CN114836395A - Thioether monooxygenase mutant and application thereof in preparation of chiral prazole medicine - Google Patents

Thioether monooxygenase mutant and application thereof in preparation of chiral prazole medicine Download PDF

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CN114836395A
CN114836395A CN202210493819.0A CN202210493819A CN114836395A CN 114836395 A CN114836395 A CN 114836395A CN 202210493819 A CN202210493819 A CN 202210493819A CN 114836395 A CN114836395 A CN 114836395A
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郁惠蕾
刘峰
许建和
潘江
耿强
赵晨
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East China University of Science and Technology
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Abstract

The invention belongs to the technical field of biological engineering, 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 application of the recombinant thioether monooxygenase mutant in preparation of chiral prazole medicines. Compared with other biocatalysts for preparing optical pure azole drugs, 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 shows wide application prospects in industrial application.

Description

Thioether monooxygenase mutant and application thereof in preparation of chiral prazole medicine
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 prazole medicines.
Background
Proton Pump Inhibitors (PPIs) are a class of first-choice drugs widely used in the treatment of gastric acid digestive diseases. The PPIs are mainly omeprazole marketed in sweden in 1988, lansoprazole marketed in japan in 1995, pantoprazole marketed in germany in 1997, rabeprazole marketed in the us in 1999, and the like, and these drugs are initially marketed in the form of racemate and play a significant role in the drug market. By 2001, the first chiral PPI esomeprazole (the levorotatory isomer of omeprazole) was marketed in the united states at heavy pounds, and thereafter, for many years, occupied the first ten worldwide drug sales. In 2009, dexlansoprazole was approved by the U.S. FDA for marketing, which is also the second chiral PPI following esomeprazole. In general, chiral PPIs have greater acid-inhibitory strength, longer acid-inhibitory time, and higher bioavailability than racemic PPIs. Therefore, chiral PPIs are becoming specific drugs for treating 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 synthesis by the chemical method cannot be avoided; large amount of peroxy acid, H used in chemical synthesis 2 O 2 Substances such as organic catalysts and organic solvents can cause serious damage to physical and psychological health of workers and human living environment; the chemical reaction conditions are relatively harsh, and the requirements on production equipment are also highHigh production input cost is increased. In response to the times of green life and green manufacturing, the biosynthesis method having the advantages of good catalytic selectivity, mild reaction conditions, green and environment-friendly reaction system and the like gradually becomes a beneficial supplement of the chemical synthesis method.
In the current biological synthesis approach of PPIs, the chiral thioether is mainly used as a substrate, and whole cells, free enzymes and the like are used as catalysts to catalyze the asymmetric oxidation of the substrate to obtain optically pure PPIs. The biocatalysts that have been reported to be useful for the synthesis of PPIs are mainly: 1) the mold Cunninghamella echinulata MK40 can catalyze the conversion of 7.5mM of rabeprazole sulfide, the conversion rate is 92% after 144 hours of reaction, the product is (S) -rabeprazole, and when omeprazole sulfide and lansoprazole sulfide are used as substrates, the conversion rates are only 45% and 0.6% respectively; 2) the bacillus Lysinibacillus sp.B71 can catalyze omeprazole thioether and pantoprazole thioether to generate esomeprazole at the concentration of 0.1g/L, but the conversion rates are only 70% and 8% respectively, and the cells have no catalytic activity on the tested ilaprazole thioether and lansoprazole thioether; 3) the cyclohexanone monooxygenase mutant from Acinetobacter calcosceticus can catalyze asymmetric oxidation of 15mM omeprazole to generate esomeprazole (99% ee); 4) the cyclohexanone monooxygenase mutant which is disclosed in the patent WO2011/071982 and is derived from Acinetobacter calcosaceicus NCIMB 9871 and is transformed by the United states Codexes company through directed evolution can efficiently catalyze omeprazole thioether to synthesize esomeprazole, but the activity to lansoprazole is very low, and the stereoselectivity is poor; 5) patent CN 112725297 a discloses thioether monooxygenase CbSMO from cupriavidius basiensis, having an oxidation activity for all 14 tested lansoprazole thioether compounds, which catalyzes the asymmetric oxidation of 10mM lansoprazole thioether to yield dexlansoprazole (99% ee). Mutant CbSMO compared to the female parent of CbSMO G266D/L313P Although having higher catalytic activity and thermal stability, it is still difficult to meet the demand of industrial production.
Although the existing biocatalyst can catalyze the substrate oxidation including lansoprazole thioether, the oxidation activity is generally low, the thermal stability is poor, the substrate specificity is poor (the product sulfoxide can be further oxidized to generate the byproduct sulfone), the loading amount on the substrate is low, and the like, so that the requirements of industrial production are difficult to meet.
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 process of synthesizing the chiral sulfoxide drug dexlansoprazole sulfoxide by a biological method, CbSMO with higher activity on lansoprazole thioether in patent CN 112725297A is selected G266D/L313P (namely, the amino acid Gly at the 266 th position in the amino acid sequence shown in SEQ ID No.3 in the patent CN 112725297A is replaced by Asp, and the amino acid Leu at the 313 th position is replaced by Pro) as a target, and the target is subjected to further molecular modification by means of protein engineering, so that a thioether monooxygenase mutant with remarkably improved Lansoprazole thioether catalytic activity, substrate specificity and thermal stability is provided, 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 lansoprazole medicaments are provided.
The purpose of the invention can be realized by the following technical scheme:
according to one technical scheme, the invention provides a thioether monooxygenase mutant with remarkably improved catalytic activity, substrate specificity and thermal stability on lansoprazole thioether. A plurality of thioether monooxygenase mutants with remarkably improved catalytic activity, substrate specificity and thermal stability on lansoprazole thioether are obtained by identifying through a random mutation and semi-rational design method and combining enzyme labeling instrument high-flux primary screening and further shake flask secondary screening by using an amino acid sequence (WT) shown as SEQ ID No. 2.
