Short-chain dehydrogenase gene for synthesizing medium-chain and long-chain aldehydes, recombinant genetically engineered bacterium and application

Abstract

The invention discloses a short-chain dehydrogenase gene for synthesizing medium-chain and long-chain aldehydes, a recombinant genetically engineered bacterium and application, the short-chain dehydrogenase gene can efficiently catalyze medium-chain and long-chain olefine aldehyde as a substrate to generate the medium-chain and long-chain aldehydes through selective hydrogenation reduction under mild conditions, and the short-chain dehydrogenase gene shows good substrate adaptability and catalytic activity. Glucose dehydrogenase and glucose are introduced to construct an NAD (P) H coenzyme cyclic regeneration system, a stable and efficient double-enzyme coupling biological catalytic reaction system is successfully established, and the coenzyme dosage is remarkably reduced while the reaction efficiency is ensured. Compared with a traditional chemical synthesis method, the biological catalysis technology has the advantages of being mild in reaction condition, high in selectivity, few in by-product, environmentally friendly and the like, a new technical approach is provided for green and sustainable preparation of medium and long-chain aldehydes such as perfume-grade dodecanal, and good industrial application prospects are achieved.

Description

(I) Technical Field

[0001] This invention belongs to the field of biochemical technology, specifically relating to a short-chain dehydrogenase for the synthesis of medium- and long-chain aldehydes. Ib The FabI gene, recombinant expression vector, and recombinant genetically engineered bacteria, and their applications, particularly in the biocatalytic preparation of the fragrance compound dodecylaldehyde. (II) Background Technology

[0002] Aliphatic aldehydes and their derivatives are important basic chemicals and intermediates in the pharmaceutical, food, fragrance, and fine chemical industries. Among them, medium- and long-chain aliphatic aldehydes have significant application value in the fragrance industry due to their unique and mellow odor characteristics. Dodecyl aldehyde, as a typical medium- and long-chain aliphatic aldehyde, has waxy, citrus, and floral aroma characteristics and is a key component in many fragrance formulations, with stable and continuous industrial demand.

[0003] Currently, dodecylaldehyde is mainly prepared through chemical synthesis methods, such as the chemical oxidation of fatty alcohols or multi-step chemical reaction routes. However, traditional chemical methods generally suffer from problems such as harsh reaction conditions, insufficient selectivity, numerous byproducts, and heavy environmental burden, making it difficult to meet the requirements of green manufacturing and sustainable development.

[0004] Biocatalysis technology, due to its advantages such as mild reaction conditions, high regioselectivity, and environmental friendliness, has gradually become an important development direction for the green preparation of aliphatic aldehydes. Dehydrogenase / reductase biocatalysts, especially the family of short-chain dehydrogenases / reductases with NAD(P)H as a cofactor, have attracted widespread attention due to their structural stability and strong substrate adaptability. However, the enzyme resources reported so far that can efficiently catalyze medium- and long-chain enaldehyde substrates and meet the needs of industrial applications are still relatively limited. In particular, for the biocatalytic conversion of dodecenal to dodecanal, there is still a lack of enzyme preparations with clearly defined sources, stable performance, and promising application prospects.

[0005] Therefore, developing a short-chain dehydrogenase that can efficiently catalyze the reduction of dodecenal to dodecaldehyde and constructing a corresponding recombinant expression system is of great technical significance and application value for achieving the green and sustainable preparation of dodecaldehyde. (III) Summary of the Invention

[0006] The purpose of this invention is to provide a short-chain dehydrogenase with a well-known source and excellent catalytic performance for the synthesis of medium- and long-chain aldehydes. Ib The FabI gene was developed, and corresponding recombinant expression vectors and recombinant genetically engineered bacteria were constructed for efficient biocatalytic reduction of medium- and long-chain enaldehydes to prepare medium- and long-chain aldehydes, especially the reduction of dodecenal to dodecaldehyde, thereby achieving the green preparation of dodecaldehyde.

[0007] The technical solution adopted in this invention is:

[0008] This invention provides a short-chain dehydrogenase for the synthesis of medium- to long-chain aldehydes. IbFabI The gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] The present invention also provides the aforementioned short-chain dehydrogenase. Ib The protein encoded by the FabI gene has the amino acid sequence shown in SEQ ID NO.2.

[0010] The present invention also provides an expression vector containing the above-mentioned genes and a recombinant genetically engineered bacterium constructed from the expression vector; preferably, the expression vector is a prokaryotic expression vector; and the host cell is *Escherichia coli*. E. coli BL21(DE3).

