CN114836395B - Thioether monooxygenase mutant and application thereof in preparation of chiral azole drugs - Google Patents
Thioether monooxygenase mutant and application thereof in preparation of chiral azole drugs Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- 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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
- C12P17/165—Heterorings having nitrogen atoms as the only ring heteroatoms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/90245—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/584—Recycling of catalysts
Abstract
The invention belongs to the technical field of bioengineering, and relates to a thioether monooxygenase mutant; nucleic acid encoding the thioether monooxygenase mutant, a recombinant expression vector containing the nucleic acid, and a recombinant expression transformant containing the recombinant expression vector; and the application of the recombinant thioether monooxygenase mutant in preparing chiral azole medicine. Compared with other biocatalysts for preparing optically pure tetrazole medicaments, the thioether monooxygenase mutant provided by the invention has the advantages of high catalytic activity, strong substrate specificity, good thermal stability, wide catalytic substrate range and high stereoselectivity, and has wide application prospect in industrial application.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a thioether monooxygenase mutant, nucleic acid for encoding the thioether monooxygenase mutant, a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the recombinant expression vector, preparation of a mutant enzyme preparation and application of the mutant enzyme preparation in preparation of chiral azole drugs.
Background
Proton pump inhibitors (Proton pump inhibitors, PPIs) are a class of drugs of choice widely used in the treatment of gastric acid digestive diseases. PPIs drugs mainly include omeprazole marketed in sweden in 1988, lansoprazole marketed in japan in 1995, pantoprazole marketed in germany in 1997, rabeprazole marketed in the united states in 1999, etc., and these drugs are initially marketed in the form of racemates and hold a significant role in the pharmaceutical market. By 2001, the first chiral PPI esomeprazole (the levoisomer of omeprazole) was marketed in the united states in weight pounds, after which it was last ten years to occupy the top ten of the global sales of drugs. In 2009, dexlansoprazole was approved by the us FDA for marketing, which is also the second chiral PPI to follow esomeprazole. Generally, chiral PPIs have stronger acid inhibition strength, longer acid inhibition time and higher bioavailability than racemic PPIs. Thus, chiral PPIs are increasingly becoming specific drugs for the treatment of gastric acid digestive diseases.
Although chiral PPIs are industrially synthesized by a chemical method at present, the problems of expensive chiral catalysts, poor stereoselectivity and the like commonly existing in the chemical method synthesis are unavoidable; the substances such as a large amount of peroxyacid, H 2O2, an organic catalyst, an organic solvent and the like used in the chemical synthesis method can cause serious injury to the physical and psychological health of workers and the living environment of human beings; the chemical reaction conditions are relatively harsh, the requirements on production equipment are high, and the production input cost is increased. In order to respond to the time call of green life and green manufacture, the biosynthesis method with the advantages of good catalytic selectivity, mild reaction condition, green and environment-friendly reaction system is gradually a beneficial supplement of the chemical synthesis method.
In the current biological synthesis route of PPIs, prochiral thioether is mainly used as a substrate, and whole cells, free enzyme and the like are used as catalysts for catalyzing asymmetric oxidation to obtain optically pure PPIs. The biocatalysts that have been reported to date as useful for PPIs synthesis are mainly: 1) Mold Cunninghamella echinulata MK, which can catalyze the conversion of 7.5mM rabeprazole thioether, the conversion rate is 92% after 144 hours of reaction, the product is (S) -rabeprazole, and when the omeprazole thioether and lansoprazole thioether are used as substrates, the conversion rate is only 45% and 0.6% respectively; 2) The bacillus Lysinibacillus sp.B71 growing cells can catalyze 0.1g/L omeprazole thioether and pantoprazole thioether to generate esomeprazole, but the conversion rate is only 70% and 8% respectively, and the bacillus Lysinibacillus sp.B71 growing cells have no catalytic activity on tested ilaprazole thioether and lansoprazole thioether; 3) Cyclohexanone monooxygenase mutants from Acinetobacter calcoaceticus, which can catalyze the asymmetric oxidation of 15mM omeprazole to produce esomeprazole (99% ee); 4) The cyclohexanone monooxygenase mutant from Acinetobacter calcoaceticus NCIMB 9871, disclosed in patent WO2011/071982, can efficiently catalyze omeprazole thioether to synthesize esomeprazole by modifying the cyclohexanone monooxygenase mutant from the company Codexis through directed evolution, but has very low activity on lansoprazole and poor stereoselectivity; 5) Patent CN 112725297A discloses that thioether monooxygenase CbSMO from Cupriavidus basilensis, which is oxidative active to all 14 measured lansoprazole thioether compounds, catalyzes the asymmetric oxidation of 10mM lansoprazole thioether to form dexlansoprazole (99% ee). Mutant CbSMO G266D/L313P has higher catalytic activity and thermal stability than the parent of CbSMO, but is still difficult to meet the requirements of industrial production.
Although the existing biocatalyst can catalyze the oxidation of substrates including lansoprazole thioether, the existing biocatalyst has the problems of low oxidation activity, poor thermal stability, poor substrate specificity (the product sulfoxide can be further oxidized to generate byproduct sulfone), low substrate loading and the like, and the requirements of industrial production are hardly met.
Disclosure of Invention
Aiming at the problems of low catalytic activity, poor thermal stability, poor substrate specificity, low substrate loading capacity and the like of a biocatalyst in the biological synthesis of a chiral sulfoxide drug dexlansoprazole sulfoxide, cbSMO G266D/L313P with higher activity on lansoprazole sulfide in a patent CN 112725297A (namely, replacement of 266 th amino acid Gly with Asp and replacement of 313 th amino acid Leu with Pro in an amino acid sequence shown in SEQ ID No.3 in a patent CN 112725297A) is selected as a target, and further molecular transformation is carried out on the target by a means of protein engineering, so as to provide a thioether monooxygenase mutant with obviously improved lansoprazole sulfide catalytic activity, substrate specificity and thermal stability, nucleic acid for encoding the thioether monooxygenase mutant, a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the recombinant expression vector, a recombinant thioether monooxygenase mutant catalyst and application of the recombinant thioether monooxygenase catalyst in preparation of chiral oxazoles.
The aim of the invention can be achieved by the following technical scheme:
According to one of the technical schemes, the invention provides a thioether monooxygenase mutant with obviously improved lansoprazole thioether catalytic activity, substrate specificity and thermal stability. The method is characterized in that (WT) with an amino acid sequence shown as SEQ ID No.2 is used, and a plurality of thioether monooxygenase mutants with obviously improved lansoprazole thioether catalytic activity, substrate specificity and thermal stability are obtained through identification by combining a high-throughput primary screening and further shaking bottle rescreening of an enzyme-labeled instrument through a random mutation and semi-rational design method.
The thioether monooxygenase mutant provided by the invention is a derivative protein of a novel amino acid sequence formed by replacing one or more amino acid residues in 98 th Asp, 252 th Gly, 253 th Met, 256 th Arg, 269 th Phe, 272 th Leu, 316 th Val, 485 th Leu, 486 th Met, 487 th Ala and 488 th Leu in an amino acid sequence shown in SEQ ID No.2 with other amino acid residues; the derivative protein has obviously improved oxidation activity to lansoprazole thioether and also has improved substrate specificity.