The thioether monooxygenase mutant provided by the invention is a derivative protein of a new amino acid sequence formed by replacing one or more amino acid residues of Asp at position 98, Gly at position 252, Met at position 253, Arg at position 256, Phe at position 269, Leu at position 272, Val at position 316, Leu at position 485, Met at position 486, Ala at position 487 and Leu at position 488 in an amino acid sequence shown in SEQ ID No.2 with other amino acid residues; the derived protein has obviously improved oxidation activity on lansoprazole thioether and is also improved in 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) the 252 th amino acid Gly in the amino acid sequence shown in SEQ ID No.2 is replaced by Met;
(4) amino acid Gly at the 252 th site in the amino acid sequence shown in SEQ ID No.2 is replaced by Pro;
(5) the 253 rd amino acid Met of 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 amino acid Arg at the 256 th site in the amino acid sequence shown in SEQ ID No.2 is replaced by Met;
(9) the amino acid Arg at the 256 th site in the amino acid sequence shown in SEQ ID No.2 is replaced by Thr;
(10) the amino acid Arg at the 256 th site in the amino acid sequence shown in SEQ ID No.2 is replaced by Trp;
(11) the 269 th amino acid Phe in the amino acid sequence shown in SEQ ID No.2 is replaced by His;
(12) the 269 th amino acid Phe 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) the 272 th amino acid Leu in the amino acid sequence shown in SEQ ID No.2 is replaced by Glu;
(15) the amino acid Val at the position 316 in the amino acid sequence shown in SEQ ID No.2 is replaced by Ala;
(16) substitution of the amino acid Val at position 316 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) the 486 th amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Leu;
(20) the 487 th amino acid of the amino acid sequence shown in SEQ ID No.2 is replaced by Ala to Asp;
(21) the 487 th amino acid Ala in the amino acid sequence shown in SEQ ID No.2 is replaced by Glu;
(22) the 488 th amino acid Leu in the amino acid sequence shown in SEQ ID No.2 is replaced by Thr;
(23) the 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Val at position 316 with Asp;
(24) the amino acid Arg at the 256 th site in the amino acid sequence shown in SEQ ID No.2 is replaced by Thr; substitution of amino acid 487 to Glu; the 488 th amino acid Leu is replaced by Thr;
(25) the 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; substitution of amino acid 269, Phe, to Ser; the 485 th amino acid Leu is replaced by Phe; the 488 th amino acid Leu is replaced by Thr;
(26) the 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Val at position 316 with Asp; the 488 th amino acid Leu is replaced by Thr;
(27) the 98 th amino acid Asp in the amino acid sequence shown in SEQ ID No.2 is replaced by Gly; (iii) replacement of Met at amino acid 253 with Gln; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Val at position 316 to Ala;
(28) the 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; the amino acid Arg at position 256 is replaced by Met; the 485 th amino acid Leu is replaced by Phe; substitution of amino acid 487 to Glu; the 488 th amino acid Leu is replaced by Thr;
(29) the 252 th amino acid Gly in the amino acid sequence shown in SEQ ID No.2 is replaced by Pro; (iii) replacement of Met at amino acid 253 with Gln; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Val at position 316 with Asp; substitution of amino acid 487 to Glu; the 488 th amino acid Leu is replaced by Thr;
(30) the 252 th amino acid Gly in the amino acid sequence shown in SEQ ID No.2 is replaced by Pro; substitution of Met to Pro at amino acid 253; substitution of amino acid Phe to Ser at position 269; substitution of amino acid Val at position 316 to Ala; substitution of amino acid 487 to Glu; the 488 th amino acid Leu is replaced by Thr;
(31) the 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; substitution of Glu for amino acid Leu at position 272; substitution of amino acid Val at position 316 with Asp; substitution of the 486 th amino acid Met with Leu; substitution of amino acid Ala to Asp at position 487; the 488 th amino acid Leu is replaced by Thr;
(32) the 252 st amino acid Gly in the amino acid sequence shown in SEQ ID No.2 is replaced by Leu; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Leu at position 272 with Glu; the 485 th amino acid Leu is replaced by Trp; substitution of the 486 th amino acid Met with Leu; the 488 th amino acid Leu is replaced by 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 to Pro; (iii) replacement of Met at amino acid 253 with Gln; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Val at position 316 to Ala; substitution of amino acid 487 to Glu; the 488 th amino acid Leu is replaced by Thr;
(34) the 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Pro; the amino acid Arg at position 256 is replaced by Met; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Val at position 316 with Asp; substitution of the 486 th amino acid Met with Leu; substitution of amino acid 487 to Glu; the 488 th amino acid Leu is replaced by Thr;
(35) the 252 th amino acid Gly in the amino acid sequence shown in SEQ ID No.2 is replaced by Pro; substitution of Met to Val at amino acid 253; the amino acid Arg at position 256 is replaced by Thr; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Leu at position 272 with Glu; the 485 th amino acid Leu is replaced by Phe; substitution of amino acid 487 to Glu; the 488 amino acid Leu is replaced by Thr.
In a second aspect, the present invention provides nucleic acids encoding the thioether monooxygenase mutants.
The nucleic acid encodes any one of the thioether monooxygenase mutants of one of the technical schemes.
The nucleotide sequence coding the thioether monooxygenase mutant is a nucleic acid sequence coding 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 of the second technical scheme of the invention.
The recombinant expression vector may be prepared by ligating the nucleic acid sequence encoding the thioether monooxygenase mutant gene of the invention to the pET28a plasmid by methods conventional in the art.
The fourth technical scheme is that the invention provides a recombinant expression transformant.
The recombinant expression transformant comprises the recombinant expression vector of 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(DE3) host cells by a conventional method in the art.
Fifth technical solution, the present 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 cells of (1), and isolating a crude enzyme solution containing the thioether monooxygenase mutant;
(3) freeze-drying the crude enzyme solution of (2) to obtain crude enzyme powder;
(4) an isolated thioether monooxygenase mutant according to one of the claims.
Wherein, the culture method and conditions for the recombinant expression transformant are the conventional method and conditions in the field in order to obtain the recombinant thioether monooxygenase mutant catalyst.
