CN114875106B - Method for generating (S) -2-methylcyclohexanone by utilizing alkene reductase in micro-flow field - Google Patents
Method for generating (S) -2-methylcyclohexanone by utilizing alkene reductase in micro-flow field Download PDFInfo
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- 150000001336 alkenes Chemical class 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 23
- LFSAPCRASZRSKS-LURJTMIESA-N (2s)-2-methylcyclohexan-1-one Chemical compound C[C@H]1CCCCC1=O LFSAPCRASZRSKS-LURJTMIESA-N 0.000 title description 17
- 238000006243 chemical reaction Methods 0.000 claims abstract description 65
- 102000004190 Enzymes Human genes 0.000 claims abstract description 41
- 108090000854 Oxidoreductases Proteins 0.000 claims abstract description 36
- LFSAPCRASZRSKS-UHFFFAOYSA-N 2-methylcyclohexan-1-one Chemical compound CC1CCCCC1=O LFSAPCRASZRSKS-UHFFFAOYSA-N 0.000 claims abstract description 35
- 102000004316 Oxidoreductases Human genes 0.000 claims abstract description 35
- LKTNAAYQZJAXCJ-UHFFFAOYSA-N 2-methylcyclohex-2-en-1-one Chemical compound CC1=CCCCC1=O LKTNAAYQZJAXCJ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000006356 dehydrogenation reaction Methods 0.000 claims abstract description 6
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
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- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-O 0.000 claims description 5
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- JVSWJIKNEAIKJW-UHFFFAOYSA-N dimethyl-hexane Natural products CCCCCC(C)C JVSWJIKNEAIKJW-UHFFFAOYSA-N 0.000 claims description 2
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- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/002—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by oxidation/reduction reactions
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
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- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
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Abstract
The invention discloses a method for generating a catalyst in a micro-flow field by utilizing alkene reductaseS) -2-methylcyclohexanone, comprising the steps of: s1: utilize%R) -selective alkene reductase catalyzed unnatural reaction-dehydrogenation reaction, resolution of racemic 2-methylcyclohexanone to obtain 2-methyl-2-cyclohexene-1-one and [ - ]S) -2-methylcyclohexanone; s2: utilize%S) -selective alkene reductase catalyzes natural reaction-reduction reaction, and reduces 2-methyl-2-cyclohexene-1-ketone to obtain the productS) -2-methylcyclohexanone. The invention uses two-step enzyme combination catalysis to racemize racemic 2-methylcyclohexanone to obtain refractory productS) 2-methylcyclohexanone has the advantages of mild reaction conditions, strong sustainability, high product yield, high optical purity and the like.
Description
Technical Field
The invention belongs to the technical field of chemical synthesis, and particularly relates to a method for generating (S) -2-methylcyclohexanone by using alkene reductase in a micro-flow field.
Background
The (S) -2-methylcyclohexanone is an important prodrug, can be used for synthesizing a plurality of high-added-value drug intermediates, and has certain application potential in the field of medicine synthesis. However, many of the current methods for synthesizing (S) -2-methylcyclohexanone involve severe conditions such as metal hydride mediation, pd ligand participation, extreme temperatures and/or extreme pH. Due to the toxicity of metal catalysts and the challenges presented by these harsh conditions to the synthesis of (S) -2-methylcyclohexanone, more and more scientists are devoted to the study of efficient green synthesis of (S) -2-methylcyclohexanone.
Disclosure of Invention
The invention aims to: in order to overcome the defects in the prior art, the invention provides a method for synthesizing (S) -2-methylcyclohexanone by combining alkene reductase and mutant strain thereof in a micro-flow field to racemize the 2-methylcyclohexanone (figure 1).
In order to solve the above technical problems, the reaction apparatus according to the present invention is shown in fig. 2. The method takes racemized 2-methylcyclohexanone as a raw material, and takes immobilized alkene reductase as a biocatalyst for catalysis, and mainly comprises two reaction steps: firstly, utilizing a non-natural reaction catalyzed by wild alkene reductase to perform dehydrogenation resolution reaction, and dehydrogenating (R) -2-methylcyclohexanone to generate 2-methyl-2-cyclohexene-1-one; under the condition of providing coenzyme circulation, the engineering alkene reductase is used for catalyzing the reduction of 2-methyl-2-cyclohexene-1-ketone to obtain (S) -2-methylcyclohexanone.
Specifically, the reaction comprises the following steps:
s1: 2-methylcyclohexanone is taken as a raw material, wild-type alkene reductase immobilized on resin is immobilized in a first micro-channel, and (R) -2-methylcyclohexanone is catalyzed into 2-methyl-2-cyclohexene-1-one in the first micro-channel by utilizing dehydrogenation resolution reaction catalyzed by the wild-type alkene reductase;
s2: adsorbing engineered ene reductase and Glucose Dehydrogenase (GDH) on a resin as biocatalysts to a second microchannel, injecting S1 reaction solution into the second microchannel, and continuously adding NADP + And glucose, reducing 2-methyl-2-cyclohexene-1-one to obtain (S) -2-methylcyclohexanone.