Preferably, the amino acid sequence of the thioether monooxygenase mutant is one of the following:
(1) The 98 th amino acid Asp in the amino acid sequence shown in SEQ ID No.2 is replaced by Met;
(2) The 98 th amino acid Asp in the amino acid sequence shown in SEQ ID No.2 is replaced by Gly;
(3) Substitution of amino acid Gly at position 252 in the amino acid sequence shown in SEQ ID No.2 with Met;
(4) Substitution of Gly at position 252 in the amino acid sequence shown in SEQ ID No.2 with Pro;
(5) The 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Val;
(6) The 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Pro;
(7) The 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln;
(8) The 256 th amino acid Arg in the amino acid sequence shown in SEQ ID No.2 is replaced by Met;
(9) The 256 th amino acid Arg in the amino acid sequence shown in SEQ ID No.2 is replaced by Thr;
(10) The 256 th amino acid Arg in the amino acid sequence shown in SEQ ID No.2 is replaced by Trp;
(11) Substitution of amino acid Phe at position 269 in the amino acid sequence shown in SEQ ID No.2 with His;
(12) The amino acid Phe at position 269 in the amino acid sequence shown in SEQ ID No.2 is replaced by Ser;
(13) The 272 th amino acid Leu in the amino acid sequence shown in SEQ ID No.2 is replaced by Lys;
(14) Substitution of Leu at amino acid 272 in the amino acid sequence shown in SEQ ID No.2 with Glu;
(15) Substitution of the 316 th amino acid Val in the amino acid sequence shown in SEQ ID No.2 with Ala;
(16) Substitution of the 316 th amino acid Val in the amino acid sequence shown in SEQ ID No.2 with Asp;
(17) The 485 th amino acid Leu in the amino acid sequence shown in SEQ ID No.2 is replaced by Phe;
(18) The 485 th amino acid Leu in the amino acid sequence shown in SEQ ID No.2 is replaced by Trp;
(19) Substitution of amino acid Met at position 486 in the amino acid sequence shown in SEQ ID No.2 with Leu;
(20) Substitution of amino acid Ala at position 487 in the amino acid sequence shown in SEQ ID No.2 with Asp;
(21) Substitution of amino acid Ala at position 487 in the amino acid sequence shown in SEQ ID No.2 with Glu;
(22) Substitution of amino acid Leu at position 488 in the amino acid sequence shown in SEQ ID No.2 with Thr;
(23) The 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Val at position 316 with Asp;
(24) The 256 th amino acid Arg in the amino acid sequence shown in SEQ ID No.2 is replaced by Thr; amino acid Ala at position 487 replaced with Glu; substitution of amino acid Leu at position 488 with Thr;
(25) The 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Leu at position 485 with Phe; substitution of amino acid Leu at position 488 with Thr;
(26) The 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Val at position 316 with Asp; substitution of amino acid Leu at position 488 with Thr;
(27) The 98 th amino acid Asp in the amino acid sequence shown in SEQ ID No.2 is replaced by Gly; substitution of amino acid Met at position 253 with gin; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Val at position 316 with Ala;
(28) The 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; amino acid Arg at position 256 is replaced by Met; substitution of amino acid Leu at position 485 with Phe; amino acid Ala at position 487 replaced with Glu; substitution of amino acid Leu at position 488 with Thr;
(29) Substitution of Gly at position 252 in the amino acid sequence shown in SEQ ID No.2 with Pro; substitution of amino acid Met at position 253 with gin; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Val at position 316 with Asp; amino acid Ala at position 487 replaced with Glu; substitution of amino acid Leu at position 488 with Thr;
(30) Substitution of Gly at position 252 in the amino acid sequence shown in SEQ ID No.2 with Pro; substitution of amino acid Met at position 253 with Pro; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Val at position 316 with Ala; amino acid Ala at position 487 replaced with Glu; substitution of amino acid Leu at position 488 with Thr;
(31) The 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; amino acid Leu at position 272 is replaced with Glu; substitution of amino acid Val at position 316 with Asp; substitution of amino acid Met at position 486 with Leu; amino acid Ala at position 487 is replaced with Asp; substitution of amino acid Leu at position 488 with Thr;
(32) Substitution of amino acid Gly at position 252 in the amino acid sequence shown in SEQ ID No.2 with Leu; amino acid Phe at position 269 is replaced by Ser; amino acid Leu at position 272 is replaced with Glu; substitution of amino acid Leu at position 485 with Trp; substitution of amino acid Met at position 486 with Leu; substitution of amino acid Leu at position 488 with Thr;
(33) The 98 th amino acid Asp in the amino acid sequence shown in SEQ ID No.2 is replaced by Gly; substitution of amino acid Gly at position 252 with Pro; substitution of amino acid Met at position 253 with gin; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Val at position 316 with Ala; amino acid Ala at position 487 replaced with Glu; substitution of amino acid Leu at position 488 with Thr;
(34) The 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Pro; amino acid Arg at position 256 is replaced by Met; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Val at position 316 with Asp; substitution of amino acid Met at position 486 with Leu; amino acid Ala at position 487 replaced with Glu; substitution of amino acid Leu at position 488 with Thr;
(35) Substitution of Gly at position 252 in the amino acid sequence shown in SEQ ID No.2 with Pro; substitution of amino acid Met at position 253 with Val; amino acid Arg at position 256 is replaced by Thr; amino acid Phe at position 269 is replaced by Ser; amino acid Leu at position 272 is replaced with Glu; substitution of amino acid Leu at position 485 with Phe; amino acid Ala at position 487 replaced with Glu; amino acid Leu at position 488 is replaced by Thr.
In a second aspect, the present invention provides a nucleic acid encoding the thioether monooxygenase mutant.
Any one of the thioether monooxygenase mutants according to one of the nucleic acid encoding schemes.
The nucleotide sequence for encoding the thioether monooxygenase mutant is a nucleic acid sequence for encoding the thioether monooxygenase mutant according to one of the technical schemes.
In a third aspect, the present invention provides a recombinant expression vector.
The recombinant expression vector comprises the nucleic acid according to the second technical scheme of the invention.
The recombinant expression vector can be connected with a pET28a plasmid through a conventional method in the field.
The invention provides a recombinant expression transformant.
The recombinant expression transformant comprises the recombinant expression vector according to the third technical scheme of the invention.
The recombinant expression transformant can be prepared by introducing the recombinant expression vector of the present invention into E.coli BL21 (DE 3) host cells by a conventional method in the art.
The invention provides a recombinant thioether monooxygenase mutant catalyst, which is in any one of the following forms:
(1) Culturing the recombinant expression transformant of the present invention, and isolating a transformant cell containing the thioether monooxygenase mutant;
(2) Disrupting the transformant cell according to (1), and separating a crude enzyme solution containing the thioether monooxygenase mutant;
(3) Lyophilizing the crude enzyme solution of (2) to obtain crude enzyme powder;
(4) An isolated thioether monooxygenase mutant according to one of the embodiments.
Wherein, for obtaining the recombinant thioether monooxygenase mutant catalyst, the culture method and the condition of the recombinant expression transformant are the methods and the conditions conventional in the field.
In some embodiments, the method of culturing the recombinant expression transformant may include the steps of: the recombinant expression transformant of the present invention is cultured to obtain a recombinant thioether monooxygenase mutant. For recombinant E.coli, the preferred medium is LB medium: 10g/L peptone, 5g/L yeast extract, 10g/L NaCl and pH 6.5-7.0. The preferred cultivation method is: recombinant E.coli constructed as described above was inoculated into LB medium containing Kanamycin (KANAMYCIN) and cultured overnight at 37℃with shaking at 180 rpm. Inoculating 1-2% (v/v) of LB culture medium (containing kanamycin) at 37 deg.C, shaking at 180rpm, culturing, adding isopropyl-beta-D-thiogalactoside (IPTG) with final concentration of 0.1-0.5 mM as inducer when OD 600 of culture solution reaches 0.6-0.8, and inducing at 16-30deg.C for 16-24 hr. The culture solution was centrifuged, and the pellet was washed twice with physiological saline to obtain recombinant expression transformant cells. And freeze-drying the obtained recombinant cells to obtain the freeze-dried cells containing the thioether monooxygenase mutant. Or suspending the obtained recombinant cells in buffer solution with the volume (v/w) of 5-10 times, carrying out ultrasonic crushing, centrifuging and collecting supernatant fluid, thus obtaining the recombinant thioether monooxygenase mutant crude enzyme solution. The collected crude enzyme solution is frozen at the temperature of minus 80 ℃, and is dried at low temperature by a vacuum freeze dryer, thus obtaining freeze-dried crude enzyme powder, and the freeze-dried crude enzyme powder is stored in a refrigerator at the temperature of 4 ℃ and can be conveniently used.
