Gcakr for efficiently degrading patulin and application thereof

Abstract

The application belongs to the technical field of biological enzyme engineering, and discloses an aldonoketoreductase GCAKR capable of degrading patulin and an application thereof. The application first proposes the aldonoketoreductase GCAKR capable of degrading patulin. The aldonoketoreductase GCAKR provided by the application solves the problems of low efficiency, secondary pollution and limited application in the existing PAT removal technology, and has the advantages of simple and efficient preparation method, wide application range, safe and efficient removal of PAT pollution in food and environment, and provides a new technical means and research basis for food safety production and environmental remediation.

Description

Technical Field

[0001] This invention belongs to the field of bioenzyme engineering technology, specifically relating to a highly efficient aldehyde-ketone reductase GCAKR for degrading patulin and its applications. Background Technology

[0002] Patulin (PAT) is a toxic secondary metabolite primarily produced by fungi such as Aspergillus and Penicillium, and is widely present in fruits and fruit processing environments. During orchard production, PAT can contaminate fresh fruit through airborne transmission of fungal spores. During post-harvest storage, transportation, and processing, the toxin further accumulates and remains, causing widespread contamination of common fruits such as apples, pears, and strawberries, as well as their processed products like juices, jams, and wines. Furthermore, PAT in orchard waste and fruit processing byproducts can seep into soil and water bodies, causing secondary environmental pollution and posing a serious threat to ecosystem stability.

[0003] PAT is chemically extremely stable and highly resistant to high temperatures, acids, and alkalis, making it highly susceptible to bioaccumulation in the food chain and ultimately entering the human body through ingestion. Numerous studies have confirmed that PAT ingestion can cause acute poisoning symptoms such as nausea, headache, and respiratory irritation. Long-term exposure can lead to chronic health problems such as liver and kidney damage and weakened immune function, seriously threatening human life and health. Furthermore, PAT contamination can cause agricultural product overstocking and product recalls, resulting in significant economic losses for the food and feed industries. Therefore, developing efficient and safe PAT degradation technologies is crucial for ensuring food safety, protecting the ecological environment, and promoting the healthy development of related industries.

[0004] Currently, methods for controlling PAT pollution mainly fall into three categories: physical methods, chemical methods, and biological methods. Physical methods (such as adsorption, filtration, and irradiation) have drawbacks such as limited degradation efficiency and the potential for nutrient loss or secondary pollution. Chemical methods (such as oxidant treatment and acid-base degradation) can degrade PAT to some extent, but they may introduce new chemical pollutants, pose food safety risks, and have limited applicability, making them difficult to meet the green and environmentally friendly requirements of actual production. In contrast, biological methods, due to their advantages of being environmentally friendly, low-cost, thorough, and without secondary pollution, have become a hot research area for PAT degradation, mainly including two technical pathways: bioadsorption and biodegradation.

[0005] Biosorption technology utilizes the cell wall components of inactivated microorganisms (such as yeast and lactic acid bacteria) to adsorb and immobilize PAT. However, this technology is limited by the limited binding sites on the microbial cell walls, resulting in low adsorption capacity, easy saturation, and difficulty in achieving complete degradation of the toxin, thus failing to fundamentally eliminate the toxicity of PAT. Biodegradation technology, on the other hand, uses active microorganisms or their produced enzymes to metabolize PAT into low-toxicity or non-toxic products, representing a more promising degradation method. Previous studies have found that some microorganisms (such as Kluyveromyces martensii and Geotrichum candida) possess PAT degradation capabilities, but the degradation efficiency of these microorganisms is greatly affected by fermentation conditions and environmental factors, exhibiting poor stability. Furthermore, the key functional enzymes mediating PAT degradation have not yet been clearly identified, significantly limiting the industrial application of this technology.

[0006] Enzymatic degradation, as an important branch of biodegradation technology, has advantages such as high specificity, mild reaction conditions, and ease of control, making it an ideal approach for the efficient degradation of PAT. However, the number of reported biodegrading enzymes is currently limited, thus necessitating the discovery of novel, highly efficient biodegrading enzymes for PAT removal.

[0007] Geotrichum candidum, belonging to the kingdom Fungi and phylum Ascomycota, is a type of fungus whose morphology is intermediate between yeast and mold. This fungus is widely used in cheese making, improving the flavor and texture of cheese. Both the European Food and Feed Culture Association (EFFCA) and the International Dairy Federation (IDF) list Geotrichum candidum as a recognized safe food fermentation microorganism. Summary of the Invention

[0008] The purpose of this invention is to address the above-mentioned problems by providing a highly efficient aldehyde-ketone reductase GCAKR for degrading patulin and its applications.

[0009] To achieve its objective, the present invention employs the following technical solution:

[0010] A first aspect of the present invention provides an aldehyde-ketone reductase GCAKR, wherein the aldehyde-ketone reductase is any one of the following proteins:

[0011] (1) A protein with an amino acid sequence as shown in SEQ ID NO.1;

[0012] (2) Proteins that have more than 90% homology with the amino acid sequence shown in SEQ ID NO.1 and have the same functional group.

[0013] A second aspect of the present invention provides a gene encoding the above-mentioned aldehyde-ketone reductase, wherein the gene is any one of the following nucleic acid molecules:

[0014] (1) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1;

[0015] (2) The nucleotide sequence as shown in SEQ ID NO.2;

[0016] (3) A nucleotide sequence that has more than 90% homology with the nucleotide sequence defined in (1) or (2) and encodes the above-mentioned aldehyde reductase;

[0017] (4) Hybridizes under strict conditions to the nucleotide sequence defined in (1) or (2) and encodes the nucleotide sequence of the above-mentioned aldehyde reductase.