[0011] This invention provides the aforementioned short-chain dehydrogenase. IbFabI Application of genes in the preparation of long-chain aldehydes from long-chain enal via biocatalytic reduction.

[0012] Furthermore, the application is to catalyze the preparation of dodecenal from dodecenal or the preparation of decanal from decenal.

[0013] Furthermore, the application is as follows: [The text abruptly shifts to a seemingly unrelated topic about short-chain dehydrogenases.] IbFabI The recombinant genetically engineered bacteria were fermented to obtain wet bacterial cells, which were then ultrasonically disrupted. The supernatant (crude enzyme solution) or the pure enzyme solution extracted from the supernatant was used as a catalyst. The reaction system was constructed using medium- and long-chain enaldehydes as substrates, NAD(P)+ as a cofactor, glucose dehydrogenase (GDH) as a coenzyme, glucose as an auxiliary substrate, isopropanol as a solubilizer, and a buffer solution with pH 7-9 as the reaction medium. The reaction was carried out in a shaker at 25-35℃ and 100-300 rpm (preferably 30℃, 200 rpm for 12 h). After the reaction was complete, the reaction solution was extracted with ethyl acetate and then dehydrated with anhydrous magnesium sulfate to obtain the product, medium- and long-chain aldehyde.

[0014] Furthermore, in the reaction system, the catalyst dosage is 1-5 mg / mL based on protein content, preferably 2 mg / mL; the substrate is first prepared into a 1M stock solution with isopropanol and then added to a final concentration of 10-50 mM, preferably 20 mM; the NAD(P)+ is added to a final concentration of 10-15 mM, preferably 12 mM; the coenzyme is added to a final concentration of 1-3 U / mL, preferably 1 U / mL; and the glucose is added to a final concentration of 30-70 mM, preferably 50 mM.

[0015] Furthermore, the reaction medium was a 50 mM potassium phosphate buffer solution with a pH of 7.

[0016] Furthermore, the preparation method of the crude enzyme solution of the short-chain dehydrogenase is as follows: wet bacterial cells are suspended in 50 mM, pH 7.0 PBS buffer and then ultrasonically disrupted in a 4°C ice-water bath for 10 min. The ultrasonic disruption conditions are: power 400 W, 3 s on, 4 s off. The ultrasonically disrupted mixture is centrifuged at 12,000 rpm for 10 min, and then the supernatant is centrifuged at 12,000 rpm for 20 min. The supernatant is then collected to obtain the crude enzyme solution containing the target short-chain dehydrogenase.

[0017] Furthermore, the pure enzyme solution is prepared as follows: the crude enzyme solution is transferred to a pre-equilibrated Ni... 2+ In the column, after sample loading, a large amount of contaminating proteins are first eluted with elution buffer containing 10 mM imidazole. Then, residual contaminating proteins and some target proteins are eluted with elution buffers containing 25 mM and 40 mM imidazole, respectively. Elution is then performed with elution buffer containing 250 mM imidazole, at which point a large amount of target protein is eluted. The eluent containing the target protein is collected. Finally, all remaining proteins in the column are eluted with elution buffer containing 250 mM imidazole. The eluent containing the target protein is desalted and concentrated by centrifugation at 4°C and 7000 rpm for 30 min using an ultrafiltration tube with a molecular weight cutoff of 10 kDa. The retentate is then used as the pure enzyme solution. The elution buffer composition is: 300 mM sodium chloride, solvent is 50 mM PBS buffer, pH 7.0.

[0018] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in the following: the present invention provides a short-chain dehydrogenase with a clearly defined source and excellent catalytic performance. Ib FabI and its encoding gene. This enzyme can efficiently catalyze the selective hydrogenation reduction of dodecenal to dodecenal under mild conditions, exhibiting good substrate adaptability and catalytic activity. By introducing glucose dehydrogenase to construct an NAD(P)H coenzyme recycling system with glucose, a stable and efficient dual-enzyme coupled biocatalytic reaction system was successfully established, significantly reducing the amount of coenzyme used while ensuring reaction efficiency. Compared with traditional chemical synthesis methods, the biocatalytic process described in this invention has the advantages of mild reaction conditions, high selectivity, few by-products, and environmental friendliness, providing a new technical route for the green and sustainable preparation of fragrance-grade dodecenal, and has good prospects for industrial application. (iv) Description of the attached drawings

[0019] Figure 1 Recombinant plasmid pET28a- of short-chain dehydrogenase Ib A schematic diagram of the FabI structure.