The invention provides a high-throughput screening method for a single oxygenase mutant of a tetrazole thioether. The high-throughput method is established according to the fact that the product of the lazole sulfoxide is degraded and developed when meeting acid, and prochiral lazole sulfide and a peroxidation byproduct of the lazole sulfone have no characteristics, and the drawing is shown in fig. 2. When the trazodone sulfide is used as a screening substrate, the thermal stability of CbSMO and the activity of the trazodone sulfide can be screened according to the yield of the product trazodone sulfone; if the lazole sulfoxide is used as a screening substrate, the substrate specificity of CbSMO can be screened according to the reduction amount of the lazole sulfoxide.
The invention provides a high-throughput screening method for the activity of the thioether monooxygenase mutant, which is shown in figure 3. Specifically, the recombinant thioether monooxygenase transformant was picked from the solid medium into a 96-well plate (primary culture plate) containing 200. Mu.L of LB medium (containing kanamycin) using a colony-picking robot, and cultured overnight at 37℃and 800 rpm. The recombinant thioether monooxygenase mutant was induced by transferring from the primary culture plate to a 96-well plate (secondary culture plate) containing 600. Mu.L of LB medium (containing kanamycin) using an automatic pipetting system, culturing at 37℃and 800rpm for 2-5 hours, then adjusting the temperature to 16-30℃and adding IPTG at a final concentration of 0.1-0.5 mM. After 12 to 24 hours, the supernatant is centrifuged, the precipitate is collected, 400 mu L of potassium phosphate buffer (0.1M, pH 8.0) containing 0.1 to 1.0g/L lysozyme is added to resuspend cells, and the cells are lysed for 1 to 3 hours at 25 to 40 ℃ and 800rpm to obtain cell lysate. Adding 0.2-1.0 mM lansoprazole thioether as a screening substrate, 10-50 mu L of methanol as a cosolvent, 50-90 mu L of potassium phosphate buffer solution dissolved with coenzyme NADPH (final concentration is 0.2-1.0 mM), and reacting at 25-35 ℃ and 800 rpm. After 2 hours, 500 mu L of ethyl acetate is added for oscillation extraction, 100 mu L of supernatant is taken after centrifugation and mixed with 100 mu L of formic acid, and the absorbance at 330nm is read on an enzyme label instrument, wherein the high absorbance indicates that the activity of the thioether monooxygenase mutant is higher.
The eighth technical scheme of the invention provides a high-throughput screening method for the thermal stability of the thioether monooxygenase mutant. Specifically, after the cell lysate is obtained by the method according to the seventh technical scheme of the invention, the cell lysate is stood and incubated for 5 hours at 40 ℃, and then the reactant is added for reaction and screening by the method according to the seventh technical scheme, wherein the heat stability of the thioether monooxygenase mutant is higher as shown by high light absorption value.
The invention provides a high-throughput screening method for substrate specificity of thioether monooxygenase mutant. Specifically, after obtaining a cell lysate according to the seventh embodiment of the present invention, 0.2-1.0 mM of dexlansoprazole sulfoxide is added as a screening substrate, 10-50. Mu.L of methanol is used as a cosolvent, 50-90. Mu.L of potassium phosphate buffer in which coenzyme NADPH (final concentration is 0.2-1.0 mM) is dissolved, and the reaction is carried out at 25-35℃and 800 rpm. After 2 hours, 500 mu L of ethyl acetate is added for oscillation extraction, 100 mu L of supernatant is mixed with 100 mu L of formic acid after centrifugation, and the absorption value at 330nm is read on an enzyme label instrument, wherein the high absorption value indicates that the oxidation amount of the oxazole sulfoxide is small, namely the substrate specificity of the thioether monooxygenase mutant to lansoprazole thioether is higher.
The invention provides a tenth technical scheme, and the application of the recombinant thioether monooxygenase mutant catalyst in catalyzing asymmetric oxidation of prochiral tetrazole thioether substrates.
In some embodiments of the invention, the enzymatic reaction is carried out in a buffer solution having a pH of 6.0 to 10.0 at 25 to 40℃and comprises lansoprazole thioether in a final concentration of 1 to 30g/L, methanol in an amount of 1 to 20% (v/v), sodium formate in an amount of 10 to 300mmol/L and NADP + in an amount of 0 to 1mmol/L, the recombinant thioether monooxygenase mutant catalyst according to claim five in an amount of 10 to 100U/L and crude dehydrogenase powder in an amount of 10 to 200U/L.
Preferably, the reaction is carried out with aeration and stirring, the aeration rate being from 0 to 2vvm, the reaction time being based on complete conversion of the substrate or no further increase in the product concentration.
In the reaction process, 0.5mL of reaction solution is sampled in a gap, 0.5mL of ethyl acetate is added for extraction, 0.3mL of extract is added with anhydrous sodium sulfate for drying, ethyl acetate is removed by volatilization, then 0.3mL of ethanol is added for dissolution, liquid phase analysis is carried out after filtration by a filter membrane with the aperture of 0.22 mu m, and the conversion rate of a substrate and the ee value of a product are determined under the following specific analysis conditions:
The chromatographic column is cellosolve CHIRALPAK IA, the mobile phase is n-heptane, ethanol=70:30 (v/v), and the flow rate is 0.5 mL-min -1; the column temperature was 40℃and the UV detection wavelength was 300nm.
Wherein the prochiral tetrazolium thioether substrate is selected from compounds shown in any one of the following chemical structural formulas:
In some embodiments of the invention, during the reaction, the coenzyme NADPH oxidizes to NADP +, catalyzing the reduction regeneration of NADP + to NADPH using a dehydrogenase.
In some embodiments of the invention, the dehydrogenase is any one of formate dehydrogenase, glucose dehydrogenase, or isopropanol dehydrogenase.
Further, the dehydrogenase of the present invention is any one of the following dehydrogenases:
(1) Formate dehydrogenase FDH (Appl Biochem Biotech 2020,192,530-543), which catalyzes the oxidation of formate with formate and NADP + as substrates, while NADP + is reduced to NADPH;
(2) Glucose dehydrogenase GDH (ChemBioChem 2020, 21:2680-2688), which catalyzes the oxidation of glucose with glucose and NADP + as substrates, while NADP + is reduced to NADPH;
(3) Alcohol dehydrogenase ADH (Tetrahedron Lett 2021, 84:153455), catalyzes the oxidation of short-chain alcohols with short-chain alcohols and NADP + as substrates, while NADP + is reduced to NADPH.
Compared with the prior art, the invention has the technical effects that:
Compared with other biocatalysts for preparing optically pure tetrazole medicaments, the thioether monooxygenase mutant provided by the invention has the advantages of high catalytic activity, strong substrate specificity, good thermal stability, wide catalytic substrate range and high stereoselectivity, and has wide application prospect in industrial application.
Drawings
FIG. 1 is a schematic diagram showing the reaction process of asymmetric oxidation and chiral sulfoxide peroxidation of a thioether monooxygenase mutant catalyzed prochiral oxazole thioether in the invention.
FIG. 2 is a schematic diagram of the high throughput screening principle of the present invention.
FIG. 3 is a schematic diagram of a high throughput screening process according to the present invention.
Detailed Description
The technical solutions and effects of the present invention will be clearly and completely described in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the scope of the present invention is not limited to these embodiments.
The media and detection methods involved in the following examples are as follows:
LB medium: peptone 10g/L, yeast extract 5g/L, naCl 10g/L (2% agar powder was added to the solid medium).
The chiral HPLC analysis method was as follows:
Instrument: shimadzu HPLC 2010A; chromatographic column model: CHIRALPAK IA; mobile phase: n-heptane: ethanol=70:30 (v/v); flow rate: 0.5mL min -1; column temperature: 40 ℃; sample injection amount: 10. Mu.L; a detector: an ultraviolet detector; detection wavelength: 300nm.
Example 1: random mutation screening CbSMO mutant with improved lansoprazole thioether activity
According to CbSMO open reading frame, the design of the upstream and downstream primers is as follows:
The upstream primer is shown as SEQ ID No. 3.
The downstream primer is shown as SEQ ID No. 4.
Wherein the upstream primer underlines the Nde I cleavage site and the downstream primer underlines the HindIII cleavage site.
A library of random mutants was created as described in example 7 of patent CN 112725297A using recombinant plasmid pET28a-CbSMO G266D/L313P as a template.
