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Dehalogenated peroxidase and preparation method and application thereof

A dehalogenated peroxide and reaction technology, applied in the field of ultra-high-efficiency artificial dehalogenated peroxidase and its preparation, to achieve the effect of improving affinity and mild reaction conditions

Active Publication Date: 2018-11-13
NANHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In previous research reports, a single point mutant F43Y Mb was designed, and its overall catalytic efficiency was 115 times that of wild-type myoglobin, but it was only 9 times higher than that of natural DHP under the same conditions.

Method used

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  • Dehalogenated peroxidase and preparation method and application thereof
  • Dehalogenated peroxidase and preparation method and application thereof
  • Dehalogenated peroxidase and preparation method and application thereof

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Experimental program
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Embodiment 1

[0035] Based on genetic engineering and protein engineering, using site-directed mutagenesis technology, tyrosine was introduced at position 43 near the heme active center of myoglobin, and histidine at position 64 was mutated into aspartic acid at the same time. Expressed in BL21(DE3), and separated and purified by guanidine hydrochloride denaturation dialysis, ion exchange, column chromatography and other methods to obtain F43Y / H64D Mb mutant protein. The protein molecular weight was shown by mass spectrometry ( figure 1 ).

[0036] The amino acid sequence (SEQ ID NO.1) of F43Y / H64D Mb is as follows:

[0037] MVLSEGEWQLVLHVWAKVEADVAGHGQDILIRLFKSHPETLEKYDRFKHLKTEAEMKASEDLKKDGVTVLTALGAILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISEAIIHVLHSRHPGDFGADAQGAMNKALELFRKDIAAKYKELGYQG

Embodiment 2

[0039] 4 μM F43Y / H64D Mb was prepared in 100 mM phosphate buffer (pH 7.0). Meanwhile, prepare 1 mM H with deionized water 2 o 2 , the concentration was calibrated by UV spectroscopy (H 2 o 2 for ε 240 nm=39.4m -1 cm -1 ). In addition, 100 mM 2,4,6-trichlorophenol (TCP) stock solution was prepared with 50% ethanol solution.

[0040] Take 1 mL of the above 4 μM F43Y / H64D Mb solution in a dry cuvette (1 cm), add 2 μL of 100 mM TCP to make the final concentration 0.1 mM, and then add 1 mL of 1 mM H 2 o 2 , start the reaction, react for 120s, collect a spectrum every 5s ( figure 2 ), where the absorbance of the product at 272nm varies with time see figure 2 illustration.

[0041] Depend on figure 2 It can be seen that the absorption peaks of the substrate TCP at 244nm and 311nm gradually disappeared, and the absorption peak of the product at 272nm gradually increased, and within 90 seconds, the substrate TCP was completely transformed into the product DCQ.

Embodiment 3

[0043] Prepare 2μM F43Y / H64D Mb and H64D Mb respectively with 100mM phosphate buffer (pH 7.0) (J.Xu, O.Shoji, T.Fujishiro, T.Ohki, T.Ueno and Y.Watanabe, Catal.Sci.Technol .,2012,2,739-744.), F43Y Mb (F.Liao,B.He,K.-J.Du,S.-Q.Gao,G.-B.Wen,Y.-W.Lin.ChemistryLetters , 2016, 45(9):1087-1089), WT Mb. At the same time, prepare 20 mM H with deionized water 2 o 2 , the concentration was calibrated by UV spectroscopy (H 2 o 2 for ε 240 nm=39.4m -1 cm -1 ), and prepare 10 mM 2,4,6-trichlorophenol (TCP) stock solution with 50% ethanol solution.

[0044] Take 2mL of the above three protein solutions respectively, add 12μL of 10mM TCP mother solution, mix evenly and place it in the injector of the rapid dwell spectrometer C, and take 20mM H 2 o 2 2mL was placed in the D injector. The catalytic reaction was carried out after injecting and mixing samples from C and D injectors, and the reaction time was 3.5s. The variation of the characteristic absorption peak at 272 nm of the p...

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Abstract

The invention belongs to the field of artificial metalloenzyme and discloses an efficient dehalogenated peroxidase and a preparation method and application thereof. The dehalogenated peroxidase utilizes myohemoglobin having an oxygen delivery function as a protein molecule design framework, the site 43 nearby the active center of myohemoglobin protoheme are mutated into tyrosine, meanwhile histidine at the site 64 is mutated into acidic aspartic acid, accordingly double-point mutant protein F43Y / H64DMb having a catalytic function is established, and its amino acid sequence is shown as SEQ ID NO.1. The artificial dehalogenated peroxidase can catalyze and oxidize halophenols into low-toxicity quinone products under the condition of low H2O2 concentration within several seconds, achieves a dehalogenation effect and greatly improves the affinity of the double-mutant protein to the halophenols under the situation that the catalytic conversion number is similar, its catalytic efficiency is obviously improved compared with a single-point mutant protein, and accordingly the dehalogenated peroxidase can be widely applied to treatment of halogenated phenol type organic pollutants.

Description

technical field [0001] The invention belongs to the field of artificial metalloenzymes, in particular to a dehalogenase and its preparation method and application, in particular to a super-efficient artificial dehaloperoxidase based on a myoglobin double-point mutant and its Preparation method and application. Background technique [0002] With the rapid development of modern industry and agriculture, halophenols have been widely used as agricultural chemicals and biocides. However, the good water solubility of halophenols also makes them highly biologically toxic to the liver and immune system of mammals. Moreover, free radicals produced by halogenated phenols can modify nucleic acid bases and cause DNA damage, which has been regarded as a class of potential carcinogens. These compounds are listed as priority organic pollutants (Priority Pollutants) by the US Environmental Protection Agency (USEPA). Therefore, it is of great significance to effectively control halogenate...

Claims

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Application Information

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IPC IPC(8): C12N9/08C12N15/53C12N15/70C02F3/34C02F101/12
CPCC02F3/342C02F2101/12C12N9/0065C12N15/70C12Y111/01
Inventor 林英武尹露露何博高淑琴
Owner NANHUA UNIV
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