CN112481224A - Baeyer-Villiger monooxygenase and application thereof - Google Patents
Baeyer-Villiger monooxygenase and application thereof Download PDFInfo
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Abstract
The invention discloses a Baeyer-Villiger monooxygenase and application thereof, belonging to the technical field of biological engineering. The invention provides another Baeyer-Villiger monooxygenase aiming at the reported problems of low catalytic activity of Baeyer-Villiger monooxygenase on substrate thioether, poor thermal stability, low ee value of a product and the like, and the Baeyer-Villiger monooxygenase has high stereoselectivity on linear ketone, cyclic ketone and thioether substrates and high catalytic activity on cyclohexanone and thioanisole, and can directly and effectively solve the problems of the conventional monooxygenase. When the S-benzyl sulfoxide is prepared by taking the thioanisole as a substrate, the conversion rate can reach more than 95 percent, and the yield can reach 99.5 percent. Provides a new idea and method for the industrialized production of the optical active sulfoxide.
Description
Technical Field
The invention relates to a Baeyer-Villiger monooxygenase and application thereof, belonging to the technical field of biological engineering.
Background
The Baeyer-Villiger monooxygenase (BVMO) belongs to a branch of flavoprotein monooxygenase, can catalyze nucleophilic oxidation of carbonyl and realize electrophilic oxidation of heteroatoms, and mainly comprises organic compounds containing heteroatoms such as boron, selenium, sulfur and the like. Due to the characteristic of wide substrate spectrum, the method is widely applied to industrial catalysis.
Optically active sulfoxides, an important chiral compound, have attracted much attention in almost every field of chemical synthesis industry in view of their great demands, involving the design and development of novel synthetic reagents, drugs and functional materials.
Chiral sulfoxides, as an extremely useful class of compounds, have a wide and important application in the fields of asymmetric synthesis and medicine. Selective sulfoxidation of thioethers is difficult to achieve by chemical methods, and thus enzyme-mediated selective sulfoxidation has been one of the hot spots of interest to organic chemists for the past decades. At present, a plurality of enzymes and microorganism whole cells as catalysts have been successfully used for catalyzing and synthesizing chiral sulfoxide, which becomes a beneficial supplement of a chemical method, and the advantages can be summarized into higher stereoselectivity. This is mainly due to the specific recognition of the substrate by the enzyme, environmental compatibility and safety. The biocatalyst is usually microorganism, plant and animal cell or separated enzyme, which is renewable, and can be easily degraded in the environment after use without pollution to the environment. The reaction conditions of the biocatalytic process are mild, usually water is used as a solvent system, and the reaction is much safer than the chemical method using organic solvent and toxic catalyst. Due to these advantages of biocatalysis, the synthesis of chiral sulfoxides by biological methods is receiving increasing attention. The report on the synthesis of chiral sulfoxide by biological method also includes that starting from easily prepared precursor thioether, the optically pure sulfoxide is obtained by oxidizing and oxygenating the thioether by using whole cells of microorganisms or free enzymes. For example, Adam et al screened a strain from soil by enrichment culture using thioanisole as the sole carbon source, and then used its whole cells to perform asymmetric oxidation on a series of aryl alkyl sulfides, and the generated sulfoxide product was S-shaped, with the highest ee value of 99% or more, but its tolerance to substrate was very low, and the substrate concentration was only 1 mM. When a series of phenylalkyl sulfides and p-methylphenylalkyl sulfides are oxidized by a strain having BV monooxygenase activity, S-configuration products can be produced, but the ee value is low (A high antibacterial biochemical reactivity by the microbial bacterium Pseudomonas front 2004). Subsequent studies have more intensively reported the asymmetric oxidation of prochiral sulfides by fungi, including arylalkyl sulfides, cycloalkylalkyl sulfides, heteroarylalkyl sulfides, and dialkyl sulfides, among others; almost all the oxidations of thioethers lead to products of S configuration with ee values of more than 80% and yields of more than 79% (Holland et al, Biotransformation of sulfides by Rhodococuccushrropolies, published in 2003). It is well known that the roots of the catalytic action in the whole cells of microorganisms are various enzymes, and the current enzymes capable of stereoselectively oxidizing thioether to the corresponding sulfoxide (or sulfone) are mainly peroxidase and monooxygenase. Among them, horseradish peroxidase, vanadium haloperoxidase, chloroperoxidase, etc. have been studied to catalyze the oxidation of a series of aliphatic and aromatic thioethers, but the enantiomeric purity of the product cannot meet the requirements. Peroxidase from different sources catalyzes an asymmetric sulfoxidation reaction of thioether, and the problems of low loading capacity on a substrate, low ee value of a product and the like exist.
