A zearalenone-degrading lactonohydrolase mutant and application thereof

Abstract

This invention relates to the field of enzyme engineering technology, and in particular to a highly efficient lactone hydrolase mutant for degrading zearalenone and its applications. This lactone hydrolase mutant uses the lactone hydrolase gene from Monosporascus sp. GIB2 as a template, and performs a site-directed mutation at amino acid position 134 to obtain L134A, L134V, L134I, and L134M mutants. Under optimal conditions of pH 9.0 and 60℃, these single-point mutants can efficiently degrade zearalenone (ZEN) within 3 minutes. The enzyme activity for degrading zearalenone is significantly improved compared to the original enzyme, with degradation activities increasing by 1.22 (L134A), 1.25 (L134V), 1.17 (L134I), and 1.14 (L134M), respectively, demonstrating significant applications and economic value in the feed industry.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, specifically to a mutant lactone hydrolase that degrades zearalenone and its applications. Background Technology

[0002] Zearalenone (ZEN) is a fungal toxin found primarily in moldy corn, wheat, barley, and other grains and their byproducts. It is one of the most widespread contaminants of the world. ZEN has a phenolic dihydroxy lactone structure, similar to estradiol, and can competitively bind to estrogen receptors, leading to disruptions in estrogen levels and reproductive systems in animals. First discovered in moldy corn in 1962, ZEN is a toxic secondary metabolite produced by Fusarium spp. and is widely found in moldy corn, wheat, barley, and other grains and grain byproducts, becoming a global food and feed contaminant. Currently, five ZEN derivatives have been identified: α / β-zearalenol (α / β-ZOL), α / β-zearalenol (α / β-ZAL), and zearalenone (ZAN). α-ZOL exhibits higher estrogenic toxicity than ZEN.

[0003] Currently, there are three main types of enzymes capable of degrading ZEN: peroxidase, laccase, and lactone hydrolase. Although peroxidase has been found to oxidize and break the phenolic dihydroxybenzene ring structure in the ZEN molecule, the reaction requires a large amount of hydrogen peroxide. Secondly, laccase removes ZEN by oxidizing the phenolic hydroxyl group in the ZEN molecule, but the site of action is uncertain and the efficiency is low, making it difficult to meet practical application requirements. In contrast, lactone hydrolase has a clear degradation mechanism of the C12' lactone bond in ZEN, the degradation products are non-toxic, and the reaction is media-independent, making it the most promising and promising ZEN-degrading enzyme. However, the reported ZEN lactone hydrolases still have many problems, such as low enzyme activity in catalyzing ZEN and its derivatives. Products contaminated with ZEN often contain high concentrations of reproductively toxic zearalenone derivatives. As a feed enzyme, it is required to be able to efficiently degrade ZEN.

[0004] Therefore, in order to obtain a ZEN lactone hydrolase that is more suitable for feed industry applications, this invention uses site-directed mutagenesis to modify the activity of a ZEN lactone hydrolase derived from Monosporascus sp. GIB2, thereby enhancing its ability to degrade ZEN and obtaining a ZEN lactone hydrolase that is more suitable for industrial applications. Summary of the Invention

[0005] The purpose of this invention is to provide a mutant lactone hydrolase that efficiently degrades zearalenone and its application.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] The nucleic acid and amino acid sequences of the wild-type ZEN lactone hydrolase derived from Monosporascus sp. GIB2 are shown in SEQ ID NO: 1-2. In this invention, a site-directed mutagenesis was performed on amino acid position 134, replacing leucine with alanine, valine, isoleucine, and methionine, yielding mutants L134A, L134V, L134I, and L134M, whose amino acid sequences are shown in SEQ ID NO: 3-6.

[0008] Compared to the wild-type enzyme, the ZEN lactone hydrolysing mutants L134A, L134V, L134I, and L134M maintained the same optimal pH (9.0) and optimal temperature (60℃) as the wild-type, but exhibited significantly enhanced ZEN degradation activity. Under optimal catalytic conditions, mutant L134A showed a 1.22-fold increase in relative ZEN degradation activity compared to the wild-type. Under the same catalytic conditions, mutant L134V showed a 1.25-fold increase in ZEN degradation activity compared to the wild-type. Mutant L134I showed a 1.17-fold increase in ZEN degradation activity compared to the wild-type, and mutant L134M showed a 1.14-fold increase. This research is of significant value for the application of lactone hydrolases in the detoxification of ZEN and its derivatives in the feed industry.