In step S1, the preparation method of the alkene reductase TsER is as follows: the gene of the alkene reductase TsER with the nucleotide sequence shown in SEQ ID No.1 is constructed on a pET-28a (+) vector, and is transferred into escherichia coli BL 21 (DE 3), cultured and purified to obtain the corresponding alkene reductase TsER.
Wherein, SEQ ID NO.1 is obtained by modifying the sequence with the sequence number of Q5SLY6, and the total length 1050 of the sequence of Q5SLY6 can be obtained by searching NCBI. The total length 1059 of the SEQ ID NO.1 sequence, wherein the front three bases belong to restriction enzyme sites and the rear six bases are restriction enzyme sites.
In step S1, the solvent for the reaction is a mixture of phosphate buffer (PBS buffer, 50mM, pH 7.4) and an organic solvent including, but not limited to, n-heptane, isooctane, methanol, ethanol, isopropanol, acetonitrile, dimethyl sulfoxide; wherein the volume of the organic solvent is less than or equal to one percent of the total reaction volume.
In step S1, the wild-type alkene reductase is TsER derived from Thermus scotoductus SA-01.
In the step S2, the engineering alkene reductase is a mutant strain TsER C25G/I66T derived from TsER of Thermus scotoductus SA-01, and the idea of mutation can be referred to journal chemBiochem.2017,18 (7), 685-691.
In step S2, the GDH is a commercial enzyme derived from Bacillus.
In steps S1 and S2, the pure enzyme-adsorbing resin includes, but is not limited to, aqueous adsorption resins ES-1, ES-103B, ES-108.
In steps S1 and S2, the resin is activated before being put into use, and then the pure enzyme is adsorbed. The method for immobilizing the alkene reductase on the resin comprises the following steps: dispersing the cultured and purified alkene reductase in phosphate buffer solution immersed with resin, slowly stirring at 20-22 ℃ and then taking out the resin, and taking out the resin after stirring for 24 hours after the resin is stirred for 24 hours in cold phosphate buffer solution of-80 ℃ as PBS buffer solution, 50mM, pH of which is 7.4.
In the steps S1 and S2, the alkene reductase is dispersed in PBS buffer solution after being purified, and is adsorbed and fixed in a micro-channel to participate in the reaction through resin, and the adsorption rate of pure enzyme on the resin can be measured by an ultraviolet spectrophotometer. The adsorption rate was calculated as (blank absorbance value-supernatant absorbance value)/blank absorbance value.
In the steps S1 and S2, the reaction temperature is 25-35 ℃, preferably 25 ℃; the inner diameter of the micro-channels is 1-3 mm, preferably 1mm.
In summary, the invention provides a method for preparing (S) -2-methylcyclohexanone by using a microchannel reaction device through catalysis of immobilized enzyme, wherein 2-methylcyclohexanone is used as a raw material, non-natural reaction-dehydrogenation reaction and natural reaction-reduction reaction of alkene reductase are used, pure enzyme is immobilized in a microfluidic field reaction technology device, and racemized (S) -2-methylcyclohexanone is prepared by using two-step enzyme combined catalysis. The method has the advantages of high optical purity, good selectivity, mild reaction conditions, strong sustainability, high product yield, high safety, quick reaction and the like.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
(1) According to the invention, the non-natural reaction-dehydrogenation resolution reaction and natural reaction-reduction reaction of the same enzyme are utilized to perform combined catalysis of two enzymes in a micro-flow field for the first time through immobilized alkene reductase and a corresponding mutant strain, so that the 2-methylcyclohexanone is racemized, and the (S) -2-methylcyclohexanone which is difficult to synthesize by a chemical method is obtained. The method avoids the preparation method with metal catalysis, high temperature and serious pollution, has simple operation, high optical purity, good selectivity, mild reaction condition, strong sustainability, high product yield, high safety and quick reaction, and meets the requirements of green chemistry;
(2) The invention adopts a method of immobilizing pure enzyme, the pure enzyme is adsorbed in water-based resin, single enzyme is immobilized, alkene reductase and glucose dehydrogenase are co-immobilized, and finally the immobilized material is filled and immobilized in a microchannel reactor, so that the immobilized material can be stored at low temperature and is convenient to reuse;
(3) The invention utilizes the micro-channel reaction technology to realize the promotion of mass transfer and heat transfer speed and reaction rate by 1-3 orders of magnitude; the real-time online reaction quantity is small, and the process is easy to control; the continuous flow and low back mixing characteristics can effectively improve the selectivity of the reaction.
Drawings
The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.
FIG. 1 is a reaction scheme of the present invention.
FIG. 2 is a schematic flow chart of the reaction apparatus of the present invention.