Wherein, the amino acid sequence of CbSMO G266D/L313P is shown as SEQ ID No.2, and the corresponding nucleotide sequence is shown as SEQ ID No. 1.
Briefly, error-prone PCR was performed with rTaq DNA polymerase to construct a random mutant pool. The target fragment was recovered after PCR amplification, and the target fragment was ligated with linearized empty plasmid pET28a and transformed into E.coli BL21 (DE 3) competent cells, which were spread evenly on LB agar plates containing 50. Mu.g/mL kanamycin, and incubated for about 12 hours in an incubator at 37 ℃. Transformants on the transformation plates were picked into 96-well deep well plates containing LB medium using a monoclonal autopick Qpix 450 and grown overnight at 37℃in a 800rpm shaker. The full-automatic liquid treatment workstation Freedom EVO is used for transferring seed liquid from a primary pore plate to a secondary pore plate for induced expression of mutant proteins. After the supernatant was removed by centrifugation of the fermentation broth, cells were lysed by adding cell lysate to each well, and then 25. Mu.L of lansoprazole thioether in methanol (lansoprazole thioether concentration: 1 mM) was added, and 75. Mu.L of potassium phosphate buffer in which coenzyme NADPH (concentration: 1 mM) was dissolved was reacted at 25℃and 800 rpm. After 2 hours, 500 mu L of ethyl acetate is added for oscillation extraction, 100 mu L of supernatant is taken after centrifugation and mixed with 100 mu L of formic acid, and the absorbance at 330nm is read on an enzyme label instrument, wherein the high absorbance indicates that the activity of the thioether monooxygenase mutant is higher. And purifying and characterizing the screened mutant, and sequencing the corresponding gene.
Example 2: random mutation screening CbSMO mutant with improved lansoprazole thioether substrate specificity
As the female parent CbSMO G266D/L313P can recognize the oxidation product, namely the dexlansoprazole sulfoxide, as a substrate in the process of catalyzing the oxidation of lansoprazole thioether to carry out peroxidation to generate a byproduct lansoprazole sulfone, the phenomenon of poor specificity to the lansoprazole thioether substrate is shown. To solve this problem, cbSMO mutants with improved substrate specificity for lansoprazole thioether were screened using a random mutagenesis approach.
Screening of CbSMO mutants with improved lansoprazole thioether substrate specificity can be achieved with minor modifications to the procedure of example 1. Specifically, the substrate lansoprazole thioether of the activity detection reaction in the example 1 is replaced by the dexlansoprazole sulfoxide, and the high absorbance value shows that the dexlansoprazole sulfoxide is more remained, namely CbSMO mutant has stronger substrate specificity on the lansoprazole thioether.
Example 3: random mutation screening for CbSMO mutants with improved thermostability
Screening of CbSMO mutants with improved thermostability was achieved with minor modifications to the procedure of example 1. Specifically, the crude enzyme solution after cleavage in example 1 was allowed to stand at 40 ℃ for 5 hours, and then subjected to activity detection reaction, and a high absorbance value indicates that the CbSMO mutant has high residual activity, i.e., the CbSMO mutant has high thermal stability.
Example 4: combination mutations of CbSMO beneficial mutants
Based on the mutations of example 1, example 2 and example 3, a plurality of dominant mutants with good activity, substrate specificity and thermal stability were obtained, respectively, and the dominant mutants were randomly combined by the method of DNA shuffling described in patent CN 112725297A to obtain DNA shuffling mutant library.
The high-throughput screening procedure of example 1 was used to screen DNA shuffling mutant libraries for a population of mutants with increased lansoprazole thioether activity (designated "mutant library a"), the high-throughput screening procedure of example 2 was used to screen mutant library a for a population of mutants with increased lansoprazole thioether substrate specificity (designated "mutant library B"), the high-throughput screening procedure of example 3 was used to screen mutant library B for a population of mutants with increased thermostability (designated "mutant library C"), and the pure enzyme specific activity, substrate specificity and thermostability of these mutants were characterized. Preferred are series of mutants with significantly increased activity of lansoprazole thioether and significantly increased ratio of activity of lansoprazole thioether to activity of dexlansoprazole sulfoxide, and good thermal stability, and the sequences of the mutants and the activity of the mutants on lansoprazole thioether and dexlansoprazole sulfoxide and the thermal melting temperature of the protein are listed in table 1, and the sequence numbers correspond to a series of sequences at the back of table 1 respectively. In the lansoprazole thioether activity column, compared with the female parent CbSMO G266D/L313P (the protein consisting of the amino acid sequence shown in SEQ ID No.2 in the sequence table), the activity of the mutant protein on lansoprazole thioether is improved by 0.5-1 times; the "++" indicates that the activity of the mutant protein on lansoprazole thioether is improved by 1-5 times; "+++" means mutant protein pair lansoprazole the activity of the azole thioether is improved by 5 to 10 times. In the activity ratio column, compared with the female parent CbSMO G266D/L313P, "+" indicates that the activity ratio of the mutant protein to lansoprazole thioether to the dexlansoprazole sulfoxide is improved by 0.1-1 times; the "++" indicates that the activity ratio of the mutant protein to lansoprazole thioether to the dextrorotatory lansoprazole sulfoxide is improved by 1-2 times; the "++ + +" indicates that the activity ratio of the mutant protein to lansoprazole thioether to the dextrorotatory lansoprazole sulfoxide is improved by 2-5 times. In the hot-melt temperature column, "+" indicates that the hot-melt temperature of the mutant protein is increased by 1-5 ℃ compared with the female parent CbSMO G266D/L313P; the "++" indicates that the hot melting temperature of the mutant protein is increased by 5-10 ℃; the "++ + +" indicates that the hot melting temperature of the mutant protein is increased by 10-20 ℃.