[0018] A third aspect of the present invention provides a recombinant expression vector containing the above-described coding gene.

[0019] Preferably, the recombinant expression vector is a recombinant expression vector obtained by inserting the above-mentioned coding gene into the multiple cloning site of the backbone vector pET-28a-SUMO.

[0020] A fourth aspect of the present invention provides a recombinant engineered bacterium containing the above-described encoding gene;

[0021] Preferably, the recombinant engineered bacteria is obtained by introducing the above-mentioned recombinant expression vector into Escherichia coli.

[0022] The fifth aspect of the invention provides a method for preparing the above-mentioned aldehyde-ketone reductase, wherein the above-mentioned recombinant engineered bacteria are cultured, induced to express, and cleaved by a tag to obtain the target protein.

[0023] The above preparation method includes the following steps:

[0024] (1) The above-mentioned recombinant expression vector was transformed into competent Escherichia coli cells, and recombinant engineered bacteria were obtained by heat shock, recovery culture and screening;

[0025] (2) Select single clones and inoculate them into the selection medium to obtain seed culture;

[0026] (3) Add the seed culture to the screening medium and culture it;

[0027] (4) Add an inducer to induce the expression of the target protein;

[0028] (5) Collect the induced bacterial cells and crush them to extract crude enzyme solution;

[0029] (6) Purify the crude enzyme solution;

[0030] (7) Remove the fusion tag from the purified protein;

[0031] (8) Purify again to obtain aldehyde-ketone reductase.

[0032] The sixth aspect of the present invention provides the use of the above-described aldehyde-ketone reductase in the degradation of patulin.

[0033] Preferably, the application steps are as follows: the above-mentioned aldehyde-ketone reductase is added to the object to be treated containing patulin, and the reaction is carried out at a temperature of 4~40℃, so that the aldehyde-ketone reductase degrades patulin.

[0034] More preferably, the application steps are as follows: The above-mentioned aldehyde-ketone reductase, coenzyme NADPH, and buffer solution are added to the object to be treated containing patulin to form a reaction system. The pH of the reaction system is controlled at 6.0~8.0 (preferably pH 7.0~8.0), the temperature at 4~40℃ (preferably 16~30℃), and the reaction time at 8~36 h, thereby achieving efficient degradation of patulin and specifically generating the product E-ascladiol. Preferably, the concentration of patulin in the reaction system is 1~40 μg / mL (preferably 1~20 μg / mL), the amount of aldehyde-ketone reductase is 25~200 μg / mL (preferably 75~100 μg / mL), and the concentration of NADPH is 1~4 mM.

[0035] The beneficial effects of this invention are:

[0036] This invention presents for the first time a novel aldehyde-ketone reductase, GCAKR, capable of degrading patulin. The GCAKR is derived from *Geotrichum candida* XG1, isolated and screened from traditional Chinese fermented foods. It is safe and reliable, suitable for the biocontrol of patulin contamination in food and agricultural products, and has significant application value. GCAKR has the following outstanding advantages:

[0037] High degradation efficiency: Under optimized conditions, 100 μg / mL GCAKR can completely degrade 10 μg / mL PAT within 12 h, with a degradation rate of 100%.

[0038] High safety: The degradation product has been structurally identified as E-ascladiol (molecular formula C7H8O4), a specific derivative of PAT generated via the reduction reaction of the α,β-unsaturated lactone ring. According to existing research, E-ascladiol is a low-toxicity derivative associated with PAT degradation, exhibiting no significant toxicity characteristics. Furthermore, the reaction system involves only enzyme catalysis and substrate transformation, without the introduction of additional chemical pollutants or the risk of secondary pollution. It is safe and reliable, possessing potential for PAT removal in food and environmental systems.

[0039] The reaction conditions are mild: the optimal reaction conditions are pH 7.0-8.0 and temperature 16-30℃, and no extreme temperature or acid / alkali environment is required.

[0040] High stability: It maintains high activity within the range of 16-30℃ and has good potential for practical applications.

[0041] Preparation is simple: high-purity products can be obtained through heterologous soluble expression in Escherichia coli and two-step Ni-NTA affinity chromatography, resulting in low production costs and high yields.

[0042] In summary, the aldehyde-ketone reductase GCAKR provided by this invention solves the problems of low efficiency, easy secondary pollution, and limited application in existing PAT removal technologies. Its preparation method is simple and efficient, and its application range is wide. It can safely and efficiently remove PAT contamination from food and the environment, providing a new technical means and research basis for food safety production and environmental remediation. Attached Figure Description

[0043] Figure 1 This is a map of the GCAKR recombinant plasmid.

[0044] Figure 2 This is the SDS-PAGE result of GCAKR.

[0045] Figure 3 Phylogenetic tree of the AKR subfamily among different species.

[0046] Figure 4 Multiple sequence alignment of AKR5 subfamily proteins across different species.

[0047] Figure 5 The effect of NADPH concentration on the degradation of PAT by GCAKR.

[0048] Figure 6 The effects of temperature, pH, substrate concentration, and enzyme dosage on the degradation of PAT by GCAKR were investigated.