FIG. 3 is a nuclear magnetic hydrogen spectrum and a nuclear magnetic carbon spectrum of 2-methyl-2-cyclohexen-1-one of the present invention.
FIG. 4 is a nuclear magnetic hydrogen spectrum and a nuclear magnetic carbon spectrum of 2-methylcyclohexanone of the invention.
Detailed Description
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.
The microchannel reaction device in the following embodiment comprises two injectors, two microchannel reactors and a product collector which are connected through a pipeline, wherein the pipeline in the microchannel reaction device is a polytetrafluoroethylene pipe, the length of the pipeline is 20cm, and the diameter of the pipeline is 1mm.
The procedure for the construction of the alkene reductase (TsER) described in the examples below was as follows, (1) the TsER alkene reductase gene derived from Thermus scotoductus SA-01 (nucleotide sequence shown in SEQ ID NO. 1) was constructed on the pET-28a (+) vector (restriction enzyme sites Ndel and HindIII) and transferred into E.coli BL 21 (DE 3); (2) And (3) inoculating 50 mu L of the TsER glycerol strain constructed in the step (1) to 5mL of LB culture medium, adding kanamycin with the final concentration of 100 mu g/mL, and carrying out shaking culture at 37 ℃ for 12 hours at 200rpm to obtain a pre-culture bacterial liquid. 5mL of the preculture broth was transferred to 500mL of TB (containing 100mL of PBS) medium, and kanamycin was added at a final concentration of 100. Mu.g/mL,shaking culture at 37℃and 200rpm for about 4 hours to OD 600 Reaching 0.6-0.7, cooling to 4 ℃, adding IPTG to the final concentration of 50mM for induction expression, and continuing to shake culture at 18 ℃ for 26h. The culture solution was concentrated by centrifugation, and the cells were collected, and suspended in a buffer solution with PBS buffer on a mixer to prepare a whole cell solution of 0.1 g/mL. (3) Crushing the whole cell liquid by using an ultrasonic cell crusher, and crushing for 20min under the conditions of power of 200W, ultrasonic on for 2s, ultrasonic off for 2s and ice water bath to obtain the whole cell lysate. (4) After the cell lysate is fully centrifuged and filtered, the supernatant is pumped into a pre-balanced Ni-NTA resin column by a peristaltic pump, after the supernatant is completely loaded, the resin is subjected to gradient elution by 60mL of lysis buffer with imidazole concentration of 10mM, 20mM, 30mM and 40mM respectively, and after the analysis of the hybrid protein by ultraviolet detection is eluted, the Ni-NTA is washed by the lysis buffer containing 250mM imidazole, and pure enzyme is collected. The collected pure enzyme contained imidazole at a high concentration, and the imidazole was eluted by ultrafiltration centrifugation using an ultrafiltration tube until the imidazole concentration was less than 10mM, and then the pure enzyme (126. Mu.g/mL) was collected.
The TsER C25G/I67T construction method described in the following examples is as follows: tsER C25G/I67T (25 th C is mutated to G and 67 th I is mutated to T) can be obtained by a series of operations such as extracting plasmids, polymerase Chain Reaction (PCR), purifying PCR reaction liquid, electrically converting to escherichia coli BL 21 (DE 3), flat plate coating, extracting single colony, single colony culture, strain preservation and the like after the TsER constructed as above is pre-cultured by the primer (the primer of C25G is 5'-TGAGCCCGATGGGTCAGTACAGC-3',3'-GCTGTACTGACCCATCGGGCTCA-5'; the primer of I67T is 5'-CGGAAGGTCGCACCAGTCCGTT-3',3 '-AACGGACTGGTGCGACCTTCCG-5'). TsER C25G/I67T culture and purification methods were consistent with TsER.
The PCR reaction system is as follows: sterilized distilled water 31. Mu.L, 10 XBuffer for KOD-Plus-Neo 5. Mu. L, dNTPs 5. Mu.L (2 mM), mgSO 4 3. Mu.L (25 mM), 1.5. Mu.L (10. Mu.M) of forward primer, 1.5. Mu.L (10. Mu.M) of reverse primer, 1. Mu.L (50 ng/. Mu.L) of template plasmid DNA, 1. Mu.L (1U/. Mu.L) of KOD-Plus-Neo, 1. Mu.L of DMSO.
Temperature program of PCR: PCR amplification was performed as follows: firstly, starting at 94 ℃ and keeping for 2min; secondly, maintaining the temperature at 98 ℃ for 10s; thirdly, maintaining the temperature at 60 ℃ for 30s; fourth, maintaining at 68 ℃ for 4min; returning to the second step, and circulating the second to fourth steps 29 times; maintaining at 68 ℃ for 10s; finally, the mixture was stored at 4 ℃.