In some embodiments, the method of culturing the recombinant expression transformant may include the steps of: culturing the recombinant expression transformant of the invention to obtain the recombinant thioether monooxygenase mutant. For recombinant E.coli, the preferred medium is LB medium: 10g/L of peptone, 5g/L of yeast extract, 10g/L of NaCl and 6.5-7.0 of pH. The preferred culture method is: the 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 LB medium (containing kanamycin) with an inoculum size of 1-2% (v/v), shaking and culturing at 37 deg.C and 180rpm, when OD of the culture solution is 600 When the concentration reaches 0.6-0.8, isopropyl-beta-D-thiogalactoside (IPTG) with the final concentration of 0.1-0.5 mM is added as an inducer to induce for 16-24 h at 16-30 ℃. The culture solution was centrifuged, and the precipitate was washed twice with physiological saline to obtain recombinant expression transformant cells. And freeze-drying the harvested recombinant cells to obtain freeze-dried cells containing the thioether monooxygenase mutant. Or suspending the harvested recombinant cells in a buffer solution with 5-10 times volume (v/w), ultrasonically crushing, centrifuging and collecting supernatant, thus obtaining the crude enzyme solution of the recombinant thioether monooxygenase mutant. Freezing the collected crude enzyme solution at-80 deg.C, and drying at low temperature with vacuum freeze dryer to obtain freeze-dried crude enzyme powder, which can be stored in 4 deg.C refrigerator for convenient use.
The sixth technical scheme provides a high-throughput screening method for a prazole thioether monooxygenase mutant. The high-throughput method is established according to the fact that the product of the prazole sulfoxide is degraded and develops color when meeting acid, and the prochiral prazole sulfide and the peroxidation byproduct of the prazole sulfone do not have the characteristic, and is shown in the attached figure 2. When the prazole thioether is used as a screening substrate, the thermal stability of the CbSMO and the activity of the CbSMO on the prazole thioether can be screened according to the generation amount of the product prazole sulfone; when the prazole sulfoxide is used as a screening substrate, the substrate specificity of CbSMO can be screened according to the reduction amount of the prazole sulfoxide.
Seventhly, 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 up from a solid medium in a 96-well plate (primary plate) containing 200. mu.L of LB medium (including kanamycin) using a colony picking robot, and cultured overnight at 37 ℃ and 800 rpm. Transferring the culture plate from the primary culture plate to a 96-deep-well plate (secondary culture plate) containing 600. mu.L LB culture medium (containing kanamycin) by using an automatic liquid transfer system, culturing at 37 ℃ and 800rpm for 2-5 hours, adjusting the temperature to 16-30 ℃, and adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.1-0.5 mM to perform inducible expression of the recombinant thioether monooxygenase mutant. Centrifuging after 12-24 hours, removing the supernatant, collecting the precipitate, adding 400 mu L of potassium phosphate buffer (0.1M, pH 8.0) containing 0.1-1.0 g/L lysozyme for resuspending the cells, and lysing the cells at 25-40 ℃ and 800rpm for 1-3 hours to obtain a cell lysate. 0.2 to 1.0mM lansoprazole thioether was added as a substrate for screening, 10 to 50. mu.L of methanol as a cosolvent, and 50 to 90. mu.L of a potassium phosphate buffer solution containing a coenzyme NADPH (final concentration: 0.2 to 1.0mM) were reacted at 25 to 35 ℃ at 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 light absorption value under 330nm is read on an enzyme-linked immunosorbent assay, and the high light absorption value indicates that the activity of the thioether monooxygenase mutant is higher.
In the eighth technical scheme, the invention provides a high-throughput screening method for the thermostability of the thioether monooxygenase mutant. Specifically, after the cell lysate is obtained by the method of the seventh technical scheme of the invention, the cell lysate is kept stand and incubated for 5 hours at 40 ℃, and then reactants are added for reaction and screening by the method of the seventh technical scheme, so that the high light absorption value indicates that the thioether monooxygenase mutant has high thermal stability.
In the ninth technical scheme, the invention provides a high-throughput screening method for the substrate specificity of the thioether monooxygenase mutant. Specifically, after cell lysate is obtained by the method according to the seventh technical scheme of the present invention, 0.2-1.0 mM D-lansoprazole sulfoxide is added as a screening substrate, 10-50 μ L of methanol is used as a cosolvent, and 50-90 μ L of potassium phosphate buffer solution in which coenzyme NADPH (final concentration 0.2-1.0 mM) is dissolved is reacted at 25-35 ℃ at 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 value under 330nm is read on an enzyme-linked immunosorbent assay, the high absorbance value indicates that the oxidized amount of the lansoprazole sulfoxide is small, namely, the thioether monooxygenase mutant has high substrate specificity on the lansoprazole thioether.
In the tenth technical scheme, the invention provides application of the recombinant thioether monooxygenase mutant catalyst in catalyzing asymmetric oxidation of prochiral prazole thioether substrates.
In some embodiments of the invention, the enzymatic reaction is performed in a buffer solution with pH of 6.0-10.0 at 25-40 ℃, and the reaction system comprises Lansoprazole sulfide with a final concentration of 1-30 g/L, 1-20% (v/v) methanol, 10-300 mmol/L sodium formate and 0-1mmol/L NADP + 10-100U/L of the recombinant thioether monooxygenase mutant catalyst and 10-200U/L of dehydrogenase crude enzyme powder.
Preferably, the reaction is carried out in the state of introducing air and stirring, the aeration rate is 0-2 vvm, and the reaction time is based on the condition that the substrate is completely converted or the concentration of the product does not continuously rise any more.
Sampling 0.5mL of reaction liquid in a gap in the reaction process, adding 0.5mL of ethyl acetate for extraction, taking 0.3mL of extract, adding anhydrous sodium sulfate for drying, volatilizing to remove ethyl acetate, then adding 0.3mL of ethanol for dissolution, filtering by using a filter membrane with a pore diameter of 0.22 mu m, performing liquid phase analysis, and determining the conversion rate of a substrate and the ee value of a product, wherein the specific analysis conditions are as follows:
the chromatographic column is xylonite Chiralpak IA, the mobile phase is n-heptane, ethanol 70:30(v/v), and the flow rate is 0.5 mL/min -1 (ii) a The column temperature was 40 ℃ and the UV detection wavelength was 300 nm.
Wherein the prochiral lazole thioether substrate is selected from compounds shown in any one of the following chemical structural formulas:
Figure BDA0003621252130000081
in bookIn some embodiments of the invention, the coenzyme NADPH is oxidized to NADP during the reaction + Catalyzing NADP using dehydrogenase + Reduced to NADPH.