[0049] Figure 7 Analysis of degradation products of PAT by GCAKR. Detailed Implementation

[0050] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0051] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0052] Example 1

[0053] I. Construction of recombinant expression plasmid pET-28a-SUMO-GCAKR

[0054] The amino acid sequence of an aldehyde-keto reductase from *Geotrichum candidum* XG1 was screened based on proteomics data, as shown in SEQ ID NO.1. Aldo-keto reductases (AKRs) are a superfamily of enzymes involved in various metabolic processes. These enzymes belong to the NAD(P)H-dependent oxidoreductase family and catalyze the reduction of carbonyl compounds such as aldehydes and ketones to their corresponding alcohols, playing a wide range of important physiological roles in carbohydrate metabolism and detoxification of exogenous substances.

[0055] The *Geotrichum candidum* strain XG1 has been disclosed in Chinese Patent ZL 202311310439.X (Authorization Announcement No. CN 117089473B). The deposit information is as follows: *Geotrichum candidum* strain XG1 was deposited in June 2023 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date was June 25, 2023, the deposit number was CGMCC No. 27705, and the classification name was *Geotrichum candidum*.

[0056] 1. Strains and vectors

[0057] Cloning host bacteria: Escherichia coli DH5α competent cells, purchased from Beijing Qingke Biotechnology Co., Ltd.;

[0058] Expression vector: pET-28a-SUMO plasmid, containing an N-terminal 6×His-SUMO fusion tag, with BamHI / XhoI restriction sites, used to construct the recombinant expression vector, purchased from Beijing Qingke Biotechnology Co., Ltd.

[0059] 2. Gene optimization and synthesis

[0060] The GCAKR gene of *Geotrichum candida* XG1, described in this invention, was cloned from *Geotrichum candida* strain XG1 using a homologous cloning strategy. First, specific primer pairs AKR-F / AKR-R were designed based on conserved regions of closely related fungi and previously reported *Geotrichum candida* aldehyde reductase genes (Genebank ID: DV453_000681). Genomic DNA was extracted from *Geotrichum candida* strain XG1, and PCR amplification was performed using this DNA as a template to obtain the target gene fragment. Subsequently, the amplified product was sequenced, and the obtained sequence was compared with databases such as NCBI for nucleotide and amino acid sequence alignment, conserved domain analysis, and phylogenetic analysis, confirming that this gene is a novel gene in the aldehyde reductase family, named the GCAKR gene, thus clarifying its gene sequence and functional classification.

[0061] The specific amplification primer pair AKR-F / AKR-R has the following sequences:

[0062] AKR-F (SEQ ID NO.3):

[0063] 5'-GGCTCACAGAGAACAGATTGGTGGATCCATGAGCTTCAAGACTAACAGCTTCGTTAAAC-3';

[0064] AKR-R (SEQ ID NO.4):

[0065] 5'-ATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTTATGCGTCCGCGTCAACTGGATG-3'.

[0066] Based on the GCAKR gene sequence of Geotrichum candidum XG1 (encoding the YJR096W protein, where YJR096W is the initial annotation number of this protein in the Geotrichum candidum proteome database, containing 300 amino acids and a gene length of 915 bp), codon optimization was performed in conjunction with E. coli codon preferences. The optimized gene was synthesized by Beijing Qingke Biotechnology Co., Ltd., and the optimized codons are shown in SEQ ID NO.2.

[0067] This study optimized the codons of the GCAKR gene to improve its expression efficiency and soluble expression level in Escherichia coli, while ensuring that the structure and function of the expressed protein remained unchanged. The codon optimization only involved synonymous codon substitution and did not alter the amino acid sequence encoded by the gene. The amino acid sequence of the expressed GCAKR protein is completely identical to that of the naturally expressed protein of Geotrichum xG1 (both are SEQ ID NO.1), and the catalytic function and spatial structure of the protein remain unchanged.

[0068] 3. Enzyme digestion and ligation

[0069] The synthesized GCAKR gene and pET-28a-SUMO vector were treated with restriction endonucleases BamHI / XhoI, respectively. After the digestion products were recovered by gel extraction, they were ligated overnight at 16°C using T4 DNA ligase to construct the recombinant expression plasmid pET-28a-SUMO-GCAKR (containing an N-terminal 6×His-SUMO fusion tag).

[0070] 4. Transformation and Identification

[0071] The ligation product was heat-shocked and transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing 50 μg / mL kanamycin sulfate, and incubated at 37°C for 12 h. Single colonies were picked for colony PCR verification. Positive clones were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were consistent with the GCAKR gene sequence, confirming successful recombinant plasmid construction. The plasmid map is attached. Figure 1 .

[0072] II. Construction of recombinant engineered bacteria, GCAKR enzyme-induced expression, and isolation and purification

[0073] 1. Construction and Induced Expression of Recombinant Engineered Bacteria

[0074] The recombinant plasmid pET-28a-SUMO-GCAKR was heat-shocked into Escherichia coli BL21 (DE3) competent cells: 1 μL of the recombinant plasmid was added to 100 μL of competent bacteria and incubated on ice for 20 min; then heat-shocked at 42℃ for 45-60 sec, immediately returned to ice to cool for 5 min, added 600 μL of LB medium, and cultured at 37℃ and 220 r / min for 1 h with shaking; after centrifugation, the entire bacterial culture was spread on LB agar plates containing 50 μg / mL kanamycin sulfate and incubated upside down at 37℃ for 12 h to obtain the recombinant engineered bacteria.

