CN115161297A - Tri-enzyme nano reactor, application thereof and synthesis of chiral tertiary alpha-phenyl cyclic alcohol - Google Patents
Tri-enzyme nano reactor, application thereof and synthesis of chiral tertiary alpha-phenyl cyclic alcohol Download PDFInfo
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- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 15
- 125000004122 cyclic group Chemical group 0.000 title claims description 16
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- 238000006243 chemical reaction Methods 0.000 claims description 51
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention provides a three-enzyme nano reactor, application thereof and synthesis of chiral tertiary alpha-phenyl cycloalcohol. The three-enzyme nano reactor can be used for divergent synthesis of tertiary alpha-phenyl cyclol, and has better stability and reusability.
Description
Technical Field
The invention relates to the technical field of enzyme reactors, in particular to a three-enzyme nano reactor, and also relates to application of the three-enzyme nano reactor and synthesis of chiral tertiary alpha-phenyl cycloalcohol by using the three-enzyme nano reactor.
Background
Different stereoisomers of chiral molecules often exhibit different or even opposite pharmacological activities, and this phenomenon is more pronounced and complicated, especially when the molecule contains multiple chiral centers. Thus, the synthesis of a complete chiral stereoisomer is of great interest for biological evaluation, but at the same time is a great challenge, especially for those chiral compounds having multiple consecutive stereocenters.
Among them, chiral tertiary alpha-phenyl cyclic alcohols having two adjacent stereocenters are an important class of synthetic molecules for use in pharmaceuticals, bioactive molecules, and natural products. In the last decades, the asymmetric hydrogenation reaction of 2-substituted cyclic ketone is realized in an alkaline environment by utilizing the dynamic kinetic resolution reaction catalyzed by chiral metal complexes such as ruthenium, iridium or copper, and the like, has higher atom economy, and is one of the most effective and most convenient methods in the preparation mode of chiral tertiary alpha-phenyl cyclic alcohol. However, due to the greater steric hindrance in the alpha-position, only cis-products are generally obtained.
In addition to the asymmetric hydrogenation of cyclic ketone catalyzed by transition metal, the asymmetric hydrogenation of 2-aryl cyclic ketene under acidic condition is carried out by using the dynamic kinetic resolution reaction catalyzed by metal Pd, which is also a currently developed way to realize the high-efficiency preparation of trans-2-phenyl cyclohexanol. However, the reaction requires the use of expensive chiral ligand and 2,2,2-trifluoroethanol, which is flammable and toxic, as a solvent, and also requires the reaction to be carried out in a high-pressure hydrogen atmosphere of 100pa, and thus has a great limitation.
Enzymes have the advantages of high efficiency, high selectivity and sustainability, and have received high attention from both academic and industrial circles. Nevertheless, the reactivity of enzymes is not as powerful as artificial catalysts, and many reactions catalyzed by artificial catalysts still do not find enzymes with the same properties. For example, although enzymatic DKR production of chiral acyclic alcohols with two adjacent stereocenters can be achieved by alcohol dehydrogenase (ADHa) and ketoreductase (KREDa), the readily available cyclic products of metal catalysts have not been reported to be catalyzed by enzymes, and with the development of enzyme application technologies, the construction of multi-enzyme cascade catalytic systems provides opportunities for solving the existing dilemma.
In particular, catalytic reactions in a multienzyme cascade combining different enantiospecific enzymes have been shown to be effective in the stereospecific synthesis of chiral cyclic alcohols containing three stereogenic centers. However, there are many limitations to constructing a multi-enzyme cascade catalytic system, such as poor stability and reusability of free enzymes, possible cross-reactions, mismatch of optimal reaction conditions of the involved enzymes, etc. All of them cause that the application of the current multi-enzyme cascade catalytic system is greatly restricted.
Disclosure of Invention
In view of the above, the present invention is directed to a three-enzyme nanoreactor, which can be applied to the synthesis of chiral tertiary α -phenyl cyclic alcohol.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
the three-enzyme nano reactor is provided with a core body and a shell layer coated outside the core body, wherein the core body is made of hydrophobic organic silicon dioxide, the shell layer is made of hydrophilic dopamine, alcohol dehydrogenase is loaded on the core body, old yellow enzyme is loaded on the shell layer, and glucose dehydrogenase is loaded on both the core body and the shell layer.