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), as formate and NADP + As substrate, catalyzing oxidation of formic acid and simultaneously NADP + Reduction to NADPH;
(2) glucose dehydrogenase GDH (ChemBiochem 2020,21: 2680-2688) with glucose and NADP + As substrate, catalyzing oxidation of glucose and NADP + Reduction to NADPH;
(3) alcohol dehydrogenase ADH (Tetrahedron Lett 2021,84:153455), with short-chain alcohols and NADP + As substrate, short-chain alcohol oxidation is catalyzed, and NADP is used simultaneously + Reduced to NADPH.
Compared with the prior art, the technical effects of the invention are mainly embodied in the following aspects:
compared with other biocatalysts for preparing optical pure azole drugs, 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 shows wide application prospects in industrial application.
Drawings
FIG. 1 is a schematic diagram of the reaction process of catalyzing asymmetric oxidation and chiral sulfoxide peroxidation of prochiral prazole thioether by a thioether monooxygenase mutant of 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 of the present invention.
Detailed Description
The technical solutions and technical effects of the present invention will be clearly and completely described below with reference to specific embodiments so that those skilled in the art can better understand the present invention and can implement the present invention, but the scope of the present invention is not limited to these embodiments.
The media and detection methods referred to in the following examples are as follows:
LB medium: 10g/L of peptone, 5g/L of yeast extract and 10g/L of NaCl (2% agar powder is added into a solid culture medium).
The chiral HPLC analysis method was as follows:
the instrument comprises the following steps: shimadzu HPLC 2010A; the type of the chromatographic column: chiralpak IA; mobile phase: n-heptane ethanol 70:30 (v/v); flow rate: 0.5 mL/min -1 (ii) a Column temperature: 40 ℃; sample introduction amount: 10 mu L of the solution; a detector: an ultraviolet detector; detection wavelength: 300 nm.
Example 1: random mutation screening CbSMO mutant with improved activity on lansoprazole thioether
According to an open reading frame of CbSMO, an upstream primer and a downstream primer are designed as follows:
the upstream primer is shown as SEQ ID No. 3.
The downstream primer is shown as SEQ ID No. 4.
The sequence of the upstream primer is shown by underlining as the restriction site of Nde I, and the sequence of the downstream primer is shown by underlining as the restriction site of Hind III.
The recombinant plasmid pET28a-CbSMO G266D/L313P For the template, a library of random mutants was created as described in example 7 of patent CN 112725297A.
Wherein CbSMO G266D/L313P The amino acid sequence of (A) is shown as SEQ ID No.2, and the corresponding nucleotide sequence is shown as SEQ ID No. 1.
Briefly, an error-prone PCR was performed using rTaq DNA polymerase to construct a random mutation library. After PCR amplification, the target fragment was recovered, ligated with linearized empty-load plasmid pET28a, transformed into E.coli BL21(DE3) competent cells, spread evenly on LB agar plates containing 50. mu.g/mL kanamycin, and cultured upside down in a 37 ℃ incubator for about 12 hours. Transformants on the transformation plates were picked into 96-well deep-well plates containing LB medium using a monoclonal autosampler Qpix 450 and cultured overnight in a shaker at 37 ℃ and 800 rpm. And transferring seed liquid from the primary pore plate to the secondary pore plate by using a full-automatic liquid treatment workstation free EVO for induced expression of the mutant protein. After the supernatant of the fermentation broth was centrifuged, cell lysate was added to each well to lyse the cells, and then 25. mu.L of lansoprazole sulfide in methanol (lansoprazole sulfide final concentration: 1mM) and 75. mu.L of potassium phosphate buffer solution in which coenzyme NADPH (final concentration: 1mM) was dissolved were added to react at 25 ℃ under 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 light absorption value under 330nm is read on an enzyme-linked immunosorbent assay, and the high light absorption value indicates that the activity of the thioether monooxygenase mutant is higher. And (4) purifying and characterizing the screened mutants, and sequencing corresponding genes.
Example 2: random mutation screening CbSMO mutant with improved lansoprazole thioether substrate specificity
Due to the female parent CbSMO G266D/L313P In the process of catalyzing the oxidation of the lansoprazole sulfide, an oxidation product, namely the dexlansoprazole sulfoxide is identified as a substrate to be subjected to peroxidation to generate a byproduct, namely the lansoprazole sulfone, and the phenomenon of poor substrate specificity to the lansoprazole sulfide is shown. To solve this problem, a random mutagenesis approach was used to screen for CbSMO mutants with improved substrate specificity for lansoprazole thioether.
The screening of the CbSMO mutant with improved lansoprazole thioether substrate specificity can be realized by slightly modifying the operation steps in the embodiment 1. Specifically, the substrate lansoprazole sulfide of the activity detection reaction in example 1 is replaced by dexlansoprazole sulfoxide, and the high absorbance value indicates that the dexlansoprazole sulfoxide remains much, i.e., the CbSMO mutant has stronger substrate specificity on the lansoprazole sulfide.
Example 3: random mutation screening of CbSMO mutant with improved heat stability
The screening of the CbSMO mutants with improved thermostability can be achieved with minor modifications to the procedure of example 1. Specifically, the crude enzyme solution after the cleavage in example 1 is allowed to stand at 40 ℃ for 5 hours, and then activity detection reaction is performed, and a high absorbance indicates that the CbSMO mutant has high residual activity, i.e., the CbSMO mutant has high thermal stability.
Example 4: combinatorial mutations of CbSMO beneficial mutants
On the basis of the mutations in the embodiment 1, the embodiment 2 and the embodiment 3, a plurality of dominant mutants with good activity, substrate specificity and thermal stability are respectively obtained, and the dominant mutants are randomly combined by adopting a DNA shuffling method described in a patent CN 112725297A to obtain a DNA shuffling mutant library.
