Lactone hydrolase mutant capable of efficiently degrading zearalenone and application of lactone hydrolase mutant

Abstract

The invention relates to the technical field of enzyme engineering, in particular to a lactone hydrolase mutant capable of efficiently degrading zearalenone and application of the lactone hydrolase mutant, the lactone hydrolase mutant takes a lactone hydrolase gene from Monosporaascus sp.GIB2 as a template, and site-specific mutagenesis is performed on the 134 amino acid of the lactone hydrolase gene to obtain L134A, L134VL134I and L134M mutants. Under the optimal conditions of pH 9.0 and 60 DEG C, the single-site mutants can efficiently degrade zearalenone (ZEN) within 3 minutes, the activity of the zearalenone degrading enzyme is remarkably improved compared with that of an original enzyme, the degradation activities are improved by 1.22 (L134A), 1.25 (L134V), 1.17 (L134I) and 1.14 (L134M) times respectively, and the single-site mutants have important feed industrial application and economic value.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, and specifically to an lactonase mutant for degrading zearalenone and its application. Background Art

[0002] Zearalenone (ZEN) is mainly present in moldy corn, wheat, barley and other grains and their by-products, and is one of the most widely polluted mycotoxins worldwide. ZEN has a lactone structure with phenolic dihydroxy groups, which is similar to the structure of estradiol, and can competitively bind to estrogen receptors, resulting in disorders of estrogen levels and reproductive systems in animals. In 1962, ZEN was first discovered in moldy corn and is a toxic secondary metabolite produced by the genus Fusarium. It is widely present in moldy corn, wheat, barley and other grains and grain by-products, and has become a food and feed contaminant worldwide. Five ZEN derivatives have been discovered so far, namely: α / β-zearalenol (α / β-ZOL), α / β-zearalanol (α / β-ZAL) and zearalanone (ZAN), among which α-ZOL exhibits higher estrogenic toxicity than ZEN.

[0003] Currently, there are mainly three kinds of bioenzymes that can degrade ZEN: peroxidase, laccase and lactonase. Although peroxidase has been found to oxidatively cleave the phenolic dihydroxy benzene ring structure in the ZEN molecule, a large amount of hydrogen peroxide is required in the reaction process. Secondly, laccase removes ZEN by oxidizing the phenolic hydroxyl group in the ZEN molecule, but the action site is uncertain and the efficiency is low, making it difficult to meet the actual application requirements. In contrast, the degradation mechanism of lactonase hydrolyzing the C12’-position lactone bond of ZEN is clear, the degradation product is non-toxic, and the reaction does not depend on the medium. It is currently the most concerned and promising ZEN degrading enzyme. In fact, there are still many problems with the reported ZEN lactonases, such as low enzyme activity in catalyzing ZEN and its derivatives. Products contaminated with ZEN often contain high concentrations of reproductive toxic ZEN derivatives. As an enzyme for feed use, it is required to be able to efficiently degrade ZEN.

[0004] Therefore, in order to obtain a ZEN lactonase more suitable for feed industry applications, the present invention uses site-directed mutagenesis to modify the activity of the ZEN lactonase derived from Monosporascus sp. GIB2, improve its degradation ability of ZEN, and obtain a ZEN lactonase more suitable for industrial applications. Summary of the Invention

[0005] The object of the present invention is to provide a lactonase mutant for efficiently degrading zearalenone and its application.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The nucleic acid sequence and amino acid sequence of the wild-type ZEN lactonase derived from Monosporascus sp. GIB2 are shown in SEQ ID NO: 1-2. The present invention performs site-directed mutagenesis on the 134th amino acid thereof, mutating the leucine at the 134th position of the amino acid sequence to alanine, valine, isoleucine, and methionine, to obtain mutants L134A, L134V, L134I, and L134M, and their amino acid sequences are shown in SEQ ID NO: 3-6.

[0008] Compared with the wild enzyme, the optimal pH 9.0 and optimal temperature 60 °C of the mutants L134A, L134V, L134I, and L134M of ZEN lactonase hydrolysis are consistent with those of the wild type, but the degradation activity against ZEN has been significantly improved. Under the optimal catalytic conditions, the relative activity of mutant L134A against ZEN is 1.22 times higher than that of the wild type. Under the same catalytic conditions, the degradation activity of mutant L134V against ZEN is 1.25 times higher than that of the wild type. The degradation activity of mutant L134I against ZEN is 1.17 times higher than that of the wild type, and the degradation activity of mutant L134M against ZEN is 1.14 times higher than that of the wild type. This has important research value for the detoxification of ZEN and its derivatives by using lactonase in the feed industry.

