A process for the preparation of 1,2-pentanediol

Abstract

This invention discloses a method for preparing 1,2-pentanediol, comprising using 2-pentanone as a substrate, adding 2-keto reductase, linalool dehydratase, formate dehydrogenase, lysozyme, coenzyme NAD+, and water to react and obtain n-pentene. The obtained n-pentene is then thoroughly mixed with ethanol. This mixture is then fed into a reaction system containing olefin monooxygenase, epoxide hydrolase, formate dehydrogenase, lysozyme, coenzyme NAD+, MgSO4, and water using a fed-batch method. After the n-pentene and ethanol mixture has been completely added, the reaction continues for a period of time, followed by extraction to obtain 1,2-pentanediol. This preparation method uses 2-pentanone as a raw material, catalyzing the synthesis of 2-pentanol via 2-keto reductase. 2-pentanol is then catalyzed by a dehydrating enzyme to generate n-pentene, which is then catalyzed by an olefin monooxygenase to generate 1,2-epoxypentane. Finally, epoxide hydrolase ring-opens the 1,2-pentanediol. This process requires only four enzymatic catalytic reactions for efficient synthesis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing 1,2-pentanediol. Background Technology

[0002] 1,2-Pentanediol is a colorless, transparent, viscous liquid. It is a diol containing two adjacent hydroxyl groups (-OH), a structure that determines its unique physicochemical properties. In terms of applications, 1,2-Pentanediol is an important organic intermediate widely used in the synthesis of the bactericide propiconazole, high-end skincare products, polyester fibers, surfactants, and pharmaceuticals. It is particularly effective in cosmetics due to its excellent solubility and coupling ability: it is soluble in water and miscible with many organic solvents, and can couple oily and watery components, acting as a solubilizer and coupling agent in formulations. It also possesses strong moisturizing properties: the two hydroxyl groups in its molecule can form hydrogen bonds with water molecules, effectively trapping moisture and providing long-lasting hydration to the skin or product system. Furthermore, it exhibits broad-spectrum antibacterial activity: this is one of the most important characteristics of 1,2-Pentanediol. It can disrupt the cell membranes of microorganisms, showing significant inhibitory effects on bacteria, fungi, and molds. Therefore, it is widely used as a key antimicrobial component in preservative-free or low-preservative formulations to reduce the use of traditional chemical preservatives; good penetration enhancement: it can reversibly change the structure of the stratum corneum, promote the penetration of other active ingredients into the skin barrier, and improve their bioavailability; low toxicity and mildness: compared with similar substances such as propylene glycol, 1,2-pentanediol is less irritating and less sensitizing to the skin, and has better skin compatibility, making it particularly suitable for sensitive skin skincare products and high-end cosmetics; low freezing point: its low freezing point helps improve the stability of products in low-temperature environments.

[0003] Industrially, the preparation of 1,2-pentanediol mainly relies on chemical synthesis. 1) Using 1-pentene as a raw material, 1,2-pentanediol is finally synthesized through epoxidative hydrolysis. The disadvantage is that 1-pentene is an important petrochemical raw material with wide applications, and its non-renewable nature makes it difficult to guarantee a stable supply of raw materials. 2) Biomass conversion, using renewable resources such as lignin, corn cobs, and starch as raw materials, synthesizes 1,2-pentanediol through high temperature, high pressure, and hydrogenolysis. The disadvantage is that this process requires high temperature, high pressure, and hydrogenolysis, which places very stringent requirements on equipment and operation. Therefore, it has not been industrialized on a large scale in China. Patent CN 119793536 A mentions using furfuryl alcohol as a raw material, with Cu-based catalyst catalysis, and hydrogenation reaction at a reaction pressure of 2.8~3.2MPa and a temperature of 145~155℃ to produce 1,2-pentanediol. In addition, patents CN120094574A, CN119701947A, and CN119897102A all use furfuryl alcohol as a raw material to prepare 1,2-pentanediol through high temperature, high pressure, and hydrogenolysis. Patent CN119753039A discloses a method for microbial synthesis of 1,2-pentanediol, which uses pyruvate or threonine as a substrate to synthesize 2-oxovalerate acid through 6 steps of metabolism, and then synthesizes 1,2-pentanediol through 3 steps of metabolism. The broad-spectrum antibacterial activity of 1,2-pentanediol means that the metabolic synthesis of 1,2-pentanediol is not an appropriate method. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing 1,2-pentanediol, so as to solve the problems mentioned in the background art.

