Enzymatic production of peracids from carboxylic acid ester substrates using non-heme haloperoxidases

一种催化制备、过酸的技术,应用在有机过/无机过化合物组合物、氧化还原酶、植物学设备和方法等方向,能够解决不能满足、过酸浓度降低、过酸不稳定等问题

Inactive Publication Date: 2012-10-10
EI DU PONT DE NEMOURS & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chemical reaction to prepare peracids has several disadvantages: a) the use of high concentrations of carboxylic acids in favor of the preparation of peracids can lead to undesirable odors when using solutions containing peracids, 2) over time Peracids are generally unstable in solution, so the concentration of peracid in solution decreases during storage before use, and 3) the resulting formulation is usually strongly acidic due to the use of concentrated sulfuric acid as a catalyst
However, the concentrations of peracids produced are often insufficient for many commercial disinfectant applications
[0017] Most known methods of preparing peracids from the corresponding carboxylates using enzyme catalysts do not produce and accumulate peracids in sufficiently high concentrations for effective disinfection in a variety of applications

Method used

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  • Enzymatic production of peracids from carboxylic acid ester substrates using non-heme haloperoxidases
  • Enzymatic production of peracids from carboxylic acid ester substrates using non-heme haloperoxidases
  • Enzymatic production of peracids from carboxylic acid ester substrates using non-heme haloperoxidases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0151] Cloning of 5B non-heme haloperoxidase from Pseudomonas putida

[0152] A series of PCR primers were designed based on published non-heme haloperoxidase sequences from Pseudomonas putida strains including KT2440, IF-3 and MR-2068 (respectively, 26991929, 6451702 and 1360922). PCR using genomic DNA from Pseudomonas putida 5B (NRRL-18668) with primer #1 (5-GATCTGG TCATCGTCGCCATGCATCAC-3'; SEQ ID NO: 1) and primer #2 (5'-GCCGA CGACTGGGACGCGCAGATG-3'; SEQ ID NO: 2) and with primer #1 and primer #3 (5'-ATGAGCTACGTCACCACCAAAGATGG-3'; SEQ ID NO: 3) generated approximately 600 bp and 700 bp products, respectively. The nucleotide sequences (SEQ ID Nos: 4 and 5) and the corresponding deduced amino acid sequences of these products were confirmed to be partially identical to non-heme haloperoxidase genes. Genome sequencing with primer #4 (5'-GGCTACGTCGTCGGCATAGTGGTC-3'; SEQ ID NO: 6) revealed the presence of a Pst| restriction site several hundred bp upstream of the non-heme ha...

Embodiment 2

[0154] Expression of Pseudomonas putida 5B non-heme haloperoxidase in Escherichia coli

[0155] The non-heme haloperoxidase from Pseudomonas putida 5B was used with primer #7 (5'-G AATTCATGAGCTATGTAACCACGAAGGACGGC-3'; SEQ ID NO: 11) and primer #8 (5'-GCGGCCGCTTAACTACGGATAAACGCCAGCAAATCCGCAT-3'; SEQ ID NO: 12) was amplified by PCR and subcloned into pTrcHis2- (Invitrogen) and 4- (Invitrogen) to generate expression plasmids pSW167 and pSW169, respectively. In addition, the EcoR| fragment from pSW169 was subcloned into pET-28a (Novagen; Madison, Wl) to generate expression plasmid pSW174. E. coli TOP10 (Invitrogen) was transformed with pSW167 or pSW169 to generate strains TOP10 / pSW167 and TOP10 / pSW169, respectively. E. coli BL21(DE3) (Novagen) was transformed with pSW174 to generate strain BL21 / pSW174. 5B non-heme haloperoxidase was expressed in TOP10 / pSW167, TOP10 / pSW169 and BL21 / pSW174 according to Invitrogen's expression protocol. Basically, at 0D 600 Cells were induc...

Embodiment 3

[0157] Agrobacterium tumefaciens (A. tumefaciens) non-heme haloperoxidase ( 16119616) expression in Escherichia coli

[0158] The non-heme haloperoxidase gene from Agrobacterium tumefaciens C58 ("C58") ( 16119616; also referred to herein as "AtuA1"; SEQ ID Nos: 13-14) was used with primer #9 (5'-ATGG GCTTCGTAACAACCAAGGACGGCAC-3'; SEQ ID NO: 15) and primer #10 (5'-TCAGCCCTTGATGAAGGCTAGCAGGTCCTG-3 '; SEQ ID NO: 16) was PCR-amplified and subcloned into 4- (Invitrogen) to generate the pSW166 plasmid. In addition, the EcoR| fragment from pSW166 was subcloned into pET-28a (Novagen) to generate expression plasmid pSW175. E. coli TOP10 (Invitrogen) was transformed with pSW166 to generate strain TOP10 / pSW166. E. coli BL21(DE3) (Novagen) was transformed with pSW175 to generate strain BL21 / pSW175. Pass C58 non-heme haloperoxidase at OD 600 Expression in TOP10 / pSW166 and BL21 / pSW175 was induced with 1 mM IPTG at =0.5-0.6 for 3 hr at 37°C with shaking. SDS-PAGE analysis co...

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Abstract

The invention relates to an enzymatic production of preacids from carboxylic acid ester substrates using non-heme haloperoxidases. A process is provided for producing peroxycarboxylic acids from carboxylic acid esters. More specifically, carboxylic acid esters are reacted in situ with an inorganic peroxide in the presence of a non-heme haloperoxidase having perhydrolysis activity to produce peroxycarboxylic acids.

Description

[0001] This application is a branch of the invention patent application PCT / US2006 / 039395 with the filing date of October 6, 2006 and the title of the invention being "Preparation of Peracid Catalyzed by Carboxylate Substrate Enzyme Using Non-heme Haloperoxidase". Case application, the Chinese patent application number of the original application is 200680037213.5. [0002] This application claims priority to US Provisional Application No. 60 / 724,106, filed October 6, 2005. technical field [0003] The invention relates to the field of peracid biosynthesis and in situ enzyme catalysis. Specifically, peracids are prepared from carboxylate substrates using non-heme haloperoxidases with perhydrolytic activity. Background technique [0004] Peracid compositions have been reported to be effective antimicrobial agents. Methods for cleaning, sterilizing and / or disinfecting hard surfaces, meat products, living plant tissue and sanitary equipment against the growth of undesired mic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/40C12N15/53C12N9/02A01N37/16A01P1/00
CPCA01N37/16C12P7/40C11D3/3945C11D3/2093C07C409/26C11D1/667C11D3/38654C07C409/24C12P7/00C11D3/38636C11D3/39A01N37/02A01N37/12A01N37/46A01N59/00A01N63/50A01N2300/00
Inventor R·迪科西莫M·S·佩恩L·沃纳J·E·加瓦根
Owner EI DU PONT DE NEMOURS & CO
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