A short-chain dehydrogenase derived from abnormal wickerham yeast, and a preparation method and application thereof

Abstract

The present application belongs to the technical field of biological enzyme engineering, and discloses a short-chain dehydrogenase WaSDR derived from abnormal williamsmyces, and a preparation method and application thereof. The short-chain dehydrogenase WaSDR enzyme in the present application is derived from abnormal williamsmyces, and the WaSDR enzyme can not only efficiently degrade patulin, but also can significantly improve the abundance of key flavor substances, the level, richness and complexity of the flavor of the fruit juice by precisely regulating the volatile component spectrum on the basis of retaining the original flavor characteristics of the fruit juice when added to the fruit juice. The present application provides an efficient and safe enzyme preparation for the biological degradation of patulin in food, effectively controls the PAT pollution in food processing, and simultaneously realizes the synergistic improvement of the nutrition and flavor quality of the product, and has important industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of bioenzyme engineering technology, specifically relating to a short-chain dehydrogenase WaSDR derived from Wickham's abnormal yeast, its preparation method, and its application. Background Technology

[0002] Patulin (PAT) is a type of secondary metabolite produced by fungi such as Penicillium, Aspergillus, and Hymenocytoxin. As a typical mycotoxin, it widely contaminates fresh fruits such as apples, pears, and peaches. It tends to accumulate in large quantities, especially during fruit rot and processing, and eventually remains in various fruit processed products such as juice, jam, and wine, becoming a significant threat to food safety.

[0003] From a toxicological perspective, the hemiacetal and lactone groups in the PAT molecule are highly reactive, specifically binding to the sulfhydryl groups of intracellular biomolecules such as DNA, RNA, enzymes, and lipids. This not only disrupts cell membrane integrity but also significantly inhibits nucleic acid and protein biosynthesis, leading to multi-organ toxicity. Studies have confirmed that ingestion of PAT-containing foods can cause a range of health problems, including immune system damage, abnormal liver and kidney function, nervous system disorders, and intestinal mucosal inflammation. The harm is particularly pronounced in vulnerable populations such as children and pregnant women. Given its serious health risks, many countries and international organizations have established strict limits. For example, China, the European Union, and the United States stipulate that the maximum permissible PAT content in fruits and their processed products should not exceed 50 μg / kg, highlighting the urgent need to control this toxin contamination.

[0004] However, the unique physicochemical properties of PAT make it difficult to remove effectively during food processing. This toxin exhibits strong thermal stability in acidic environments, and conventional processing techniques such as pasteurization of fruit juices and high-temperature sterilization of canned goods not only fail to degrade it but may also lead to toxin concentration accumulation due to water evaporation, posing a significant challenge to food safety. To address this problem, researchers have developed various PAT removal technologies, including physical, chemical, and biological methods, but all have significant limitations: physical methods (such as adsorption, membrane separation, and irradiation), while convenient to operate, easily damage the color, texture, and nutritional components of food and may lead to toxin transfer rather than complete degradation; chemical methods (such as ozone oxidation and hydrogen peroxide treatment) can achieve partial degradation of PAT, but easily produce potentially toxic degradation products and may introduce new chemical contaminants, posing secondary food safety risks; traditional biological methods, such as microbial adsorption technology, are limited by the number of adsorption sites on microbial cell walls, resulting in low removal efficiency and susceptibility to the influence of the food system environment, making it difficult to meet the needs of industrial applications.

[0005] Enzymatic degradation technology, as a core branch of biological methods, has become a research hotspot in the field of PAT pollution control due to its advantages such as mild reaction conditions, environmental friendliness, high specificity, and no secondary pollution. This technology uses specific degrading enzymes produced by microorganisms to convert PAT into non-toxic products, fundamentally eliminating its hazards. Previous studies have found that microorganisms such as *Kluyveromyces martensii* can degrade PAT into non-toxic substances through intracellular enzymes. However, the enzyme production efficiency of these natural microorganisms is low, and the process of isolating and purifying degrading enzymes from fermentation broth is complex, time-consuming, labor-intensive, and costly, resulting in a very limited number of identified PAT-specific degrading enzymes. The scarcity of degrading enzyme resources and the bottlenecks in preparation technology severely restrict the large-scale application of enzymatic degradation technology in the food industry.

[0006] Therefore, exploring new and efficient PAT-degrading enzyme resources, establishing simple and efficient enzyme preparation processes, and developing application technologies suitable for food processing scenarios are key to breaking through the current predicament of PAT pollution control. This has important practical significance and application value for ensuring food safety and protecting public health.

[0007] *Wickerhamomyces anomalus*, belonging to the Ascomycota, is a single-celled fungus widely distributed in soil, fruits, and fermented foods. It exhibits strong resistance to environmental stresses such as high sugar, high salt, and pH fluctuations, and can grow in environments ranging from 10–35°C to pH 3.0–7.0. Its unique physiological characteristics enable it to produce antifungal metabolites, effectively inhibiting mold growth in food and reducing the accumulation of mycotoxins, thus acting as a natural barrier for food preservation. In terms of safety, *Wickerhamomyces anomalus* is classified as a Biosafety Level 1 (BSL-1) strain, posing no health threat to individuals with healthy immune systems. However, there are currently few reports on its use in degrading patulin.

