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6-aminopenicillanic acid degrading bacterium and screening method thereof

An aminopenicillanic acid, bacterial technology, applied in the direction of microbial-based methods, bacteria, chemical instruments and methods

Active Publication Date: 2014-09-17
CHINA THREE GORGES CORPORATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • 6-aminopenicillanic acid degrading bacterium and screening method thereof
  • 6-aminopenicillanic acid degrading bacterium and screening method thereof
  • 6-aminopenicillanic acid degrading bacterium and screening method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The separation of the functional bacteria of embodiment 1 degradation 6-APA

[0025] Materials and Methods

[0026] 1. Medium

[0027] ① Enrichment medium:

[0028] Glucose, 20g; Peptone, 4g; Yeast powder, 1g; Beef extract, 2g; NaCl, 4g; K 2 HPO 4 , 1.5g; MgCl 2 ·6H 2 O, 0.1g; FeSO 4 ·7H 2 O, 0.1g; vitamin solution 10ml (cobalamin, 0.01g; ascorbic acid, 0.025g; riboflavin, 0.025g; citric acid, 0.02g; pyridoxal, 0.05g; folic acid, 0.01g; p-aminobenzoic acid , 0.01g; creatine, 0.025g); trace element solution (MnSO 4 ·7H 2 O, 0.01g; ZnSO 4 ·7H 2 O, 0.05g; H 3 BO 3 , 0.01g; N(CH 2 COOH) 3 , 4.5g; CaCl 2 2H 2 O, 0.01g; Na 2 MoO 4 , 0.01g; CoCl 2 ·6H 2 O, 0.2g; AlK(SO4 ) 2 , 0.01g) 10ml, finally dissolved in 1L.

[0029] ②Separation medium:

[0030] Glucose 10g; 6-APA 5g; NaCl, 4g; K 2 HPO 4 , 1.5g; MgCl 2 ·6H 2 O, 0.1g; FeSO 4 ·7H 2 O, 0.1g; vitamin solution 10ml (cobalamin, 0.01g; ascorbic acid, 0.025g; riboflavin, 0.025g; citric acid, 0.02g; p...

Embodiment 2

[0033] The strain identification of embodiment 2 bacterial strain L2

[0034] 1. Genomic DNA extraction

[0035] The above-mentioned strain L2 was mass-cultured according to conventional technical means, and then its genomic DNA was obtained.

[0036] 2. Identification method of 16S rDNA of strain L2

[0037] 2. PCR amplification and sequencing of 116S rRNA gene sequence and construction of phylogenetic tree

[0038] 2.1. PCR amplification of 116S rRNA gene sequence

[0039] The primers at both ends of the amplified 16S rRNA gene sequence are universal primers: forward primer BSF8 / 20: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 1) and reverse primer BSR1541 / 20: 5'-AAGGAGGTGATCCAGCCGCA-3' (SEQ ID NO: 2). The PCR reaction system was 50 μL, and the reaction conditions were denaturation at 94°C for 5 minutes; followed by 35 cycle reactions: denaturation at 94°C for 45 s, annealing at 50°C for 45 s, and extension at 72°C for 90 s; then extension at 72°C for 10 min, and finally store...

Embodiment 36

[0073] The degradation rate of embodiment 36-APA

[0074] In this study, high performance liquid chromatography was used to determine the concentration of 6-APA. Agilent1100 high performance liquid chromatography of Agilent Technologies Co., Ltd. was used for determination. Chromatographic column: XDB-C18 (4.6×150mm, 5μm). The detection conditions are flow: 0.5mL / min, injection volume: 10μL, temperature: 30°C, wavelength: 210nm, 220nm, 230nm, 240nm, 260nm, mobile phase: methanol: water = 50:50.

[0075] Inoculate 0.1 ml of strain L2 bacterial solution with 100 ml of isolation medium (the content of 6-APA is 300 mg / L), culture at 37°C and 170 rpm for 72 hours, measure the concentration of 6-APA, and calculate its removal rate. The removal rate of 6-APA of strain L2 was calculated to be 28%.

[0076]

[0077]

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Abstract

The invention relates to a 6-aminopenicillanic acid (6-APA) degrading bacterium and a screening method thereof. Particularly, the invention relates to a strain of 6-APA degrading bacterium Bordetella sp.L2, which is preserved in China General Microbiological Culture Collection Center (CGMCC) on October 18, 2012, with the preservation number of CGMCC No. 6691; and the GenBank registration number of the 16S rRNA gene sequence of the 6-APA degrading bacterium Bordetella sp.L2 is HQ840720. The 6-APA degrading bacterium Bordetella sp.L2 is an aerobic bacillus brevis; and when the pH value is 8.0, the 6-APA degradation rate of the 6-APA degrading bacterium Bordetella sp.L2 is 28%. The strain achieves higher 6-APA degradation capability and has a practical significance and an important engineering application value for treatment of waste water containing 6-APA cephalosporin.

Description

technical field [0001] The invention relates to bacteria degrading 6-aminopenicillanic acid and a screening method thereof. Background technique [0002] Antibiotic wastewater is a kind of high-concentration organic wastewater containing refractory substances and biotoxic substances. In recent years, due to the rapid development of the pharmaceutical industry, especially the emergence of antibiotics, the water resources in China and even the world have been seriously polluted. It is imminent to carry out reasonable and effective treatment of antibiotic wastewater, which is related to the development of society and the future of human beings. [0003] The composition of antibiotic wastewater is very complex, containing a variety of refractory organic and inorganic substances, which are generally difficult to deal with. For example, bacteria are generally used to degrade antibiotics, but the residual substance zimycin contained in it is harmful to Gram-positive bacteria. and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N1/02C02F3/34C12R1/01C02F101/38C02F103/36
Inventor 林海龙陈先明关旸陈兆波王晓雨王锐范阳
Owner CHINA THREE GORGES CORPORATION
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