Carboxylesterase D-1CarE3 cell surface display system and whole-cell catalyst as well as preparation method and application of whole-cell catalyst

A whole-cell catalyst and cell surface technology, applied in biochemical equipment and methods, methods based on microorganisms, bacteria, etc., can solve the problems of limited popularization and application, complicated collection, limited enzyme production ability of wild strains, etc., and achieve the preparation method and Ease of use and broad application prospects

Inactive Publication Date: 2014-06-25
YUNNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Microbial degradation of pesticide residues mostly depends on the enzymatic hydrolysis of intracellular enzymes, but wild strains have limited enzyme p

Method used

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  • Carboxylesterase D-1CarE3 cell surface display system and whole-cell catalyst as well as preparation method and application of whole-cell catalyst
  • Carboxylesterase D-1CarE3 cell surface display system and whole-cell catalyst as well as preparation method and application of whole-cell catalyst
  • Carboxylesterase D-1CarE3 cell surface display system and whole-cell catalyst as well as preparation method and application of whole-cell catalyst

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: Construction of pET28NCD3 plasmid

[0023] Alicyclobacillus D-1 genomic DNA extraction: CTAB lysis method, the specific steps are: centrifuge the fresh bacterial liquid cultured in liquid for 12 hours, and add 800 μL solution Ⅰ (20mM Tris, pH8.0, 2mMNa 2 -EDTA, final concentration 20mg / mL lysozyme), keep warm at 37℃ for 30min, add 100μL 10% SDS and mix upside down, add 10μL 10mg / mL proteinase K, keep warm at 37℃ for 30min, add 150μL 5MNaCl and 150μL 10%CTAB solution and mix upside down Homogenize, keep warm at 65°C for 20min, add an equal volume of phenol / chloroform / isopropanol (volume ratio 25:24:1) for extraction, centrifuge at 12000rpm at 4°C for 10min, take the supernatant in chloroform / isopropanol (volume Ratio 24:1) to extract again to remove impurity proteins, centrifuge at 12000rpm at 4°C for 10min, then take the supernatant and add 2 times the volume of absolute ethanol and 1 / 10 volume of pH5.23M NaAc, precipitate at -70°C for 1h, 4°C Centrifuge ...

Embodiment 2

[0034] Embodiment 2: the preparation of engineering bacteria BL21 (DE3) / NCD3 whole cell catalyst

[0035] Take the engineered bacteria BL21(DE3) / NCD3 and the empty plasmid Escherichia coliBL21(DE3) strain containing only pET-28a(+), and inoculate the LB culture medium containing 50 μg / mL kana resistance at a volume ratio of 0.1% In this method, shake rapidly at 37°C for 12 hours, and then inoculate the activated bacterial solution into fresh LB induction culture medium with an inoculum of 1% volume ratio, and culture with shaking at 37°C for 3-4 hours, until the cell density OD600=0.5 or so, Add a final concentration of 0.05mMIPTG to induce culture at 20°C for 20 hours, centrifuge at 12,000rpm for 5 minutes, collect the bacteria, and freeze them into dry powder in an ultra-low temperature freezer vacuum dryer, which is the whole-cell catalyst D-1CarE3.

[0036] For the separation of cell components, take an appropriate amount of the above-mentioned centrifuge to collect the ce...

Embodiment 3

[0038] Embodiment 3: the application of engineering bacteria BL21 (DE3) / NCD3 whole cell catalyst

[0039] Weigh 1 mg of the whole-cell catalyst D-1CarE3 described in Example 2 and add 100 μl of 100 mg / ml cyfluthrin in acetone solution to shake for 60 minutes at room temperature, add an equal volume of n-hexane for extraction, centrifuge to take an appropriate amount of the upper layer and put it on a silica gel G plate. Thin-layer chromatography analysis, developed with n-hexane: acetone volume ratio 4:1 as the developing agent, naturally air-dried silica gel G plate, fumigated with bromine steam for 5 seconds, sprayed with 0.1% benzylidine acetone solution, and after air-dried, irradiated with ultraviolet light for 2 minutes to develop color ( image 3 ), the results show that the whole-cell catalyst D-1CarE3 can catalyze the pyrethroid pesticide cyfluthrin to form two products 1 and 2, which can be used in the treatment of pyrethroid pesticide pollution.

[0040] Put the wh...

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Abstract

The invention discloses a carboxylesterase D-1CarE3 cell surface display system and a whole-cell catalyst as well as a preparation method and an application of the whole-cell catalyst. A construction method of the surface display system comprises the following steps: constructing a prokaryotic expression vector pET28NCD3 by utilizing an NC end of synthetic truncated ice nucleoprotein of source pseudomonas syringae as ankyrin to be fused with target protein carboxylesterase D-1CarE3, and converting the prokaryotic expression vector into escherichia coli BL21 to construct escherichia coli cell surface display engineering bacterium BL21/pET28NCD3 of the carboxylesterase D-1CarE3. Under the effect of a strong promoter T7, through IPTG inducible expression, the display of carboxylesterase D-1CarE3 cell surface activity is successfully realized, and the whole-cell catalyst D-1CarE3 is prepared by using an ultra-low temperature freeze drying method. The whole-cell catalyst D-1CarE3 can be applied to the treatment of environmental pollution of pesticide residues.

Description

technical field [0001] The invention belongs to the field of bioengineering technology and analysis technology, and relates to a carboxylesterase D-1CarE3 cell surface display system, a whole-cell catalyst and its preparation method and application. Background technique [0002] Carboxylesterases (Carboxylesterases, EC3.1.1.1) are a class of non-specific esterases that can catalyze the hydrolysis of carboxylic esters to generate carboxylic acids and alcohols. They are widely found in animals, plants, and microorganisms to participate in life metabolism activities. The enzyme belongs to the α / β hydrolase family, and has a Ser-Asp-His triplet structure catalytic active center. Carboxylesterase derived from microorganisms is an important industrial enzyme, which has important application value in catalyzing ester hydrolysis, ester synthesis, transesterification, etc., and has good regioselectivity and stereoselectivity. These unique properties make carboxylesterase have broad ...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21A62D3/02C12R1/19A62D101/04A62D101/28A62D101/26
Inventor 黄遵锡谢振荣丁俊美唐湘华李俊俊许波杨云娟周峻沛
Owner YUNNAN NORMAL UNIV
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