[0005] This invention provides a novel green method for synthesizing 1,2-pentanediol. Using 2-pentanone as a raw material, 2-pentanol is synthesized by 2-keto reductase catalysis. 2-pentanol is then catalyzed by dehydrating enzyme to generate n-pentene, which is then catalyzed by olefin monooxygenase to generate 1,2-epoxypentane. Finally, 1,2-pentanediol is generated by ring-opening with epoxyhydrolase. This process requires only 4 enzymatic catalytic reactions to efficiently synthesize 1,2-pentanediol, and the required 2-pentanone raw material can be obtained in one step by ethanol condensation.

[0006] The reaction route is as follows:

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing 1,2-pentanediol, comprising the following steps: S1: In a reactor connected to a condenser, 2-pentanone is used as a substrate, and 2-keto reductase, linalool dehydratase, formate dehydrogenase, lysozyme, coenzyme NAD+ and water are added to react and obtain n-pentene. S2: The above-obtained n-pentene is thoroughly mixed with ethanol. Then, the mixture of n-pentene and ethanol is added to a reaction system containing olefin monooxygenase, epoxide hydrolase, formate dehydrogenase, lysozyme, coenzyme NAD+, MgSO4 and water by a feed method. After the mixture of n-pentene and ethanol has been completely added, the reaction continues for a period of time. Finally, the mixture is extracted to obtain 1,2-pentanediol.

[0008] Preferably, in step S1, the condensation device is set to a temperature of -10°C.

[0009] In any of the above embodiments, it is preferred that the reaction temperature is set to 35°C.

[0010] In any of the above embodiments, the preferred reaction temperature is 25°C.

[0011] Preferably, in any of the above schemes, the flow rate in the feeding method is controlled at 5 mL / h.

[0012] In any of the above schemes, the extraction is preferably performed by extracting twice with ethyl acetate, combining the ethyl acetate, adding activated carbon for decolorization at room temperature in a sealed environment, filtering to remove the activated carbon, and then rotary evaporating to remove the ethyl acetate.

[0013] In any of the above schemes, it is preferred that the reaction continues for 5-10 h after the mixture of n-pentene and ethanol is completely added.

[0014] An application of 1,2-pentanediol in the preparation of cosmetics.

[0015] Considering that 2-pentanone can be obtained by one-step condensation of ethanol, this invention uses 2-pentanone as a substrate for the holoenzymatic synthesis of 1,2-pentanediol. First, 2-ketoreductase is used. The literature Nanduri, VB, A. Banerjee, JM Howell, DB Brzozowski, RF Eiring, and RN Patel. "Purification of astereospecific 2-ketoreductase from Gluconobacter oxydans." Journal of Industrial Microbiology and Biotechnology 25, no. 3 (2000): 171-175. reports that 2-ketoreductase derived from Gluconobacter oxydans can catalyze the conversion of 2-pentanone to 2-pentanol. This is in conjunction with the literature Liu, Xu, Rong Chen, Zhongwei Yang, Jiale Wang, Jinping Lin, and Dongzhi Wei. "Characterization of a putative stereoselective oxidoreductase from Gluconobacter oxydans and its application in producing ethyl (R)-4-chloro-3-hydroxybutanoate ester. Molecular biotechnology 56, no. 4 (2014): 285-295. The 2-keto reductase Gox2036 was obtained. This enzyme is an NADH-dependent reductase. The amino acid sequence of Gox2036 is as follows: MSLSGKIAAVTGAAQGIGKAIALRLAKDGADVILLDVKQDTLAETAKEVEALGRRAVALTADISNRDQFRSTLADAAKTLGGLDIMVNNAGICQVKPILDIEPAEIEKIFSINVQGVLWGMQAAATLFKE KGTKGKIINACSIAGHEGYPLLGAYSATKFAVRALTQSAAKELASSGITVNSYCPGIVGTDMWVTIDKRMAEITGTEIGATYKKYVEGIALGRVETADDVAGFVAYLSSSDADYMTGQSVLIDGGLVFR.