Further, the preparation method of the three-enzyme nano reactor comprises the following steps:
a1. synthesizing hydrophobic organic silicon dioxide as a nucleus body;
a2. loading an alcohol dehydrogenase and a portion of a glucose dehydrogenase onto the core;
a3. coating the surface of a core body loaded with the alcohol dehydrogenase and the glucose dehydrogenase with hydrophilic dopamine serving as a shell layer;
a4. and loading old yellow enzyme and the other part of the glucose dehydrogenase on the shell layer to obtain the three-enzyme nano reactor.
Further, in step a1, the step of synthesizing the hydrophobic organic silica as a nucleus body comprises:
a11, adding cetyl trimethyl ammonium bromide, n-butyl alcohol and cyclohexane into a urea solution, and performing ultrasonic dispersion to obtain a mixed solution;
a12, dropwise adding a mixed solution of tetraethoxysilane and 1,2-bis (triethoxysilyl) ethane into the mixed solution;
a13, reacting the mixed solution at 70 ℃;
a14, centrifuging the mixed solution after the reaction is finished, drying to obtain white powder, and removing the template by using a Soxhlet extractor;
and a15, removing the template, and then centrifugally drying to obtain the hydrophobic organic silicon dioxide serving as a nuclear body.
Further, in step a2, the alcohol dehydrogenase and a portion of the glucose dehydrogenase are loaded on the core, comprising the steps of:
a21, ultrasonically dispersing the hydrophobic organic silicon dioxide prepared in the step a1 into a buffer solution, and continuously stirring;
a22, adding alcohol dehydrogenase and glucose dehydrogenase freeze-dried powder, and reacting;
a23, after the reaction is finished, centrifuging the reaction solution, and drying the precipitate in vacuum to obtain a nucleus body loaded with the alcohol dehydrogenase and the glucose dehydrogenase.
Further, in the step a3, coating the hydrophilic dopamine serving as a shell layer on the surface of the core body loaded with the alcohol dehydrogenase and the glucose dehydrogenase comprises the following steps:
a31, ultrasonically dispersing the core body loaded with the alcohol dehydrogenase and the glucose dehydrogenase prepared in the step a2 into a buffer solution, and continuously stirring;
a32, adding a dopamine solution into the solution;
and a33, after the reaction is finished, centrifuging the reaction solution to obtain black precipitate, and drying the precipitate in vacuum to obtain a shell-coated nucleus body.
Further, in step a4, loading old yellow enzyme and another part of the glucose dehydrogenase on the shell layer comprises the following steps:
a41, ultrasonically dispersing the core body coated with the shell layer prepared in the step a3 into a buffer solution, and continuously stirring;
a42, adding old yellow enzyme and freeze-dried powder of glucose dehydrogenase for reaction;
and a43, after the reaction is finished, centrifuging the reaction solution, and drying the precipitate in vacuum to obtain the three-enzyme nano reactor.
Further, the old yellow enzyme adopts YqjM with (R) -selectivity or YqjM mutant with (S) -selectivity; the alcohol dehydrogenase employs ADH-A with (S) -selectivity, or DR-ADH with (R) -selectivity.
Compared with the prior art, the invention has the following advantages:
according to the three-enzyme nano reactor, hydrophobic organic silicon dioxide is used as a core body, hydrophilic dopamine is used as a shell layer, alcohol dehydrogenase is loaded on the core body, old yellow enzyme is loaded on the shell layer, glucose dehydrogenase is loaded on both the core body and the shell layer, and the old yellow enzyme and the alcohol dehydrogenase have different enantioselectivities.
Therefore, biomolecules are co-immobilized in the amphiphilic core-shell structure nano material, old yellow enzyme is utilized to catalyze asymmetric reduction of 2-phenylcyclohexenone (C = C), alcohol dehydrogenase is utilized to catalyze asymmetric reduction of tertiary alpha-phenylcyclic ketone (C = O), glucose dehydrogenase is utilized to catalyze regeneration of coenzyme, and through combination of old yellow enzyme and alcohol dehydrogenase with different enantioselectivities, efficient and high-selectivity preparation of chiral tertiary alpha-phenylcyclic alcohol containing two continuous chiral centers can be realized.
