CN108998430B - Interface self-assembly carbonyl reductase and application thereof in synthesis of (R) -3-hydroxy-3-ethyl phenylpropionate - Google Patents

Interface self-assembly carbonyl reductase and application thereof in synthesis of (R) -3-hydroxy-3-ethyl phenylpropionate Download PDF

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CN108998430B
CN108998430B CN201810696272.8A CN201810696272A CN108998430B CN 108998430 B CN108998430 B CN 108998430B CN 201810696272 A CN201810696272 A CN 201810696272A CN 108998430 B CN108998430 B CN 108998430B
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ethyl
assembly
carbonyl reductase
carbonyl
phenylpropionate
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CN108998430A (en
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欧志敏
王莹
泮佳莹
杜理华
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Zhejiang University of Technology ZJUT
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01184—Carbonyl reductase (NADPH) (1.1.1.184)

Abstract

The invention discloses an interface self-assembly carbonyl reductase and application thereof in synthesis of (R) -3-hydroxy-3-ethyl phenylpropionate, wherein carbonyl reductase is added into Tirs-HCl buffer solution, then toluene solution of polystyrene is added, oscillation reaction is carried out for 1-4 hours in a shaking table under the conditions of darkness, 40-100R/min and 20-40 ℃, centrifugation is carried out to obtain a reaction system with an organic phase as an upper layer, an interface self-assembly carbonyl reductase as a middle layer and a water phase as a lower layer, and the middle layer is taken out and washed by toluene and Tris-HCl buffer solution to obtain the interface self-assembly carbonyl reductase; the interface self-assembly carbonyl reductase constructed by the invention has a better catalytic function on an organic phase interface and a water two-phase interface, and can convert 3-carbonyl-3-ethyl phenylpropionate into (R) -3-hydroxy-3-ethyl phenylpropionate, wherein the enantiomer excess value of the (R) -3-hydroxy-3-ethyl phenylpropionate is more than 99%.

Description

Interface self-assembly carbonyl reductase and application thereof in synthesis of (R) -3-hydroxy-3-ethyl phenylpropionate
(I) technical field
The invention relates to preparation of a microbial-derived interface self-assembly carbonyl reductase and application of the microbial-derived interface self-assembly carbonyl reductase in preparation of a non-central excitation drug R-tomoxetine key chiral intermediate (R) -3-hydroxy-3-phenylpropionic acid ethyl ester for treating ADHD.
(II) background of the invention
The interfacial self-assembly enzyme is a polymer-enzyme complex, combines the enzyme with macromolecular polymer to enable the modified enzyme to have an amphiphilic structure, the structure is similar to a protein-based surfactant, the interfacial self-assembly enzyme not only contains a hydrophilic protein head but also contains a hydrophobic polymer tail, when the interfacial self-assembly enzyme is put into an oil-water system, the polymer part in the polymer-enzyme extends to an oil phase, the enzyme part is embedded into a water phase, and the enzyme is fixed on an oil-water interface to catalyze the chemical reaction in the oil-water two phases. The carbonyl reductase and the polymer are combined at a proper oil-water interface to form the interface self-assembly carbonyl reductase, and the application of the carbonyl reductase in the preparation of the chiral drug intermediate by the asymmetric reduction of the carbonyl compound catalyzed by the oil-water two-phase system has positive significance for improving the biological catalysis efficiency of the oil-water two-phase system.
The selective carbonyl reductase can asymmetrically reduce carbonyl compounds to prepare chiral compounds. Carbonyl reductase exists widely in microbe, and chiral alcohol may be prepared through asymmetric reduction of latent chiral carbonyl compound with microbe containing carbonyl reductase. Microbial cells are generally capable of exerting higher activity in aqueous solution, while organic substrates are less water soluble, so microbial cells catalyze less water soluble substrates in aqueous phase with less efficient conversion. The conversion efficiency is hopeful to be improved by adopting a water/organic phase two-phase system to convert a substrate with lower water solubility. The carbonyl reductase existing in microbial cells is separated, extracted and prepared into the interface self-assembly enzyme, which is favorable for realizing the reutilization of the enzyme, and the quick conversion at an oil-water interface is realized, thereby being favorable for improving the biotransformation efficiency of water-insoluble substrates.
The tomoxetine has a chiral carbon atom in its molecular structure, and has two enantiomers, the R-form and the S-form. Wherein, the (R) -tomoxetine has 9 times of the drug effect of the (S) -tomoxetine, and the medicines sold in the market are all (R) -tomoxetine. Ethyl (R) -3-hydroxy-3-phenylpropionate ((R) -ethyl-3-hydroxy-3-phenylpropanoate) is a key chiral intermediate for the synthesis of R-tomoxetine. Of the formula C11H14O3Molecular weight 194.23, CAS number 72656-47-4, boiling point 315.3. + -. 20 ℃ and density 1.119. Ethyl (R) -3-hydroxy-3-phenylpropionate can be prepared by asymmetrically reducing ethyl 3-carbonyl-3-phenylpropionate.
(R) -tomoxetine, english name: atomoxetine, CAS accession number: 83015-26-3, molecular formula C17H21NO, molecular weight 255.355. (R) -tomoxetine is a nervous system drug and is used clinically primarily for the treatment of attention deficit disorder (ADHD). Attention deficit disorder (ADHD), also known as attention deficit hyperactivity disorder, is currently believed to be associated with a reduced turnover of the catecholamines neurotransmitters dopamine and norepinephrine. Tomoxetine optionsInhibit the presynaptic movement of noradrenaline, enhance the noradrenaline function, thereby improving the symptoms of ADHD and indirectly promoting the completion of cognition and concentration. Have little affinity for other neurotransmitter receptors, such as cholinergic, histamine, dopamine, 5-hydroxytryptamine, and alpha-adrenergic receptors.
The (R) -3-hydroxy-3-phenylpropionic acid ethyl ester can be obtained by chemically and asymmetrically reducing 3-carbonyl-3-phenylpropionic acid ethyl ester, the method needs to prepare a chemical catalyst, and the catalyst with asymmetric reduction capability often contains noble metal and is expensive. The ethyl 3-hydroxy-3-phenylpropionate can also be prepared by asymmetric resolution of ethyl 3-hydroxy-3-phenylpropionate by a chemical method or an enzymatic method, and the highest conversion rate of the method can only reach 50% theoretically. The (R) -3-hydroxy-3-ethyl phenylpropionate can also be obtained by asymmetrically reducing 3-carbonyl-3-ethyl phenylpropionate by adopting a whole microbial cell, the highest conversion rate can reach 100% theoretically, but the substrate treatment amount is lower, and the production efficiency is not high. In order to further improve the production efficiency, the invention separates and extracts carbonyl reductase in microbial cells, carries out interface self-assembly on the carbonyl reductase to prepare the interface self-assembly carbonyl reductase, converts the 3-carbonyl-3-ethyl phenylpropionate in a water/organic phase two-phase system to prepare the (R) -3-hydroxy-3-ethyl phenylpropionate, and improves the treatment capacity and the production efficiency of a substrate.
The 3-carbonyl-3-ethyl phenylpropionate is used as a substrate, the interfacial self-assembly carbonyl reductase is used as a catalyst, and the catalytic reaction is carried out in an organic phase/water two-phase system to convert the 3-carbonyl-3-ethyl phenylpropionate into the (R) -3-hydroxy-3-ethyl phenylpropionate. The hydrophilic part of the interfacial self-assembly enzyme is distributed in the water phase, the hydrophobic part is distributed in the organic phase, and the interfacial self-assembly enzyme is anchored at the organic phase/water interface. The substrate 3-carbonyl-3-ethyl phenylpropionate and the product (R) -3-hydroxy-3-ethyl phenylpropionate are distributed in an organic phase, and the substrate is contacted with an interfacial self-assembly enzyme at an organic phase/water interface to realize a catalytic reaction. The reduction reaction needs coenzyme NADH to provide hydrogen for the reaction process, therefore, NADH is added in the water phase, and simultaneously alcohol dehydrogenase and alcohol are added to realize the regeneration of NADH, so as to provide a continuous hydrogen donor for the reduction reaction, and the reaction mechanism is shown in figure 1.