[0008] Short-chain dehydrogenases / reductases (SDRs) are one of the most widely distributed and numerous enzyme superfamilies in nature. They are a class of NAD(P)H coenzymes-dependent oxidoreductases that play a central role in metabolic regulation, signal transduction, and biosynthesis in organisms. Currently, there are few reports on the use of SDRs derived from *Saccharomyces aberrantis* for PAT degradation. Summary of the Invention

[0009] The purpose of this invention is to address the above-mentioned problems by providing a short-chain dehydrogenase WaSDR derived from *Wickhamia esculenta*, its preparation method, and its applications.

[0010] To achieve its objective, the present invention employs the following technical solution:

[0011] A first aspect of the present invention provides a short-chain dehydrogenase WaSDR, said short-chain dehydrogenase being any of the following proteins:

[0012] (1) A protein with an amino acid sequence as shown in SEQ ID NO.1;

[0013] (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.

[0014] A second aspect of the present invention provides a gene encoding a short-chain dehydrogenase as described above, wherein the gene is any of the following nucleic acid molecules:

[0015] (1) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1;

[0016] (2) The nucleotide sequence as shown in SEQ ID NO.2;

[0017] (3) A nucleotide sequence that has more than 90% homology with the nucleotide sequence defined in (1) or (2) and encodes the above-mentioned short chain dehydrogenase;

[0018] (4) Hybridizes under strict conditions to the nucleotide sequence defined in (1) or (2) and encodes the nucleotide sequence of the short chain dehydrogenase described above.

[0019] A third aspect of the present invention provides a recombinant expression vector containing the above-described coding gene.

[0020] 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.

[0021] A fourth aspect of the present invention provides a recombinant engineered bacterium containing the above-described encoding gene;

[0022] Preferably, the recombinant engineered bacteria is obtained by introducing the above-mentioned recombinant expression vector into Escherichia coli.

[0023] The fifth aspect of the present invention provides a method for preparing the above-mentioned short-chain dehydrogenase, wherein the above-mentioned recombinant engineered bacteria are cultured, induced to express, and cleaved by a tag to obtain the target protein.

[0024] Preferably, the preparation method includes the following steps:

[0025] (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;

[0026] (2) Select single clones and inoculate them into the selection medium to obtain seed culture;

[0027] (3) Add the seed culture to the screening medium and culture it;

[0028] (4) Add an inducer to induce the expression of the target protein;

[0029] (5) Collect the induced bacterial cells and crush them to extract crude enzyme solution;

[0030] (6) Purify the crude enzyme solution;

[0031] (7) Remove the fusion tag from the purified protein;

[0032] (8) Purify again to obtain short-chain dehydrogenase.

[0033] The sixth aspect of the present invention provides the application of the above-described short-chain dehydrogenase in the degradation of patulin;

[0034] The preferred application is as follows: adding the above-mentioned short-chain dehydrogenase to the object to be treated containing patulin, and reacting to degrade patulin by the short-chain dehydrogenase.

[0035] Preferably, the application steps are as follows: The above-mentioned short-chain dehydrogenase, 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 7-8, and the temperature is controlled at 4-40℃ (preferably 4-30℃) for 12-36 h. Preferably, the concentration of patulin in the reaction system is 1-40 μg / mL, the amount of short-chain dehydrogenase is 10-100 μg / mL, and the concentration of NADPH is 1-4 mM.

[0036] The seventh aspect of the present invention provides the application of the above-described short-chain dehydrogenase as a food additive in fruit juice; preferably for enhancing the flavor of fruit juice, wherein the fruit juice is apple juice;

[0037] The preferred application is as follows: WaSDR enzyme and coenzyme NADPH are added to the juice so that the juice contains a final concentration of 25~35 μg / mL of WaSDR enzyme and 0.7~1.3 mmol / L of NADPH, and the system is reacted at 25~35℃ for 12~48 h.

[0038] The beneficial effects of this invention are:

[0039] (1) High degradation efficiency and strong specificity: The short-chain dehydrogenase (WaSDR) of the present invention can efficiently degrade PAT. Under suitable conditions, it can completely degrade PAT in buffer solution within 24 hours and completely degrade PAT in apple juice within 48 hours. The degradation rate can reach 100%, effectively reducing the PAT content in food and ensuring food safety.

[0040] (2) Strong adaptability: The enzyme has good stability in the medium and low temperature range (4~30℃) and is tolerant to most common metal ions in food. For complex low pH systems such as apple juice (pH 3.0-4.5), although the degradation activity is slightly lower than that under neutral to weakly alkaline conditions (pH 7.0-8.0), it can still maintain an effective degradation rate of 63%-88%. Furthermore, by optimizing process parameters (such as increasing the enzyme concentration to 40-50 μg / mL or extending the reaction time to 72 hours), the PAT degradation rate can be ≥95%, which is fully adaptable to the actual process requirements of low pH food processing.

[0041] (3) It can optimize food quality: While degrading PAT, the enzyme can promote the release of bound functional components (such as vitamin C and total phenols) in food into free state, improve the nutritional value of food, enhance the release of specific volatile flavor components, optimize food flavor, and maintain the stability of basic physicochemical indicators and color of food.

[0042] (4) High efficiency of preparation process: Recombinant engineered bacteria are constructed by using the Escherichia coli exogenous expression system. Combined with Ni-NTA affinity chromatography, dialysis and tag cutting, the target protein with a purity of ≥95% can be obtained. The preparation process is simple and efficient, suitable for large-scale production, and provides a guarantee for its industrial application.

