 4 .
[0162] 4.2 CAT protein expression in BL21(DE3)
[0163] After IPTG induction, SDS-PAGE electrophoresis analysis was performed to evaluate the expression of mutant stable CAT. Figure 5 As shown in the figure, CAT protein was expressed in large quantities after IPTG induction. A large amount of CAT protein was expressed in the supernatant of the fragmentation, and a small amount was present in the fragmentation precipitate. Therefore, CAT protein was expressed in a soluble form.
[0164] 4.3 CAT crude enzyme solution and standard enzyme activity detection and grayscale analysis
[0165] Take the above supernatant and 0.1g of the standard and dissolve them in 1*PBS to detect the enzyme activity. The CAT enzyme activity of the crude CAT enzyme solution was 4474800U / mL, and the standard was 542400U / mL. Then take the same batch of samples for protein electrophoresis to obtain Figure 6 It can be seen that the band size of CAT crude enzyme solution is larger than that of the standard.
[0166] Image J was then used to analyze the grayscale value, and the CAT enzyme activity / grayscale value was used to compare the enzyme activity. Figure 7 , the enzyme activity of CAT crude enzyme solution is greater than that of the standard.
[0167] 4.4 Test of acidic and alkaline environment tolerance of CAT crude enzyme solution and standard products
[0168] The crude enzyme solution was tested for tolerance to acidic and alkaline environments to evaluate the stability of CAT protein under extreme pH conditions. Figure 3 , 4 As shown in the figure, after being treated in an acidic environment of pH 3 for 30 minutes, both the crude CAT enzyme and the standard maintained more than 60% of their activity, showing good tolerance to the acidic environment. The CAT activity of the crude CAT enzyme reached a high point when treated in an acidic environment of pH 5, exceeding the CAT enzyme activity at pH 7.
[0169] In the alkaline environment test, such as Figure 8As shown in the figure, after being treated in an alkaline environment of pH 11 for 30 minutes, the crude CAT enzyme and the standard maintained more than 60% of their activity, showing a high tolerance to alkaline environments. The crude CAT enzyme had a high point of CAT activity when treated in a weakly alkaline environment of pH 8, which exceeded the CAT enzyme activity at pH 7.
[0170] 4.5 High temperature environment tolerance test of CAT crude enzyme solution and standard product
[0171] Then, the CAT crude enzyme solution and the standard product were subjected to a high temperature environment tolerance test by heating them at different temperatures for 10 minutes. Fig. 9 CAT crude enzyme solution can maintain stable enzyme activity at 25℃-75℃, and the standard product can maintain stable enzyme activity at 35℃
[0172] The enzyme activity reached its highest level and then dropped rapidly.
[0173] Then, heat at 50℃ or 60℃ and observe the enzyme activity of the crude CAT enzyme solution and the standard product within 100 min. Figure 10-11 When heated at 50℃, the activity of CAT crude enzyme solution tends to be stable within 100min. It is the same as the standard. When heated at 60℃, the activity of CAT crude enzyme solution tends to be stable within 100min. It is the same as the stability of the standard and the activity of CAT crude enzyme solution increases significantly after heating at 60℃ for 20min.
[0174] V. Test Purpose and Field of Application
[0175] Through the above specific embodiment experiments, it is found that the present invention has a mutant highly stable catalase and a construction method thereof. The catalase has a high prokaryotic expression level, can be directly synthesized, is suitable for large-scale preparation, is simple to operate, and has good solubility. The effective range of CAT standard products on the market is: temperature: 30°C-40°C, pH: 6.0-8.0, and the optimal range is: temperature: 35°C-40°C, pH: 6.5-7.0. The mutant highly stable catalase of the present application can still have stable and highest enzyme activity at 60 degrees Celsius.
[0176] In the food production process, CAT is often used in cake baking because it can decompose H2O2 into 02 and H20. It makes the cake fluffy and pore-filled. Before milk preservation and cheese making, H2O2 is used to sterilize cow milk and cheese raw milk, and then CAT is used to remove residual H2O2. Unlike heat sterilization, this sterilization can be carried out at low temperatures, which will not kill useful lactic acid bacteria, and will not affect the action of lipase, protease and phosphatase.
