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Pseudomonas allowing (+/-)gamma-lactam to be split to obtain (-)gamma-lactam and screening and application thereof

A technology of pseudomonas and lactam, applied in the field of microorganisms, can solve the problems of expensive raw materials, environmental pollution, complicated steps, etc., and achieve the effects of high stereoselectivity, high product concentration, and high reaction yield

Active Publication Date: 2016-05-18
INST OF MICROBIOLOGY JIANGXI ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of using this method are: the raw materials are expensive, the steps are cumbersome, the cost is high, and it will cause serious environmental pollution, and the product usually contains metal impurities
There are currently no reports of Pseudomonas granadai screening

Method used

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  • Pseudomonas allowing (+/-)gamma-lactam to be split to obtain (-)gamma-lactam and screening and application thereof
  • Pseudomonas allowing (+/-)gamma-lactam to be split to obtain (-)gamma-lactam and screening and application thereof
  • Pseudomonas allowing (+/-)gamma-lactam to be split to obtain (-)gamma-lactam and screening and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1. Screening of Pseudomonas bacteria that can split (+ / -) γ-lactam to obtain (-) γ-lactam:

[0037] (1) Initial culture: Take sludge from the bottom of Aixi Lake, dilute it with sterile water, spread it on the culture medium, and cultivate it at 30°C for 2 days;

[0038] The primary screening medium is: NH 4 CL2.0g, KH 2 PO 4 1.5g, Na 2 HPO 4 1.5g, MgSO 4 0.2g, CaCl 2 0.1g, (+ / -) γ-lactam 2g, trace elements 100μL, agar powder 20g; the trace elements are calculated according to the amount added per liter, including: CaCL 2 2H 2 O3.6g, ZnO2.0g, CuCl 2H 2 O0.85g, NaMoO 2H 2 O4.8g, MnCl 2 4H 2 O2.0g, FeCl3 ·6H 2 O5.4g, CoCL 2 ·6H 2 O2.4g;

[0039] (2) Pick a single colony and streak again on the screening medium to separate and purify the single colony;

[0040] The morphological characteristics of a single colony picked are: the colony is round, with neat edges, raised and moist, white with a slight yellowish color, the inside of the colony is mucus-like, a...

Embodiment 2

[0051] With embodiment 1, difference is:

[0052] 1. Screening of Pseudomonas bacteria that can split (+ / -) γ-lactam to obtain (-) γ-lactam:

[0053] The primary screening medium is: NH 4 CL10g, KH 2 PO 4 0.01g, Na 2 HPO 4 0.01g, MgSO 4 0.01g, CaCl 2 0.01g, (+ / -) γ-lactam 1g.

[0054] The Pseudomonas was cultured for microbial fermentation and enzyme production for 1 day at a culture temperature of 40° C. and pH 3. The composition of the microbial fermentation and enzyme production medium was in g / L: glucose 5.0 g, NH 4 CL10g, KH 2 PO 4 0.01g, Na 2 HPO 4 0.01g, MgSO 4 0.01g, CaCl 2 0.01g, (+ / -) γ-lactam 2g, FeSO 4 0.08g; then centrifuge the above fermented broth to obtain wet cells for later use.

[0055] 2. The method of splitting (+ / -) γ-lactam to obtain (-) γ-lactam:

[0056] (3) Preparation of the resolution system: 600 g / L (+ / -) γ-lactam was prepared with phosphate buffer; the pH of the phosphate buffer was 3, and the concentration was 0.01 mol / L.

[0057...

Embodiment 3

[0060] With embodiment 1, difference is:

[0061] 1. Screening of Pseudomonas bacteria that can split (+ / -) γ-lactam to obtain (-) γ-lactam:

[0062] The primary screening medium is: NH 4 CL2.0g, KH 2 PO 4 10g, Na 2 HPO 4 10g, MgSO 4 ~1g, CaCl 2 0.01g, (+ / -) γ-lactam 1g.

[0063] The Pseudomonas is cultured for 1 to 3 days by microbial fermentation and enzyme production at a culture temperature of 20° C. and pH 10. The composition of the microbial fermentation and enzyme production medium is in g / L: glucose 5.0 g, NH 4 CL10g, KH 2 PO 4 10g, Na 2 HPO 4 0.01g, MgSO 4 0.01g, CaCl 2 0.5g, (+ / -) γ-lactam 2g, FeSO 4 0.08g; then centrifuge the above fermented liquid to obtain wet thallus for subsequent use;

[0064] 2. The method of splitting (+ / -) γ-lactam to obtain (-) γ-lactam:

[0065] (3) Preparation of the resolution system: 2 g / L (+ / -) γ-lactam was prepared with phosphate buffer; the pH of the phosphate buffer was 10, and the concentration was 0.5 mol / L.

[00...

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Abstract

The invention belongs to the technical field of microorganisms and particularly relates to pseudomonas allowing (+ / -)gamma-lactam to be split to obtain (-)gamma-lactam and a method using pseudomonas to split (+ / -)gamma-lactam. According to the method, a microorganism strain allowing (+ / -)gamma-lactam to be split to obtain (-)gamma-lactam is screened from soil and identified as pseudomonas granadensis B6; (+ / -)gamma-lactam is hydrolyzed in a water-phase system selectively in a three-dimensional mode to prepare (-)gamma-lactam. Under the condition that concentration of a substrate is 300 g / L, extracting, drying, evaporating, concentrating, cooling and crystallizing are carried out to obtain the product (-)gamma-lactam, optical purity (Ee value) ranges from 95.0% to 99.9%, and chemical purity ranges from 95% to 99%. The method of catalyzing and asymmetrically hydrolyzing (+ / -)gamma-lactam through organisms to prepare (-)gamma-lactam has the advantages of being high in stereoselectivity, large in reaction yield and high in product concentration.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a pseudomonas capable of splitting (+ / -) gamma-lactam to obtain (-) gamma-lactam, the pseudomonas being Pseudomonas granada (Pseudomonas granadensis B6). Background technique [0002] Abacavir and peramivir are specific drugs for the treatment of AIDS, bird flu and influenza A respectively. The current research results show that the enzymatic chemical synthesis method using optically pure (-) γ-lactam as the starting compound is the most convenient and economical method for synthesizing these two specific drugs. Before it was discovered that microorganisms could selectively split (+ / -)γ-lactams to obtain (-)γ-lactams, the preparation of optically pure (-)γ-lactams reported in the literature generally used chemical asymmetric synthesis. The disadvantage of using this method is that the raw materials are expensive, the steps are cumbersome, the cost is high, an...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N1/02C12P41/00C12P17/10C12R1/38
CPCC12N1/02C12N1/20C12P17/10C12P41/001C12N1/205C12R2001/38
Inventor 王金昌王建军靳亮
Owner INST OF MICROBIOLOGY JIANGXI ACADEMY OF SCI
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