[0009] The present invention provides a coding gene encoding the above lactonase mutant.

[0010] The present invention also provides a recombinant expression vector pET-22b(+) carrying the above coding gene.

[0011] The present invention also provides a recombinant expression host bacterium Escherichia coli E. coli BL21(DE3), and the expression host bacterium contains the above recombinant expression vector.

[0012] Compared with the prior art, the beneficial effects of the present invention are:

[0013] (1) Through a large number of experimental explorations, the present invention obtained ZEN lactonase mutants L134A, L134V, L134I, and L134M derived from Monosporascus sp. GIB2, with significantly improved degradation activity against ZEN. Among them, the enzyme activity of mutant L134A against ZEN is 454 U / mg, which is 1.22 times higher than that of the wild enzyme (371 U / mg). The degradation enzyme activity of mutant L134V against ZEN is 466 U / mg, which is 1.25 times higher than that of the wild enzyme (371 U / mg). The degradation enzyme activity of mutant L134I against ZEN is 434 U / mg, which is 1.17 times higher than that of the wild enzyme (371 U / mg). The degradation enzyme activity of L134M against ZEN is 424 U / mg, which is 1.14 times higher than that of the wild enzyme (371 U / mg). According to the enzyme reaction kinetics study, the kcat / Km values of mutants L134A and L134V against ZEN are 0.356 min -1 μM -1 and 0.367 min -1 μM -1 respectively, both higher than that of the wild enzyme, indicating that the catalytic efficiency is significantly improved after mutation.

[0014] (2) The mutants L134A, L134V, L134I, and L134M obtained in the present invention have important research value for the industrial treatment of zearalenone pollution in feeds and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is an SDS-PAGE electrophoresis diagram of the recombinant expression host bacterium BL21(DE3) induced to express ZEN lactonase and its mutants according to an embodiment of the present invention. Among them, M: Marker; Lane 1: purified mutant enzyme L134A; Lane 2: purified mutant enzyme L134V; Lane 3: purified mutant enzyme L134I; Lane 4: purified mutant enzyme L134M.

[0016] Figure 2 is the absolute activity diagram of the wild enzyme WT of ZEN lactonase and mutants L134A, L134V, L134I, and L134M against ZEN.

[0017] Figure 3 shows the enzymatic reaction rates of the wild enzyme, L134A, and L134V. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0018] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0019] Example 1 Preparation of ZEN Lactonase Mutants

[0020] (1) Obtaining the wild-type plasmid: Select pET-22b(+) as the backbone for expressing the target gene, and introduce the zearalenone lactonase ZENM gene (nucleotide sequence and amino acid sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2) from Monosporascus sp. GIB2 between two restriction enzyme sites NdeI and XhoI to generate a linearized vector. The vector is introduced into Escherichia coli competent cells through chemical transformation to obtain an engineered bacterium. The positive clone is cultured in large quantities in the medium, and a high-purity plasmid is extracted using a commercial kit.

[0021] (2) Construction of mutant plasmids: Site-directed mutagenesis is carried out according to the QuikChange TM method. Using the wild-type plasmid as a template, at the L134 position, leucine is mutated to alanine, valine, isoleucine, and methionine respectively to construct 4 single-point mutants (L134A, L134V, L134I, L134M), and the mutation results are verified as correct mutants by sequencing.

[0022] Using the recombinant wild-type plasmid as a template, forward and reverse primers for mutants L134A, L134V, L134I, and L134M are designed. The mutant primers are shown in Table 1 below (sequences are shown in SEQ ID NO: 7-14), and the lowercase letters are the mutation sites.

