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A strain producing polyvinyl alcohol degrading enzyme and polyvinyl alcohol degrading enzyme

A polyvinyl alcohol and degrading enzyme technology, which is applied in the direction of enzymes, bacteria, oxidoreductases, etc., can solve the problems of single species, unfavorable biodegradation of degrading bacteria, and the promotion of the scope of application.

Active Publication Date: 2020-03-20
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The single species of PVA-degrading bacteria isolated in the existing research, because the optimal growth environment of microorganisms in the same genus is relatively uniform, so the single species of degrading bacteria is not conducive to the promotion of the scope of biodegradation, which makes different species It is necessary to isolate microorganisms capable of degrading polyvinyl alcohol in

Method used

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  • A strain producing polyvinyl alcohol degrading enzyme and polyvinyl alcohol degrading enzyme
  • A strain producing polyvinyl alcohol degrading enzyme and polyvinyl alcohol degrading enzyme
  • A strain producing polyvinyl alcohol degrading enzyme and polyvinyl alcohol degrading enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Screening of strains of the invention

[0040] 1.1 Strain screening

[0041] The strain screening sample is the Qinling Plot (sample collection location: Qinling virgin forest; sample collection date: 2008.8; altitude, latitude and longitude: altitude: 1500-2500 meters; latitude and longitude: 108°E, 34°N; separation source: deciduous forest; Date of separation: 2008.8) Fallen leaves sample.

[0042] Weigh the sample into a centrifuge tube, use sterile distilled water according to the mass fraction ratio of 1:10 2 , 1:10 3 And 1:10 4 Dilute the sample, vortex and mix thoroughly, spread it on a polyvinyl alcohol strain screening medium plate with a spreader, invert the culture at 30°C for 48 hours, pick a single colony that can grow on the medium and store it on the slope of the test tube.

[0043] 1.2 I 2 -KI color method to determine the concentration of polyvinyl alcohol

[0044] Take 1.2 mL of the culture solution, centrifuge at 8000 rpm, 4°C for 10 min, take the s...

Embodiment 2

[0056] Example 2: S. rhizophila QL-P4 whole genome sequencing, PVA degradation pathway related gene prediction and function annotation, and protein domain analysis

[0057] 2.1 S. rhizophila QL-P4 whole genome sequencing

[0058] Using Paific Biosciences' third-generation single molecule real-time sequencing (SMRT) platform RSⅡ sequencer (Pacific Biosciences, Menlo Park, CA, USA), using P6 / C4 reagents to perform whole-genome sequencing of Stenotrophomonasrhizophila QL-P4: 1 was tested for this strain A SMRT cell with a sequencing depth above 80X. De novo splicing by SMRT Portal (version 2.2.0) Hierarchical Genome Assembly Process (HGAP.3) algorithm (HGAP is a software for high-quality de novo splicing for PacBio data); then use PBjelly or PCR to splice the results of SMRT Portal HGAP Fill holes; finally, based on the Blast results, manually remove the overlapping regions at both ends of the contig to circularize the genome.

[0059] Since the third-generation SMRT sequencing is pro...

Embodiment 3

[0077] Example 3: In vitro expression of PVA degradation pathway gene-encoded protein and detection of enzyme kinetic parameters

[0078] 3.1 In vitro expression of genes related to PVA degradation pathway in E. coli BL21DE3

[0079] Design primers, use PCR technology to amplify the target gene, and use the SanPrep column DNA gel recovery kit to recover the amplified target gene; then use HindⅢHF and NdeI to double the recovered target gene and plasmid vector pET-28a(+) Enzyme digestion, use T4DNA ligase to ligate the double-enzyme digested target gene and the plasmid vector overnight at 4℃; the ligation product is transformed into Trans 5α, the positive clones are picked by colony PCR, and the recombinant plasmid is extracted and transformed into expression bacteria E after the sequencing is correct. coliBL21 DE3; culture E.coli BL21 DE3 containing recombinant plasmid at 37℃ to OD 600 When it reaches the range of 0.6-0.8, add 1M IPTG to a final concentration of 1mM, induce a suita...

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Abstract

The invention relates to a bacterial strain producing polyvinyl alcohol degrading enzyme and the polyvinyl alcohol degrading enzyme. The strain involved is named: Stenotrophomonas rhizophila, which was deposited on March 25, 2016 in the General Microorganism Center of the China Committee for the Collection of Microbial Cultures, and the preservation number is: CGMCC 1.15515. The polyvinyl alcohol degrading enzyme involved in the present invention is the enzyme produced by the above strain. The polyvinyl alcohol degrading enzyme of the invention has remarkable effects on the degradation of high poly PVA in sewage.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and relates to a strain producing polyvinyl alcohol degrading enzyme and polyvinyl alcohol degrading enzyme. Background technique [0002] Polyvinyl alcohol (PVA) is a widely used water-soluble polymer. Due to the poor biodegradability of polyvinyl alcohol (BOD / COD=0.07), entering the water body will cause water pollution, increase the surface foam and viscosity of the polluted water body, affect the activity of aerobic microorganisms, and affect the water body. The visual performance and reoxygenation of the water body are extremely disadvantageous. The degree of damage to water quality is no less than that of other organic pollutants. [0003] At present, physical and chemical degradation and separation methods are often used for the pollution of water body polyvinyl alcohol. Although such methods are simple and easy to operate, they have disadvantages such as high cost and serious secondary environment...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N15/53C02F3/34C12R1/01C02F101/34
CPCC02F3/342C02F2101/34C12N9/0006C12Y101/03018C12N1/205C12R2001/01
Inventor 卫亚红陈非胡小平周云横武建荧付靖贾鑫淼
Owner NORTHWEST A & F UNIV
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