A laccase mutant, its encoding gene, expression strain and application thereof

Abstract

The present invention discloses a laccase mutant, its encoding gene, expression strain and application thereof. The present invention searches for homologous sequences based on the laccase LacB of Trametes sp., analyzes its evolutionary relationship, and obtains mutant genes through ancestral sequence reconstruction. After the engineered bacteria containing the mutant gene are induced to express, a laccase mutant LacANC with improved specific enzyme activity and improved stability is obtained, and the specific enzyme activity of the mutant is increased by 3.5 times. While the specific activity is improved, the stability of the mutant is improved by 20 times, and the mutant has potential application value in the oxidation and conversion of aflatoxin B1.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a laccase mutant, a coding gene, an expression strain and an application thereof. Background Art

[0002] Laccase is a copper-containing polyphenol oxidase that can catalyze the oxidation of a variety of phenolic and non-phenolic substances, while reducing oxygen to water with the transfer of electrons. Laccase has a wide range of substrates, including phenols and their derivatives, aromatic amines and their derivatives. Laccase has potential application value in many fields such as bioremediation, toxin oxidation and transformation, and degradation of environmental pollutants.

[0003] Aflatoxin B1 is a secondary metabolite produced by fungi such as Aspergillus flavus, Aspergillus parasiticus and Aspergillus tertius. A large number of studies have confirmed that aflatoxin B1 is the most toxic and carcinogenic pollutant found today. The main target organ of this toxin is the liver, which can cause liver necrosis, the formation of liver cancer, cause animal immunosuppression and reduce egg and milk production.

[0004] Currently, laccases used in the oxidation and transformation of aflatoxin B1 are mostly natural laccases produced by fungal fermentation. In the actual toxin oxidation and transformation system, natural laccases are more easily inactivated, so it is necessary to transform natural laccases to screen for mutants with higher stability. Based on bioinformatics technology, mining laccases with good enzyme activity and stability and applying them to toxin oxidation and transformation is also an effective strategy to obtain high-quality detoxification enzymes. Based on the analysis of the amino acid sequence and protein structure of laccases, using bioinformatics technology, it is expected to accurately screen and obtain new target enzyme proteins from massive databases. Summary of the invention

[0005] The present invention provides a laccase mutant, its encoding gene, expression strain and application thereof. Trametes sp.) as the starting point to search for homologous sequences, analyze their evolutionary relationships, and reconstruct ancestral sequences to obtain mutant genes. After the engineered bacteria containing the mutant gene were induced to express, a laccase mutant LacANC with improved specific enzyme activity and improved stability was obtained. When ABTS was used as a substrate, the specific enzyme activity of the mutant increased by 3.5 times. While the specific activity was improved, the half-life of the mutant increased to 350min at 60°C, which was 20 times that of the starting enzyme. The mutant has potential application value in the oxidation and conversion of aflatoxin B1. The experimental results showed that the mutant LacANC oxidized 17.7% of aflatoxin B1, and the starting enzyme LacB oxidized 7.9% of aflatoxin B1, and the oxidation conversion efficiency increased by 1.24 times. The mutant has potential application value in the efficient oxidation and conversion of aflatoxin B1.

[0006] The laccase mutant LacANC of the present invention is a laccase mutant obtained by mutating the 27th alanine to valine, the 89th serine to proline, the 241st serine to glycine, the 253rd serine to proline, the 286th isoleucine to proline, the 291st alanine to threonine, the 341st glutamic acid to valine, the 343rd leucine to phenylalanine, the 368th threonine to proline, the 376th leucine to proline, the 410th serine to tryptophan, the 449th alanine to proline, and the 475th proline to glutamic acid. The amino acid sequence of the laccase mutant LacANC is shown in SEQ ID No: 1.

[0007] The amino acid sequence of the laccase mutant of the present invention may also include a combination of nonsense mutations or synonymous mutations in the sequence.

[0008] The nucleotide sequence of the coding gene of the laccase mutant of the present invention is shown in SEQ ID No:2.

[0009] The mutant gene of the present invention is a gene encoding a laccase mutant as described in SEQ ID No:2.

[0010] The expression strain of the laccase mutant of the present invention contains the mutant gene.

[0011] The expression strain of the laccase mutant of the present invention is classified and named Shepherd's pie GS115 / pPic9k(+)- lacANC , has been sent to the China Center for Type Culture Collection for preservation. The preservation date is March 3, 2023, and the preservation number is CCTCC NO: M2023238.