Meanwhile, the hydrophilic dopamine shell layer can ensure the dispersibility of the nano reactor in water, and the hydrophobic organic silicon dioxide core body can build a hydrophobic microenvironment with the capability of enriching substrates, so that the stability and the catalytic efficiency of the enzyme can be improved.
The three-enzyme nano reactor can enhance the stability and reusability of enzyme molecules, can also improve the cascade catalytic performance by matching the arrangement of different enzyme positions with the reaction sequence, has no toxicity and harm in the reaction process, and has the advantages of high efficiency, high selectivity and environmental friendliness.
The invention also provides the application of the three-enzyme nano reactor, which is used for carrying out the divergent synthesis of the chiral tertiary alpha-phenyl cycloalcohol under mild conditions.
In addition, the invention also provides a synthetic method of chiral tertiary alpha-phenyl cyclic alcohol, which comprises the following steps:
b1, sequentially adding 2-phenylcyclohexenone and NADH into a reaction vessel, preparing a three-enzyme nano reactor, glucose, a buffer solution and an ionic liquid, and reacting at 30 ℃;
b2. after the reaction is finished, dichloromethane is used for extraction, anhydrous magnesium sulfate is used for drying, and after suction filtration and desolventization, the chiral tertiary alpha-phenyl cyclic alcohol which is white solid is obtained through silica gel column chromatography.
Further, the silica gel column contains petroleum ether: ethyl acetate =10:1.
the invention utilizes the three-enzyme nano reactor to catalyze the asymmetric reduction reaction of the 2-phenylcyclohexenone by old yellow enzyme, catalyze the asymmetric reduction reaction of the tertiary alpha-phenylcyclohexenone to carry out cascade connection in a pot by alcohol dehydrogenase, and carry out coenzyme circulation in the reaction process by glucose dehydrogenase, thereby realizing the divergent synthesis of the chiral tertiary alpha-phenylcycloalkanol.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is SEM and TEM images of a three-enzyme nanoreactor prepared according to example one of the present invention;
FIG. 2 is a schematic diagram of a three-enzyme cascade catalytic reaction process of a three-enzyme nanoreactor according to a second embodiment of the present invention;
FIG. 3 is a substrate spectrum of a divergent synthesis of 2-phenylcyclohexanol in example 2 of the second embodiment of the present invention;
FIG. 4 is SEM and TEM images of a three-enzyme nanoreactor used repeatedly 5 times in example III of the present invention.
Detailed Description
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
In addition, unless otherwise specified in the examples of the present invention, terms and processes used in the examples of the present invention should be understood in accordance with common knowledge and conventional methods in the art.
The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.
Example one
The present example relates to a three-enzyme nanoreactor, which has a core and a shell coated outside the core, wherein the core is hydrophobic organic silica (DON), the shell is hydrophilic dopamine (PDA), alcohol Dehydrogenase (ADHs) is loaded on the core, yellow-light enzyme (OYEs) is loaded on the shell, and Glucose Dehydrogenase (GDH) is also loaded on both the core and the shell.
Specifically, for old yellow enzyme and alcohol dehydrogenase having different enantioselectivities, when embodied, for example, old yellow enzyme may employ YqjM having (R) -selectivity, or YqjM mutant having (S) -selectivity. Alcohol dehydrogenases may be used, for example, as ADH-A which is (S) -selective or as DR-ADH which is (R) -selective.
Incidentally, yqjM as described above may be derived from Bacillus subtilis, for example, and A60G/I69G may be used as the YqjM mutant. While the DR-ADH may, for example, be derived from deinococcus radiodurans, and ADH-A may, for example, be derived from Rhodococcus ruber.