[0016] Furthermore, through the BRENDA enzyme library, we learned about etnD, an olefin monooxygenase derived from Mycolicibacterium chubuense NBB4. This enzyme is an NADH-dependent reductase, as reported in the literature McCall, Victoria, et al. "Heterologous expression of mycobacterium alkenemonooxygenases in gram-positive and gram-negative bacterial hosts." Applied and Environmental Microbiology 84.15 (2018): e00397-18. This enzyme is reported to be active against C3-C8 ortho-olefins. The amino acid sequence of etnD is as follows: MGDTVTVQPFGDTFPVESGETVLSAILRNGRFVKYGCKHGGCSTCRAQVVEGEFTQSDGTSFSLSDADRDAGVVLLCSTYADGLVDVDVGETMADLTEDEYNAGQDIVEFVGTVDRIVDYTADIKGIEIALDEPSAISFVPGQYVEVLVPGSDDAWRSFSMANRPSDNSRVH LVVRVIPDGRFTSQIGTTISAGTRLNLRGPLGQFAIRLSHRPIIFIAGGSGIAPVLSMLADLIEQNNQRRTTFLYGARTVADLPMLDELRQLSDELDWFTFIPALSQPDDTPWDGETGLITEVYHRNFPSGHGHEAYLCGPPGMIDAALESLIASGCKERHIFFDRFVPSG.

[0017] Huang Rui reported on the expression and substrate selectivity of the epoxide hydrolase SfEH from *Streptomyces fischeri* in his paper "Study on the Expression and Stereoselectivity of Epoxide Hydrolase SfEH" [D]. Anhui University of Technology, 2023. 1,2-Epoxypentane was used as a substrate for activity testing, showing that SfEH1 has the ability to hydrolyze 1,2-epoxypentane to generate vicinal diol. The amino acid sequence of SfEH1 is as follows: MENNTPDGIRPFRVDVPQDRLDDLRARLAATRWPDELPGVGWSRGVPVAYLKELADHWRTAYDWRAHEAELNALPQYTATLDGQNVHFLHVRSPRPDATPLVLLHGWPG SVADFLDVIGPLTDPEAHGGAATDAFHLVIPSLPGFGFSTPLAGPGMDTSRMAGLFFTRLMERLGYTRYGVQGYDTGSWVAPHMARQAPDRVIGVHVNALLTFPVGAEGE RDGLTEAERRRLHRMENFNDGYLQCNSKRPQTVAYALTDSPAGQLAWMVEKYKELTDPEDALPEESIHRDRVLTQVSLYWLTATAASAAQIYYEEISASAWGGDGADGG DGADGRDGEGWSAGGTGDTGGADAGGTGWGGADGGGWGDGHEDWAAAARGTVPTAVLLSTHDVTIRRWAERDHNVVRWTELDRGGHFLAMEAPDLLVGDVRAFFAGLR.

[0018] 2-Ketoreductase, olefin monooxygenase, and epoxide hydrolase have all been reported in the literature, but there are no reports directly on dehydrating enzymes using 2-pentanol as a substrate to generate 1-pentene. However, through in-depth research, it was found that linalool dehydratase has the ability to catalyze the dehydration of adjacent hydroxyl groups and methyl groups to form double bonds. Therefore, we rationally modified the linalool dehydratase LinD, derived from *Castellaniella defragrans*, using protein engineering. Its original sequence is as follows: MRFTLKTTAIVSAAALLAGFGPPPRAAELPPGRLATTEDYFAQQAKQAVTPDVMAQLAYMNYIDFISPFYSRGCSFEAWELKHTPQRVIKYSIAFYAYGLASVALIDPKLRALAGHDLDIAVSKMKCKRVWGDWEEDGFGTDPIEKENIMYKGHLNLMYGLYQLVTGSRRYEAEHAHLTRIIHDEIAANPFAGIVCEPD NYFVQCNSVAYLSLWVYDRLHGTDYRAATRAWLDFIQKDLIDPERGAFYLSYHPESGAVKPWISAYTTAWTLAMVHGMDPAFSERYYPRFKQTFVEVYD EGRKARVRETAGTDDADGGVGLASAFTLLLAREMGDQQLFDQLLNHLEPPAKPSIVSASLRYEHPGSLLFDELLFLAKVHAGFGALLRMPPPAAKLAGK.