Disclosure of the invention
The invention aims to provide an interface self-assembly carbonyl reductase, and the interface self-assembly carbonyl reductase is adopted to asymmetrically reduce 3-carbonyl-3-ethyl phenylpropionate in an organic phase/water two-phase system to prepare (R) -3-hydroxy-3-ethyl phenylpropionate, so that a new way is provided for biosynthesis of (R) -tomoxetine, and the method is favorable for separation and extraction of a substrate and a product in the organic phase/water two-phase system; the reaction condition is mild; the interface self-assembly enzyme can be recycled, and the biotransformation efficiency of the substrate is improved.
The technical scheme adopted by the invention is as follows:
in a first aspect, the present invention provides an interfacial self-assembly carbonyl reductase, which is prepared as follows: adding carbonyl reductase into Tirs-HCl buffer solution with the pH value of 6-9 (preferably 7.9) and the pH value of 0.05M, adding toluene solution of polystyrene, oscillating and reacting for 1-4 hours (preferably 2 hours) in a shaking table under the conditions of darkness, 40-100r/min (preferably 80r/min) and 20-40 ℃ (preferably 30 ℃), centrifuging (preferably centrifuging reaction solution 10000r/min for 10 minutes) to obtain a reaction system with an organic phase at the upper layer, an interface self-assembly carbonyl reductase at the middle layer and an aqueous phase at the lower layer, and washing the middle layer (namely the interface self-assembly carbonyl reductase distributed on an oil-water interface) with toluene, Tris-HCl buffer solution with the pH value of 6-9 (preferably 7.9) and the Tris-HCl buffer solution with the pH value of 0.05M for 3-5 times (preferably 3 times) respectively to obtain the interface self-assembly carbonyl reductase; the addition amount of the carbonyl reductase is 20-200U/ml (preferably 144U/ml) in terms of the volume of the buffer solution, the concentration of the toluene solution of the polystyrene is 1.0-4.0 g/L (preferably 3.75g/L), and the volume ratio of the toluene solution of the polystyrene to the buffer solution is 1-3:1, preferably 1.6: 1.
Further, the carbonyl reductase is from Candida tropicalis (CGMCC No. 15016), is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.15016, has the preservation date of 2017, 12 months and 4 days, and has the preservation address: western road No.1, north chen west road, north kyo, chaoyang, institute of microbiology, china academy of sciences, zip code 100101.
Further, the carbonyl reductase is prepared as follows: (1) dispersing wet thalli obtained by fermentation culture of Candida tropicalis CGMCC No.15016 in 50ml of Tris-HCl (0.05M, pH 7.9.9) buffer solution, wherein the concentration of the thalli is 2g/L (dry weight of the thalli/volume of the buffer solution), and placing the thalli in an ice water bath (0-4 ℃) for ultrasonic cell disruption; immersing an ultrasonic probe into the liquid level of the bacterial suspension for 1-1.5 cm, the power is 405W, the work time is 2s, the pause time is 4s, the cell is broken by ultrasonic for 20min, and the broken sample is centrifuged for 10min at 4 ℃ in a centrifuge of 8000r/min to obtain supernatant, namely crude enzyme liquid I;
(2) carrying out ammonium sulfate fractional precipitation on the crude enzyme solution I: placing the enzyme solution I in an ice water bath for precooling to 0-4 ℃, gently stirring while slowly adding ammonium sulfate which is pre-ground into powder until the saturation of the ammonium sulfate is 40%, continuously stirring in the ice bath for 1h after the ammonium sulfate is completely dissolved, centrifuging for 10min at 10000 Xg and 4 ℃, and redissolving the obtained precipitate in Tris-HCl buffer solution with the volume of 1-2 times of that of the precipitate to obtain a crude enzyme solution II;
(3) and (3) carrying out ultrafiltration concentration on the crude enzyme solution II: and (3) placing the crude enzyme solution II in a 10kD Millipore ultrafiltration centrifugal tube, centrifuging for 20min at 3500 Xg and 4 ℃, wherein the concentrated solution in the sleeve is carbonyl reductase enzyme solution, the enzyme activity is preferably 180U/mg, and the solution is stored at 4 ℃ for later use.
Further, the wet cells were prepared as follows: 1) inoculating Candida tropicalis (CGMCC No. 15016) into slant culture medium, and culturing at 30 deg.C for 3-5 days to obtain slant thallus; the slant culture medium comprises: 20g/L glucose, 1g/L ammonium sulfate, 0.5g/L potassium dihydrogen phosphate, 1.5g/L dipotassium hydrogen phosphate, 1g/L sodium chloride, 0.1g/L magnesium sulfate, 20g/L agar and deionized water as a solvent, wherein the pH value is natural; 2) inoculating the thalli on the inclined plane into a 100mL triangular flask containing 25mL seed culture medium, and culturing at 30 ℃ at 120r/min for 24h to obtain a seed solution; the seed culture medium comprises the following components: 46g/L glucose, 25g/L yeast juice, 4g/L potassium dihydrogen phosphate, 4g/L dipotassium hydrogen phosphate, 0.1g/L sodium chloride, deionized water as a solvent and natural pH value; 3) inoculating the seed solution cultured for 24h into a 1000mL triangular flask containing 250mL fermentation medium with the inoculation amount of 10% of volume concentration, culturing for 24h at 30 ℃ at 120r/min, centrifuging the obtained fermentation liquid in a centrifugal machine at 8000r/min for 20min, and discarding the supernatant to obtain wet thalli; the fermentation medium comprises the following components: 46g/L glucose, 25g/L yeast juice, 4g/L potassium dihydrogen phosphate, 4g/L dipotassium hydrogen phosphate, 0.1g/L sodium chloride and deionized water as a solvent, and the pH value is natural.
In a second aspect, the invention also provides an application of the interfacial self-assembly carbonyl reductase in preparation of (R) -3-hydroxy-3-ethyl phenylpropionate by asymmetrically reducing 3-carbonyl-3-ethyl phenylpropionate.
Further, the application method comprises the following steps: taking an interface self-assembly carbonyl reductase as a catalyst, taking 3-carbonyl-3-phenylpropionic acid ethyl ester as a substrate, taking NADH as a coenzyme, taking ethanol as an auxiliary substrate, adding ethanol dehydrogenase for in-situ regeneration of coenzyme NADH, taking a Tris-HCl/toluene two-phase system formed by Tris-HCl buffer solution with the pH value of 3.0-8.0 (preferably 7.9) and toluene as a reaction medium, reacting in a shaking table with the temperature of 20-40 ℃ (preferably 35 ℃) and the temperature of 80-120R/min (preferably 120R/min), and after the reaction is completed, separating and purifying the reaction liquid to obtain the (R) -3-hydroxy-3-phenylpropionic acid ethyl ester; the dosage of the catalyst is 5-200U/mL (preferably 31-36U/mL) in terms of the volume of the buffer solution, the dosage of the substrate is 0.010-0.070 mol/L (preferably 0.037-0.062mol/L, most preferably 0.056mol/L) in terms of the volume of the buffer solution, and the dosage of the ethanol is 1-12% (preferably 6-10%, most preferably 10%) in terms of the volume of the buffer solution respectively; the amount of NADH is 0.02-0.14 mol/L (preferably 0.04-0.14mol/L, most preferably 0.10mol/L) based on the volume of the buffer, the amount of the alcohol dehydrogenase (preferably 300U/mg) is 0.01-0.4 g/L or 3000-120000U/L (preferably 6000U/L) based on the volume of the buffer, and the volume ratio of the toluene to the buffer is 2.5-20:10 (preferably 10-15:10, most preferably 12.5: 10).
Further, after the reaction is finished, centrifuging 10000R/min of the reaction solution for 10min to obtain an organic phase, an intermediate layer and a water phase containing the ethyl 3-carbonyl-3-phenylpropionate and the ethyl (R) -3-hydroxy-3-phenylpropionate, wherein the ethyl (R) -3-hydroxy-3-phenylpropionate exists in the organic phase, extracting the water phase for three times by using equal amount of ethyl acetate, combining the extract liquor, performing rotary evaporation to remove most of the ethyl acetate, combining the extract liquor with the organic phase, and further separating the ethyl 3-carbonyl-3-phenylpropionate and the ethyl (R) -3-hydroxy-3-phenylpropionate by silica gel column chromatography to obtain the ethyl (R) -3-hydroxy-3-phenylpropionate; the intermediate layer recovers the interface self-assembly carbonyl reductase for recycling.