As an important organic synthesis intermediate, epsilon-caprolactone can be used for synthesizing polycaprolactone or blending and modifying with substances such as starch and the like, and the application of epsilon-caprolactone is very wide. The synthesis of epsilon-caprolactone has certain difficulty in the aspects of production safety, product stability and the like, and the synthesis technology has higher difficulty. Along with the continuous expansion of the application of caprolactone, the market needs to be larger and larger, and the research on epsilon-caprolactone is increasingly paid attention by researchers. The synthesis method of epsilon-caprolactone includes 1, 6-hexanediol dehydrogenation method, 6-hydroxycaproic acid intramolecular condensation method, adipic acid acidification method, cyclohexanone oxidation method and the like according to the synthesis process of epsilon-caprolactone and the difference of raw materials. The oxidation of cyclohexanone is currently the most efficient process, both from the starting materials and from the various factors such as the reaction apparatus and the reaction conditions, and is currently the most commercially practiced process for the production of epsilon-caprolactone. A peroxy acid method is used in the traditional epsilon-caprolactone production, but the peroxy acid has great potential safety hazard in the transportation and storage processes, a large amount of waste acid is generated by reaction, and environmental pollution is easily caused.
The biological oxidation method is a method for synthesizing epsilon-caprolactone by oxidizing cyclohexanone by adopting biological enzyme or microbial fermentation. The enzyme has the characteristics of high efficiency and specificity, so the biological enzyme oxidation method has the specific advantages. CHMO is the abbreviation of cyclohexanone monooxygenase, and the Baeyer-Villiger oxidation reaction for catalyzing and oxidizing cyclohexanone by using biological enzyme as a catalyst has been reported. The use of A.calcoaceticus for catalyzing the oxidation of cyclohexanone has been reported to increase the rate of oxidation of cyclohexanone (Mandal et al, "biological transformation of cyclohexanone by fusarium SP," 2002). Although the Baeyer-Villiger oxidation reaction of cyclohexanone can be effectively catalyzed by Geotrichum candidum NCYC49, the yield of epsilon-caprolactone can reach more than 90% at most, but the oxygen concentration in the reaction system is required to be high, and the reaction needs to be carried out in a high-concentration oxygen reaction system under strict control (Carbilleria et al, "Biotransformation of cyclohexanone using immobilized geotrichum candidum NCYC49 semiconductors that have been disclosed in 2004). Furthermore, when cyclohexanone is oxidized by catalysis of the genus Pseudomonas, the conversion of cyclohexanone and the yield of epsilon-caprolactone are not high (Zheng Aifang, et al, "study on biosynthesis of caprolactone from cyclohexanone", published in 2006).
Disclosure of Invention
The invention aims to solve the technical problems of low catalytic activity of the reported Baeyer-Villiger monooxygenase on substrate thioether, poor thermal stability, low ee value of a product, low cyclohexanone conversion rate and product yield and the like, and provides another Baeyer-Villiger monooxygenase which has good solubility, high stereoselectivity shown on linear ketone, cyclic ketone and thioether substrates, high catalytic activity on cyclohexanone and methyl sulfide and can directly and effectively solve the problems of the conventional monooxygenase.
The first purpose of the invention is to provide a method for preparing an optically active sulfoxide, which takes monooxygenase with an amino acid sequence shown as SEQ ID NO.2 as a catalyst and thioether as a substrate.
In one embodiment of the invention, the nucleotide sequence encoding the monooxygenase is shown in SEQ ID NO. 1.
In one embodiment of the present invention, the optically active sulfoxide includes benzyl sulfoxide, p-chlorobenzyl sulfoxide, p-methoxybenzyl sulfoxide, p-aminobenzyl sulfoxide.
In one embodiment of the invention, S-benzyl sulfoxide is generated by catalysis by taking the benzyl sulfide as a substrate.
In one embodiment of the present invention, the concentration of the thioanisole in the reaction system is 30-100 mmol.L-1。
In one embodiment of the invention, the monooxygenase enzyme is present in the range of 30-100 kU.L-1The amount of (b) is added to the reaction system.
