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Baeyer-Villiger monooxygenase and application of Baeyer-Villiger monooxygenase

A monooxygenase and reaction system technology, applied in the field of bioengineering, can solve the problems of low conversion rate of cyclohexanone and product yield, low product ee value, low catalytic activity, etc., and achieve good application development prospects, three-dimensional Strong selectivity and good catalytic effect

Inactive Publication Date: 2021-03-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved in the present invention is the low catalytic activity to the substrate thioether of reported Baeyer-Villiger monooxygenase, poor thermal stability, low product ee value, low cyclohexanone conversion rate and product yield, etc. problem, to provide another Baeyer-Villiger monooxygenase, which has good solubility, high stereoselectivity for linear ketones, cyclic ketones and thioether substrates, and high sensitivity to cyclohexanone and benzyl The high catalytic activity of thioether can directly and effectively solve the problems existing in monooxygenase

Method used

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Embodiment 1

[0036] Embodiment 1: Construction of recombinant escherichia coli

[0037] The chemically synthesized nucleotide sequence is shown in SEQ ID NO.1 as the coding gene sequence of Baeyer-Villiger monooxygenase, which is named Rabvmo, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0038] With the restriction endonuclease NdeI and BamHI after plasmid pET28a digested and the nucleotide fragment of Rabvmo, gene Rabvmo is connected with digested plasmid pET28 by T4 DNA ligase, obtains recombinant expression vector pET28a-Rabvmo( figure 1 ). Transform the constructed recombinant expression vector pET28a-Rabvmo into Escherichia coli BL21(DE3) competent, coat a solid plate containing kanamycin-resistant LB, and perform colony PCR verification after overnight culture. The positive clone is the recombinant large intestine Bacillus BL21(DE3) / pET28a-Rabvmo. Pick positive clones and culture them overnight in LB medium, transfer them into fresh LB culture at th...

Embodiment 2

[0040] Example 2: Properties of Baeyer-Villiger monooxygenases

[0041] (1) Separation and purification of Baeyer-Villiger monooxygenase

[0042] Suspend the recombinant cells in solution A (20mmol·L -1 Sodium phosphate, 500mmol·L -1 NaCl, 20mmol·L -1 imidazole, pH 7.4), the crude enzyme solution was obtained after sonication and centrifugation. The column used for purification is an affinity column HisTrap FF crude (nickel column), which is accomplished by using the histidine tag on the recombinant protein for affinity binding. First, use solution A to equilibrate the nickel column, load the crude enzyme solution, continue to use solution A to elute the breakthrough peak, and after equilibrium, use solution B (20mmol L -1 Sodium phosphate, 500mmol·L - 1 NaCl, 1000mmol·L -1 imidazole, pH 7.4) for gradient elution to elute the recombinant protein bound to the nickel column to obtain recombinant Baeyer-Villiger monooxygenase. The purified protein was assayed for enzyme a...

Embodiment 3

[0072] Embodiment 3: Baeyer-Villiger monooxygenase is applied to the preparation of S-phenylmethyl sulfoxide

[0073] Take 2-500 U of the obtained recombinant Baeyer-Villiger monooxygenase and 2-500 U of glucose dehydrogenase (GDH, purchased from Novizyme, 5 U / g) in Tris-HCl buffer (pH 9, 100 mmol L -1), add 5% methanol, the concentration in the reaction system is 30~100mmol·L -1 The sulfide anisole (Table 5), the total volume of the reaction solution is 10mL. The reaction is placed at 30°C, and the conversion process is detected by sampling. The conditions are as follows: chiral OD-H chromatographic column (250mm×4.6mm×5μm), the detection wavelength is 254nm, and the mobile phase is n-hexane:isopropanol (90:10 ), the flow rate is 0.2-1mL / min.

[0074] The reaction process is sampled and detected in real time, and when it is detected that the product no longer increases, the reaction ends.

[0075] The results are shown in Table 5, Baeyer-Villiger monooxygenase can maintain...

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Abstract

The invention discloses a Baeyer-Villiger monooxygenase and an application of the Baeyer Villiger monooxygenase, and belongs to the technical field of bioengineering. The invention provides another Baeyer-Villiger monooxygenase aiming at the problems of reported Baeyer-Villiger monooxygenase, such as low catalytic activity to substrate thioether, poor thermal stability, low ee value of a product and the like. The enzyme shows high stereoselectivity to linear ketone, cyclic ketone and thioether substrates, has high catalytic activity to cyclohexanone and thioanisole, and can be used for preparing high-purity products. Therefore, the problems of the existing monooxygenase can be directly and effectively solved. When diphenyl sulfide is used as a substrate to prepare S-benzosulfoxide, the conversion rate can reach 95% or above, and the yield can reach 99.5%. According to the present invention, a new thought and a new method are provided for industrial production of the optically active sulfoxide.

Description

technical field [0001] The invention relates to a Baeyer-Villiger monooxygenase and its application, belonging to the technical field of bioengineering. Background technique [0002] Baeyer-Villiger monooxygenase (BVMO) belongs to a branch of flavoprotein monooxygenase, in addition to catalyzing the nucleophilic oxidation of carbonyl groups, it can also realize the electrophilic oxidation of heteroatoms. atoms of organic compounds. Due to its broad substrate spectrum, it has been widely used in industrial catalysis. [0003] As an important chiral compound, optically active sulfoxide has attracted a lot of attention due to its great demand in almost every field of the chemical synthesis industry, involving the design and development of new synthetic reagents, drugs and functional materials. [0004] As a class of extremely useful compounds, chiral sulfoxides have extensive and important applications in the fields of asymmetric synthesis and medicine. It is difficult to re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12P11/00
CPCC12N9/0073C12Y114/13008C12P11/00
Inventor 倪晔魏世誉许国超韩瑞枝周婕妤
Owner JIANGNAN UNIV
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