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Preparation method and application of EDDS lyase immobilized enzymes

A technology of immobilizing enzymes and lyases, applied in biochemical equipment and methods, immobilized on or in inorganic carriers, immobilized on/in organic carriers, etc., can solve the problem of reducing the synthesis efficiency and transformation capacity of EDDS. problems such as poor enzyme activity, high conversion rate, and improved operability are achieved.

Active Publication Date: 2020-07-28
TAIZHOU UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition to EDDS lyase, microbial cells also contain high fumarase activity, which can convert fumaric acid, one of the substrates of the reaction, into malic acid, which will greatly reduce the synthesis efficiency of EDDS, and the conversion ability is poor

Method used

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  • Preparation method and application of EDDS lyase immobilized enzymes
  • Preparation method and application of EDDS lyase immobilized enzymes
  • Preparation method and application of EDDS lyase immobilized enzymes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Escherichia coli BL21 (DE3) / pTZU-27 (genetically engineered bacteria with EDDS lyase of His-tag) single colony was inoculated in a 15mL test tube, the plasmid pTZU-27 contained the nucleic acid sequence SEQ ID NO.1, and 30 μg / mL ampicillin 5ml-LB, cultured at a constant temperature of 37°C, after overnight culture, inoculated 1mL cells in 40mL TB medium (2.4% yeast extract, 1.2% trypsin, 0.4% glycerol, 17mmol / L K 2 PO 4 、72mmol / L KH 2 PO 4 ), and 50mg / mL ampicillin was added to a 500mL flask. After 4 hours, the culture was transferred to 2.0L TB medium in a 3L fermenter. After 600 reaches about 1, lower the temperature to 25°C, then add 150mL 20% (w / v) lactose solution to induce culture, keep the air flow at 1.0vvm, adjust the stirring speed to keep the dissolved oxygen (DO) above 20%, At the end of 20 hours of fermentation, centrifuge the fermentation broth at 10000×g, store the cells at -20°C, suspend 30 grams of cells in 270mL water, homogenize the bacterial su...

Embodiment 2

[0037] Take 1 mL of the crude enzyme solution obtained from the cultivation in Example 1 above and 1 mL of Binding / wash buffer (50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 20 mM imidazole, pH 8.0), mix them evenly, and flow through them with 2 mL of Binding / wash buffer in advance. Buffer equilibrated column prepacked with 1mL Ni-IDA resin;

[0038] Then wash the column with 2mL Binding / wash buffer; then, elute with 2mL Elution Buffer (50mM sodium dihydrogen phosphate, 300mM sodium chloride, 500mM imidazole, pH=8.0), collect the eluate to obtain the corresponding purified enzyme liquid.

[0039] The standard EDDS lyase activity detection method for the above-mentioned purified enzyme solution can be used as follows: take 500uL enzyme solution, add 1.0mL reaction substrate solution (50mmol / L tris, 50mmol / L sodium dihydrogen phosphate; 600mmol / L fumaric acid; 300mmol / L ethylenediamine hydrochloride; pH8.0), the temperature is controlled at 30°C, the rotation speed...

Embodiment 3

[0044] Take 20g of LX-1000HA resin, wash with 100mL of deionized water and 100mL of 100mmol / L sodium phosphate buffer solution with a pH of 8.0, filter and dry, then add 100mL of 100mmol / L with a pH of 8.0 containing 2.0wt% glutaraldehyde Sodium phosphate buffer solution, the temperature was controlled at 30° C., and the rotation speed was 200 rpm for 2 hours, the resin was recovered, washed 3 times with deionized water, and the activated LX-1000HA resin carrier was obtained.

[0045] Take 20ml of the purified enzyme liquid obtained by the method of Example 2 above, add 1.0g of glutaraldehyde-activated amino carrier, control the temperature at 30°C, and carry out immobilization treatment at 200rpm for 1 hour. During the fixation process, take 20uL of the clear liquid and add 80uL Coomassie Brilliant Blue solution (25mg G250, 12.5mL ethanol, 25mL 85% phosphoric acid, after dissolving, add deionized water to 250mL) to detect protein residues, and control residues by gradually inc...

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Abstract

The invention relates to a preparation method and application of EDDS lyase immobilized enzymes, and belongs to the technical field of biology. In order to solve existing problems of being poor in purity and low in conversion rate, the invention provides a preparation method of the EDDS lyase immobilized enzymes. The method comprises the steps of performing fermentation and coarse extraction on EDDS lyase genetic engineering bacteria having His-tag to obtain EDDS lyase coarse enzyme liquid; enabling the coarse enzyme liquid to be in contact with a metal compatible carrier, enabling EDDS lyaseto be adsorbed onto the metal compatible carrier, and performing purification; eluting the EDDS lyase on the metal compatible carrier to obtain purified enzyme liquid; and immobilizing the enzyme liquid and the immobilized carrier to obtain EDDS lyase immobilized enzymes having His-tag for synthetizing (S,S)-EDDS by using fumaric acid and ethylenediamine as substrates. According to the preparationmethod disclosed by the invention, fumarase in the enzyme liquid can be effectively removed, the efficient purification effect can be achieved, and the preparation method has efficient enzyme activity capacity and high reutilization rate.

Description

technical field [0001] The invention relates to a preparation method and application of an EDDS lyase immobilized enzyme, belonging to the field of biotechnology. Background technique [0002] Ethylenediamine disuccinic acid (Ethylenediamine N,N'-disuccinic acid, EDDS, CAS: 2084691-7) is a natural bio-sourced amino polycarboxylic acid chelating agent, widely used in detergents, cosmetics and heavy metal contaminated soil remediation . The molecular structure of EDDS contains two chiral centers and has three isomers (S,S)-EDDS, (R,S)-EDDS and (R,R)-EDDS, of which only (S,S)-EDDS Can be completely biodegradable. [0003] At present, (S,S)-EDDS is mainly produced by chemical methods, and there are many reports on the synthesis of (S,S)-EDDS by biological methods. For example, in 1984, nishikiori et al. isolated (S,S)-EDDS from fungus culture. EDDS, ENDO, etc. use aspartate ammonia lyase to synthesize R,S-EDDS. More reports are using microbial EDDS lyase to catalyze the synt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/08C12N11/10C12N11/14C12N9/88
CPCC12N9/88C12N11/08C12N11/10C12N11/14C12Y403/02001
Inventor 杨仲毅陶宇翔陈良明汪怡璐
Owner TAIZHOU UNIV
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