The high-throughput screening program of example 1 is adopted to screen a DNA shuffling mutant library to obtain a batch of mutants with improved activity on the lansoprazole thioether (named as 'mutant library A'), the high-throughput screening program of example 2 is adopted to screen the mutant library A to obtain a batch of mutants with improved substrate specificity on the lansoprazole thioether (named as 'mutant library B'), the high-throughput screening program of example 3 is adopted to screen the mutant library B to obtain a batch of mutants with improved thermal stability (named as 'mutant library C'), and then the pure enzyme specific activity, substrate specificity and thermal stability of the mutants are characterized. The mutants are preferably a series of mutants with obviously improved activity to the lansoprazole thioether and obviously improved activity ratio to the lansoprazole thioether and the dexlansoprazole sulfoxide, the mutants are good in thermal stability, the sequences of the mutants, the activity of the mutants to the lansoprazole thioether and the dexlansoprazole sulfoxide and the hot melting temperature of the protein are listed in table 1, and the sequence numbers respectively correspond to a series of sequences behind table 1. In the lansoprazole thioether activity column, the parent CbSMO G266D/L313P Compared with the protein consisting of the amino acid sequence shown in SEQ ID No.2 in the sequence table, the plus represents that the activity of the mutant protein on the lansoprazole thioether is improved by 0.5-1 time; the activity of the mutant protein on the lansoprazole thioether is improved by 1-5 times by '+ +'; the activity of the mutant protein on the lansoprazole thioether is improved by 5-10 times by +++. In the activity ratio column, the parent CbSMO G266D/L313P Compared with the results, the activity ratio of the mutant protein to the lansoprazole thioether and the dexlansoprazole sulfoxide is improved by 0.1-1 time by '+'; "+ +" indicates mutant protein para-orchidThe activity ratio of the sorazole thioether to the dexlansoprazole sulfoxide is improved by 1-2 times; the activity ratio of the mutant protein to the lansoprazole thioether and the dexlansoprazole sulfoxide is improved by 2-5 times by +++. In the hot melting temperature series, with the parent CbSMO G266D/L313P Compared with the method, the plus indicates that the hot melting temperature of the mutant protein is increased by 1-5 ℃; the "+" shows that the hot melting temperature of the mutant protein is increased by 5-10 ℃; "+ + + +" indicates that the hot melting temperature of the mutant protein is increased by 10-20 ℃.
TABLE 1 thioether monooxygenase mutant sequences, fold increase in activity of lansoprazole sulfide, fold increase in activity ratio of lansoprazole sulfide to dexlansoprazole sulfoxide, degree increase in hot melt temperature
Figure BDA0003621252130000111
Figure BDA0003621252130000121
Figure BDA0003621252130000131
The amino acid sequences of the thioether monooxygenase mutants corresponding to the sequence numbers are respectively 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) the substitution of the 252 th amino acid Gly into Met in the amino acid sequence shown in SEQ ID No. 2;
(4) the amino acid Gly at the 252 th site in the amino acid sequence shown as SEQ ID No.2 is replaced by Pro;
(5) the 253 rd amino acid Met in the amino acid sequence shown as SEQ ID No.2 is replaced by Val;
(6) the 253 rd amino acid Met in the amino acid sequence shown as SEQ ID No.2 is replaced by Pro;
(7) the 253 rd amino acid Met in the amino acid sequence shown as SEQ ID No.2 is replaced by Gln;
(8) the amino acid Arg at the 256 th site in the amino acid sequence shown as SEQ ID No.2 is replaced by Met;
(9) the amino acid Arg at the 256 th site in the amino acid sequence shown as SEQ ID No.2 is replaced by Thr;
(10) the amino acid Arg at the 256 th site in the amino acid sequence shown as SEQ ID No.2 is replaced by Trp;
(11) the 269 th amino acid Phe in the amino acid sequence shown in SEQ ID No.2 is replaced by His;
(12) the amino acid Phe at the 269 th amino acid position in the amino acid sequence shown as SEQ ID No.2 is replaced by Ser;
(13) the 272 th amino acid Leu in the amino acid sequence shown as SEQ ID No.2 is replaced by Lys;
(14) the 272 th amino acid Leu in the amino acid sequence shown in SEQ ID No.2 is replaced by Glu;
(15) the amino acid Val at the 316 th position in the amino acid sequence shown in SEQ ID No.2 is replaced by Ala;
(16) the amino acid Val at the 316 th position in the amino acid sequence shown as SEQ ID No.2 is replaced by Asp;
(17) the 485 th amino acid Leu in the amino acid sequence shown as 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) the 486 th amino acid Met in the amino acid sequence shown as SEQ ID No.2 is replaced by Leu;
(20) the 487 th amino acid of the amino acid sequence shown as SEQ ID No.2 is replaced by Ala to Asp;
(21) the 487 th amino acid Ala in the amino acid sequence shown as SEQ ID No.2 is replaced by Glu;
(22) the 488 th amino acid Leu in the amino acid sequence shown as SEQ ID No.2 is replaced by Thr;
(23) the 253 rd amino acid Met in the amino acid sequence shown as SEQ ID No.2 is replaced by Gln; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Val at position 316 with Asp;
(24) the amino acid Arg at the 256 th site in the amino acid sequence shown as SEQ ID No.2 is replaced by Thr; substitution of amino acid 487 to Glu; the 488 th amino acid Leu is replaced by Thr;
(25) the 253 rd amino acid Met in the amino acid sequence shown as SEQ ID No.2 is replaced by Gln; substitution of amino acid Phe to Ser at position 269; the 485 th amino acid Leu is replaced by Phe; the 488 th amino acid Leu is replaced by Thr;
(26) the 253 rd amino acid Met in the amino acid sequence shown as SEQ ID No.2 is replaced by Gln; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Val at position 316 with Asp; the 488 th amino acid Leu is replaced by Thr;
(27) the 98 th amino acid Asp in the amino acid sequence shown in SEQ ID No.2 is replaced by Gly; (iii) replacement of Met at amino acid 253 with Gln; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Val at position 316 to Ala;
(28) the 253 rd amino acid Met in the amino acid sequence shown as SEQ ID No.2 is replaced by Gln; the amino acid Arg at position 256 is replaced by Met; the 485 th amino acid Leu is replaced by Phe; substitution of amino acid 487 to Glu; the 488 th amino acid Leu is replaced by Thr;