Construction of the mutant strain: after completion of PCR amplification, 1. Mu.L of DpnI (20U/. Mu.L) was added to the PCR reaction solution, and the template plasmid DNA was digested overnight at 37 ℃. After purification of the PCR reaction solution, the mixture was introduced into E.coli BL 21 (DE 3) competence by an electrotransformation apparatus, the electrotransformation mixture was dispersed in 1mL of LB liquid medium, incubated at 37℃and 200rpm for 1 hour, then coated on agarose solid medium containing 100. Mu.g/mL kanamycin, incubated at 37℃overnight, single colonies on the plates were picked up and incubated at 5mL of LB medium (containing 100. Mu.g/mL kanamycin) at 37℃overnight, and the production of mutants was confirmed by sequencing.
Example 1
400mg of aqueous resin ES-1 was added to 1mL of TsER pure enzyme solution (126. Mu.g/mL, PBS buffer, 50mM, pH 7.4), and after stirring slowly at 20℃and 700rpm for 24 hours, the immobilized enzyme and supernatant were obtained by centrifugation. The absorbance of the supernatant was measured at 340nm using an ultraviolet spectrophotometer and the absorbance of the corresponding blank was 0.071, which was calculated to be 29% for the aqueous resin ES-1 over TsER pure enzyme. The absorptivity calculation formula is: absorbance= (blank absorbance-detection absorbance)/blank absorbance.
Example 2
400mg of aqueous resin ES-103B was added to 1mL of TsER pure enzyme solution (126. Mu.g/mL), and after stirring slowly at 20℃and 700rpm for 24 hours, the immobilized enzyme and supernatant were obtained by centrifugation. The absorbance of the supernatant was measured at 340nm using an ultraviolet spectrophotometer and found to be 0.014, and the absorbance of the corresponding blank was found to be 0.071, which means that the absorbance of the aqueous resin ES-103B for TsER pure enzyme was 81% under this condition.
Example 3
400mg of aqueous resin ES-108 was added to 1mL of TsER pure enzyme solution (126. Mu.g/mL), and after stirring slowly at 20℃and 700rpm for 24 hours, the immobilized enzyme and supernatant were obtained by centrifugation. The absorbance of the supernatant was measured at 340nm using an ultraviolet spectrophotometer and found to be 0.001, and the absorbance of the corresponding blank was found to be 0.071, which means that the absorbance of the aqueous resin ES-108 for the TsER pure enzyme was found to be 98% under this condition.
Example 4
12g of aqueous resin ES-108 was added to 30mL of TsER pure enzyme solution (126. Mu.g/mL), the resin was taken out after being slowly stirred at 20℃and 700rpm for 24 hours, the resin was frozen at-80℃for 12 hours, then dehydrated in a freeze dryer, and the dried immobilized resin was filled into a microchannel reactor A having an inner diameter of 1mm. 10mL of 2-methylcyclohexanone solution with the concentration of 10mM is placed in an injector A, the solution is injected into a microchannel reactor A through the injector A, the reaction temperature is 25 ℃, the flow rate is controlled, after about 1 hour of reaction, the reaction conversion rate of (R) -2-methylcyclohexanone in a substrate 2-methylcyclohexanone is greater than or equal to 99% after the reaction is monitored by Gas Chromatography (GC) to generate 2-methyl-2-cyclohexene-1-one. The nuclear magnetic spectrum is shown in figure 3.HRMS (ESI) m/z calc for C 7 H 10 O[M+Na]+:133.0624,found:133.0622。
Example 5
12g of aqueous resin ES-108 was added to 30mL of TsER pure enzyme solution (126. Mu.g/mL), the resin was taken out after being slowly stirred at 20℃and 700rpm for 24 hours, the resin was frozen at-80℃for 12 hours, then dehydrated in a freeze dryer, and the dried immobilized resin was filled into a microchannel reactor A having an inner diameter of 1mm. 10mL of 2-methylcyclohexanone solution with the concentration of 10mM is placed in an injector A, the solution is injected into a microchannel reactor A through the injector A, the reaction temperature is 25 ℃, the flow rate is controlled, after the reaction is carried out for about 45min, 2-methyl-2-cyclohexene-1-one is generated through monitoring by GC, the (R) -2-methylcyclohexanone in the substrate 2-methylcyclohexanone is converted into 2-methyl-2-cyclohexene-1-one, and the reaction conversion rate of the (R) -2-methylcyclohexanone is more than or equal to 99%. HRMS (ESI) m/z calc for C 7 H 10 O[M+K]+:149.0363,found:149.0365.