[0043] The short-chain dehydrogenase involved in this invention is derived from *Saccharomyces cerevisiae* XL1, and its encoding gene was obtained through recombinant expression and purification. This enzyme is safe and highly efficient, effectively degrading patulin in food and improving the safety of related products. Complete degradation of patulin in apple juice can be achieved within 48 hours of treatment with this enzyme, while simultaneously increasing the content of vitamin C and total phenols. Vitamin C can inhibit the oxidation of phenolic substances, maintaining their appearance and quality stability. This enzyme shows good application potential in the food industry and is suitable for the control and quality improvement of patulin contamination in fruit juices and liquid foods. Attached Figure Description

[0044] Figure 1 This is a map of the WaSDR recombinant plasmid.

[0045] Figure 2 This is the SDS-PAGE result of WaSDR.

[0046] Figure 3 The effect of NADPH concentration on the degradation of PAT by WaSDR.

[0047] Figure 4 The effects of temperature, pH, substrate concentration, and enzyme dosage on the degradation of PAT by WaSDR were investigated.

[0048] Figure 5 The effect of metal ions on the degradation of PAT by WaSDR.

[0049] Figure 6 The effect of WaSDR on the degradation of patulin in apple juice.

[0050] Figure 7 The effects of WaSDR treatment on the nutritional components, physicochemical properties and sensory quality of apple juice.

[0051] Figure 8 Results of the electronic nose after WaSDR processing. Detailed Implementation

[0052] 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.

[0053] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0054] Example 1

[0055] I. Construction of recombinant expression plasmid pET-28a-SUMO-WaSDR

[0056] The abnormal Wickham yeast strain XL1 has been disclosed in Chinese patent application 202510965743.0 (publication number CN120843305 A), and its deposit information is as follows:

[0057] The *Wickerhamomyces anomalus* strain XL1 was deposited at the China General Microbiological Culture Collection Center (CGMCC) in May 2025, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date was May 12, 2025, the accession number was CGMCC No. 34500, and the classification name was *Wickerhamomyces anomalus*.

[0058] The short-chain dehydrogenase WaSDR gene of *Saccharomyces cerevisiae* XL1 in this invention was obtained and identified through a strategy of proteomics screening combined with homologous cloning and functional characterization verification. The specific process was as follows: First, quantitative proteomics analysis was performed on *Saccharomyces cerevisiae* XL1 after patulin (PAT) stress treatment to screen for differentially upregulated redox-related proteins and obtain their amino acid sequences. Then, based on these amino acid sequences and combined with the conserved region characteristics of short-chain dehydrogenase (SDR) genes (NCBI accession number: ncbi_30200513) from closely related species of *Saccharomyces cerevisiae* reported in the NCBI database, specific amplification primer pair SDR-F / SDR-R was designed. Genomic DNA of *Saccharomyces cerevisiae* XL1 was extracted as a template for PCR amplification. The amplified gene fragment was sequenced to obtain its nucleotide sequence. Homology comparison of this sequence with SDR family genes in the GenBank database showed that the encoded amino acid sequence contained the core conserved domain of the SDR family and also possessed the SDR family's NAD-dependent amino acid sequence. Based on the oxidoreductase functional characteristics of (P)H, the gene was identified as a short-chain dehydrogenase gene and named WaSDR (Wickerhamomyces anomalus XL1 Short-chain Dehydrogenase / Reductase). Subsequently, codon optimization and prokaryotic expression verification were completed based on the natural gene sequence, and the complete functional sequence of the WaSDR gene was finally obtained.

[0059] The specific amplification primer pair SDR-F / SDR-R sequences are as follows:

[0060] SDR-F (SEQ ID NO.3):

[0061] 5'-ATTATTGAGGCTCACAGAGAACAGATTGGTGGATCCATGTCTGGTAAAGTTTACTTCGT-3';

[0062] SDR-R (SEQ ID NO.4):

[0063] 5'-TCAGTGGTGGTGGTGGTGGTGCTCGAGTTAGAACACCTGTTCCACACC-3'.

[0064] Based on proteomics data, a short-chain dehydrogenase amino acid sequence from *Wickerhamomyces anomalus* XL1 was screened, as shown in SEQ ID NO.1. Its amino acid composition contains four methionines (1.6%), no cysteine, and a relatively abundant proline content (nine, 3.5%). The predicted solubility score in *E. coli* is 0.7641, indicating that this protein possesses highly efficient soluble expression characteristics and is suitable for recombinant expression using a prokaryotic expression system.

[0065] 1. Strains and vectors

[0066] Cloning host bacteria: Escherichia coli DH5α competent cells, purchased from Beijing Qingke Biotechnology Co., Ltd.;

[0067] 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.

[0068] 2. Gene optimization and synthesis

[0069] Based on the WaSDR gene sequence (containing 254 amino acids) of Wickerhamomyces anomalus XL1, codon optimization was performed considering E. coli codon bias. The optimized gene was synthesized by Beijing Qingke Biotechnology Co., Ltd. The optimized codon is shown in SEQ ID NO.2.

[0070] This study optimized the codons of the WaSDR gene, with the core objective of improving 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 expressed WaSDR protein has the same amino acid sequence as the naturally expressed protein of Wickham yeast XL1 (both are SEQ ID NO.1), and the protein's catalytic function and spatial structure remain unchanged.