[0012] The method for constructing the expression strain of the laccase mutant of the present invention comprises the following steps:

[0013] Trametes Trametes sp.) laccase LacB was used as a starting point to search for homologous sequences, analyze their evolutionary relationships, and reconstruct ancestral sequences through RAxML to obtain multiple mutants. According to the calculation results of ProtParam, the protein stability parameters (Instability index), aliphatic index, and GRAVY value were selected as stable mutants. Molecular docking simulation was performed on the determined stable mutants, and the mutant with the highest molecular docking score with aflatoxin B1 was selected to determine the mutants with improved detoxification performance and stability of the laccase LacANC. The whole gene was synthesized and connected to the vector pPic9k, and the linearized plasmid pPic9k was electroporated into the expression host Pichia pastoris. Shepherd's Pie GS115, the mutant gene is integrated into the Pichia pastoris alcohol oxidase genome, and after MD and BMM plate screening, an engineered strain containing the mutant gene of the present invention is obtained.

[0014] The laccase mutant protein of the present invention can be obtained by fermenting the engineering strain.

[0015] The application of the laccase mutant of the present invention is to use the laccase mutant for the oxidation conversion of aflatoxin B1. After 3 days of reaction in a constant temperature water bath shaker at pH 6.5, 45°C, and 150r / min, the oxidation conversion rate of the mutant LacANC was 17.7%, and the oxidation conversion of the starting enzyme LacB was 7.9%. The mutant was 1.24 times more efficient than the starting enzyme. The mutant has potential application value in the oxidation conversion of aflatoxin B1.

[0016] The present invention measured and compared the specific enzyme activity, optimal temperature, stability, etc. of the mutant protein and the original starting enzyme protein. The measurement results show that the specific enzyme activity of the mutant obtained by the present invention is increased to 3.5 times. While the specific activity is improved, the stability of the mutant is also improved. Under the conditions of 60°C and pH 4.0, the half-life of the mutant is increased by 20 times. Compared with the starting enzyme, the optimal temperature is changed from 50°C to 70°C, which improves the catalytic ability in high temperature environment (65-75°C). BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The SDS-PAGE profiles of the purified starting enzyme LacB and the mutant LacANC of the present invention are shown in the figure below: the lanes are Marker, pure LacB enzyme, and pure LacANC enzyme of the present invention, respectively.

[0018] Figure 2 Figure a in the middle shows the results of the determination of the optimum temperature of the starting enzyme LacB; Figure b shows the results of the determination of the temperature of the mutant LacANC of the present invention.

[0019] Figure 3 Figure a in the middle shows the temperature stability of the starting enzyme LacB at 50°C, 60°C, 70°C, pH 4.0; Figure b shows the temperature stability of the mutant LacANC of the present invention at 50°C, 60°C, 70°C, pH 4.0.

[0020] Figure 4 This is an experiment on the oxidation conversion of aflatoxin B1 by the starting enzyme LacB and the mutant LacANC of the present invention. DETAILED DESCRIPTION

[0021] The implementation methods in the following embodiments are all conventional methods unless otherwise specified.

[0022] (I) Construction of an expression strain containing the laccase mutant gene of the present invention

[0023] 1. Obtaining laccase gene mutants

[0024] Invention of Trametes tricholoma ( Trametes sp.) as the starting point to search for homologous sequences and obtain 11 starting sequences. RAxML was used to construct a phylogenetic tree to analyze its evolutionary relationship. Paxml's CodeML was used to complete the calculation of ancestral sequence reconstruction (important parameters were set to: ncatG8, Small-diff5e-6, fixblength2:fixed), and 9 ancestral sequences were obtained. According to the calculation results of ProtParam, the protein stability parameter (Instability index) was 25-30, the fat coefficient (Aliphaticindex) was 68-92, and the GRAVY value was negative, and the parameters all showed that the mutant was stable. Molecular docking simulation was performed on the determined stable mutants, and the mutant with the highest docking score with aflatoxin B1 was selected to determine the laccase LacANC with improved detoxification performance and stability of the mutant, and obtain the mutant gene.

[0025] The laccase mutant LacANC of the present invention is a laccase mutant obtained by mutating the 27th alanine to valine, the 89th serine to proline, the 241st serine to glycine, the 253rd serine to proline, the 286th isoleucine to proline, the 291st alanine to threonine, the 341st glutamic acid to valine, the 343rd leucine to phenylalanine, the 368th threonine to proline, the 376th leucine to proline, the 410th serine to tryptophan, the 449th alanine to proline, and the 475th proline to glutamic acid. The amino acid sequence of the laccase mutant LacANC is shown in SEQ ID No: 1.

[0026] 2. Construction of laccase mutant genetic engineering strain

[0027] The laccase mutant gene in step 1 was fully synthesized and connected to the expression vector pPic9k, and the expression host was Pichia pastoris.

