WO2019169695A1 - 一种慢生根瘤菌单加氧酶及其在制备手性亚砜中的应用 - Google Patents

一种慢生根瘤菌单加氧酶及其在制备手性亚砜中的应用 Download PDF

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WO2019169695A1
WO2019169695A1 PCT/CN2018/083060 CN2018083060W WO2019169695A1 WO 2019169695 A1 WO2019169695 A1 WO 2019169695A1 CN 2018083060 W CN2018083060 W CN 2018083060W WO 2019169695 A1 WO2019169695 A1 WO 2019169695A1
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amino acid
acid sequence
monooxygenase
seq
botemo
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French (fr)
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郁惠蕾
张龑
许建和
赵骞
潘江
刘峰
林国强
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华东理工大学
江苏奥赛康药业股份有限公司
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Priority to CN201880003603.3A priority Critical patent/CN110573605B/zh
Priority to US16/620,525 priority patent/US10787651B2/en
Publication of WO2019169695A1 publication Critical patent/WO2019169695A1/zh

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    • C12Y114/13022—Cyclohexanone monooxygenase (1.14.13.22)

Definitions

  • the invention belongs to the field of bioengineering technology, in particular to a slow-growing rhizobium monooxygenase, a gene encoding the same, a recombinant expression vector containing the same, and a recombinant expression transformant, and the recombinant expression transformant is used to prepare the single
  • Chiral sulfoxides have a wide range of important applications, and their uses can be broadly classified into several types: chiral auxiliary reagents, chiral ligands, chiral catalysts, and chiral drugs and pharmaceutical intermediates.
  • Some chiral sulfoxides are important intermediates or drugs themselves that contain a sulfur atomic chiral center drug.
  • a series of benzopyrazole proton pump inhibitors represented by Esomeprazole are all chiral heterocyclic sulfoxides.
  • Proton Pump Inhibitors are the treatment of choice for many gastroesophageal diseases (eg, gastric and duodenal ulcers, gastroesophageal reflux disease) as H + /K + -ATPase inhibitors. Fast onset, strong in action, high in specificity and long in duration.
  • Lansoprazole alias Takepron, chemical name 2-[3-methyl-4-(2,2,2-trifluoroethoxy)-2-piperidinyl] Methyl sulfinyl-1H-benzimidazole, as a new generation of proton inhibitor antacids and anti-ulcer drugs, strongly inhibits gastric acid secretion.
  • the bioavailability of lansoprazole is higher than that of omeprazole, and the single right-handed lansoprazole sustained-release capsule has several aspects such as cure rate, duration of acid control, and heartburn control rate. Both have better performance than racemic lansoprazole.
  • the single d-lansolinazole was developed by Takeda Pharmaceutical Co., Ltd., and was approved by the FDA in the United States in 2009. Since then, it has been listed in Europe, Asia, and the Americas.
  • biocatalytic synthesis of chiral sulfoxide has the advantages of high stereoselectivity, mild and safe reaction conditions, and environmental friendliness. It is a useful supplement to the chemical synthesis of chiral sulfoxides, and has become the current research with the development of biotechnology. hot spot.
  • biocatalysts can asymmetrically catalyze the oxidation of thioether compounds
  • existing biocatalysts have extremely low catalytic efficiency for large hindered thioether substrates.
  • Babiak et al. screened a wild strain from soil contaminants and identified it as Lysinibacillus. The growth cells of this strain were used to catalyze the conversion of omeprazole thioether.
  • WO2011/071982 discloses Codexis's directed evolution of cyclohexanone monooxygenase CHMO from Acinetobacter sp. NCIMB 9871, and the obtained engineering enzyme can efficiently catalyze the oxidation of omeprazole thioether (S)-Ogilvy
  • S omeprazole thioether
  • the engineering enzyme obtained by Codexis can catalyze the production of (R)-lansoprazole by lansoprazole thioether, but the catalytic efficiency of the engineering enzyme is extremely low.
  • the substrate loading amount is 1.5g/L
  • the conversion rate is only 1.2 at 17h. %, and the optical purity of the product could not be determined due to the extremely low conversion.
  • dexlansoprazole is still produced by chemical methods.
  • the asymmetric catalytic oxidation process has poor selectivity and low conversion rate.
  • the amount of the chiral metal titanium reagent and the auxiliary agent tartaric acid is large, the residual amount of the thioether is about 10 to 15%, and the content of the impurity sulfone is about 2%.
  • Due to the large amount of impurities in the reaction product multiple extraction and crystallization are required in the post-treatment process, the yield is low, and a large amount of three wastes (waste water, waste gas, waste liquid) is produced.
  • the existing biocatalyst has a low conversion rate although the reaction conditions are mild, safe and environmentally friendly.
  • the invention aims to solve the problem that the sulfoxide compound produced by the chemical method has poor selectivity, low conversion rate and low conversion rate of the biocatalytic synthesis of the large sulfoxide compound, and provides a Bradyrhizobium oligotrophicum ECU1212 obtained by screening.
  • the present invention provides a Bradyrhizobium oligotrophicum which is Bradyrhizobium oligotrophicum ECU 1212, deposited at the General Microbiology Center of the China Collection of Microorganisms and Cultures, and has a deposit number of CGMCC No. 15208.
  • the Bradyrhizobium oligotrophicum can produce a Bradyrhizobium oligotrophicum ECU 1212 thioether monooxygenase having the amino acid sequence set forth in SEQ ID No. 2.
  • the present invention also provides a monooxygenase comprising the amino acid sequence shown in SEQ ID No. 2; or
  • the monooxygenase contains a mutant amino acid sequence in which the amino acid sequence shown in SEQ ID No. 2 is mutated.
  • the mutated amino acid sequence is a mutated amino acid sequence which is produced by substitution of any one to five amino acids in the amino acid sequence shown in SEQ ID No. 2.
  • the mutated amino acid sequence is any one of amino acids 295, 357, 394, 395, and 396 of the amino acid sequence set forth in SEQ ID No. 2.
  • a mutated amino acid sequence generated after one or more substitutions have occurred.
  • the mutated amino acid sequence comprises any one or more of the following characteristics:
  • amino acid Asp at position 295 in the amino acid sequence shown in SEQ ID No. 2 is replaced with Cys;
  • the monooxygenase comprises a mutated amino acid sequence set forth in SEQ ID No. 4.
  • the monooxygenase comprises a mutated amino acid sequence set forth in SEQ ID No. 6.
  • the invention also provides an isolated nucleic acid encoding the monooxygenase of any of the above.
  • the invention also provides a recombinant expression vector comprising the nucleic acid described above.
  • the present invention also provides a recombinant expression transformant comprising the recombinant expression vector as described above.
  • the present invention also provides a method for preparing a monooxygenase as described above, comprising the steps of:
  • the recombinant expression transformant as described above is cultured, from which the monooxygenase is isolated.
  • the invention also provides the use of Bradyrhizobium oligotrophicum or monooxygenase as described above for asymmetric catalytic oxidation of latent chiral thioether compounds.
  • the latent chiral thioether compound is selected from the group consisting of compounds of any of the following chemical formulas:
  • the latent chiral thioether compound is asymmetrically catalytically oxidized to a sulfoxide compound.
  • a positive progressive effect of the present invention is that the present invention provides a monooxygenase comprising BoTEMO, a BoTEMO mutant, which can efficiently catalyze asymmetric oxidation of thioether to prepare optics. Pure chiral sulfoxide.
  • concentration of lansoprazole thioether substrate reaches 10 g/L
  • the conversion rate still exceeds 99%
  • the ee value reaches 99% or more
  • the product sulfoxide is not further oxidized to a sulfone by-product.
  • the product obtained by the method of the invention has high concentration, good optical purity, no by-product formation, mild reaction conditions, environmental friendliness, simple operation, easy industrial amplification, and therefore good industrialization. Application prospects.
  • FIG. 1 is a schematic view showing the reaction process of the asymmetrically catalyzed oxidation of a thioether compound to an optically pure chiral sulfoxide by a monooxygenase of the present invention.
  • the Bradyrhizobium oligotrophicum provided by the present invention is obtained by the inventors for large-scale soil microbial screening. Among them, the collection of soil is mainly divided into two parts, directly collecting soil sample and soil sample after pre-buried substrate, a total of 252 soil samples. Guided by lansoprazole thioether, the concentration of lansoprazole thioether was continuously increased by four rounds of gradient enrichment culture. By priming and rescreening, a lansoprazole sulphur can be catalyzed from the soil.
  • the ether-oxidized strain is named Bradyrhizobium oligotrophicum ECU1212; the nomenclature is named after the generic name (genus) + species name + strain code, wherein Bradyrhizobium represents the generic name, oligotrophicum represents the species name, and ECU 1212 represents the strain code; In the invention, Bradyrhizobium is translated as Slow Rhizobium, or Slow Rhizobium.
  • CGMCC China General Microbiological Culture Collection Center
  • the Bradyrhizobium oligotrophicum ECU1212 has the following physiological and biochemical characteristics:
  • the culture method and culture conditions of the Bradyrhizobium oligotrophicum ECU 1212 are not particularly limited as long as the strain of Bradyrhizobium oligotrophicum ECU 1212 can be grown to produce the monooxygenase of the present invention.
  • a preferred medium formulation is: peptone 1 g / L, broth extract 1 g / L, NaCl 0.5 g / L, culture conditions: 28 ° C.
  • Another preferred medium formulation is glucose 15g/L, peptone 5g/L, yeast powder 5g/L, K 2 HPO 4 ⁇ 3H 2 O 0.5g/L, KH 2 PO 4 0.5g/L, NaCl 1.0g /L, MgSO 4 0.5 g / L, culture conditions: 28 ° C.
  • the Bradyrhizobium oligotrophicum ECU1212 is used to culture the harvested resting cells for asymmetric catalytic oxidation of lansoprazole thioether to prepare (R)-lansoprazole, the thioether substrate concentration is: 0.1 g/L, and the catalyst is uploaded as 10 g/L, the reaction was carried out at 30 ° C and stirring at 180 rpm.
  • the conversion of lansoprazole thioether can reach 80%, and the optical purity of the product reaches 99% ee (R).
  • the Bradyrhizobium oligotrophicum ECU1212 contains a functional enzyme having an asymmetric catalytic oxidation function, and has high catalytic conversion rate, and can solve the problem of low conversion rate of the conventional biocatalyst in the asymmetric catalytic oxidation process.
  • the Bradyrhizobium oligotrophicum ECU 1212 provided by the present invention can produce a monooxygenase comprising the amino acid sequence shown in SEQ ID No. 2.
  • the inventors of the present invention have found through experiments that the resting cells of the Bradyrhizobium oligotrophicum ECU1212 can asymmetrically oxidize the thioether compound to form a sulfoxide compound, thereby indicating that the Bradyrhizobium oligotrophicum ECU1212 can produce an enzyme having the catalytic function. And there is a gene encoding this enzyme. Therefore, based on the Bradyrhizobium oligotrophicum ECU 1212, the present invention also provides a monooxygenase, a preparation method and application thereof, an isolated gene encoding the monooxygenase, a recombinant expression vector containing the isolated gene, and a recombinant expression transformant .