[0071] 3. Enzyme digestion and ligation

[0072] The synthesized WaSDR gene and pET-28a-SUMO vector were treated with restriction endonucleases BamHI / XhoI, respectively. After the digestion products were recovered by gel electrophoresis, they were ligated overnight at 16°C using T4 DNA ligase to construct the recombinant expression plasmid pET-28a-SUMO-WaSDR (containing an N-terminal 6×His-SUMO fusion tag).

[0073] 4. Transformation and Identification

[0074] 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 optimized WaSDR gene sequence, confirming the successful construction of the recombinant plasmid. The plasmid map is shown below. Figure 1 .

[0075] The positive clone (E. coli DH5α / pET-28a-SUMO-WaSDR) that was verified by sequencing was inoculated into LB liquid medium containing 50 μg / mL kanamycin sulfate and cultured at 37°C and 220 rpm for 12 h with shaking. The recombinant plasmid pET-28a-SUMO-WaSDR was extracted using a plasmid miniprep kit for subsequent construction of recombinant engineered bacteria.

[0076] II. Construction of recombinant engineered bacteria, WaSDR enzyme-induced expression, and isolation and purification

[0077] 1. Construction and Induced Expression of Recombinant Engineered Bacteria

[0078] The recombinant plasmid pET-28a-SUMO-WaSDR, extracted after cloning and amplification of E. coli DH5α, was heat-shocked into E. 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, it was heat-shocked at 42℃ for 45-60 sec, immediately returned to ice to cool for 5 min, 600 μL of LB medium was added, and the cells were 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 (pET-28a-SUMO-WaSDR / BL21(DE3)).

[0079] 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. Isopropyl-β-D-thiogalactoside (IPTG) was added to one system to a final concentration of 0.2 mM, and the system was incubated overnight at 18°C ​​with shaking at 220 rpm. IPTG was added to the other system to a final concentration of 0.2 mM, and the system was incubated at 30°C with shaking at 220 rpm for 16 h to induce soluble expression of the WaSDR-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 pellet was collected for later use.

[0080] 2. Cell disruption and preparation of crude enzyme solution

[0081] 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.

[0082] 3. Ni-NTA affinity purification

[0083] 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 WaSDR-SUMO fusion protein.

[0084] 4. SUMO tag cleavage and secondary purification

[0085] 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 is the WaSDR pure enzyme with the SUMO tag removed. 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.

[0086] 5. Purity Verification and Result Analysis

[0087] Enzyme purity was assessed using SDS-PAGE electrophoresis with a 12% separating gel and a 5% stacking gel. After electrophoresis, the bands were observed after Coomassie brilliant blue staining and destaining. Figure 2 As shown, the electrophoresis results of the finally purified WaSDR enzyme were verified. With 2 μg of purified enzyme sample loaded into each lane, a single sharp band appeared in the 25-27 kDa range, with no detectable contaminating proteins, indicating that the final WaSDR enzyme had a purity ≥95% and a molecular weight of 27.7 kDa, meeting the requirements for subsequent experiments. The obtained WaSDR enzyme was used in subsequent Examples 2-6.

[0088] Example 2: Effect of coenzyme NADPH concentration on the degradation of PAT by WaSDR

[0089] 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 WaSDR (final concentration 100 μg / mL) purified in Example 1 in 2 mL enzyme-free sterile centrifuge tubes to form four systems. Control group: The difference was that the system did not contain NADPH and WaSDR.

[0090] 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.

[0091] 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.

[0092] Degradation rate calculation formula:

[0093]

[0094] In the formula, C0 is the initial concentration of PAT, and C is the residual concentration of PAT after the reaction.

[0095] Figure 3 The effect of NADPH concentration on the degradation of PAT by WaSDR was investigated: In the control group, the PAT degradation rate only increased from 16.21% at 4 hours to 55.96% within 24 hours, indicating low degradation efficiency. In contrast, the degradation rate in the NADPH-treated groups was significantly higher than that in the control group at all time points, and there was no significant difference in degradation rate among different NADPH-treated groups. All NADPH-treated groups achieved 100% complete degradation of PAT within 24 hours. This clearly demonstrates that NADPH is a key cofactor for PAT degradation by WaSDR, significantly improving the efficiency of this degradation process. Further experiments confirmed that when the NADPH concentration in the reaction system is ≥1.0 mM, 30 μg / mL WaSDR can completely degrade 10 μg / mL PAT within 24 hours, and the optimal NADPH concentration in this reaction system was determined to be 1.0 mM.

[0096] Example 3: Effects of temperature, pH, PAT concentration, and WaSDR dosage on the degradation of PAT by WaSDR

[0097] Basic reaction system: 1 mL of the system contained 10 μg / mL PAT, 30 μg / mL WaSDR enzyme, 1 mM NADPH, and TBS buffer (pH 8.0), and was cultured at 30℃ with shaking at 150 rpm for 24 h. Based on this basic reaction system, the effects of temperature, pH, PAT concentration, and WaSDR dosage on the degradation of PAT by WaSDR were investigated.

[0098] 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 24 h of reaction.

[0099] Temperature effect: The reaction temperature was set at 4, 10, 20, 30 and 40℃, and other conditions were the same as the basic system. The PAT degradation rate was detected after 24 h of reaction.

[0100] Effect of enzyme concentration: Enzyme concentrations of 10, 20, 30, 40, 50, 80, and 100 μg / mL were set, and PAT concentration of 10 μg / mL was set. Other conditions were the same as the basic system. The PAT degradation rate was detected after 24 h of reaction.