[0075] A single colony of the recombinant engineered bacteria was picked and inoculated into 5 mL of LB broth containing 50 μg / mL kanamycin sulfate. The culture was incubated at 37°C and 220 rpm with shaking for 12 h to obtain the seed culture. The seed culture was then transferred at a ratio of 1:100 to 100 mL of LB broth containing 50 μg / mL kanamycin sulfate and incubated at 37°C and 220 rpm until OD (dose elongation) was reached. 600 =0.5-0.8, two parallel culture systems were set up. IPTG was added to one system to a final concentration of 0.2 mM, and the system was incubated overnight at 18°C ​​and 220 rpm with shaking. IPTG was added to the other system to a final concentration of 0.2 mM, and the system was incubated at 30°C and 220 rpm with shaking for 16 h to induce soluble expression of the GCAKR-SUMO fusion protein. After incubation, the bacterial cells were collected by centrifugation at 4°C and 4000 rpm for 10 min, washed twice with TBS buffer, and the remaining culture was centrifuged and the bacterial cell pellet was collected for later use.

[0076] 2. Cell disruption and preparation of crude enzyme solution

[0077] The collected bacterial cells were resuspended in TBS buffer containing 1 mM PMSF and disrupted using an ultrasonic cell disruptor under ice bath conditions (power 40%, working for 2 seconds, intermittent for 3 seconds, total duration 20 min). After disruption, the cells were centrifuged at 4°C and 10,000 rpm for 20 min. The supernatant was collected as the crude enzyme solution, and a portion of the precipitate was resuspended in TBS for later use.

[0078] 3. Ni-NTA affinity purification

[0079] Affinity purification was performed using a low-pressure chromatography system: the crude enzyme solution was loaded at a flow rate of 1 mL / min onto a Ni-NTA affinity chromatography column pre-equilibrated with Ni-NTA binding-Buffer (20 mM Tris-HCl, 0 mM imidazole, 0.5 M NaCl, pH 8.0); subsequently, the column was washed with the same binding-Buffer at a flow rate of 1 mL / min until the effluent OD was reached. 280 Once the value reaches baseline, wash with Ni-NTA Washing-Buffer (20mM Tris-HCl, 50mM imidazole, 0.5M NaCl, pH 8.0) at a flow rate of 1mL / min to remove contaminating proteins until the effluent OD reaches baseline. 280 The value was returned to baseline; finally, the target protein was eluted sequentially with Ni-NTA Elution-Buffer (20mM Tris-HCl, 0.5M NaCl, pH 8.0) containing 100mM, 250mM, and 500mM imidazole at a flow rate of 1mL / min, and the eluents of each imidazole concentration were collected. The 500mM imidazole eluent contained high-purity GCAKR-SUMO fusion protein.

[0080] 4. SUMO tag cleavage and secondary purification

[0081] The purified fusion protein collected in 500 mM imidazole elution buffer was mixed with SUMO protease at a mass ratio of 100:1, placed in a dialysis bag, and dialyzed overnight with TBS buffer at 4°C for enzyme digestion. After digestion, the dialysate was loaded at a flow rate of 1 mL / min onto a Ni-NTA affinity chromatography column pre-equilibrated with Binding-Buffer (20 mM Tris-HCl, 0.5 M NaCl, pH 8.0), and the flow-through was collected, which was the GCAKR pure enzyme without the SUMO tag. At the same time, the chromatography column was washed with buffer containing 25 mM imidazole, and the washing buffer containing impurities was collected for later use.

[0082] 5. Purity Verification and Result Analysis

[0083] The purity of samples at each step was determined by SDS-PAGE electrophoresis using a 12% separating gel and a 5% stacking gel. After electrophoresis, the samples were stained with Coomassie Brilliant Blue and destained before observing the bands. Figure 2 The image shows the SDS-PAGE results of GCAKR enzyme expression, purification, and tag cleavage. Detailed explanations of each sub-figure are as follows:

[0084] Figure 2 A: Lane 1 is the supernatant of uninduced recombinant engineered bacterial cell lysis, with no obvious specific band, indicating that the target protein is not expressed in the uninduced state; Lane 2 is the supernatant of cell lysis after IPTG induction, with a clear specific band at approximately 55 kDa, corresponding to the GCAKR-SUMO fusion protein; Lanes 3 and 4 are the soluble components of the bacterial cells induced at 18℃ and 30℃, respectively, both showing the same intensity of the target band at 55 kDa, proving that the fusion protein can achieve efficient soluble expression at both temperatures.

[0085] Figure 2 B: The results of Ni-NTA affinity chromatography purification are shown. Lanes 1-3 contain elution buffers of 100 mM, 250 mM, and 500 mM imidazole, respectively. Only lane 3 (500 mM imidazole elution buffer) shows a single band at 55 kDa, with no other contaminating protein bands, indicating that high-purity separation of the fusion protein was achieved through gradient elution.

[0086] Figure 2 C: Results of SUMO tag cleavage and secondary purification. Lane 1 shows the GCAKR-SUMO fusion protein before cleavage (55 kDa); Lane 2 shows the flow-through buffer after tag cleavage through a Ni-NTA chromatography column, where a single, clear band appears at approximately 33 kDa, consistent with the theoretical molecular weight of the GCAKR purified enzyme (33.9 kDa), proving that the SUMO tag has been completely removed and the target protein has not been adsorbed by the chromatography column; Lane 3 shows the washing buffer of impurities after secondary purification, with no obvious bands, further verifying the separation effect of the purified enzyme.