Example 6
12g of aqueous resin ES-108 was added to 30mL of TsER pure enzyme solution (126. Mu.g/mL), the resin was taken out after stirring slowly at 20℃and 700rpm for 24 hours, the resin was frozen at-80℃for 12 hours, and then lyophilizedAnd (3) dewatering in the machine, and filling the dried immobilized resin into a microchannel reactor A with the inner diameter of 1mm. 10mL of 2-methylcyclohexanone solution with the concentration of 10mM is placed in an injector A, the solution is injected into a microchannel reactor A through the injector A, the reaction temperature is 25 ℃, the flow rate is controlled, after about 30 minutes of reaction, 2-methyl-2-cyclohexene-1-one is generated through GC monitoring, the (R) -2-methylcyclohexanone in the substrate 2-methylcyclohexanone is converted into 2-methyl-2-cyclohexene-1-one, and the reaction conversion rate of the (R) -2-methylcyclohexanone is more than or equal to 99%. HRMS (ESI) m/z calc for C 7 H 10 O[M+K]+:149.0363,found:149.0366.
Example 7
12g of aqueous resin ES-108 was added to 30mL of TsER pure enzyme solution (126. Mu.g/mL), the resin was taken out after being slowly stirred at 20℃and 700rpm for 24 hours, the resin was frozen at-80℃for 12 hours, then dehydrated in a freeze dryer, and the dried immobilized resin was filled into a microchannel reactor A having an inner diameter of 1mm. 10mL of 2-methylcyclohexanone solution with the concentration of 10mM is placed in an injector A, the solution is injected into a microchannel reactor A through the injector A, the reaction temperature is 25 ℃, the flow rate is controlled, after the reaction is carried out for about 15min, 2-methyl-2-cyclohexene-1-one is generated through monitoring by GC, the (R) -2-methylcyclohexanone in the substrate 2-methylcyclohexanone is converted into 2-methyl-2-cyclohexene-1-one, and the reaction conversion rate of (R) -2-methylcyclohexanone is 89%. HRMS (ESI) m/z calc for C 7 H 10 O[M+K]+:149.0363,found:149.0361.
Example 8
12g of aqueous resin ES-108 was added to 30mL of TsER pure enzyme solution (126. Mu.g/mL), the resin was taken out after being slowly stirred at 20℃and 700rpm for 24 hours, the resin was frozen at-80℃for 12 hours, then dehydrated in a freeze dryer, and the dried immobilized resin was filled into a microchannel reactor A having an inner diameter of 2 mm. 10mL of 2-methylcyclohexanone solution with the concentration of 10mM is placed in an injector A, the solution is injected into a microchannel reactor A through the injector A, the reaction temperature is 25 ℃, the flow rate is controlled, after about 30 minutes of reaction, 2-methyl-2-cyclohexene-1-one is generated through monitoring by GC, the (R) -2-methylcyclohexanone in the substrate 2-methylcyclohexanone is converted into 2-methyl-2-cyclohexene-1-one, and the reaction conversion rate of (R) -2-methylcyclohexanone is 95%. HRMS (ESI) m/z calc for C 7 H 10 O[M+K]+:149.0363,found:149.0365.
Example 9
12g of aqueous resin ES-108 was added to 30mL of TsER pure enzyme solution (126. Mu.g/mL), the resin was taken out after being slowly stirred at 20℃and 700rpm for 24 hours, the resin was frozen at-80℃for 12 hours, then dehydrated in a freeze dryer, and the dried immobilized resin was filled into a microchannel reactor A having an inner diameter of 3 mm. 10mL of 2-methylcyclohexanone solution with the concentration of 10mM is placed in an injector A, the solution is injected into a microchannel reactor A through the injector A, the reaction temperature is 25 ℃, the flow rate is controlled, after about 30 minutes of reaction, 2-methyl-2-cyclohexene-1-one is generated through monitoring by GC, the (R) -2-methylcyclohexanone in the substrate 2-methylcyclohexanone is converted into 2-methyl-2-cyclohexene-1-one, and the reaction conversion rate of (R) -2-methylcyclohexanone is 90%. HRMS (ESI) m/z calc for C 7 H 10 O[M+K]+:149.0363,found:149.0363.
Example 10
12g of aqueous resin ES-108 was added to 30mL of TsER pure enzyme solution (126. Mu.g/mL), the resin was taken out after being slowly stirred at 20℃and 700rpm for 24 hours, the resin was frozen at-80℃for 12 hours, then dehydrated in a freeze dryer, and the dried immobilized resin was filled into a microchannel reactor A having an inner diameter of 1mm. 10mL of 2-methylcyclohexanone solution with the concentration of 10mM is placed in an injector A, the solution is injected into a microchannel reactor A through the injector A, the reaction temperature is 20 ℃, the flow rate is controlled, after the reaction is carried out for about 30min, 2-methyl-2-cyclohexene-1-one is generated through monitoring by GC, the (R) -2-methylcyclohexanone in the substrate 2-methylcyclohexanone is converted into 2-methyl-2-cyclohexene-1-one, and the reaction conversion rate of (R) -2-methylcyclohexanone is 87%. HRMS (ESI) m/z calc for C 7 H 10 O[M+Na]+:133.0624,found:133.0622.