[0101] Effect of substrate concentration: PAT concentrations of 1, 5, 10, 20, and 40 μg / mL and enzyme concentration of 30 μg / mL were set, with other conditions the same as the basic system. The PAT degradation rate was detected after 24 h of reaction.

[0102] Figure 4 The effects of temperature, pH, substrate concentration, and enzyme dosage on the degradation of PAT by WaSDR (wherein) Figure 4 A corresponds to the effect of pH. Figure 4 B corresponds to the effect of temperature. Figure 4 C corresponds to the effect of PAT concentration. Figure 4 D corresponds to the effect on WaSDR content). The results are as follows: Figure 4 As shown, regarding enzyme concentration, the degradation rate of PAT significantly increased with increasing enzyme concentration (10-30 μg / mL), and complete degradation of PAT could be achieved when the enzyme concentration reached 30 μg / mL. Regarding PAT concentration, when the PAT concentration was ≤10 μg / mL, 100% degradation could be achieved within 24 hours, and the degradation rate remained above 40% when the PAT concentration was increased to 20-40 μg / mL. Regarding pH, the degradation rate was 63%-88% under acidic conditions (pH 3-6), while it reached 100% under neutral to weakly alkaline conditions (pH 7.0-8.0). Regarding temperature, the degradation rate of PAT by this enzyme remained stable at 100% in the range of 4-30℃, and the degradation rate decreased to 80.94% at 40℃.

[0103] In summary, the recommended concentration of WaSDR short-chain dehydrogenase is 30 μg / mL, and the final concentration of coenzyme NADPH should not be less than 1.0 mM. The preferred pH of the reaction system is 7.0-8.0, and the reaction temperature range is 4-40℃. The enzyme activity is stable between 4-30℃, at which temperature the PAT degradation rate is ≥93%, achieving efficient degradation. The acceptable enzyme activity range is between 31-40℃, at which temperature the PAT degradation rate is ≥80%, still meeting the toxin removal requirements in practical applications. WaSDR can be adapted to some food processing scenarios with high temperature requirements, further broadening the practical application range of this enzyme.

[0104] Example 4: Effect of metal ions on the degradation of PAT by WaSDR

[0105] To investigate the effect of metal ions on the degradation rate of PAT, a series of metal ions (Fe) were selected. 3+ K + Mg 2+ Zn 2+ Cu 2 + Ca 2+ Na + Mn 2+ Al 3+ The study was conducted using TBS buffer (pH 8) containing 30 μg / mL WaSDR enzyme, 10 μg / mL PAT, and 1 mmol / L NADPH. Target metal ions were added to each reaction system to a final concentration of 1 mmol / L. All systems were reacted at 30℃ and 150 rpm for 24 h with shaking. After the reaction, an equal volume of acetonitrile solution containing 1% formic acid was added to terminate the reaction. The solution was vortexed, filtered through a 0.22 μm filter, and residual PAT was quantitatively analyzed by HPLC.

[0106] The results are as follows Figure 5 As shown, for Mg 2+ Cu 2+ Fe 3+ Al 3+ Mn 2+ Even with metal ions, WaSDR maintained a 100% degradation rate of PAT, indicating that these ions had no significant inhibitory effect on the enzyme's degradation efficiency; however, the addition of K... + Zn 2+ Na + Ca 2+ At that time, the degradation rate was between 93.41% and 98.54%, with only a slight inhibitory effect on the degradation effect. Tolerance experiments to common metal ions showed that when Fe was present in the system... 3+ K + Mg 2+ Zn 2+ Cu 2+ Ca 2+ Na + Mn 2+ Al 3+Even with metal ions present at a final concentration not exceeding 1 mmol / L, WaSDR maintains over 93% of its PAT degradation activity, indicating stable degradation performance in food and environmental systems with complex ionic compositions, ensuring its reliability in real-world applications. WaSDR exhibits good tolerance to most common metal ions, with only a few causing slight inhibition, maintaining high overall degradation activity. This characteristic makes it suitable for practical scenarios in food processing (such as juice containing trace amounts of metal ions), providing compatibility for its industrial applications.

[0107] Example 5: Effect of WaSDR on the degradation rate of PAT in apple juice

[0108] The experimental system consisted of 5 mL apple juice containing a final concentration of 30 μg / mL WaSDR enzyme, 10 μg / mL PAT, and 1 mmol / L NADPH. All systems were reacted at 30℃ and 150 rpm with shaking, and samples were taken at 12, 24, 48, 72, and 96 h. The reaction was terminated by adding an equal volume of acetonitrile solution containing 1% formic acid. After vortexing, the solution was filtered through a 0.22 μm filter, and residual PAT was quantitatively analyzed by HPLC.

[0109] The results are as follows Figure 6 As shown, the PAT degradation rate was only 32.18% after 12 hours, rising to 62.07% after 24 hours, and remaining stable at 100% within 48-96 hours, achieving complete PAT degradation. The low pH environment of apple juice inhibits the degradation efficiency of WaSDR to some extent, but even with this inhibitory factor, WaSDR can still complete the complete degradation of PAT in apple juice within 48 hours. Therefore, WaSDR is suitable for application scenarios in complex low-pH systems such as apple juice in the food industry and has good potential for industrial application in food processing.

