CN106492396A - A kind of antibiotic bacterium dregs laccase enzyme solution - Google Patents

Abstract

The invention provides a method for treating antibiotic scum by using laccase, wherein the antibiotic slag is treated with laccase, wherein: the antibiotic slag is thioerythromycin slag; The slag was treated at a temperature of 40-55°C, a pH of 6-7, and an enzyme concentration of 0.005-0.02g/mL for 4-6h, and the potency of the thioerythromycin residue was reduced by more than 90%. The invention is simple and feasible, has low processing cost, no secondary pollution, energy saving and environmental protection, can remove most of the antibiotic residues in antibiotic residues, facilitates the subsequent treatment of antibiotic residues, and provides a certain method for enzymatic treatment of antibiotic residues. Data basis.

Description

A kind of laccase enzymatic hydrolysis method of antibiotic bacteria residue

technical field

The invention belongs to the field of environmental protection, and in particular relates to a method for enzymatic hydrolysis of residual antibiotic residues of antibiotic bacteria, in particular to a method for enzymatic hydrolysis of residual antibiotic residues of antibiotic bacteria by using laccase.

Background technique

my country is a major producer of antibiotics. A large amount of solid waste, namely antibiotic residue, will be produced in the fermentation production of antibiotics. The main components of antibiotic residues are: microbial mycelia that produce specific antibiotics, reagents added during the extraction of antibiotics, incompletely utilized medium, degradation products of the medium, metabolites produced during fermentation, and unknown growth factors and small amounts of residual antibiotics. During the open-air stacking of antibiotic residues, on the one hand, it will cause air pollution and directly affect the lives of the surrounding residents; Medicinal properties indirectly affect human health. And, in 2008, antibiotic residues were included in the "National List of Hazardous Wastes" as hazardous wastes. Therefore, it is very necessary to find an environmentally friendly and economical method to deal with antibiotic residues.

CN105457968A discloses a method for harmless treatment of antibiotic residues, which inactivates antibiotic residues under conditions of pH 8-11 and temperature 90-100°C. The method deactivates the fungus residue, degrades the antibiotics therein and loses its activity, and the inactivated fungus residue mycelium contains microbial cells, coagulation aids and remaining nutrients, and can be used as an organic fertilizer or a feed additive. However, if the degradation of antibiotics is not complete, it is easy to cause antibiotics to remain in organic fertilizers or feed additives, and indirectly endanger human health.

CN101380509A discloses a method for harmless treatment of macrolide antibiotic bacteria residue, which belongs to the technical field of solid waste treatment. The method adopts countercurrent mechanical agitation to effectively break the mycelium mass, and then uses spiramycin harmless treatment bacteria agent to carry out harmless treatment on the macrolide antibiotic bacteria residue, and the treated antibiotic bacteria residue is used as a plant fertilizer additive . However, if the spiramycin harmless treatment bacteria agent is added improperly, it is easy to cause secondary pollution of antibiotics, and mechanical stirring has a certain cost loss.

Therefore, it is hoped to propose a fast, economical and effective method for removing residual antibiotics in antibiotic residues.

Contents of the invention

The purpose of the present invention is mainly to propose a new method for treating antibiotic scum, which uses laccase to treat antibiotic slag and reduces the residual antibiotics in antibiotic slag. It is used to solve technical problems such as difficult treatment of antibiotic bacteria residue, high cost, easy secondary pollution, long treatment cycle and complicated operation.

The invention provides a method for laccase enzymolysis of antibiotic bacteria residue, comprising the following steps:

a. Prepare a standard sample of erythromycin with a concentration of 0.001-0.005g/mL;

b. Prepare a suspension of antibiotic bacteria residue with a concentration of 0.01-0.04g/mL, and adjust the pH to 6-7;

c. Add laccase suspension to the bacterial residue suspension described in step b, and digest the remaining antibiotics in the antibiotic bacterial residue at a temperature of 40-55° C. and a time of 4-6 hours;

Preferably, the titer of the erythromycin standard sample described in step a is 920-4600 U/mL.

Thierythromycin bacteria residue is selected as the antibiotic residue, and there is no special requirement for its source.