In one embodiment of the present invention, the reaction system further contains an alcohol dehydrogenase and methanol.
In one embodiment of the present invention, the amount of glucose dehydrogenase added is 10 to 100 kU.L-1。
In one embodiment of the present invention, the concentration of methanol in the reaction system is 3 to 8%.
In one embodiment of the present invention, a Tris-HCl buffer is added to the reaction system to maintain the pH, and the concentration of the Tris-HCl buffer is 50 to 150 mmol.L-1。
In one embodiment of the invention, the reaction is carried out at 25-40 ℃ for 2-30 h.
The second purpose of the invention is to provide the application of monooxygenase in the preparation of S-benzyl sulfoxide, wherein the amino acid sequence of the monooxygenase is shown as SEQ ID NO. 2.
In one embodiment of the invention, S-benzylsulfoxide is prepared by using thioanisole as a substrate and the monooxygenase as a catalyst.
In one embodiment of the present invention, the reaction system further contains an alcohol dehydrogenase.
The third purpose of the invention is to provide a method for preparing epsilon-lactone, which takes monooxygenase as a catalyst, and the amino acid sequence of the monooxygenase is shown as SEQ ID NO. 2.
In one embodiment of the invention, cyclohexanone is used as the substrate.
In one embodiment of the invention, the concentration of cyclohexanone is from 50 to 200 mmol.L-1。
In one embodiment of the invention, the monooxygenase is added in an amount of 10-50 kU.L-1。
In one embodiment of the present invention, the reaction system further contains alcohol dehydrogenase and methanol.
In one embodiment of the present invention, the alcohol dehydrogenase includes glucose dehydrogenase, and 20 to 50 kU.L is added to the reaction system-1The glucose dehydrogenase of (1).
In one embodiment of the invention, the concentration of methanol in the reaction is 3-8%.
In one embodiment of the invention, the reaction is carried out at a pH of 8 to 10 and at a temperature of 25 to 35 ℃.
The invention also provides application of the method in preparing the optically active sulfoxide.
The invention also provides the application of the method in the preparation of epsilon-lactone.
Has the advantages that: the Baeyer-Villiger monooxygenase provided by the invention can be used as a catalyst to be applied to preparation of optically pure S-benzyl sulfoxide, and the enzyme has good solubility, mild applicable reaction conditions and environmental friendliness. The Baeyer-Villiger monooxygenase has good catalytic effect and wide substrate applicability, can catalyze cyclic ketone and thioether, and can tolerate high-concentration substrates. Can convert high-concentration thioanisole into S-benzyl sulfoxide, the conversion rate can reach more than 95%, the stereoselectivity is strong (e.e. > 96%), a new method is provided for the industrial large-scale production of the optically active sulfoxide, and the method has good application and development prospects.
Drawings
FIG. 1 is a physical map of pET28a-Rabvmo recombinant plasmid.
FIG. 2 is a protein electrophoresis image of recombinant Baeyer-Villiger monooxygenase enzyme; m, Marker; lanes 1, 2 and 3 are the supernatant, precipitate and pure enzyme of the recombinant genetically engineered bacterium BL21(DE3)/pET28a-Rabvmo after induction, respectively.
FIG. 3 is a liquid phase assay of a Baeyer-Villiger monooxygenase enzyme selectivity assay.
Detailed Description
Example 1: construction of recombinant Escherichia coli
The Baeyer-Villiger monooxygenase coding gene sequence shown in SEQ ID NO.1 is chemically synthesized and named as Rabvmo, and the amino acid sequence of the coded protein is shown in SEQ ID NO. 2.
After the plasmid pET28a is cut by restriction enzymes NdeI and BamHI, the nucleotide fragment of Rabvmo is ligated with the gene Rabvmo and the cut plasmid pET28 by T4 DNA ligase, and then the recombinant expression vector pET28a-Rabvmo (figure 1) is obtained. Transferring the constructed recombinant expression vector pET28a-Rabvmo into escherichia coli BL21(DE3) competence, coating an LB solid plate containing kanamycin resistance, performing colony PCR verification after overnight culture, and obtaining a positive clone, namely the recombinant escherichia coli BL21(DE3)/pET28 a-Rabvmo. Selecting positive clones, culturing overnight in LB culture medium, transferring into fresh LB culture medium according to transfer amount of 1mL/100mL the next day, and culturing to OD600When the concentration reaches 0.6-0.8, 0.2 mmol/L is added-1IPTG, induced culture at 30 ℃ for 6 hours, and centrifugation at 8000r/min for 10min at 4 ℃ to collect the thalli. The collected cells were suspended in potassium phosphate buffer (100 mmol. multidot.L)-1pH 8.0), sonicated and the protein expression was analyzed by SDS-PAGE (fig. 2).