[0087] Figure 2 D: Electrophoresis verification results of the final purified GCAKR enzyme. 3 μg of purified enzyme sample was loaded into the lane. A single sharp band appeared in the 33-35 kDa range, with no detectable contamination from other proteins, indicating that the purity of the final GCAKR enzyme was ≥95%, meeting the requirements for subsequent experiments.

[0088] III. Phylogenetic Tree Analysis of GCAKR

[0089] Using the amino acid sequence of GCAKR (AKR protein derived from Geotrichum candidum) as the target sequence, AKR family protein sequences from different species, including bacteria (such as Streptomyces fradiae), fungi (such as Saccharomyces cerevisiae), plants, and animals, were obtained through sequence homology search. After multiple sequence alignment using ClustalW, a phylogenetic tree was constructed using the neighbor-joining (NJ) method with Mega7 software. The bootstrap replication count was set to 1000 times, and the phylogenetic tree was visualized and enhanced using the ITOL website. Figure 3 .

[0090] like Figure 3 As shown, this phylogenetic tree clearly demonstrates the molecular evolutionary relationships among AKR family members of different species: the clustering pattern of branches in the tree is highly consistent with the classification of AKR subfamilies (such as AKR12, AKR16, AKR7, etc.). AKR proteins within the same subfamily (such as Streptomyces fradiae AKR12A1 and Saccharopolyspora erythraea AKR12B1 of the AKR12 subfamily) all cluster under independent branches with a spread support ≥100, reflecting the evolutionary specificity of AKR subfamilies. Simultaneously, AKR family members are widely distributed in prokaryotes (such as Streptomyces bacteria) and eukaryotes (such as Saccharomyces cerevisiae fungi), confirming that this family is an evolutionarily highly conserved ancient functional protein family. Furthermore, different AKR members from human origins (such as AKR7A3 and AKR1C1) exhibit branching dispersion in the tree, reflecting intraspecific AKR distribution. The evolutionary characteristics formed by gene replication and functional differentiation within a family, and the clustering of AKR subfamilies across species, indicate that the functions of AKR subfamilies are selectively conserved during evolution.

[0091] This phylogenetic tree clarifies the correspondence between the subfamily classifications of the AKR family and their molecular evolutionary relationships, providing a molecular evolutionary basis for tracing the functional origins of AKR proteins and classifying and identifying family members.

[0092] IV. Multiple sequence alignment analysis using GCAKR

[0093] Using the amino acid sequence of GCAKR as the core, AKR5 isotype protein sequences from different species, including Leishmania major AKR5A1, Gluconobacteroxydans AKR5C3, and Bacillus subtilis AKR5G1, were selected. Multiple sequence alignment was performed using ClustalW, and the alignment results were visualized using Espript (including secondary structure annotation). Figure 4 .

[0094] like Figure 4 As shown, the sequence alignment results, combined with conserved secondary structure elements (β-sheets: β1~β10; α-helices: α1~α11; random coils: η1~η5), exhibit the following characteristics: The secondary structure framework of AKR family proteins is strictly evolutionarily conserved. The β-sheet regions (corresponding to positions 1~10 (β1), 11~20 (β2), 151~160 (β7), and 201~210 (β9)) show nearly 100% amino acid residue identity—for example, the "GLY-VAL" continuous motif in the β1 region, the "VAL-ALA" residues in the β2 region, the "KVV-IGVS" sequence in the β7 region, and the "Gly-XX-Gly (GXXG)" motif in the β9 region (specifically "Gly-Pro-Thr-Gly") are completely identical in all aligned proteins, forming the core conserved backbone of the AKR protein spatial structure; the α-helices and random coil regions (such as positions 31~40 in the α1 region) are also highly conserved. Although there are a few amino acid residue substitutions in the η1 region (positions 71-80), the distribution patterns of hydrophobic amino acids (such as Leu and Ile) and polar functional residues (such as Ser and Thr) remain stable, ensuring the spatial arrangement stability of secondary structural elements. Furthermore, the above-mentioned conserved sequence regions precisely correspond to the catalytic active site domain of AKR family proteins, and the high consistency of their residues directly supports the evolutionary conservation of the catalytic function of this family of proteins.

[0095] The alignment results clearly define the correspondence between core conserved sequence sites and secondary structural elements of AKR family proteins, providing a direct reference for the precise identification of functional sites, three-dimensional structural homology modeling, and specific screening of target homologous proteins in this family of proteins.

[0096] Figure 3 , Figure 4 The results show that the GCAKR gene of Geotrichum xG1 described in this invention is a new gene in the aldehyde-ketone reductase family.

[0097] Example 2: Effect of coenzyme NADPH concentration on the degradation of PAT by GCAKR

[0098] GCAKR belongs to the aldehyde-ketone reductase (AKR) family. The catalytic reactions of this type of enzyme rely on NAD(P)H coenzymes to donate hydrogen protons to complete the reduction of carbonyl compounds. NADPH is the core essential coenzyme for its redox catalytic function. This experiment investigated the effect of NADPH concentration on the degradation of PAT by GCAKR. Firstly, it aimed to verify the consistency of GCAKR's catalytic characteristics with the functions of the aldehyde-ketone reductase family, clarifying that its PAT degradation reaction depends on NADPH. Secondly, it aimed to screen the optimal NADPH concentration for efficient PAT degradation by GCAKR, providing key process parameters for the practical application of this enzyme in the degradation of PAT contamination in food and the environment. It also demonstrated that exogenous addition of NADPH can significantly improve the PAT degradation efficiency of GCAKR, providing experimental support for achieving efficient detoxification through coenzyme regulation in practical applications.

