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

Abstract

The present invention discloses a laccase mutant enzyme, its encoding gene, expression strain and application thereof. The present invention uses the laccase of Coprinopsis cinerea as the starting enzyme to search for homologous sequences, analyze their evolutionary relationships, and reconstruct the ancestral sequence to obtain the mutant gene. After inducing expression of the engineered bacteria containing the mutant gene, a laccase mutant enzyme Lcc5-1 with improved stability is obtained. When ABTS is used as the substrate, the mutant enzyme Lcc5-1 is stably improved to 4 times that of the starting enzyme at 45°C and pH 6-8. The mutant enzyme has potential application value in the oxidative conversion of zearalenone.

Description

Technical Field

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

[0002] Laccase is a copper-containing polyphenol oxidase that catalyzes the oxidation of various phenolic and non-phenolic compounds to the corresponding quinones. Simultaneously, electrons are transferred to reduce the molecules to water, without generating any other byproducts. Laccase has a wide range of substrates, including phenols and their derivatives, and aromatic amines and their derivatives. Due to its environmentally friendly, efficient, and broad substrate spectrum, laccase has potential applications in bioremediation, toxin oxidation, and degradation of environmental pollutants.

[0003] Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin primarily produced by Fusarium spp. and widely found in cereals such as corn, barley, wheat, and sorghum, as well as their by-products. ZEN enters the food chain through contaminated cereal agricultural products and feed. Once absorbed by animals, ZEN causes estrogen syndrome in livestock, leading to excess estrogen in the body and causing infertility, miscarriage, and stillbirth. It is also highly carcinogenic, severely endangering both livestock and human health. Zearalenone, primarily found in crops such as corn and peanuts, has severely impacted my country's exports of peanuts, corn, and their products, causing significant losses to my country's economy and trade.

[0004] Thanks to the rapid development of high-throughput sequencing technology, a large amount of nucleotide and protein data from different samples have been sequenced and uploaded to public databases. Through bioinformatics, such as database search, potential enzyme sequences can be analyzed and discovered. Based on the analysis of the amino acid sequence and protein structure of the enzyme, many efficient enzymes can be mined from these data using highly conserved sequences, seed models and other means to provide high-quality enzymes for biocatalysis. Therefore, based on bioinformatics technology, mining laccases with good enzyme activity and stability and applying them to toxin oxidation conversion is also an effective strategy for obtaining high-quality detoxification enzymes. Based on the analysis of the amino acid sequence and protein structure of the enzyme, the use of bioinformatics technology circumvents the shortcomings of traditional natural screening methods such as high cost, long time consumption and low success rate. On the contrary, it can reduce costs and increase efficiency, and accurately screen out new target enzyme proteins with expected specificity from massive databases with high success rate in a short period of time. Summary of the Invention

[0005] The present invention provides a laccase mutant, its encoding gene, expression strain, and application thereof. Starting from the laccase Lcc5 of Coprinus cinerea (C. cinerea), the present invention searches for homologous sequences, analyzes their evolutionary relationships, and reconstructs the ancestral sequence to obtain the mutant gene. After inducing expression in the expression strain containing the mutant gene, a laccase mutant Lcc5-1 with improved stability is obtained. The half-life of the mutant enzyme at pH 6 and 45°C is increased to 10 hours, four times that of the original enzyme. The mutant enzyme Lcc5-1 has potential application value in the oxidation and conversion of zearalenone. Lcc5-1 oxidizes 54% of zearalenone, while the original enzyme Lcc5 only oxidizes 34% of zearalenone. The oxidation conversion rate is increased to 1.6 times that of the original enzyme, indicating that Lcc5-1 has application prospects in the oxidation and conversion of zearalenone.

[0006] The laccase mutant enzyme of the present invention has an amino acid sequence as shown in SEQ ID No: 1, and the amino acid sequence changes compared to the starting enzyme Lcc5 are as follows: glycine at position 40 is mutated to alanine, glutamic acid at position 54 is mutated to glutamine, aspartic acid at position 74 is mutated to threonine, glutamic acid at position 127 is mutated to glutamine, serine at position 190 is mutated to proline, glutamine at position 191 is mutated to aspartic acid, glycine at position 207 is mutated to histidine, aspartic acid at position 217 is mutated to glycine, serine at position 218 is mutated to lysine, aspartic acid at position 232 is mutated to phenylalanine, alanine at position 277 is mutated to proline and glycine at position 296 is mutated to asparagine, threonine at position 402 is mutated to proline, methionine at position 492 is mutated to valine, and proline at position 513 is mutated to aspartic acid.

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

[0008] The nucleotide sequence of the gene encoding the laccase mutant enzyme 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 enzyme as described in SEQ ID No: 2.

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

[0011] The engineered strain of the laccase mutant enzyme of the present invention is classified and named Pichia pastoris GS115 / pPic9k(+)-Lcc5-1, and has been sent to the China Center for Type Culture Collection for preservation with the preservation number CCTCC NO: M 20231112. The preservation date is June 27, 2023, and the preservation address is Wuhan University, Wuhan, China.

