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Amidase gene of Variovorax boronicumulans CGMCC 4969, and its application in biological degradation of acrylamide

A technology of acrylamide and biodegradation, applied in the field of microorganisms, can solve the problem of no greedy phagocytosis degrading acrylamide

Inactive Publication Date: 2013-10-09
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently Arthrobacter Arthrobacter sp.J1, Bacillus sphaericu, Pseudomonas putrefaciens, Rhodococcus Rhodococcus sp., Nocardia Nocardia rhodochrous, Rhodopseudomonas palustris, Enterobacter aerogenes Enterobacter aerogenes, etc. can be degraded Acrylamide, however, there is no report on the degradation of acrylamide by phages (Buranasip K, Charoenpanich J, 2011. Biodegradation of acrylamide by Enterobacter aerogenes isolated from wastewater in Thailand. Journal of Environmental Sciences, 23 ( 3): 396–403)

Method used

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  • Amidase gene of Variovorax boronicumulans CGMCC 4969, and its application in biological degradation of acrylamide
  • Amidase gene of Variovorax boronicumulans CGMCC 4969, and its application in biological degradation of acrylamide
  • Amidase gene of Variovorax boronicumulans CGMCC 4969, and its application in biological degradation of acrylamide

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0015] Example one: V. boronicumulans Degradation of Acrylamide by CGMCC 4969 Strain

[0016] to be preserved V. boronicumulans CGMCC 4969 strain was streaked in a petri dish containing LB solid medium. The composition of LB medium is: peptone 10g / L, yeast extract 5g / L, NaCl 10g / L, solid medium plus agar powder 15g / L, pH7.2. After a single colony grows on the plate, pick a ring of single colony and inoculate it into LB liquid medium, shake it at 220 rpm for 24 h at 30°C, and collect the cells by centrifugation. Cells were suspended in 20 mmol / L sodium phosphate buffer (pH 7.0) containing 5.60 g / L acrylamide, and the degradation of acrylamide was detected by the HPLC method reported by Prabu and Thattheyus (2007). Such as figure 1 As shown, the retention time of the substrate acrylamide is 3.0 min, and the peak time of the product acrylic acid is 2.7 min. After 24 hours of transformation, the concentration of the remaining substrate acrylamide was 1.16g / L, the degra...

example 2

[0017] Example two: V. boronicumulans Acrylamide Degradation Kinetics of CGMCC 4969 Strain

[0018] The experimental method is the same as that of Example 1, and the initial concentration of the substrate acrylamide is 0.5 g / L. Take samples regularly to detect the reduction of acrylamide. get as figure 2 In the acrylamide degradation curve shown, the degradation half-life is 45 minutes, the reaction time is 120 minutes, the degradation amount of acrylamide is 0.46 g / L, and the degradation rate is 92%; while the substrate control hardly degrades.

example 3

[0019] Example three: V. boronicumulans CGMCC 4969 Amidase Gene Cloning

[0020] Pick up from the LB-containing solid medium with a sterile toothpick V. boronicumulans A single colony of CGMCC 4969 was inoculated into a 100 mL Erlenmeyer flask filled with 20 mL of LB liquid medium, and cultured in a shaker at 30°C and 220 rpm for 16 hours. The culture solution was centrifuged at 13,000 rpm for 5 min, the cells were collected and washed, and the genomic DNA was extracted using the MiniBEST Bacterial Genome Extraction Kit from TaKaRa Company.

[0021] Using greedy phagocytosis published in the Genbank database V. paradoxus S110 (accession number: CP001635.1) and V. paradoxus The amidase gene of EPS (accession number: CP002417.1) was homologously compared, and primers P3-1: 5'-ATGAGACATGGT GATATTTCGAGC-3' (SEQ ID No: 2) and P3-2: 5'-ATGAGAGTTCGG were designed CGTAGTTG-3' (SEQ ID No: 3), the primer was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. Synthe...

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Abstract

The invention discloses Variovorax boronicumulans CGMCC 4969 capable of biologically degrading acrylamide. The amidase gene of the Variovorax boronicumulans CGMCC 4969 is composed of a DNA sequence represented by SEQ ID No:1 in a sequence table; DNA fragments composed of the sequence represented by the SEQ ID No:1 are inserted into an Escherichia coli expression vector pET28a plasmid to obtain a recombinant pET28a-amidase; and the recombinant pET28a-amidase is transformed into an Escherichia coli Rosetta strain, the amidase is expressed through induction, and Escherichia coli cells containing expression proteins can degrade acrylamide.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, in particular to greedy phagocytic bacteria V. boronicumulans The amidase gene produced by CGMCC 4969 was applied to the biodegradation of acrylamide. Background technique [0002] Acrylamide is widely used in oil extraction, agriculture and chemical industry. Polyacrylamide, a polymer of acrylamide, is used as oil displacement agent to improve the recovery rate of oil recovery; it is used as flocculant in paper industry, factory wastewater treatment and sewage treatment; As a soil conditioner, it can aggregate the soil, improve air circulation, water permeability and water retention. The extensive use of acrylamide in industry and agriculture causes soil and water pollution. Since acrylamide is a nerve agent and carcinogen, eliminating the pollution of acrylamide can help improve the environment and reduce its potential risk to the ecological environment. [0003] Microbial degradatio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N15/70C12N1/21A62D3/02C12R1/19A62D101/28
Inventor 戴亦军曹玉敏周倩雯翟闪王颖袁生
Owner NANJING NORMAL UNIVERSITY
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