[0009] This invention provides a coding gene that encodes the above-mentioned lactone hydrolase mutant.

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

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

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

[0013] (1) Through extensive experimental exploration, this invention obtained ZEN lactone hydrolase mutants L134A, L134V, L134I, and L134M derived from Monosporascus sp. GIB2, which significantly enhanced the ZEN degradation activity. Specifically, mutant L134A exhibited a ZEN degradation activity of 454 U / mg, a 1.22-fold increase compared to the wild-type enzyme's 371 U / mg. Mutant L134V showed a ZEN degradation activity of 466 U / mg, a 1.25-fold increase compared to the wild-type enzyme's 371 U / mg. Mutant L134I achieved a ZEN degradation activity of 434 U / mg, a 1.17-fold increase compared to the wild-type enzyme's 371 U / mg. L134M demonstrated a ZEN degradation activity of 424 U / mg, a 1.14-fold increase compared to the wild-type enzyme's 371 U / mg. Enzyme reaction kinetics studies showed that the kcat / Km ratios of mutants L134A and L134V for ZEN were 0.356 min. -1 μM -1 and 0.367min -1 μM -1 The levels were all higher than those of the wild-type enzyme, indicating that the catalytic efficiency was significantly improved after mutation.

[0014] (2) The mutants L134A, L134V, L134I and L134M obtained in this invention have important research value for industrial treatment of corn juglans regrinone contamination, such as feed. Attached Figure Description

[0015] Figure 1 This is an SDS-PAGE electrophoresis image of ZEN lactone hydrolase and its mutants induced by recombinant expression host bacterium BL21(DE3) according to one embodiment of the present invention. Wherein, 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 This is a graph showing the absolute activities of ZEN lactone hydrolase wild-type enzyme WT and mutants L134A, L134V, L134I, and L134M with ZEN.

[0017] Figure 3 The enzymatic reaction rates of wild-type enzyme, L134A, and L134V are given. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Example 1: Preparation of ZEN lactone hydrolase mutant

[0020] (1) Obtaining the wild-type plasmid: pET-22b(+) was selected as the backbone for expressing the target gene. The ZENM gene of zearalenone lactone hydrolase derived from Monosporascus sp. GIB2 (nucleotide and amino acid sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2) was introduced into two restriction enzyme sites, NdeI and XhoI, to generate a linearized vector. The vector was introduced into competent E. coli cells through chemical transformation to obtain engineered bacteria. Positive clones were cultured in large quantities in culture medium, and high-purity plasmids were extracted using a commercial kit.

[0021] (2) Construction of mutant plasmids: according to QuikChange TM Site-directed mutagenesis was performed using a wild-type plasmid as a template. At the L134 position, leucine was mutated to alanine, valine, isoleucine, and methionine, respectively, to construct four single-point mutants (L134A, L134V, L134I, and L134M). Sequencing confirmed that the mutation results were correct.

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

[0023] Table 1. Upstream and downstream primer sequences of the mutant.

[0024] name 5'-3' base sequence 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 was 50 μL. The reaction program was as follows: 95℃, 5 min; 95℃, 30 s; 57℃, 30 s; 72℃, 3 min; 28 cycles; 72℃, 5 min; 4℃.

[0026] Table 2 Composition of PCR reaction system

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

[0028] (3) Agarose gel electrophoresis verification and template digestion: The size of the PCR product bands was verified by agarose gel electrophoresis according to the marker. After verification, 1 μL of Q.cut DpnI and 2 μL of Q.cut Buffer (10×) were added to the PCR product system and the enzyme digestion reaction was carried out in a 37℃ water bath for 120 min to remove the template DNA in the PCR reaction system.

[0029] (4) Transformation: The digested PCR product was introduced into *E. coli* DH5α competent cells and plated on LB agar plates containing 50 μg / mL ampicillin, and incubated overnight at 37°C. Positive clones were then picked for plasmid extraction and DNA sequencing. The successfully sequenced mutant plasmid was introduced into *E. coli* BL21(DE3) competent cells to construct a mutant gene recombinant expression strain for the induction of mutant enzyme expression.