  • the present invention provides a monooxygenase, which is based on the bioinformatics analysis strategy of the inventors of the present invention by screening and obtaining Bradyrhizobium oligotrophicum ECU1212, and predicting the monooxygenase which may have significant oxidation activity to thioether.
  • the genes are sorted and cloned for expression to verify their function.
  • a monooxygenase with high-efficiency asymmetric catalytic oxidation of thioether substrate to obtain chiral sulfoxide was obtained, which catalyzed the oxidation products of five major hindered azole thioether substrates.
  • the ee value was as high as 99%, and the monooxygenase was determined to contain the amino acid sequence as shown in SEQ ID No. 2.
  • the monooxygenase is named BoTEMO ( B radyrhizobium o ligotrophicum ECU1212 T hio e ther M ono o xygenase).
  • the present invention can also obtain a mutant amino acid sequence by mutating the amino acid sequence shown in SEQ ID No. 2 to modify the amino acid sequence shown in SEQ ID No. 2, that is, to modify BoTEMO, and obtain an activity-enhanced Monooxygenase.
  • the modified BoTEMO which is named as a BoTEMO mutant in the present invention, contains a mutant amino acid sequence in which the amino acid sequence shown in SEQ ID No. 2 is mutated.
  • BoTEMO was mutated by a random mutation strategy, and lansoprazole thioether was used as a screening substrate. After priming and rescreening the 10,000 mutant library, the amino acid sequence shown by SEQ ID No. 2 was subjected to mutation. , a derivative protein-boTEMO mutant obtained by substitution, deletion or addition of one or more amino acids.
  • the amino acid sequence of the BoTEMO mutant ie, the mutated amino acid sequence after mutation of the amino acid sequence shown in SEQ ID No. 2, that is, the mutated amino acid sequence relative to the amino acid sequence of BoTEMO
  • the amino acid sequence which is produced by substitution of any one to five amino acids in the amino acid sequence shown by SEQ ID No. 2 is used.
  • amino acid sequence of the BoTEMO mutant is preferably one or more of amino acid positions 295, 357, 394, 395, and 396 in the amino acid sequence shown in SEQ ID No. 2. And the resulting amino acid sequence.
  • amino acid sequence of the BoTEMO mutant is preferably an amino acid sequence which is produced by substitution of one or more of amino acid positions 295, 395, and 396 in the amino acid sequence shown in SEQ ID No. 2; Preferably, the amino acid sequence which is produced by substitution of one or both of the 357th and 394th amino acids in the amino acid sequence shown by SEQ ID No. 2 is used.
  • the Asp at position 295 in the amino acid sequence shown in SEQ ID No. 2 may be replaced with Cys; the Ser at position 357 in the amino acid sequence shown in SEQ ID No. 2 may be replaced with Ile; as SEQ ID No .2, the Phe at position 394 in the amino acid sequence shown is replaced by Ala; the Ser at position 395 in the amino acid sequence shown in SEQ ID No. 2 is replaced with Leu; in the amino acid sequence shown in SEQ ID No. Replace the Trp at position 396 with Ala.
  • nucleotide sequence corresponding to the amino acid sequence of the BoTEMO mutant which is the nucleotide sequence encoding the corresponding BoTEMO mutant, based on the basic knowledge of biology.
  • amino acid sequence of an activity-enhanced BoTEMO mutant provided by the present invention is the amino acid sequence shown in SEQ ID No. 4, and correspondingly, the amino acid sequence can be passed through the nucleoside as shown in SEQ ID No. 3. The acid sequence is encoded.
  • amino acid sequence of an activity-enhanced BoTEMO mutant further provided by the present invention is the amino acid sequence shown in SEQ ID No. 6, and correspondingly, the amino acid sequence can pass through the nucleus as shown in SEQ ID No. 5.
  • the nucleotide sequence is encoded.
  • the monooxygenase refers to any one or more of BoTEMO or BoTEMO mutants.
  • the present invention also provides an isolated gene comprising the nucleotide sequence shown in SEQ ID No. 1; or the isolated gene comprising the nucleotide sequence shown in SEQ ID No. 1 after mutation Mutant nucleotide sequence. Accordingly, the above isolated gene is capable of encoding the above monooxygenase.
  • the source of the isolated gene encoding BoTEMO of the present invention is obtained by using the genomic DNA of Bradyrhizobium oligotrophicum ECU1212 as a template, and obtaining a complete DNA core encoding the above BoTEMO by a conventional method in the art, such as polymerase chain reaction (PCR). Glycosylate molecule.
  • PCR polymerase chain reaction
  • the primer pair of the isolated gene was designed based on genomic analysis.
  • the forward primer and the reverse primer of the primer pair used to prepare the above isolated gene contain the nucleotide sequence shown below:
  • the Nde I cleavage site is underlined in the forward primer, and the Hind III cleavage site is underlined in the reverse primer. Then, the genomic DNA of Bradyrhizobium oligotrophicum ECU1212 was used as a template, and gene amplification was carried out by PCR to obtain a PCR product of the BoTEMO full-length gene.
  • nucleotide sequence of an isolated gene provided by the present invention is as shown in SEQ ID No. 1, and has a full length of 1461 bp, a start codon of ATG, a stop codon of TGA, and a coding sequence (CDS) from the first From 1 base to 1461 bases, the amino acid sequence of the encoded protein BoTEMO is shown in SEQ ID No. 2.
  • the nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 2 is not limited to the nucleotide sequence shown in SEQ ID No. 1.
  • a mutant nucleotide sequence obtained by mutating a nucleotide sequence as shown in SEQ ID No. 1 can be obtained by a person skilled in the art by appropriately introducing substitution, deletion, alteration, insertion or addition, that is, the present invention encompasses these mutant nucleosides.
  • the acid sequence is as long as the monooxygenase expressed therein maintains the asymmetric catalytic oxidation activity of the thioether.
  • the mutated nucleotide sequence of the nucleotide sequence shown in SEQ ID No. 1 in the present invention may be passed through one or more nucleotides in the nucleotide sequence as shown in SEQ ID No. 1. It is prepared by making substitutions, deletions or additions within the range of activity.
  • the nucleotide sequence shown in SEQ ID No. 1 of the present invention is capable of encoding BoTEMO, and the mutated nucleotide sequence mutated by the nucleotide sequence shown in SEQ ID No. 1 can encode a BoTEMO or BoTEMO mutant. Any of them.
  • the present invention also provides a recombinant expression vector comprising the isolated gene as described above.
  • the recombinant expression vector can be constructed by ligating the nucleotide sequence of the isolated gene of the present invention to various suitable vectors by a conventional method in the art.
  • the vector may be various conventional vectors in the art, such as a commercially available plasmid, cosmid, phage or viral vector; further, the vector is preferably a plasmid, and the recombinant expression vector prepared according to the conventional techniques in the art is recombinantly expressed.
  • a plasmid, a more preferred plasmid is plasmid pET28a.
  • An isolated gene of the invention can be operably linked to express a suitable regulatory sequence to effect constitutive or inducible expression of the monooxygenase.
  • the recombinant expression vector of the present invention can be produced by the following exemplary method: a PCR product comprising an isolated gene obtained by PCR amplification, and digested with restriction endonucleases NdeI and HindIII to form a complementary viscosity.
  • the cloning vector gene fragment and the expression vector pET28a were digested with restriction endonucleases NdeI and HindIII, and the digested gene fragment and expression vector were ligated with T4 DNA ligase to form a BoTEMO containing the present invention.
  • the recombinant expression plasmid pET-BoTEMO The recombinant expression plasmid pET-BoTEMO.
  • the present invention also provides a recombinant expression transformant comprising the recombinant expression vector as described above.
  • a recombinant expression transformant can be produced by transforming the recombinant expression vector of the present invention into a host cell.
  • the host cell may be various conventional host cells in the art, provided that the recombinant expression vector is stably self-replicating, and the isolated gene of the monooxygenase carried thereby can be efficiently expressed.
  • Escherichia coli is preferred in the present invention, and E. coli BL21 (DE3) or E. coli DH5 ⁇ is more preferred.
  • the recombinant expression plasmid pET-BoTEMO can be transformed into E. coli BL21 (DE3) to obtain a preferred genetically engineered strain of the present invention, namely recombinant Escherichia coli E. coli BL21(DE3)/pET -BoTEMO.
  • the culture method and culture conditions of the recombinant expression transformant of the present invention are not particularly limited, and may be appropriately selected according to the common knowledge in the field according to factors such as host cell type and culture method, as long as the recombinant expression transformant can grow and The monooxygenase of the present invention may be produced.
  • the recombinant expression transformant is Escherichia coli
  • an LB medium containing peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH 7.0 is preferred.
  • the recombinant Escherichia coli preferably E.
  • coli BL21 (DE3)) of the present invention is inoculated into LB medium containing kanamycin.
  • Culture when the optical density OD600 of the culture solution reaches 0.5-0.7 (preferably 0.6), the isopropyl- ⁇ -D-thiopyran half at a final concentration of 0.1-1.0 mmol/L (preferably 0.2 mmol/L)
  • IPTG lactobionide
  • the expressed monooxygenase can be isolated by conventional methods in biology.
  • the present invention discloses a method for producing the above monooxygenase, which comprises culturing the above recombinant expression transformant, and then separating the monooxygenase therefrom.
  • the invention also provides the use of the above Bradyrhizobium oligotrophicum ECU1212 or monooxygenase in asymmetric catalytic oxidation of latent chiral thioether compounds.
  • Bradyrhizobium oligotrophicum ECU 1212 can be used in the form of a resting whole cell for the asymmetric catalytic oxidation of latent thioether compounds.
  • the latent thioether compound is selected from the compounds of any of the following chemical formulas:
  • the Chinese names of the above Chemical Formulas I to IX are respectively phenyl sulfide, p-methyl benzo sulfide, p-methoxy thioanisole, 5-methoxy-2-(methyl sulphur) Generation) benzimidazole, omeprazole thioether, lansoprazole thioether, pantoprazole thioether, rabeprazole thioether, iprazol thioether, of course, in other literature It can also be named in a different way.
  • the monooxygenase asymmetrically catalyzes the latent chiral thioether compound to a sulfoxide compound.
  • asymmetric oxidation of the thioether to the optically active sulfoxide is carried out using the Bradyrhizobium oligotrophicum ECU 1212 or monooxygenase of the present invention.
  • the specific reaction conditions involved such as substrate concentration, pH, buffer composition, enzyme dosage, and the like, can be selected according to conventional conditions for such reactions in the art.
  • the asymmetric catalytic oxidation reaction can be carried out under shaking or stirring.
  • the reaction can be carried out by the following exemplary method: as shown in FIG. 1, the concentration of the Tris-HCl buffer can be 0.05 to 0.2 mol/L in a Tris-HCl buffer having a pH of 8.0 to 10.0. .
  • An optically active sulfoxide is obtained by asymmetric oxidation of thioether in the presence of glucose dehydrogenase, glucose and NADP+ under the action of the monooxygenase of the present invention.
  • the concentration of the substrate in the reaction solution is from 0.1 to 37 g/L.
  • the enzyme living unit (U) of the monooxygenase of the present invention is defined as the amount of enzyme required to catalyze the production of 1 ⁇ mol of substrate per minute.