[0099] TBS buffer containing 0, 1, 2, and 4 mM NADPH (reduced nicotinamide adenine dinucleotide phosphate, also known as reduced coenzyme II) was thoroughly mixed with PAT (final concentration 10 μg / mL) and GCAKR (final concentration 100 μg / mL) purified in Example 2 in 1.5 mL enzyme-free sterile centrifuge tubes to form 4 systems.

[0100] Control group: The difference is that NADPH and GCAKR are not present in the system.

[0101] All samples were placed in a shaking incubator at 30℃ and 150 rpm. After incubation for 0, 4, 8, 16, and 24 hours, an equal volume of acetonitrile (1% formic acid) was added to the samples and the reaction was terminated by thorough mixing. The resulting supernatant was filtered through a 0.22 μm pore size filter before detection, and the concentration of PAT was detected by high performance liquid chromatography.

[0102] HPLC detection conditions: Agilent 1260 was used for analysis; the column was Agilent SB-C18 (250×4.6mm, 5 μm); the mobile phase was acetonitrile:water = 10:90 (v / v); the flow rate was 1 mL / min; the detection wavelength was 276 nm; the injection volume was 8 μL; and the elution time was 12 min.

[0103] Degradation rate calculation formula:

[0104]

[0105] In the formula, C0 is the initial concentration of PAT, and C is the residual concentration of PAT after the reaction.

[0106] Figure 5 The effect of NADPH concentration on the degradation of PAT by GCAKR was investigated: when the NADPH concentration was ≥1 mM, 10 μg / mL PAT could be completely degraded within 12 h; without NADPH, the degradation rate was only 52.76% after 36 h, indicating that NADPH can significantly enhance the degradation activity of GCAKR enzyme.

[0107] Example 3: Effects of temperature, pH, PAT concentration, and GCAKR dosage on the degradation of PAT by GCAKR

[0108] The experimental conditions are set as follows:

[0109] Basic reaction system: 1 mL of the system contained 10 μg / mL PAT, 100 μg / mL GCAKR purified enzyme, 1 mM NADPH, and TBS buffer (pH 8.0), and was cultured at 30℃ with shaking at 150 rpm for 12 h. Based on this basic reaction system, the effects of temperature, pH, PAT concentration, and GCAKR dosage on the degradation of PAT by GCAKR were investigated.

[0110] pH effect: The reaction system was set at pH 3.0-8.0 (citric acid-sodium citrate buffer pH 3.0-6.0, TBS buffer pH 7.0-8.0), and other conditions were the same as the basic system. The PAT degradation rate was detected after 12 h of reaction.

[0111] Temperature effect: The reaction temperature was set at 4, 16, 20, 30 and 40℃, and other conditions were the same as the basic system. The PAT degradation rate was detected after 12 h of reaction.

[0112] Effect of enzyme concentration: The enzyme concentration was set at 25, 50, 75, 100, and 125 μg / mL, and the PAT concentration was 10 μg / mL. Other conditions were the same as the basic system. The PAT degradation rate was detected after 12 h of reaction.

[0113] Effect of substrate concentration: PAT concentrations of 1, 5, 10, 20, and 40 μg / mL and enzyme concentration of 100 μg / mL were set, with other conditions the same as the basic system. The PAT degradation rate was detected after 12 h of reaction.

[0114] Figure 6 The effects of temperature, pH, substrate concentration, and enzyme dosage on the degradation of PAT by GCAKR (wherein) Figure 6 A corresponds to the influence of GCAKR content. Figure 6 B corresponds to the effect of PAT concentration. Figure 6 C corresponds to pH effect Figure 6 D corresponds to the effect of temperature.

[0115] The results are as follows Figure 6 As shown, temperature plays a crucial role in enzyme activity. Within the range of 16–30℃, GCAKR maintains high activity, with a degradation rate of PAT exceeding 81.96%; however, at 4℃ and 40℃, the degradation rate decreases to 64.10% and 62.81%, respectively.

[0116] pH also has a significant effect on enzyme activity. Under acidic conditions of pH 3–5, the degradation rate of PAT is below 47.91%; when the pH rises to 6.0, the degradation rate increases to 79.93%; and in the pH range of 7.0–8.0, the degradation rate reaches its peak, approximately 95%.

[0117] The initial substrate concentration significantly modulates the degradation effect. When the initial PAT concentration is 1–10 μg / mL, the target substrate can be completely degraded within 12 h; however, when the concentration is increased to 20 μg / mL and 40 μg / mL, the degradation rate decreases to 66.08% and 48.42%, respectively.

[0118] Enzyme concentration is a crucial factor affecting degradation efficiency. When the GCAKR concentration increased from 25 μg / mL to 100 μg / mL, the PAT degradation rate significantly increased from 20.29% to 100%. However, once the enzyme concentration exceeded 100 μg / mL, the degradation efficiency did not improve further, indicating that the enzyme-substrate binding had reached saturation.

[0119] Example 4 Determination of PAT degradation products

[0120] I. Experimental Methods

[0121] The reaction system consists of: adding PAT standard (final concentration 10 μg / mL), GCAKR enzyme (final concentration 100 μg / mL), and NADPH coenzyme (final concentration 1 mM) to a 2 mL system.

