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Method for producing (-)gamma-lactam from marine low-temperature gamma-lactamase

A technology of lactamase and lactam, which is applied in the field of bioengineering to achieve the effects of reducing safety hazards, high efficiency, and reducing production costs

Inactive Publication Date: 2018-06-22
DALIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no research reports on the production of low-temperature γ-lactamases by fermentation of marine microorganisms at home and abroad

Method used

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  • Method for producing (-)gamma-lactam from marine low-temperature gamma-lactamase
  • Method for producing (-)gamma-lactam from marine low-temperature gamma-lactamase
  • Method for producing (-)gamma-lactam from marine low-temperature gamma-lactamase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Step 1, microbial fermentation produces low-temperature γ-lactamase

[0034] Select Bacillus sp.DL-8 bacterial strain as seed, inoculate into culture medium (yeast extract 0.5%, glucose 0.5%, KH 2 PO 4 0.7%, Na 2 HPO 4 0.2%, MgSO 4 0.04%, FeSO 4 0.002%, CaCl 2 0.002%, NH 4 Cl0.5%, adjusted to pH7.0), cultured at 26°C and 150r / min for 16h, and then inserted into the enzyme-producing fermentation medium (glucose 0.4%, peptone 1.0%, Tween-80 0.1%, KH 2 PO 4 0.7%, Na 2 HPO 4 0.15%, Mg 2 SO 4 0.04%, pH7.63) 7.5L fermenter, liquid volume 2.5L, culture temperature 26°C, rotation speed 150rpm, ventilation rate 1vvm, initial pH8.0, fermented liquid was collected after 24 hours of fermentation.

[0035] Step 2, the acquisition of crude enzyme solution

[0036] Centrifuge the fermented broth obtained in step 1 at 4°C and 12000rpm, separate the bacteria, collect the bacteria, wash the obtained wet bacteria three times with sodium phosphate buffer (0.05mol / L, p...

Embodiment 2

[0042] Step 1, microbial fermentation produces low-temperature γ-lactamase

[0043] Select Bacillus sp.DL-8 bacterial strain as seed, inoculate into culture medium (yeast extract 0.5%, glucose 0.5%, KH 2 PO 4 0.7%, Na 2 HPO 4 0.2%, MgSO 4 0.04%, FeSO 4 0.002%, CaCl 2 0.002%, NH 4 Cl0.5%, adjusted to pH7.0), cultured at 26°C and 150r / min for 16h, and then inserted into the enzyme-producing fermentation medium (glucose 0.4%, peptone 1.0%, Tween-80 0.1%, KH 2 PO 4 0.7%, Na 2 HPO 4 0.15%, Mg 2 SO 4 0.04%, pH7.63) 7.5L fermenter, liquid volume 2.5L, culture temperature 26°C, rotation speed 150rpm, ventilation rate 2vvm, initial pH8.0, fermented liquid was collected after 24 hours of fermentation.

[0044] Step 2, the acquisition of crude enzyme solution

[0045] Centrifuge the fermented broth obtained in step 1 at low temperature and high speed, separate the thallus, collect the thallus, wash the obtained wet thallus three times with sodium phosphate buffer (0...

Embodiment 3

[0051] Step 1, microbial fermentation produces low-temperature γ-lactamase

[0052] Select Bacillus sp.DL-8 bacterial strain as seed, inoculate into culture medium (yeast extract 0.5%, glucose 0.5%, KH 2 PO 4 0.7%, Na 2 HPO 4 0.2%, MgSO 4 0.04%, FeSO 4 0.002%, CaCl 2 0.002%, NH 4 Cl0.5%, adjusted to pH7.0), cultured at 26°C and 150r / min for 16h, and then inserted into the enzyme-producing fermentation medium (glucose 0.4%, peptone 1.0%, Tween-80 0.1%, KH 2 PO 4 0.7%, Na 2 HPO 4 0.15%, Mg 2 SO 4 0.04%, pH7.63) 7.5L fermenter, liquid volume 2.5L, culture temperature 26°C, rotation speed 180rpm, air flow 2vvm, initial pH8.0, fermented for 24 hours and collected the fermented liquid.

[0053] Step 2, the acquisition of crude enzyme solution

[0054] Centrifuge the fermented broth obtained in step 1 at low temperature and high speed, separate the thallus, collect the thallus, wash the obtained wet thallus three times with sodium phosphate buffer (0.05mol / L, pH...

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Abstract

The invention discloses a method for producing (-)gamma-lactam from marine low-temperature gamma-lactamase. The method comprises the following steps: 1, production of low-temperature gamma-lactamase by microbial fermentation; 2, obtaining of a crude enzyme solution; 3, concentration of an enzyme solution; 4, production of (-)gamma-lactam. The method for producing (-)gamma-lactam from marine low-temperature gamma-lactamase is raised for the first time. According to the method, the cost of an enzyme production medium is low, and the production period is short; materials used for concentration ofthe enzyme solution can be recycled, the operation process is simple, and the enzyme activity recovery rate is high; bacterial contamination prevention measures are taken in the degradation process,the purpose of efficiently and rapidly producing (-)gamma-lactam with high medical value from (+ / -)gamma-lactam with low value is achieved, and the method has remarkable economic and social benefits.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for producing (-) gamma-lactam by marine low-temperature gamma-lactamase. Background technique [0002] γ-lactamase (γ-lactamses, EC 3.5.2.-) is a novel amidase that catalyzes the hydrolysis of the amide bond of amide compounds. (+)γ-lactamase can kinetically resolve racemic γ-lactam with high efficiency to obtain optically pure (-)γ-lactam. Optically pure (-) γ-lactam is a chiral intermediate for the preparation of antiviral drugs. synthetic raw materials. In addition, γ-lactamase can catalyze the racemic γ-lactam to obtain γ-aminobutyric acid analogs (GABA) with different configurations. GABA is an inhibitory neurotransmitter, which participates in various metabolic activities and has high physiological activity. Before the discovery of (+) γ-lactamase, the conventional method to prepare optically pure (-) γ-lactam generally adopts asymmetric syn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/86C12P41/00C12P13/02
CPCC12N9/86C12P13/02C12P41/001
Inventor 张庆芳迟乃玉王晓辉
Owner DALIAN UNIV
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