[0020] Figure 2 Short-chain dehydrogenases IbSDS-PAGE images of FabI crude enzyme solution and purified enzyme solution; L1 is standard protein, L2 is crude enzyme solution of raw coli BL21, and L3 is... Ib Precipitation of FabI rupture fluid, L4 is Ib FabI crude enzyme solution (supernatant), L5 is Ib FabI unfiltered purified enzyme, L6 is Ib FabI pure enzyme solution.

[0021] Figure 3 Short-chain dehydrogenases Ib FabI's optimal temperature (A) and temperature stability (B) curves.

[0022] Figure 4 Short-chain dehydrogenases Ib FabI's optimal pH (A) and pH stability (B) curves.

[0023] Figure 5 Gas chromatogram of the substrate dodecenal standard sample.

[0024] Figure 6 Gas chromatogram of the standard sample of the product dodecylaldehyde.

[0025] Figure 7 Short-chain dehydrogenases Ib Gas chromatogram of the reaction solution for the hydrogenation of dodecenal to dodecenal catalyzed by FabI crude enzyme solution.

[0026] Figure 8 Short-chain dehydrogenases Ib Conversion curves of dodecenal catalyzed by FabI pure enzyme solution under different catalyst dosages (A) and different reaction times (B).

[0027] Figure 9 Gas chromatogram of the substrate decenal standard sample.

[0028] Figure 10 Gas chromatogram of the standard sample of the product decanal.

[0029] Figure 11 Short-chain dehydrogenases Ib Gas chromatogram of the reaction solution for the hydrogenation of decenal to decanal catalyzed by FabI crude enzyme solution. (V) Detailed Implementation Methods

[0030] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0031] Experimental materials and reagents used in the embodiments of this invention:

[0032] 1. Enzymes and other biochemical reagents: Plasmid miniprep kit and BCA protein concentration assay kit were purchased from Beyotime Biotechnology Co., Ltd., and ultrafiltration tubes were purchased from Millipore. All other reagents were analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0033] 2. Culture medium:

[0034] LB liquid medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, solvent: deionized water, pH 6.8–7.0. LB solid medium is prepared by adding 20 g / L agar to the LB liquid medium.

[0035] Unless otherwise specified in the following examples, the molecular biology experimental methods were performed in accordance with the specific methods listed in the book "Experimental Guide to Biochemical Techniques" (Chemical Industry Press), edited by Sun Peilong and Wu Shijin, or according to the kit and product instructions.

[0036] Example 1: Short-chain dehydrogenase Ib Screening, expression and enzyme activity detection of FabI

[0037] 1. Short-chain dehydrogenases Ib FabI screening

[0038] The protein sequences involved in this invention were obtained through gene mining and bioinformatics screening methods. First, using marine invertebrates... Idotea baltica Using publicly available genomic and transcriptomic data as the research object, the encoded protein sequence information was retrieved from the public sequence database NCBI GenBank. For hypothetical protein sequences in the database that lack clear functional annotations, sequence homology analysis was used for preliminary screening. BLAST was used to perform homology alignment analysis on candidate proteins, and protein sequences with certain sequence similarity to reported oxidoreductases were selected as potential functional enzyme candidates. Based on this, conserved domain analysis of the candidate sequences was performed using Pfam protein domain data, confirming that they contain typical coenzyme binding sites and catalytically conserved motifs typical of the short-chain dehydrogenase / reductase family, thus preliminarily determining that the protein belongs to the short-chain dehydrogenase / reductase family.

[0039] Based on the above screening and analysis, the present invention identified the target protein sequence and synthesized or amplified its encoding gene artificially, constructed a recombinant expression vector, and expressed it in host cells. Subsequent enzymatic experiments verified that the protein can catalyze the reduction of unsaturated aldehyde substrates, particularly exhibiting significant catalytic activity for the double bond reduction of dodecenal, thus identifying it as a short-chain dehydrogenase with potential application value.

[0040] 2. Short-chain dehydrogenases IbExpression of FabI

[0041] (1) Recombinant plasmid: The plasmid derived from NCBI Idotea baltica The hypothetical protein GTU68_059282 (MCL4136035.1) was subjected to codon optimization. The codon-optimized gene is denoted as... Ib The FabI gene (nucleotide sequence shown in SEQ ID NO.1, amino acid sequence encoding the protein shown in SEQ ID NO.2) was synthesized and inserted between NdeI and BamHI in pET28a to obtain the recombinant plasmid pET28a- Ib FabI ( Figure 1 ).