[0019] Considering that both 2-keto reductase Gox2036 and olefin monooxygenase etnD require NADH as a cofactor, we introduced formate dehydrogenase CbFDH from *Candida boidinii* to regenerate NADH. Secondly, the product n-pentene synthesized by 2-keto reductase Gox2036 and the linalool dehydratase LinD mutant has a boiling point of only 30℃. We improved the reaction apparatus to collect n-pentene by condensing the product during the reaction at 35℃. The advantage of this method is that it avoids substrate inhibition, allows for sustained enzyme-catalyzed reactions, and yields high-purity n-pentene. Then, n-pentene is further catalyzed by olefin monooxygenase etnD and epoxide hydrolase SfEH1 to finally synthesize 1,2-pentanediol. The amino acid sequence of formate dehydrogenase is as follows: MKIVLVLYDAGKHAADEEKLYGCTENKLGIANWLKDQGHELITTSDKEGETSELDKHIPDADIIITTPFHPAYITKERLDKAKNLKLVVVAGVGSDHIDLDYINQTGKKISVLEVTGSNVVSVAEHVVMTMLVLVRNFVPAHEQIINHDWEVAAIAKDAYDIEGKTIATIGAGRIGYRVLER LLPFNPKELLYYDYQALPKEAEEKVGARRVENIEELVAQADIVTVNAPLHAGTKGLINKELLSKFKKGAWLVNTARGAICVAEDVAAALESGQLRGYGGDVWFPQPAPKDHPWRDMRNKYGAGNAMTPHYSGTTLDAQTRYAEGTKNILESFFTGKFDYRPQDIILLNGEYVTKAYGKHDKK.

[0020] The above gene source information is as follows: The technical effects and advantages of this invention are as follows: The preparation method of 1,2-pentanediol uses 2-pentanone as raw material. 2-Pentanol is synthesized by 2-keto reductase catalysis. 2-Pentanol is then catalyzed by a dehydrating enzyme to generate n-pentene, which is then catalyzed by an olefin monooxygenase to generate 1,2-epoxypentane. Finally, 1,2-pentanediol is generated by ring-opening with an epoxide hydrolase. This process requires only four enzymatic catalytic reactions to efficiently synthesize 1,2-pentanediol. The required 2-pentanone raw material can be obtained in one step through ethanol condensation. This production route is green and pollution-free. Furthermore, the n-pentene product synthesized by 2-keto reductase Gox2036 and the linalool dehydratase LinD mutant has a boiling point of only 30°C. Under reaction conditions of 35°C, the n-pentene product is collected by condensation while reacting, which eliminates substrate inhibition problems, allows for sustained long-term enzymatic catalysis, and yields high-purity n-pentene. Attached Figure Description Figure 1 This is a chromatogram of pET30a-2-keto reductase Gox2036 of the present invention; Figure 2 This is a schematic diagram of the structure of pET30a-linalool dehydratase LinD of the present invention; Figure 3 This is a schematic diagram of the structure of pET30a-olefin monooxygenase etnD of the present invention; Figure 4 This is a schematic diagram of the structure of pET30a-epoxide hydrolase SfEH1 of the present invention; Figure 5 This is a schematic diagram of the structure of the pET30a-formate dehydrogenase CbFDH of the present invention; Figure 6 This is a diagram showing the molecular docking results of the linalool dehydrogenase of this invention. Figure 7 This is a primer design map for constructing the combined mutant library of this invention; Figure 8 This is a gas chromatogram of the reaction product from the first step of this invention; Figure 9 This is a gas chromatogram of the reaction product from the second step of this invention; Figure 10 This is a diagram illustrating the antibacterial effect of 1,2-pentanediol in this invention. Detailed Implementation

