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A high-temperature polyphenol oxidase and its application in the treatment of phenolic wastewater

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

Active Publication Date: 2021-11-26
玉凰生态科技股份有限公司
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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 temperature resistance, and polyphenols in current applications normal temperature enzymes
In addition, the expression level of naturally occurring polyphenol oxidase is low, and its catalytic performance is usually for the stress response or nutritional needs of microorganisms, and the enzyme activity is low. Therefore, in order to improve the application value of polyphenol oxidase, it is necessary to discover Gene resources, and random or directional enzyme engineering optimization, so as to develop industrial-grade recombinant polyphenol oxidase that is easy to immobilize and enhance catalytic performance

Method used

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  • A high-temperature polyphenol oxidase and its application in the treatment of phenolic wastewater
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  • A high-temperature polyphenol oxidase and its application in the treatment of phenolic wastewater

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1 Mutation mutation of polyphenol oxidase and screening of polyphenol oxidation enzyme mutants

[0035] 1) Splicing of polyphenol oxidase gene

[0036]Since the environmental soil sample macro base is a DNA template, the primer is expanded using a polyphenol oxide primer, and the primers are SEQ ID NO.1 and SEQ ID NO. 2, and the product after amplification of PCR is agarose. Glue electrophoresis, the strip size is a DNA product cutting rubber between 1200-2000 bp, and uses the kit for DNA recovery, and then uses molecular biology techniques to connect the intercepted PCR product cell (DNA Pool) to Thermo Fisher PET101 TOPO. In the plasmid system, then transformed into E. coli BL21-DE3, the coating, overnight growth, obtained resistant positive clones; picked 20 large coli orders, inoculated into 3 ml of liquid medium, using overnight Cultured E. coli cells were extracted by plasmid. The extracted plasmid was extracted from positive clones by XMAI. The obtained posit...

Embodiment 2

[0043] Example 2 recombinant expression of Bacillus subtilis of polyphenol oxide enzyme

[0044] In order to reduce the expression and purification cost of polyphenol oxidase, foreign proteins can generally be introduced into the microbial host cell to secrete expression, and the commonly used secreted bacteria expressions are mainly subtilis. To this end, a codon optimization is performed on the DNA sequence SEQ ID NO. 3 encoding polyphenol oxidase YHL21 to obtain an optimized YHL21 gene sequence such as SEQ IDNO.5. The DNA fragment fuses with the PXYLA promoter fragment of Bacillus recombinant expression to obtain a complete expression reading frame (SEQ ID No.6). Sequence shown in SEQ ID NO.6 is synthesized by gene, and then via ECO Ri After the enzyme digestion was inserted into the plasmid PMK4, the expression plasmid pMK-YHL21 was obtained, and the sequence SEQ ID NO. 7 was obtained. Constructed recombinant expression plasmid transformed to Bacillus subtilis Bacillus Subtili...

Embodiment 3

[0045] Example 3 Recombination of recombinant polyphenol oxide enzymes, degradation of artificial phenolic wastewater

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Abstract

The invention discloses a polyphenol oxidase YHL21 gene and polypeptide sequence. The polyphenol oxidase is confirmed by gene splicing and high-throughput screening of enzyme performance. It has a wide range of temperature resistance and strong high-temperature catalytic performance. Single peptide construction, easy membrane immobilization and other advantages. The present invention also discloses a method for recombinant expression and secretion of polyphenol oxidase YHL21 in Bacillus subtilis strain 168, and a method for using polyvinylidene fluoride (PVDF) membrane to immobilize recombinant polyphenol oxidase on the membrane surface , the membrane-enzyme composite system can stably and efficiently degrade organic substances such as phenol and p-chlorophenol in aqueous solution. The present invention carries out enzyme engineering optimization through gene splicing, and obtains a highly efficient polyphenol oxidase that degrades phenolic pollutants, uses Bacillus subtilis to carry out recombinant secretion expression, and uses PVDF to carry out enzyme immobilization, and improves the enzyme catalytic efficiency.

Description

Technical field [0001] The present invention belongs to the field of environmental biotechnology, and more particularly to a high temperature polyphenol oxidase and its application in wastewater treatment of phenolic contaminants. Background technique [0002] Polyphenol oxidase is a copper-containing ionic oxidase which is first isolated from the active substance that cures the polyphenol oxidation, and is also referred to as polyphenol oxidation enzyme (laccase). Polyphenol oxidase can be synthesized from various cell synthesis, plants, animals, bacteria and fungi can produce polyphenol oxidases, and its oxidized substrate object is very broad spectrum, more than hundreds of different structural or type of complex organic matter, so It is widely used in the fields of food, paper, textile, industrial catalysis, pollutant treatment. [0003] In the food industry, polyphenol oxidase can be used to treat the phenomenon of aromaine and phenolic substances in alcoholic juice, improve...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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 玉凰生态科技股份有限公司
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