  • glucose and glucose dehydrogenase from Bacillus megaterium are additionally added to the reaction system (for preparation, see: Journal of Industrial Microbiology and Biotechnology 2011, 38, 633-641).
  • NADP+ is converted to NADPH by glucose oxidation catalyzed by glucose dehydrogenase.
  • the unit of activity of the glucose dehydrogenase may be comparable to the unit of activity of the monooxygenase of the present invention.
  • the amount of glucose may be 2 to 20 mmol/L, and the amount of additional NADP+ may be 0 to 1 mmol/L.
  • the temperature of the asymmetric oxidation reaction may be from 20 to 35 ° C, preferably 25 ° C.
  • the chiral sulfoxide product can be extracted from the reaction mixture by conventional methods in the art.
  • the invention provides a Bradyrhizobium oligotrophicum ECU1212, which can derive a monooxygenase from the Bradyrhizobium oligotrophicum ECU1212, which can efficiently catalyze the oxidation of thioether compounds to form optically pure chiral sulfoxide compounds, and has high efficiency and high stereoselection. Sex and high conversion rate characteristics. For example, when the concentration of lansoprazole thioether substrate reaches 10 g/L, the conversion rate still exceeds 99%, the ee value reaches 99% or more, and the product sulfoxide does not further oxidize to a sulfone by-product. Compared with other asymmetric oxidation preparation methods, the product obtained by the method of the invention has high concentration, good optical purity, no by-product formation, mild reaction conditions, environmental friendliness, simple operation, easy industrial amplification, and therefore good industrialization. Application prospects.
  • the expression plasmid pET28a was purchased from Shanghai Novagen.
  • E.coli DH5 ⁇ and E.coli BL21(DE3) competent cells were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.
  • the collection of soil is mainly divided into two parts.
  • the soil samples collected directly after collecting soil samples and embedded substrates, a total of 252 soil samples.
  • Direct soil sample collection collect more moist soil, usually water source, plant, contaminated substrate, etc., dig the soil 2-3cm away from the ground, about 3-5g, the soil sample after use is placed at low temperature, Store in a dry place or directly in a 1.5mL Eppendorf tube and store in a 4°C freezer.
  • the collection locations are as follows: Shanghai Fengxian Chemical Zone, Xinhua Hospital, Orchard, Vegetable Farm, Garbage Bin, Riverside, Green Belt, Campus (East China University of Technology Xuhui or Fengxian Campus), residential greening, botanical garden, etc.
  • Lansoprazole thioether is white powder, hardly soluble in water but soluble in dimethyl sulfoxide (DMSO), so it is pre-buried in multiple locations using two different forms. Usually vegetation is abundant, and the microbial population may have the potential to be domesticated. The first is to directly pre-bury the white powder to the soil about 5 cm away from the ground. The second is to dissolve the lansoprazole thioether in dimethyl sulfoxide (DMSO) and then pour it onto the soil surface.
  • DMSO dimethyl sulfoxide
  • All reactions were carried out at 30 ° C before the selection of the selected strains and optimization of the culture conditions.
  • the loading of the medium in the test tubes was 4 mL, and the rotational speed of the shaker was set to 180 r/min.
  • the screening process was carried out in four rounds of enrichment and a gradient culture method was used in which the concentration of yeast powder was halved in each round of culture, and the substrate concentration was doubled during each round of cultivation.
  • the plate culture was carried out in an incubator at 30 °C.
  • Bradyrhizobium oligotrophicum ECU1212 obtained by screening in Example 1 was inoculated to rich medium (glucose 15 g/L, peptone 10 g/L, yeast extract 5 g/L, NaH 2 PO 4 0.5 g/L, MgSO 4 0.5 g/L, NaCl In 10 g/L, pH 7.0), the cells were shake-cultured at 28 ° C and 180 rpm for 24 hours, and then centrifuged at 5000 ⁇ g for 10 min to collect wet cells. The collected wet cells were frozen at -80 ° C for 12 h, and then dried by a freeze dryer for 20 h to obtain freeze-dried cells, which were stored in a refrigerator at 4 ° C.
  • rich medium glucose 15 g/L, peptone 10 g/L, yeast extract 5 g/L, NaH 2 PO 4 0.5 g/L, MgSO 4 0.5 g/L, NaCl In 10 g/L, pH 7.0
  • the present invention analyzes the genes of enzymes which may have significant asymmetric catalytic oxidation activity for thioethers through bioinformatics analysis strategies, and sorts them for cloning and expression. Its function. Using this method, an isolated gene of BoTEMO capable of producing a highly efficient asymmetric catalytic oxidation of thioether substrate was cloned from Bradyrhizobium oligotrophicum ECU1212.
  • BoTEMO present invention (Bradyrhizobium oligotrophicum ECU1212 sulfide monooxygenase, B radyrhizobium o ligotrophicum ECU1212 T hio e ther M ono o xygenase) catalytic five kinds of sterically hindered omeprazole sulfide oxidation product substrate ee of 99%.
  • the optical purity of the product can be significantly increased compared to conventional biocatalysts.
  • a preferred source of the BoTEMO isolated gene of the present invention is the genomic DNA of Bradyrhizobium oligotrophicum ECU1212 as a template, and a complete nucleic acid molecule encoding the BoTEMO is obtained by a conventional method in the art (such as polymerase chain reaction, PCR).
  • nucleotide sequence of the primer pair is as follows:
  • the underlined portion of the forward primer is an NdeI restriction site
  • the underlined portion of the reverse primer is a HindIII restriction site.
  • the genomic DNA of Bradyrhizobium oligotrophicum ECU1212 prepared as described in Example 1 was used as a template, and gene amplification was carried out by polymerase chain reaction (PCR).
  • the PCR system is: 2 ⁇ Taq PCR MasterMix 25 ⁇ l, 1.5 ⁇ l (0.3 ⁇ mol/L) of the forward and reverse primers, 1.5 ⁇ l (0.1 ⁇ g) of the DNA template, 2 ⁇ l of DMSO and 19 ⁇ l of ddH 2 O.
  • the PCR amplification steps were: (1) 95 ° C, pre-denaturation for 3 min; (2) 94 ° C, denaturation for 1 min; (3) annealing at 55 ° C for 30 s; (4) extension at 72 ° C for 2 min; steps (2) - (4) repeated 30 times; (5) 72 ° C continued to extend for 10 min, cooled to 12 ° C.
  • the PCR product was purified by agarose gel electrophoresis, and the target band in the range of 1400 to 1600 bp was recovered by using an agarose gel DNA recovery kit.
  • the nucleotide sequence of the isolated gene contained therein is 1461 bp in length, and the nucleotide sequence thereof is shown in SEQ ID No. 1.
  • the start codon is ATG
  • the stop codon is TGA
  • the coding sequence (CDS) is from the first base to the 1461th base
  • the amino acid sequence of the encoded monooxygenase is as shown in SEQ ID No. 2. Show.
  • the PCR product containing the isolated gene cloned as described in Example 3 was digested with restriction endonucleases NdeI and HindIII for 12 hours at 37 ° C, and purified by agarose gel electrophoresis using agarose gel DNA recovery reagent. The box recycles the target segment. Under the effect of T 4 DNA ligase, the fragment of the same target was digested plasmid pET28a after NdeI and Hindlll, connected at 16 deg.] C overnight to give the recombinant plasmid pET-BoTEMO.
  • the above recombinant expression plasmid was transformed into E. coli DH5 ⁇ competent cells, and the positive recombinants were screened on a kanamycin-containing resistant plate, and the monoclonal clones were picked, and the colonies were verified by colony PCR.
  • the recombinant strain was cultured, and after the plasmid was amplified, the plasmid was extracted and re-transformed into E. coli BL21 (DE3) competent cells, and the transformant was applied to an LB plate containing kanamycin, and cultured at 37 ° C overnight. That is, a positive recombinant expression transformant Escherichia coli E. coli BL21(DE3)/pET-BoTEMO was obtained, and colony PCR confirmed a positive clone.
  • the recombinant expression transformant E. coli BL21(DE3)/pET-BoTEMO prepared by the method described in Example 4 was inoculated into LB medium (peptone 10 g/L, yeast containing 50 ⁇ g/mL kanamycin). 5g/L, NaCl 10g/L, pH 7.0), shaking culture at 37 ° C, 180 rpm shaker, when the OD 600 of the culture solution reached 0.6, adding IPTG with a final concentration of 0.2 mmol/L as an inducer, 16 After induction for 16 hours at ° C, the culture solution was centrifuged, and the cells were collected and washed twice with physiological saline to obtain resting cells.
  • LB medium peptone 10 g/L, yeast containing 50 ⁇ g/mL kanamycin
  • 5g/L, NaCl 10g/L, pH 7.0 shaking culture at 37 ° C, 180 rpm shaker, when the OD 600 of the culture solution reached 0.6, adding IP
  • the resting cells obtained from 100 mL of the fermentation broth were suspended in 10 mL of pH 7.0 buffer, and sonicated in an ice water bath (the setting power of the ultrasonic breaker was 400 W, working for 4 s, intermittent 6 s, total circulation 99 times) .
  • the crushed liquid was centrifuged at 15,000 rpm for 40 min in a 4 ° C low temperature centrifuge to obtain a supernatant enzyme solution for viability determination and protein purification.
  • the collected crude enzyme solution was frozen at -80 ° C for 12 h, and then dried at a low temperature for 20 h in a vacuum freeze dryer to obtain a freeze-dried crude enzyme powder, which was stored in a refrigerator at 4 ° C.
  • the activity of the lyophilized crude enzyme powder to thioanisole was 0.2 U/mg.
  • the crude enzyme solution was analyzed by polyacrylamide gel electrophoresis. The recombinant protein was partially soluble in the cells, and some proteins were present in the cell debris.
  • the purification experiments were all performed using a nickel affinity self-packing column.
  • the buffer used in the purification process was: liquid A: 50 mM KPB, 500 mM NaCl, 10 mM imidazole, 2 mM ⁇ -mercaptoethanol, pH 8.0; liquid B: 50 mM KPB, 500 mM NaCl, 300 mM imidazole, 2 mM ⁇ -mercaptoethanol, pH 8.0; C solution: 50 mM KPB, 150 mM NaCl, 1 mM DTT, pH 9.0.
  • the purification method is as follows:
  • Ni column is pre-equilibrated with 5 to 10 column volumes of liquid A;
  • the collected target protein is concentrated with a 30kDa ultrafiltration tube. When concentrated to 500 ⁇ l, add 5ml of C solution and continue to concentrate by ultrafiltration. Repeat 3 ⁇ 5 times to remove the imidazole in the enzyme solution and reduce the salt concentration. Replacement
  • BoTEMO and glucose dehydrogenase were determined by measuring the change in absorbance at 340 nm using a spectrophotometer.
  • BoTEMO activity was as follows: 1 mL reaction system (100 mmol/L Tris-HCl buffer, pH 9.0), 1 mmol/L thioanisole, 0.2 mmol/L NADPH was added, and the mixture was incubated at 30 ° C for 2 minutes, and then added to Example 5 Prepare the appropriate amount of crude enzyme solution, mix quickly, and detect the change in absorbance at 340 nm. The specific activity of the crude enzyme solution was measured to be 101 mU/mL.
