Method for increasing activity and stability of microbial surface-displayed organophosphorus hydrolase

A surface display, microbial technology, applied in the fields of enzyme engineering and material science, can solve problems such as activity and stability that are not involved, achieve the effect of improving activity and stability, improving the activity or stability of enzymes, and simple preparation method

Active Publication Date: 2017-06-20
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this study did not involve reports on the interaction of OPH wi

Method used

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  • Method for increasing activity and stability of microbial surface-displayed organophosphorus hydrolase
  • Method for increasing activity and stability of microbial surface-displayed organophosphorus hydrolase
  • Method for increasing activity and stability of microbial surface-displayed organophosphorus hydrolase

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0018] Example 1

[0019] Preparation of microorganisms displaying organophosphorus hydrolase on the surface:

[0020] Preparation method reference: X. Tang, B. Liang, T. Yi, G. Manco, I. Palchetti, A. Liu, Cell Surface Display of Organophosphorus Hydrolase for Sensitive Spectrophotometric Detection of p-Nitrophenol Substituted Organophosphates. Enzyme and Microbial Technology, 2014, 55, 107−112. Specifically, the anchor protein and OPH genes were inserted into the plasmid and transformed into E. coli competent cells ( E. coli BL21), the engineered bacteria were cultured in LB medium containing kanamycin resistance at 37 °C with shaking; when the culture medium had an absorbance at 600 nm (abbreviated as OD 600 nm ) Is about 0.6, add isopropyl-β-d-thiogalactoside to induce the expression of anchoring protein and OPH and display on the cell surface; after shaking culture at 37 °C for 10 hours, centrifugation (rotating speed 6000 rpm, 5 Minutes) Collect the bacteria.

Example Embodiment

[0021] Example 2

[0022] Preparation of microbial-cobalt phosphate octahydrate hybrid material displaying organophosphorus hydrolase on the surface:

[0023] (1) E. coli with organophosphorus hydrolase displayed on the surface (final OD 600 nm = 6) Add to phosphate buffer (pH 7.4) and mix well; (2) Add cobalt chloride aqueous solution (0.5 mM) to the above mixed solution and mix well. After standing at room temperature for one day, the resulting cobalt phosphate salt is uniform Distributed on the surface of microorganisms to form a microorganism-cobalt phosphate octahydrate hybrid material displaying organophosphorus hydrolase on the surface; (3) Centrifuge the above reaction solution to remove the supernatant containing unreacted components, and collect the precipitate at 4°C Keep it for later use. The quantity of materials is E. coli OD 600 nm Remember.

[0024] Do another control experiment: take the unshown E. coli Replacement surface displays organophosphorus hydrolase E. c...

Example Embodiment

[0025] Example 3

[0026] Morphology and elemental analysis of the microbial-cobalt phosphate octahydrate hybrid material with organophosphorus hydrolase on the surface:

[0027] After drying the suspension of the hybrid material at room temperature, its morphology is analyzed by scanning electron microscope and transmission electron microscope. figure 1 A is a scanning electron microscope photo of the hybrid material. It can be seen from the figure that the prepared hybrid material has a spindle-shaped structure with a length of about 1 µm and a diameter of about 450 nm. It basically maintains the microbial cells before biomineralization. size. figure 1 B and C are transmission electron micrographs of the hybrid material. The photos further confirm the spindle-shaped structure of the hybrid material, and it can be seen from the figure that the surface of the hybrid material has a layered structure.

[0028] The above-mentioned hybrid materials were analyzed by X-ray energy spectru...

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Abstract

The invention relates to the fields of enzyme engineering and material science, and discloses a method for increasing activity and stability of microbial surface-displayed organophosphorus hydrolase. The method biomineralizes microbes displaying organophosphorus hydrolase on the surfaces to form a microbe-cobaltous phosphate hybrid material, and the material has higher activity and stability than microbial surface-displayed organophosphorus hydrolase before biomineralization. The method is simple, environment-friendly and effective.

Description

technical field [0001] The invention relates to the fields of enzyme engineering and material science, and specifically includes a method for improving the activity and stability of organophosphate hydrolase displayed on the surface of microorganisms. Background technique [0002] Microbial surface display technology refers to the use of genetic engineering to fuse the target protein (foreign protein or protein domain) with the anchor protein, and use the mutual recognition between the anchor protein and the specific area on the cell surface to express the target protein in the microbial host. And displayed on the surface of the host cell, the displayed target protein still basically retains the original spatial structure and biological function. This technology is often used to display enzymes as target proteins on the cell surface, and has many advantages: Exogenous enzymes anchored on the cell surface can directly interact with substrates in the reaction solution, thereby...

Claims

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Application Information

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IPC IPC(8): C12N1/38
CPCC12N1/38
Inventor 韩磊
Owner QINGDAO AGRI UNIV
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