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Production method of halide alcohol dehalogenase

The technology of a halohydrin dehalogenase and a production method, which is applied in the field of bioengineering, can solve the problem of low activity of the halohydrin dehalogenase, and achieve the effects of increasing yield and improving enzyme activity.

Inactive Publication Date: 2012-08-15
ENZYMEWORKS
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to overcome the problems of low halohydrin dehalogenase activity in the prior art, and provide a kind of enzyme activity and yield higher, production Lower cost method for producing halohydrin dehalogenase

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  • Production method of halide alcohol dehalogenase

Examples

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specific Embodiment approach

[0025] A specific embodiment of halohydrin dehalogenase produced by the present invention is:

[0026] (1) Construct genetically engineered bacteria and prepare shake flask seed medium and fermentation medium.

[0027] (2) Activate the slant of the genetically engineered bacteria for 24 hours, then inoculate the activated genetically engineered bacteria into a 300 mL shake flask with a seed medium filling volume of 50 mL, and inoculate at 37°C at 220 r / The rotation speed of min was oscillating for 10 hours, and then the above-mentioned oscillating culture solution was inserted into the secondary shaking flask at an inoculum volume ratio of 25% according to the volume ratio of the inoculum / shaking flask seed medium, and then oscillating for 10 hours under the same conditions to obtain Seed liquid; two times of shaking flask culture is just to expand the culture amount of the strain, which is a routine operation.

[0028] (3) Put the seed liquid after shaking culture i...

Embodiment 1

[0033] Under other conditions being the same, this example uses three common carbon sources instead of glycerol as the carbon source in the fermentation medium to compare the effects of different carbon sources on the enzymatic activity of the produced halohydrin dehalogenase (see Table 1). The results showed that when lactose, sucrose and glucose were used as carbon sources, the enzyme activity was low when the addition amount was 0.6% (v / v), and the enzyme activity did not increase significantly or even decreased when the addition amount increased to 5.0%.

[0034] Table 1 The effect of carbon source on bacterial growth and enzyme production

[0035]

Embodiment 2

[0037] Glycerol was selected as the carbon source of the fermentation medium. Under other conditions being the same, three organic nitrogen sources including yeast powder and four inorganic nitrogen sources were used as the nitrogen sources of the fermentation medium respectively. The moles are equal to compare the effects of different nitrogen sources on the enzymatic activity of the produced halohydrin dehalogenase (see Table 2). It can be seen from Table 2 that when yeast powder is used as nitrogen source, the enzyme activity is as high as 545.7U / mL, while the biomass is only 1.78g / L; when peptone and beef extract are used as nitrogen source, although the unit enzyme activity is higher than Inorganic nitrogen sources, but the bacteria grow too fast, and a large amount of nutrients are consumed in the increase of biomass; while using inorganic nitrogen sources, although the enzyme production capacity of the bacteria is high, the total enzyme content is not high due to the s...

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Abstract

The invention relates to a production method of a halide alcohol dehalogenase, which utilizes microbial fermentation to generate halide alcohol dehalogenase, and enhances the output and the activity of the halide alcohol dehalogenase trough optimal fermentation process and fermentation medium. The production method provided by the invention utilizes a genet engineering strain to express recombined escherichia coli of the halide alcohol dehalogenase, the fermentation medium is prepared from 9-15g / L of yeast powder, 4.5-7.5g / L of glycerol, 8-11g / L of NaCl, 1-2g / L of NaNO3, 2-3g / L of KH2PO4, 12-13g / L of K2HPO4 and 2-5mg / L of trace element, and the trace element comprises CoCl12.6H2O, MnCl2.4H2O, CuCl2.4H2O, biotin and the like. The production method provided by the invention can obviously enhance the output of the halide alcohol dehalogenase, and the enzymatic activity can be enhanced to 900U / ml.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a method for fermenting and producing halohydrin dehalogenase with genetically engineered bacteria. Background technique [0002] Organohalogen compounds have become one of the important environmental pollutants in today's society, mainly due to industrial waste and the wide application of artificially synthesized halides in chemical synthesis and agriculture. In nature, most xenobiotic halides have poor self-degradation ability, and many compounds are suspected to be carcinogenic or highly mutagenic substances. Therefore, the application of microorganisms to degrade organic halides has attracted widespread attention. Since Castro first discovered the Flavobacterium strain that uses 2,3-dibromopropanol as the only carbon source in 1968, people have successively screened a variety of microorganisms that can degrade o-halohydrins, including those isolated from freshwater ...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12R1/19
Inventor 谢磊郑飞剑张秀兰李斌
Owner ENZYMEWORKS
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