[0042] SEQ ID NO.1:

[0043] 5’-GGATGGTGAGCGGGATACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGTTCGAAGGTAAAAAGGTGCTGATTCTGGGCGTTGCAAATAAAAAAAGCATTGCCTGGGCAATTACCGAAGCACTGGTTAAAAATGGTGCAAAAGTGGCCCTGACCTATGCCAATGAAGCAATTGAAAAACGCGTTCGTCCGCTGGCAGAAAGCATTCAGTGCGAAAATGTGTATGAATGTGATGTTCAGAGCGATGAACAGATTGATAAACTGTTTGTGGATCTGAAAAACGATTTTGGTAATCTGGATGCCGTGGTTCATAGTGTGGCATTTGCCAATAAAGAAGATCTGGCCAATGATTTTAGTGAAACCACCCGCGCAGGTTTTACCATGGCCCTGGATATTAGTGCCTATAGTCTGATTGCCGTTGCCCGCGGCGCCAAAAGCCTGATGAATGAAGGCGGCAGTATTATTACCCTGAGTTATCTGGGTGCAGTTCGTGTGGTTGATAATTATAATGTTATGGGTGTGGCAAAAGCAGCCCTGGAAAGTAGTGTTCGCTATCTGGCCGCAGATCTGGGTCGTACCAATATTCGCGTGAATGCAATTAGCGCCGGTCCGATTAAAACCCTGGCCGCCAGTGGTATTCCGAAATTTCGTGAACTGCTGAATAATTTTGCAGAATGAGCCCGCTGAAACGTAATGTGGAACTGGAAGATGTGGTATACCGCATGTTTCTGCTGGGTCATCTGGGTAGTGTGGTACTGCGGAGTGATTTATACCCGATTGCGGCTTTTAGCCCAGATGGGGCAC -3’。

[0044] SEQ ID NO.2:

[0045] MFEGKKVLIL GVANKKSIAWAITEALVKNGAKVALTYANEAIEKRVRPLAESIQCENVYECDVQSDEQIDKLFVDLKNDFGNLDAVVHSVAFANKEDLANDFSETTRAGFTMALDISAYSLIAVARGAKSLMNEGGSIITLSYLGAVRVVDN YNVMGVAK AALESSVRYLAADLGRTNIRVNAISAGPIKTLAASGIPKFRELLNNFAEMSPLKRNVELEDVANTAMFLLGHLGSGVTGEVIYTDCGFSQMGTA.

[0046] The underlined amino acids above represent key conserved amino acid segments in short-chain dehydrogenases, with amino acids 12-21 (GVANKKSIA) primarily controlling the conformation of the coenzyme NADH. The sequence of amino acids 153-160 (YNVMGVAK) contains the key amino acids for catalytic activity.

[0047] (2) Recombinant genetically engineered bacteria: 6 μL of recombinant plasmid pET28a- Ib FabI added to 50 μL E. coli In BL21(DE3) competent cells, gently tap the tube wall to mix, and place on ice for 30 min. Heat shock in a 42°C water bath for 1 min, then immediately place on ice for 2 min. Add 1 mL of LB liquid medium to the tube and incubate in a 37°C metal bath for 30 min. Centrifuge the culture at 8000 rpm for 4 min and discard the supernatant. Resuspend the cells in the remaining medium, and spread 100 μL onto LB solid medium containing 50 μg / mL kanamycin. Incubate overnight at 37°C for 12–14 h to obtain cells containing short-chain dehydrogenase. Ib FabI recombinant genetically engineered bacteria E. coli BL21(DE3) / pET28a- Ib FabI. Control strains were constructed using the same method. E. coli BL21(DE3) / pET28a.

[0048] (3) Wet bacterial cells: Recombinant genetically engineered bacteria E. coli BL21(DE3) / pET28a- IbFabI was inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C for 14 h. Then, it was inoculated again into LB liquid medium containing 50 μg / mL kanamycin at a volume concentration of 1% and cultured at 37°C for 3 h. Next, isopropyl-β-D-thiogalactoside (IPTG) was added to the medium to a final concentration of 0.2 mM, and expression was induced at 18°C ​​for 15 h to obtain the fermentation broth. The obtained fermentation broth was centrifuged at 12000 rpm for 10 min at 4°C, and the precipitate was collected as wet cells.

