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Decolorizing peroxidase and preparation method and application thereof

A technology of peroxidase and tryptophan, which is applied in the field of artificial metalloenzymes, can solve the problems of long catalysis time and achieve the effects of simple operation, increased catalytic conversion number, and reduced dependence

Active Publication Date: 2018-10-19
NANHUA UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Previous research reports have designed a double-point mutant F43Y / F138W Mb based on the myoglobin (Mb) molecule with the function of transporting oxygen. Although it has significant dye decolorization peroxidase activity, it has a certain amount of hydrogen peroxide (H 2 o 2 ) dependence, if necessary in 50mM H 2 o 2 Concentration, to show significant catalytic activity
Moreover, the catalytic time is relatively long, for example, it takes 100s to completely decolorize 0.1mM RB19

Method used

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  • Decolorizing peroxidase and preparation method and application thereof
  • Decolorizing peroxidase and preparation method and application thereof
  • Decolorizing peroxidase and preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0034]Based on genetic engineering and protein engineering, using site-directed mutation technology, tyrosine was introduced at the 43rd position near the heme active center of myoglobin, tryptophan was introduced at the 138th position, and tryptophan was introduced at the 88th position outside the heme active center at the same time, Furthermore, it was expressed in Escherichia coli BL21(DE3), and separated and purified by ion exchange column (DEAE 52 resin) and gel column (Superdex75) to obtain F43Y / F138W / P88W Mb mutant protein. The protein molecular weight was shown by mass spectrometry ( figure 1 ).

[0035] The amino acid sequence (SEQ ID NO.1) of the F43Y / F138W / P88W Mb mutant is as follows:

[0036] VLSEGEWQLVLHVWAKVEADVAGHGQDILIRLFKSHPETLEKYDRFKHLKTEAEMKASEDLKKHGVTVLTALGAILKKKGHHEAELKWLAQSHATKHKIPIKYLEFISEAIIHVLHSRHPGDFGADAQGAMNKALELWRKDIAAKYKELGYQG.

Embodiment 2

[0038] 7 μM F43Y / F138W / P88W Mb was prepared in 100 mM phosphate buffer (pH 7.0). At the same time, prepare 10mM H with deionized water 2 o 2 and 10mM active blue substance RB19 (Reactive blue, RB19) mother solution, the concentration is calibrated with ultraviolet spectrum (H 2 o 2 for ε 240 nm=39.4m -1 cm -1 , RB19 is ε 595 nm=10mM -1 cm -1 ).

[0039] Take 2 mL of the above F43Y / F138W / P88W Mb solution, add 20 μL of 10 mM RB19 mother solution, mix evenly and place it in the injector C of the rapid dwell spectrometer, and take 10 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 10s, and a total of 500 spectra were collected ( figure 2 ). Monitor the change of RB19 characteristic absorption 595nm absorption peak with time ( figure 2 illustration).

[0040] The results showed that F43Y / F138W / P88W Mb could make the absorption peak of dye RB19 at 595nm...

Embodiment 3

[0042] 7 μM F43Y / F138W / P88W Mb was prepared in 100 mM phosphate buffer (pH 7.0). At the same time, prepare 10mM H with deionized water 2 o 2 and 10mM active blue substance RB19 mother solution (Reactive blue, RB19), the concentration is calibrated with ultraviolet spectrum (H 2 o 2 for ε 240 nm=39.4m -1 cm -1 , RB19 is ε 595 nm=10mM -1 cm -1 ).

[0043] Take two cuvettes of the same specification (clean and dry), add 1mL of the above F43Y / F138W / P88W Mb solution to each, and add 10μL of 10mM RB19 mother solution and mix well. Add 1mL 100mM phosphate buffer to one of the cuvettes, and add 1mL 10mM H to the other cuvette 2 o 2 , the operation requires simultaneous execution. React for 5s and take pictures quickly. The color of the solution before and after the reaction is as follows: image 3 .

[0044] The results showed that F43Y / F138W / P88W Mb could decolorize the dye RB19 within a few seconds (5s).

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Abstract

The invention belongs to the field of manual metalloenzyme and discloses high-efficiency decolorizing peroxidase and a preparation method and application thereof. According to the decolorizing peroxidase disclosed by the invention, myohemoglobin with an oxygen carrying function is utilized as protein molecules to design a framework, tyrosine is introduced to a 43 site near the heme active center of the myohemoglobin, and tryptophan is introduced to a 138 site; meanwhile, tryptophan is introduced to an 88 site at the periphery of the heme active center to obtain three-point mutant protein, andan amino acid sequence of the three-point mutant protein is shown as SEQ ID NO.1. According to the decolorizing peroxidase disclosed by the invention, a catalytic conversion number is improved, and dependence on hydrogen peroxide is reduced; the preparation method of the decolorizing peroxidase has the advantages of simpleness in operation and suitability for large-scale industrial production. Experiments show that the decolorizing peroxidase disclosed by the invention can degrade dye molecules under low-concentration hydrogen peroxide and within several seconds to achieve a decolorizing effect, a catalytic efficiency is increased by 130% compared with that of two-point mutant protein, and the decolorizing peroxidase can be widely applied to pollution treatment of industrial dye wastewater.

Description

technical field [0001] The invention belongs to the field of artificial metalloenzymes, in particular to a decolorizing peroxidase and its preparation method and application, in particular to a high-efficiency artificial decolorizing peroxidase based on a myoglobin three-point mutant and its preparation method and application application. Background technique [0002] Dye-decolorizing peroxidases (DyPs) are a new class of heme-containing peroxidases, which widely exist in bacteria, fungi and other microorganisms, and can be applied to the degradation of various industrial dyes. It has a certain application prospect in the fields of water pollution control and so on. However, it is relatively difficult to obtain natural decolorizing peroxidases, and the catalytic conditions of these biological enzymes are relatively harsh, and they usually have certain biocatalytic functions only under relatively acidic conditions. For example, the decolorizing peroxidase extracted from Vib...

Claims

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Application Information

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IPC IPC(8): C12N9/08C12N15/53C02F3/34C02F101/30
CPCC02F3/342C02F2101/308C12N9/0065C12Y111/01019
Inventor 林英武李乐乐何博张萍
Owner NANHUA UNIV
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