[0177] In the pulp and paper industry, the traditional chlorine-containing bleaching agents that have been used react with the residual lignin in the pulp to produce toxic pollutants such as chlorophenol compounds. Then, countries began to ban the use of chlorine-containing bleaching agents in the pulp and paper industry. Therefore, in recent years, the world's papermaking industry has successively used H2O2 bleaching to replace traditional bleaching methods. The H2O2 remaining in the paper can be neutralized with CAT to meet the production standards and put on the market.
[0178] After the clothes are bleached, CAT is often used to neutralize the residual H2O2. Compared with the traditional high-temperature water washing process to remove residual H2O2, it has the advantages of saving water, electricity, gas, and labor, and greatly reduces costs, improves production efficiency, and reduces pollution to the environment.
[0179] CAT protects cells from the toxic effects of H2O2. Its enzyme provides antioxidant defense. Since antioxidant complexes containing superoxide dismutase can be used for treatment, breaking down superoxide anions into H2O2, and CAT can break down H2O2 into non-toxic substances, it is believed that CAT can also be used to treat oxidative damage diseases.
[0180] CAT has a large market demand, so how to quickly and cheaply produce a large amount of high-enzyme activity CAT is a very important thing. The present invention project discovered a mutant highly stable catalase, which has high specific enzyme activity, heat resistance, acid and alkali resistance. It can save more costs during production, transportation and storage. The company can use fermentation purification to quickly produce a large amount of CAT. This not only saves costs, but also greatly shortens the output time and increases production efficiency. Therefore, the present invention is an invention of great significance that is close to practical application.
[0181] Finally, it should be noted that the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.
Claims
1. A mutant highly stable catalase, characterized in that: The protein sequence of the catalase is: MEARKAPRLTADGRPIGDNQNALTAGPRGPLLIQDVQLLEQIQHFNRERIPERVVHAKGSGAYGTFTVTNDVTRYTKAAFLSEVGKQTEVFVRFSTVAGERGAADAERDVRGFAVKFYTEEGN FDLVGNNTPVFFVRDPYKFQMFIHSQKRHPKTNLRDPDMQWDFWSLCPESLHQVTILFSDRGIPASYRHMNGYGSHTYSMYNDRGELFWVKFHFKTQQGIKCLTDEAARLIGEDRETHQRDLYE AIERGDYPRWTLYIQVMTPEQAENFRWNPFDLTKVWPHAEFPLIEVGVLELNRNPENYFAEVEQAAFKPSAFVPGIGPSPDKMLQARLMSYADAHRYRLGVNYQQLKVNRPRCPVHHYQRDGFMA QIEEGSGHPNYFPNSIPGSPQDDPIYKEPAWHLGEVIVDRYDSRKDHDDYTQAGNLYRLFDEGQKDRLARAIAASLGQARLEVQKRQLGHFYRADVDYGRRVARALGFDPAAAEAELGIDASVAG.
2. A method for constructing a mutant highly stable catalase as claimed in claim 1, characterized in that The construction method includes the following steps: Step 1) obtaining a catalase sequence from a thermophilic bacteria in the NCBI database; Step 2) synthesizing the mutated amino acid sequence using codon optimization; Step 3) amplifying the target gene and pET-28a vector using PCR; Step 4) Clone the target gene into the pET-28a vector using seamless cloning; Step 5) Transform into BL21(DE3) for expression.
3. The method for constructing a mutant highly stable catalase according to claim 2, characterized in that: Step 1)-step 2) specifically include: obtaining the catalase sequence WP_012845258.1 of thermophilic bacteria from the NCBI database, mutating the amino acids, and then optimizing the codons using Escherichia coli as the host, placing the gene synthesis sequence on pGEX-4T-1, using TOP10 as the host bacteria, and obtaining a plasmid containing the optimized target gene.
