Mutation coprinus cinereus peroxidase and preparing method and application thereof

A peroxidase and ghost umbrella technology, which is applied in the fields of genetic engineering and environment, can solve the problems of narrow pH range of high activity, difficult to adapt to the needs of high temperature reaction, etc., and achieve the effect of enhanced stability

Inactive Publication Date: 2016-03-30
NANYANG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the optimum reaction temperature of CIPmt6-2 is only 32°C, and the pH range of high activity is narrow, so it is difficult to meet the high temperature reaction requirements in industrial wastewater treatment.

Method used

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  • Mutation coprinus cinereus peroxidase and preparing method and application thereof
  • Mutation coprinus cinereus peroxidase and preparing method and application thereof
  • Mutation coprinus cinereus peroxidase and preparing method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0029] Molecular directed evolution of mutant Coprinus cinerea peroxidase, including:

[0030] 1) Synthesis of CIPmt4 gene

[0031] Use DNAworks3.1 software to design and optimize the primers for gene assembly, search for the DNA sequence of CIP in genebank (Genebank accession number: X69457), translate it into amino acid sequence (as shown in SEQIDNO: 1), replace the 49th, 53rd, 166 and 242 amino acids, the parameter selection is: primer length 40bp, Pichia pastoris codon preference, melting temperature of the overlapping region 60°C, knocking out the internal SacI site, adding SacII and EcoRI restriction enzymes at both ends site; after entering the software, a series of oligonucleotide primers with a length of about 40 bp are obtained, and 50 primers with an overlapping region of no less than 12 bp, a difference of melting temperature of no more than 2 °C, and the least repetition and mismatch are selected as primers for gene synthesis (CIP1~CIP50 are as shown in SEQIDNO:4...

Embodiment 2

[0045] Preparation and purification of mutant Coprinus cinerea peroxidase, including:

[0046] 1) Massive expression of mutant enzymes

[0047] Inoculate CIPmt5 / GS115 into a 20mL vial containing 5mL of BMGY medium for overnight culture, transfer to a 500mL Erlenmeyer flask containing 100mL of BMGY medium, seal with 4 layers of gauze, and culture overnight on a shaker at 30°C (to OD 600 2 to 6), the cells were collected by centrifugation and resuspended in BMMY medium to adjust the OD 600 10, cultured on a shaker at 29°C, and added methanol every 24 hours to a final concentration of 0.5% to maintain induction;

[0048] 2) Purification of protein

[0049] Collect 20 mL of the culture cultured for 120 h, centrifuge twice at 10,000 r / min to take the supernatant, put it into a dialysis bag, and concentrate it to 1.5 mL with PEG-4000; use Qiagen’s Ni-NTASpincolumn to purify the protein, and refer to the kit manual for operation : Take 2 Ni-NTA centrifugal adsorption columns for e...

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Abstract

The invention discloses mutation coprinus cinereus peroxidase and the preparing method and application thereof and belongs to the technical fields of genetic engineering and the environment. CIPmt4 is synthesized by means of a gene synthesis site-specific mutagenesis platform, error-prone PCR and resistance screening are conducted three times to obtain the coprinus cinereus peroxidase CIPmt5 containing five amino acid mutations (I49S, V53A, T121A, M166F and Y272F), and the amino acid sequence is shown in SEQ ID NO:3. The optimal reaction temperature of the mutation coprinus cinereus peroxidase is increased to 45 DEG C from 25 DEG C corresponding to the wild type, the optimal reaction pH is increased to 6.5 from the original 5.0, the high-activity reaction pH becomes larger, stability is improved, and the mutation coprinus cinereus peroxidase is more suitable for industrialized application. Except methylene blue, the optimal pH of the mutation coprinus cinereus peroxidase CIPmt5 for degradation of dye amino black, methyl orange, Congo red, aniline blue, crystal violet and bromophenol blue tends to be neutral and alkali, and the degradation ability on the five kinds of dye is remarkably higher than that of wild-type enzyme.

Description

technical field [0001] The invention relates to a mutant Coprinus cinerea peroxidase, and also relates to a preparation method and application of the mutant Coprinus cinerea peroxidase, which belongs to the field of genetic engineering and environmental technology. Background technique [0002] With the development of the dyeing and weaving industry, a large amount of dyes are discharged into the surrounding environment in the form of wastewater, which seriously damages the ecosystem. The treatment of toxic dye wastewater has become a problem that plagues environmental protection. Azo dyes in the dyeing and weaving industry have complex aromatic ring structures, are stable to physical and chemical factors, heat and light, and are difficult to be biodegraded. Currently used dye wastewater treatment methods mainly include physical, chemical and biological methods. The cost of physical and chemical methods is relatively high, and the structure of the dye is not destroyed, but ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N1/21C02F3/34C12R1/19
CPCC02F3/342C12N9/0065C12Y111/01007
Inventor 董冰雪张磊方金涛张伟石建州李玉英夏敏
Owner NANYANG NORMAL UNIV
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