Compared with the prior art, the invention has the following beneficial effects:
the interface self-assembly carbonyl reductase constructed by the invention has a better catalytic function on an organic phase interface and a water two-phase interface, and can convert 3-carbonyl-3-ethyl phenylpropionate into (R) -3-hydroxy-3-ethyl phenylpropionate, wherein the enantiomer excess value of the (R) -3-hydroxy-3-ethyl phenylpropionate is more than 99%. In a water phase, coenzyme NADH can smoothly realize in-situ regeneration, which is beneficial to improving the conversion rate of a substrate, and the substrate conversion rate can be improved to 24 percent of the original substrate conversion rate by adding the coenzyme NADH under the same reaction condition. The substrate and the product are dissolved in the organic phase, and the catalytic reaction is carried out at the interface of the organic phase and water, which is beneficial to the subsequent separation and extraction of the product. Compared with the traditional free enzyme catalysis in a two-phase system, the method can reduce the enzyme activity loss and improve the enzyme catalysis efficiency. The interfacial self-assembly enzyme can be repeatedly utilized, which is an advantage incomparable with free enzyme. The interface self-assembly carbonyl reductase plays a catalytic function at the interface of an organic phase and water, and a new way is provided for the synthesis of (R) -3-hydroxy-3-ethyl phenylpropionate.
(IV) description of the drawings
FIG. 1 is a schematic diagram of the reaction mechanism of the present invention.
FIG. 2 photomicrograph of strain 412.
FIG. 3 phylogenetic tree between the test strain and other genera of yeast.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1 screening and identification of Candida tropicalis (Candida tropicalis) CGMCC No.15016
1. And (3) strain screening:
(1) culture medium
Enrichment plate medium (g/L): glucose 20, (NH)4)2SO4 1，KH2PO4 0.5，K2HPO4 1.5，NaCl1，MgSO40.1, agar 20 and deionized water as solvent, and the pH value is natural.
Slant medium (g/L): glucose 20, (NH)4)2SO4 1，KH2PO4 0.5，K2HPO4 1.5，NaCl 1，MgSO40.1, agar 20 and deionized water as solvent, and the pH value is natural.
Seed medium (g/L): glucose 20, yeast powder 3, (NH)4)2SO4 1，KH2PO4 0.5，K2HPO4 1.5，NaCl 1，MgSO40.1, deionized water as solvent, and natural pH value. The fermentation medium is composed of the same seed culture medium.
(2) Culture method
Slant culture: culturing at 30 deg.C for 3-5 days.
Seed culture: the cultured slant is inoculated into a 100mL triangular flask containing 25mL seed culture medium, and cultured for 24h at 30 ℃ at 120 r/min.
Fermentation culture: the seed solution cultured for 24 hours is inoculated into a 100mL triangular flask containing 25mL fermentation medium by the inoculation amount with the volume concentration of 10 percent, and cultured for 24 hours at the temperature of 30 ℃ and at the speed of 120 r/min.
(3) Dilution of soil samples
Weighing 1g of soil sample, adding 50mL of distilled water, shaking uniformly, standing, and clarifying the supernatant for later use.
(4) Strain screening
The substrate beta-carbonyl ethyl phenylpropionate can volatilize along with water vapor and is insoluble in water, so that a solid culture medium taking the beta-carbonyl ethyl phenylpropionate as a unique carbon source cannot be prepared, and a fumigation method is selected for primary screening. And (4) carrying out plate streaking on the fumigated soil supernatant, wherein strains which are tolerant to the substrate can grow on the plate, and strains which can utilize the substrate and convert into products are possible.
1) Preliminary screening
5mL of soil supernatant is added into a lower cover of a watch glass, and an appropriate amount of a substrate, namely ethyl benzoylacetate, is coated on an upper cover of the watch glass, and the watch glass is closed and then stands for 24 hours. The soil supernatant was then inoculated into enrichment plates every 24h under sterile conditions for streaking until no colonies grew on the plates, for a total of 6 d. The plate was placed upside down in a 30 ℃ biochemical incubator for 3 days and the growth was observed.
Colonies growing on the plate were screened, and single colonies with different morphologies and colors were selected as much as possible. After slant culture, the screened strain is preserved at 4 ℃ for observation and subsequent experiments.
2) Double sieve
And (4) carrying out seed and fermentation culture on the strains obtained by preliminary screening, and centrifuging the fermentation liquor for 10min at 8000 r/min. And (3) discarding the supernatant, washing the thalli twice by using water, dispersing the thalli into 25mL of phosphate buffer solution, adding 0.056mol/L substrate beta-carbonyl ethyl phenylpropionate, and carrying out reduction reaction at 30 ℃ at 120r/min for 48 h. And after the reaction is finished, centrifuging the conversion solution, taking a proper amount of supernatant, extracting with 5mL of ethyl acetate, detecting the conversion rate and the enantiomeric excess value of the target product, and screening to obtain the strain zjutss412 with higher conversion rate and enantiomeric excess value.
The concentrations of the substrate and product were determined using Shimadzu GC-2014 gas chromatograph and a chiral column supelco beta-120 (250X 2.5 mm). GC detection conditions are as follows: the sample volume is 1 mu L; the column temperature is 125 ℃; the temperature of a sample inlet is 250 ℃; the detection port temperature is 255 ℃; the carrier gas is nitrogen; the carrier gas flow is 2 mL/min; the split ratio is 1: 15; the detector is a hydrogen Flame Ionization Detector (FID).
2. Strain identification
After morphological identification, the strain zjutss412 is handed to Shanghai biological engineering company for sequencing to identify the strain, and is analyzed through an adjacent phylogenetic tree. The strain identification experiment process is as follows:
1) genomic DNA was extracted according to the SK8257 (yeast) kit.
2) PCR amplifies the 26S rDNNAD 1/D2 region sequence of the test bacteria.
3) And (4) performing gel electrophoresis. Electrophoresis in 1% agarose, 150V, 100mA, 20 min.
4) And (5) purifying and recovering. And cutting the band of the required DNA by using the electrophoresis band of the PCR product, purifying according to an SK8131 kit method, and directly sequencing the PCR product by using a PCR primer.
(1) Morphological identification of strains
After the strain zjutss412 (yeast) is activated for one generation, the colony morphology of a single colony is observed through zonal streaking separation. Taking the bacterial suspension which is diluted properly, and observing the shape of the bacteria through microscopic examination after the steps of smear, fixation, crystal violet staining and the like.
Colony characteristics: the strain zjutss412 is oval and large on the solid culture medium; the color is light yellow; neat edges, smooth and flat surface, sticky and wet.
Microstructure of the strain: the cell morphology of the strain zjutss412 is shown in fig. 2 as being oval and large when observed under a microscope of 10 × 100 times. It is preliminarily judged to be yeast.
(2) Identification of conserved rDNA sequence of strain
A26S rDNA sequence (shown in SEQ ID NO. 1) of the strain zjutss412 screened from the soil was BLAST-aligned with the NCBI database, and the sequence of the strain was found to have 99% homology with Candida tropicalis. According to BLAST homology comparison results and MEGA6.0 software analysis and calculation, a phylogenetic tree (figure 3) of the test strain zjutss412 is obtained, and the phylogenetic position of the test strain is determined. The phylogenetic relationship between the strain and other species of yeasts is shown as a phylogenetic tree, and the strain is determined to belong to the genus Candida by combining the morphological characteristics of bacterial colonies, is named as Candida tropicalis (Candida tropicalis) zjutss412 and is preserved in the China general microbiological culture Collection center (CGMCC), the preservation date is 2017, 12 and 4, the preservation number is CGMCC No.15016, and the preservation address is as follows: western road No.1, north chen west road, north kyo, chaoyang, institute of microbiology, china academy of sciences, zip code 100101.
Example 2: preparation of carbonyl reductase
1. Wet thallus
(1) Inoculating Candida tropicalis (CGMCC No. 15016) into slant culture medium, and culturing at 30 deg.C for 3-5 days to obtain slant thallus; slant culture medium composition: 20g/L glucose, 1g/L ammonium sulfate, 0.5g/L potassium dihydrogen phosphate, 1.5g/L dipotassium hydrogen phosphate, 1g/L sodium chloride, 0.1g/L magnesium sulfate, 20g/L agar and deionized water as a solvent, and the pH value is natural.
