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

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...

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

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