[0122] Reaction conditions control: The reaction system was incubated in a constant temperature shaking incubator at 30℃ and 150 rpm, and sampling time points were set at 0 h, 4 h, 8 h, 12 h and 16 h respectively.

[0123] Sample pretreatment: After sampling at each time point, immediately add an equal volume of acetonitrile solution containing 1% formic acid, mix well to terminate the enzymatic reaction, take the supernatant and filter it through a 0.22 μm organic phase filter membrane, store it at -20℃ for later use, and analyze it by UPLC-Q-TOF / MS.

[0124] UPLC-Q-TOF / MS detection conditions:

[0125] Chromatographic conditions: A Poroshell C18 column (100 × 2.1 mm, 1.9 μm) was used; mobile phase A was ultrapure water containing 0.1% formic acid, and mobile phase B was acetonitrile; the flow rate was 0.3 mL / min; the column temperature was 40℃; the injection volume was 20 μL; the elution program was isocratic elution, maintaining mobile phase A at 95% and mobile phase B at 5% throughout the elution process.

[0126] Mass spectrometry conditions: Electrospray ionization (ESI) source, negative ion detection mode; capillary voltage 3000 V; end plate offset voltage 500 V; nebulizing gas pressure 3 bar; drying gas flow rate 8 L / min; drying gas temperature 220℃; scanning range m / z 50-1500; collision energy 7 eV; ion energy 4 eV; sodium formate was used for mass calibration to ensure detection accuracy.

[0127] System suitability verification: Use PAT standards to conduct system suitability tests to confirm the stability of retention time (RSD≤2%) and the repeatability of peak area (RSD≤3%). Sample analysis can only be performed after the system meets the testing requirements.

[0128] II. Experimental Results

[0129] 1. Dynamic monitoring of PAT degradation

[0130] UPLC chromatographic analysis showed that the retention time of the PAT standard was 4.1 min (e.g., Figure 7 (As shown in A). In the enzymatic reaction system, as the reaction time increased, the chromatographic peak intensity of PAT at 4.1 min gradually decreased: the peak intensity was highest at 0 h, decreased significantly at 4 h, was extremely low at 8 h, and completely disappeared at 12 h, indicating that the GCAKR enzyme can completely degrade PAT in the system.

[0131] 2. Separation and identification of degradation products

[0132] The emergence of a new product: While the PAT peak gradually disappeared, a new chromatographic peak appeared at a retention time of 2.8 min (e.g., Figure 7 As shown in Figure B, the intensity of this peak gradually increases with the extension of reaction time: it begins to appear at 4 h, the intensity increases significantly at 8 h, and remains stable at 12-16 h, indicating that the substance corresponding to this new peak is a product of PAT catalyzed by the degradation of PAT by GCAKR enzyme.

[0133] Molecular weight determination: High-resolution Q-TOF / MS analysis was performed on the new peak at 2.8 min, and its deprotonated ion peak was detected at m / z 155.0343 (e.g., Figure 7 (As shown in C). Based on precise molecular weight calculations, the molecular formula of this ion is C7H8O4, which has two more hydrogen atoms than the molecular formula of PAT (C7H6O4), suggesting that PAT may generate this degradation product through a reduction reaction.

[0134] Structural confirmation: The degradation product was analyzed by MS / MS secondary mass spectrometry, such as... Figure 7 As shown in Figure C, characteristic fragment ion peaks were obtained: m / z 137.0243 (corresponding to C7H6O3, a neutral fragment indicating the loss of one molecule of H2O, confirming the presence of a primary hydroxyl group in the molecule), m / z 127.0415 (corresponding to C6H8O3, a neutral fragment indicating the loss of CO from the side chain carbonyl group, confirming the open-ring structure of the metabolite), and m / z 111.0748 (corresponding to C6H8O2, a characteristic fragment indicating the loss of CO2 after decarboxylation of the lactone after ring opening). These characteristic fragments are completely consistent with the characteristic fragments of the known PAT degradation product E-ascladiol, confirming that the product of PAT degradation catalyzed by GCAKR enzyme is E-ascladiol. E-ascladiol, chemically named (5E)-5-(2-hydroxyethylmethylene)-4-(hydroxymethyl)furan-2(5H)-one, is a fungal metabolite closely related to the biosynthesis and degradation of patulin.

[0135] like Figure 7 As shown in D, the GCAKR enzyme can catalyze the cleavage of the CO bond in the dihydropyran ring and the opening of the lactone ring in the PAT molecule, specifically converting PAT into E-ascladiol. This degradation process is highly efficient and specific, and can completely degrade PAT in the system within 12 hours. Moreover, the degradation products have a well-defined structure, providing core experimental evidence for the application of GCAKR enzyme in the detoxification of PAT contamination in food and the environment.

[0136] The amino acid sequence of the aldehyde-ketone reductase GCAKR of the present invention is as follows (SEQ ID NO.1):

[0137] MSFKTNSFVKLNSGYNIPRIGLGVFKAESSEATDSVLVALEAGYRHVDSAKYYNNERDSVEGILKFLEKPGQTVKREDIFYTTKIWNDDHGYEKATAAIDAALDKLSGYSKEDQTTRNLEYIDLVLIHSPLSNREKRLGTWKALQEAVKA GKIKSIGVSNYGIPHLEELLNWDGLVIKPAVDQIELHPWLQRSELVGYLRNKDIVPVAYSPLTRGQRLDDPQLAEYAKRYNKSPAQILIKWSLQAGFVSLPKSTNPQRIKDNIDIDDFELSAEDFKKLGDKNENGVTGWDPTVHPVDADA.