[0110] In summary, this embodiment systematically verifies the PAT degradation efficiency of WaSDR short-chain dehydrogenase in the actual application scenario of apple juice, clarifying its degradation efficiency and stability in complex food systems. To simulate the apple juice processing environment in industrial production, an apple juice experimental system containing PAT was constructed: PAT with a final concentration of 10 μg / mL was added to apple juice, along with WaSDR short-chain dehydrogenase with a final concentration of 30 μg / mL and 1.0 mM coenzyme NADPH, and the mixture was shaken under mild conditions of 30℃ and 150 rpm. The residual PAT concentration of apple juice samples at different time points (12 h, 24 h, 48 h, 72 h, and 96 h) was detected by high-performance liquid chromatography (HPLC). The results showed that the PAT degradation rate was only 32.18% after 12 h of treatment, increased to 62.07% after 24 h, and achieved 100% complete PAT degradation after 48 h, with the degradation rate remaining at 100% after 72 h and 96 h. Even though the low pH acidity of apple juice has a certain inhibitory effect on enzyme activity, WaSDR short-chain dehydrogenase can still completely degrade the target concentration (10 μg / mL) of PAT within 48 hours, which fully demonstrates its application potential in complex food systems such as apple juice and provides an efficient and reliable technical solution for the industrial removal of PAT from processed fruit products.

[0111] Example 6: Effects of WaSDR treatment on the nutritional components, physicochemical properties, sensory quality, and flavor of apple juice.

[0112] I. Effects of WaSDR on the nutritional components, physicochemical properties, and sensory quality of apple juice

[0113] The experimental system consisted of 10 mL apple juice containing WaSDR enzyme (final concentration 30 μg / mL), PAT (final concentration 10 μg / mL), and NADPH (final concentration 1 mmol / L). The control group consisted of 10 mL apple juice without WaSDR enzyme, PAT, or NADPH. All systems were reacted at 30℃ and 150 rpm with shaking, and the reaction was terminated after 48 h. Three replicates were set up for each group, along with three blank control groups (containing only apple juice, without any reagents). All systems were processed simultaneously to ensure the reproducibility of the experimental results.

[0114] The physicochemical properties of apple juice were determined as follows: pH was measured directly using a pH meter; soluble solids content was measured using a handheld saccharimeter, and the corresponding Brix degree (°Brix) was calculated by measuring the refractive index of the juice; titratable acid content was determined by acid-base titration, using a 0.1 mol / L sodium hydroxide standard solution as the titrant and phenolphthalein as the acid-base indicator. The titratable acid content was calculated by utilizing the neutralization reaction of sodium hydroxide with organic acids in the juice and the volume of alkali consumed at the endpoint, combined with a conversion factor. Vitamin C content was determined by 2,6-dichlorophenolindophenol titration, with the result expressed as milligrams of vitamin C per 100 g of apple juice; browning degree was determined by colorimetry. After filtration, the absorbance of the sample at 420 nm was measured using an ELISA reader, and this absorbance value characterized the degree of browning; juice clarity was determined by measuring the transmittance of the uniformly mixed apple juice at 625 nm. The determination of total phenol content was based on the Folin-Ciocalteu method with slight modifications. The specific procedure was as follows: 0.4 mL of apple juice was accurately transferred and thoroughly mixed with 1.0 mL of Folin-Ciocalteu reagent, and the mixture was allowed to react in the dark for 5 min. Then, 2.0 mL of 5% sodium carbonate solution was added to bring the volume to 10 mL, and the mixture was shaken well. The reaction was continued for 60 min at room temperature in the dark. After the reaction was complete, the absorbance of the reaction system was measured at a wavelength of 765 nm to calculate the total phenol content in the sample. A colorimeter was used to measure the color parameters of the juice.

[0115] The results are as follows Figure 7 As shown, in terms of functional components, the control group apple juice contained less vitamin C ( Figure 7 The vitamin C content was 3.54±0.14 mg / L, and the total phenol content was 205.85±1.61 mg / L; the vitamin C content in the enzyme-treated group was significantly increased to 23.23±0.21 mg / L (p<0.05), and the total phenol content was 205.85±1.61 mg / L. Figure 7 The content of vitamin C (G) increased to 257.17±3.75 mg / L (p<0.05). This is because WaSDR can degrade macromolecules such as pectin and cellulose, releasing bound vitamin C and total phenols from the complex into free states and retaining them in the juice, while not damaging the original structure and physicochemical stability of the apple juice.

[0116] In terms of color and basic physicochemical indicators, the color parameters of the enzyme-treated juice compared to the control group ( Figure 7 D, brightness L*, red-green value a*, yellow-blue value b*), pH value ( Figure 7 A) Light transmittance ( Figure 7 B) Soluble solids content (°Brix, Figure 7 F), titration acidity ( Figure 7H) and browning degree ( Figure 7 C) No statistically significant changes were observed (p > 0.05). This indicates that WaSDR effectively degrades PAT and enhances nutritional components without causing common quality deterioration problems in fruit juice, such as acidification, decreased clarity, browning, or sugar-acid imbalance, thus ensuring the stability of the fruit juice in terms of sensory properties and basic physical properties. This result confirms that WaSDR can maintain or even optimize the basic quality of fruit juice while improving its functional components (vitamin C, total phenols), demonstrating its application advantages in the food processing field.

[0117] II. The effect of WaSDR on apple juice flavor

[0118] The experimental system was prepared using 5 mL of apple juice as a base, with a final concentration of 30 μg / mL WaSDR enzyme, 10 μg / mL PAT, and 1 mmol / L NADPH. The control group consisted of 10 mL of apple juice without WaSDR enzyme, PAT, or NADPH. All systems were placed in a constant temperature shaking incubator and reacted at 30℃ and 150 r / min for 48 h. After the reaction, the results were analyzed by electronic nose and gas chromatography-mass spectrometry (GC-MS).

