High-temperature polyphenol oxidase and application thereof in phenol-containing wastewater treatment

A polyphenol oxidase and application technology, applied in the field of environmental biotechnology applications, can solve the problems of low enzyme activity, unfavorable fermentation production, low expression of polyphenol oxidase, etc., and achieve the effect of improving catalytic efficiency

Active Publication Date: 2021-10-01
FOSHAN YUHUANG ECOLOGICAL ENVIRONMENT TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, fungal-derived enzymes have several major application drawbacks compared to their bacterial counterparts
1) Its protein is easily modified by glycosylation, which is not conducive to the use of genetically engineered bacteria for fermentation and production; 2) The optimal pH value of the fungal enzyme's catalysis is more acidic, while the bacterial-derived enzyme has a broader spectrum, neutral and alkaline environments 3) The distribution of extreme bacteria is wider, and it is easy to find some industrial application enzymes with better resistance, while fungal enzymes have poor temperatu

Method used

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  • High-temperature polyphenol oxidase and application thereof in phenol-containing wastewater treatment
  • High-temperature polyphenol oxidase and application thereof in phenol-containing wastewater treatment
  • High-temperature polyphenol oxidase and application thereof in phenol-containing wastewater treatment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Splicing mutation of polyphenol oxidase and screening of polyphenol oxidase mutant strain

[0035] 1) Splicing of polyphenol oxidase gene

[0036]Using the metagenome from the environmental soil sample as a DNA template, using the designed polyphenol oxidase primers to amplify, the primers are SEQ ID NO.1 and SEQ ID NO.2 respectively, and the products amplified by PCR were subjected to agarose gelation Gel electrophoresis, cutting the DNA product with a band size between 1200-2000 bp, and using the kit for DNA recovery, and then using molecular biology techniques to connect the intercepted PCR product pool (DNA pool) to Thermo Fisher pET101 Topo Plasmid system, then transformed into Escherichia coli BL21-DE3, plated, and grown overnight to obtain resistant positive clones; pick about 20 Escherichia coli single clones, inoculate them into 3 ml of liquid medium, and use them overnight Cultured E. coli cells were subjected to plasmid extraction. The extracted p...

Embodiment 2

[0043] Example 2 Recombinant expression of polyphenol oxidase in Bacillus subtilis

[0044] In order to reduce the cost of expression and purification of polyphenol oxidase, foreign proteins can usually be introduced into microbial host cells for secreted expression, and the commonly used secreted bacterial expression hosts mainly include Bacillus subtilis. Therefore, codon optimization was performed on the DNA sequence SEQ ID NO.3 encoding polyphenol oxidase YHL21, and the optimized YHL21 gene sequence was obtained as shown in SEQ ID NO.5. The DNA fragment is fused with the PxylA promoter fragment used for recombinant expression in Bacillus subtilis to obtain a complete expression reading frame (SEQ ID NO.6). The sequence shown in SEQ ID NO.6 was synthesized by gene, and then passed Eco RI After digestion, it was inserted into plasmid pMK4 to obtain expression plasmid pMK-YHL21, the sequence of which was SEQ ID NO.7. The constructed recombinant expression plasmid was trans...

Embodiment 3

[0045] Example 3 Recombinant production and immobilization of recombinant polyphenol oxidase and degradation of artificial phenolic wastewater

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Abstract

The invention discloses a gene and a polypeptide sequence of polyphenol oxidase YHL21. The polyphenol oxidase is confirmed by gene splicing and high-throughput screening of enzyme performance, and has the advantages of wide temperature-resistant range, strong high-temperature catalytic performance, easy membrane immobilization and the like, wherein functional enzyme is constructed by a single peptide fragment. The invention also discloses a method for carrying out recombinant expression and secretion production on the polyphenol oxidase YHL21 in a bacillus subtilis strain 168, and a method for immobilizing the recombinant polyphenol oxidase on the surface of a membrane by utilizing a polyvinylidene fluoride (PVDF) membrane, and the membrane enzyme composite system can stably and efficiently degrade organic matters such as phenol, parachlorophenol and the like in an aqueous solution. Enzyme engineering optimization is carried out through gene splicing, polyphenol oxidase capable of efficiently degrading phenolic pollutants is obtained, recombinant secretory expression is carried out through bacillus subtilis, enzyme immobilization is carried out through PVDF, and the catalytic efficiency of enzyme is improved.

Description

technical field [0001] The invention belongs to the application field of environmental biotechnology, and in particular relates to a high-temperature polyphenol oxidase and its application in the treatment of wastewater containing phenolic pollutants. Background technique [0002] Polyphenol oxidase is an oxidase containing copper ions. It is firstly separated from the active substances oxidized and solidified by raw polyphenols, so it is also called polyphenol oxidase (Laccase). Polyphenol oxidase can be synthesized by various cells. Plants, animals, bacteria and fungi can produce polyphenol oxidase, and its oxidized substrate objects are very broad-spectrum, which can exceed hundreds of complex organic substances with different structures or types. Therefore, It is widely used in food, paper making, textile, industrial catalysis, pollutant treatment and other fields. [0003] In the food industry, polyphenol oxidase can be used to deal with the turbidity of aromatic amine...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/75C12N1/21C12N11/082C02F3/34C12R1/125C02F101/34
CPCC12N9/0061C12N15/75C12N11/082C02F3/342C12Y110/03002C02F2101/345
Inventor 邱建贺翁雯梁锡伟周国彪易旭超
Owner FOSHAN YUHUANG ECOLOGICAL ENVIRONMENT TECH
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