[0049] (4) Crude enzyme solution: The wet bacterial cells obtained in step (3) were resuspended in 50 mL of 50 mM phosphate buffer at pH 7 and then sonicated. The sonicated mixture was then centrifuged at 12,000 rpm for 10 min and the supernatant was centrifuged at 12,000 rpm for 20 min to obtain the precipitate and supernatant. The supernatant was collected to obtain the solution containing short-chain dehydrogenase. Ib The protein concentration of the crude enzyme solution from FabI was determined using a BCA kit and was 8 mg / mL. The crude enzyme solution of the control strain was prepared using the same method.

[0050] (5) Pure enzyme solution: Transfer the crude enzyme solution to the pre-equilibrated Ni using a chromatography apparatus. 2+ In the column, after sample loading, a large amount of contaminating protein was first eluted with elution buffer containing 10 mM imidazole for 2 column volumes. Then, residual contaminating protein and some target protein were eluted with elution buffers containing 25 mM and 40 mM imidazole, respectively, for 2 column volumes each. Next, elution was performed with elution buffer containing 250 mM imidazole for 3 column volumes. At this point, a large amount of target protein was eluted, and the eluent containing the target protein was collected. Finally, all remaining protein in the column was eluted with elution buffer containing 250 mM imidazole for 5 column volumes. The eluent containing the target protein (i.e., the unpurified enzyme) was desalted and concentrated by centrifugation at 4°C and 7000 rpm for 30 min using an ultrafiltration tube with a molecular weight cutoff of 10 kDa. The retentate was taken as the pure enzyme solution with a protein concentration of 6 mg / mL. The elution buffer composition was: 300 mM sodium chloride, solvent was 50 mM PBS buffer, pH 7.0.

[0051] (6) SDS gel electrophoresis: The short-chain dehydrogenases prepared above were subjected to SDS gel electrophoresis. Ib Crude enzyme solutions of FabI and control strains, Ib Precipitation of FabI rupture fluid Ib FabI enzymes were not purified by ultrafiltration. Ib The FabI pure enzyme solution was analyzed by SDS gel electrophoresis, and the results are shown in the figure. Figure 2.

[0052] (7) Enzyme activity assay: The enzyme activity of the purified enzyme solution from step (5) was measured using a Biotec Synergy H1 multi-functional microplate reader. The total reaction volume was 200 μL, consisting of 190 μL of pH 7, 50 mM potassium phosphate buffer solution, 4 μL of the purified enzyme solution prepared in step (5), 4 μL of 20 mM isopropanol solution of substrate (dodecenal), and 2 μL of 20 mM NADH aqueous solution. The absorbance of the above system at 340 nm was measured over time.

[0053] Enzyme activity is calculated as follows: Activity (U) = ΔA·V / ε

[0054] Specific enzyme activity is calculated as follows: Specific activity (U / mg) = ΔA·V / ε·m

[0055] In the formula, ΔA represents the decrease in absorbance at a wavelength of 340 nm per unit time;

[0056] V - Volume of the reaction system (0.2 mL);

[0057] The molar absorptivity of ε-NADH at 340 nm is 6.22 mM. -1 cm -1 );

[0058] M - The amount of enzyme added in the actual reaction (mg).

[0059] Upon testing, the short-chain dehydrogenase prepared in step (4) was found to be... Ib The specific enzyme activity of FabI pure enzyme solution against the substrate dodecenal is 4.5 U / mg protein.

[0060] Example 2: Short-chain dehydrogenase Ib Enzymatic properties determination of FabI

[0061] The enzyme activity of the pure enzyme solution (protein concentration of 6 mg / mL) prepared by the method in Example 1 was determined using a Biotec Synergy H1 multi-functional microplate reader.

[0062] 1. Optimal temperature

[0063] The total reaction volume was 200 μL: 190 μL of pH 7, 50 mM potassium phosphate buffer, 4 μL of purified enzyme solution, 4 μL of 20 mM substrate (dodecenal) isopropanol solution, and 2 μL of 50 mM coenzyme NADH aqueous solution. The absorbance of the above system at 340 nm was measured over time.

[0064] Enzyme activity assay method: 198 μL of reaction system without added coenzyme NADH was incubated at 20℃, 25℃, 30℃, 35℃, 40℃, 45℃ and 50℃ for 5 min respectively. Then, 2 µL of 50 mM NADH aqueous solution was added and enzyme activity was measured using a Biotec microplate reader according to the method in Example 1. The highest enzyme activity was taken as 100%.