TABLE 1 thioether monooxygenase mutant sequences, fold increase in lansoprazole thioether activity, fold increase in lansoprazole thioether to dextral lansoprazole sulfoxide ratio, and degree increase in hot melt temperature
The amino acid sequences of thioether monooxygenase mutants corresponding to the sequence numbers are as follows:
(1) The 98 th amino acid Asp in the amino acid sequence shown in SEQ ID No.2 is replaced by Met;
(2) The 98 th amino acid Asp in the amino acid sequence shown in SEQ ID No.2 is replaced by Gly;
(3) Substitution of amino acid Gly at position 252 in the amino acid sequence shown in SEQ ID No.2 with Met;
(4) Substitution of Gly at position 252 in the amino acid sequence shown in SEQ ID No.2 with Pro;
(5) The 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Val;
(6) The 253 th amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Pro;
(7) The 253 th amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln;
(8) The 256 th amino acid Arg in the amino acid sequence shown in SEQ ID No.2 is replaced by Met;
(9) The 256 th amino acid Arg in the amino acid sequence shown in SEQ ID No.2 is replaced by Thr;
(10) The 256 th amino acid Arg in the amino acid sequence shown in SEQ ID No.2 is replaced by Trp;
(11) Substitution of amino acid Phe at position 269 in the amino acid sequence shown in SEQ ID No.2 with His;
(12) The amino acid Phe at position 269 in the amino acid sequence shown in SEQ ID No.2 is replaced by Ser;
(13) The 272 th amino acid Leu in the amino acid sequence shown in SEQ ID No.2 is replaced by Lys;
(14) Substitution of Leu at amino acid 272 in the amino acid sequence shown in SEQ ID No.2 with Glu;
(15) Substitution of the 316 th amino acid Val to Ala in the amino acid sequence shown in SEQ ID No. 2;
(16) Substitution of the 316 th amino acid Val with Asp in the amino acid sequence shown in SEQ ID No. 2;
(17) Substitution of the 485 th amino acid Leu in the amino acid sequence shown in SEQ ID No.2 with Phe;
(18) The 485 th amino acid Leu in the amino acid sequence shown in SEQ ID No.2 is replaced by Trp;
(19) Substitution of amino acid Met at position 486 in the amino acid sequence shown in SEQ ID No.2 with Leu;
(20) Substitution of amino acid Ala at position 487 with Asp in the amino acid sequence shown in SEQ ID No. 2;
(21) Substitution of amino acid Ala at position 487 in the amino acid sequence shown in SEQ ID No.2 with Glu;
(22) Substitution of amino acid Leu at position 488 in the amino acid sequence shown in SEQ ID No.2 with Thr;
(23) The 253 th amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Val at position 316 with Asp;
(24) The 256 th amino acid Arg in the amino acid sequence shown in SEQ ID No.2 is replaced by Thr; amino acid Ala at position 487 replaced with Glu; substitution of amino acid Leu at position 488 with Thr;
(25) The 253 th amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Leu at position 485 with Phe; substitution of amino acid Leu at position 488 with Thr;
(26) The 253 th amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Val at position 316 with Asp; substitution of amino acid Leu at position 488 with Thr;
(27) The 98 th amino acid Asp in the amino acid sequence shown in SEQ ID No.2 is replaced by Gly; substitution of amino acid Met at position 253 with gin; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Val at position 316 with Ala;
(28) The 253 th amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; amino acid Arg at position 256 is replaced by Met; substitution of amino acid Leu at position 485 with Phe; amino acid Ala at position 487 replaced with Glu; substitution of amino acid Leu at position 488 with Thr;
(29) Substitution of Gly at position 252 in the amino acid sequence shown in SEQ ID No.2 with Pro; substitution of amino acid Met at position 253 with gin; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Val at position 316 with Asp; amino acid Ala at position 487 replaced with Glu; substitution of amino acid Leu at position 488 with Thr;
(30) Substitution of Gly at position 252 in the amino acid sequence shown in SEQ ID No.2 with Pro; substitution of amino acid Met at position 253 with Pro; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Val at position 316 with Ala; amino acid Ala at position 487 replaced with Glu; substitution of amino acid Leu at position 488 with Thr;
(31) The 253 th amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; amino acid Leu at position 272 is replaced with Glu; substitution of amino acid Val at position 316 with Asp; substitution of amino acid Met at position 486 with Leu; amino acid Ala at position 487 is replaced with Asp; substitution of amino acid Leu at position 488 with Thr;
(32) Substitution of amino acid Gly at position 252 in the amino acid sequence shown in SEQ ID No.2 with Leu; amino acid Phe at position 269 is replaced by Ser; amino acid Leu at position 272 is replaced with Glu; substitution of amino acid Leu at position 485 with Trp; substitution of amino acid Met at position 486 with Leu; substitution of amino acid Leu at position 488 with Thr;
(33) The 98 th amino acid Asp in the amino acid sequence shown in SEQ ID No.2 is replaced by Gly; substitution of amino acid Gly at position 252 with Pro; substitution of amino acid Met at position 253 with gin; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Val at position 316 with Ala; amino acid Ala at position 487 replaced with Glu; substitution of amino acid Leu at position 488 with Thr;
(34) The 253 th amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Pro; amino acid Arg at position 256 is replaced by Met; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Val at position 316 with Asp; substitution of amino acid Met at position 486 with Leu; amino acid Ala at position 487 replaced with Glu; substitution of amino acid Leu at position 488 with Thr;
(35) Substitution of Gly at position 252 in the amino acid sequence shown in SEQ ID No.2 with Pro; substitution of amino acid Met at position 253 with Val; amino acid Arg at position 256 is replaced by Thr; amino acid Phe at position 269 is replaced by Ser; amino acid Leu at position 272 is replaced with Glu; substitution of amino acid Leu at position 485 with Phe; amino acid Ala at position 487 replaced with Glu; amino acid Leu at position 488 is replaced by Thr.
Example 5: activity determination of thioether monooxygenase mutant CbSMO M34 on lansoprazole thioether
The viability of CbSMO M34 on a series of tetrazolium thioether substrates was determined as follows: 0.5mL of the reaction system (0.1M KPB buffer, pH 8.0) was added with 0.2mM of the substrate of the tetrazole sulfide, 0.2mM of NADPH, the mixture was incubated at 30℃for 2 minutes, then added with an appropriate amount of purified pure enzyme solution, and the mixture was rapidly and uniformly mixed, reacted at 30℃and 1000rpm for 10 minutes, quenched with 0.5mL of ethyl acetate and extracted, and the supernatant after centrifugation was filtered with a filter membrane having a pore size of 0.22 μm and subjected to liquid phase analysis to determine the conversion of the substrate and the ee value of the product.
Specific analytical conditions for substrate conversion and ee value are as follows:
Instrument: shimadzu HPLC 2010A; chromatographic column model: CHIRALPAK IA; mobile phase: n-heptane: ethanol=70:30 (v/v); flow rate: 0.5mL min -1; column temperature: 40 ℃; sample injection amount: 10. Mu.L; a detector: an ultraviolet detector; detection wavelength: 300nm.
The enzyme activity unit is defined as: under the conditions, the amount of enzyme required to catalyze the conversion of 1. Mu. Mol of the substrate of the tetrazole thioether to form the sulfoxide of the tetrazole is reduced per minute.
The specific activity of the mutant CbSMO M34 to a series of trazosulfide substrates and the ee value of the products and the substrate specificity are shown in Table 2.
TABLE 2 determination of pure enzyme Activity, stereoselectivity and substrate specificity of CbSMO M34
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a“+”:500～1000U/g；“++”:1000～2000U/g；“+++”:>2000U/g
b“+”:10～20；“++”:20～50；“+++”:>50
Example 6: preparation of thioether monooxygenase crude enzyme solution
The preparation of a crude enzyme solution of thioether monooxygenase CbSMO M34 is exemplified. Recombinant E.coli containing CbSMO M34 was inoculated into LB medium containing Kanamycin (KANAMYCIN) and cultured overnight at 37℃with shaking at 180 rpm. Inoculating 1% (v/v) of the recombinant expression transformant into a 500mL Erlenmeyer flask containing 100mL of LB medium (containing kanamycin), shaking at 37℃and 180rpm, culturing, adding isopropyl-beta-D-thiogalactoside (IPTG) with a final concentration of 0.2mM as an inducer when the OD 600 of the culture solution reaches 0.6, inducing at 16℃for 24 hours, centrifuging the culture solution, collecting the precipitate, and washing twice with physiological saline to obtain the recombinant expression transformant cell. Suspending the obtained recombinant cells in buffer solution with the volume of 10 times (v/w), carrying out ultrasonic crushing, centrifuging and collecting supernatant fluid to obtain crude enzyme liquid of the recombinant thioether monooxygenase CbSM M34.
Example 7: preparation of thioether monooxygenase crude enzyme powder
The preparation of a crude enzyme powder of thioether monooxygenase CbSMO M34 is exemplified. The crude enzyme solution of the thioether monooxygenase mutant CbSMO M34 obtained in example 6 is collected, frozen at-80 ℃, and then dried at low temperature by using a vacuum freeze dryer, so that the thioether monooxygenase mutant CbSMO M34 crude enzyme powder with the specific activity of 122U/g for lansoprazole thioether and 198U/g for rabeprazole thioether can be obtained. The obtained crude enzyme powder is stored in a refrigerator at 4 ℃ and can be conveniently used.
Example 8: cbSMO G266D/L313P catalyzes 20g/L lansoprazole thioether asymmetric oxidation
The reaction process of the thioether monooxygenase mutant for catalyzing asymmetric oxidation of prochiral oxazole thioether and peroxidation of chiral sulfoxide is shown in figure 1.
To 100mL of the reaction system (potassium phosphate buffer, 200mmol/L, pH 8.0), cbSMO G266D/L313P crude enzyme powder 6U (crude enzyme powder prepared as described in example 7) and formate dehydrogenase FDH crude enzyme powder 12U were added, and lansoprazole sulfide, methanol, sodium formate, and NADP + were continuously added to the above system to final concentrations of 20g/L, 10% (v/v), 150mmol/L, and 0.2mmol/L, respectively. The reaction was shaken at 30℃and 220 rpm. 0.5mL of the reaction solution was intermittently sampled, 0.5mL of ethyl acetate was added for extraction, 0.3mL of the extract was dried over anhydrous sodium sulfate, ethyl acetate was removed by evaporation, and then 0.3mL of ethanol was added for dissolution, and the substrate conversion and the ee value of the product were determined by the HPLC analysis method described in example 5. The substrate conversion rate is more than 97% after the reaction is carried out for 32 hours, the ee value of the product is more than 99% (R), and the proportion of the byproduct lansoprazole sulfone accounting for the total product is 10.5%.
