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Preparation method of crosslinking halohydrin dehalogenase aggregate

A halohydrin dehalogenase, aggregate technology, applied in biochemical equipment and methods, halocarbon lyase, immobilized on/in organic carriers, etc. possible, low fixing efficiency, etc., to achieve the effect of high reuse rate, low cost, and high mechanical strength

Inactive Publication Date: 2016-10-26
UNIV OF ELECTRONICS SCI & TECH OF CHINA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] As we all know, one of the main problems in the field of enzyme application is: the lack of immobilized enzymes that are stable in a wide range of pH and temperature and have high tolerance to organic solvents; and the cost of reagents and carriers used for immobilization is high and the immobilization efficiency On the low side, enzymes with industrial application value are limited when they are actually put into industrial applications
[0006] At present, there is no general method applicable to the immobilization of all enzymes, and the changes in the activity and stability of immobilized enzymes are usually contradictory, that is to say, the activity is improved while the lower stability is exchanged. And it is almost impossible to recycle and reuse; on the contrary, if the stability is improved, the activity will be limited

Method used

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  • Preparation method of crosslinking halohydrin dehalogenase aggregate
  • Preparation method of crosslinking halohydrin dehalogenase aggregate
  • Preparation method of crosslinking halohydrin dehalogenase aggregate

Examples

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Embodiment 1

[0040] A method for preparing cross-linked halohydrin dehalogenase aggregates, comprising the following steps:

[0041] Step A: Prepare the crude enzyme solution for halohydrin dehalogenation; specifically, the crude enzyme solution is prepared by constructing recombinant Escherichia coli, and the steps are as follows:

[0042] A1: Transfer the recombinant halohydrin dehalogenase gene into the host cell E.coli MC1061 to obtain the recombinant strain E.coliMC1061, such as figure 1 Shown is a schematic diagram of the recombinant expression of the recombinant halohydrin dehalogenase gene;

[0043] (1) Preparation of competent state:

[0044] Under sterile conditions, the host bacteria E.coli MC1061 was streak-grown on solid LB medium and cultured overnight at 37°C upside down. Pick a single clone from the bacterial plate, inoculate it into fresh LB liquid medium, and culture it on a shaker at a temperature of 37°C and a speed of 180rpm for about 5 hours. 600 =0.3~0.6, take 1ml...

Embodiment 2

[0056] Effect of different concentrations of precipitant (ammonium sulfate solution) on the enzyme activity of cross-linked halohydrin dehalogenase aggregates:

[0057] In this example, the following reaction system was adopted, and the concentration of ammonium sulfate solution was used as a single variable to complete five parallel experiments of immobilized halohydrin dehalogenases:

[0058] Under the condition of ice bath, take five groups of 1ml of haloalcohol dehalogenation crude enzyme solution with a concentration of 20mg / ml, add 20mg of bovine serum albumin (BSA) powder and stir for 15 minutes, slowly add 5ml to the five groups of mixed solutions respectively Concentration is 50%, 60%, 70%, 80%, 90% ammonium sulfate solution, then stirs for 1 hour, then slowly adds glutaraldehyde dropwise so that the concentration of glutaraldehyde in the solution is 1%, crosslinks for 2 hours, and prepares Cross-linked halohydrin dehalogenase aggregates were obtained.

[0059] In th...

Embodiment 3

[0061] Effects of different immobilization temperatures on the activity of cross-linked halohydrin dehalogenase aggregates

[0062] In this example, the following reaction system was adopted, and three sets of parallel experiments of immobilized halohydrin dehalogenases were completed with the immobilization temperature as a single variable:

[0063] At the three temperatures of 0°C, 15°C and 30°C, take 1ml of halohydrin dehalogenation crude enzyme solution with a concentration of 20mg / ml, add 20mg of bovine serum albumin (BSA) powder and stir for 15 minutes, and then separately Slowly add 5ml of ammonium sulfate solution with a concentration of 70%, and after stirring for 1 hour, slowly add glutaraldehyde dropwise so that the concentration of glutaraldehyde in the solution is 1%. Enzyme aggregates.

[0064] In this embodiment, the enzyme activity of the three groups of cross-linked halohydrin dehalogenase aggregates prepared above is determined by using the method of measuri...

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Abstract

The invention discloses a preparation method of a crosslinking halohydrin dehalogenase aggregate, and belongs to the field of biological chemical engineering. The preparation method comprises the following steps: adding a bovine serum albumin preparation serving as a protecting agent into coarse halohydrin dehalogenase liquid, and preparing the crosslinking halohydrin dehalogenase aggregate from an ammonium sulfate solution serving as a precipitant and glutaraldehyde serving as a crosslinking agent, wherein the coarse halohydrin dehalogenase liquid is prepared by building recombinant bacteria for prokaryote external induction expression. The preparation method is simple in experimental operation and low in cost; the prepared crosslinking halohydrin dehalogenase aggregate is higher in mechanical strength and heat resistance and can be repeatedly used; an experiment shows that the immobilized enzyme amount can be over 80 percent of the total enzyme amount under the optimal preparation condition; the activity of the crosslinking halohydrin dehalogenase aggregate is 470.2 U / g; compared with the enzyme activity of free coarse enzyme, the relative enzyme activity is up to 91.36 percent, so that the crosslinking halohydrin dehalogenase aggregate can be widely applied to degrading of environment pollutants such as organic halide and catalysis in a synthesis process of chiral drugs.

Description

technical field [0001] The invention belongs to the field of biochemical industry, and specifically discloses a preparation method of a cross-linked halohydrin dehalogenase aggregate. Background technique [0002] With the rapid development of modern social industry, a large amount of improper discharge of industrial waste, and the wide application of artificially synthesized halides in chemical synthesis and agriculture, organic halides have become one of the important environmental pollutants today. In nature, organic halides mainly exist in soil and groundwater, and have strong stability. Most xenogeneic halides have poor natural degradation ability and can exist in nature for hundreds of years. The treatment effect of traditional physical and chemical methods is often low, and it is easy to cause secondary pollution. Therefore, the application of biodegradation of such compounds has become a research hotspot at home and abroad. [0003] Biodegradation refers to the proc...

Claims

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Application Information

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IPC IPC(8): C12N11/02
CPCC12N9/88C12N11/02C12Y405/01
Inventor 汤丽霞廖茜冯娟仝亚沛邓文凤辛东民
Owner UNIV OF ELECTRONICS SCI & TECH OF CHINA
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