  • the glucose dehydrogenase activity was measured as follows: 1 mL of the reaction system (100 mmol/L sodium phosphate buffer, pH 7.0), 10 mmol/L glucose, 1 mmol/L NADP + was added, and the mixture was incubated at 30 ° C for 2 minutes, and then dehydrogenated with glucose.
  • the enzyme (see Preparation: Journal of Industrial Microbiology and Biotechnology 2011, 38, 633-641) was rapidly mixed to detect changes in absorbance at 340 nm in real time.
  • enzyme activity (U) EW ⁇ V ⁇ 10 3 / (6220 ⁇ 1)
  • EW is the change in absorbance at 340 nm in 1 minute; V is the volume of the reaction solution in ml; 6220 is the molar extinction coefficient of NADPH in units of L/(mol ⁇ cm); 1 is the optical path distance, unit Is cm.
  • Per unit of BoTEMO is defined as the amount of enzyme required to catalyze the oxidation of 1 ⁇ mol of NADPH per minute under the above conditions.
  • Each unit of glucose dehydrogenase is defined as the amount of enzyme required to catalyze the reduction of 1 ⁇ mol of NADP + per minute under the above conditions.
  • BoTEMO pure enzyme BoTEMO pure enzyme prepared by the method described in Example 5 was added to 0.5 mL of potassium phosphate buffer (100 mmol/L, pH 9.0), and a thioether substrate dissolved in DMSO was added to terminate the thioether. The concentration was 0.2 to 2 mmol/L, the final concentration of DMSO was 2% (v/v), and the final concentration was 0.2 to 2 mmol/L of NADPH. The reaction was shaken at 1000 ° C for 1 hour at 25 °C. After the completion of the reaction, 0.6 mL of ethyl acetate was added for extraction, and the extract was dried over anhydrous sodium sulfate. The organic phase was taken up and evaporated to remove the solvent overnight, and then dissolved in 0.5 mL of isopropanol, and the ee value of the product was analyzed.
  • AS-H column 250 mm ⁇ 4.6 mm, 5 ⁇ m particle size, Daicel
  • Bradyrhizobium oligotrophicum ECU1212 a resting cell asymmetric catalytic oxidation of thioanisole
  • lyophilized cells (freeze-dried cells prepared as described in Example 2) of Bradyrhizobium oligotrophicum ECU1212 was added to 100 mL of Tris-HCl buffer (100 mmol/L, pH 9.0), and thioanisole and methanol were added to a final concentration of 37 g/L. , 10% (v/v). The reaction was stirred at 28 ° C, 180 rpm, and 100 ⁇ L was sampled intermittently.
  • BoTEMO crude enzyme solution the crude enzyme solution prepared by the method described in Example 5
  • glucose dehydrogenase crude enzyme solution to 0.5 mL potassium phosphate buffer (100 mmol/L, pH 9.0), and add thioanisole.
  • the final concentrations of methanol, NADP + and glucose were 2 mmol/L, 10% (v/v), 0.2 mmol/L and 3.6 g/L, respectively.
  • the reaction was shaken at 1000 ° C for 1 hour at 25 °C.
  • BoTEMO crude enzyme solution the crude enzyme solution prepared by the method described in Example 5
  • glucose dehydrogenase crude enzyme solution to 0.5 mL of potassium phosphate buffer (100 mmol/L, pH 9.0), and add omeprazole.
  • the final concentrations of thioether, methanol, NADP + and glucose were 0.2 mmol/L, 10% (v/v), 0.2 mmol/L and 3.6 g/L, respectively.
  • the reaction was shaken at 1000 ° C for 1 hour at 25 °C.
  • BoTEMO lyophilized crude enzyme powder (the crude enzyme powder prepared by the method described in Example 5) and 0.2 g of glucose dehydrogenase lyophilized enzyme powder to 100 mL of Tris-HCl buffer (100 mmol/L, pH 9.0).
  • the final concentrations of omeprazole thioether, methanol, NADP + and glucose were 1 g/L, 10% (v/v), 0.2 mmol/L and 3.6 g/L, respectively.
  • the reaction was stirred at 180 ° C at 180 ° C, and after 3 h of reaction, the substrate conversion and product ee were determined as described in Example 7, the substrate conversion was greater than 99%, and the product ee value was greater than 99% (R).
  • BomethO lyophilized crude enzyme powder 0.2 g (the crude enzyme powder prepared as described in Example 5) and glucose dehydrogenase lyophilized enzyme powder 0.02 g were added to 10 mL of Tris-HCl buffer (100 mmol/L, pH 9.0), and added.
  • the reaction was carried out at 25 ° C, 180 rpm for 24 hours.
  • the substrate conversion and product ee values were determined as described in Example 7.
  • the conversion of the substrate of (R)-lansoprazole obtained by asymmetric catalytic oxidation of lansoprazole sulphide is greater than 99%, and the ee value of the product is greater than 99% (R).
  • the G at position 883 of the nucleotide sequence encoding BoTEMO was mutated to T, the A mutation at position 884 was G, the C mutation at position 1184 was T, and the T mutation at position 1186 was G, the 1187th position.
  • the G mutation is C, thereby obtaining the nucleotide sequence of the mutated gene as shown in SEQ ID No. 3.
  • the encoded amino acid sequence is SEQ ID No. 4, that is, the Asp at position 295 of BoTEMO (such as the amino acid sequence of SEQ ID No. 2) is mutated to Cys, and the Ser at position 395 is mutated to Leu, at position 396.
  • the Trp mutation is Ala, and the monooxygenase encoded by the mutant gene is named BoTEMO-M1.
  • the G at position 1070 of the nucleotide sequence encoding BoTEMO was mutated to T, the T mutation at position 1180 was G, the T mutation at position 1181 was C, and the C mutation at position 1182 was A, thereby obtaining The nucleotide sequence of the mutated gene shown in SEQ ID No. 5.