(2) The thalli on the inclined plane is inoculated into a 100mL triangular flask containing 25mL seed culture medium, and cultured for 24h at 30 ℃ at 120r/min to obtain seed liquid. Seed culture medium components: 46g/L glucose, 25g/L yeast juice, 4g/L potassium dihydrogen phosphate, 4g/L dipotassium hydrogen phosphate, 0.1g/L sodium chloride and deionized water as a solvent, and the pH value is natural.
(3) The seed solution cultured for 24 hours is inoculated into a 1000mL triangular flask containing 250mL fermentation medium by the inoculation amount with the volume concentration of 10 percent, and cultured for 24 hours at 30 ℃ at 120 r/min. And centrifuging the obtained fermentation liquor in a centrifugal machine at 8000r/min for 20 minutes, and discarding the supernatant to obtain wet thalli. Fermentation medium components: 46g/L glucose, 25g/L yeast juice, 4g/L potassium dihydrogen phosphate, 4g/L dipotassium hydrogen phosphate, 0.1g/L sodium chloride and deionized water as a solvent, and the pH value is natural.
2. Candida tropicalis CGMCC No.15016 wet thalli is dispersed in 50ml of Tris-HCl (0.05M, pH 7.9.9) buffer solution, the concentration of the thalli is 2g/L (dry weight of the thalli/volume of the buffer solution), and the thalli is placed in an ice water bath (0-4 ℃) for ultrasonic cell disruption. Immersing an ultrasonic probe into the liquid level of the bacterial suspension for 1-1.5 cm, the power is 405W, the work time is 2s, the pause time is 4s, the cell is broken by ultrasonic for 20min, and centrifuging the broken sample in a centrifugal machine with the speed of 8000r/min at the temperature of 4 ℃ for 10min to obtain supernatant, namely crude enzyme liquid I.
3. And carrying out ammonium sulfate fractional precipitation on the crude enzyme solution I. And (3) placing the crude enzyme solution I in an ice water bath for precooling to 0-4 ℃, gently stirring while slowly adding the ammonium sulfate which is ground into powder in advance until the saturation of the ammonium sulfate is 40%, continuously stirring in the ice bath for 1h after the ammonium sulfate is completely dissolved, and centrifuging for 10min at 10000 Xg and 4 ℃. And re-dissolving the obtained precipitate in a Tris-HCl buffer (0.05M, pH 7.9.9) with the volume of 1-2 times of the precipitate to obtain a crude enzyme solution II.
4. And (4) carrying out ultrafiltration concentration on the crude enzyme solution II. The crude enzyme solution II was centrifuged at 3500 Xg at 4 ℃ for 20min in a 10kD Millipore ultrafiltration tube. The concentrated solution in the sleeve is carbonyl reductase enzyme solution, the specific activity of the enzyme is 180U/mg, and the solution is stored at 4 ℃ for later use.
The method for measuring the content of the carbonyl reductase protein comprises the following steps: the Bradford method was used, and bovine serum albumin was used as a standard protein.
The method for measuring the enzyme activity of the carbonyl reductase comprises the following steps: the enzyme activity is defined as catalyzing 1 mu mol NADH to generate NAD within 1 minute at 25 DEG C+The amount of carbonyl reductase required. mu.L of the enzyme was added to 5ml of a pH7.9 50mmol/L LTris-HCl buffer solution containing 0.25mmol/L NADH and 0.25mmol/L acetophenone, and reacted at 25 ℃ for 5 min. The absorbance of the sample at 340nm was measured. The enzyme activity is calculated by a formula (1) and the relative enzyme activity is calculated by a formula (2).
Enzyme activity (U) ═ E × V × 103/(6220×1) (1)
Specific activity of enzyme (U/mg) ═ enzyme activity/protein content (2)
E represents the change in absorbance at 340nm for 1 min. V represents the volume of the reaction solution. 6220 represents the molar absorptivity (L. mol)-1·cm-1). 1 represents an optical path length (cm).
Example 3 preparation and application of interfacial self-assembly carbonyl reductase
(1) Preparation of interface self-assembly carbonyl reductase
5mg of the carbonyl reductase enzyme solution (180U/mg) prepared in example 2 was added to 6.25ml of Tris-HCl buffer solution (pH 7.9, 0.05M) to prepare 0.8g/L of the enzyme solution, 10ml of a 3.75g/L polystyrene (Mw 10000Da) toluene solution was added thereto, and the mixture was reacted at 30 ℃ for 1 hour in the dark at a shaker rotation speed of 80 r/min. After the reaction is finished, centrifuging at 1200r/min for 10min, and distributing the interfacial self-assembly enzyme on a two-phase interface of water and an organic phase (namely, toluene is arranged at the upper layer, interfacial self-assembly enzyme is arranged in the middle layer, and Tris-HCl buffer solution is arranged at the lower layer). The buffer and the organic solvent were removed, and the intermediate layer was washed successively with toluene and 0.05M Tris-HCl buffer solution at pH7.9 for 3 times to remove free carbonyl reductase and polystyrene, to obtain 2mg of interfacial self-assembly carbonyl reductase having a surface area of 16.6cm2The specific activity was 155U/mg.
The method for measuring the content of the interface self-assembly carbonyl reductase protein comprises the following steps: the method for measuring the protein content of the interfacial self-assembly carbonyl reductase adopts a material balance method for calculation. The difference value between the protein content of the free carbonyl reductase for the interface self-assembly and the protein content of the residual protein in the phosphate buffer after the interface self-assembly is the protein content of the carbonyl reductase subjected to the interface self-assembly. Protein content was determined using the Bradford method.
The method for measuring the activity of the interface self-assembly carbonyl reductase comprises the following steps: the interfacial self-assembly carbonyl reductase was added to 5mL of Tris-HCl buffer, pH7.9, 0.05mol/L, containing 0.10mmol/L NADH and 0.25mmol/L acetophenone, and the light absorption of the sample at 340nm was examined. The enzyme activity was calculated using the formulas (1) and (2) of example 2.
(2) Asymmetric reduction of ethyl 3-carbonyl-3-phenylpropionate
In eight 50ml Erlenmeyer flasks were added 0.10mol/LNADH, 0.2mg alcohol dehydrogenase (enzyme activity 300U/mg), 10ml Tris-HCl (0.05mol/L, pH7.9) buffer, 10% ethanol by volume and 2mg interfacial self-assembly carbonyl reductase prepared in step (1) (155U/mg), respectively. 2.5ml, 5ml, 7.5ml, 10ml, 12.5ml, 15ml, 17.5ml and 20ml of toluene were added to the above eight flasks, and 0.056 mol/L3-carbonyl-3-phenylpropionic acid ethyl ester was added thereto in terms of buffer volume, followed by reaction for 8 hours at 35 ℃ on a 120r/min shaker. After the reaction is finished, the reaction solution is centrifuged at 10000r/min for 10min and divided into three layers, wherein the upper layer is an organic phase (toluene), the middle layer is an interface self-assembly enzyme, the lower layer is a buffer solution, and the toluene phase and the Tris-HCl buffer solution can be well separated. After centrifugation, an organic phase containing the substrate ethyl 3-carbonyl-3-phenylpropionate and the product ethyl (R) -3-hydroxy-3-phenylpropionate can be obtained. Extracting the Tris-HCl buffer solution obtained after centrifugation for three times by using equal amount of ethyl acetate, combining extraction liquid, combining a sample with an organic phase after the ethyl acetate is removed by rotary evaporation, further analyzing the content of the ethyl 3-carbonyl-3-phenylpropionate and the content of the ethyl (R) -3-hydroxy-3-phenylpropionate in the combined sample by adopting a gas chromatography GC, and determining the conversion rate and the enantiomer excess value of the ethyl (R) -3-hydroxy-3-phenylpropionate.