Example 11
12g of aqueous resin ES-108 was added to 30mL of TsER pure enzyme solution (126. Mu.g/mL), the resin was taken out after being slowly stirred at 20℃and 700rpm for 24 hours, the resin was frozen at-80℃for 12 hours, then dehydrated in a freeze dryer, and the dried immobilized resin was filled into a microchannel reactor A having an inner diameter of 1mm. 10mL of a 10mM 2-methylcyclohexanone solution was placed in syringe A and injectedInjecting an injector A into a micro-channel reactor A, controlling the reaction temperature to be 30 ℃, controlling the flow rate, reacting for about 30min, and then monitoring by GC to obtain 2-methyl-2-cyclohexene-1-one, wherein the reaction conversion rate of (R) -2-methylcyclohexanone in a substrate 2-methylcyclohexanone is 94 percent. HRMS (ESI) m/z calc for C 7 H 10 O[M+K]+:149.0363,found:149.0367.
Example 12
12g of aqueous resin ES-108 was added to 30mL of TsER pure enzyme solution (126. Mu.g/mL), the resin was taken out after being slowly stirred at 20℃and 700rpm for 24 hours, the resin was frozen at-80℃for 12 hours, then dehydrated in a freeze dryer, and the dried immobilized resin was filled into a microchannel reactor A having an inner diameter of 1mm. 10mL of 2-methylcyclohexanone solution with the concentration of 10mM is placed in an injector A, the solution is injected into a microchannel reactor A through the injector A, the reaction temperature is 35 ℃, the flow rate is controlled, after the reaction is carried out for about 30min, 2-methyl-2-cyclohexene-1-one is generated through monitoring by GC, the (R) -2-methylcyclohexanone in the substrate 2-methylcyclohexanone is converted into 2-methyl-2-cyclohexene-1-one, and the reaction conversion rate of (R) -2-methylcyclohexanone is 88%. HRMS (ESI) m/z calc for C 7 H 10 O[M+K]+:149.0363,found:149.0363.
Example 13
12g of aqueous resin ES-108 was added to 30mL of TsER pure enzyme solution (126. Mu.g/mL), the resin was taken out after being slowly stirred at 20℃and 700rpm for 24 hours, the resin was frozen at-80℃for 12 hours, then dehydrated in a freeze dryer, and the dried immobilized resin was filled into a microchannel reactor A having an inner diameter of 1mm. 10mL of 2-methylcyclohexanone solution with the concentration of 10mM is placed in an injector A, the solution is injected into a microchannel reactor A through the injector A, the reaction temperature is 25 ℃, the flow rate is controlled, after about 30 minutes of reaction, 2-methyl-2-cyclohexene-1-one is generated through GC monitoring, the (R) -2-methylcyclohexanone in the substrate 2-methylcyclohexanone is converted into 2-methyl-2-cyclohexene-1-one, and the reaction conversion rate of the (R) -2-methylcyclohexanone is more than or equal to 99%. After the reaction is finished, the pipeline is washed by PBS buffer solution, 10mL of 2-methylcyclohexanone solution with the concentration of 10mM is introduced again under the same reaction conditions, and the reaction conversion rate of (R) -2-methylcyclohexanone is more than or equal to 99 percent. After repeating this step ten times, the conversion rate is still 95% or more.
Example 14
12g of aqueous resin ES-108 was added to 30mL of TsER pure enzyme solution (126. Mu.g/mL), 12g of aqueous resin ES-108 was added to 30mL of mixed pure enzyme solution of TsER C25G/I67T (126. Mu.g/mL) and 30U of GDH, and the resins were taken out after being slowly stirred at 20℃and 700rpm for 24 hours, frozen at-80℃for 12 hours, and then dehydrated in a freeze dryer, and the dried immobilized resins were filled into a 1 mm-inner diameter microchannel reactor A and a microchannel reactor B, respectively. 10mL of a 10mM 2-methylcyclohexanone solution was placed in syringe A, injected into the microchannel reactor A via syringe, at a reaction temperature of 25℃and at a controlled flow rate, and reacted for about 30min. Buffer (12. Mu.M, NADP) was added to syringe B + The method comprises the steps of carrying out a first treatment on the surface of the 40mM, glucose) and the reaction liquid flowing out of the micro-channel reactor A enter the micro-channel reactor B together through the Y-type adapter, and the two-phase flow rate ratio of the flow rate is controlled to be 1:1, reacting for 1h at 25 ℃, monitoring the conversion of 2-methyl-2-cyclohexene-1 ketone into (S) -2-methylcyclohexanone by GC, obtaining the conversion rate and ee value by high performance Gas Chromatography (GC), wherein the reaction conversion rate is more than or equal to 99%, and the enantiomeric excess (ee) value is more than or equal to 99%. The nuclear magnetic spectrum is shown in figure 4.HRMS (ESI) m/z calc for C 7 H 12 O[M+K]+:151.0520,found:151.0522。