Example 9: cbSMO M31 catalyzes 20g/L lansoprazole thioether asymmetric oxidation
To 100mL of the reaction system (potassium phosphate buffer, 200mmol/L, pH 8.0), cbSMO M31 crude enzyme powder 6U (crude enzyme powder prepared as described in example 7) and formate dehydrogenase FDH crude enzyme powder 12U were added, and lansoprazole sulfide, methanol, sodium formate, and NADP + were continuously added to the above system to final concentrations of 20g/L, 10% (v/v), 150mmol/L, and 0.2mmol/L, respectively. The reaction was shaken at 30℃and 220 rpm. 0.5mL of the reaction solution was intermittently sampled, 0.5mL of ethyl acetate was added for extraction, 0.3mL of the extract was dried over anhydrous sodium sulfate, ethyl acetate was removed by evaporation, and then 0.3mL of ethanol was added for dissolution, and the substrate conversion and the ee value of the product were determined by the HPLC analysis method described in example 5. The substrate conversion rate is more than 99% after 24 hours of reaction, the ee value of the product is more than 99% (R), and the proportion of the byproduct lansoprazole sulfone is 1.8% of the total product.
Example 10: cbSMO M31 catalyzes 20g/L rabeprazole thioether asymmetric oxidation
To 100mL of the reaction system (potassium phosphate buffer, 200mmol/L, pH 8.0), cbSMO M31 crude enzyme powder 6U (crude enzyme powder prepared as described in example 7) and glucose dehydrogenase GDH crude enzyme powder 12U were added, and rabeprazole sulfide, methanol, sodium formate and NADP + were continuously added to the above system to a final concentration of 20g/L, 10% (v/v), 150mmol/L and 0.2mmol/L, respectively. The reaction was shaken at 30℃and 220 rpm. 0.5mL of the reaction solution was intermittently sampled, 0.5mL of ethyl acetate was added for extraction, 0.3mL of the extract was dried over anhydrous sodium sulfate, ethyl acetate was removed by evaporation, and then 0.3mL of ethanol was added for dissolution, and the substrate conversion and the ee value of the product were determined by the HPLC analysis method described in example 5. The substrate conversion rate is more than 99% after 19h reaction, the ee value of the product is more than 99% (R), and the proportion of the byproduct rabeprazole sulfone to the total product is 2.1%.
Example 11: cbSMO M31 catalyzes 20g/L pantoprazole sulfide asymmetric oxidation
To 100mL of the reaction system (potassium phosphate buffer, 200mmol/L, pH 8.0), cbSMO M31 crude enzyme powder 6U (crude enzyme powder prepared as described in example 7) and alcohol dehydrogenase ADH crude enzyme powder 12U were added, and pantoprazole sulfide, methanol, sodium formate and NADP + were continuously added to the above system to final concentrations of 20g/L, 10% (v/v), 150mmol/L and 0.2mmol/L, respectively. The reaction was shaken at 30℃and 220 rpm. 0.5mL of the reaction solution was intermittently sampled, 0.5mL of ethyl acetate was added for extraction, 0.3mL of the extract was dried over anhydrous sodium sulfate, ethyl acetate was removed by evaporation, and then 0.3mL of ethanol was added for dissolution, and the substrate conversion and the ee value of the product were determined by the HPLC analysis method described in example 5. The substrate conversion rate is more than 99% after 16h reaction, the ee value of the product is more than 99% (R), and the proportion of the byproduct pantoprazole sulfone to the total product is 2.4%.
Example 12: cbSMO M27 catalyzes 30g/L lansoprazole thioether asymmetric oxidation
To 100mL of the reaction system (potassium phosphate buffer, 200mmol/L, pH 8.0), cbSMO M27 crude enzyme powder 6U (crude enzyme powder prepared as described in example 7) and formate dehydrogenase FDH crude enzyme powder 12U were added, and lansoprazole sulfide, methanol, sodium formate, and NADP + were continuously added to the above system to final concentrations of 30g/L, 10% (v/v), 200mmol/L, and 0.2mmol/L, respectively. The reaction was shaken at 30℃and 220 rpm. 0.5mL of the reaction solution was intermittently sampled, 0.5mL of ethyl acetate was added for extraction, 0.3mL of the extract was dried over anhydrous sodium sulfate, ethyl acetate was removed by evaporation, and then 0.3mL of ethanol was added for dissolution, and the substrate conversion and the ee value of the product were determined by the HPLC analysis method described in example 5. The substrate conversion rate is more than 98 percent after 30 hours of reaction, the ee value of the product is more than 99 percent (R), and the proportion of the byproduct lansoprazole sulfone accounting for the total product is 1.3 percent.
Example 13: cbSMO M27 catalyzes 30g/L lansoprazole thioether asymmetric oxidation
To a 1L reaction system (potassium phosphate buffer, 200mmol/L, pH 8.0), cbSMO M27 crude enzyme powder 100U (crude enzyme powder prepared as described in example 7) and formic acid dehydrogenase FDH crude enzyme powder 200U were added, and lansoprazole sulfide, methanol, sodium formate and NADP + were continuously added to the above system to a final concentration of 30g/L, 10% (v/v), 200mmol/L and 0.2mmol/L, respectively. The reaction was stirred at 30℃at 150rpm with an aeration rate of 0.4 vvm. 0.5mL of the reaction solution was intermittently sampled, 0.5mL of ethyl acetate was added for extraction, 0.3mL of the extract was dried over anhydrous sodium sulfate, ethyl acetate was removed by evaporation, then 0.3mL of ethanol was added for dissolution, and the substrate conversion, the ee value of the product and the content of by-product sulfone were determined by the HPLC analysis method described in example 5. The substrate conversion rate is more than 99% after the reaction is carried out for 32 hours, the ee value of the product is more than 99% (R), and the proportion of the byproduct lansoprazole sulfone accounting for the total product is 1.8%.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Sequence listing
<110> University of Industy of Huadong
<120> Thioether monooxygenase mutant and application thereof in preparation of chiral trazole drugs
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1569
<212> DNA
<213> Artificial Sequence
<400> 1
atgctggcgg gtaacgcgga taaagcgcac ccgccggcgc gtcaggcgcc gcacgagcaa 60
gttcaccact tcgatgttct ggtggtgggt gcgggtctga gcggcatctg cgcggcgtac 120
cacctgcaga ccagctgccc gggtaaacgt tatgcgattc tggaaggtcg tgatgcggtg 180
ggtggcacct gggacctgtt tcgttacccg ggtgttcgta gcgacagcga tatgtatacc 240
ctgggcttca gctttcgtcc gtggcgtagc gataagagca tcgcggcggg tgacgcgatc 300
ctggagtaca ttcgtgatac cgcgcgtacc ttcgacatcg aacgtcacat tcgttttggt 360
catcgtgcga cccgtgcgag ctggagcagc gagaccgcgc gttggaccgt tgatgcggcg 420
gttggtccgc aaggtgaacc ggcgcgtttc acctgcaact ttctgtacct gtgcagcggt 480
tactatgatt atgcggatgg ttatatgccg ggttggccgg gtatggagcg ttttggtggc 540
cgtgtggttc acccgcagca ctggccggaa gatctggttt acgacgatca acgtgtggtt 600
gtgatcggta gcggtgcgac cgcggtgacc ctgctgccgg agatggcgaa acgtgcggcg 660