  • the amino acid sequence encoded by the gene is SEQ ID No. 6, that is, the Ser at position 357 of BoTEMO (such as the amino acid sequence shown in SEQ ID No. 2) is mutated to Ile, and the Phe at position 394 is mutated to Ala.
  • the encoded monooxygenase was named BoTEMO-M2.
  • Recombinant transformants were prepared by the methods described in Example 4 using the above-mentioned BoTEMO-M1 and BoTEMO-M2 mutant genes, respectively, and resting cells and lyophilized crude enzyme powder were prepared according to the method as described in Example 5, further The enzyme activities of BoTEMO-M1 and BoTEMO-M2 were determined according to the enzyme activity assay method as described in Example 6.
  • the enzyme activities of BoTEMO-M1 and BoTEMO-M2 were 7.6 times (BoTEMO-M1) and 1.6 times (BoTEMO), respectively.
  • -M2 the activity of lansoprazole thioether reached 20 U/g (BoTEMO-M1) and 4.2 U/g (BoTEMO-M2).
  • BoTEMO-M1 asymmetric catalytic oxidation of lansoprazole sulphide
  • BoTEMO-M1 asymmetric catalytic oxidation of lansoprazole sulphide

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Abstract

一种慢生根瘤菌单加氧酶、编码该酶的基因、含有该基因的重组表达载体和重组表达转化体，利用重组表达转化体制备所述单加氧酶的方法，以及利用所述单加氧酶制备光学纯手性亚砜，特别是催化拉唑类前体硫醚不对称氧化制备拉唑类药物的方法。

Description

一种慢生根瘤菌单加氧酶及其在制备手性亚砜中的应用 技术领域
本发明属于生物工程技术领域，尤其是涉及一种慢生根瘤菌单加氧酶、编码该酶的基因、含有该基因的重组表达载体和重组表达转化体，利用重组表达转化体制备所述单加氧酶的方法，以及利用所述单加氧酶制备光学纯手性亚砜，特别是催化拉唑类前体硫醚氧化制备拉唑类药物的方法。
背景技术
手性亚砜具有广泛而重要的应用价值，它的用途大致可以分为几类：手性辅助试剂、手性配体、手性催化剂以及手性药物和药物中间体。
一些手性亚砜是含有硫原子手性中心药物的重要中间体或药物本身。例如：艾司奥美拉唑为代表的一系列苯并吡唑类质子泵抑制剂，都是手性杂环基亚砜类化合物。质子泵抑制剂(PPIs,Proton Pump Inhibitors)是许多胃食管疾病(如胃及十二指肠溃疡，胃食管反流病)的首选治疗药物，作为H +/K +-ATP酶抑制剂，具有起效快、作用强、特异性高和持续时间长的特点。目前临床上广泛应用的质子泵抑制剂有奥美拉唑(1988年在瑞典上市)，兰索拉唑(1995年在日本上市)，泮托拉唑(1997年在德国上市)，雷贝拉唑(1999年在美国上市)以及艾司奥美拉唑(2001年在美国上市)。其中兰索拉唑(Lansoprazole)，别名达克普隆(Takepron)，化学名2-[3-甲基-4-(2,2,2-三氟乙氧基)-2-哌啶基]甲基亚硫酰基-1H-苯并咪唑，作为新一代质子抑制剂类抗酸药及抗溃疡病药物，强力抑制胃酸分泌。在临床应用中，兰索拉唑的生物利用度较奥美拉唑更高，而单一右旋兰索拉唑缓释胶囊在治愈率、控酸作用持续时间、烧心控制率等几个方面，都比消旋兰索拉唑具有更好的表现。单一右旋兰索拉唑由日本武田制药公司研发，于2009年获得FDA批准在美国上市，此后在欧、亚、美洲等多个国家相继上市。
利用生物催化的方法合成手性亚砜具有立体选择性高、反应条件温和安全、环境友好等优点，是化学方法合成手性亚砜的有益补充，并且随着生物技术的发展已经成为目前研究的热点。虽然可以不对称催化氧化硫醚化合物的生物催化剂种类繁多，但是现有的生物催化剂对大位阻硫醚底物的催化效率极低。亚克(Babiak)等 从土壤污染物中筛选到了一株野生菌，经鉴定为赖氨酸芽孢杆菌(Lysinibacillus)，使用该菌株生长细胞催化奥美拉唑硫醚的转化，底物上载量为0.1g/L时，发酵培养48h后，转化率仅为77％。WO2011/071982中公开了Codexis公司对来自Acinetobacter sp.NCIMB 9871的环己酮单加氧酶CHMO进行了定向进化，得到的工程酶能够高效催化奥美拉唑硫醚氧化制备(S)-奥美拉唑，尽管如此，目前仍然缺乏能够不对称催化氧化兰索拉唑硫醚的高效催化剂。Codexis公司得到的工程酶能够催化兰索拉唑硫醚生成(R)-兰索拉唑，但是工程酶的催化效率极低，底物上载量为1.5g/L时，17h转化率仅为1.2％，且由于极低的转化率导致无法测定产物的光学纯度。
目前，右旋兰索拉唑仍然采用化学法生产，在现有生产工艺中，不对称催化氧化过程的选择性差、转化率低。手性金属钛试剂和辅剂酒石酸用量大，硫醚剩余约10～15％，杂质砜的含量约2％。由于反应产物杂质多，后处理过程中需要多次萃取结晶，产率低，并且产生大量三废(废水、废气、废液)。而现有的生物催化剂虽然反应条件温和、安全且环境友好，但是转化率较低。
发明内容
本发明针对现有技术中化学法生产亚砜类化合物选择性差、转化率低以及生物催化合成大位阻亚砜类化合物转化率低的问题，提供了一株筛选获得的Bradyrhizobium oligotrophicum ECU1212。
本发明提供了一种Bradyrhizobium oligotrophicum，其为Bradyrhizobium oligotrophicum ECU1212，保藏在中国微生物菌种保藏委员会普通微生物中心，保藏编号为CGMCC No.15208。
在其中一个实施例中，所述的Bradyrhizobium oligotrophicum可以产生氨基酸序列如SEQ ID No.2所示的Bradyrhizobium oligotrophicum ECU1212硫醚单加氧酶。
本发明还提供了一种单加氧酶，所述单加氧酶含有如SEQ ID No.2所示的氨基酸序列；或，
所述单加氧酶含有如所述SEQ ID No.2所示的氨基酸序列发生突变后的突变氨基酸序列。
在其中一个实施例中，所述突变氨基酸序列为所述SEQ ID No.2所示的氨基酸序列中任意1至5个氨基酸发生替换后而生成的突变氨基酸序列。
在其中一个实施例中，所述突变氨基酸序列为所述SEQ ID No.2所示的氨基酸序列中第295位、第357位、第394位、第395位以及第396位的氨基酸中的任意一个或多个发生替换后而生成的突变氨基酸序列。
在其中一个实施例中，所述突变氨基酸序列包括以下任意一项或多项特征：
(1)所述SEQ ID No.2所示的氨基酸序列中第295位的氨基酸Asp替换为Cys；
(2)所述SEQ ID No.2所示的氨基酸序列中第357位的氨基酸Ser替换为Ile；
(3)所述SEQ ID No.2所示的氨基酸序列中第394位的氨基酸Phe替换为Ala；
(4)所述SEQ ID No.2所示的氨基酸序列中第395位的氨基酸Ser替换为Leu；
(5)所述SEQ ID No.2所示的氨基酸序列中第396位的氨基酸Trp替换为Ala。
在其中一个实施例中，，所述单加氧酶含有如SEQ ID No.4所示的突变氨基酸序列。
在其中一个实施例中，所述单加氧酶含有如SEQ ID No.6所示的突变氨基酸序列。
本发明还提供了一种分离的核酸，所述的核酸编码上述任一项所述的单加氧酶。
本发明还提供了一种重组表达载体，所述重组表达载体包含上述的核酸。
本发明还提供了一种重组表达转化体，所述重组表达转化体包含如上所述的重组表达载体。
本发明还提供了一种如上所述的单加氧酶的制备方法，包括以下步骤：
培养如上所述的重组表达转化体，从中分离所述单加氧酶。
本发明还提供了一种如上所述的Bradyrhizobium oligotrophicum或单加氧酶在不对称催化氧化潜手性硫醚化合物中的应用。
在其中一个实施例中，所述潜手性硫醚化合物选自以下任一化学式所示的化合物：
Figure PCTCN2018083060-appb-000001
Figure PCTCN2018083060-appb-000002
在其中一个实施例中，将所述潜手性硫醚化合物不对称催化氧化为亚砜化合物。
本发明的积极进步效果在于：本发明提供了一种单加氧酶，所述单加氧酶包括BoTEMO、BoTEMO突变体，所述单加氧酶可以高效催化硫醚的不对称氧化以制备光学纯手性亚砜。在兰索拉唑硫醚底物浓度达到10g/L时，转化率仍达到99％以上，ee值达到99％以上，并且产物亚砜不会进一步氧化为砜副产物。相对于其他不对称氧化制备方法，使用本发明方法制备所得的产物浓度高，光学纯度好，无副产物生成，反应条件温和，对环境友好，操作简便，易于工业放大，因此具有很好的工业应用前景。
附图说明
图1为本发明的单加氧酶不对称催化氧化硫醚化合物生成光学纯手性亚砜的反应过程示意图。
具体实施例
本发明提供的一种Bradyrhizobium oligotrophicum，是发明人进行了大规模土壤微生物筛选获得的。其中，土壤的采集主要分为两个部分，直接采集土样和预埋底物之后的土样采集，共252份土样。以兰索拉唑硫醚为导向，通过四轮的梯度富集培养不断提高兰索拉唑硫醚的浓度，通过初筛和复筛，从土壤中分离获得一种可以催化兰索拉唑硫醚氧化的菌株，命名为Bradyrhizobium oligotrophicum ECU1212； 该命名采用属名(genus)+种名(species)+菌株代号的命名方式，其中，Bradyrhizobium表示属名，oligotrophicum表示种名，ECU1212表示菌株代号；本发明中将Bradyrhizobium译为慢生根瘤菌，或慢生根瘤菌属。
目前，该Bradyrhizobium oligotrophicum已经保藏于中国微生物菌种保藏管理委员会普通微生物中心(China General Microbiological Culture Collection Center，CGMCC)，保藏时间为：2018-01-15，其保藏编号为：CGMCC No.15208。
该Bradyrhizobium oligotrophicum ECU1212具有以下生理生化特征：
在显微镜下观察呈杆状，不产生芽孢，革兰氏阴性，好氧，以氧为末端电子受体的严格呼吸型，以一根极毛或亚极毛运动；菌落呈圆形，不透明，罕见半透明，白色和凸起，有颗粒状结构，最适温度25～30℃，最适pH 6.0～8.0；在酵母膏-甘露醇-无机盐琼脂上5～7天的菌落不超过lmm，在5～7天或更长时间的液体培养物呈中度混浊。
该Bradyrhizobium oligotrophicum ECU1212的培养方法和培养条件没有特殊的限制，只要能使该Bradyrhizobium oligotrophicum ECU1212的菌株生长并产生本发明所述的单加氧酶即可。一种优选的培养基配方为：蛋白胨1g/L，肉汤提取物1g/L，NaCl 0.5g/L，培养条件为：28℃。另一种优选的培养基配方为葡萄糖15g/L，蛋白胨5g/L，酵母粉5g/L，K 2HPO 4·3H 2O 0.5g/L，KH 2PO 40.5g/L，NaCl 1.0g/L，MgSO 40.5g/L，培养条件为：28℃。
