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Bacterial strain for generating 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: 2017-12-15
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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  • Bacterial strain for generating polyvinyl alcohol degrading enzyme, and polyvinyl alcohol degrading enzyme
  • Bacterial strain for generating polyvinyl alcohol degrading enzyme, and polyvinyl alcohol degrading enzyme
  • Bacterial strain for generating 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 present invention

[0040] 1.1 Strain screening

[0041] The strain screening sample is the Qinling plot (sample collection site: Qinling virgin forest; sample collection date: 2008.8; altitude, latitude and longitude: altitude: 1500-2500 meters; longitude and latitude: 108°E, 34°N; isolation source: virgin forest leaves; Separation date: 2008.8) Leaf samples.

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

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

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

Embodiment 2

[0056] Example 2: Whole genome sequencing of S. rhizophila QL-P4, prediction and functional annotation of genes related to PVA degradation pathway, and protein domain analysis

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

[0058] Using the third-generation single-molecule real-time sequencing (SMRT) platform RSⅡ sequencer of Paific Biosciences (Pacific Biosciences, Menlo Park, CA, USA), the whole genome of Stenotrophomonasrhizophila QL-P4 was sequenced using P6 / C4 reagents: the strain was tested for 1 SMRT cell, the sequencing depth is above 80X. De novo assembly by SMRT Portal (version 2.2.0) Hierarchical Genome Assembly Process (HGAP.3) algorithm (HGAP is a high-quality de novo assembly software for PacBio data); then use PBjelly or PCR to assemble the results of SMRT Portal HGAP Carry out hole filling; 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-g...

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] The primers were designed, the target gene was amplified by PCR technology, and the amplified target gene was recovered with SanPrep column DNA gel recovery kit; then, the recovered target gene and plasmid vector pET-28a(+) were doubled with HindIIIHF and NdeI. Enzymatic digestion, using T4 DNA ligase to connect the double-digested target gene and plasmid vector overnight at 4°C; the ligated product was transformed into Trans 5α, positive clones were picked by colony PCR, and the recombinant plasmid was extracted after correct sequencing and transformed into expression bacteria E. coliBL21 DE3; E. coli BL21 DE3 containing the recombinant plasmid was grown to OD at 37°C 600 When it reaches the range of 0.6-0.8, add 1M IPTG to a final concentration...

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Abstract

The invention relates to a bacterial strain for generating polyvinyl alcohol degrading enzyme, and the polyvinyl alcohol degrading enzyme. The related bacterial strain is named as stenotrophomonas rhizophila, the bacterial strain was preserved in China General Microbiological Culture Collection Center (CGMCC) on March 25, 2016, and the preservation number is CGMCC 1.15515. The related polyvinyl alcohol degrading enzyme is enzyme generated by the bacterial strain. The polyvinyl alcohol degrading enzyme provided by the invention has remarkable effect of degrading high polymer PVA in sewage.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a strain producing a polyvinyl alcohol degrading enzyme and a polyvinyl alcohol degrading enzyme. Background technique [0002] Polyvinyl alcohol (PVA) is a water-soluble polymer with a wide range of uses. Due to the poor biodegradability of polyvinyl alcohol (BOD / COD=0.07), entering the water body will cause water pollution, increase the foam on the surface of the polluted water body, increase the viscosity, affect the activity of aerobic microorganisms, and affect the water body. It is extremely unfavorable for the visual performance and the reoxygenation of the water body. Destruction of water quality is no less harmful than other organic pollutants. [0003] At present, physical and chemical degradation and separation methods are still widely used in view of the pollution of polyvinyl alcohol in water bodies. Although such methods are simple and easy to operate, they have disadvan...

Claims

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Application Information

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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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