[0030] Example 2: Expression and Nickel Column Affinity Purification of the Mutant Enzyme

[0031] The mutant plasmids pET-22b(+)-L134A, pET-22b(+)-L134V, pET-22b(+)-L134I, and pET-22b(+)-L134M, whose mutations were verified to be successful after sequencing, were introduced into competent cells of the expression host strain *Escherichia coli* BL21(DE3) and plated on LB agar plates containing 50 μg / mL ampicillin and incubated overnight at 37°C. Positive colonies were picked and incubated overnight at 37°C and 200 rpm in LB liquid medium containing 50 μg / mL ampicillin. The seed culture was then expanded to 200 mL of LB liquid medium containing 50 μg / mL ampicillin at an inoculum size of 2 / 1000 and incubated at 37°C and 200 rpm for 2–3 h until the OD value reached 0.6–0.8. Subsequently, 1 mM IPTG was added to the expansion medium and the culture was incubated at 25°C in a shaker for 6 h.

[0032] The induced fermentation broth was centrifuged at 8000 rpm for 5 min to collect the bacterial cells. The cells were resuspended in 15 mL of lysis buffer (50 mM Tris, 200 mM NaCl, pH adjusted to 7.0 with HCl). The resuspended cells were placed in an ice-water mixing bath, and the system was then placed in an ultrasonic cell disruptor. The cells were sonicated at 450 kHz for 1 second, paused for 2 seconds, and then for 20 min to obtain intracellular products. The disrupted broth was centrifuged at 8000 rpm for 5 min in a low-temperature centrifuge at 4°C, and the supernatant was collected. The supernatant was filtered through a 0.45 μm microporous membrane and used for later use.

[0033] Protein purification of the crude enzyme solution was performed using nickel column affinity purification. First, the constant flow peristaltic pump and Ni column were connected to the protein purification system.2+ The tubing connecting the affinity chromatography column, 280nm detector, and other instruments was set to a constant flow rate of 1.5 mL / min and leak checked. 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 equilibration, the crude lysate was pumped into the column at a flow rate of 0.5 mL / min. The column was then continuously washed with equilibration buffer at a flow rate of 1.5 mL / min. Once the UV detector reading stabilized, wash buffer (30 mM imidazole, 50 mM Tris, 500 mM NaCl, pH 7.5) was used to wash away contaminating proteins with weak nickel ion binding affinity. After the UV detector reading stabilized again, 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 wash buffer (target protein) was collected in a 10 mL centrifuge tube. The target protein was aspirated and transferred to a dialysis bag with a molecular weight cutoff of 14 kDa. The bag was clamped with double dialysis clamps and placed in dialysis solution A (10 mM EDTA·2Na, 50 mM Tris, pH 7.5). Dialysis was performed overnight, followed by dialysis with dialysis solution B (50 mM Tris, pH 7.0) for 6 hours. Dialysis with dialysis solution A was performed once, and dialysis with dialysis solution B was repeated twice. The enzyme solution was then collected and stored at 4°C for later use.

[0034] The purified target recombinant protein and mutant protein obtained above were analyzed by protein electrophoresis. Figure 1 The image shows the electrophoretic images of the purified target recombinant wild-type enzyme protein, mutant protein L134A, mutant protein L134V, mutant protein L134I, and mutant protein L134M. Electrophoresis analysis showed that the bands for all four proteins were single, indicating electrophoretic purity, and they are ready for further processing. The electrophoresis also showed that the molecular weight of each subunit of the wild-type enzyme protein, mutant protein L134A, mutant protein L134V, mutant protein L134I, and mutant protein L134M is approximately 29 kDa.

[0035] Example 3: Determination of ZEN degradation ability by ZEN lactone hydrolase mutant

[0036] This example compares the changes in the ZEN degradation ability of the mutant and wild-type enzymes before and after mutation. A 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 enzyme reaction. ZEN residue was detected by high-performance liquid chromatography (HPLC) to determine enzyme activity. HPLC conditions: mobile phase: acetonitrile:water = 6:4, flow rate 1.0 mL / min, column temperature 30°C, detection wavelength 254 nm. Hypersi1 was used. TM ODS C18 column (250×4.6mm, 5μm) chromatography column. Enzyme activity is defined as the amount of enzyme required to degrade 1μg of substrate per minute, which is 1U.