[0021] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0022] Gene synthesis and vector construction: The above genes were codon-optimized and synthesized by Nanjing Qingke Biotechnology. The optimized base sequence is as follows: 2-Ketoreductase Gox2036 base sequence: Atgagcctgagcggcaaaattgcggcggtgaccggcgcggcgcagggcattggcaaagcgattgcgctgcgcctggcgaaagatggcgcggatgtgattctgctggatgtgaaacaggataccctggcggaaaccgcgaaagaagtggaagcgctgggccgccgcgcggtggcgctgaccgcggatattagcaaccgcgatcagtttcgcagcaccctggcggatgcggcgaaaaccctgggcggcctggatattatggtgaacaacgcgggcatttgccaggtgaaaccgattctggatattgaaccggcggaaattgaaaaaatttttagcattaacgtgcagggcgtgctgtggggcatgcaggcggcggcgaccctgtttaaagaaaaaggcaccaaaggcaaaattattaacgcgtgcagcattgcgggccatgaaggctatccgctgctgggcgcgtatagcgcgaccaaatttgcggtgcgcgcgctgacccagagcgcggcgaaagaactggcgagcagcggcattaccgtgaacagctattgcccgggcattgtgggcaccgatatgtgggtgaccattgataaacgcatggcggaaattaccggcaccgaaattggcgcgacctataaaaaatatgtggaaggcattgcgctgggccgcgtggaaaccgcggatgatgtggcgggctttgtggcgtatctgagcagcagcgatgcggattatatgaccggccagagcgtgctgattgatggcggcctggtgtttcgctaa。

[0023] Base sequence of linalool dehydratase mutant LinD (K82R / P363G / S365E / D369Q):

[0024] olefin monooxygenase etnD base sequence:

[0025] Epoxide hydrolase SfEH1 base sequence:

[0026] Formate dehydrogenase CbFDH base sequence:

[0027] The above genes were synthesized by Nanjing Qingke Biotechnology and cloned into the multiple cloning site NdeI and XhoI of the pET30a vector. The vector map is attached to the instruction manual. Figure 1-5 .

[0028] To realize the enzymatic synthesis route of 1,2-pentanediol, we used 2-pentanol as a substrate to perform molecular docking with linalool dehydrogenase and established a combinatorial mutant library to achieve the enzymatic catalytic activity for the synthesis of 2-pentanol from n-pentene. The molecular docking software used was Discovery Studio, and the results are shown below. Figure 6 As shown.

[0029] Combinatorial mutant libraries were constructed using molecular docking, targeting sites that interact with or are partially similar to the substrate 2-pentanol. These sites primarily involved Q56, Y59, K82, T84, P85, V88, L358, R359, Y360, E361, P363, S365, L366, L367, F368, D369, and E370. The primer design map for the combinatorial mutant library construction is shown below. Figure 7 As shown.

[0030] A combinatorial mutant library was constructed using the above primer map: PCR amplification was performed using primers Backboon-F / Backboon-R, Flancking-F / NNK5659-R, NNK5659-F / MNN828485-R, MNN828485-F / MNN585960-R, and NNK(358-370)-F / Flancking-F, with plasmid pET30a-LinD as the template. The polymerase used was KOD-Plus-Neo (KOD-401). The PCR program was: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 10 s, 58℃ annealing for 10 s, 68℃ extension for 1 min, 68℃ final extension for 5 min, and 95℃ denaturation for 10 s. Annealing at 58℃ for 10 s and extension at 68℃ for 3 min were performed for 25 cycles. The template was then removed by digestion with the nuclease DpnI. The product was recovered using a DNA purification and recovery kit, and finally, fragment ligation was performed using a seamless cloning kit. The fragments were then electroporated into *E. coli* expression strain BL21(DE3), plated into kanamycin-resistant plates, and screened using high-throughput 96-well plates. MNN and NNK are degenerate bases in the primer design. MNN encodes 32 codons covering 19 amino acids, and NNK also encodes 32 codons covering 20 amino acids. Based on this, we achieved random combinations of different amino acids through the design of degenerate primers and random ligation between fragments. The primers were synthesized by Nanjing Qingke Biotechnology, and their sequences are as follows: Construction of combined mutant libraries: 1. PCR amplification and recovery products: Polymerase KOD-Plus-Neo was purchased from Toyobo Shanghai Biotechnology Co., Ltd., catalog number KOD-401; PCR product purification and recovery kit was purchased from Magen Biotech, catalog number D2141; Seamless cloning kit Gibson Assembly was purchased from Thermo Fisher Scientific, catalog number A46228; Electroporation competent expression strain Escherichia coli BL21(DE3) was purchased from Shanghai Weidi Biotechnology, catalog number EE1002M.

