Cyclohexanone monooxygenase and application thereof

A cyclohexanone monooxygenase and cyclohexanone mono technology, which is applied in the field of biological engineering, can solve the problems of poor thermal stability and low catalytic activity of sulfide, and achieve the effects of thorough reaction, good catalytic effect and good solubility

Pending Publication Date: 2021-02-26
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 to provide a kind of cyclohexanone monooxygenase, which has Good solubility, exhibits high stereoselectivity for thioethers, and high catalytic activity for cyclohexanone and thioanisole

Method used

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  • Cyclohexanone monooxygenase and application thereof
  • Cyclohexanone monooxygenase and application thereof
  • Cyclohexanone monooxygenase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Construction and cultivation of recombinant Escherichia coli BL21(DE3) / pET28a-Amchmo

[0042] Using the gene sequence shown in SEQ ID NO.2 as a template, using upstream primers and downstream primers (F and R) to clone the target gene, the system is as follows (μL): 10×PCR Mix 10, upstream primer 0.2 , downstream primer 0.2, genome 0.2, DNA polymerase 0.2, ddH 2 O 9.2. The PCR program was: pre-denaturation at 95°C for 10 min, cleavage at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min 30 s, 30 cycles, and extension at 72°C for 10 min. The PCR product was purified by agarose gel electrophoresis, and the band between 1500 and 2000 bp was recovered by using an agarose gel recovery kit ( figure 1 ), the cyclohexanone monooxygenase gene. The obtained cyclohexanone monooxygenase gene is named Amchmo, and the encoded protein sequence is shown in SEQ ID NO.1.

[0043] F: TGGGTCGCGGATCCTCAG ACGGCCGCCG (SEQ ID NO. 3),

[0044] R: TCGCGGATCCT...

Embodiment 2

[0047] Embodiment 2: substrate spectrum analysis

[0048] The enzyme expressed in Escherichia coli was isolated and purified, and the enzyme activity of cyclohexanone monooxygenase (AmCHMO) catalyzing different thioethers was determined. The enzyme activity measured with thioanisole as the substrate was 100% control, and other substrates The enzyme activities measured by the substances were calculated as the percentage of the two. The measurement results are shown in Table 1.

[0049] Table 1 AmCHMO substrate spectrum

[0050]

[0051] Table 1 shows that AmCHMO has a broad substrate spectrum and can catalyze thioanisole, cyclic ketones and linear ketones.

Embodiment 3

[0052] Example 3: Properties of cyclohexanone monooxygenase

[0053] (1) The optimal pH of the enzyme

[0054] Prepare 100mmol·L -1 Buffers with different pH: phosphate buffer (pH 6.0-8.0), Tris-HCl (8.0-9.0), glycine-NaOH buffer (pH 9.0-11.0). Then, using thioanisole as a substrate, the relative enzymatic activity of AmCHMO in different pH buffers was determined. The optimal reaction pH of AmCHMO is Tris-HCl, 8.0-9.0, and the enzyme activity is 0.64-0.95U·mg -1 . In a phosphate buffer solution with a pH of 6.0-7.0, the enzyme activity drops below 20%.

[0055] (2) The optimal temperature of the enzyme

[0056] Using thioanisole as the substrate, the enzyme activity of AmCHMO was measured at different temperatures (20-55°C) for 15 minutes. The highest enzyme activity measured was defined as 100%. Calculated as a percentage of maximum viability. The results show that the optimum reaction temperature of AmCHMO is 35℃, which is 0.21~0.56U·mg –1 .

[0057] Table 2 Optimum...

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Abstract

The invention discloses a cyclohexanone monooxygenase and application thereof, and belongs to the technical field of bioengineering. The cyclohexanone monooxygenase disclosed by the invention is derived from Amycolatopsis methanolica, and can be used as a catalyst for efficient synthesis of thioether. According to the invention, thioether such as cyclic ketone and linear ketone can be taken as a substrate, and the prepared cyclohexanone monooxygenase can tolerate high concentration of substrates, and has high catalytic activity and strong stereoselectivity, which is environmentally friendly and has a good application and development prospect.

Description

technical field [0001] The invention relates to a cyclohexanone monooxygenase and its application, belonging to the technical field of bioengineering. Background technique [0002] Cyclohexanone monooxygenase (CHMO) is a FAD and NADPH-dependent Baeyer-Villiger monooxygenase, and CHMO is the most extensively studied among many BVMOs. Cyclohexanone monooxygenase not only catalyzes the insertion of an oxygen atom near the carbonyl carbon atom of ketone substrates to generate corresponding esters or lactones, but also catalyzes the oxidation of heteroatoms including nitrogen, boron, selenium, phosphorus and sulfur, and ring oxidation reaction. This class of enzymes has been well-applied in stereoselectively catalyzing the asymmetric oxidation of thioethers. [0003] As an important chiral compound, optically active sulfoxides are increasingly used in asymmetric synthesis. Chiral sulfoxide has extensive and important application value, and its use can be roughly divided into t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12P11/00C12P17/08
CPCC12N9/0071C12Y114/13022C12P11/00C12P17/08
Inventor 倪晔魏世誉许国超韩瑞枝周婕妤
Owner JIANGNAN UNIV
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