[0065] Short-chain dehydrogenases were determined Ib The enzyme activities of FabI at 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, and 50℃ were 50%, 69%, 100%, 95%, 81%, 58%, and 40% of the maximum enzyme activity, respectively. Figure 3 As shown in Figure A. The short-chain dehydrogenase was detected. Ib FabI exhibits the highest enzyme activity at 30℃, meaning its optimal conversion temperature is 30℃.

[0066] 2. Temperature stability

[0067] The pure enzyme solution was incubated at 20℃, 25℃, 30℃, 35℃, 40℃, 45℃ and 50℃ for one hour respectively.

[0068] The total reaction volume was 200 µL: 190 µL of pH 7, 50 mM potassium phosphate buffer, 4 µL of purified enzyme solution after incubation, 4 µL of 20 mM substrate (dodecenal) isopropanol solution, and 2 µL of 50 mM coenzyme NADH aqueous solution. Enzyme activity was detected using the method described in Example 1.

[0069] Determination of short-chain dehydrogenases Ib After FabI was incubated at 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, and 50℃ for one hour, its enzyme activity at the optimum temperature (30℃) was 91%, 83%, 81%, 78%, 66%, 52%, and 32% of the initial enzyme activity, respectively. Figure 3 As shown in Figure B, the short-chain dehydrogenase IbFabI has good thermostability; after incubation at 40°C for one hour, the enzyme activity is still as high as 66%.

[0070] 3. Optimal pH

[0071] The total reaction volume was 200 μL: 190 μL of buffer solutions at different pH, 4 μL of pure enzyme solution, 4 μL of 20 mM substrate (dodecenal) isopropanol solution, and 2 μL of 50 mM coenzyme NADH aqueous solution.

[0072] The buffer solutions were: 50 mM sodium acetate buffer (pH 4-5), 50 mM potassium phosphate buffer (pH 6-8), and 50 mM Tirs-HCl buffer (pH 8-9).

[0073] Enzyme activity assay: A 198 μL reaction mixture without NADH was incubated at 30 °C for 5 min. Then, 2 µL of 50 mM NADH aqueous solution was added, and enzyme activity was detected using the method described in Example 1. Experimental results are as follows: Figure 4 As shown in Figure A, pH 7 is the optimal pH value, at which enzyme activity is at its maximum. This pH value is defined as 100% of the enzyme activity.

[0074] 4. pH stability

[0075] Four μL of pure enzyme solution was placed in 190 μL of buffer solutions at different pH values ​​(pH 4, 5, 6, 7, 8, 9) and incubated at 30°C for 4 h. Then, four μL of 20 mM substrate (dodecenal) isopropanol solution and two μL of 50 mM coenzyme NADH aqueous solution were added. Enzyme activity was detected using the method described in Example 1. The results are as follows: Figure 4 As shown in Figure B, the results indicate that the enzyme exhibits excellent pH stability and high residual activity in neutral to weakly alkaline (pH 6-8) PBS buffer, demonstrating good operational tolerance and application adaptability.

[0076] Example 3: Short-chain dehydrogenase Ib Conversion rate of FabI-catalyzed substrate dodecenal to dodecenal

[0077] The reaction system (1 mL) used crude enzyme solution prepared according to the method in Example 1 as a catalyst, with an addition amount of 1 mg / mL based on protein content, resulting in a final NAD concentration of 12 mM. + As a cofactor, glucose dehydrogenase (purchased from Aladdin) at a final concentration of 1 U / mL was used as the coenzyme, and glucose at a final concentration of 50 mM was used as the auxiliary substrate. 20 μL of 1 M substrate dodecenaldehyde in isopropanol solution was added to achieve a final concentration of 20 mM. A 1 mL reaction system was prepared using 50 mM, pH 7 PBS buffer as the reaction medium. The reaction was carried out at 30℃ and 200 rpm for 12 h in a shaker. After the reaction, an equal volume of ethyl acetate was added for extraction. The upper organic phase was dehydrated with anhydrous MgSO4, allowed to stand for 10 min, and centrifuged at 12,000 rpm for 10 min. The supernatant was collected, and the contents of the substrate dodecenaldehyde and the product dodecenaldehyde were determined by gas chromatography. The product yield was calculated, and the results are shown in [Figure number missing]. Figure 7 As shown. Gas chromatograms of the substrate dodecenal and the product dodecenal standard are shown below. Figure 5 and Figure 6 As shown.