[0031] 2. PCR product recovery and seamless Gibson cloning were performed strictly in accordance with the kit instructions. After ligation, the product was purified again using a PCR product recovery kit and finally eluted with 20 μL of deionized water.

[0032] 3. Electroconversion: Electroconversion parameters were set as follows: C=25 μF, PC=200 Ω, V=1.8 kV. The electroconversion cuvettes with a 0.2 cm spacing were removed from ice, their surfaces wiped dry with absorbent paper, and placed in the electroconversion tank. After electroconversion, the cuvettes were removed and allowed to reach room temperature. The lids were opened, and within 15 seconds, 0.9 ml of preheated SOC medium was added. The bottom of the cuvette was aspirated 2-3 times with a 1 ml pipette to mix thoroughly. The mixture was then transferred to a 50 ml centrifuge tube, and SOC medium was added to bring the total volume to 10 ml. The mixture was incubated at 37°C and 225 rpm for 60 minutes.

[0033] 4. Plate Spreading: Centrifuge at 5000 rpm for one minute to collect the bacteria. Resuspend the bacteria in an appropriate amount of SOC and spread them onto SOC plates containing the corresponding antibiotic (due to the large bacterial count, if spreading all the bacteria, please use 2-5 15cm diameter petri dishes). Invert the plates and incubate overnight at 37℃ for 13-17 hours. SOC medium formulation: 2% tryptone, 0.5% yeast extract, 0.05% NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM D-glucose, pH 7.5.

[0034] High-throughput screening of mutant libraries: Single clones were selected and cultured in 96-well deep-plates with 500 μL of LB medium at 37°C and 800 rpm for 6–8 h on a high-speed plate shaker. The cells were then resuspended by repeated pipetting and reacted at 16°C and 80 rpm for 12 h on a shaker. Afterward, the plates were centrifuged at 4°C and 8000 rpm. 100 μL of the reaction solution was transferred to an ELISA plate, and 100 μL of bromine in carbon tetrachloride solution was added. The plate was incubated at room temperature for 20 min, and the color change was observed. The principle is that alkenes and alkynes decolorize with bromine in carbon tetrachloride solution, thus this can be used for high-throughput screening of n-pentene synthase mutant libraries.

[0035] Through high-throughput screening of mutant libraries, we obtained that the linalool dehydratase LinD (K82R / P363C / S365E / D369Q) can catalyze the synthesis of n-pentene from 2-pentanol.

[0036] Enzyme protein fermentation preparation (5L fermenter): LB medium formulation: 1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.5 Fermentation medium preparation: 10g / L yeast extract (Angel FM902), 15g / L peptone, 10g / L glycerol (food grade), 10g / L dipotassium hydrogen phosphate (food grade), 5g / L potassium dihydrogen phosphate (food grade), 0.1g / L ferric ammonium citrate, 1g / L citric acid, 5g / L ammonium sulfate, 1g / L magnesium sulfate heptahydrate, 2ml / L trace elements, 1ml / L polyether defoamer, pH 7.5, autoclaved at 121℃.