The concentrations of the substrate and product were determined using Shimadzu GC-2014 gas chromatograph and a chiral column supelco beta-120 (250X 2.5 mm). GC detection conditions are as follows: the sample volume is 1 mu L; the column temperature is 125 ℃; the temperature of a sample inlet is 250 ℃; the detection port temperature is 255 ℃; the carrier gas is nitrogen; the carrier gas flow is 2 mL/min; the split ratio is 1: 15; the detector is a hydrogen Flame Ionization Detector (FID).
TABLE 1 influence of toluene and Tris-HCl buffer solution volume ratio on the reduction reaction
Figure BDA0001713546460000101
The volume ratio of the organic solvent to the buffer during the biotransformation process influences the biotransformation efficiency of the ethyl 3-carbonyl-3-phenylpropionate. Table 1 shows that the reaction conversion increases with increasing toluene volume, and reaches a maximum when the volume ratio of toluene to Tris-HCl buffer solution reaches 12.5: 10. Thereafter, the conversion started to decrease rapidly as the volume of toluene increased. This is probably due to the fact that an excess of toluene generates some poisoning of the interfacial self-assembly enzyme. The change of the volume ratio of the two phases hardly affects the enantiomeric excess of the ethyl (R) -3-hydroxy-3-phenylpropionate, and the enantiomeric excess of the product ethyl (R) -3-hydroxy-3-phenylpropionate is 100%. Therefore, the most suitable volume ratio of toluene to Tris-HCl buffer solution is 12.5:10, the reaction conversion is 96.2%, and the enantiomeric excess of the product is 100%.
Example 4:
(1) preparing an interfacial self-assembly enzyme: the procedure was the same as in example 3.
(2) Asymmetric reduction of ethyl 3-carbonyl-3-phenylpropionate: NADH with the concentration of 0.02, 0.04, 0.08, 0.10 and 0.14mol/L respectively is added into five 50ml Erlenmeyer flasks, and then 0.2mg of alcohol dehydrogenase (enzyme activity is 300U/mg), 10ml of Tris-HCl (0.05mol/L, pH7.9) buffer, 10% ethanol with the concentration of 10% by volume of buffer, 2mg (155U/mg) of interfacial self-assembly carbonyl reductase prepared in the step (1), 12.5ml of toluene and 0.056 mol/L3-carbonyl-3-ethyl phenylpropionate with the concentration of 0.056mol/L by volume of buffer are added into each Erlenmeyer flask for biotransformation reaction. The reaction was carried out for 8 hours at 35 ℃ in a shaker at 120 r/min. After the reaction is finished, the reaction solution is centrifuged at 10000r/min for 10min, and the toluene phase and the Tris-HCl buffer solution can be well separated. After centrifugation, an organic phase containing the substrates ethyl 3-carbonyl-3-phenylpropionate and ethyl (R) -3-hydroxy-3-phenylpropionate was obtained. The Tris-HCl buffer obtained after centrifugation is extracted three times by using equal amount of ethyl acetate, the extract liquid is combined, most of the ethyl acetate is removed by rotary evaporation, and the sample is combined with the organic phase. The combined samples were further analyzed for the content of ethyl 3-carbonyl-3-phenylpropionate and ethyl (R) -3-hydroxy-3-phenylpropionate by GC to determine the conversion and the enantiomeric excess of ethyl (R) -3-hydroxy-3-phenylpropionate.
TABLE 2 influence of the amount of NADH added on the reduction reaction
Figure BDA0001713546460000111
The amount of NADH added affects the biotransformation efficiency of ethyl 3-carbonyl-3-phenylpropionate. The conversion rate increases with increasing NADH concentration, and when the NADH concentration reaches 0.10mol/L, the conversion rate does not increase. The preferred amount of NADH added is 0.10 mol/L. The amount of NADH added did not greatly affect the enantiomeric excess of ethyl (R) -3-hydroxy-3-phenylpropionate, and the enantiomeric excess of ethyl (R) -3-hydroxy-3-phenylpropionate was maintained at 100%.
Example 5:
(1) preparing an interfacial self-assembly enzyme: the procedure was the same as in example 3.
(2) Asymmetric reduction of ethyl 3-carbonyl-3-phenylpropionate: in six 50ml Erlenmeyer flasks were added NADH at a concentration of 0.10mol/L, 0.2mg of alcohol dehydrogenase (enzyme activity 300U/mg), 10ml of Tris-HCl (0.05mol/L, pH7.9) buffer, 0%, 4%, 6%, 8%, 10% and 12% ethanol and 2mg (155U/mg) of interfacial self-assembly carbonyl reductase prepared in step (1), 12.5ml of toluene, 0.056 mol/L3-carbonyl-3-phenylpropionic acid ethyl ester, respectively, by volume of buffer. The reaction was carried out for 8 hours at 35 ℃ in a shaker at 120 r/min. After the reaction is finished, the reaction solution is centrifuged at 10000r/min for 10min, and the toluene phase and the Tris-HCl buffer solution can be well separated. After centrifugation, an organic phase containing the substrates ethyl 3-carbonyl-3-phenylpropionate and ethyl (R) -3-hydroxy-3-phenylpropionate was obtained. The Tris-HCl buffer obtained after centrifugation is extracted three times by using equal amount of ethyl acetate, the extract liquid is combined, most of the ethyl acetate is removed by rotary evaporation, and the sample is combined with the organic phase. The combined samples were further analyzed for the content of ethyl 3-carbonyl-3-phenylpropionate and ethyl (R) -3-hydroxy-3-phenylpropionate by GC to determine the conversion and the enantiomeric excess of ethyl (R) -3-hydroxy-3-phenylpropionate.
TABLE 3 influence of the amount of ethanol added on the reduction reaction
Figure BDA0001713546460000112
Ethanol is converted into acetaldehyde under the action of alcohol dehydrogenase, and the generated NADH provides continuous coenzyme for reduction reaction, so that the regeneration of the coenzyme is realized. The appropriate amount of ethanol added helps to increase conversion. The optimum ethanol addition amount is 10%. The enantiomeric excess value of ethyl (R) -3-hydroxy-3-phenylpropionate was kept constant at 100% at various addition levels of ethanol.
Example 6:
(1) preparing an interfacial self-assembly enzyme: the procedure was the same as in example 3.
(2) Asymmetric reduction of ethyl 3-carbonyl-3-phenylpropionate: in five 50ml Erlenmeyer flasks were added NADH at a concentration of 0.10mol/L, alcohol dehydrogenase at 0.2mg (enzyme activity 300U/mg), Tris-HCl (0.05mol/L, pH7.9) at 10ml, interfacial self-assembly carbonyl reductase prepared in step (1) at 2mg (155U/mg), ethanol at 10% concentration by volume, and toluene at 12.5ml, respectively. Five conical flasks were charged with the substrate ethyl 3-carbonyl-3-phenylpropionate at concentrations of 0.037mol/L, 0.044mol/L, 0.050mol/L, 0.056mol/L and 0.062mol/L, respectively, based on the volume of the buffer, to conduct the biotransformation reaction. The reaction was carried out for 8 hours at 35 ℃ in a shaker at 120 r/min. After the reaction is finished, the reaction solution is centrifuged at 10000r/min for 10min, and the toluene phase and the Tris-HCl buffer solution can be well separated. After centrifugation, an organic phase containing the substrates ethyl 3-carbonyl-3-phenylpropionate and ethyl (R) -3-hydroxy-3-phenylpropionate was obtained. The Tris-HCl buffer obtained after centrifugation is extracted three times by using equal amount of ethyl acetate, the extract liquid is combined, most of the ethyl acetate is removed by rotary evaporation, and the sample is combined with the organic phase. The combined samples were further analyzed for the content of ethyl 3-carbonyl-3-phenylpropionate and ethyl (R) -3-hydroxy-3-phenylpropionate by GC to determine the conversion and the enantiomeric excess of ethyl (R) -3-hydroxy-3-phenylpropionate.