In addition, when the three-enzyme nano-reactor of the embodiment is prepared, the preparation method related to the three-enzyme nano-reactor comprises the following steps:
step a1. synthesizes hydrophobic organosilica as a nucleus and this step a1 also specifically includes the steps of:
a11, adding cetyl trimethyl ammonium bromide, n-butyl alcohol and cyclohexane into a urea solution, and performing ultrasonic dispersion to obtain a mixed solution;
a12, dropwise adding a mixed solution of tetraethoxysilane and 1,2-bis (triethoxysilyl) ethane into the mixed solution;
a13, reacting the mixed solution at 70 ℃, wherein the reaction time can be 24 hours generally;
a14, centrifuging the mixed solution after the reaction is finished, drying to obtain white powder, and removing the template by using a Soxhlet extractor;
a15, removing the template, and then centrifugally drying to obtain hydrophobic organic silica (DON) as a nucleus body.
Step a2. Loading an alcohol dehydrogenase and a portion of a glucose dehydrogenase onto the core, and this step a2 also specifically includes the steps of:
a21, ultrasonically dispersing the hydrophobic organic silicon dioxide prepared in the step a1 into a buffer solution, and continuously stirring;
a22, adding alcohol dehydrogenase and glucose dehydrogenase freeze-dried powder, and reacting;
a23, centrifuging the reaction solution after the reaction is finished, and vacuum-drying the precipitate to obtain nucleus bodies (DON @ ADHs) loaded with the alcohol dehydrogenase and the glucose dehydrogenase.
A3. coating the surface of the core body loaded with the alcohol dehydrogenase and the glucose dehydrogenase with hydrophilic dopamine as a shell layer, and the step a3 specifically includes the following steps:
a31, ultrasonically dispersing the core body loaded with the alcohol dehydrogenase and the glucose dehydrogenase prepared in the step a2 into a buffer solution, and continuously stirring;
a32, adding a dopamine solution into the solution;
a33, centrifuging the reaction solution after the reaction is finished to obtain black precipitate, and vacuum drying the precipitate to obtain core body coated with shell layer (DON @ ADHs @ PDA).
A4. loading old yellow enzyme and another part of the glucose dehydrogenase on the shell layer to obtain a three-enzyme nano reactor, wherein the step a4 also specifically comprises the following steps:
a41, ultrasonically dispersing the core body coated with the shell layer prepared in the step a3 into a buffer solution, and continuously stirring;
a42, adding old yellow enzyme and freeze-dried powder of glucose dehydrogenase for reaction;
a43, centrifuging the reaction solution after the reaction is finished, and vacuum-drying the precipitate to obtain the Trienzyme nanoreactor (DON @ ADHs @ PDA @ OYEs).
Based on the above preparation method, yqjM is used as Old Yellow Enzyme (OYEs), ADH-A is used as Alcohol Dehydrogenase (ADHs), and a specific preparation example of the three-enzyme nanoreactor is further illustrated in this example.
In this preparation example, the specific procedure is as follows:
first, 20mg DON was dispersed in 4mL KPi buffer (100mM, pH 7) by sonication, 2mL protein solution containing 2mg ADH-A and 26.4. Mu.g GDH was added, stirred at room temperature for 6h, and centrifuged to obtain DON @ ADH-A after the reaction.
Next, 10mg of the thus-prepared DON @ ADH-A was dispersed in 10mL of KPi buffer (100mM, pH 7), 2mg/mL of PDA solution was added thereto, and the mixture was reacted at room temperature for 6 hours, followed by completion of the reaction and centrifugation to obtain DON @ ADH-A @ PDA.
Finally, 20mg of DON @ ADH-A @ PDA was dispersed in 15mL of KPi buffer (100mM, pH 7) containing 1.2mg of YqjM and 26.4. Mu.g of GDH, stirred at room temperature for 1 hour, and after completion of the reaction, centrifuged to obtain DON @ ADH-A @ PDA @ YqjM.
SEM and TEM images of the fabricated three-enzyme nanoreactor of the preparation example of this example are shown in fig. 1, and the nano-morphology of the fabricated three-enzyme nanoreactor can be well seen from fig. 1.
Example two
This example relates first to the application of the above three-enzyme nanoreactor, which is specifically used for the divergent synthesis of chiral tertiary α -phenyl cycloalkanol under mild conditions, where the mild conditions are usually pH of the reaction system of 7-9 and reaction temperature of 20-40 ℃.