[0023] Table 1 Upstream and downstream primer sequences of mutants

[0024] Name Base sequence 5'-3' L134A-F <![CDATA[5’-CTAGCCCT gcg TTCGGTCAGCTCTTAAAACT-3’]]> L134A-R <![CDATA[5’-GACCGAA cgc AGGGCTAGCGGCCGTCGGAAC-3’]]> L134V-F <![CDATA[5’-CTAGCCCT gtg TTCGGTCAGCTCTTAAAACT-3’]]> L134V-R <![CDATA[5’-GACCGAA cac AGGGCTAGCGGCCGTCGGAAC-3’]]> L134I-F <![CDATA[5’-CTAGCCCT att TTCGGTCAGCTCTTAAAACT-3’]]> L134I-R <![CDATA[5’-GACCGAA aat AGGGCTAGCGGCCGTCGGAAC-3’]]> L134M-F <![CDATA[5’-CTAGCCCT atg TTCGGTCAGCTCTTAAAACT-3’]]> L134M-R <![CDATA[5’-GACCGAA cat AGGGCTAGCGGCCGTCGGAAC-3’]]>

[0025] PCR amplification: The total volume of the reaction system is 50 μL. The reaction program is: 95 °C, 5 min; 95 °C, 30 s; 57 °C, 30 s; 72 °C, 3 min; cycle 28 times; 72 °C, 5 min; 4 °C.

[0026] Table 2 Composition of the PCR reaction system

[0027] Component Added volume (μL) PrimeSTAR Max Premix (2×) 25 Forward primer F (20 μM) 1 Reverse primer R (20 μM) 1 Wild-type recombinant plasmid (10 ng / μL) 0.5 <![CDATA[Sterile water (ddH2O)]]> 22.5

[0028] (3) Agarose gel nucleic acid electrophoresis verification and template digestion: Through agarose gel electrophoresis, verify whether the size of the PCR product band is correct according to the Marker. After verification, add 1 μL of Q.cut DpnI and 2 μL of Q.cut Buffer (10×) to the PCR product system, and carry out an enzymatic digestion reaction in a 37°C water bath for 120 min to remove the template DNA in the PCR reaction system.

[0029] (4) Transformation: Introduce the digested PCR product into Escherichia coli DH5α competent cells and coat it on an LB solid plate containing 50 μg / mL ampicillin, and culture it overnight at 37°C. Subsequently, pick positive clones for plasmid extraction and DNA sequencing. Introduce the successfully sequenced mutant plasmid into Escherichia coli BL21(DE3) competent cells to construct a mutant gene recombinant expression strain for the induced expression of the mutant enzyme.

[0030] Example 2 Expression and nickel column affinity purification of mutant enzyme

[0031] Introduce the mutant plasmids pET-22b(+)-L134A, pET-22b(+)-L134V, pET-22b(+)-L134I, and pET-22b(+)-L134M, which are verified to be mutant successfully after sequencing, into the competent Escherichia coli BL21(DE3) of the expression host strain, and coat them on an LB solid plate containing 50 μg / mL ampicillin and culture them overnight at 37°C. Pick positive colonies into a liquid LB medium containing 50 μg / mL ampicillin, culture them overnight at 37°C and 200 rpm, then expand the seed liquid to 200 mL of liquid LB medium containing 50 μg / mL ampicillin at an inoculation amount of 2 / 1000, and culture them at 37°C and 200 rpm for 2 - 3 h until the OD value is 0.6 - 0.8. Subsequently, add 1 mM IPTG to the expanded medium and place it in a shaker at 25°C for induction for 6 h.

[0032] Centrifuge the induced fermentation broth at 8000 rpm for 5 min to collect the bacteria. Add 15 mL of lysis buffer (50 mM Tris, 200 mM NaCl, adjusted to pH 7.0 with HCl) to resuspend the bacteria. Place the resuspended bacteria in an ice-water bath mixture, and put this system into an ultrasonic cell disruptor, and carry out ultrasonic treatment at 450 kHz, ultrasonic for 1 s, stop for 2 s, for 20 min to break the fermentation cells to obtain intracellular products. Place the disrupted solution in a low-temperature centrifuge (4°C), centrifuge at 8000 rpm for 5 min, and collect the supernatant. Filter it with a 0.45 μm microporous filter membrane for standby.