cacgttacca tgctgcaacg tagcccgacc tatattgttg cgcgtccgag cagcgatgcg 720
gttagcgcgt ggctgcgtcg taagctgccg gcgggtatgg cgcaccgtgt gacccgttgg 780
aaaaacgttc tgttcgatat gtacttttat cacctggcgc gtcgtaagcc ggagctggtg 840
aagcgtaaaa ttctggaagc ggcgcgtgcg cagctgggtc cggactacga tgttgacaag 900
cacttcagcc cggcgtataa accgtgggat caacgtccgt gcctggtgcc ggatagcgac 960
ctgttcaaaa gcatccgtgc gggtcgtgcg agcgttgtga ccgaccacat tgagagcttt 1020
accgaaaccg gtctgcagct gcgtagcggc caaaagctgg atgcggacgt tatcgtgacc 1080
gcgaccggtc tgcagctgaa agtggcgggt ggcatgcgta ttgaagttga tggtgtgccg 1140
gcggacccgg cgcaagcgtt tatgtacaag ggcatgatgt atagcgatgt tccgaacctg 1200
gcggtggcga tgggttacgt taacgcgagc tggaccctga aagcggagct gagcagcatg 1260
tatgtttgcc gtctgatcaa ccacatggag gcgaacggtc atgactggtg cgcgccgcgt 1320
cgtggtcatg cggcgggtga cgatgagccg agcctgagcc tgaccagcgg ttatgtgcag 1380
cgtgcgagcg gtattctgcc gcgtcaaggt agcaagcgtc cgtggcgtgt gcaccagaac 1440
tacctgttcg acctgatggc gctgaagttt ggtaaagttg aggacgatgc gatggaattt 1500
ggtcgtgcgg gcccggcggc gccgcgtgcg ccggcgccgg cgcgtgcggc ggaaagcgcg 1560
cacggctaa 1569
<210> 2
<211> 522
<212> PRT
<213> Cupriavidus basilensis
<400> 2
Met Leu Ala Gly Asn Ala Asp Lys Ala His Pro Pro Ala Arg Gln Ala
1 5 10 15
Pro His Glu Gln Val His His Phe Asp Val Leu Val Val Gly Ala Gly
20 25 30
Leu Ser Gly Ile Cys Ala Ala Tyr His Leu Gln Thr Ser Cys Pro Gly
35 40 45
Lys Arg Tyr Ala Ile Leu Glu Gly Arg Asp Ala Val Gly Gly Thr Trp
50 55 60
Asp Leu Phe Arg Tyr Pro Gly Val Arg Ser Asp Ser Asp Met Tyr Thr
65 70 75 80
Leu Gly Phe Ser Phe Arg Pro Trp Arg Ser Asp Lys Ser Ile Ala Ala
85 90 95
Gly Asp Ala Ile Leu Glu Tyr Ile Arg Asp Thr Ala Arg Thr Phe Asp
100 105 110
Ile Glu Arg His Ile Arg Phe Gly His Arg Ala Thr Arg Ala Ser Trp
115 120 125
Ser Ser Glu Thr Ala Arg Trp Thr Val Asp Ala Ala Val Gly Pro Gln
130 135 140
Gly Glu Pro Ala Arg Phe Thr Cys Asn Phe Leu Tyr Leu Cys Ser Gly
145 150 155 160
Tyr Tyr Asp Tyr Ala Asp Gly Tyr Met Pro Gly Trp Pro Gly Met Glu
165 170 175
Arg Phe Gly Gly Arg Val Val His Pro Gln His Trp Pro Glu Asp Leu
180 185 190
Val Tyr Asp Asp Gln Arg Val Val Val Ile Gly Ser Gly Ala Thr Ala
195 200 205
Val Thr Leu Leu Pro Glu Met Ala Lys Arg Ala Ala His Val Thr Met
210 215 220
Leu Gln Arg Ser Pro Thr Tyr Ile Val Ala Arg Pro Ser Ser Asp Ala
225 230 235 240
Val Ser Ala Trp Leu Arg Arg Lys Leu Pro Ala Gly Met Ala His Arg
245 250 255
Val Thr Arg Trp Lys Asn Val Leu Phe Asp Met Tyr Phe Tyr His Leu
260 265 270
Ala Arg Arg Lys Pro Glu Leu Val Lys Arg Lys Ile Leu Glu Ala Ala
275 280 285
Arg Ala Gln Leu Gly Pro Asp Tyr Asp Val Asp Lys His Phe Ser Pro
290 295 300
Ala Tyr Lys Pro Trp Asp Gln Arg Pro Cys Leu Val Pro Asp Ser Asp
305 310 315 320
Leu Phe Lys Ser Ile Arg Ala Gly Arg Ala Ser Val Val Thr Asp His
325 330 335
Ile Glu Ser Phe Thr Glu Thr Gly Leu Gln Leu Arg Ser Gly Gln Lys
340 345 350
Leu Asp Ala Asp Val Ile Val Thr Ala Thr Gly Leu Gln Leu Lys Val
355 360 365
Ala Gly Gly Met Arg Ile Glu Val Asp Gly Val Pro Ala Asp Pro Ala
370 375 380
Gln Ala Phe Met Tyr Lys Gly Met Met Tyr Ser Asp Val Pro Asn Leu
385 390 395 400
Ala Val Ala Met Gly Tyr Val Asn Ala Ser Trp Thr Leu Lys Ala Glu
405 410 415
Leu Ser Ser Met Tyr Val Cys Arg Leu Ile Asn His Met Glu Ala Asn
420 425 430
Gly His Asp Trp Cys Ala Pro Arg Arg Gly His Ala Ala Gly Asp Asp
435 440 445
Glu Pro Ser Leu Ser Leu Thr Ser Gly Tyr Val Gln Arg Ala Ser Gly
450 455 460
Ile Leu Pro Arg Gln Gly Ser Lys Arg Pro Trp Arg Val His Gln Asn
465 470 475 480
Tyr Leu Phe Asp Leu Met Ala Leu Lys Phe Gly Lys Val Glu Asp Asp
485 490 495
Ala Met Glu Phe Gly Arg Ala Gly Pro Ala Ala Pro Arg Ala Pro Ala
500 505 510
Pro Ala Arg Ala Ala Glu Ser Ala His Gly
515 520
<210> 3
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 3
ccgcgcggca gccatatgct ggcgggtaac 30
<210> 4
<211> 37
<212> DNA
<213> Artificial Sequence
<400> 4
cgagtgcggc cgcaagcttt tagccgtgcg cgctttc 37
Claims (9)
1. A thioether monooxygenase mutant, characterized in that the amino acid sequence of the thioether monooxygenase mutant is one of the following:
(1) The 98 th amino acid Asp in the amino acid sequence shown in SEQ ID No.2 is replaced by Met;
(2) The 98 th amino acid Asp in the amino acid sequence shown in SEQ ID No.2 is replaced by Gly;
(3) Substitution of amino acid Gly at position 252 in the amino acid sequence shown in SEQ ID No.2 with Met;
(4) Substitution of Gly at position 252 in the amino acid sequence shown in SEQ ID No.2 with Pro;
(5) The 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Val;
(6) The 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Pro;
(7) The 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln;
(8) The 256 th amino acid Arg in the amino acid sequence shown in SEQ ID No.2 is replaced by Met;
(9) The 256 th amino acid Arg in the amino acid sequence shown in SEQ ID No.2 is replaced by Thr;
(10) The 256 th amino acid Arg in the amino acid sequence shown in SEQ ID No.2 is replaced by Trp;
(11) Substitution of amino acid Phe at position 269 in the amino acid sequence shown in SEQ ID No.2 with His;
(12) The amino acid Phe at position 269 in the amino acid sequence shown in SEQ ID No.2 is replaced by Ser;
(13) The 272 th amino acid Leu in the amino acid sequence shown in SEQ ID No.2 is replaced by Lys;
(14) Substitution of Leu at amino acid 272 in the amino acid sequence shown in SEQ ID No.2 with Glu;
(15) Substitution of the 316 th amino acid Val in the amino acid sequence shown in SEQ ID No.2 with Ala;
(16) Substitution of the 316 th amino acid Val in the amino acid sequence shown in SEQ ID No.2 with Asp;
(17) The 485 th amino acid Leu in the amino acid sequence shown in SEQ ID No.2 is replaced by Phe;
(18) The 485 th amino acid Leu in the amino acid sequence shown in SEQ ID No.2 is replaced by Trp;
(19) Substitution of amino acid Met at position 486 in the amino acid sequence shown in SEQ ID No.2 with Leu;
(20) Substitution of amino acid Ala at position 487 in the amino acid sequence shown in SEQ ID No.2 with Asp;
(21) Substitution of amino acid Ala at position 487 in the amino acid sequence shown in SEQ ID No.2 with Glu;
(22) Substitution of amino acid Leu at position 488 in the amino acid sequence shown in SEQ ID No.2 with Thr;
(23) The 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Val at position 316 with Asp;
(24) The 256 th amino acid Arg in the amino acid sequence shown in SEQ ID No.2 is replaced by Thr; amino acid Ala at position 487 replaced with Glu; substitution of amino acid Leu at position 488 with Thr;
(25) The 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Leu at position 485 with Phe; substitution of amino acid Leu at position 488 with Thr;
(26) The 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Val at position 316 with Asp; substitution of amino acid Leu at position 488 with Thr;
(27) The 98 th amino acid Asp in the amino acid sequence shown in SEQ ID No.2 is replaced by Gly; substitution of amino acid Met at position 253 with gin; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Val at position 316 with Ala;