In addition, based on the application of the three-enzyme nanoreactor, the embodiment also relates to a method for synthesizing chiral tertiary alpha-phenyl cyclic alcohol, which comprises the following steps:
b1, sequentially adding 2-phenylcyclohexenone and NADH, the three-enzyme nano-reactor prepared in the first embodiment, glucose, a buffer solution and an ionic liquid into a reaction vessel, and reacting at 30 ℃ for 16-24 hours;
b2. after the reaction is finished, dichloromethane is used for extraction, anhydrous magnesium sulfate is used for drying, and after suction filtration and desolventization, the chiral tertiary alpha-phenyl cyclic alcohol which is white solid is obtained through silica gel column chromatography.
In this embodiment, when the three-enzyme nano-reactor is used for the divergent synthesis of chiral tertiary alpha-phenyl cyclic alcohol, the three-enzyme cascade catalytic reaction process of the three-enzyme nano-reactor can be as shown in fig. 2.
The ionic liquid may be [ BMIm ]][NTF 2 ]The ratio between petroleum ether and ethyl acetate in the silica gel column is petroleum ether: ethyl acetate =10:1.
in addition, in the case of carrying out the asymmetric reduction reaction catalyzed by the three-enzyme cascade, the concentration of the substrate may be 25 to 35mM, preferably 30mM, the amount of the three-enzyme nanoreactor may be 80 to 120mg, preferably 100mg, the pH of the reaction system is preferably pH 8, the reaction temperature is preferably 30 ℃, and the optimum reaction time is 20 hours.
The enantioselective divergent synthesis of chiral tertiary alpha-phenyl cyclanols in a three-enzyme nanoreactor is further illustrated by several specific preparation examples in connection with FIG. 3. In each of the following preparation examples, the preparation method of the three-enzyme nanoreactor was carried out by the preparation method of example one.
Preparation of example 1
To the reactor were added 0.3mmol of 2-phenylcyclohexenone, 1.5. Mu. Mol of NADH,100mg of DON @ ADH-A @ PDA @ YqjM and an excess of glucose, using 9.5mL of KPi buffer solution (50mM, pH = 8) and 0.5mL of BMIm [ ([ lambda ] M ], [ solution ] K][NTf 2 ]Dissolving and reacting for 20 hours at 30 ℃. After the reaction is finished, dichloromethane is used for extraction, anhydrous magnesium sulfate is used for drying, and after suction filtration and desolventization, the chiral tertiary alpha-phenyl cyclic alcohol in white solid is obtained by silica gel column chromatography.
Preparation of example 2
To the reactor were added 0.3mmol of 2-phenylcyclohexenone, 1.5. Mu. Mol of NADH,100mg of DON @ DR-ADH @ PDA @ YqjM and an excess of glucose, using 9.5mL of KPi buffer solution (50mM, pH = 8) and 0.5mL of BMIm [ ([ lambda ] M ], [ solution ] K][NTf 2 ]Dissolving and reacting for 20 hours at 30 ℃. After the reaction is finished, dichloromethane is used for extraction, anhydrous magnesium sulfate is used for drying, and after suction filtration and desolventization, silica gel column chromatography is used for obtaining the chiral tertiary alpha-phenyl cycloalcohol in a white solid state.
Preparation of example 3
To the reactor were added 0.3mmol of 2-phenylcyclohexenone, 1.5. Mu. Mol of NADH,100mg of DON @ ADH-A @ PDA @ A60G/I69G and an excess of glucose, using 9.5mL of KPi buffer solution (50mM, pH = 8) and 0.5mL of BMIm [, ]][NTf 2 ]Dissolving and reacting for 20 hours at 30 ℃. After the reaction is finished, dichloromethane is used for extraction, anhydrous magnesium sulfate is used for drying, and after suction filtration and desolventization, the chiral tertiary alpha-phenyl cyclic alcohol in white solid is obtained by silica gel column chromatography.
Preparation of example 4
To the reactor were added 0.3mmol of 2-phenylcyclohexenone, 1.5. Mu. Mol of NADH,100mg of DON @ DR-ADH @ PDA @ A60G/I69G and an excess of glucose, using 9.5mL of KPi buffer solution (50mM, pH = 8) and 0.5mL of BMIm [, ]][NTf 2 ]Dissolving and reacting for 20 hours at 30 ℃. After the reaction is finished, dichloromethane is used for extraction, anhydrous magnesium sulfate is used for drying, and after suction filtration and desolventization, chiral tertiary alpha-phenyl cycloalcohol in a white solid state is obtained through silica gel column chromatography.