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

[0013] Using the laccase of Coprinus cinerea as a starting enzyme, we searched for homologous sequences, analyzed their evolutionary relationships, and reconstructed their ancestral sequences using RAxML to obtain multiple mutant enzymes. Using ProtParam, we screened for gene sequences capable of stable expression in Pichia pastoris. Molecular docking simulations were performed, and the mutant enzymes with the highest molecular docking scores for zearalenone were selected. Laccase Lcc5-1, which may have improved detoxification performance and stability, was identified. The entire gene was synthesized and ligated into the vector pPic9k. The linearized plasmid pPic9k was electroporated into the expression host, Pichia pastoris GS115. The mutant gene was integrated into the Pichia pastoris alcohol oxidase genome. After plate screening, an engineered strain containing the mutant gene of the present invention was obtained.

[0014] The laccase mutant enzyme of the present invention can be obtained by fermentation of the engineered strain.

[0015] The present invention discloses a mutant laccase enzyme for the oxidative conversion of zearalenone. When reacted in a constant-temperature water bath shaker at 150 rpm and pH 7 for 6 hours at 45°C, the mutant enzyme Lcc5-1 achieved an oxidative conversion rate of 54% for zearalenone, compared to 34% for the original enzyme Lcc5. This mutant enzyme exhibits potential application in the oxidative conversion of zearalenone.

[0016] The present invention measured and compared the temperature adaptability and pH stability of the mutant enzyme and the original enzyme. The results showed that while the mutant's optimal temperature remained unchanged compared to the original enzyme, its enzyme activity at high temperatures was significantly improved. Furthermore, the mutant enzyme's stability was also enhanced. At 45°C and pH 6, the half-life of the mutant enzyme was four times that of the original enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The SDS-PAGE profiles of the purified starting enzyme Lcc5 and the mutant enzyme Lcc5-1 of the present invention are shown in FIG. 1 : the lanes are respectively marker, pure Lcc5 enzyme, and pure Lcc5-1 enzyme of the present invention.

[0018] Figure 2 These are the results of determining the optimum temperatures for the starting enzyme Lcc5 and the mutant enzyme Lcc5-1 of the present invention.

[0019] Figure 3 Figure a shows the pH stability of the starting enzyme Lcc5 at 45°C; Figure b shows the pH stability of the mutant enzyme Lcc5-1 of the present invention at 45°C.

[0020] Figure 4 This is an experiment on the oxidation conversion of zearalenone by the starting enzyme Lcc5 and the mutant enzyme Lcc5-1 of the present invention. DETAILED DESCRIPTION

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

[0022] (1) Construction of an engineered strain containing the laccase mutant gene of the present invention

[0023] 1. Obtaining laccase gene mutant enzyme

[0024] This study used Lcc5 from Coprinus cinerea as a starting enzyme, searched for homologous sequences, and obtained 150 sequences. A phylogenetic tree was constructed using RAxML to analyze their evolutionary relationships. Paxml's CodeML was used to perform ancestral sequence reconstruction, resulting in 132 ancestral sequences. ProtParam was then used to screen for gene sequences that could be stably expressed in Pichia pastoris. Molecular docking simulations were then performed, and the mutant enzyme with the highest docking score with zearalenone was selected as a candidate mutant enzyme, named Lcc5-1.

[0025] The amino acid sequence of the laccase mutant enzyme of the present invention is shown in SEQ ID No: 1, and the changed positions compared with the original enzyme Lcc5 are as follows: glycine at position 40 is mutated to alanine, glutamate at position 54 is mutated to glutamine, aspartic acid at position 74 is mutated to threonine, glutamate at position 127 is mutated to glutamine, serine at position 190 is mutated to proline, glutamine at position 191 is mutated to aspartic acid, glycine at position 207 is mutated to histidine, aspartic acid at position 217 is mutated to glycine, serine at position 218 is mutated to lysine, aspartic acid at position 232 is mutated to phenylalanine, alanine at position 277 is mutated to proline and glycine at position 296 is mutated to asparagine, threonine at position 402 is mutated to proline, methionine at position 492 is mutated to valine, and proline at position 513 is mutated to aspartic acid.

[0026] 2. Construction of laccase mutant genetically engineered strains

[0027] The laccase mutant gene in step 1 is fully synthesized and ligated into the expression vector pPic9k, with the expression host being Pichia pastoris, to obtain an engineered strain containing the mutant gene of the present invention.

[0028] The engineered strain of the laccase mutant enzyme of the present invention is classified and named Pichia pastoris GS115 / pPic9k(+)-Lcc5-1, and has been sent to the China Center for Type Culture Collection for preservation with the preservation number CCTCC NO: M 20231112. The preservation date is June 27, 2023, and the preservation address is Wuhan University, Wuhan, China.