4. The method for constructing a mutant highly stable catalase according to claim 3, characterized in that: Plasmid construction: Using the synthesized plasmid containing pGEX-4T-1-CAT as a template, the target gene CAT was amplified by PCR using primers Cat-f and Cat-r, and the linearized vector fragment of pET28a containing homology arms was amplified by PCR using XF and XR. The product was electrophoresed through 1% agarose gel, and the size of the electrophoresis band of the product was detected by ultraviolet light and compared with the expected size. If they were consistent, the gel recovery kit was used for recovery.
5. The method for constructing a mutant highly stable catalase according to claim 4, characterized in that: Cat-f primer sequence (5'-3') GAGATATACCATGGAAGCGCGCAAAGCGCCGCG; Cat-r primer sequence (5'-3') TTCTTAGCCCGCCACGCTCGCATCAATGCCCA; XF primer sequence (5′-3′) GCGTGGCGGGCTAAGAATTCGAGCTCCGTC; XR primer sequence (5'-3') GCGCTTCCATGGTATATCTCCTTCTTAAAGTTAAAC.
6. A method for constructing a mutant highly stable catalase according to claim 4 or 5, characterized in that: Seamless cloning: The target gene CAT obtained by PCR recovery and the linearized vector of pET28a containing homology arms were connected by seamless cloning. The reaction conditions were 50°C for 30 minutes.
7. The method for constructing a mutant highly stable catalase according to claim 2, characterized in that: Step 5) specifically includes heat shock transformation of DH5α, plasmid extraction, heat shock transformation of BL21 (DE3), and strain induction expression; heat shock transformation of DH5α: transforming the seamless cloning product into DH5α competent cells, and the specific steps are: 1) Take out the DH5α competent cells from the -80℃ freezer and thaw them on ice; 2) Add the seamless cloning product to the thawed DH5α competent cells, tap the wall of the centrifuge tube to fully mix the seamless cloning product and DH5α competent cells, place on ice for 30 minutes, and turn on the 42°C water bath; 3) Heat shock in a 42°C water bath for 45 seconds, place on ice for 3-5 minutes, add 700uL culture medium, and culture at 37°C, 220rpm for 60 minutes; 4) Centrifuge at 12000rpm, 4℃ for 1min, discard the supernatant, keep 200uL for resuspending, apply on LB plate of corresponding resistance, and culture at 37℃ for 12h; 4) Plaque verification: colonies were picked from the above LB plate and placed in 5 mL LB medium. They were cultured at 37°C and 220 rpm for 12 h for identification. If the sequence was correct, the bacteria and plasmid were returned.
8. The method for constructing a mutant highly stable catalase according to claim 7, characterized in that: Plasmid extraction: transfer the returned plasmid, culture at 37°C, 220 rpm for 12 h, then use a plasmid small-scale extraction kit to extract the plasmid, measure the plasmid concentration by ultraviolet spectrophotometer, and store at -20°C.
9. The method for constructing a mutant highly stable catalase according to claim 7 or 8, characterized in that: Specific steps for heat shock transformation of BL21(DE3): 1) Take out the BL21(DE3) competent cells from the -80℃ freezer and thaw them on ice; 2) Add 10uL of the above plasmid to 100uL of thawed BL21(DE3) competent cells, flick the wall of the centrifuge tube to mix the plasmid and BL21(DE3) competent cells thoroughly, and place on ice for 30 minutes; 3) Turn on the 42°C water bath, heat shock for 45 seconds at 42°C, place on ice for 3-5 minutes, add 700uL culture medium, and culture at 37°C, 220rpm for 60 minutes; 4) Centrifuge at 12000 rpm and 4°C for 1 min, discard the supernatant, keep 200uL for resuspending, spread on LB plates with corresponding resistance, and then culture at 37°C for 12h.
10. The method for constructing a mutant highly stable catalase according to claim 9, characterized in that: Strain induction expression: 1) Pick a single colony into 5 mL LB medium, add 5 uL 50 mg / mL Kan solution, and culture at 37°C, 220 rpm until OD600 is 0.6-0.8; 2) Add 5 uL 1 M IPTG and culture at 37°C, 220 rpm for 12 h to obtain catalase protein.
Citation Information
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