[0037] Trace element preparation: 10 g / L FeSO4·7H2O, 1.53 g / L CaCl2, 2.2 g / L ZnSO4·7H2O, 1 g MnSO4·4H2O, 1 g / L CuSO4·5H2O, 0.1 g / L (NH4)6Mo7O24·4H2O, 0.2 g / L Na2B4O7·10H2O, 1 g / L NiCl2, 1 g / L H3BO3, and 10 mL / L HCl. The trace elements were first dissolved in HCl, then water was added to make up the volume, the pH was adjusted to 7.5, and the mixture was filtered to remove bacteria.

[0038] Feeding medium preparation: 500 g / L glucose, 7 g / L MgSO4, 1 mL / L trace elements.

[0039] Fermentation process 1. Prepare the corresponding culture medium according to the above formula. Seed culture: Take 10ul of the low-temperature preserved strain and inoculate it into a 500ml shake flask with 100ml of LB medium. Incubate overnight at 37℃ and 220r on a shaker.

[0040] 2. Take 1 ml of overnight culture and inoculate it into a 1000 ml shake flask containing 300 ml of fermentation medium. Incubate at 37°C and 220 rpm for 3-4 hours on a shaker. During this period, monitor the OD600. When the OD600 reaches approximately 2, inoculate the fermenter with 300 ml of the culture solution using a flame inoculation.

[0041] 3. During the fermentation process, the initial aeration rate is controlled at 1 VVM, dissolved oxygen (DO) is controlled at 100%, tank pressure is set at 0.02 MPa, temperature is controlled at 37℃, and rotation speed is 200 r.

[0042] 4. During the growth of the microbial strain, dissolved oxygen will continuously decrease. If it falls below 90%, adjust the rotation speed to maintain dissolved oxygen above 90%. Once the rotation speed reaches its maximum, adjust the aeration rate to maintain dissolved oxygen above 90% and the aeration rate to its maximum. Adjust the tank pressure to maintain dissolved oxygen (DO) at around 30%. When the carbon source in the culture medium is depleted and DO rebounds, start the feeding process. During this period, control dissolved oxygen at around 30% by adjusting the aeration rate and tank pressure. Monitor the glucose concentration and adjust the feeding rate to control the glucose concentration at around 3g / L. Measure the OD600 of the microbial growth. When the OD600 reaches around 20, add the inducing agent IPTG to a final concentration of 0.3mM. Adjust the temperature to 25℃ and the fermentation time to 14-16 hours.

[0043] After fermentation, the cells were collected by centrifugation and used for enzyme catalysis experiments.

[0044] Two-step multi-enzyme catalyzed synthesis of 1,2-pentanediol Step 1, 3L reaction system: Weigh 80g of 2-keto reductase Gox2036 fermentation wet cells, 200g of linalool dehydratase mutant LinD (K82R / P363G / S365E / D369Q) fermentation wet cells, and 150g of formate dehydrogenase CbFDH fermentation wet cells. Add these to a 5L reactor connected to a condenser. Also add 120g of substrate 2-pentanone, 0.3g of lysozyme, and 3g of coenzyme NAD. + Add water to a volume of 3L, set the reaction temperature to 35℃, and the condenser temperature to -10℃. Since the boiling point of n-pentene is 30℃, the catalytically synthesized n-pentene will evaporate at 35℃. Collect the evaporated product via condensation to obtain 37.5ml of colorless liquid. The product was analyzed by gas chromatography (see [link to product description]). Figure 8 The purity of the sample was 99.78%. Detection conditions: Gas chromatograph, Agilent 8860, with flame ionization detector (FID) (Agilent); DB-WAXetr column, 30m × 320μm × 1μm; initial temperature 25℃, held for 1 min, then increased to 180℃ at a rate of 15℃ / min, held for 18 min; split ratio 100:1; injector temperature 230℃, detector temperature 250℃; high-purity air flow rate 300 mL / min; high-purity hydrogen flow rate 30 mL / min; carrier gas flow rate 1.0 mL / min; injection volume 1 μL.

