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Azo red dye decolorized enzyme and preparation method and application thereof

A dye decolorization and azo red technology, which is applied in the field of artificial metalloenzymes, can solve the problems of poor catalytic effect of Amaranth, and achieve the effects of improving catalytic efficiency, reducing dependence and mild reaction conditions

Active Publication Date: 2019-07-16
NANHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004] Previous research reports have designed a three-point mutant F43Y / F138W / P88W Mb based on the myoglobin (Mb) molecule. Although it has significant dye decolorization peroxidase activity, it has certain limitations in the catalytic range. , such as under the condition of pH 7, the catalytic effect on the azo dye Amaranth is poor

Method used

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  • Azo red dye decolorized enzyme and preparation method and application thereof
  • Azo red dye decolorized enzyme and preparation method and application thereof
  • Azo red dye decolorized enzyme and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Based on genetic engineering and protein engineering, using site-directed mutagenesis, tyrosine was introduced at position 43, tryptophan at position 138, and arginine at position 67 near the active center of myoglobin heme, and then in Escherichia coli BL21(DE3) Expressed in , and separated by ion exchange column (DEAE 52 resin) and gel column (Superdex 75) to separate and purify the protein to obtain the F43Y / F138W / T67R Mb mutant protein.

[0040] The amino acid sequence (SEQ ID NO.1) of the F43Y / F138W / T67R Mb mutant is as follows:

[0041] VLSEGEWQLVLHVWAKVEADVAGHGQDILIRLFKSHPETLEKYDRFKHLKTEAEMKASEDLKKHGVRVLTALGAILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISEAIIHVLHSRHPGDFGADAQGAMNKALELWRKDIAAKYKELGYQG.

Embodiment 2

[0043] 10 μM F43Y / F138W / T67R Mb was prepared in 100 mM phosphate buffer (pH 7.0). Meanwhile, prepare 6 mM H with deionized water 2 o 2 and 100mM amaranth (Amaranth) mother solution, the concentration is demarcated with ultraviolet spectrum (H 2 o 2 for ε 240nm =39.4M - 1 cm -1 , Amaranth is ε 521nm =13.6mM -1 cm -1 ).

[0044] Take 2 mL of the above F43Y / F138W / T67R Mb solution, add 2.4 μL of 100 mM Amaranth mother solution, mix evenly and place it in the C sampler of the rapid dwell spectrometer, and take 6 mM H 2 o 2 2mL was placed in the D injector. The catalytic reaction was carried out after sample injection and mixing by C and D injectors, the reaction time was 100s, and a total of 500 spectra were collected ( figure 1 ). Monitor the change of Amaranth characteristic absorption 521nm absorption peak with time ( figure 2 ).

[0045] The results showed that F43Y / F138W / T67R Mb could make the absorption peak of the dye Amaranth at 521nm almost disappear with...

Embodiment 3

[0047] 5 μM F43Y / F138W / T67R Mb was prepared in 100 mM phosphate buffer (pH 7.0). Meanwhile, prepare 200 mM H with deionized water 2 o 2 and 100mM amaranth mother solution (Amaranth), the concentration was demarcated by ultraviolet spectrum (H 2 o 2 for ε 240nm =39.4M - 1 cm -1 , Amaranth is ε 521nm =13.6mM -1 cm -1 ).

[0048] Take two cuvettes of the same specification (clean and dry), add 2 mL of the above F43Y / F138W / T67R Mb solution to each, and add 1.2 μL of 100 mM Amaranth mother solution and mix well. Then add 30 μL 200mM H to the cuvette 2 o 2 , the operation requires simultaneous execution. React for 100s and take pictures. The color of the solution before and after the reaction is as follows: image 3 .

[0049] The results show that F43Y / F138W / T67R Mb can completely decolorize the dye Amaranth in about 100s (note: the color of the solution after the reaction is the color of F43Y / F138W / T67R Mb protein, not the color of the dye).

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Abstract

The invention belongs to the field of artificial metalloenzyme, and discloses an efficient azo red dye decolorized enzyme and a preparation method and application thereof. According to the azo red dyedecolorized enzyme, myoglobin having the function of transporting oxygen is used as a protein molecule design framework, tyrosine is introduced to the 43rd site near the active center of myoglobin hemachrome, tryptophan is introduced to the 138th site, arginine is introduced to the 67th site, three-point mutant protein F43Y / F138W / T67R Mb is obtained, and the amino acid sequence is as shown in SEQID NO.1. The azo red dye decolorized enzyme disclosed by the invention can degrade azo red dye molecules under low hydrogen peroxide concentration and neutral condition so that decolourizing effects can be achieved; and the azo red dye decolorized enzyme can be widely applied to pollution control of industrial dye wastewater, and a method which is easy to implement, efficient and harmless to environment is provided for degradation of azo type dyes. The preparation method of the azo red dye decolorized enzyme is simple to operate, and is suitable for large-scale industrialization production.

Description

technical field [0001] The invention belongs to the field of artificial metalloenzymes, in particular to an azored dye decolorizing enzyme and its preparation method and application, in particular to a high-efficiency artificial azored dye decolorizing enzyme based on myoglobin three-point mutant and its preparation Methods and applications. Background technique [0002] The world produces more than 1 million tons of dye wastewater every year, of which azos account for about 70%. Azo dyes are the most common synthetic colorants in the textile, paint, varnish, paper, plastic, food, pharmaceutical and cosmetic industries. But azo cleavage products (aromatic amines) are carcinogenic. [0003] The molecular design of artificial decolorizing peroxidase is particularly important for understanding the structure-function relationship of natural enzymes and preparing artificial metalloenzymes with potential applications. Dye-decolorizing peroxidases (DyPs) are a new class of heme ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/08C12N15/53C12N15/70C02F3/34
CPCC02F3/342C12N9/0065C12N15/70
Inventor 林英武张萍何博高淑琴
Owner NANHUA UNIV
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