The yields of the above preparation examples were determined by nuclear magnetic resonance and the enantioselectivities of the compounds were determined by liquid chromatography, as specified in table 1 below:
TABLE 1 yield and enantioselectivity of the Compounds
It can be seen from table 1 that the tri-enzyme nanoreactor prepared in the first embodiment can realize efficient and highly selective preparation of chiral tertiary alpha-phenyl cyclic alcohol, and has better preparation effects when YqjM is used as Old Yellow Enzyme (OYEs), ADH-A is used as Alcohol Dehydrogenase (ADHs), yqjM is used as Old Yellow Enzyme (OYEs), and DR-ADH is used as Alcohol Dehydrogenase (ADHs).
EXAMPLE III
This example relates to the stability and reusability tests of the three-enzyme nanoreactor prepared in example one, as follows.
In this example to investigate the thermal stability of the three-enzyme nanoreactors, the three-enzyme nanoreactors and the free enzymes (YqjM, ADH-a and GDH having the same enzyme activity but not subjected to the immobilization treatment) were incubated in KPi (50mm, ph = 7.5) buffer at 40 and 50 ℃ for 90min, and the three-enzyme nanoreactors and the free enzyme yields were determined.
The yields of the three-enzyme nanoreactors were determined to be 60% (40 ℃) and 44% (50 ℃) and the yields of the free enzyme were 18% (40 ℃) and 0% (50 ℃).
Detection 2
In this example to investigate the pH stability of the three-enzyme nanoreactors, the three-enzyme nanoreactors and the same free enzyme were incubated in Tris-HCl buffer (50mm, pH = 4.0) or acetate buffer (50mm, pH = 10.0) for 90min at room temperature, and the yields of the three-enzyme nanoreactors and the free enzyme were determined.
The yields of the three-enzyme nanoreactors were determined to be 42% (pH = 10.0) and 37% (pH = 4.0) and the yield of the free enzyme was 18% (pH = 10.0) and 0% (pH = 4.0).
In order to investigate the organic solvent resistance of the three-enzyme nanoreactor, the three-enzyme nanoreactor and the same free enzyme were incubated in 10mL of acetone solution for 3h, and the yields of the three-enzyme nanoreactor and the free enzyme were determined.
The measured yield of the three-enzyme nanoreactor was 42% and the yield of the free enzyme was 12%.
Detection 4
In order to investigate the storage stability of the three-enzyme nanoreactor, the three-enzyme nanoreactor and the same free enzyme were left at room temperature for 12d, and the yields of the three-enzyme nanoreactor and the free enzyme were measured.
The yield of the three-enzyme nanoreactor was determined to be 45% and the yield of the free enzyme was determined to be 5%.
Detection 5
In this example, in order to study the reusability of the three-enzyme nano-reactor, the three-enzyme nano-reactor was centrifugally separated after each reaction, and the separated three-enzyme nano-reactor was reused in a new round of catalytic reaction.
SEM and TEM images of the three-enzyme nanoreactor after 5-time reuse are shown in fig. 4, which can be seen to still maintain a good morphology.
Through the above tests, it can be seen that the three-enzyme nanoreactor of the present invention also has good stability and reusability.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.
Claims (10)
1. A three-enzyme nanoreactor, characterized in that: the three-enzyme nano reactor is provided with a core body and a shell layer coated outside the core body, wherein the core body is made of hydrophobic organic silicon dioxide, the shell layer is made of hydrophilic dopamine, alcohol dehydrogenase is loaded on the core body, old yellow enzyme is loaded on the shell layer, and glucose dehydrogenase is loaded on both the core body and the shell layer.
2. The three-enzyme nanoreactor according to claim 1, wherein the preparation method of the three-enzyme nanoreactor comprises the following steps:
a1. synthesizing hydrophobic organic silicon dioxide as a nucleus body;
a2. loading an alcohol dehydrogenase and a portion of a glucose dehydrogenase onto the core;
a3. coating the surface of a core body loaded with the alcohol dehydrogenase and the glucose dehydrogenase with hydrophilic dopamine serving as a shell layer;
a4. and loading old yellow enzyme and the other part of the glucose dehydrogenase on the shell layer to obtain the three-enzyme nano reactor.
