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Method for efficiently degrading tetracyclic and sulfonamide antibiotics by laccase

A technology of antibiotics and tetracyclics, applied in the field of enzyme engineering and technology and fermentation, can solve the problems of high price, unsatisfactory degradation effect, and difficulty in obtaining intellectual property rights, etc., and achieve the effect of broad application prospects

Inactive Publication Date: 2019-12-17
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the reaction time of the existing research needs several hours or even more than a few days, and the domestic laccases used for antibiotic degradation are mainly imported commercial laccases, which are expensive and the degradation effect is not ideal, so it is difficult to achieve Large-scale application, and it is difficult for us to obtain independent intellectual property rights

Method used

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  • Method for efficiently degrading tetracyclic and sulfonamide antibiotics by laccase

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Experimental program
Comparison scheme
Effect test

Embodiment

[0024] The experimental materials involved in the embodiments of the present invention are as follows:

[0025] strain

[0026] Pycnoporus sp.SYBC-L10 has been deposited in China Center for Type Culture Collection in 2015 with the deposit number CCTCC#NO: M2015020, address: Wuhan University, Wuhan, China.

[0027] culture medium

[0028] PDA medium composition: potatoes 200g L -1 , Glucose 20g L -1 , agar powder 20g L -1 , pH natural;

[0029] PDB medium composition: potatoes 200g L -1 , Glucose 20g L -1 , pH natural;

[0030] The composition of the improved basal enzyme production medium: glucose 15g L -1 , soybean meal 5g L -1 , diammonium hydrogen citrate 0.5g L -1 , CuSO 4 ·5H 2 O 0.125g L -1 , KCl 0.5g L -1 , KH 2 PO 4 1g L -1 , MgSO 4 ·7H 2 O 0.5g L -1 , yeast extract 1g L -1 , MnSO4·H 2 O 0.01g L -1 , the initial pH is 4.0;

[0031] main reagent

[0032] Tetracycline (TC, 95%, CAS: 60-54-8) and sulfamethoxazole (SMX, 98%, CAS 723-46-6) were pur...

example 1

[0035] Application of Example 1 Laccase Mediator System in the Degradation of Tetracyclic Antibiotics

[0036] 1.1 Reaction system

[0037] 1.5mL contains a final concentration of 50mg L -1 TC,5.0U mL -1 Lac-Q, 0.5 mmol L -1 ABTS

[0038] 1.2 Reaction conditions

[0039] 25°C, pH 6.0, static incubation.

[0040] 1.3 Operation steps

[0041] (1) Use a pipette to draw 1.05mL of 0.1mol L -1 Phosphate-sodium hydrogen phosphate buffer solution with pH 6.0 was added to a 1.5 centrifuge tube, 0.15mL tetracycline mother solution was drawn into the centrifuge tube, and 0.15mL ABTS mother solution was added to the centrifuge tube, and incubated at 25°C for 10 minutes.

[0042] (2) Take 0.15mL of pure enzyme solution with a pipette gun, add it to a centrifuge tube, start the reaction, start the timer, and react for 0.5-5 minutes. Sample 0.25 mL at 0.5, 1, 2, 3, 4, and 5 minutes respectively, and immediately add 0.5 mL of methanol to terminate the reaction. Each reaction was perf...

example 2

[0047] Application of example 2 laccase mediator system in the degradation of tetracyclic antibiotics

[0048] 2.1 Reaction system

[0049] 1.5mL contains a final concentration of 50mg L -1 TC,10.0U mL -1 Lac-Q, 1.0 mmol L -1 ABTS

[0050] 2.2 Reaction conditions

[0051] 0°C, pH 6.0, static incubation.

[0052] 2.3 Operation steps

[0053] (1) Use a pipette to draw 0.75mL of 0.1mol L -1 Phosphate-disodium hydrogen phosphate buffer solution with pH 6.0 was added to a 1.5 centrifuge tube, 0.15mL tetracycline mother solution was drawn into the centrifuge tube, and 0.3mL ABTS mother solution was added to the centrifuge tube, and incubated at 0°C for 10 minutes.

[0054] (2) Take 0.3mL of pure enzyme solution with a pipette gun, add it to a centrifuge tube, start the reaction, start the timer, and react for 0.5-5 minutes. Sample 0.25 mL at 0.5, 1, 2, 3, 4, and 5 minutes respectively, and immediately add 0.5 mL of methanol to terminate the reaction. Each reaction was perfo...

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Abstract

The invention discloses a method for efficiently degrading tetracyclic and sulfonamide antibiotics by laccase. The method comprises the following steps: S1, fermenting by adopting Pycnoporus sp. SYBC-L10 to produce laccase; and S2, firstly adjusting the pH value of a waste liquid containing tetracyclic antibiotics or sulfonamide antibiotics to 3.0-6.0, then adding the laccase obtained in the stepS1, simultaneously adding a small molecular mediator ABTS or syringaldehyde, standing and carrying out incubation for a period of time. The invention provides laccase capable of efficiently degradingtetracyclic and sulfanilamide antibiotics in the environment and a method. Compared with reported similar laccase, the laccase of the invention has the advantages that the effect is obviously quicker(generally, 50 mg L<-1> of antibiotics are degraded only within 5-10 min). Compared with a traditional physical-chemical method, the method of the invention is green, safe and environmentally friendly, meets the sustainable development requirement and has wide application prospects in the application of antibiotics to the future environment treatment.

Description

technical field [0001] The present invention relates to the fields of enzyme engineering and technology and fermentation technology, in particular to a method for efficiently degrading tetracyclic and sulfonamide antibiotics by fermenting a strain of Mycoporeus to produce laccase Background technique [0002] Antibiotics left in the environment are considered to be a new class of pollutants, which pose a great risk to human health and the stability of the ecosystem. In recent years, how to remove antibiotics left in the environment has attracted widespread attention. Antibiotics are widely used in medicine, aquaculture and other industries, and are currently one of the most widely used drugs in the world. In recent years, laccases have played an important role in the degradation of antibiotics. However, the reaction time of the existing research needs several hours or even more than a few days, and the domestic laccases used for antibiotic degradation are mainly imported c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N1/14A62D3/02C12R1/645A62D101/28A62D101/26
CPCA62D3/02A62D2101/26A62D2101/28C12N1/14C12N9/0061C12Y110/03002
Inventor 廖祥儒田乔鹏赖邓婷孟迪翟李欣管政兵蔡宇杰
Owner JIANGNAN UNIV
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