Preferably, the original potency of the suspension of fungal residues in step b is 73.4-293.7 U/mL.

Preferably, the enzyme concentration of the laccase suspension in step c is 0.005-0.02 g/mL.

Preferably, the laccase in step c has an enzyme activity of 35-140 U/mL.

In step c, the ratio of the laccase suspension to the bacteria residue suspension is 1:1.

Among them, antibiotics have a diffusion and penetration effect in the agar plate medium. According to the derivation of the law of diffusion, the logarithmic value of the total amount of antibiotics is in a linear relationship with the square of the diameter of the inhibition zone. Comparing the size of the inhibition zone of the standard antibiotic and the test product, The potency of the tested antibiotic can be calculated. In the present invention, the titer of thioerythromycin slag can be calculated by comparing the inhibition zone of erythromycin standard substance with known potency and unknown potency thierythromycin test product.

Compared with the prior art, the present invention has the following significant advantages:

The invention takes the thioerythromycin slag as the research object, and uses laccase to treat the thioerythromycin slag, which overcomes the bottleneck of specific enzyme treatment of specific substrates. In general, enzymes have strict selectivity for the substrate they act on. An enzyme can only act on one substance, or a class of substances with similar molecular structures, to promote a certain chemical reaction and produce a certain reaction product. This selective action is called the specificity of the enzyme. Usually an enzyme can only catalyze one chemical reaction or a class of similar reactions. We have conducted a lot of experiments to find the treatment method of thioerythromycin residue, but we were surprised to find that laccase has an excellent digestion effect on the residual antibiotics in thioerythromycin residue, which can make thioerythromycin bacteria The potency of slag is reduced by more than 90%.

At present, the domestic and foreign methods of treating antibiotic bacteria residues are mainly to use high-temperature and strong alkali inactivation treatment of antibiotic bacteria residues as feed additives or as organic fertilizers after physical and chemical treatment, but if chemical preparations are added improperly, secondary pollution will easily occur. Moreover, if the residual antibiotics in the bacteria residues are not degraded completely, it is easy to cause antibiotic residues, which indirectly endangers human health. At the same time, high temperature and strong alkali treatment, physical treatment, etc. have certain energy losses, and the treatment cost is relatively high. The enzymatic hydrolysis method of antibiotic bacteria residue is based on the characteristics of small amount and high efficiency of enzymes to treat antibiotic residues, which will not cause secondary pollution, is more environmentally friendly, and is simple to operate, short reaction cycle, low cost, and can remove most of the residues of antibiotic bacteria residues The antibiotics are convenient for the subsequent treatment of antibiotic residues, and at the same time, a new method for enzymatic treatment of antibiotic residues is provided.

detailed description

In order to illustrate the technical characteristics of the present invention more clearly, the present invention will be described in detail below through specific embodiments.

The research on the antibiotic laccase enzymatic hydrolysis technology of antibiotic slag residue of the present invention adopts non-specific enzyme to treat the antibiotic slag, comprising the following steps:

a. Prepare a standard sample of erythromycin with a concentration of 0.001-0.005g/mL;

b. Prepare a suspension of antibiotic bacteria residue with a concentration of 0.01-0.04g/mL, and adjust the pH to 6-7;

c. Add laccase suspension to the bacteria residue suspension, and digest the residual antibiotics in the antibiotic bacteria residue at a temperature of 40-55° C. for 4-6 hours.

Wherein, the antibiotic residue is selected from thioerythromycin residue, and the original potency is 73.4-293.7U/mL;

Wherein, the enzyme concentration of the laccase suspension is 0.005-0.02g/mL, and the enzyme activity is 35-140U/mL

The potency of antibiotic bacteria residue and the potency of standard products were determined by tube and plate method; the enzyme activity was determined by high performance liquid chromatography.

Example 1

Prepare a standard sample of erythromycin with a concentration of 0.001g/mL, and its potency is 920U/mL;

The preparation concentration is 0.01g/mL suspension of thiorubicin bacteria residue, its original potency is 73.4U/mL, and the pH is adjusted to be 6;

Add laccase suspension to the thioerythromycin residue suspension, the enzyme concentration is 0.015g/mL, and the enzyme activity is 105U/mL; Residual antibiotics were digested. After digestion, the titer of thioerythromycin bacteria residue was 3.67U/mL, and the titer of thioerythromycin bacteria residue was reduced by 95%.

