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Lactamase, application of lactamase and method for preparing (1R,4S)-gamma-lactam through enzymatic resolution

A technology of lactamase and vince lactone, applied in the field of bioengineering, can solve the problems of low tolerance and activity, reduced production efficiency, low substrate concentration, etc., to reduce production costs, provide production efficiency, The effect of high substrate tolerance

Active Publication Date: 2020-09-04
安徽红杉生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, optically pure (1R, 4S)-venslides are usually obtained by biocatalysis, but the tolerance and activity of enzymes currently used in biocatalysis are generally not high, resulting in a normal reaction system The substrate concentration is small, which reduces the production efficiency and the cost is high

Method used

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  • Lactamase, application of lactamase and method for preparing (1R,4S)-gamma-lactam through enzymatic resolution
  • Lactamase, application of lactamase and method for preparing (1R,4S)-gamma-lactam through enzymatic resolution
  • Lactamase, application of lactamase and method for preparing (1R,4S)-gamma-lactam through enzymatic resolution

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Effect test

Embodiment 1

[0041] The method for enzymatic resolution and preparation of (1R,4S)-venslide provided in this example specifically includes the following steps: dissolving 500 g of the substrate venslide with 1 L of phosphate buffer at a concentration of 10 mM, and then adding lactam Enzyme 3g, start stirring, react at 25°C, take samples in the middle and control in the middle, after the reaction ≥ 99% (see figure 2), start post-processing, add 4% dichloromethane to the reaction system, stir for 10 minutes, then concentrate to 3 / 5 volume, use 2.5 times volume of dichloromethane to extract 3 times (1 times, 1 times, 0.5 times ), the concentrated dichloromethane phase after the extraction was completed, the solvent was recovered, and evaporated to dryness to obtain the target product (1R, 4S)-2-azabicyclo[2.2.1]hept-5-en-3-one 135.9g, yield 48.8%, chiral purity 100% (see image 3 ).

[0042] in, figure 2 and image 3 They are the detection result diagrams of the high performance liquid ...

Embodiment 2

[0054] The method for the enzymatic resolution and preparation of (1R,4S)-venslide provided in this example specifically includes the following steps: dissolving 500 g of the substrate venslide with 1 L phosphate buffer solution with a concentration of 10 mM, and then adding lactam Enzyme 3g, start stirring, react at 15°C, take samples in the middle and control in the middle, after the reaction is ≥99%, start post-processing, add 4% dichloromethane to the reaction system, stir for 10 minutes, then concentrate to 3 / 5 volume, use 2.5 times the volume of dichloromethane was extracted 3 times (1 times, 1 times, 0.5 times), after the extraction, the dichloromethane phase was concentrated, the solvent was recovered, and the target product (1R,4S)-2-azabicyclo was obtained after evaporation to dryness [2.2.1] 135.9 g of hept-5-en-3-one.

Embodiment 3

[0056] The method for the enzymatic resolution and preparation of (1R,4S)-venslide provided in this example specifically includes the following steps: dissolving 500 g of the substrate venslide with 1 L phosphate buffer solution with a concentration of 10 mM, and then adding lactam Enzyme 3g, start stirring, react at 20°C, take samples in the middle and control in the middle, after the reaction is ≥99%, start post-processing, add 4% dichloromethane to the reaction system, stir for 10 minutes, then concentrate to 3 / 5 volume, use 2.5 times the volume of dichloromethane was extracted 3 times (1 times, 1 times, 0.5 times), after the extraction, the dichloromethane phase was concentrated, the solvent was recovered, and the target product (1R,4S)-2-azabicyclo was obtained after evaporation to dryness [2.2.1] 135.9 g of hept-5-en-3-one.

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Abstract

The invention discloses lactamase, an application of the lactamase and a method for preparing (1R,4S)-gamma-lactam through enzymatic resolution. An amino acid sequence of the lactamase is as shown inSEQ ID NO.1. A nucleotide sequence of a nucleic acid molecule for encoding the lactamase is as shown in SEQ ID NO. 2. The method for preparing the (1R,4S)-gamma-lactam through enzymatic resolution comprises the following step of in a buffer solution system, mixing a substrate and the lactamase to react, wherein the substrate is 2-azabicyclo[2.2.1]hept-5-ene-3-one. The lactamase has the characteristics of high substrate tolerance and high activity, and when the lactamase is applied to the method for preparing the (1R,4S)-gamma-lactam through enzymatic resolution, the substrate with relatively high concentration can fully react under the action of the lactamase to obtain the (1R,4S)-gamma-lactam with relatively high yield and chiral purity, so that the production efficiency is improved, andthe production cost is reduced.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a lactamase and its application and a method for enzymatic resolution and preparation of (1R, 4S)-vins lactone. Background technique [0002] γ-lactam is the abbreviation of the compound 2-azabicyclo[2.2.1]hept-5-en-3-one (also commonly known as Wens lactone), and its molecular formula is C 6 h 7 NO. One of the isomers of γ-lactam is (-)-γ-lactam (1R, 4S)-wens lactone, which is mainly synthesized from optically pure (1R, 4S)-wens lactone There is the anti-HIV drug abacavir, and the anti-influenza drug peramivir is mainly synthesized from (1S,4R)-vinslide. Therefore, the separation and preparation of (1R, 4S)-venslide has important economic and social value. [0003] At present, optically pure (1R, 4S)-venslides are usually obtained by biocatalysis, but the tolerance and activity of enzymes currently used in biocatalysis are generally not high, resulting in a normal rea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/86C12N15/55C12P41/00C12P17/10
CPCC12N9/86C12P17/10C12P41/001C12P41/006
Inventor 高仰哲吴法浩李钢王志航
Owner 安徽红杉生物医药科技有限公司
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