[0078] Gas chromatography detection conditions: Determination was performed using an HP-5 capillary column equipped with an FID detector; FID detection temperature 250℃, injector temperature 250℃; column inlet pressure 60 kPa; the flow rate and split ratio of N2 as carrier gas were set to 1 mL / min and 1:100, respectively, with an injection volume of 1 µL. The sample was separated using an HP-5 column (25 m × 250 µm) under the following conditions: constant temperature at 80℃ for 3 min, followed by a ramp-up to 260℃ at 10℃ / min and holding for 2 min. The retention times for the product dodecylaldehyde and the substrate dodecenal were 15.813 min and 17.203 min, respectively.

[0079] The short-chain dehydrogenase prepared in Example 1 was tested and found to be effective. Ib The crude FabI enzyme solution can catalyze the reduction of dodecenal to dodecenal, and the substrate conversion rate is 63% after the reaction is completed.

[0080] Example 4: Short-chain dehydrogenase Ib Conversion rate of dodecenal to dodecenal catalyzed by different amounts of FabI pure enzyme solution and different reaction times

[0081] 1. Catalyst dosage

[0082] The reaction system consisted of 1 mL of pure enzyme solution prepared according to the method in Example 1, with the catalyst added at an amount of 0.5-4 mg / mL (0.5, 1, 2, 3, 4 mg / mL) based on protein content, resulting in a final NAD concentration of 12 mM. + As a cofactor, glucose dehydrogenase (purchased from Aladdin) at a final concentration of 1 U / mL was used as the coenzyme, and glucose at a final concentration of 50 mM was used as the auxiliary substrate. 20 μL of 1 M substrate dodecenaldehyde in isopropanol solution was added to achieve a final concentration of 20 mM. A 1 mL reaction system was prepared using 50 mM, pH 7 PBS buffer as the reaction medium. The reaction was carried out at 30°C and 200 rpm for 12 h in a shaker. After the reaction, an equal volume of ethyl acetate was added to the reaction solution for extraction. The upper organic phase was dehydrated with anhydrous MgSO4, allowed to stand for 10 min, and centrifuged at 12,000 rpm for 10 min. The supernatant was collected, and the contents of the substrate dodecenaldehyde and the product dodecenaldehyde were determined by gas chromatography as described in Example 3. The product yield was calculated, and the catalytic efficiency results are shown in [Figure 3]. Figure 8 As shown in A.

[0083] 2. Reaction time

[0084] The catalyst dosage in step 1 was fixed at 1 mg / mL, and the reaction time was changed to 6, 8, 12, 16, 18, and 24 h. All other operations remained the same. The results are shown in [Figure number missing]. Figure 8 B.

[0085] It can be seen that short-chain dehydrogenases Ib The optimal reaction conditions for FabI pure enzyme solution to catalyze the substrate dodecenal are a reaction time of 12 h and an enzyme concentration of 1 mg / mL, which yields the highest catalytic efficiency and a substrate conversion rate of 89.3%. Too low a protein concentration results in insufficient active sites, while too high a concentration leads to aggregation or steric hindrance, causing the conversion rate to initially rise and then fall. The highest catalytic efficiency is achieved at a reaction time of 12 h. The substrate is gradually converted in the early stages, while the conversion of products and byproducts increases in the later stages, resulting in a single-peak trend in the conversion rate—first rising and then falling.

[0086] Example 5: Short-chain dehydrogenase Ib FabI catalyzes the preparation of decanal from decenal substrates.

[0087] The reaction system consisted of 1 mL of the following: crude enzyme solution prepared in Example 1 was used as the catalyst, with an addition amount of 1 mg / mL (based on protein content) of NAD+ at a final concentration of 12 mM as the cofactor; glucose dehydrogenase (purchased from Aladdin) at a final concentration of 1 U / mL as the coenzyme; glucose at a final concentration of 50 mM as the auxiliary substrate; and 20 μL of 1 M decenal isopropanol solution to bring the final concentration to 20 mM. The reaction medium was 50 mM, pH 7 PBS buffer. The reaction was carried out at 30°C and 200 rpm for 12 h in a shaker. After the reaction, an equal volume of ethyl acetate was added for extraction. The upper organic phase was dehydrated with anhydrous MgSO4, allowed to stand for 10 min, and centrifuged at 12,000 rpm for 10 min. The supernatant was collected and the contents of the substrate decenal and the product decenal were determined by gas chromatography. The product yield was calculated. The chromatogram is shown in [Figure number missing]. Figure 11 As shown. Gas chromatograms of the substrate decenal and the product decenal standard are shown below. Figure 9 and Figure 10 As shown.