[0119] Electronic nose detection method: 5 mL of apple juice was placed in a 20 mL headspace vial, sealed, and equilibrated in a 40℃ water bath for 15 min. The injection needle was then inserted into the headspace vial. The following parameters were set: gas flow rate of 400 mL / min; injection waiting time of 5 s; sampling time of 90 s; and sample washing time of 60 s. Each sample was sampled six times. The electronic nose sensor type and responding substance are shown in Table 1.

[0120] Table 1 Performance Description of Electronic Nose Sensor

[0121]

[0122] GC-MS Detection Method: Volatile aroma components were extracted using solid-phase microextraction (SPME) and analyzed by GC-MS. 5 mL of fruit juice sample was placed in a 20 mL brown headspace vial along with 1.0 g sodium chloride (NaCl) and 6 μL of 2-octanol internal standard solution (concentration 200 μg / mL). The mixture was incubated at 40 °C for 40 min to achieve headspace gas equilibration and allow volatile components to adsorb onto the surface of the extraction fiber.

[0123] After extraction, the adsorbed volatile components were thermally desorbed and then introduced into a Shimadzu QP2010 Ultra gas chromatograph-mass spectrometer. The separation process was performed on a DB-5ms capillary column (30 m length, 0.25 mm inner diameter, 0.25 μm film thickness) using helium as the carrier gas at a constant flow rate of 1.66 mL / min. Mass spectrometry detection was performed in electron ionization mode (ionization energy 70 eV), with a mass-to-charge ratio (m / z) range of 35–600; the ion source and quadrupole temperature were maintained at 250 °C. All samples (including the control group) were analyzed in triplicate.

[0124] From the results of the electronic nose radar map ( Figure 8 As can be seen, compared with untreated apple juice, the response values ​​of sensors 1, 2, and 5 were significantly higher in enzyme-treated apple juice. Sensor 1 corresponds to short-chain alkanes, sensor 2 corresponds to nitrogen oxides and aldehydes, ketones, and esters, and sensor 5 corresponds to ammonia aromatic compounds. This indicates that enzyme treatment can effectively promote the release of key volatile flavor components such as short-chain alkanes, aldehydes, ketones, esters, and ammonia aromatic compounds in apple juice, thereby enhancing the flavor complexity of the juice. However, the response values ​​of other core flavor components such as benzene aromatic compounds and sulfur compounds corresponding to sensors 3-4 and 6-10 did not differ significantly between the two groups of samples. This result shows that enzyme treatment can specifically promote the release of specific volatile flavor components while preserving the original basic flavor characteristics of apple juice.

[0125] Based on the qualitative and quantitative analysis results of key volatile flavor components of apple juice using GC-MS (Table 2), the flavor change patterns revealed by the electronic nose were further confirmed. Specifically, the contents of typical flavor compounds such as aldehydes (e.g., hexanal, nonanal), ketones (1-octen-3-one, 2-octanone), and esters (butyl acetate, hexyl acetate) in the enzyme-treated group were significantly increased compared with the control group (p<0.05). This is highly consistent with the increasing trend of the response value of sensor 2 (aldehydes, ketones, esters) in the electronic nose, indicating that enzyme treatment can effectively promote the release and enrichment of these volatile flavor compounds. At the same time, the contents of characteristic terpenoid aroma compounds (D-limonene, linalool, caryophyllene) also increased significantly after enzyme treatment, further enriching the fruity and floral aroma layers of apple juice.

[0126] It is noteworthy that enzyme treatment did not lead to an imbalance in the overall flavor profile of apple juice. On the one hand, the content of some original flavor components, such as hexyl formate and nonanal, remained stable; on the other hand, the content of components with special aroma profiles, such as trans-α-ionone, decreased slightly, possibly related to their transformation or changes in volatility balance under enzymatic action. This reflects that while enzyme treatment enhances some aromas, it may also have a fine-tuning effect on individual high-threshold flavor substances. Overall, while significantly increasing the content of key aldehydes, ketones, esters, and terpenes, enzyme treatment did not have a significant negative impact on the basic flavor profile of apple juice, which is consistent with the results from the electronic nose. In conclusion, WaSDR enzyme treatment can effectively enhance the abundance of key volatile flavor components such as aldehydes, esters, and terpenes in apple juice, enriching the flavor complexity of the juice and thus optimizing and improving the flavor quality of apple juice. This characteristic fully demonstrates the application value of WaSDR in the food processing field.

[0127] Table 2. Effects of WaSDR on volatile aroma compounds in apple juice.