[0037] The pure enzyme solution obtained in Example 2 was subjected to an enzymatic reaction under optimal conditions, namely pH 9.0 and a temperature of 60°C, and the reaction was analyzed by high-performance liquid chromatography to compare the difference in degradation ability between the mutant enzyme of the present invention and the wild-type enzyme. Figure 2 As shown. Mutant L134A can degrade 91.7% of ZEN within 3 minutes, while the wild-type enzyme's degradation capacity for ZEN within 3 minutes is 74.9%, representing a 1.22-fold increase compared to the wild-type. Mutant L134V can degrade 94.2% of ZEN within 3 minutes, representing a 1.25-fold increase compared to the wild-type. Mutant L134I can degrade 87.6% of ZEN within 3 minutes, representing a 1.17-fold increase compared to the wild-type. Mutant L134M can degrade 85.4% of ZEN within 3 minutes, representing a 1.14-fold increase compared to the wild-type. Therefore, for the amino acid position 134, a small hydrophobic side chain helps to enhance the degradation capacity of lactone hydrolases for ZEN. Additionally, as... Figure 2 As shown, the mutant L134V, which exhibited the highest ZEN enzyme activity, increased from 317 U / mg to 466 U / mg relative to the wild type. The mutant L134A, on the other hand, increased from 317 U / mg to 454 U / mg relative to the wild type. The remaining two mutants, L134I and L134M, had absolute enzyme activities of 434 U / mg and 424 U / mg, respectively. This demonstrates that the four mutants obtained in Example 2 all showed significantly enhanced ZEN enzyme activity, enabling efficient ZEN degradation in a short time. This proves that introducing a small hydrophobic side chain residue at position 134 of the enzyme's spatial structure greatly enhances the degradation activity of lactone hydrolases against ZEN.

[0038] Example 4: Determination of substrate kinetic parameters of ZEN lactone hydrolase mutant

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

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

[0041]

[0042] like Figure 3 As shown, under the same reaction conditions, the reaction rates of mutants L134A and L134V with ZEN were significantly higher than those of the wild type. At low substrate concentrations, the enzyme's active site is vacant, awaiting substrate binding; at this point, the enzyme's reaction rate increases with increasing substrate concentration. The enzyme's active site gradually becomes saturated with increasing substrate concentration, at which point the reaction rate is determined by the enzyme concentration. Once the enzyme's active site is saturated with substrate, the enzyme-catalyzed reaction rate reaches equilibrium, i.e., the maximum enzyme-catalyzed reaction rate V0. max The maximum enzymatic reaction rates of mutants L134A and L134V were significantly higher than those of the wild-type enzyme, indicating that the mutants have a stronger ability to transform substrates and higher catalytic efficiency.

[0043] As shown in Table 3, the wild-type enzyme's kJ / kJ of ZEN cat and k cat / K m 70min -1 and 0.292min -1 μM -1 The mutant L134A has a k-type effect on ZEN. cat and k cat / K m 96min respectively -1 and 0.356min -1 μM -1 The L134V mutant's k-type response to ZEN cat and k cat / K m 113min respectively -1and 0.367min -1 μM -1 The levels were higher than those of the wild-type enzyme. This indicates that mutants L134A and L134V can convert ZEN more efficiently at lower substrate concentrations. This invention yields a more efficient and specific zearalenone lactone hydrolase.

[0044] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A mutant lactone hydrolase that efficiently degrades zearalenone, characterized in that: From Monosporascus sp. Using the lactone hydrolase gene in GIB2 as a template, a site-directed mutation was performed on amino acid position 134 to obtain mutants, specifically any one of mutants L134A, L134V, L134I, and L134M, whose amino acid sequences are shown in SEQ ID NO: 3-6.

2. A coding gene encoding the lactone hydrolase mutant of claim 1.

3. A recombinant expression vector pET-22b(+) carrying the encoding gene of claim 2.

4. A recombinant expression host bacterium, characterized in that: Escherichia coli E. coli BL21(DE3), the expression host bacterium contains the recombinant expression vector as described in claim 3.

5. The application of the lactone hydrolase mutant of claim 1 in the degradation of zearalenone.

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