可选地，使用该Bradyrhizobium oligotrophicum ECU1212培养收获的静息细胞不对称催化氧化兰索拉唑硫醚制备(R)-兰索拉唑，硫醚底物浓度为：0.1g/L，催化剂上载为10g/L，反应在30℃，180rpm搅拌条件下进行。兰索拉唑硫醚的转化率能够达到80％，产物的光学纯度达99％ee(R)。由此可知，该Bradyrhizobium oligotrophicum ECU1212中含有具有不对称催化氧化功能的功能性酶，且催化的转化率高，能够解决传统的生物催化剂在不对称催化氧化过程中转化率低的问题。
进一步地，本发明提供的Bradyrhizobium oligotrophicum ECU1212可以产生一种单加氧酶，该单加氧酶含有如SEQ ID No.2所示的氨基酸序列。
本发明的发明人通过实验发现，该Bradyrhizobium oligotrophicum ECU1212的静息细胞能够对硫醚化合物进行不对称催化氧化生成亚砜化合物，从而能够表明该Bradyrhizobium oligotrophicum ECU1212能够产生一种具有该催化功能的酶，并且具有编码该种酶的基因。因此，基于该Bradyrhizobium oligotrophicum ECU1212，本发明还分别提供了一种单加氧酶及其制备方法和应用、编码该单加氧酶的分离基 因、含有该分离基因的重组表达载体以及重组表达转化体。
本发明提供的一种单加氧酶，是本发明发明人在筛选获得Bradyrhizobium oligotrophicum ECU1212的基础上，通过生物信息学分析的策略，分析预测其中可能对硫醚具有明显氧化活性的单加氧酶的基因，并将其分选出来进行克隆表达，验证其功能获得的。采用这种方法，从中克隆获得一种具有高效不对称催化氧化硫醚底物获得手性亚砜的单加氧酶，该单加氧酶催化5种大位阻拉唑硫醚底物氧化产物的ee值高达99％，经测定，该单加氧酶含有如SEQ ID No.2所示的氨基酸序列。本发明中将该单加氧酶命名为BoTEMO( Bradyrhizobium  oligotrophicum ECU1212  Thio ether  Mono oxygenase)。
进一步地，本发明还可以通过使如SEQ ID No.2所示的氨基酸序列发生突变获得突变氨基酸序列，以改造如SEQ ID No.2所示的氨基酸序列，即改造BoTEMO，获得了活性提高的单加氧酶。改造后的BoTEMO，本发明将其命名为BoTEMO突变体，该BoTEMO突变体含有如所述SEQ ID No.2所示的氨基酸序列发生突变后的突变氨基酸序列。
可选地，采用随机突变策略对BoTEMO进行突变改造，以兰索拉唑硫醚为筛选底物，经过对10,000突变体库初筛、复筛获得由SEQ ID No.2所示氨基酸序列经过突变，即经过取代、缺失或添加一个或多个氨基酸而得到的酶活性提高的衍生蛋白质--BoTEMO突变体。
突变改造后的BoTEMO，即BoTEMO突变体的氨基酸序列(即如SEQ ID No.2所示的氨基酸序列的发生突变后的突变氨基酸序列，也就是说相对于BoTEMO的氨基酸序列的突变氨基酸序列)，优选地为如SEQ ID No.2所示的氨基酸序列中任意1～5个氨基酸发生替换而生成的氨基酸序列。
进一步地，BoTEMO突变体的氨基酸序列优选为如SEQ ID No.2所示的氨基酸序列中第295位、第357位、第394位、第395位、第396位氨基酸中一个或多个发生替换而生成的氨基酸序列。
更进一步地，BoTEMO突变体的氨基酸序列优选为如SEQ ID No.2所示的氨基酸序列中第295位、第395位、第396位氨基酸中一个或多个发生替换而生成的氨基酸序列；或者优选为如SEQ ID No.2所示的氨基酸序列中第357位、第394位氨基酸中一个或两个发生替换而生成的氨基酸序列。
例如，可以是如SEQ ID No.2所示的氨基酸序列中第295位的Asp替换为Cys；如SEQ ID No.2所示的氨基酸序列中第357位的Ser替换为Ile；如SEQ ID No.2 所示的氨基酸序列中第394位的Phe替换为Ala；如SEQ ID No.2所示的氨基酸序列中第395位的Ser替换为Leu；如SEQ ID No.2所示的氨基酸序列中第396位的Trp替换为Ala。
相应地，依据BoTEMO突变体的氨基酸序列，本领域技术人员可以根据生物学的基本知识，确定编码相应BoTEMO突变体的核苷酸序列，即BoTEMO突变体的氨基酸序列对应的核苷酸序列。
可选地，本发明提供的一种活性提高的BoTEMO突变体的氨基酸序列如SEQ ID No.4所示的氨基酸序列，相应地，该氨基酸序列可以通过如SEQ ID No.3所示的核苷酸序列编码获得。
可选地，本发明还提供的一种活性提高的BoTEMO突变体的氨基酸序列如SEQ ID No.6所示的氨基酸序列，相应地，该氨基酸序列可以通过如SEQ ID No.5所示的核苷酸序列编码。
需要说明的是，在本发明中没有特别指出时，单加氧酶指的是BoTEMO或BoTEMO突变体中的任意一种或多种。
本发明还提供了一种分离基因，该分离基因含有如SEQ ID No.1所示的核苷酸序列；或，该分离基因含有如SEQ ID No.1所示的核苷酸序列发生突变后的突变核苷酸序列。相应地，上述分离基因能够编码上述单加氧酶。
可选地，编码本发明的BoTEMO的分离基因的来源有：以Bradyrhizobium oligotrophicum ECU1212的基因组DNA为模板，采用本领域常规技术方法，如聚合酶链反应(PCR)，获得编码上述BoTEMO的完整DNA核苷酸分子。并根据基因组分析设计的合成该分离基因的引物对。
可选地，用于制备上述分离基因的引物对的正向引物以及反向引物含有如下所示的核苷酸序列：
正向引物：CCG  CATATG TCAACTGAGCATGTCGAC
反向引物：CCG  AAGCTT TCACGAATACCGCATCACCC
其中，正向引物中以下划线标出的为Nde I酶切位点，反向引物中以下划线标出的为Hind III酶切位点。然后以Bradyrhizobium oligotrophicum ECU1212的基因组DNA为模板，利用PCR进行基因扩增，获得BoTEMO全长基因的PCR产物。
具体地，本发明提供的一种分离基因的核苷酸序列如SEQ ID No.1所示，全长1461bp，其起始密码子为ATG，终止密码子为TGA，编码序列(CDS)从第1个碱基起至第1461个碱基，所编码的蛋白质BoTEMO的氨基酸序列如SEQ ID No.2 所示。
进一步地，由于密码子的简并性，编码如SEQ ID No.2所示的氨基酸序列的核苷酸序列不仅仅局限于如SEQ ID No.1所示的核苷酸序列。本领域技术人员可以通过适当引入替换、缺失、改变、插入或增加来获得如SEQ ID No.1所示的核苷酸序列发生突变后的突变核苷酸序列，即本发明涵盖这些突变核苷酸序列，只要其表达的单加氧酶保持硫醚不对称催化氧化活性即可。
本发明中如SEQ ID No.1所示核苷酸序列的发生突变后的突变核苷酸序列可以通过对如SEQ ID No.1所示的核苷酸序列中的一个或多个核苷酸在保持活性范围内进行替换、缺失或增加来制得。
本发明提供的如SEQ ID No.1所示的核苷酸序列能够编码BoTEMO，如SEQ ID No.1所示的核苷酸序列发生突变后的突变核苷酸序列能够编码BoTEMO或BoTEMO突变体中的任意一种。
相应地，本发明还提供了一种重组表达载体，该重组表达载体包含如上所述的分离基因。
具体可选地，重组表达载体可通过本领域常规方法将本发明的分离基因的核苷酸序列连接于各种合适载体上构建而成。其中，载体可以是本领域的各种常规载体，如市售的质粒、粘粒、噬菌体或病毒载体等；进一步地，载体优选质粒，根据本领域常规的技术手段制备的重组表达载体为重组表达质粒，更优选的质粒为质粒pET28a。本发明的分离基因可以操作性连接于表达合适的调控序列，以实现所述单加氧酶的组成型或诱导型表达。
可选地，本发明的重组表达载体可通过下述示例性方法制得：通过PCR扩增获得的包含分离基因的PCR产物，用限制性内切酶NdeI和HindIII双酶切，形成互补的黏性末端，同时将克隆载体基因片段和表达载体pET28a用限制性内切酶NdeI和HindIII双酶切，经T4 DNA连接酶连接经过酶切的基因片段和表达载体，形成含有本发明的BoTEMO的分离基因的重组表达质粒pET-BoTEMO。
进一步相应地，本发明还提供了一种重组表达转化体，该重组表达转化体包含如上所述的重组表达载体。
具体可选地，可通过将本发明的重组表达载体转化至宿主细胞中来制得重组表达转化体。其中，宿主细胞可以是本领域的各种常规宿主细胞，前提是能使该重组表达载体稳定地自行复制，且其所携带的单加氧酶的分离基因可被有效表达。本发明优选大肠杆菌，更优选大肠杆菌(E.coli)BL21(DE3)或大肠杆菌(E.coli)DH5α。
可选地，将重组表达质粒pET-BoTEMO转化至大肠杆菌(E.coli)BL21(DE3)中，即可获得本发明优选的基因工程菌株，即重组大肠杆菌E.coli BL21(DE3)/pET-BoTEMO。
本发明所述重组表达转化体的培养方法和培养条件没有特殊的限制，可以根据宿主细胞类型和培养方法等因素的不同按本领域普通知识进行适当的选择，只要使重组表达转化体能够生长并产生本发明所述的单加氧酶即可。所述重组表达转化体是大肠杆菌时，优选LB培养基，该培养基含有蛋白胨10g/L，酵母膏5g/L，NaCl10g/L，pH 7.0。重组表达转化体的培养和单加氧酶的产生，可优选下述方法：将本发明涉及的重组大肠杆菌(优选E.coli BL21(DE3))接种至含卡那霉素的LB培养基中培养，当培养液的光密度OD600达到0.5-0.7(优选0.6)时，在终浓度为0.1-1.0mmol/L(优选0.2mmol/L)的异丙基-β-D-硫代吡喃半乳糖苷(IPTG)的诱导下，即可高效表达本发明所述的单加氧酶。表达的单加氧酶可以通过生物学的常规方法分离获得。
即本发明公开了一种上述单加氧酶的制备方法，培养上述的重组表达转化体，然后从中分离单加氧酶。
本发明还提供了一种上述Bradyrhizobium oligotrophicum ECU1212或单加氧酶在不对称催化氧化潜手性硫醚化合物中的应用。可选地，Bradyrhizobium oligotrophicum ECU1212可以是以其静息整细胞的形式用于不对称催化氧化潜手性硫醚化合物的应用中。
进一步可选地，潜手性硫醚化合物选自以下任一化学式所示的化合物：
Figure PCTCN2018083060-appb-000003
Figure PCTCN2018083060-appb-000004
其中，本发明中将上述化学式Ⅰ至Ⅸ的中文名称分别以苯甲硫醚、对甲基苯甲硫醚、对甲氧基苯甲硫醚、5-甲氧基-2-(甲基硫代)苯并咪唑、奥美拉唑硫醚、兰索拉唑硫醚、泮托托拉唑硫醚、雷贝拉唑硫醚、艾普拉唑硫醚进行叙述，当然，在其他文献中也可以以不同的命名方式命名。
进一步可选地，所述单加氧酶将所述潜手性硫醚化合物不对称催化氧化为亚砜化合物。
可选地，利用本发明的Bradyrhizobium oligotrophicum ECU1212或单加氧酶将硫醚不对称催化氧化生成光学活性的亚砜。涉及的具体反应条件如底物浓度、pH、缓冲液组成、酶用量等可按本领域此类反应的常规条件进行选择。进一步地，不对称催化氧化反应可在振荡或搅拌条件下进行。
具体而言，可按下述示例性方法进行：反应历程如附图1所示，在pH 8.0～10.0的Tris-HCl缓冲液中，Tris-HCl缓冲液的浓度可以为0.05～0.2mol/L。在葡萄糖脱氢酶、葡萄糖和NADP+的存在下，在本发明的单加氧酶作用下，对硫醚进行不对称氧化反应，制得光学活性亚砜。优选如下：底物在反应液中的浓度为0.1-37g/L。本发明所述单加氧酶的酶活单位(U)定义为每分钟催化1μmol底物生成产物所需的酶量。在硫醚的不对称氧化时，为了进行辅酶循环，向反应体系中额外添加葡萄糖和来自巨大芽孢杆菌的葡萄糖脱氢酶(制备方法参见：Journal of Industrial Microbiology and Biotechnology 2011,38,633-641)。通过葡萄糖脱氢酶催化葡萄糖氧化，使NADP+转化为NADPH。取决于不同反应体系，葡萄糖脱氢酶的活力单位可以与本发明所述单加氧酶的活力单位相当。葡萄糖的用量可以为2～20mmol/L，额外添加的NADP+的用量可以为0～1mmol/L。所述的不对称氧化反应的温度可以 为20～35℃，优选25℃。不对称氧化反应结束后，可按本领域常规方法，从反应混合液中提取手性亚砜产物。