[0033] Use nickel column affinity purification to purify the protein from the crude enzyme solution. First, connect the constant flow peristaltic pump in the protein purification system, Ni2+ The pipelines between instruments such as the affinity chromatography column and the 280 nm detector were set with a constant flow rate of 1.5 mL / min for the constant flow pump and leak detection was carried out. Two column volumes of equilibration buffer (50 mM Tris, 500 mM NaCl, pH 7.5) were prepared to equilibrate the nickel ion affinity chromatography column. After equilibrating the column, the crude lysed enzyme solution was pumped into the chromatography column at a flow rate of 0.5 mL / min. Subsequently, the equilibration buffer was continued to be used to wash the chromatography column at a flow rate of 1.5 mL / min. After the UV detector reading was stable, the washing buffer (30 mM imidazole, 50 mM Tris, 500 mM NaCl, pH 7.5) was used to wash away the miscellaneous proteins with weak binding ability to nickel ions. After the UV detector reading was stable again, the elution buffer (500 mM imidazole, 50 mM Tris, 500 mM NaCl, pH 7.5) was pumped in at a flow rate of 1 mL / min, and the washing solution, which was the target protein, was collected with a 10 mL centrifuge tube. The target protein was aspirated and transferred into a dialysis bag with a molecular weight cut-off of 14 kDa. After clamping it with a double-layer dialysis clip, it was placed in dialysis solution A (10 mM EDTA·2Na, 50 mM Tris, pH 7.5). After dialysis overnight, it was changed to dialysis solution B (50 mM Tris, pH 7.0) for dialysis for 6 h. After dialysis with dialysis solution A once and dialysis with dialysis solution B twice, the enzyme solution was collected and stored in a refrigerator at 4°C for standby.

[0034] SDS-PAGE analysis was performed on the purified target recombinant protein and mutant protein prepared above. Figure 1 The SDS-PAGE patterns of the purified target recombinant wild enzyme protein, mutant protein L134A, mutant protein L134V, mutant protein L134I, and mutant protein L134M are shown. After SDS-PAGE determination, the bands of the recombinant wild enzyme protein, mutant protein L134A, mutant protein L134V, mutant protein L134I, and mutant protein L134M were single, reaching SDS-PAGE purity, and the next step could be carried out. At the same time, SDS-PAGE showed that the molecular weight of the single subunit of the wild enzyme protein, mutant protein L134A, mutant protein L134V, mutant protein L134I, and mutant protein L134M was about 29 kDa.

[0035] Example 3 Determination of the Degradation Ability of ZEN Lactonohydrolase Mutants to ZEN

[0036] This example compared the changes in the degradation ability of the mutant and the wild enzyme to ZEN before and after mutation. The 250 μL enzymatic reaction system contained 195 μL of buffer (50 mM Tris-HCl, pH 9.0), 5 μL of substrate (4 mg / mL), and 50 μL of enzyme solution (0.2 mg / mL). After reacting at 60 °C for 3 min, 750 μL of acetonitrile was added to terminate the enzymatic reaction. The residual amount of ZEN was detected by high performance liquid chromatography to detect the enzyme activity. High performance liquid chromatography conditions: The mobile phase was acetonitrile: water = 6:4, the flow rate was 1.0 mL / min, the column temperature was 30 °C, and the detection wavelength was 254 nm. Using Hypersi1 TM ODS C18 column (250×4.6 mm, 5 μm) chromatographic column. The definition of enzyme activity is: The amount of enzyme added required to degrade 1 μg of substrate per minute is 1 U.

[0037] The pure enzyme solution obtained in Example 2 was subjected to an enzymatic reaction under the optimal conditions of pH 9.0 and temperature 60 °C, and was determined by high performance liquid chromatography to compare the degradation ability differences between the mutant enzyme and the wild enzyme of the present invention, as Figure 2 shown. The mutant L134A was able to degrade 91.7% of ZEN within 3 min, while the degradation ability of the wild-type enzyme to ZEN within 3 min was 74.9%, which was 1.22 times higher than that of the wild-type. The mutant L134V was able to degrade 94.2% of ZEN within 3 min, which was 1.25 times higher than that of the wild-type. The mutant L134I was able to degrade 87.6% of ZEN within 3 min, which was 1.17 times higher than that of the wild-type. The mutant L134M was able to degrade 85.4% of ZEN within 3 min, which was 1.14 times higher than that of the wild-type. Thus, for the amino acid position 134, small side-chain hydrophobic amino acids will help to improve the degradation ability of lactone hydrolase to ZEN. Additionally, as Figure 2 shown, the mutant L134V with the highest enzyme activity against ZEN had its absolute enzyme activity increased from the original 317 U / mg to 466 U / mg compared to the wild-type. The absolute enzyme activity of the mutant L134A relative to the wild-type increased from the original 317 U / mg to 454 U / mg. The absolute enzyme activities of the remaining two mutants L134I and L134M were 434 U / mg and 424 U / mg respectively. Thus, it can be seen that the four mutants obtained in Example 2 all had a significant increase in enzyme activity against ZEN and were able to efficiently degrade ZEN in a short time, proving that introducing small side-chain hydrophobic residues at position 134 of the enzyme's spatial structure will greatly improve the degradation activity of lactone hydrolase to ZEN.