TABLE 43 influence of the amount of Ethyl carbonyl-3-phenylpropionate added on the reduction reaction
Figure BDA0001713546460000121
The catalytic efficiency of the interfacial self-assembly carbonyl reductase is different due to different substrate concentrations. The concentration of the substrate is too high, and certain toxicity exists on the interfacial self-assembly enzyme. The concentration of the substrate is too low, and the production efficiency is not high. Therefore, a suitable substrate concentration is critical to the efficiency of the catalytic reaction. When the concentration of the substrate is increased from 0.037mol/L to 0.056mol/L, the conversion rate of the reaction tends to increase, and when the concentration exceeds 0.056mol/L, the conversion rate is significantly decreased. Therefore, the optimum substrate concentration is 0.056 mol/L. The enantiomeric excess value of the product (R) -ethyl 3-hydroxy-3-phenylpropionate is not affected by the concentration of the substrate, and the enantiomeric excess value of the ethyl (R) -3-hydroxy-3-phenylpropionate is 100% in the range of the substrate concentration of 0.037-0.062 mmol/L.
Example 7:
(1) preparing an interfacial self-assembly enzyme: the procedure was the same as in example 3.
(2) Asymmetric reduction of ethyl 3-carbonyl-3-phenylpropionate: five 50ml Erlenmeyer flasks were charged with NADH at a concentration of 0.10mol/L, alcohol dehydrogenase at 0.2mg (enzyme activity 300U/mg), Tris-HCl (0.05mol/L, pH7.9) at 10ml, interfacial self-assembly carbonyl reductase prepared in step (1) at 2mg (180U/mg), ethanol at 10% at 12.5ml, toluene at 0.056 mol/L3-carbonyl-3-phenylpropionic acid ethyl ester at 0.056mol/L at buffer volume, and the five Erlenmeyer flasks were placed in a shaker at 20 ℃, 25 ℃, 30 ℃, 35 ℃ and 40 ℃, 120r/min, respectively, and reacted for 8 hours. After the reaction is finished, the reaction solution is centrifuged at 10000r/min for 10min, and the toluene phase and the Tris-HCl buffer solution can be well separated. After centrifugation, an organic phase containing the substrates ethyl 3-carbonyl-3-phenylpropionate and ethyl (R) -3-hydroxy-3-phenylpropionate was obtained. The Tris-HCl buffer obtained after centrifugation is extracted three times by using equal amount of ethyl acetate, the extract liquid is combined, most of the ethyl acetate is removed by rotary evaporation, and the sample is combined with the organic phase. The combined samples were further analyzed for the content of ethyl 3-carbonyl-3-phenylpropionate and ethyl (R) -3-hydroxy-3-phenylpropionate by GC to determine the conversion and the enantiomeric excess of ethyl (R) -3-hydroxy-3-phenylpropionate.
TABLE 5 Effect of reaction temperature on the reduction reaction
Figure BDA0001713546460000131
The temperature has a great influence on the conversion rate of the reduction reaction. Temperature affects not only the stability and activity of the enzyme, but also the equilibrium of the reaction. The conversion of ethyl 3-carbonyl-3-phenylpropionate increased with increasing temperature. The reaction conversion reached a maximum when the temperature reached 35 ℃, and then the conversion began to decrease as the temperature increased. While the change in temperature had little effect on the enantiomeric excess of ethyl (R) -3-hydroxy-3-phenylpropionate. Therefore, the optimum temperature for the reaction is 35 ℃.
Example 8:
(1) preparing an interfacial self-assembly enzyme: the procedure was the same as in example 3.
(2) Asymmetric reduction of ethyl 3-carbonyl-3-phenylpropionate: six 50ml Erlenmeyer flasks are respectively added with 0.10mol/L NADH, 0.2mg alcohol dehydrogenase (enzyme activity 300U/mg), 10ml Tris-HCl (0.05mol/L pH7.9) buffer solution and 2mg interfacial self-assembly carbonyl reductase (155U/mg) prepared in the step (1), 10% ethanol, 12.5ml toluene and 0.056 mol/L3-carbonyl-3-ethyl phenylpropionate, the six Erlenmeyer flasks are respectively placed in a shaker at 35 ℃ and 120r/min for reaction for 3h, 4h, 5h, 6h, 8h and 10 h. After the reaction is finished, the reaction solution is centrifuged at 10000r/min for 10min, and the toluene phase and the Tris-HCl buffer solution can be well separated. After centrifugation, an organic phase containing ethyl 3-carbonyl-3-phenylpropionate and ethyl (R) -3-hydroxy-3-phenylpropionate was obtained. The Tris-HCl buffer obtained after centrifugation is extracted three times by using equal amount of ethyl acetate, the extract liquid is combined, most of the ethyl acetate is removed by rotary evaporation, and the sample is combined with the organic phase. The combined samples were further analyzed for the content of ethyl 3-carbonyl-3-phenylpropionate and ethyl (R) -3-hydroxy-3-phenylpropionate by GC to determine the conversion and the enantiomeric excess of ethyl (R) -3-hydroxy-3-phenylpropionate.
TABLE 6 Effect of reaction time on the reduction reaction
Figure BDA0001713546460000141
The results showed that the biotransformation efficiency was gradually increased with the increase of the reaction time, and the transformation efficiency was not increased much when the reaction time was more than 8 hours, so that the optimum reaction time was determined to be 8 hours. The reaction time has little influence on the enantiomeric excess value of the ethyl (R) -3-hydroxy-3-phenylpropionate, and the enantiomeric excess value is kept at 100 percent.
Example 9: (Recycling)
(1) Preparing an interfacial self-assembly enzyme: the procedure was the same as in example 3.
(2) Asymmetric reduction of ethyl 3-carbonyl-3-phenylpropionate: six 50ml Erlenmeyer flasks were charged with 0.10mol/L NADH, 0.2mg alcohol dehydrogenase (enzyme activity 300U/mg), 10ml Tris-HCl (0.05mol/L, pH7.9) buffer and 2mg interfacial self-assembly carbonyl reductase (155U/mg) prepared in step (1), 10% ethanol, 12.5ml toluene, 0.056 mol/L3-carbonyl-3-phenylpropionic acid ethyl ester, respectively, by buffer volume, and the six Erlenmeyer flasks were placed in a shaker at 35 ℃ and 120r/min for reaction for 8 hours. After the reaction is finished, the reaction solution is centrifuged at 10000r/min for 10min, and the toluene phase and the Tris-HCl buffer solution can be well separated. After centrifugation, an organic phase containing ethyl 3-carbonyl-3-phenylpropionate and ethyl (R) -3-hydroxy-3-phenylpropionate was obtained. The contents of ethyl 3-carbonyl-3-phenylpropionate and ethyl (R) -3-hydroxy-3-phenylpropionate in the sample were further analyzed by gas chromatography GC to determine the conversion and the enantiomeric excess of ethyl (R) -3-hydroxy-3-phenylpropionate. The interfacial self-assembly enzyme obtained after centrifugation was redispersed in 10ml Tris-HCl (0.05mol/L, pH7.9) buffer system to repeat the above reaction process, and the recycling condition of the interfacial self-assembly enzyme was investigated. The results are shown in Table 7.
TABLE 7 Effect of reaction time on the reduction reaction
Figure BDA0001713546460000151
The result shows that the enzyme activity is gradually reduced and the conversion rate is gradually reduced along with the repeated utilization of the interface self-assembly enzyme. After six times of recycling, the conversion rate of the substrate is still 68.3 percent of the first conversion rate. The reaction times have little influence on the enantiomeric excess value of the ethyl (R) -3-hydroxy-3-phenylpropionate, and the enantiomeric excess value is kept at 100 percent. Indicating that the interfacial self-assembly enzyme could be well recycled 6 times.
Example 10: (comparative test)
(1) Preparing an interfacial self-assembly enzyme: the procedure was the same as in example 3.
(2) Respectively catalyzing asymmetric reduction reaction of 3-carbonyl-3-phenylpropionic acid ethyl ester in a water phase and a Tris-HCl/toluene two-phase system by using free carbonyl reductase and interfacial self-assembly carbonyl reductase as catalysts, and performing a comparative test. 5mg of carbonyl reductase (180U/mg) was prepared according to the method of example 2, and 2mg of interfacial self-assembly carbonyl reductase prepared according to the method of example 3 had a specific activity of 155U/mg. In a comparative experiment, 5mg (180U/mg) of carbonyl reductase and 2mg (155U/mg) of interfacial self-assembly enzyme prepared from 5mg (180U/mg) of carbonyl reductase were dispersed in an aqueous phase or a Tris-HCl/toluene two-phase system, respectively, to catalyze the asymmetric reduction of ethyl 3-carbonyl-3-phenylpropionate.