本发明提供的一种Bradyrhizobium oligotrophicum ECU1212，由该Bradyrhizobium oligotrophicum ECU1212能够衍生出一种单加氧酶，可以高效不对称催化氧化硫醚化合物以生成光学纯手性亚砜化合物，具有高效、高立体选择性以及高转化率的特点。例如，在兰索拉唑硫醚底物浓度达到10g/L时，转化率仍达到99％以上，ee值达到99％以上，并且产物亚砜不会进一步氧化为砜副产物。相对于其他不对称氧化制备方法，使用本发明方法制备所得的产物浓度高，光学纯度好，无副产物生成，反应条件温和，对环境友好，操作简便，易于工业放大，因此具有很好的工业应用前景。
进一步的，本发明下列实施例中的材料来源为：
Bradyrhizobium oligotrophicum ECU1212，CGMCC No.15208。
表达质粒pET28a购自上海Novagen公司。
E.coli DH5α和E.coli BL21(DE3)感受态细胞，2×Taq PCR MasterMix，琼脂糖凝胶DNA回收试剂盒均购自北京天根生化科技有限公司。
除非另有说明，本发明所用的试剂和原料均为市售可得。
本说明书中，除非另有注明具体条件，实施例中各实验方法均按照常规方法和条件或按照试剂说明书进行。
实施例1
Bradyrhizobium oligotrophicum ECU1212的筛选
土壤的采集主要分为两个部分，直接采集土样和预埋底物之后的土样采集，共252份土样。
直接土样采集：采集较为湿润的土壤，一般多为水源、植物、受污染底物等处，挖取距地面2-3cm处的土壤，大约3-5g，使用后的土样放于低温、干燥处保存，也可直接放在1.5mL Eppendorf管中后置于4℃冰箱中保存。采集地点如下：上海奉贤化工区、新华医院、果园、菜场、垃圾箱附近、河道附近、绿化带、校园(华东理工大学徐汇或奉贤校区)、居民区绿化、植物园等。
预埋底物：兰索拉唑硫醚为白色粉末状，难溶于水但溶于二甲基亚砜(DMSO)，因此使用了两种不同的形式在多个地点进行预埋，地点选择多为植被丰富，微生物群可能具有被驯化的潜力处。第一种是直接将白色粉末预埋至土壤离地大约5cm 处，第二种是将兰索拉唑硫醚溶于二甲基亚砜(DMSO)后，浇至土壤表面。
在对选定的菌种进行表征和培养条件优化之前，所有的反应都在30℃下进行，试管中培养基的装载量为4mL，摇床的转速设置为180r/min。筛选的过程采取了四轮富集培养，并且采用了梯度培养的方法，即酵母粉的浓度在每一轮培养过程中减半，而底物浓度在每一轮培养过程中翻倍。平板培养在30℃的恒温箱内进行。
由于非天然底物兰索拉唑硫醚对大部分野生菌来说都是难以利用的，252份土样进行四轮富集培养之后，菌体生长良好的的样品只有124份，经过初筛有21份培养液中含有产物兰索拉唑，且转化率>1％。初筛得到的21份培养液中，划线得到了81个单菌，单独培养后用0.33g/L底物浓度做24h的转化反应之后，将活力最高的菌株进行16S rDNA验证，测序结果在NCBI数据库中检索并与同源序列比对后发现，该菌株和Bradyrhizobium oligotrophicum的序列一致性为99％，因此命名为Bradyrhizobium oligotrophicum ECU1212。
实施例2
Bradyrhizobium oligotrophicum ECU1212静息细胞的制备
将如实施例1筛选获得的Bradyrhizobium oligotrophicum ECU1212接种至丰富培养基(葡萄糖15g/L，蛋白胨10g/L，酵母膏5g/L，NaH 2PO 40.5g/L，MgSO 40.5g/L，NaCl 10g/L，pH 7.0)中，28℃、180rpm摇床振摇培养24小时后，5000×g离心10min，收集湿细胞。所收集的湿细胞在-80℃下冷冻12h后，用冷冻干燥机低温干燥20h，得到冻干细胞，储存在4℃冰箱内。
实施例3
BoTEMO的分离基因的克隆
在筛选获得Bradyrhizobium oligotrophicum ECU1212的基础上，本发明通过生物信息学分析的策略，分析预测其中可能对硫醚具有明显不对称催化氧化活性的酶的基因，并将其分选出来进行克隆表达，验证其功能。采用这种方法，从Bradyrhizobium oligotrophicum ECU1212中克隆获得一个种能够产生高效不对称催化氧化硫醚底物的BoTEMO的分离基因。
本发明的BoTEMO(Bradyrhizobium oligotrophicum ECU1212硫醚单加氧酶， Bradyrhizobium  oligotrophicum ECU1212  Thio ether  Mono oxygenase)催化5种大位阻拉唑硫醚底物氧化产物的ee值高达99％。相比于传统的生物催化剂，能够显著 提高产物的光学纯度。
可选地，本发明的BoTEMO分离基因较佳的来源为Bradyrhizobium oligotrophicum ECU1212的基因组DNA为模板，采用本领域常规技术方法(如聚合酶链反应，PCR)，获得编码所述BoTEMO的完整核酸分子。
可选地，在本实施例中，引物对的核苷酸序列如下：
正向引物：CCG  CATATG TCAACTGAGCATGTCGAC
反向引物：CCG  AAGCTT TCACGAATACCGCATCACCC
其中，正向引物中带下划线部分为NdeI酶切位点，反向引物中带下划线部分为HindIII酶切位点。以如实施例1所述方法制备的Bradyrhizobium oligotrophicum ECU1212的基因组DNA为模板，利用聚合酶链式反应(PCR)进行基因扩增。
可选地，PCR体系为：2×Taq PCR MasterMix 25μl，正向引物和反向引物各1.5μl(0.3μmol/L)，DNA模板1.5μl(0.1μg)，DMSO 2μl和ddH 2O 19μl。PCR扩增步骤为：(1)95℃，预变性3min；(2)94℃，变性1min；(3)55℃退火30s；(4)72℃延伸2min；步骤(2)～(4)重复30次；(5)72℃继续延伸10min，冷却至12℃。PCR产物经琼脂糖凝胶电泳纯化，利用琼脂糖凝胶DNA回收试剂盒回收1400～1600bp区间的目标条带。
其中包含的分离基因核苷酸序列，全长1461bp，其核苷酸序列如SEQ ID No.1所示。其起始密码子为ATG，终止密码子为TGA，编码序列(CDS)从第1个碱基起至第1461个碱基，所编码的单加氧酶的氨基酸序列如SEQ ID No.2所示。
实施例4
重组表达质粒和重组表达转化体的制备
将如实施例3所示方法克隆的包含分离基因的PCR产物在37℃用限制性内切酶NdeI和HindIII双酶切12小时，经琼脂糖凝胶电泳纯化，利用琼脂糖凝胶DNA回收试剂盒回收目标片段。在T 4DNA连接酶的作用下，将目标片段与同样经NdeI和HindIII酶切后的载体质粒pET28a，在16℃下连接过夜得到重组表达质粒pET-BoTEMO。
将上述重组表达质粒转化到大肠杆菌E.coli DH5α感受态细胞中，在含有卡那霉素的抗性平板上对阳性重组体进行筛选，挑取单克隆，菌落PCR验证阳性克隆。培养重组菌，待质粒扩增后提取质粒，重新转化至大肠杆菌E.coli BL21(DE3)感受态细胞中，转化液涂布到含有卡那霉素的LB平板上，37℃倒置培养过夜，即 获得阳性重组表达转化体大肠杆菌E.coli BL21(DE3)/pET-BoTEMO，菌落PCR证实为阳性克隆。
实施例5
重组BoTEMO的表达
将如实施例4所述的方法制备的重组表达转化体大肠杆菌E.coli BL21(DE3)/pET-BoTEMO，接种至含50μg/mL卡那霉素的LB培养基(蛋白胨10g/L，酵母膏5g/L，NaCl 10g/L，pH 7.0)中，37℃、180rpm摇床振摇培养，当培养液的OD 600达到0.6时，加入终浓度为0.2mmol/L的IPTG作为诱导剂，16℃诱导16小时后，将培养液离心，收集细胞，并用生理盐水洗涤两次，得静息细胞。将100mL发酵液所得的静息细胞悬浮于10mL pH 7.0的缓冲液中，在冰水浴的条件下进行超声破碎(超声破碎仪的设定功率为400W，工作4s，间歇6s，共循环99次)。破碎液在4℃低温离心机里15000rpm离心40min，获得上清粗酶液进行活力测定以及蛋白纯化。
所收集的粗酶液在-80℃下冷冻12h后，用真空冷冻干燥机低温干燥20h，即可得到冻干粗酶粉，储存在4℃冰箱内。冻干粗酶粉对苯甲硫醚的活力为0.2U/mg。粗酶液经聚丙烯酰胺凝胶电泳图分析，重组蛋白在细胞中以部分可溶的形式存在，另外有部分蛋白存在于细胞碎片中。
纯化实验全部使用镍亲和自装柱完成，纯化过程中使用的缓冲液为：A液：50mM KPB，500mM NaCl，10mM咪唑，2mMβ-巯基乙醇，pH 8.0；B液：50mM KPB，500mM NaCl，300mM咪唑，2mMβ-巯基乙醇，pH 8.0；C液：50mM KPB，150mM NaCl，1mM DTT，pH 9.0。纯化方法如下：
1)菌体用A液重新悬浮后进行超声破碎，破碎后的粗酶液用低温高速离心机在4℃离心，12000rpm离心30min，离心后的上清液暂时保存在4℃冰箱或冷库里；
2)Ni柱用5～10倍柱体积的A液预先平衡；
3)保存的上清液上样；
4)上样完成后用5～10倍柱体积的A和B混合液(5％B液)洗去杂蛋白；
5)用1个柱体积的B液洗脱目的蛋白并收集；
6)收集到的目的蛋白用30kDa的超滤管进行浓缩，浓缩至500μl时添加5ml C液继续超滤浓缩，重复3～5次以除去酶液中的咪唑并降低盐浓度，完成缓冲液的 置换；
7)置换后的酶液用液氮速冻后保存于-80℃冰箱。
实施例6
重组BoTEMO和葡萄糖脱氢酶活力的测定
利用分光光度计，通过检测340nm处吸光值的变化来测定BoTEMO和葡萄糖脱氢酶的活力。
BoTEMO活力的测定方法如下：1mL反应体系(100mmol/L Tris-HCl缓冲液，pH 9.0)中，加入1mmol/L苯甲硫醚，0.2mmol/L NADPH，30℃保温2分钟后加入实施例5制备的适量粗酶液，迅速混匀，检测340nm处吸光值的变化。测得该粗酶液的比活为101mU/mL。
葡萄糖脱氢酶活力的测定方法如下：1mL反应体系(100mmol/L磷酸钠缓冲液，pH7.0)中，加入10mmol/L葡萄糖，1mmol/L NADP +，30℃保温2分钟后加入葡萄糖脱氢酶(制备方法参见：Journal of Industrial Microbiology and Biotechnology 2011,38,633-641)，迅速混匀，实时检测340nm处吸光值的变化。
酶活力的计算公式为：酶活力(U)＝EW×V×10 3/(6220×1)
式中，EW为1分钟内340nm处吸光度的变化；V为反应液的体积，单位为ml；6220为NADPH的摩尔消光系数，单位为L/(mol·cm)；1为光程距离，单位为cm。每单位BoTEMO的定义为在上述条件下每分钟催化1μmol NADPH氧化所需的酶量。每单位葡萄糖脱氢酶的定义为在上述条件下，每分钟催化1μmol NADP +还原所需的酶量。
实施例7
重组BoTEMO对一系列硫醚的活力测定
在0.5mL磷酸钾缓冲液(100mmol/L，pH 9.0)中加入BoTEMO纯酶(如实施例5所述方法制备的BoTEMO纯酶)，加入溶解于DMSO中的硫醚底物，硫醚的终浓度为0.2～2mmol/L，DMSO的终浓度为2％(v/v)、加入终浓度为0.2～2mmol/L的NADPH。在25℃，1000rpm振荡反应1小时。反应结束后加入0.6mL乙酸乙酯进行萃取，萃取液加入无水硫酸钠干燥，吸取有机相清液并过夜挥发除去溶剂，然后加入0.5mL异丙醇溶解，分析测定产物的ee值。
产物转化率及ee值的具体分析条件如下：
使用高效液相色谱仪进行分析，色谱柱为手性OD-H柱(250mm×4.6mm,5μm particle size,Daicel)，流动相为正己烷：异丙醇＝93:7，流速1mL/min，在254nm处紫外检测。或AS-H柱(250mm×4.6mm,5μm particle size,Daicel)，流动相为正己烷：异丙醇＝55:45，流速0.5mL/min，在254nm处紫外检测。