[0045] Step 2: In a 3L reaction system, thoroughly mix the substrate n-pentene with 30ml of ethanol, add 80g of olefin monooxygenase etnD fermentation wet cells, 80g of epoxide hydrolase SfEH1 fermentation wet cells, 50g of formate dehydrogenase CbFDH fermentation wet cells, 0.1g of lysozyme, and 1g of coenzyme NAD. + 1 g MgSO4 was added, and water was added to a final volume of 900 mL. The reaction temperature was set at 25℃. A mixture of n-pentene and 30 mL ethanol was added to the reaction system via a feed-feed method at a rate of 5 mL / h until the feed was complete. The reaction continued for 5 h. After the reaction was complete, the mixture was extracted twice with 300 mL ethyl acetate. The ethyl acetate was combined, and 0.5 g activated carbon was added for decolorization at room temperature under sealed conditions. The activated carbon was removed by filtration, and then the ethyl acetate was removed by rotary evaporation at 70℃, yielding 32 mL of a colorless liquid. The product was analyzed by gas chromatography, and the purity was 99.62%. Detection conditions: Product was analyzed by gas chromatography (see [link to gas chromatography analysis]). Figure 9 Detection conditions: Gas chromatograph, Agilent 8860, with flame ionization detector (FID) (Agilent), DB-WAXetr column, specifications 30m × 320μm × 1μm; initial temperature 130℃, held for 1 min, then increased to 180℃ at a rate of 30℃ / min, held for 18 min; split ratio 100:1; injector temperature 230℃, detector temperature 250℃; high-purity air flow rate 300mL / min; high-purity hydrogen flow rate 30mL / min; carrier gas flow rate 1.0mL / min; injection volume 1μL.

[0046] 1,2-Pentanediol efficacy verification: Since 1,2-pentanediol is primarily used in cosmetics for its antibacterial and moisturizing effects, we further verified the antibacterial effect of enzymatically synthesized 1,2-pentanediol. We selected representative prokaryotic lactic acid bacteria and eukaryotic yeasts for inhibition zone experiments. These experiments allowed for a more direct observation of the antibacterial effect of the enzymatically synthesized 1,2-pentanediol. (See attached image.) Figure 10 As shown, the left side contains lactic acid bacteria, and the right side contains yeast.

[0047] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing 1,2-pentanediol, characterized in that, Includes the following steps: S1: In a reactor connected to a condenser, 2-pentanone is used as a substrate, and 2-keto reductase, linalool dehydratase mutant, formate dehydrogenase, lysozyme, coenzyme NAD+ and water are added to react and obtain n-pentene. S2: The above-obtained n-pentene is thoroughly mixed with ethanol. Then, the mixture of n-pentene and ethanol is added to a reaction system containing olefin monooxygenase, epoxide hydrolase, formate dehydrogenase, lysozyme, coenzyme NAD+, MgSO4 and water by a feed method. After the mixture of n-pentene and ethanol has been completely added, the reaction continues for a period of time. Finally, the mixture is extracted to obtain 1,2-pentanediol.

2. The method for preparing 1,2-pentanediol according to claim 1, characterized in that, In step S1, the condensation device is set to a temperature of -10°C.

3. The method for preparing 1,2-pentanediol according to claim 1, characterized in that, In step S1, the reaction temperature is set to 35°C.

4. The method for preparing 1,2-pentanediol according to claim 1, characterized in that, In step S2, the reaction temperature is 25°C.

5. The method for preparing 1,2-pentanediol according to claim 1, characterized in that, In step S2, the flow rate in the feeding method is controlled at 5 mL / h.

6. The method for preparing 1,2-pentanediol according to claim 1, characterized in that, In step S2, the extraction is performed twice with ethyl acetate, the ethyl acetate is combined, activated carbon is added for decolorization at room temperature in a sealed environment, the activated carbon is removed by filtration, and then the ethyl acetate is removed by rotary evaporation.

7. The method for preparing 1,2-pentanediol according to claim 1, characterized in that, In step S2, the reaction continues for 5-10 h after the mixture of n-pentene and ethanol is completely added.

8. The use of 1,2-pentanediol prepared according to any one of claims 1-7 in the preparation of cosmetics.

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