Comparative reaction conditions in the two-phase system were as follows: 2mg (155U/mg) and 5mg (180U/mg) of interfacial self-assembly carbonyl reductase was added to two 50ml Erlenmeyer flasks, respectively. 0.10mol/L ADH, 0.2mg of alcohol dehydrogenase (enzyme activity 300U/mg) and 10ml of Tris-HCl (0.05mol/L pH7.9) buffer solution, 10% ethanol, 12.5ml of toluene and 0.056 mol/L3-carbonyl-3-ethyl phenylpropionate are respectively added into two erlenmeyer flasks, the two erlenmeyer flasks are placed in a shaker at 35 ℃ and 120r/min for reaction for 8 hours, and a comparison test is carried out.
Comparative reaction conditions in the aqueous phase were as follows: 2mg (155U/mg) and 5mg (180U/mg) of interfacial self-assembly carbonyl reductase was added to two 50ml Erlenmeyer flasks, respectively. The concentration of NADH of 0.10mol/L, the concentration of alcohol dehydrogenase of 0.2mg (enzyme activity of 300U/mg) and the concentration of Tris-HCl (0.05mol/L pH7.9) buffer solution of 10ml are respectively added into two erlenmeyer flasks, the concentration of ethanol of 10 percent and the concentration of ethyl 3-carbonyl-3-phenylpropionate of 0.056mol/L are respectively added into the two erlenmeyer flasks, the two erlenmeyer flasks are placed in a shaker at 35 ℃ and 120r/min for reaction for 8 hours, and a comparison test is carried out.
After the reaction is finished, the reaction solution is centrifuged for 10min at 10000r/min in a centrifuge, and the toluene phase and the Tris-HCl buffer solution can be well separated. After centrifugation, an organic phase containing ethyl 3-carbonyl-3-phenylpropionate and ethyl (R) -3-hydroxy-3-phenylpropionate was obtained. The contents of ethyl 3-carbonyl-3-phenylpropionate and ethyl (R) -3-hydroxy-3-phenylpropionate in the sample were further analyzed by gas chromatography GC to determine the conversion and the enantiomeric excess of ethyl (R) -3-hydroxy-3-phenylpropionate. The comparative reaction results are shown in Table 8.
TABLE 8 free carbonyl reductase and interfacial self-assembling carbonyl reductase in aqueous and Tris-HCl/toluene two-phase systems
Comparison of the catalytic reaction of 3-carbonyl-3-phenylpropionic acid Ethyl ester by asymmetric reduction
Figure BDA0001713546460000161
The result shows that the interfacial self-assembly carbonyl reductase has the best reduction effect in a Tris-HCl/toluene two-phase system. Free carbonyl reductase has better catalytic activity in water phase, but a substrate is not dissolved in water, carbonyl reductase conversion substrates need to be in full contact with the substrate for continuous reaction, the substrate has low solubility in water, the contact between the substrate and the enzyme is limited, on the other hand, the organic substrate has activity inhibition effect on enzyme protein molecules, the enzyme activity is lost, and therefore the catalytic activity of the free carbonyl reductase in the water phase does not achieve the optimal effect. The free carbonyl reductase has the lowest catalytic activity in a Tris-HCl/toluene two-phase system, and the conversion rate is only 42.5 percent, mainly because the organic phase has an inhibiting effect on the activity of the free enzyme. The reason that the catalytic activity of the interfacial self-assembly carbonyl reductase in a Tris-HCl/toluene two-phase system is the highest is that the enzyme activity stability is enhanced after the enzyme is immobilized by self-assembly, and meanwhile, the enzyme can be continuously and fully contacted with a substrate at an interface to carry out biotransformation reaction, so that the conversion rate is higher.
Example 11 interfacial self-Assembly carbonyl reductase (IACR) preparation Condition optimization
1) Selection of organic solvents
The toluene in example 3 was replaced with n-hexane, n-heptane, dichloromethane, chloroform, n-butanol, and dibutyl phthalate, respectively, and the interfacial self-assembly method was the same as in example 3. The interface self-assembly efficiency of the interface self-assembly enzyme is the ratio of the specific activity of the interface self-assembly carbonyl reductase to the specific activity of the free carbonyl reductase.
TABLE 9 influence of different organic solvents on the efficiency of IACR interfacial self-assembly
Figure BDA0001713546460000171
The interfacial self-assembly efficiency of interfacial self-assembly enzymes is affected by organic solvents. The low solubility of polystyrene in n-hexane, n-heptane, n-butanol and dibutyl phthalate leads to a low specific activity of the IACR. The polystyrene is easy to dissolve in benzene, toluene, dichloromethane and chloroform, so that the enzyme can be easily assembled with the polystyrene at a Tris-HCl/organic solvent interface, and the specific activity of the interfacial self-assembly carbonyl reductase is higher. The interfacial self-assembly efficiency is the ratio of the specific activities of the interfacial self-assembly carbonyl reductase and the free carbonyl reductase. The specific activity of the free carbonyl reductase was 180U/mg. Table 9 shows that the interfacial self-assembly efficiency can reach 87% by taking toluene as an organic solvent, and the specific activity of the interfacial self-assembly carbonyl reductase is 155U/mg. Therefore, toluene is the best organic solvent for preparing the interfacial self-assembly carbonyl reductase, and toluene/Tris-HCl is selected as the best two-phase reaction system.
2) Influence of concentration of free carbonyl reductase on preparation of interface self-assembly carbonyl reductase
1.5mL of free carbonyl reductase with different concentrations of 0.4g/L, 0.8g/L, 1.2g/L, 1.6g/L and 2.0g/L was prepared, 10mL of an organic solvent dissolving 10mg of polystyrene was added, the concentrations of the enzyme solutions in step (1) of example 3 were changed to 0.4, 0.8, 1.2, 1.6 and 2.0g/L, respectively, and the interfacial self-assembly method was the same as example 3.
TABLE 10 Effect of free carbonyl reductase concentration on interfacial self-assembling carbonyl reductase preparation
Figure BDA0001713546460000172
Figure BDA0001713546460000181
In the preparation process of the interfacial self-assembly carbonyl reductase, the initial concentration of the free carbonyl reductase in the water phase has obvious influence on the interfacial assembly efficiency of the interfacial self-assembly carbonyl reductase. The optimal initial concentration of free carbonyl reductase will facilitate its assembly with polystyrene at the Tris-HCl and toluene biphasic interface. The protein self-assembly rate is defined as the ratio of the protein content of the interfacial self-assembly carbonyl reductase and the free carbonyl reductase in the Tris-HCl buffer. A high rate of protein self-assembly indicates that more carbonyl reductase is assembled with polystyrene, which decreases as the initial concentration of free carbonyl reductase increases. The interfacial assembly efficiency of the interfacial self-assembly carbonyl reductase is basically kept at 86 percent by adding free carbonyl reductase with different initial protein concentrations. Therefore, the optimum initial concentration of free carbonyl reductase was selected to be 0.8 g/L.
3) Effect of polystyrene concentration on IACR preparation
1.5mL of carbonyl reductase was added to 10mL of an organic solvent having polystyrene concentrations of 1.25g/L, 2.50g/L, 3.75g/L, 5.00g/L, 7.50g/L, and 10.00g/L, respectively, and the polystyrene concentrations in step (1) of example 3 were changed to 1.25, 2.5, 3.75, 5.0, 7.5, and 10g/L, respectively, in the same manner as in example 3. The protein self-assembly ratio refers to the ratio of the content of protein in the interfacial self-assembly enzyme to the content of protein in the free carbonyl reductase.
TABLE 11 influence of polystyrene concentration on the preparation of IACR
Figure BDA0001713546460000182
Polymer-enzyme conjugates can self-assemble at the oil/water interface and affect interfacial biotransformation. The surfactant-like structure consisting of a hydrophilic protein head and a hydrophobic polymer tail facilitates the construction of interfacial self-assembling enzymes. In the process of preparing IACR, when the concentration of the carbonyl reductase is 0.8g/L, polystyrene with proper concentration is beneficial to better combining with the carbonyl reductase and increasing the catalytic activity of the reaction, and the experimental result is shown in Table 11. As the concentration of polystyrene increases, the self-assembly rate of protein also increases, but when the concentration of PS exceeds 5.00g/L, the self-assembly rate of protein begins to keep stable. Therefore, when the concentration of carbonyl reductase is 0.8g/L, the most suitable concentration of polystyrene is 5.00 g/L.