(28) The 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; amino acid Arg at position 256 is replaced by Met; substitution of amino acid Leu at position 485 with Phe; amino acid Ala at position 487 replaced with Glu; substitution of amino acid Leu at position 488 with Thr;
(29) Substitution of Gly at position 252 in the amino acid sequence shown in SEQ ID No.2 with Pro; substitution of amino acid Met at position 253 with gin; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Val at position 316 with Asp; amino acid Ala at position 487 replaced with Glu; substitution of amino acid Leu at position 488 with Thr;
(30) Substitution of Gly at position 252 in the amino acid sequence shown in SEQ ID No.2 with Pro; substitution of amino acid Met at position 253 with Pro; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Val at position 316 with Ala; amino acid Ala at position 487 replaced with Glu; substitution of amino acid Leu at position 488 with Thr;
(31) The 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; amino acid Leu at position 272 is replaced with Glu; substitution of amino acid Val at position 316 with Asp; substitution of amino acid Met at position 486 with Leu; amino acid Ala at position 487 is replaced with Asp; substitution of amino acid Leu at position 488 with Thr;
(32) Substitution of amino acid Gly at position 252 in the amino acid sequence shown in SEQ ID No.2 with Leu; amino acid Phe at position 269 is replaced by Ser; amino acid Leu at position 272 is replaced with Glu; substitution of amino acid Leu at position 485 with Trp; substitution of amino acid Met at position 486 with Leu; substitution of amino acid Leu at position 488 with Thr;
(33) The 98 th amino acid Asp in the amino acid sequence shown in SEQ ID No.2 is replaced by Gly; substitution of amino acid Gly at position 252 with Pro; substitution of amino acid Met at position 253 with gin; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Val at position 316 with Ala; amino acid Ala at position 487 replaced with Glu; substitution of amino acid Leu at position 488 with Thr;
(34) The 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Pro; amino acid Arg at position 256 is replaced by Met; amino acid Phe at position 269 is replaced by Ser; substitution of amino acid Val at position 316 with Asp; substitution of amino acid Met at position 486 with Leu; amino acid Ala at position 487 replaced with Glu; substitution of amino acid Leu at position 488 with Thr;
(35) Substitution of Gly at position 252 in the amino acid sequence shown in SEQ ID No.2 with Pro; substitution of amino acid Met at position 253 with Val; amino acid Arg at position 256 is replaced by Thr; amino acid Phe at position 269 is replaced by Ser; amino acid Leu at position 272 is replaced with Glu; substitution of amino acid Leu at position 485 with Phe; amino acid Ala at position 487 replaced with Glu; amino acid Leu at position 488 is replaced by Thr.
2. An isolated nucleic acid encoding any one of the thioether monooxygenase mutants of claim 1.
3. A recombinant expression vector comprising the nucleic acid of claim 2.
4. A recombinant expression transformant comprising the recombinant expression vector of claim 3.
5. A recombinant thioether monooxygenase mutant catalyst, characterized in that the recombinant thioether monooxygenase mutant catalyst is in any one of the following forms:
(1) Culturing the recombinant expression transformant of claim 4, and isolating a transformant cell containing the thioether monooxygenase mutant;
(2) Disrupting the transformant cells of (1), and separating a crude enzyme solution containing the thioether monooxygenase mutant;
(3) Freeze-drying the crude enzyme liquid of the step (2) to obtain crude enzyme powder;
(4) The thioether monooxygenase mutant of claim 1.
6. Use of the recombinant thioether monooxygenase mutant catalyst according to claim 5, for catalyzing the asymmetric oxidation of a prochiral tetrazolium thioether substrate, wherein the prochiral tetrazolium thioether substrate is selected from the group consisting of compounds represented by any one of the following chemical formulas:
7. The use according to claim 6, wherein the recombinant thioether monooxygenase mutant catalyst catalyzes the oxidation of a prochiral trazosulfide substrate to the corresponding chiral sulfoxide using oxygen as an oxidant.
8. The use according to claim 6, characterized in that during the reaction the coenzyme NADPH is oxidized to NADP +, and NADP + is catalytically reduced and regenerated to NADPH using a dehydrogenase.
9. The use according to claim 8, wherein the dehydrogenase is any one of formate dehydrogenase, glucose dehydrogenase or isopropanol dehydrogenase.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102834386A (en) * | 2009-09-02 | 2012-12-19 | 帝斯曼知识产权资产管理有限公司 | Polypeptides having oxidoreductase activity and their uses |
| CN108570425A (en) * | 2018-03-08 | 2018-09-25 | 华东理工大学 | A kind of slow raw rhizobium monooxygenase and its application in preparing chiral sulfoxide |
| CN111218431A (en) * | 2018-11-26 | 2020-06-02 | 华东理工大学 | Monooxygenase and application thereof in preparation of optically pure sulfoxide |
| CN112725297A (en) * | 2021-01-29 | 2021-04-30 | 华东理工大学 | Thioether monooxygenase and application thereof in preparation of chiral prazole medicine |
| CN113430216A (en) * | 2021-07-26 | 2021-09-24 | 福州大学 | Propiophenone monooxygenase and application thereof in preparation of prazole drugs |
-
2022
- 2022-04-28 CN CN202210493819.0A patent/CN114836395B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102834386A (en) * | 2009-09-02 | 2012-12-19 | 帝斯曼知识产权资产管理有限公司 | Polypeptides having oxidoreductase activity and their uses |
| CN108570425A (en) * | 2018-03-08 | 2018-09-25 | 华东理工大学 | A kind of slow raw rhizobium monooxygenase and its application in preparing chiral sulfoxide |
| CN110573605A (en) * | 2018-03-08 | 2019-12-13 | 华东理工大学 | Slow rhizobium monooxygenase and application thereof in preparation of chiral sulfoxide |
| CN111218431A (en) * | 2018-11-26 | 2020-06-02 | 华东理工大学 | Monooxygenase and application thereof in preparation of optically pure sulfoxide |
| CN112725297A (en) * | 2021-01-29 | 2021-04-30 | 华东理工大学 | Thioether monooxygenase and application thereof in preparation of chiral prazole medicine |
| CN113430216A (en) * | 2021-07-26 | 2021-09-24 | 福州大学 | Propiophenone monooxygenase and application thereof in preparation of prazole drugs |
Non-Patent Citations (3)
| Title |
|---|
| A Baeyer-Villiger monooxygenase from Cupriavidus basilensis catalyzes asymmetric synthesis of (R)-lansoprazole and other pharmaco-sulfoxides;Feng Liu等;Applied microbiology and biotechnology(第08期);全文 * |
| Synthetic construct NAD(P)/FAD-dependent oxidoreductase gene, complete cds;GenBank;GenBank;MT992076 * |
| 假单胞菌单加氧酶基因的克隆表达及其在手性亚砜生物催化合成中的活性分析;袁志美;李林艳;邓成念;郝丽颖;仇治梅;欧刚卫;杨加伟;;基因组学与应用生物学(第04期);全文 * |
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