(29) the amino acid Gly at the 252 th site in the amino acid sequence shown as SEQ ID No.2 is replaced by Pro; (iii) replacement of Met at amino acid 253 with Gln; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Val at position 316 with Asp; substitution of amino acid 487 to Glu; the 488 th amino acid Leu is replaced by Thr;
(30) the amino acid Gly at the 252 th site in the amino acid sequence shown as SEQ ID No.2 is replaced by Pro; substitution of Met to Pro at amino acid position 253; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Val at position 316 to Ala; substitution of amino acid 487 to Glu; the 488 th amino acid Leu is replaced by Thr;
(31) the 253 rd amino acid Met in the amino acid sequence shown as SEQ ID No.2 is replaced by Gln; substitution of amino acid Leu at position 272 with Glu; substitution of amino acid Val at position 316 with Asp; substitution of the 486 th amino acid Met with Leu; substitution of amino acid Ala to Asp at position 487; the 488 th amino acid Leu is replaced by Thr;
(32) the 252 th amino acid Gly in the amino acid sequence shown in SEQ ID No.2 is replaced by Leu; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Leu at position 272 with Glu; the 485 th amino acid Leu is replaced by Trp; substitution of the 486 th amino acid Met with Leu; the 488 th amino acid Leu is replaced by 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 to Pro; (iii) replacement of Met at amino acid 253 with Gln; substitution of amino acid 269, Phe, to Ser; replacement of amino acid Val at position 316 with Ala; substitution of amino acid 487 to Glu; the 488 th amino acid Leu is replaced by Thr;
(34) the 253 rd amino acid Met in the amino acid sequence shown as SEQ ID No.2 is replaced by Pro; the amino acid Arg at position 256 is replaced by Met; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Val at position 316 with Asp; substitution of the 486 th amino acid Met with Leu; substitution of amino acid 487 to Glu; the 488 th amino acid Leu is replaced by Thr;
(35) the amino acid Gly at the 252 th site in the amino acid sequence shown as SEQ ID No.2 is replaced by Pro; substitution of Met to Val at amino acid 253; the amino acid Arg at position 256 is replaced by Thr; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Leu at position 272 with Glu; the 485 th amino acid Leu is replaced by Phe; substitution of amino acid 487 to Glu; the 488 amino acid Leu is replaced by Thr.
Example 5: thioether monooxygenase mutant CbSMO M34 Activity assay for Lansoprazole thioether
Determination of CbSMO M34 The activity of a series of prazole thioether substrates was determined as follows: 0.5mL of a reaction system (0.1M KPB buffer solution, pH 8.0) is added with 0.2mM of a prazole thioether substrate and 0.2mM of NADPH, the mixture is subjected to heat preservation at 30 ℃ for 2 minutes, then a proper amount of purified pure enzyme solution is added, the mixture is rapidly and uniformly mixed, the reaction is carried out for 10 minutes at 30 ℃ and 1000rpm, the mixture is stopped and extracted by 0.5mL of ethyl acetate, centrifugal supernatant is filtered by a filter membrane with the aperture of 0.22 mu M, then liquid phase analysis is carried out, and the conversion rate of the substrate and the ee value of a product are determined.
The specific analysis conditions for the substrate conversion and ee value were as follows:
the instrument comprises the following steps: shimadzu HPLC 2010A; the type of the chromatographic column: chiralpak IA; mobile phase: n-heptane, ethanol 70:30 (v/v); flow rate of flow：0.5mL·min -1 (ii) a Column temperature: 40 ℃; sample introduction amount: 10 mu L of the solution; a detector: an ultraviolet detector; detection wavelength: 300 nm.
The enzyme activity unit is defined as: the amount of enzyme required to catalyze the conversion of 1 μmol of the prazole thioether substrate to prazole sulfoxide per minute under the conditions described above.
Determination of the mutant CbSMO M34 The specific activity and ee value of the product and the substrate specificity results for a series of prazole thioether substrates are shown in table 2.
TABLE 2 CbSMO M34 Determination of pure enzyme activity, stereoselectivity and substrate specificity
Figure BDA0003621252130000151
Figure BDA0003621252130000161
Figure BDA0003621252130000171
a “+”:500～1000U/g；“++”:1000～2000U/g；“+++”:>2000U/g
b “+”:10～20；“++”:20～50；“+++”:>50
Example 6: preparation of crude enzyme solution of thioether monooxygenase
With thioether monooxygenase CbSMO M34 The preparation of the crude enzyme solution is exemplified. Will contain CbSMO M34 The recombinant Escherichia coli of (1) was inoculated into LB medium containing Kanamycin (Kanamycin) and cultured overnight at 37 ℃ with shaking at 180 rpm. Inoculating to 500mL Erlenmeyer flask containing 100mL LB medium (containing kanamycin) at an inoculum size of 1% (v/v), shaking and culturing at 37 deg.C and 180rpm, when OD of the culture solution is 600 When the concentration reaches 0.6, adding isopropyl-beta-D-thiogalactoside (IPTG) with final concentration of 0.2mM as inducer, inducing at 16 deg.C for 24 hr, centrifuging the culture solution, collecting precipitate, and adding physiological salineAnd washing twice to obtain recombinant expression transformant cells. Suspending the harvested recombinant cells in 10 times (v/w) volume of buffer solution, ultrasonically crushing, centrifuging and collecting supernatant to obtain the recombinant thioether monooxygenase CbSM M34 The crude enzyme solution of (1).
Example 7: preparation of thioether monooxygenase crude enzyme powder
With thioether monooxygenase CbSMO M34 The preparation of crude enzyme powder is exemplified. Collection of thioether monooxygenase mutant CbSMO obtained in example 6 M34 Freezing the crude enzyme solution at-80 deg.C, and drying at low temperature with vacuum freeze drier to obtain thioether monooxygenase mutant CbSMO M34 The specific activity of the crude enzyme powder on the lansoprazole thioether is 122U/g, and the specific activity on the rabeprazole thioether is 198U/g. The obtained crude enzyme powder is stored in a refrigerator at 4 ℃ and can be conveniently used.
Example 8: CbSMO G266D/L313P Catalyzing 20g/L lansoprazole thioether asymmetric oxidation
The reaction process of catalyzing asymmetric oxidation and chiral sulfoxide peroxidation of prochiral prazole thioether by the thioether monooxygenase mutant is shown in figure 1.