进一步按实施例6所述方法测定BoTEMO催化一系列硫醚底物不对称氧化生成光学活性亚砜的活力，测定结果如表1所示。
表1.BoTEMO纯酶比活测定
Figure PCTCN2018083060-appb-000005
a“+”：<10U/g；“++”：10～100U/g；“+++”：>100U/g
实施例8
Bradyrhizobium oligotrophicum ECU1212整静息细胞不对称催化氧化苯甲硫醚
在100mLTris-HCl缓冲液(100mmol/L，pH 9.0)中加入Bradyrhizobium oligotrophicum ECU1212的1g冻干细胞(如实施例2所述制备的冻干细胞)，加入苯甲硫醚、甲醇至终浓度为37g/L、10％(v/v)。在28℃，180rpm搅拌反应，间歇取样100μL。取样后加入0.6mL乙酸乙酯进行萃取，萃取液加入无水硫酸钠干燥，挥发除去溶剂，然后加入0.5mL异丙醇溶解，按如实施例7所述方法分析测定底物转化率和产物ee值。24h时底物的转化率大于99％，产物的ee值大于99％(S)。
实施例9
重组BoTEMO不对称催化氧化苯甲硫醚
在0.5mL磷酸钾缓冲液(100mmol/L，pH 9.0)中加入BoTEMO粗酶液100μL(如实施例5所述方法制备的粗酶液)和葡萄糖脱氢酶粗酶液，加入苯甲硫醚、甲醇、NADP +和葡萄糖至终浓度分别为2mmol/L、10％(v/v)、0.2mmol/L和3.6g/L。在25℃，1000rpm振荡反应1小时。反应结束后加入0.6mL乙酸乙酯进行萃取，萃取液加入无水硫酸钠干燥后，挥发除去溶剂，然后加入0.5mL异丙醇溶解，按如实施例7所述方法分析测定底物转化率和产物ee值，底物的转化率大于99％，产物的ee值大于99％(S)。
实施例10
重组BoTEMO不对称催化氧化奥美拉唑硫醚
在0.5mL磷酸钾缓冲液(100mmol/L，pH 9.0)中加入BoTEMO粗酶液100μL(如实施例5所述方法制备的粗酶液)和葡萄糖脱氢酶粗酶液，加入奥美拉唑硫醚、甲醇、NADP +和葡萄糖至终浓度分别为0.2mmol/L、10％(v/v)、0.2mmol/L和3.6g/L。在25℃，1000rpm振荡反应1小时。反应结束后加入0.6mL乙酸乙酯进行萃取，萃取液加入无水硫酸钠干燥后，挥发除去溶剂，然后加入0.5mL异丙醇溶解，按如实施例7所述方法分析测定底物转化率和产物ee值。底物转化率大于99％，产物的ee值大于99％(R)。
实施例11-15
重组BoTEMO不对称催化氧化系列拉唑硫醚
在10mL磷酸钾缓冲液(100mmol/L，pH 9.0)中加入BoTEMO冻干粗酶粉0.1g(如实施例5所述方法制备的粗酶粉和葡萄糖脱氢酶冻干酶粉(15U/mg)0.02g，加入奥美拉唑硫醚/兰索拉唑硫醚/泮托拉唑硫醚/雷贝拉唑硫醚/艾普拉唑硫醚1～3g/L、甲醇10％(v/v)、NADP +0.2mmol/L和葡萄糖10g/L。在25℃，180rpm反应，间歇取样100μL。取样后加入0.6mL乙酸乙酯进行萃取，萃取液加入无水硫酸钠干燥，挥发除去溶剂，然后加入0.5mL异丙醇溶解，按如实施例7所述方法分析测定底物转化率和产物ee值。
测得BoTEMO以上述条件不对称催化氧化5种拉唑前体硫醚-大位阻硫醚化合 物，反应24h所得底物的转化率大于90％，产物ee值大于99％。测定结果如表2所示：
表2
Figure PCTCN2018083060-appb-000006
实施例16
重组BoTEMO不对称催化氧化奥美拉唑硫醚
在100mLTris-HCl缓冲液(100mmol/L，pH 9.0)中加入BoTEMO冻干粗酶粉1g(如实施例5所述方法制备的粗酶粉)和葡萄糖脱氢酶冻干酶粉0.2g，加入奥美拉唑硫醚、甲醇、NADP +和葡萄糖至终浓度分别为1g/L、10％(v/v)、0.2mmol/L和3.6g/L。在25℃，180rpm搅拌反应，反应3h后，按如实施例7所述方法分析测定底物转化率和产物ee值，底物转化率大于99％，产物ee值大于99％(R)。
实施例17
重组BoTEMO不对称催化氧化兰索拉唑硫醚
在10mLTris-HCl缓冲液(100mmol/L，pH 9.0)中加入BoTEMO冻干粗酶粉0.2g(如实施例5所述制备的粗酶粉)和葡萄糖脱氢酶冻干酶粉0.02g，加入兰索拉唑硫醚、甲醇、NADP +和葡萄糖至终浓度分别为2g/L、10％(v/v)、0.2mmol/L和3.6g/L。在25℃，180rpm反应24小时。按如实施例7所述方法分析测定底物转化率和产物ee值。不对称催化氧化兰索拉唑硫醚所得(R)-兰索拉唑的底物的转化率大于99％，产物的ee值大于99％(R)。
实施例18
BoTEMO的突变
对实施例3所得的BoTEMO全长基因序列(如SEQ ID No.1所述的核苷酸酸序列)进行定点突变，得到两个突变体：
1)将编码BoTEMO的核苷酸序列的第883位的G突变为T，第884位的A突变为G，第1184位的C突变为T，第1186位的T突变为G，第1187位的G突变为C，从而得到如SEQ ID No.3所示的突变基因的核苷酸序列。其编码的氨基酸序列为SEQ ID No.4，即将BoTEMO(如SEQ ID No.2所述的氨基酸序列)的第295位的Asp突变为Cys，第395位的Ser突变为Leu，第396位的Trp突变为Ala，该突变基因所编码的单加氧酶命名为BoTEMO-M1。
2)将编码BoTEMO的核苷酸序列的第1070位的G突变为T，第1180位的T突变为G，第1181位的T突变为C，第1182位的C突变为A，从而得到如SEQ ID No.5所示的突变基因的核苷酸序列。其编码的氨基酸序列为SEQ ID No.6，即将BoTEMO(如SEQ ID No.2所示的氨基酸序列)的第357位的Ser突变为Ile，第394位的Phe突变为Ala，该突变基因所编码的单加氧酶命名为BoTEMO-M2。
按如实施例4所述的方法分别用上述BoTEMO-M1以及BoTEMO-M2两个突变基因制备重组转化体，并按照如实施例5所述的方法制备静息细胞和冻干粗酶粉，进一步按照如实施例6所述的酶活力测定方法测定BoTEMO-M1以及BoTEMO-M2的酶活力，BoTEMO-M1和BoTEMO-M2的酶活力分别是BoTEMO的7.6倍(BoTEMO-M1)和1.6倍(BoTEMO-M2)，对兰索拉唑硫醚的活力达到20U/g(BoTEMO-M1)和4.2U/g(BoTEMO-M2)。
实施例19
BoTEMO-M1不对称催化氧化兰索拉唑硫醚
在100mLTris-HCl缓冲液(100mmol/L，pH 9.0)中加入BoTEMO-M1冻干粗酶粉1g和葡萄糖脱氢酶粗酶0.2g，加入兰索拉唑硫醚、甲醇、NADP +和葡萄糖至终浓度分别为10g/L、10％(v/v)、0.2mmol/L和15g/L。在25℃，180rpm搅拌反应24小时。不对称氧化兰索拉唑硫醚所得(R)-兰索拉唑的底物的转化率大于99％，产物的ee值大于99％(R)。
实施例20
BoTEMO-M2不对称催化氧化兰索拉唑硫醚
在100mL Tris-HCl缓冲液(100mmol/L，pH 9.0)中加入BoTEMO-M2冻干粗酶粉1g和葡萄糖脱氢酶粗酶0.2g，加入兰索拉唑硫醚、甲醇、NADP +和葡萄糖至终浓度分别为3g/L、10％(v/v)、0.2mmol/L和5.4g/L。在25℃，180rpm搅拌反应24小时。不对称氧化兰索拉唑硫醚所得(R)-兰索拉唑的底物的转化率大于99％，产物的ee值大于99％(R)。
实施例21
BoTEMO-M1不对称催化氧化兰索拉唑硫醚
在2L Tris-HCl缓冲液(100mmol/L，pH 9.0)中加入BoTEMO-M1冻干粗酶粉20g和葡萄糖脱氢酶粗酶4g，加入兰索拉唑硫醚、甲醇、NADP +和葡萄糖至终浓度分别为10g/L、10％(v/v)、0.2mmol/L和15g/L。在25℃，180rpm搅拌反应24小时。不对称氧化兰索拉唑硫醚所得(R)-兰索拉唑的底物的转化率大于99％，产物的ee值大于99％(R)。经萃取分离得到产物(R)-兰索拉唑17.2g，得率为86％。
应理解，在阅读了本发明的上述内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
Figure PCTCN2018083060-appb-000007
Figure PCTCN2018083060-appb-000008
Figure PCTCN2018083060-appb-000009
Figure PCTCN2018083060-appb-000010
Figure PCTCN2018083060-appb-000011
Figure PCTCN2018083060-appb-000012

Claims (15)

  1. 一种Bradyrhizobium oligotrophicum，其为Bradyrhizobium oligotrophicum ECU1212，保藏在中国微生物菌种保藏委员会普通微生物中心，保藏编号为CGMCC No.15208。
  2. 根据权利要求1所述的Bradyrhizobium oligotrophicum，其特征在于可以产生氨基酸序列如SEQ ID No.2所示的Bradyrhizobium oligotrophicum ECU1212硫醚单加氧酶。
  3. 一种单加氧酶，其特征在于，所述单加氧酶含有如SEQ ID No.2所示的氨基酸序列；或，
    所述单加氧酶含有如所述SEQ ID No.2所示的氨基酸序列发生突变后的突变氨基酸序列。
  4. 根据权利要求3所述的单加氧酶，其特征在于，所述突变氨基酸序列为所述SEQ ID No.2所示的氨基酸序列中任意1至5个氨基酸发生替换后而生成的突变氨基酸序列。
  5. 根据权利要求4所述的单加氧酶，其特征在于，所述突变氨基酸序列为所述SEQ ID No.2所示的氨基酸序列中第295位、第357位、第394位、第395位以及第396位的氨基酸中的任意一个或多个发生替换后而生成的突变氨基酸序列。
  6. 根据权利要求5所述的单加氧酶，其特征在于，所述突变氨基酸序列包括以下任意一项或多项特征：
    (1)所述SEQ ID No.2所示的氨基酸序列中第295位的氨基酸Asp替换为Cys；
    (2)所述SEQ ID No.2所示的氨基酸序列中第357位的氨基酸Ser替换为Ile；
    (3)所述SEQ ID No.2所示的氨基酸序列中第394位的氨基酸Phe替换为Ala；
    (4)所述SEQ ID No.2所示的氨基酸序列中第395位的氨基酸Ser替换为Leu；
    (5)所述SEQ ID No.2所示的氨基酸序列中第396位的氨基酸Trp替换为Ala。
  7. 根据权利要求6所述的单加氧酶，其特征在于，所述单加氧酶含有如SEQ ID No.4所示的突变氨基酸序列。
  8. 根据权利要求6所述的单加氧酶，其特征在于，所述单加氧酶含有如SEQ ID No.6所示的突变氨基酸序列。
  9. 一种分离的核酸，其特征在于，所述的核酸编码权利要求3-8中任一项所述的单加氧酶。
  10. 一种重组表达载体，其特征在于，所述重组表达载体包含如权利要求9 所述的核酸。
  11. 一种重组表达转化体，其特征在于，所述重组表达转化体包含如权利要求10所述的重组表达载体。
  12. 一种如权利要求3至8任一项所述的单加氧酶的制备方法，其特征在于，包括以下步骤：
    培养如权利要求11所述的重组表达转化体，从中分离所述单加氧酶。
  13. 一种如权利要求1-8任一项所述的Bradyrhizobium oligotrophicum或单加氧酶在不对称催化氧化潜手性硫醚化合物中的应用。
  14. 根据权利要求13所述的应用，其特征在于，所述潜手性硫醚化合物选自以下任一化学式所示的化合物：
    Figure PCTCN2018083060-appb-100001
  15. 根据权利要求13所述的应用，其特征在于，将所述潜手性硫醚化合物不对称催化氧化为亚砜化合物。
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