Example 15
12g of aqueous resin ES-108 was added to 30mL of TsER pure enzyme solution (126. Mu.g/mL), 12g of aqueous resin ES-108 was added to 30mL of a mixed pure enzyme solution of TsER C25G/I67T (126. Mu.g/mL) and 30U of GDH, the resin was taken out after slowly stirring at 20℃and 700rpm for 24 hours, the resin was frozen at-80℃for 12 hours, and then water was removed in a freeze dryer, and the dried immobilized resin was filled into a 1mm inner diameter microchannel reactor A and a microchannel reactor B, respectively. 10mL of a 10mM 2-methylcyclohexanone solution was placed in syringe A, injected into the microchannel reactor A via syringe, at a reaction temperature of 25℃and at a controlled flow rate, and reacted for about 30min. Buffer (12. Mu.M, NADP) was added to syringe B + The method comprises the steps of carrying out a first treatment on the surface of the 40mM, glucose) and the reaction solution flowing out of the microchannel reactor A are jointly introduced into the microchannel through the Y-type adapterIn the reactor B, the control flow rate and the two-phase flow rate ratio are 1:1, reacting for 45min at 25 ℃, monitoring the conversion of 2-methyl-2-cyclohexene-1 ketone into (S) -2-methylcyclohexanone by GC, wherein the reaction conversion rate is more than or equal to 99%, and the ee value is 97%. HRMS (ESI) m/z calc for C 7 H 12 O[M+K]+:151.0520,found:151.0519.
Example 16
12g of aqueous resin ES-108 was added to 30mL of TsER pure enzyme solution (126. Mu.g/mL), 12g of aqueous resin ES-108 was added to 30mL of a mixed pure enzyme solution of TsER C25G/I67T (126. Mu.g/mL) and 30U of GDH, the resin was taken out after slowly stirring at 20℃and 700rpm for 24 hours, the resin was frozen at-80℃for 12 hours, and then water was removed in a freeze dryer, and the dried immobilized resin was filled into a 1mm inner diameter microchannel reactor A and a microchannel reactor B, respectively. 10mL of a 10mM 2-methylcyclohexanone solution was placed in syringe A, injected into the microchannel reactor A via syringe, at a reaction temperature of 25℃and at a controlled flow rate, and reacted for about 30min. Buffer (12. Mu.M, NADP) was added to syringe B + The method comprises the steps of carrying out a first treatment on the surface of the 40mM, glucose) and the reaction liquid flowing out of the micro-channel reactor A enter the micro-channel reactor B together through the Y-type adapter, and the two-phase flow rate ratio of the flow rate is controlled to be 1:1, reacting for 30min at 25 ℃, monitoring the conversion of 2-methyl-2-cyclohexene-1 ketone into (S) -2-methylcyclohexanone by GC, wherein the reaction conversion rate is 89%, and the ee value is more than or equal to 99%. HRMS (ESI) m/z calc for C 7 H 12 O[M+Na]+:135.0780,found:135.0782.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Sequence listing
<110> university of Nanjing Industrial science
<120> a method for producing (S) -2-methylcyclohexanone using alkene reductase in a micro-flow field
<140> 2022103485600
<141> 2022-04-01
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<170> SIPOSequenceListing 1.0
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<213> Gene of TsER (Artificial Sequence)
<400> 1
catatgggcc tgctgtttac gccgctggaa ctgcgtgggc tgcgtctgaa aaatcgtctg 60
gcaatgagcc cgatgtgtca gtacagcgcg acggaagggg gggaagttac ggactggcac 120
ctgctgcatt atcctacccg tgcactgggt ggggttggtc tggttatcgt ggaagcaaca 180
gcggttctgc cggaaggtcg cattagtccg tttgatctgg gaatttggag cgaagaccat 240
ctgccgggcc tgcgcgaact ggcacgtcgt attcgtgaag caggggcagt gcctggcatt 300
cagctggcac atgcaggtcg caaagcaggt accgcccgtc cgtgggaagg aggacgccct 360
ctgggttgga aagtggttgg tccgagtccg attccgtttg cagaaggtta tccggtgccg 420
gaagcactgg atgaggcagg catggcacgc gttctggaag cgtttgttga gggtgccaaa 480
cgtgccctgc gtgcaggttt tctggttgtt gagctgcata tggcgcatgg ttatctgctg 540
agcagttttc tgagtccgct ggcaaatcgt cgtgaggatg cctatggtgg tagtcgtgaa 600
aatcgtatgc gttttccgct ggaagttgcg cgtgcagttc gtgaagcaat tcctccggaa 660
ctgcctctgt ttgttcgtgt tagcgccacc gattgggcag aaggggggtg gggtctggag 720
gataccctgg catttgcaga acgtctgaaa gctctgggtg tggatctgct ggatctgtct 780