3. The triase nanoreactor as claimed in claim 2, wherein the step of synthesizing the hydrophobic organo-silica as a core in step a1 comprises:
a11, adding cetyl trimethyl ammonium bromide, n-butyl alcohol and cyclohexane into a urea solution, and performing ultrasonic dispersion to obtain a mixed solution;
a12, dropwise adding a mixed solution of tetraethoxysilane and 1,2-bis (triethoxysilyl) ethane into the mixed solution;
a13, reacting the mixed solution at 70 ℃;
a14, centrifuging the mixed solution after the reaction is finished, drying to obtain white powder, and removing the template by using a Soxhlet extractor;
and a15, removing the template, and then centrifugally drying to obtain the hydrophobic organic silicon dioxide serving as a nucleus body.
4. The three-enzyme nanoreactor according to claim 2, wherein in step a2, alcohol dehydrogenase and a portion of glucose dehydrogenase are loaded on the core body, comprising the steps of:
a21, ultrasonically dispersing the hydrophobic organic silicon dioxide prepared in the step a1 into a buffer solution, and continuously stirring;
a22, adding alcohol dehydrogenase and glucose dehydrogenase freeze-dried powder, and reacting;
a23, after the reaction is finished, centrifuging the reaction solution, and drying the precipitate in vacuum to obtain a nucleus body loaded with the alcohol dehydrogenase and the glucose dehydrogenase.
5. The three-enzyme nanoreactor according to claim 2, wherein coating the surface of the core body loaded with the alcohol dehydrogenase and the glucose dehydrogenase with hydrophilic dopamine as a shell layer in step a3 comprises the following steps:
a31, ultrasonically dispersing the nucleosomes which are prepared in the step a2 and are loaded with the alcohol dehydrogenase and the glucose dehydrogenase into a buffer solution, and continuously stirring;
a32, adding a dopamine solution into the solution;
and a33, after the reaction is finished, centrifuging the reaction solution to obtain black precipitate, and drying the precipitate in vacuum to obtain a shell-coated nucleus body.
6. The three-enzyme nanoreactor according to claim 2, wherein the step a4 of loading old yellow enzyme and another part of the glucose dehydrogenase onto the shell layer comprises the following steps:
a41, ultrasonically dispersing the core body coated with the shell layer prepared in the step a3 into a buffer solution, and continuously stirring;
a42, adding old yellow enzyme and freeze-dried powder of glucose dehydrogenase for reaction;
and a43, after the reaction is finished, centrifuging the reaction solution, and drying the precipitate in vacuum to obtain the three-enzyme nano reactor.
7. The triase nanoreactor according to any one of claims 1 to 6, characterized in that: the old yellow enzyme adopts YqjM with (R) -selectivity or YqjM mutant with (S) -selectivity; the alcohol dehydrogenase employs ADH-A with (S) -selectivity, or DR-ADH with (R) -selectivity.
8. Use of a three-enzyme nanoreactor according to any of claims 1 to 7, characterized in that: the three-enzyme nano reactor is used for carrying out divergent synthesis of chiral tertiary alpha-phenyl cyclol under mild conditions.
9. A synthetic method of chiral tertiary alpha-phenyl cyclic alcohol is characterized by comprising the following steps:
b1, sequentially adding 2-phenylcyclohexenone, NADH, the three-enzyme nano-reactor of any one of claims 1 to 7, glucose, a buffer solution and an ionic liquid into a reaction vessel, and reacting at 30 ℃;
b2. after the reaction is finished, dichloromethane is used for extraction, anhydrous magnesium sulfate is used for drying, and after suction filtration and desolventization, the chiral tertiary alpha-phenyl cyclic alcohol which is white solid is obtained through silica gel column chromatography.
10. The method of synthesizing a chiral tertiary alpha-phenyl cyclic alcohol of claim 9, wherein: petroleum ether in the silica gel column: ethyl acetate =10:1.
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