As can be seen from FIG. 2, all of the target proteins were found in the supernatant, indicating that the recombinant enzyme was successfully expressed in E.coli.
Example 2: properties of the Baeyer-Villiger monooxygenase
(1) Separation and purification of Baeyer-Villiger monooxygenase
Suspension of recombinant cells in solution A (20 mmol. L)-1Sodium phosphate, 500 mmol. L-1NaCl，20mmol·L-1Imidazole, pH 7.4), and obtaining a crude enzyme solution after ultrasonic crushing and centrifugation. The column used for purification was an affinity column HisTrap FF column (Nickel column), and the recombinant protein was usedThe histidine tag on the whites was used for affinity binding. First, the nickel column is equilibrated with solution A, the crude enzyme solution is loaded, the penetration peak is eluted with solution A, after equilibration, solution B (20 mmol. L) is used-1Sodium phosphate, 500 mmol. L- 1NaCl， 1000mmol·L-1Imidazole, pH 7.4) and eluting the recombinant protein bound on the nickel column to obtain the recombinant Baeyer-Villiger monooxygenase. The purified protein was subjected to enzyme activity assay (thioanisole as substrate) and SDS-PAGE analysis (FIG. 2). As can be seen from FIG. 2, after the nickel column purification, a single band is shown at about 61kDa, and there are few hetero-proteins, indicating that the nickel column purification effect is good. The purified cyclohexanone monooxygenase was then replaced with Tris-HCl (100 mmol. L.) using a HiTrap Desainting Desalting column (GE Healthcare)-1pH 9.0) buffer, and the next enzymatic property analysis was performed.
(2) Enzyme activity assay for Baeyer-Villiger monooxygenase
And (3) measuring the enzyme activity of the Baeyer-Villiger monooxygenase on the substrate of the thioanisole. The measuring system is as follows: appropriate amount of enzyme solution, 5 mmol. L-1Methyl sulfide. Standing and reacting for 15min at 30 ℃. After the reaction is finished, sampling and carrying out liquid phase detection. Liquid phase detection conditions: the chromatographic column is a chiral OD-H chromatographic column (250mm × 4.6mm × 5 μm), and the mobile phase is n-hexane: isopropyl alcohol (90: 10) at a flow rate of 0.2 to 1mL/min-1The detection wavelength is 254 nm.
Definition of enzyme activity unit (U):
the amount of enzyme required by Baeyer-Villiger monooxygenase to catalyze the formation of 1. mu. mol of benzylthionine at 30 ℃ is defined as one enzyme activity unit (U).
(3) Substrate profiling
And (3) measuring the enzyme activity of the Baeyer-Villiger monooxygenase (RaBVMO) for catalyzing different thioethers, wherein the enzyme activity measured by taking the thiobenzol as a substrate is 100 percent of a control, and the enzyme activity measured by other substrates is calculated by the percentage of the two. The measurement results are shown in table 1.
TABLE 1 substrate spectra of RaBVMO
Table 1 shows that the ravbvmo substrate spectrum is broad and can catalyze thioethers, cyclic ketones and linear ketones to form sulfoxides, lactones and esters, respectively.
(4) Optimum pH of enzyme
Preparation of 100 mmol. L-1Buffers at different pH: phosphate buffer (pH 6.0-9.0), Tris-HCl (8.0-9.0), glycine-NaOH buffer (pH 9.0-11.0). Then taking the thioanisole as a substrate to determine the relative enzyme activity of RaBVMO in buffers with different pH values. The optimum reaction pH of RaBVMO is Tris-HCl, 8.0-9.0, and the enzyme activity is 0.31-0.92 U.mg–1. In a glycine-NaOH buffer solution with the pH value of 1.0-11, the enzyme activity is reduced to below 25%.
