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Method for preparing (-) gamma-lactam by catalysis asymmetry of microorganism

A microorganism and lactam technology, applied in the field of bioengineering, can solve the problems of unfavorable industrial production and low substrate concentration, and achieve the effects of high stereoselectivity, high reaction yield and high product concentration

Active Publication Date: 2013-06-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

And cell immobilization research has been carried out to mutagenic strain, has improved the stability of cell, can reuse [Zheng Guojun etc.: CN 101285059A], although enzyme activity has been greatly improved, the substrate concentration that can tolerate is still not enough. High, not conducive to the application of industrial production

Method used

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  • Method for preparing (-) gamma-lactam by catalysis asymmetry of microorganism
  • Method for preparing (-) gamma-lactam by catalysis asymmetry of microorganism
  • Method for preparing (-) gamma-lactam by catalysis asymmetry of microorganism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Screening of microbial strains with lactamase activity

[0025] More than 20 soil samples collected near the local chemical factory were diluted with sterile water and left to stand, then the supernatant was inoculated on the enrichment medium, and the enriched cultured bacteria solution was spread on the plate screening medium, at 30°C Cultivate for 2-3 days, pick a single colony, and measure the activity.

[0026]The single colony obtained by screening was inserted into the fermentation medium, and cultured at 30°C and 110 r / min for 48 h. Then the wet cells obtained by centrifugation were suspended in phosphate buffer solution (0.05 mol / L, pH 7.0), the concentration of wet cells was 50 g / L, and the concentration of substrate (±) γ-lactam was 10 g / L. React at 30°C and 180 r / min, take samples within 48 h, remove the bacteria from the transformation solution by centrifugation, extract the supernatant with ethyl acetate, add an appropriate amount of anhydrous ...

Embodiment 2

[0029] Embodiment 2: the impact of different carbon sources on fermentation enzyme production

[0030] With peptone (10%) and yeast extract (5%) as nitrogen sources, the following carbon sources were added at a concentration of 3%: glucose, α-lactose, inulin, sucrose, soluble starch, citric acid, trisodium citrate, glycerin , corn steep liquor, as a fermentation medium, and for the hydrolysis of racemic γ-lactam. The transformation solution after the microbial catalytic reaction was centrifuged to separate the bacteria, and the supernatant was extracted with ethyl acetate, dried by adding an appropriate amount of anhydrous magnesium sulfate, and then detected by chiral HPLC. To investigate the effect of different carbon sources on bacterial strains Delftia Sp. CGMCC 5755 Fermentation Enzyme Production and Effect of Product Optical Purity.

[0031] It can be seen from Table 2 that the carbon source has little influence on the biomass of the bacteria, but has a great influen...

Embodiment 3

[0034] Embodiment 3: the impact of different nitrogen sources on fermentation enzyme production

[0035] Add yeast extract, corn steep liquor, beef extract, urea, peptone, ammonium bicarbonate, diammonium hydrogen citrate, and ammonium chloride at a concentration of 30 g / L on the medium with sucrose as the carbon source , ammonium acetate, and diammonium hydrogen phosphate as the nitrogen source of the fermentation medium, for Delftia The biomass and enzyme production of sp. CGMCC 5755 strain were studied. The results are shown in Table 3. Compared with the control (beef extract 3 g / L, peptone 10 g / L) when a single nitrogen source was used, the ee values ​​were lower. Therefore, beef extract and peptone were selected as the compound nitrogen source for bacterial fermentation to produce enzyme.

[0036] Table 3 Effect of nitrogen source on fermentation enzyme production

[0037]

[0038] "-" indicates that the ee value and enzyme activity were not determined because the...

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Abstract

The invention discloses a method for preparing (-) gamma-lactam by the catalysis asymmetry of microorganism, belonging to the technical field of biological engineering. Racemic modification 2-aza-second ring-(2.2.1)-heptane-5-alkene-3-ketone is hereafter referred as (+ / -) gamma-lactam. The method disclosed by the invention comprises the following steps of: screening from soil to obtain a microorganism bacterial strain having high-enantioselectivity (+) gamma-lactam, identifying to be Delftiasp, wherein the preservation No. is CGMCCNo.5755; and hydrolyzing (+ / -) gamma-lactam in a water phase system in a stereoselectivity way, to prepare the (-) gamma-lactam. Under the condition that the concentration of substrate is 200g / L, the (-) gamma-lactam is extracted, concentrated in an evaporating way, and crystallized in a cooling way to obtain the (-) gamma-lactam, wherein the optical purity of the (-) gamma-lactam is 95.0%-99.9%, and the chemical purity of the (-) gamma-lactam is 95%-99%. The method for preparing the (-) gamma-lactam by the catalysis asymmetry of the microorganism has the characteristics of being high in stereoselectivity, high in reaction yield and high in product concentration.

Description

technical field [0001] The invention relates to a method for preparing (-) gamma-lactam by utilizing microorganism stereoselective hydrolysis racemate (±) gamma-lactam, which belongs to the technical field of bioengineering. Background technique [0002] Optically pure (-)-γ-lactam is an important pharmaceutical intermediate. The anti-AIDS drugs (-)abacavir and (-)carbovir can be synthesized from (-)-γ-lactam as raw material, and the anti-influenza drug Para Mivir [Wang Jianjun et al.: Acta Microbiology, 50(8):988-994, 2010]. Due to the increasing demand of the above-mentioned chiral drugs, the research on the preparation of optically pure (-)-γ-lactam as an important chiral intermediate has attracted more and more attention. Currently available preparation methods include chemical synthesis and biological resolution. The main problems of preparing (-)γ-lactam by chemical method are many by-products, low yield, high preparation cost and cumbersome steps, etc., so that the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P41/00C12P17/10C12R1/01
Inventor 倪晔陈红干薛天芸
Owner JIANGNAN UNIV
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