To a 100mL reaction system (potassium phosphate buffer, 200mmol/L, pH 8.0), CbSMO was added G266D/L313P Crude enzyme powder 6U (crude enzyme powder prepared by the method described in example 7) and crude formate dehydrogenase FDH powder 12U were added to the above system, and Lansoprazole sulfide, methanol, sodium formate and NADP were further added to the 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 mixture was sampled intermittently, extracted with 0.5mL of ethyl acetate, and 0.3mL of the extract was dried over anhydrous sodium sulfate, evaporated to remove ethyl acetate, and dissolved in 0.3mL of ethanol, and the conversion of the substrate and the ee value of the product were measured by the HPLC analysis method described in example 5. The conversion rate of the substrate is more than 97 percent in 32 hours, the ee value of the product is more than 99 percent (R), and the proportion of the by-product lansoprazole sulfone in the total product is 10.5 percent.
Example 9: CbSMO M31 Catalyzing 20g/L lansoprazole thioether asymmetric oxidation
At 100mTo the L reaction system (potassium phosphate buffer, 200mmol/L, pH 8.0), CbSMO was added M31 Crude enzyme powder 6U (crude enzyme powder prepared by the method described in example 7) and crude formate dehydrogenase FDH powder 12U were added to the above system, and Lansoprazole sulfide, methanol, sodium formate and NADP were further added to the 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 mixture was sampled intermittently, extracted with 0.5mL of ethyl acetate, and 0.3mL of the extract was dried over anhydrous sodium sulfate, evaporated to remove ethyl acetate, and dissolved in 0.3mL of ethanol, and the conversion of the substrate and the ee value of the product were measured by the HPLC analysis method described in example 5. The conversion rate of the substrate is more than 99 percent after 24 hours of reaction, the ee value of the product is more than 99 percent (R), and the proportion of the byproduct lansoprazole sulfone in the total product is 1.8 percent.
Example 10: CbSMO M31 Catalyzing 20g/L rabeprazole thioether asymmetric oxidation
To a 100mL reaction system (potassium phosphate buffer, 200mmol/L, pH 8.0), CbSMO was added M31 Crude enzyme powder 6U (crude enzyme powder prepared as described in example 7) and glucose dehydrogenase GDH crude enzyme powder 12U, rabeprazole 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 mixture was sampled intermittently, extracted with 0.5mL of ethyl acetate, and 0.3mL of the extract was dried over anhydrous sodium sulfate, evaporated to remove ethyl acetate, and dissolved in 0.3mL of ethanol, and the conversion of the substrate and the ee value of the product were measured by the HPLC analysis method described in example 5. The substrate conversion rate is more than 99 percent after the reaction for 19 hours, the ee value of the product is more than 99 percent (R), and the proportion of the byproduct rabeprazole sulfone in the total product is 2.1 percent.
Example 11: CbSMO M31 Catalyzing 20g/L pantoprazole thioether asymmetric oxidation
To a 100mL reaction system (potassium phosphate buffer, 200mmol/L, pH 8.0), CbSMO was added M31 Crude enzyme powder 6U (crude enzyme powder prepared as described in example 7) and crude alcohol dehydrogenase ADH powder 12U, to which pantoprazole sulfide, methanol, sodium formate and NADP were further added + 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 mixture was sampled intermittently, extracted with 0.5mL of ethyl acetate, and 0.3mL of the extract was dried over anhydrous sodium sulfate, evaporated to remove ethyl acetate, and dissolved in 0.3mL of ethanol, and the conversion of the substrate and the ee value of the product were measured by the HPLC analysis method described in example 5. The substrate conversion rate is more than 99 percent after 16 hours of reaction, the ee value of the product is more than 99 percent (R), and the proportion of the byproduct pantoprazole sulfone in the total product is 2.4 percent.
Example 12: CbSMO M27 Catalyzing asymmetric oxidation of 30g/L lansoprazole thioether
To a 100mL reaction system (potassium phosphate buffer, 200mmol/L, pH 8.0), CbSMO was added M27 Crude enzyme powder 6U (crude enzyme powder prepared by the method described in example 7) and crude formate dehydrogenase FDH powder 12U were added to the above system, and Lansoprazole sulfide, methanol, sodium formate and NADP were further added to the 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 mixture was sampled intermittently, extracted with 0.5mL of ethyl acetate, and 0.3mL of the extract was dried over anhydrous sodium sulfate, evaporated to remove ethyl acetate, and dissolved in 0.3mL of ethanol, and the conversion of the substrate and the ee value of the product were measured by the HPLC analysis method described in example 5. The conversion rate of the substrate is more than 98 percent after the reaction for 30 hours, the ee value of the product is more than 99 percent (R), and the proportion of the byproduct lansoprazole sulfone in the total product is 1.3 percent.