[0138] The codon-optimized nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1 (SEQ ID NO.2):

[0139] ATGAGCTTCAAGACTAACAGCTTCGTTAAACTGAACTCTGGTTACAACATCCCGCGTATCGGTCTGGGTGTATTCAAAGCGGAATCTAGCGAAGCGACCGATTCCGTTCTGGTTGCACTGGAAGCTGGTTACCGTCACGTTGACTCTGCGAAATACTACAACAACGAACGTGATTCCGTTGAAGGCATTCTGAAATTTCTGGAGAAACCGGGTCAGACCGTTAAACGTGAAGACATCTTCTACACCACCAAGATCTGGAACGACGACCACGGCTACGAGAAAGCGACCGCAGCAATCGATGCAGCACTGGACAAACTGTCCGGTTACTCCAAAGAAGACCAGACCACTCGTAACCTGGAATACATCGACCTGGTGCTGATCCACTCTCCACTGTCTAACCGTGAGAAACGTCTGGGTACCTGGAAAGCGCTGCAAGAAGCAGTTAAAGCGGGTAAGATCAAATCCATCGGTGTTTCCAACTACGGTATTCCGCATCTGGAAGAACTGCTGAACTGGGACGGTCTGGTTATCAAACCAGCTGTTGACCAGATCGAACTGCATCCGTGGCTGCAGCGTTCTGAACTGGTTGGTTACCTGCGTAACAAAGACATCGTTCCGGTTGCTTACAGTCCACTGACTCGTGGTCAGCGTCTGGACGATCCACAACTGGCGGAATACGCTAAACGTTACAACAAATCCCCAGCTCAGATCTTGATCAAATGGTCTCTGCAGGCTGGTTTCGTAAGCCTGCCGAAATCCACCAATCCGCAGCGTATCAAAGATAACATCGACATCGACGACTTCGAACTGTCTGCTGAAGACTTCAAGAAACTGGGTGATAAGAACGAGAACGGTGTTACCGGTTGGGATCCAACCGTACATCCAGTTGACGCGGACGCA。

Claims

1. An aldehyde-ketone reductase GCAKR, characterized in that, The aldehyde-ketone reductase is any one of the following proteins: (1) A protein with an amino acid sequence as shown in SEQ ID NO.1; (2) Proteins that have more than 90% homology with the amino acid sequence shown in SEQ ID NO.1 and have the same functional group.

2. The gene encoding aldehyde-ketone reductase as described in claim 1, characterized in that, The encoding gene is any one of the following nucleic acid molecules: (1) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1; (2) The nucleotide sequence as shown in SEQ ID NO.2; (3) Having more than 90% homology with the nucleotide sequence defined in (1) or (2) and encoding the nucleotide sequence of the aldehyde-ketone reductase of claim 1; (4) Hybridizes under stringent conditions to the nucleotide sequence defined in (1) or (2) and encodes the nucleotide sequence of the aldehyde-ketone reductase of claim 1.

3. A recombinant expression vector, characterized in that, It contains the encoding gene as described in claim 2.

4. The recombinant expression vector as described in claim 3, characterized in that: The recombinant expression vector is a recombinant expression vector obtained by inserting the coding gene of claim 2 into the multiple cloning site of the backbone vector pET-28a-SUMO.

5. A recombinant engineered bacterium, characterized in that: Contains the encoding gene as described in claim 2; Preferably, the recombinant engineered bacteria is obtained by introducing the recombinant expression vector of claim 4 into Escherichia coli.

6. A method for preparing the aldehyde-ketone reductase according to claim 1, characterized in that: The recombinant engineered bacteria described in claim 5 are cultured, induced to express, and the target protein is obtained after being cleaved by a tag.

7. The preparation method according to claim 6, characterized in that, Includes the following steps: (1) The recombinant expression vector described in claim 3 is transformed into competent Escherichia coli cells, and recombinant engineered bacteria are obtained by heat shock, recovery culture and screening; (2) Select single clones and inoculate them into the selection medium to obtain seed culture; (3) Add the seed culture to the screening medium and culture it; (4) Add an inducer to induce the expression of the target protein; (5) Collect the induced bacterial cells and crush them to extract crude enzyme solution; (6) Purify the crude enzyme solution; (7) Remove the fusion tag from the purified protein; (8) Purify again to obtain aldehyde-ketone reductase.

8. The application of the aldehyde-ketone reductase as described in claim 1 in the degradation of patulin.

9. The application according to claim 8, characterized in that, The steps are as follows: Add the aldehyde-ketone reductase described in claim 1 to the object to be treated containing patulin, and react at a temperature of 4~40℃, so that the aldehyde-ketone reductase degrades patulin.

10. The application according to claim 9, characterized in that, The steps are as follows: Add the aldehyde-ketone reductase, coenzyme NADPH, and buffer solution as described in claim 1 to the object to be treated containing patulin to form a reaction system. Control the pH of the reaction system to 6.0-8.0, the temperature to 4-40℃, and the reaction time to 8-36 h to achieve efficient degradation of patulin and generate the degradation product E-ascladiol. Preferably, the concentration of patulin in the reaction system is 1-40 μg / mL, the amount of aldehyde-ketone reductase is 25-200 μg / mL, and the concentration of NADPH is 1-4 mM.

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