(5) Optimum temperature of enzyme
And respectively taking the thiobenzol as a substrate, determining the enzyme activity of the RaBVMO reacting for 15min at different temperatures (20-55 ℃), determining the highest enzyme activity to be 100%, and calculating the enzyme activities measured at other temperatures according to the percentage relative to the highest activity. The result shows that the optimum reaction temperature of RaBVMO is 30-40 ℃, and the enzyme activity can be kept at 0.90-1.16 U.mg–1。
TABLE 2 enzyme Activity of RaBVMO at different temperatures
(6) Thermostability of the enzyme
The thermal stability of RaBVMO at 30 ℃ was determined using thioanisole as substrate, and the initial activity of 0 hour was defined as 100%, and the measured activities at other time points were calculated as percentages relative to the initial activity of 0 hour. The result shows that the enzyme activity of the RaBVMO can be kept above 98% after 8 hours at 30 ℃, 86% after 16 hours and reduced to 50% of the initial activity after 25 hours.
TABLE 3 thermal stability of RaBVMO
(7) Analysis of kinetic parameters
Kinetic parameters of RaBVMO on the substrate thioanisole are determined. The enzyme activity assay system is listed as follows: Tris-HCl buffer (100 mmol. L)-1pH 9.0), thiobenzole (0-15 mmol. multidot.L)-1). The reaction rate was characterized by calculating the specific enzyme activity, and thus the kinetic parameters were calculated. The kinetic parameters of the RaBVMO to the substrate of the thioanisole are respectively Km0.158 mmol. multidot.L-1，VmaxIs 1.890. mu. mol/min–1·mg–1。
(8) Effect of Metal ions on enzyme Activity
The final concentration is 1 mmol.L-1The metal ions in the form of chloride salt of (2) were added to a pure enzyme solution, incubated at 30 ℃ for 15min, and then added to PBS buffer (100 mmol. multidot.L)-1And pH 8.0) and taking the thiobenzol as a substrate to determine the residual enzyme activity. Under the same condition, the enzyme activity measured without adding any metal ion is 100%, and the enzyme activity measured with adding metal ion is calculated by the percentage of the contrast. The results are shown in Table 2.
TABLE 4 Effect of Metal ions on the enzyme Activity of Baeyer-Villiger monooxygenase
As can be seen from Table 4, the activity of the Baeyer-Villiger monooxygenase enzyme was not inhibited when EDTA was added, and thus the enzyme was a non-metal ion dependent enzyme.
(9) Stereoselective analysis
The selectivity of the RaBVMO catalytic substrate, thioanisole, was determined. The reaction system (10mL) was: purified enzyme solution of appropriate amount, 5 mmol. multidot.L-1Methyl sulfide. Standing and reacting for 15min at 30 ℃. After the reaction is finished, sampling and carrying out liquid phase detection. Detection conditions are as follows:chiral OD-H chromatographic column (250mm × 4.6mm × 5 μm), detection wavelength 254nm, mobile phase n-hexane: isopropanol (90: 10) at a flow rate of 0.2-1 mL/min. As can be seen from FIG. 3, the enzyme has very good stereoselectivity, and only S-benzylsulfoxide is produced.
Example 3: application of Baeyer-Villiger monooxygenase in preparation of S-benzyl sulfoxide
Taking 2-500U of the obtained recombinant Baeyer-Villiger monooxygenase and 2-500U of glucose dehydrogenase (GDH, obtained from Novonoprazan, 5U/g) in Tris-HCl buffer (pH 9, 100 mmol. L)-1) Adding 5% methanol into the mixture, wherein the concentration of the mixture in a reaction system is 30-100 mmol.L-1The total volume of the reaction mixture was 10mL (Table 5). The reaction was placed at 30 ℃ and samples were taken to examine the conversion process under the following conditions: chiral OD-H chromatographic column (250mm × 4.6mm × 5 μm), detection wavelength 254nm, mobile phase n-hexane: isopropanol (90: 10) at a flow rate of 0.2-1 mL/min.
And sampling and detecting the reaction process in real time, and finishing the reaction when the product is detected not to increase any more.
The results are shown in Table 5, the Baeyer-Villiger monooxygenase can keep good catalytic performance under high substrate concentration, the conversion rate can be kept above 95%, and the yield can reach 99.5%.