[0128]

[0129] In summary, this embodiment aims to comprehensively evaluate the impact of WaSDR short-chain dehydrogenase on the core quality indicators and flavor characteristics of apple juice while degrading PAT, ensuring that the food's nutritional value is not impaired or even improved while guaranteeing food safety. Through multi-dimensional analysis of apple juice from the enzyme-treated group and the untreated control group, the following results were observed: Regarding functional nutrients, the vitamin C content in the treated apple juice significantly increased from 3.54 mg / L to 23.23 mg / L, and the total phenolic content increased from 205.85 mg / L to 257.17 mg / L. The increase in concentration (mg / L) stems from the ability of WaSDR short-chain dehydrogenase to gently degrade some of the pectin, cellulose, and other macromolecular complexes in apple juice, converting the originally bound vitamin C and total phenols into free states that remain stably present in the system, significantly enhancing the nutritional value of the juice. Regarding basic physicochemical and color indicators, tests showed no significant differences between the treated and control groups in pH value, soluble solids content (°Brix), titratable acidity, transmittance, browning degree, and color parameters (L*, a*, b*). The overall color of the juice did not exhibit browning, darkening, or other negative deterioration, maintaining the quality of the apple juice. Regarding appearance and flavor characteristics, electronic nose technology was used to detect volatile components. The results showed that after enzyme treatment, the release of characteristic flavor components such as short-chain alkanes, nitrogen oxides, aldehydes, ketones, esters, and ammonia aromatics in apple juice significantly increased (corresponding to significantly higher response values ​​for sensors 1, 2, and 5). However, the response values ​​of main flavor components such as benzene aromatics and sulfur compounds showed no significant difference compared to the control group. This indicates that WaSDR short-chain dehydrogenase can specifically enhance some characteristic flavor components without destroying the original main flavor of apple juice, resulting in a richer and more distinct flavor profile. In summary, WaSDR short-chain dehydrogenase achieves dual optimization of apple juice's nutrition and flavor while efficiently degrading PAT, fully meeting the core requirements of the food industry for processing aids: "safety, efficiency, and quality improvement."

[0130] The sequence of SEQ ID NO.1 is as follows:

[0131] MSGKVYFVSGGNRGIGFQFVKILSSNQENTVIASARDPVKATELQALADTQKNVKIVKLD

[0132] VSDKASVDALDVQLKEVAKDGIDVLIANAGISQSVLPAIDTAEDIYLRHYRTNVLGPIFL

[0133] TKALYPYLKLKETRHLTYVSSLAGSIGGFIPFTSSAYGQSKAALNYSIKEISFELGAEGF

[0134] TAVAMQPGLVDTDMSRAGLKALKEVNPDVVEILKDYPNIPAEESAKSQLENIIFKLNKDM

[0135] NGKFFDYHGVEQVF。

[0136] The sequence of SEQ ID NO.2 is as follows:

[0137] ATGTCTGGTAAAGTTTACTTCGTTTCCGGTGGCAACCGTGGTATCGGTTTCCAGTTCGTGAAGATTCTGAGCTCTAACCAAGAGAACACCGTAATCGCATCTGCGCGCGATCCGGTTAAAGCAACTGAACTGCAGGCGCTGGCTGACACTCAGAAGAACGTTAAGATCGTTAAACTGGACGTTTCTGATAAAGCTTCCGTTGATGCACTGGACGTTCAGCTGAAAGAAGTGGCTAAAGACGGTATCGACGTGCTGATCGCAAACGCTGGTATCAGCCAGTCTGTGCTGCCGGCGATCGATACCGCTGAAGACATCTACCTGCGTCACTACCGTACCAACGTGCTGGGTCCGATCTTCTTGACCAAAGCACTGTATCCGTATCTGAAGCTGAAAGAAACTCGCCACCTGACTTACGTGTCTAGCCTGGCTGGCTCTATCGGTGGTTTCATTCCATTCACCAGCAGCGCGTATGGCCAGTCCAAAGCTGCTCTGAACTACTCCATCAAAGAAATCTCTTTCGAACTGGGTGCTGAAGGTTTCACTGCGGTTGCTATGCAGCCGGGTCTGGTGGACACTGACATGAGCCGTGCTGGTCTGAAAGCGCTGAAAGAAGTTAATCCGGATGTGGTGGAAATTCTGAAAGACTATCCAAACATTCCAGCTGAAGAATCTGCTAAATCTCAGCTGGAGAACATCATCTTCAAACTGAACAAAGACATGAACGGCAAATTCTTCGACTACCACGGTGTGGAACAGGTGTTC。

Claims

1. A short-chain dehydrogenase WaSDR, characterized in that, The short-chain dehydrogenase 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 encoding gene for the short-chain dehydrogenase 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 short-chain dehydrogenase of claim 1; (4) Hybridizes under stringent conditions to the nucleotide sequence defined in (1) or (2) and encodes the nucleotide sequence of the short-chain dehydrogenase 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 short-chain dehydrogenase 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 short-chain dehydrogenase.

8. The application of the short-chain dehydrogenase as described in claim 1 in the degradation of patulin; The preferred application is as follows: adding the short-chain dehydrogenase of claim 1 to the object to be treated containing patulin, and reacting to degrade patulin by the short-chain dehydrogenase.

9. The application according to claim 8, characterized in that, The steps are as follows: Add the short-chain dehydrogenase, 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 be 7-8 and the temperature to be 4-40℃ for 12-36 h. Preferably, the concentration of patulin in the reaction system is 1-40 μg / mL, the amount of short-chain dehydrogenase is 10-100 μg / mL, and the concentration of NADPH is 1-4 mM.

10. The application of the short-chain dehydrogenase as described in claim 1 as a food additive in fruit juice; preferably used to enhance the flavor of fruit juice, wherein the fruit juice is apple juice; The preferred application is as follows: WaSDR enzyme and coenzyme NADPH are added to the juice so that the juice contains a final concentration of 25~35 μg / mL of WaSDR enzyme and 0.7~1.3 mmol / L of NADPH, and the system is reacted at 25~35℃ for 12~48 h.

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