Sequence listing
<110> Zhejiang industrial university
<120> interface self-assembly carbonyl reductase and application thereof in synthesis of (R) -3-hydroxy-3-ethyl phenylpropionate
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 584
<212> DNA
<213> Candida tropicalis (Candida tropicalis)
<400> 1
gcggaggaaa agaaaccaac agggattgcc ttagtagcgg cgagtgaagc ggcaaaagct 60
caaatttgaa atctggctct ttcagagtcc gagttgtaat ttgaagaagg tatctttggg 120
tctggctctt gtctatgttt cttggaacag aacgtcacag agggtgagaa tcccgtgcga 180
tgagatgatc caggcctatg taaagttcct tcgaagagtc gagttgtttg ggaatgcagc 240
tctaagtggg tggtaaattc catctaaagc taaatattgg cgagagaccg atagcgaaca 300
agtacagtga tggaaagatg aaaagaactt tgaaaagaga gtgaaaaagt acgtgaaatt 360
gttgaaaggg aagggcttga gatcagactt ggtattttgt atgttacttc ttcgggggtg 420
gcctctacag tttatcgggc cagcatcagt ttgggcggta ggagaattgc gttggaatgt 480
ggcacggctt cggttgtgtg ttatagcctt cgtcgatact gccagcctag actgaggact 540
gcggtttata cctaggatgt tggcataatg atcttaagtc gccc 584

Claims (7)

1. The application of the interface self-assembly carbonyl reductase in the preparation of (R) -3-hydroxy-3-ethyl phenylpropionate by asymmetrically reducing 3-carbonyl-3-ethyl phenylpropionate is characterized in that the interface self-assembly carbonyl reductase is prepared by the following method: adding carbonyl reductase into 0.05M of Tirs-HCl buffer solution with the pH value of 6.0-9.0, adding a toluene solution of polystyrene, carrying out oscillation reaction for 1-4 hours in a shaking table under the conditions of darkness, 40-100r/min and 20-40 ℃, centrifuging to obtain a reaction system with an organic phase as an upper layer, an interface self-assembly carbonyl reductase as a middle layer and a water phase as a lower layer, taking out the middle layer, and washing the middle layer with toluene and 0.05M of Tris-HCl buffer solution with the pH value of 6.0-9.0 and 0.05M for 3-5 times respectively to obtain the interface self-assembly carbonyl reductase; the carbonyl reductase is derived from Candida tropicalis (A)Candida tropicalis) zjutss412 deposited with the general microorganisms of the China Committee for culture Collection of microorganismsThe preservation center has a preservation number of CGMCC No.15016, a preservation date of 2017, 12 months and 4 days, and a preservation address: western road No.1, north chen west road, north kyo, chaoyang, institute of microbiology, china academy of sciences, zip code 100101.
2. The use according to claim 1, wherein the carbonyl reductase is added in an amount of 20 to 200U/ml based on the volume of the buffer, the concentration of the toluene solution of polystyrene is 1.0 to 4.0g/L, and the volume ratio of the toluene solution of polystyrene to the buffer is 1-3: 1.
3. The use as claimed in claim 1, characterized in that the carbonyl reductase is prepared as follows: (1) candida tropicalis (C.tropicalis) (C.tropicalis)Candida tropicalis) Dispersing wet thalli obtained by fermentation culture of CGMCC No.15016 in 0.05M, pH 7.9.9 Tris-HCl buffer solution, placing in ice water bath at 0-4 ℃, carrying out ultrasonic crushing under the condition of power 405W for 2s, carrying out intermittent 4s, carrying out ultrasonic cell crushing for 20min, and centrifuging at 8000r/min and 4 ℃ for 10min after crushing to obtain supernatant, namely crude enzyme solution I; (2) placing the enzyme solution I in an ice-water bath for precooling to 0-4 ℃, gently stirring while slowly adding ammonium sulfate which is pre-ground into powder until the saturation of the ammonium sulfate is 40%, continuously stirring for 1h in the ice bath after the ammonium sulfate is completely dissolved, centrifuging for 10min at 10000 Xg and 4 ℃, and redissolving the obtained precipitate in 0.05M, pH 7.9.9 Tris-HCl buffer solution with the volume of 1-2 times of the precipitate to obtain a crude enzyme solution II; (3) and (3) placing the crude enzyme solution II in a 10kD Millipore ultrafiltration centrifugal tube, and centrifuging for 20min at 3500 Xg and 4 ℃, wherein the concentrated solution in the sleeve is carbonyl reductase enzyme solution.
4. The use according to claim 3, wherein the wet biomass is prepared by: 1) inoculating Candida tropicalis CGMCC No.15016 into slant culture medium, and culturing at 30 deg.C for 3-5 days to obtain slant thallus; slant culture medium composition: 20g/L glucose, 1g/L ammonium sulfate, 0.5g/L potassium dihydrogen phosphate, 1.5g/L dipotassium hydrogen phosphate, 1g/L sodium chloride, 0.1g/L magnesium sulfate, 20g/L agar and deionized water as a solvent, wherein the pH value is natural; 2) inoculating the thalli on the inclined plane into a 100mL triangular flask containing 25mL seed culture medium, and culturing at 30 ℃ at 120r/min for 24h to obtain a seed solution; the seed culture medium comprises the following components: 46g/L glucose, 25g/L yeast juice, 4g/L potassium dihydrogen phosphate, 4g/L dipotassium hydrogen phosphate, 0.1g/L sodium chloride, deionized water as a solvent and natural pH value; 3) inoculating the seed solution cultured for 24h into a 1000mL triangular flask containing 250mL fermentation medium with the inoculation amount of 10% of volume concentration, culturing for 24h at 30 ℃ at 120r/min, centrifuging the obtained fermentation liquid in a centrifugal machine at 8000r/min for 20min, and discarding the supernatant to obtain wet thalli; the fermentation medium comprises the following components: 46g/L glucose, 25g/L yeast juice, 4g/L potassium dihydrogen phosphate, 4g/L dipotassium hydrogen phosphate, 0.1g/L sodium chloride and deionized water as a solvent, and the pH value is natural.
5. The use according to claim 1, characterized in that the method of application is: the method comprises the steps of taking an interface self-assembly carbonyl reductase as a catalyst, taking 3-carbonyl-3-phenylpropionic acid ethyl ester as a substrate, taking NADH as a coenzyme, taking ethanol as an auxiliary substrate, adding ethanol dehydrogenase for in-situ regeneration of coenzyme NADH, taking a Tris-HCl/toluene two-phase system formed by Tris-HCl buffer solution with the pH value of 3.0-8.0 and toluene as a reaction medium, reacting in a shaking table at the temperature of 20-40 ℃ and at the speed of 80-120R/min, and separating and purifying reaction liquid after complete reaction to obtain the (R) -3-hydroxy-3-phenylpropionic acid ethyl ester.
6. The use of claim 5, wherein the amount of the catalyst is 5-200U/mL based on the volume of the buffer solution, the amount of the substrate is 0.010-0.070 mol/L based on the volume of the buffer solution, and the amount of the ethanol is 1-12% based on the volume of the buffer solution; the NADH dosage is 0.02-0.14 mol/L based on the volume of the buffer solution, the alcohol dehydrogenase 3000-120000U/L, and the volume ratio of the toluene to the buffer solution is 2.5-20: 10.
7. The application of claim 5, wherein after the reaction is finished, 10000R/min of the reaction solution is centrifuged for 10min, the centrifugation is carried out to obtain an organic phase, a middle layer and an aqueous phase containing substrates of the ethyl 3-carbonyl-3-phenylpropionate and the ethyl (R) -3-hydroxy-3-phenylpropionate, the aqueous phase is extracted for three times by using equal amount of ethyl acetate, the extracts are combined, most of the ethyl acetate is removed by rotary evaporation, and then the combined organic phase is combined with the extract, and silica gel column chromatography is carried out to obtain the ethyl (R) -3-hydroxy-3-phenylpropionate; the intermediate layer recovers the interface self-assembly carbonyl reductase for recycling.
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