Transgenic Pichia yeast engineering strain and construction method thereof

Abstract

The invention discloses a transgenic Pichia pastoris engineering strain and a construction method thereof, and aims to provide an engineering strain which can safely perform extracellular secretory expression on phenylmethane dye decolorizing enzyme in a large amount. The technical proposal of the invention comprises that a triphenylmethane dye decolorizing enzyme TpmD gene is cloned from Aeromonas hydrophlla subsp DN322; the triphenylmethane dye decolorizing enzyme TpmD gene is connected with an expression vector, namely a pPICZaA vector to construct a plasmid pPICZaA-tpmD; the constructed plasmid pPICZaA-tpmD is subjected to enzyme digestion linearization and then is led into Pichia pastoris X-33; and the transformation of the triphenylmethane dye decolorizing enzyme TpmD gene into the Pichia pastoris X-33 is determined, thereby obtaining the transgenic Pichia pastoris engineering strain. The transgenic Pichia pastoris engineering strain is mainly used for environmental remediation and treatment.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a genetically modified Pichia pastoris engineering strain and a construction method thereof. Background technique: [0002] Triphenylmethane dyes are a large class of dyes second only to azo dyes and are widely used. Due to their biological toxicity, carcinogenicity, teratogenicity and mutagenicity, they have seriously threatened human health and ecological balance. . Among them, malachite green has attracted widespread attention in the world because it was widely used as an antifungal agent in aquaculture, resulting in residues in water and fish. [0003] In recent years, many studies have been carried out on the physical, chemical and biological treatment methods of printing and dyeing wastewater, and some progress has been made. Physical and chemical methods mainly include flocculation based on inorganic and organic polymer flocculants, ozone, H 2 o 2 and Fenton's...

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Problems solved by technology

The molecular structure of the enzyme contains a heme porphyrin ring, the catalytic activity is strongly inhibited by cytochrome P450 specific inhibitors, and free radicals participate in the catalytic reaction. These three characteristics are very similar to those of P450 monooxygenase, but the spectrum In terms of characteristics, there is no characteristic characteristic absorption peak at 450nm of typical P450 monooxygenase ("Study on the enzymatic characteristics of bacterial decolorizing enzyme TpmD on the decolorization of triphenylmethane dyes", Ren Suizhou, Guo Jun, Wang Yali, Cen Yinghua, Sun Guoping Acta Microbiology, Volume 46, Issue 3, Pages 385-389, April 2006)

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