TABLE 5 BV monooxygenase preparation of S-benzyl sulfoxide
Example 4: application of Baeyer-Villiger monooxygenase in preparation of epsilon-lactone
Taking 2-500U of the obtained recombinant Baeyer-Villiger monooxygenase and 2-500U of glucose dehydrogenase (GDH, obtained from Novonoprazan, 5U/g) in Tris-HCl buffer (pH 9, 100 mmol. L)-1) Adding 5% methanol into the mixture, wherein the concentration of the methanol in the reaction system is 50-200 mmol.L-1The total volume of the reaction solution was 10 mL. The reaction was placed at 30 ℃ and samples were taken to examine the conversion process under the following conditions: shimadzu gas GC-2014, achiral column: AT-SE-54(30 m.times.0.25 mm. times.0.33 μm), in parts by weightThe analytical procedure was as follows: maintaining at 100 deg.C for 1min, increasing to 180 deg.C at a speed of 10 deg.C/min, and maintaining at 180 deg.C for 3 min. And sampling and detecting the reaction process in real time, and finishing the reaction when the product is detected not to increase any more.
The results are shown in Table 6, the Baeyer-Villiger monooxygenase enzyme can keep good catalytic performance under high substrate concentration, and the conversion rate can be kept above 95%.
TABLE 6 BV monooxygenase preparation of epsilon-lactones
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> Baeyer-Villiger monooxygenase and application thereof
<130> BAA201283A
<160> 2
<170> PatentIn version 3.3
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tacccgggcg cgcggaccga ctgcgagggg tactactact gctactcgtt cgacccggag 240
atgctgcagc agtggaactg gacggagcgg tatcccactc agccggagat gcgggcctac 300
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gcgcgttacg acgaggtatg ggccaaagtg cgccggcact ggttctcgtt cccgttcgac 780
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ggctgggcaa acggcggatt cccgttcctg ttcaccttcg acgatctgct gttcgatccc 900
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Met Ser Ala Ser Ala Thr Gly Glu Ile Thr Ala Gln Gln Pro Gly Arg
1 5 10 15
Glu Val Asp Ala Val Val Val Gly Ala Gly Phe Gly Gly Leu Tyr Met
20 25 30
Val His Arg Leu Arg Glu Met Gly Leu Thr Val Gln Gly Tyr Glu Ser
35 40 45
Ala Pro Asp Val Gly Gly Thr Trp Cys Trp Asn Gly Tyr Pro Gly Ala
50 55 60
Arg Thr Asp Cys Glu Gly Tyr Tyr Tyr Cys Tyr Ser Phe Asp Pro Glu
65 70 75 80
Met Leu Gln Gln Trp Asn Trp Thr Glu Arg Tyr Pro Thr Gln Pro Glu
85 90 95
Met Arg Ala Tyr Phe Gly Tyr Val Ala Asp Lys Leu Asp Leu Arg Arg
100 105 110
Ser Tyr Arg Phe Gly Thr Arg Val Glu Ala Ala Val Phe Asp Glu Asp
115 120 125
Ser Gly Arg Trp His Val Arg Thr Glu Ala Gly Glu Arg Thr Ser Ala
130 135 140
Thr Tyr Leu Ile Thr Ala Val Gly Ile Leu Ser Ala Pro Asn Leu Pro
145 150 155 160
Asn Phe Pro Gly Val Glu Ser Phe Glu Gly Gln Trp Tyr His Thr Ala
165 170 175
Tyr Trp Pro Glu Glu Gly Val Asp Leu Ala Gly Lys Arg Val Gly Ile
180 185 190
Ile Gly Thr Gly Ser Thr Gly Val Gln Ala Ile Pro Leu Leu Ala Glu
195 200 205
Gln Ala Glu His Leu Thr Val Phe Gln Arg Thr Pro Asn Tyr Val Ile
210 215 220
Pro Ala Arg Asn Arg Pro Val Ser Gln Gln Glu Met Asp Glu Val Lys
225 230 235 240
Ala Arg Tyr Asp Glu Val Trp Ala Lys Val Arg Arg His Trp Phe Ser
245 250 255
Phe Pro Phe Asp Met Ala Asn Met Leu Ala Gly Ser Thr Glu Glu Glu
260 265 270
Glu Arg Thr Arg Ile Tyr Glu Gln Gly Trp Ala Asn Gly Gly Phe Pro
275 280 285
Phe Leu Phe Thr Phe Asp Asp Leu Leu Phe Asp Pro Val Ala Asn Glu
290 295 300
Ser Ala Ala Glu Phe Val Arg Thr Lys Ile Arg Ala Ala Val Glu Asp
305 310 315 320
Pro Ala Val Ala Glu Leu Leu Cys Pro Arg Tyr Pro Phe Gly Ala Lys