[0088] Gas chromatography detection conditions: Determination was performed using an HP-5 capillary column equipped with an FID detector; FID detection temperature 250℃, injector temperature 250℃; column inlet pressure 60 kPa; the flow rate and split ratio of N2 as carrier gas were set to 1 mL / min and 1:100, respectively, with an injection volume of 1 µL. The sample was separated using an HP-5 column (25 m × 250 µm) under the following conditions: constant temperature at 50℃ for 3 min, followed by a ramp-up to 250℃ at 10℃ / min and holding for 2 min. The retention times of the product decanal and the substrate decenal were 12.081 min and 12.817 min, respectively.

[0089] The short-chain dehydrogenase prepared in Example 1 was tested and found to be effective. Ib The crude FabI enzyme solution can catalyze the reduction of decenal to decanal, and the substrate conversion rate is 58.8% after the reaction is completed.

[0090] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these examples without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A short-chain dehydrogenase for the synthesis of medium- to long-chain aldehydes. Ib The FabI gene is characterized by, The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A short-chain dehydrogenase according to claim 1 Ib The protein encoded by the FabI gene is characterized by, The amino acid sequence is shown in SEQ ID NO.

2.

3. A recombinant genetically engineered bacterium expressing the gene of claim 1.

4. A short-chain dehydrogenase according to claim 1 Ib Application of the FabI gene in the preparation of long-chain aldehydes from long-chain enaldehydes through biocatalytic reduction.

5. The application as described in claim 4, characterized in that, The application is to catalyze the preparation of dodecenal from dodecenal or the preparation of decanal from decenal.

6. The application as described in claim 5, characterized in that, The application is as follows: [This refers to the use of] short-chain dehydrogenases. Ib The recombinant genetically engineered bacteria of the FabI gene were fermented and the resulting wet cells were ultrasonically disrupted. The supernatant of the disrupted mixture after centrifugation or the pure enzyme solution extracted from the supernatant was used as a catalyst. The reaction system was constructed using medium- and long-chain enaldehydes as substrates, NAD(P)+ as a cofactor, glucose dehydrogenase as a coenzyme, glucose as an auxiliary substrate, isopropanol as a cosolvent, and a buffer solution with pH 7-9 as the reaction medium. The reaction was carried out in a shaker at 25-35℃ and 100-300 rpm. After the reaction was complete, the reaction solution was extracted with ethyl acetate and then dehydrated with anhydrous magnesium sulfate to obtain the product, medium- and long-chain aldehyde.

7. The application as described in claim 6, characterized in that, In the reaction system, the amount of catalyst used is 1-5 mg / mL based on protein content; the substrate is first prepared into a 1M stock solution with isopropanol and then added to the system, with a final concentration of 10-50 mM; the NAD(P)+ is added to a final concentration of 10-15 mM; the coenzyme is added to a final concentration of 1-3 U / mL; and the glucose is added to a final concentration of 30-70 mM.

8. The application as described in claim 6, characterized in that, The reaction medium was a 50 mM potassium phosphate buffer solution with a pH of 7.

9. The application as described in claim 6, characterized in that, The supernatant was prepared as follows: wet bacterial cells were suspended in 50 mM, pH 7.0 PBS buffer and then ultrasonically disrupted for 10 min in a 4°C ice-water bath. The ultrasonic disruption conditions were 400 W power, 3 s on, 4 s off. The ultrasonically disrupted mixture was centrifuged at 12,000 rpm for 10 min, and then the supernatant was centrifuged at 12,000 rpm for 20 min. The supernatant was collected to obtain a crude enzyme solution containing the target short-chain dehydrogenase.

10. The application as described in claim 6, characterized in that, The pure enzyme solution was prepared as follows: the supernatant was transferred to a pre-equilibrated Ni... 2+ In the column, after sample loading, a large amount of contaminating proteins are first eluted with elution buffer containing 10 mM imidazole. Then, residual contaminating proteins and some target proteins are eluted with elution buffers containing 25 mM and 40 mM imidazole, respectively. Elution is then performed with elution buffer containing 250 mM imidazole, at which point a large amount of target protein is eluted. The eluent containing the target protein is collected. Finally, all remaining proteins in the column are eluted with elution buffer containing 250 mM imidazole. The eluent containing the target protein is desalted and concentrated by centrifugation at 4°C and 7000 rpm for 30 min using an ultrafiltration tube with a molecular weight cutoff of 10 kDa. The retentate is then used as the pure enzyme solution. The elution buffer composition is: 300 mM sodium chloride, solvent is 50 mM PBS buffer, pH 7.0.

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