[0038] Example 4 Determination of Substrate Kinetic Parameters of ZEN Lactone Hydrolase Mutants

[0039] Using ZEN at final concentrations of 0, 10, 30, 50, 60, 70, 80, 90, and 100 μg / mL as variables, the kinetic parameters of 40 μg / mL of recombinant wild-type enzyme, mutant enzymes L134A and L134V were investigated at the optimal pH of 9.0 and optimal temperature of 60 °C of the wild-type enzyme. Then, the Michaelis-Menten fitting model in Origin 2021 was used to obtain the kinetic curves of the enzymes, and the Michaelis constant (K m ), catalytic constant (k cat ), and second-order rate constant (k cat / K m ) were calculated respectively. The catalytic efficiencies of the mutants L134A and L134V studied in this example for ZEN were both improved, as shown in Figure 3 and Table 3.

[0040] Table 3 Kinetic parameters of wild-type enzyme and mutant enzymes for ZEN

[0041]

[0042] As shown in Figure 3 , under the same reaction conditions, the reaction rates of mutants L134A and L134V for ZEN were significantly higher than those of the wild-type. At low substrate concentrations, the active sites of the enzyme were vacant, waiting for substrate binding, and at this time, the reaction rate of the enzyme increased with the increase in substrate concentration. The active sites of the enzyme were gradually saturated with the increase in substrate concentration, and at this time, the reaction rate was determined by the enzyme concentration. When the active sites of the enzyme were saturated with the substrate, the enzyme-catalyzed reaction rate reached equilibrium, that is, the maximum enzyme-catalyzed reaction rate V max . The maximum enzyme-catalyzed reaction rates of mutants L134A and L134V were both significantly higher than those of the wild-type enzyme, indicating that after mutation, their ability to convert substrates was stronger and the catalytic efficiency was higher.

[0043] As shown in Table 3, the k cat and k cat / K m of the wild-type enzyme for ZEN were 70 min -1 and 0.292 min -1 μM -1 . While the k cat and k cat / K m of mutant L134A for ZEN were 96 min -1 and 0.356 min -1 μM -1 , and the k cat and k cat / K m of mutant L134V for ZEN were 113 min -1and 0.367 min -1 μM -1 , both higher than those of the wild enzyme. This indicates that the mutants L134A and L134V can convert ZEN more efficiently at lower substrate concentrations, and the present invention obtains a more efficient and specific zearalenone lactonohydrolase.

[0044] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An inner esterase mutant for efficiently degrading zearalenone, characterized in that: Using the lactone hydrolase gene from Monosporascus sp. GIB2 as a template, site-directed mutagenesis was performed on the 134th amino acid to obtain mutants.

2. The lactonohydrolase mutant for efficiently degrading zearalenone according to claim 1, characterized in that: Including mutant L134A, whose amino acid sequence is shown in SEQ ID NO:

3.

3. The lactonase mutant for highly efficient degradation of zearalenone according to claim 1, wherein: Including mutant L134V, whose amino acid sequence is shown in SEQ ID NO:

4.

4. The lactonohydrolase mutant for highly efficient degradation of zearalenone according to claim 1, characterized in that: Including mutant L134I, whose amino acid sequence is shown in SEQ ID NO:

5.

5. The lactonohydrolase mutant for efficiently degrading zearalenone according to claim 1, characterized in that: Including mutant L134M, whose amino acid sequence is shown in SEQ ID NO:

6.

6. A coding gene encoding the lactone hydrolase mutant according to any one of claims 2-5.

7. A recombinant expression vector pET-22b(+) carrying the coding gene according to claim 6.

8. A recombinant expression host bacterium, characterized in that: The expression host bacterium is Escherichia coli BL21(DE3), which contains the recombinant expression vector according to claim 7.

9. Use of the lactone hydrolase mutant according to any one of claims 1-5 in degrading zearalenone.

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