325 330 335
Arg Pro Pro Ser Gly Thr Gly Tyr Tyr Glu Thr Phe Asn Arg Asp Asn
340 345 350
Val Ala Leu Val Asp Val Ala Thr Asn Ala Ile Ala Glu Ile Thr Pro
355 360 365
Arg Gly Val Arg Leu Ala Asp Gly Thr Glu His Gln Val Asp Val Leu
370 375 380
Val Phe Ala Thr Gly Phe Asp Ala Ser Thr Gly Ala Leu Thr Arg Met
385 390 395 400
Asn Ile Val Gly Arg Asp Gly Arg Val Leu Ala Asp Lys Trp Ala Pro
405 410 415
Gly Pro Ser Thr His Leu Gly Ile Gly Thr His Gly Phe Pro Asn Met
420 425 430
Phe Met Ile Thr Gly Pro Gln Ser Pro Phe Thr Asn Ile Pro Pro Cys
435 440 445
Ala Gln Asn Thr Ala Asp Trp Ile Ala Glu Ala Ile Ala His Leu Arg
450 455 460
Arg Glu Gly Ala Thr Arg Met Glu Ala Thr Glu Ala Ala Glu Gln Ala
465 470 475 480
Trp Thr Glu Gln Ile Thr Ala Ile Ala Glu Gln Thr Leu Leu Thr Ala
485 490 495
Gly Lys Asp Val His Ser Trp Phe Thr Gly Thr Asn Val Asp Gly Lys
500 505 510
Ala Ala Val Ile Asn Val Phe Phe Gly Gly Ala Asp Lys Tyr Met Asp
515 520 525
Ile Cys Glu Gln Val Ala Ala Asp Asn Tyr Ser Gly Phe Glu Ile Thr
530 535 540
Thr Thr Glu Pro Ala Tyr Ala Arg
545 550
Claims (10)
1. A method for preparing optically active sulfoxide is characterized in that monooxygenase with an amino acid sequence shown as SEQ ID NO.2 is used as a catalyst to catalyze thioether to generate optically active sulfoxide.
2. The method of claim 1, wherein the optically active sulfoxide comprises benzyl sulfoxide, p-chlorobenzyl sulfoxide, p-methoxybenzyl sulfoxide, p-aminobenzyl sulfoxide.
3. The method of claim 2, wherein S-benzylsulfoxide is catalytically produced using thioanisole as a substrate.
4. The method according to claim 3, wherein the concentration of the thioanisole in the reaction system is 30-100 mmol-L-1。
5. The method of claim 4, wherein the monooxygenase enzyme is present at 30-100 kU-L-1The amount of (B) is added to the reaction system.
6. The process according to claim 3, wherein the reaction is carried out at 25 to 40 ℃ for 2 to 30 hours.
7. The application of monooxygenase in preparing S-benzyl sulfoxide is characterized in that the amino acid sequence of the monooxygenase is shown as SEQ ID NO. 2.
8. The use according to claim 7, wherein S-benzylsulfoxide is prepared using thioanisole as substrate and the monooxygenase as catalyst.
9. The use according to claim 8, wherein the reaction system further comprises an alcohol dehydrogenase.
10. Use of the method according to any one of claims 1 to 6 for the preparation of an optically active sulfoxide.
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| CN113430216A (en) * | 2021-07-26 | 2021-09-24 | 福州大学 | Propiophenone monooxygenase and application thereof in preparation of prazole drugs |
| CN114425136A (en) * | 2022-01-11 | 2022-05-03 | 江南大学 | Method for treating polyethylene by combining peroxide and cutinase |
| CN114480315A (en) * | 2022-02-16 | 2022-05-13 | 成都栩哲医药科技有限公司 | Baeyer-Villiger monooxygenase and application thereof in brivaracetam synthesis |
| CN115141814A (en) * | 2022-06-28 | 2022-10-04 | 江南大学 | Application of 4-hydroxyacetophenone monooxygenase |
| CN115305243A (en) * | 2022-06-28 | 2022-11-08 | 江南大学 | Baeyer-Villiger monooxygenase mutant and application thereof |
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| CN115141814A (en) * | 2022-06-28 | 2022-10-04 | 江南大学 | Application of 4-hydroxyacetophenone monooxygenase |
| CN115305243A (en) * | 2022-06-28 | 2022-11-08 | 江南大学 | Baeyer-Villiger monooxygenase mutant and application thereof |
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