tcaggtggtg ttgttccggg agttcgcgtt ccggttgcgc cgggttttca ggttccgttt 840
gcggatgcag ttcgtaaacg tgttggtatg cctacgggtg cagttggtct gctgacgacc 900
ccggagcagg cagaaacagt tctgcaggca ggtagtgcag atctggttct gctgggtcgt 960
gttctgctgc gtgatccgta ttttccgctg aaagcagcaa aagcactggg tgcagaagca 1020
ccggttccgc cgcagtatca gcgtgcattt taaaagctt 1059
<210> 2
<211> 23
<212> DNA
<213> forward primer of C25G (Artificial Sequence)
<400> 2
tgagcccgat gggtcagtac agc 23
<210> 3
<211> 22
<212> DNA
<213> reverse primer of C25G (Artificial Sequence)
<400> 3
gctgtactga cccatcgggc tc 22
<210> 4
<211> 21
<212> DNA
<213> i67t forward primer (Artificial Sequence)
<400> 4
cggaaggtcg caccagtccg t 21
<210> 5
<211> 22
<212> DNA
<213> i67t reverse primer (Artificial Sequence)
<400> 5
aacggactgg tgcgaccttc cg 22
Claims (4)
1. Using alkene reductase in micro-flow field to generate%S) -2-methylcyclohexanone, comprising the steps of:
s1: 2-methylcyclohexanone is used as a raw material, wild type alkene reductase immobilized on resin is immobilized in a first micro-channel, and a dehydrogenation resolution reaction catalyzed by the wild type alkene reductase is utilized to carry out a reaction in the first micro-channelR) Catalytic formation of 2-methyl-2-cyclohexene-1 from 2-methylcyclohexanoneA ketone;
s2: adsorbing the engineered alkene reductase and glucose dehydrogenase on the resin as biocatalyst to fix into the second micro-channel, injecting the reaction solution obtained in the step S1 into the second micro-channel, and continuously adding NADP + And glucose, reducing 2-methyl-2-cyclohexene-1-one to obtainS) -2-methylcyclohexanone;
wherein in step S1, the wild-type alkene reductase is derived fromThermus scotoductus SA-01TsER, tsER is prepared by the following steps: constructing a gene of the alkene reductase TsER with a nucleotide sequence shown as SEQ ID NO.1 on a pET-28a (+) vector, transferring the gene into escherichia coli BL 21 (DE 3), culturing and purifying to obtain the corresponding alkene reductase TsER;
in step S1, the steps of fixing to the first micro channel are: adding 12g of aqueous resin ES-108 into 30 mLTsER enzyme solution, fixing into a micro-channel reactor with an inner diameter of 1mm, wherein the reaction temperature is 25 ℃ and the reaction time is 30 min;
the method for adsorbing and fixing the alkene reductase on the resin comprises the following steps: dispersing the cultured and purified alkene reductase in phosphate buffer solution immersed with resin, slowly stirring at 20-22 ℃, taking out the resin, freezing at-80 ℃ for 12h, then dewatering in a freeze dryer, and filling the dried immobilized resin into the micro-channels; in step S2, the engineered alkene reductase is derived fromThermus scotoductus SA-01Mutant strain of TsER C25G/I66T.
2. The method according to claim 1, wherein in step S1, the reaction solvent is a mixture of PBS buffer and an organic solvent, and the organic solvent is any one or more of n-heptane, isooctane, methanol, ethanol, isopropanol, acetonitrile, and dimethyl sulfoxide; wherein the volume of the organic solvent is less than or equal to one percent of the total volume of the reaction solvent.
3. The method of claim 1, wherein in step S2, the glucose dehydrogenase is a commercial enzyme derived from bacillus.
4. The method according to claim 1, wherein the 2-methylcyclohexanone, tsER C25G/I66T, glucose dehydrogenase dosage ratio in steps S1 and S2 is 5-20 mM: 3-5 mg: 3-5 mg: 30-50U.
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| CN110951718A (en) * | 2019-12-02 | 2020-04-03 | 吉林凯莱英医药化学有限公司 | Co-immobilized enzyme, preparation method and application thereof |
| CN113444752A (en) * | 2021-07-15 | 2021-09-28 | 南京工业大学 | Method for continuously preparing 2-benzyl isoindolinone compound by adopting microchannel reactor |
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| CN110951718A (en) * | 2019-12-02 | 2020-04-03 | 吉林凯莱英医药化学有限公司 | Co-immobilized enzyme, preparation method and application thereof |
| CN113444752A (en) * | 2021-07-15 | 2021-09-28 | 南京工业大学 | Method for continuously preparing 2-benzyl isoindolinone compound by adopting microchannel reactor |
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| A robust and stereocomplementary panel of ene-reductase variants for gram-scale asymmetric hydrogenation;Nathalie N等;Molecular Catalysis;第502卷;111404 * |
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