Example 2

Prepare a standard sample of erythromycin with a concentration of 0.003g/mL, and its potency is 2760U/mL;

The preparation concentration is 0.02g/mL suspension of thioerythromycin bacteria residue, its original potency is 220.1U/mL, and the pH is adjusted to be 7;

Add laccase suspension to the thioerythromycin residue suspension, the enzyme concentration is 0.007g/mL, and the enzyme activity is 49U/mL; Residual antibiotics were digested. After digestion, the titer of thioerythromycin residue was 15.4U/mL, and the titer of thioerythromycin residue was reduced by 93%.

Example 3

Prepare a standard sample of erythromycin with a concentration of 0.005g/mL, and its potency is 4600U/mL;

The preparation concentration is 0.03g/mL suspension of thioerythromycin bacteria residue, its original potency is 78.15U/mL, and the pH is adjusted to be 7;

Add laccase suspension to the thioerythromycin residue suspension, the enzyme concentration is 0.02g/mL, and the enzyme activity is 140U/mL; Residual antibiotics were digested. After digestion, the titer of thioerythromycin residue was 3.9 U/mL, and the titer of thioerythromycin residue was reduced by 95%.

Example 4

Prepare a standard sample of erythromycin with a concentration of 0.004g/mL, and its potency is 3680U/mL;

The preparation concentration is 0.04g/mL the suspension of thiorubicin bacteria residue, its original potency is 293.7U/mL, adjust pH to be 6.5;

Add laccase suspension to the thioerythromycin residue suspension, the enzyme concentration is 0.015g/mL, and the enzyme activity is 105U/mL; Residual antibiotics were digested. After digestion, the titer of thioerythromycin bacteria residue was 5.9 U/mL, and the titer of thioerythromycin bacteria residue was reduced by 98%.

The invention discloses a method for enzymatic hydrolysis of antibiotic scum, which is a new exploration of using enzymes to treat antibiotics. Starting from the data, the present invention verifies the feasibility of using enzymes to treat antibiotic residues, and overcomes the bottleneck of specific enzymes for treating specific bacteria residues. At the same time, the method is simple in operation, short in reaction cycle, low in cost, and does not produce secondary Secondary pollution, has very good environmental protection benefits.

Claims (7)

1. A method for laccase enzymolysis of antibiotic bacteria residue, comprising the following steps: a. Prepare a standard sample of erythromycin with a concentration of 0.001-0.005g/mL; b. Prepare a suspension of antibiotic bacteria residue with a concentration of 0.01-0.04g/mL, and adjust the pH to 6-7; c. Add laccase suspension to the bacteria residue suspension described in step b, and digest the remaining antibiotics in the antibiotic bacteria residue at a temperature of 40-55° C. and a time of 4-6 hours. 2. the method for laccase enzymolysis of antibiotic bacteria residue as claimed in claim 1, is characterized in that, the potency of the erythromycin standard sample described in step a is 920-4600U/mL. 3. The method for laccase enzymatic hydrolysis of antibiotic slag as claimed in claim 1, wherein said antibiotic slag is selected from thioerythromycin slag. 4. The method for laccase enzymatic hydrolysis of antibiotic residues according to any one of claims 1 to 3, characterized in that the original potency of the suspension of bacteria residues in step b is 73.4-293.7 U/mL. 5. The method for laccase enzymatic hydrolysis of antibiotic bacteria residue according to any one of claims 1 to 4, wherein the enzyme concentration of the laccase suspension in step c is 0.005-0.02g/mL. 6. The method for laccase enzymatic hydrolysis of antibiotic bacteria residue according to any one of claims 1 to 5, characterized in that the laccase in step c has an enzyme activity of 35-140 U/mL. 7. The method for laccase enzymatic hydrolysis of antibiotic residues according to any one of claims 1 to 6, characterized in that the ratio of the laccase suspension to the suspension of bacteria residues in step c is 1:1.