Example 13: CbSMO M27 Catalyzing asymmetric oxidation of 30g/L lansoprazole thioether
To a 1L reaction system (potassium phosphate buffer, 200mmol/L, pH 8.0), CbSMO was added M27 100U crude enzyme powder (prepared as described in example 7) and 200U crude formate dehydrogenase FDH powder were added to the above system, and Lansoprazole sulfide, methanol, sodium formate and NADP were further added to the system + To final concentrations of 30g/L, 10% (v/v), 200mmol/L and 0.2mmol/L, respectively. The reaction was stirred at 30 ℃ and 150rpm with an aeration rate of 0.4 vvm. Intermittently sampling 0.5mL of the reaction solution, adding 0.5mL of ethyl acetate to extract, adding anhydrous sodium sulfate to 0.3mL of the extract, and dryingThereafter, the ethyl acetate was evaporated and dissolved in 0.3mL of ethanol, and the conversion of the substrate, the ee value of the product and the content of the by-product sulfone were measured by the HPLC analysis method described in example 5. The conversion rate of the substrate is more than 99 percent after 32 hours of reaction, the ee value of the product is more than 99 percent (R), and the proportion of the byproduct lansoprazole sulfone in the total product is 1.8 percent.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, 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 embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Sequence listing
<110> university of east China's college of science
<120> thioether monooxygenase mutant and application thereof in preparation of chiral prazole medicine
<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 (10)

1. A thioether monooxygenase mutant which is a derivative protein of a new amino acid sequence formed by replacing one or more amino acid residues of Asp at position 98, Gly at position 252, Met at position 253, Arg at position 256, Phe at position 269, Leu at position 272, Val at position 316, Leu at position 485, Met at position 486, Ala at position 487 and Leu at position 488 in the amino acid sequence shown in SEQ ID No.2 with other amino acid residues;
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) the 252 th amino acid Gly in the amino acid sequence shown in SEQ ID No.2 is replaced by Met;
(4) the 252 th amino acid Gly in the amino acid sequence shown in SEQ ID No.2 is replaced by Pro;
(5) the 253 rd amino acid Met of 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 amino acid Arg at the 256 th site in the amino acid sequence shown in SEQ ID No.2 is replaced by Met;
(9) the amino acid Arg at the 256 th site in the amino acid sequence shown in SEQ ID No.2 is replaced by Thr;
(10) the amino acid Arg at the 256 th site in the amino acid sequence shown in SEQ ID No.2 is replaced by Trp;
(11) the 269 th amino acid Phe in the amino acid sequence shown in SEQ ID No.2 is replaced by His;
(12) the 269 th amino acid Phe 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) the 272 th amino acid Leu in the amino acid sequence shown in SEQ ID No.2 is replaced by Glu;
(15) the amino acid Val at the position 316 in the amino acid sequence shown in SEQ ID No.2 is replaced by Ala;
(16) substitution of the amino acid Val at position 316 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) the 486 th amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Leu;
(20) the 487 th amino acid of the amino acid sequence shown in SEQ ID No.2 is replaced by Ala to Asp;
(21) the 487 th amino acid Ala in the amino acid sequence shown in SEQ ID No.2 is replaced by Glu;
(22) the 488 th amino acid Leu in the amino acid sequence shown in SEQ ID No.2 is replaced by Thr;
(23) the 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Val at position 316 with Asp;
(24) the amino acid Arg at the 256 th site in the amino acid sequence shown in SEQ ID No.2 is replaced by Thr; substitution of amino acid 487 to Glu; the 488 th amino acid Leu is replaced by Thr;
(25) the 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; substitution of amino acid 269, Phe, to Ser; the 485 th amino acid Leu is replaced by Phe; the 488 th amino acid Leu is replaced by Thr;
(26) the 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Val at position 316 with Asp; the 488 th amino acid Leu is replaced by Thr;
(27) the 98 th amino acid Asp in the amino acid sequence shown in SEQ ID No.2 is replaced by Gly; (iii) replacement of Met at amino acid 253 with Gln; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Val at position 316 to Ala;
(28) the 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; the amino acid Arg at position 256 is replaced by Met; the 485 th amino acid Leu is replaced by Phe; substitution of amino acid 487 to Glu; the 488 th amino acid Leu is replaced by Thr;
(29) amino acid Gly at the 252 th site in the amino acid sequence shown in SEQ ID No.2 is replaced by Pro; (iii) replacement of Met at amino acid 253 with Gln; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Val at position 316 with Asp; substitution of amino acid 487 to Glu; the 488 th amino acid Leu is replaced by Thr;
(30) the 252 th amino acid Gly in the amino acid sequence shown in SEQ ID No.2 is replaced by Pro; substitution of Met to Pro at amino acid 253; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Val at position 316 to Ala; substitution of amino acid 487 to Glu; the 488 th amino acid Leu is replaced by Thr;
(31) the 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Gln; substitution of amino acid Leu at position 272 with Glu; substitution of amino acid Val at position 316 with Asp; substitution of the 486 th amino acid Met with Leu; substitution of amino acid Ala to Asp at position 487; the 488 th amino acid Leu is replaced by Thr;
(32) replacing the 252 th amino acid Gly in the amino acid sequence shown in SEQ ID No.2 with Leu; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Leu at position 272 with Glu; the 485 th amino acid Leu is replaced by Trp; substitution of the 486 th amino acid Met with Leu; the 488 th amino acid Leu is replaced by 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 to Pro; (iii) replacement of Met at amino acid 253 with Gln; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Val at position 316 to Ala; substitution of amino acid 487 to Glu; substitution of amino acid Leu at position 488 to Thr;
(34) the 253 rd amino acid Met in the amino acid sequence shown in SEQ ID No.2 is replaced by Pro; the amino acid Arg at position 256 is replaced by Met; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Val at position 316 with Asp; substitution of the 486 th amino acid Met with Leu; substitution of amino acid 487 to Glu; the 488 th amino acid Leu is replaced by Thr;
(35) the 252 th amino acid Gly in the amino acid sequence shown in SEQ ID No.2 is replaced by Pro; substitution of Met to Val at amino acid 253; the amino acid Arg at position 256 is replaced by Thr; substitution of amino acid 269, Phe, to Ser; substitution of amino acid Leu at position 272 with Glu; the 485 th amino acid Leu is replaced by Phe; substitution of amino acid 487 to Glu; the 488 amino acid Leu 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 said 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 isolating a crude enzyme solution containing the thioether monooxygenase mutant;
(3) freeze-drying the crude enzyme solution obtained in the step (2) to obtain crude enzyme powder;
(4) the thioether monooxygenase mutant according to claim 1.
6. Use of a recombinant thioether monooxygenase mutant catalyst as claimed in claim 5 for catalyzing asymmetric oxidation of a prochiral prazole thioether substrate.
7. The use of claim 6, wherein the recombinant thioether monooxygenase mutant catalyst catalyzes the oxidation of a prochiral prazole thioether substrate to the corresponding chiral sulfoxide with oxygen as an oxidant.
8. The use according to claim 7, wherein the prochiral lazole thioether substrate is selected from compounds of any one of the following chemical formulae:
Figure FDA0003621252120000041
9. the use according to claim 6, characterized in that, during the reaction, the coenzyme NADPH is oxidized to NADP + Catalyzing NADP using dehydrogenase + Reduced to NADPH.
10. The use of claim 9, wherein the dehydrogenase is any one of formate dehydrogenase, glucose dehydrogenase or isopropanol dehydrogenase.
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