[0029] (II) Expression of genetically engineered bacteria containing laccase mutations and protein purification

[0030] The expression strain Pichia pastoris GS115 / pPic9k(+)-Lcc5-1 obtained in (1) 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 conical flask containing 50 mL of BMGY and cultured until the OD 600 When it reaches 2.0, centrifuge at 4°C and 3000rpm for 20min, and resuspend the bacteria in BMM liquid; the resuspended bacteria are placed in a 500mL triangular flask with a baffle (containing 200mL BMM liquid culture medium). Samples are taken every 24 hours, and an equal amount of methanol is added to the sample and induced at 28°C. The enzyme activity of the sample is detected, and the bacterial liquid is collected after 7 days of culture in BMM culture medium to purify the target protein. The crude enzyme solution after centrifugation and dialysis is filtered with a 0.22μm filter membrane to remove impurities and bubbles in the supernatant, and loaded onto a DEAE-SpharoseFastFlow anion exchange column. The obtained protein is tested by SDS-PAGE to achieve the purity of enzymatic properties detection ( Figure 1 ).

[0031] (III) Detection of temperature adaptability of the mutant enzyme of the present invention

[0032] Laccase enzyme activity assay: The total reaction system is 1 mL, including 33 μL ABTS (final concentration 15 mM), 950 μL sodium tartrate buffer solution (pH 4.0), incubated at 30°C for 5 minutes, and 17 μL enzyme solution is added to the EP tube to make the reaction system 1 mL. After reacting at 30°C for 3 minutes, the reaction is allowed to stand on ice for 30 seconds, and the OD is measured by spectrophotometer. 420 value.

[0033] The enzyme activity calculation formula is: enzyme activity (U / L) = 555.56 × dilution factor × OD 420

[0034] Test results such as Figure 2It was shown that when ABTS was used as the substrate, the optimum temperature of the mutant enzyme obtained by the present invention was 60°C, the enzyme could show more than 50% enzyme activity in the range of 50°C-80°C, and the enzyme activity was higher than that of the starting enzyme Lcc5 in the environment of 70°C-80°C. The temperature adaptability of the mutant enzyme of the present invention was improved.

[0035] (IV) Stability of the Laccase Mutant Enzyme Containing the Present Invention at Different pH

[0036] Using ABTS as substrate, the mutant enzyme was incubated at 45°C in different pH buffers, and samples were taken at intervals to detect the enzyme activity. The initial enzyme activity was taken as 100%, and the remaining enzyme activity after a certain period of time was calculated. Figure 3 It was shown that the half-life of the mutant enzyme obtained in the present invention at 45° C. and pH 6 was 10 h, which was 4 times that of the original enzyme.

[0037] (V) Application of the Laccase Mutant Enzyme Containing the Present Invention in Toxin Oxidation and Conversion Experiments

[0038] The detoxification reaction system is 1 mL, of which the enzyme amount is 2 U, 950 μL of disodium hydrogen phosphate citric acid buffer, and the final concentration of zearalenone is 10 μg / mL. It is shaken and mixed, and placed in a constant temperature water bath shaker at 45°C and 150 r / min for reaction. Samples are taken at intervals, and 200 μL of methanol is added to terminate the reaction. Then, it is vortexed and shaken for 30 seconds using a vortex shaker. After the shaking is completed, it is centrifuged at 12000 r / min in a refrigerated centrifuge for 10 minutes, and the supernatant is taken. The methanol containing zearalenone is filtered with a 0.22 μm organic needle filter for preparation for machine detection. HPLC detection conditions: mobile phase: acetonitrile-water-methanol = 46:46:8 (V / V / V), chromatographic column selected Agilent 5TC-C18 (2) chromatographic column (column length 250 mm, column inner diameter 4.6 mm, particle size 5 μm, Sun Fire); set the flow rate to 1 mL / min; column temperature to 25 °C; detector set fluorescence detection excitation wavelength to 274 nm; emission wavelength to 440 nm.

[0039] Test results such as Figure 4 It showed that under the same conditions, with zearalenone as the substrate, the oxidation conversion rate of the mutant enzyme Lcc5-1 was 54%, and the oxidation conversion rate of the starting enzyme Lcc5 was 34%. The oxidation conversion efficiency of zearalenone by the mutant enzyme was 60% higher than that of the starting enzyme.

Claims

1. A laccase mutant enzyme, characterized in that: The laccase mutant enzyme is abbreviated as Lcc5-1, and its amino acid sequence is shown in SEQ ID No:

1.

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

2.

3. The engineered strain of the laccase mutant enzyme according to claim 1, characterized in that: The engineering strain is classified and named Pichia pastoris GS115 / pPic9k(+)-Lcc5-1, and has been sent to the China Center for Type Culture Collection for preservation, with the preservation number CCTCC NO: M 20231112, the preservation date is June 27, 2023, and the preservation address is: Wuhan University, Wuhan, China.

4. Use of the laccase mutant enzyme according to claim 1 in the oxidation conversion of zearalenone, characterized in that: The laccase mutant enzyme was applied to directly oxidize zearalenone.

5. The use according to claim 4, characterized in that: Using zearalenone as substrate, the oxidative conversion of the mutant enzyme increased by 60% compared with the original enzyme at pH 7 and 45℃.

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