[0028] 2. Expression of genetically engineered bacteria containing laccase mutations and protein purification

[0029] The engineered strain obtained in (a) Shepherd's PieGS115 / pPic9k(+)-LacANC was inoculated into a test tube containing 5 mL of BMGY, cultured overnight in a shaker at 28°C and 200 rpm, and then transferred to a flask containing 50 mL of BMGY and cultured until OD 600 When it reaches 2.0, centrifuge at 4℃ and 3000rpm for 20min, and resuspend the bacteria in BMM liquid; the resuspended bacteria are connected to a 500mL triangular flask with baffles (containing 200mL of BMM liquid culture medium). Take samples every 24h, take a sample and add an equal amount of methanol, and induce culture at 28℃. By detecting the changes in sample enzyme activity, culture in BMM culture medium for 7 days, collect the bacterial liquid and purify the target protein. After centrifugation and dialysis, the crude enzyme solution is filtered with a 0.22μm filter membrane to remove impurities and bubbles in the supernatant, and loaded on a DEAE-SpharoseFastFlow anion exchange column. The obtained protein is tested by SDS-PAGE to achieve the purity of enzymatic property detection.

[0030] (III) Detection of enzyme activity and temperature adaptability of the laccase mutant

[0031] Laccase enzyme activity assay method: The total reaction system is 1 mL. Take 17 μL ABTS (15 mM) and 950 μL sodium tartrate buffer solution (pH 4.0) and add them to a 2.0 mL EP tube. Incubate at 30°C for 5 min. Take 33 μL enzyme solution and add it to the EP tube to make the reaction system 1 mL. After reacting at 30°C for 3 min, place on ice for 30 s and measure OD by spectrophotometer. 420 value.

[0032] The enzyme activity calculation formula is: enzyme activity (U / L) = 555.6 × dilution factor × OD 420 .

[0033] The test results show that when ABTS is used as a substrate, the mutant obtained by the present invention has improved adaptability in a high temperature environment.

[0034] (IV) Testing of the stability of the laccase mutant of the present invention at 50°C, 60°C and 70°C

[0035] Using ABTS as substrate, the mutant enzyme was incubated at 50°C, 60°C, 70°C, pH 4.0, and samples were taken every 0.5h. The initial enzyme activity was taken as 100%, and the residual enzyme activity after a certain period of time was calculated. The formula is as follows: residual enzyme activity = (initial enzyme activity - loss enzyme activity) / initial enzyme activity × 100%. The measurement results show that the half-life of the mutant obtained by the present invention at 60°C and pH 4.0 is 350min, which is 20 times that of the starting enzyme.

[0036] (V) Application of the Laccase Mutant Containing the Present Invention in Toxin Oxidation and Transformation Experiments

[0037] The detoxification reaction system is 1mL, in which the amount of enzyme added is 2U, that is, the enzyme activity is 2U / mL, and the citric acid disodium hydrogen phosphate buffer (pH6.5) is added to 950μL, the final concentration of aflatoxin B1 is 0.2μg / mL, and the mixture is shaken and mixed, and placed in a constant temperature water bath shaker at 45℃ and 150r / min for 3 days. Every 24h, 200μL of the reaction system is added with 300uL methanol to terminate the reaction, and the enzyme activity is measured, and then vortexed and shaken for 30s with a vortex shaker. After the shaking is completed, the supernatant is taken and the methanol containing aflatoxin B1 is filtered with a 0.22μm organic needle filter for machine detection. The control is a mixed system of 950μL buffer and 50μL of aflatoxin B1 standard working solution. Three parallels are set for the control group and the experimental group.

[0038] HPLC detection conditions:

[0039] Aflatoxin: Mobile phase: methanol-water = 45:55 (V / V) Set the flow rate to 1 mL / min; Column temperature to 25°C; Detector set fluorescence detection excitation wavelength to 360 nm; Emission wavelength to 440 nm. Post-column photochemical derivatization device.

[0040] The experimental results showed that under the same conditions, with aflatoxin B1 as the substrate, the oxidation conversion rate of the mutant LacANC was 17.7%, and the oxidation conversion rate of the starting enzyme LacB was 7.9%. The oxidation conversion efficiency of the mutant was 1.24 times higher than that of the starting enzyme.

Claims

1. A laccase mutant, characterized in that: The amino acid sequence of the laccase mutant is shown in SEQ ID No:

1.

2. The gene encoding the laccase mutant according to claim 1, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID No:

2.

3. An expression strain of the laccase mutant according to claim 1, characterized in that: The expression strain is Pichia pastoris GS115 / pPic9k(+)- lacANC , has been sent to the China Center for Type Culture Collection for preservation. The preservation date is March 3, 2023, and the preservation number is CCTCC NO: M2023238.

4. A use of the laccase mutant according to claim 1, characterized in that: The laccase mutant was used for oxidative conversion of aflatoxin B1.

5. The use according to claim 4, characterized in that: After reacting in a constant temperature water bath shaker at pH 6.5, 45° C., and 150 r / min for 3 days, the laccase mutant had an oxidation conversion rate of 17.7% for aflatoxin B1.

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