Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for preparing stable zymoprotein ring through precise regulation and control assembly

An enzyme protein and stabilization technology, applied in the direction of enzyme stabilization, oxidoreductase, recombinant DNA technology, etc., can solve the problems of unpredictability and lack of ligand, and achieve the effect of separation, good catalytic activity and stability

Pending Publication Date: 2021-12-17
HANGZHOU NORMAL UNIVERSITY
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a major drawback to overcome in precisely controlling protein assembly is that protein surfaces do not provide complex ligands that can interact and always assemble in unpredictable ways

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for preparing stable zymoprotein ring through precise regulation and control assembly
  • Method for preparing stable zymoprotein ring through precise regulation and control assembly
  • Method for preparing stable zymoprotein ring through precise regulation and control assembly

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] A method of preparing a stable enzyme protein regulation ring assembly precision, includes the following steps:

[0037] (1) E. coli (MG1655) becomes host gene expression induced keto reductase, and the cells were harvested by centrifugation to obtain the precipitate is centrifuged 8000 rpm for the number of revolutions, time of 5min, and washed with PBS buffer (0.02mol·L -1 , PH 7.0) was washed precipitate; pellet was resuspended in PBS and lysed by sonication cell, wherein an amount of PBS was added 1 / 5 volume of the original cell suspension, ultrasonication using an ice bath, 400W power, crushing time 10min, wherein each of 10s set stop 7S broken 3s; soluble and insoluble fraction after cell disruption by centrifugation, also, the number of revolutions of 10000 rpm for centrifugal, time 15min, such isolated cell homogenate supernatant;

[0038] (2) the crosslinking agent bis alkyne (5,6,11,12-tetrahydro-dibenzo [a, e] cyclooctene was dissolved in isopropanol, at a concen...

Embodiment 2

[0044] Two mutant AKR incorporated unnatural amino acid sites of regulation of the morphology of enzyme protein assembly

[0045] The three-dimensional view AKR gene, the active site of the gene is Asp53, Tyr58, Lys84 and His117, select the appropriate mutation sites need to avoid the active site and the active site of NADP +. The present invention is selected six sites away from the active site and the active site, and then obtained 2.5 AKR different spatial distance mutants, a three-dimensional map of the mutation site mutants such two AKR figure 2 Distance figure 2 A is the active site of the gene is Asp53, Tyr58, Lys84 and His117, figure 2 B-F, respectively, represent the distance between the AKR-114Y-189Q, AKR-198Y-232W, AKR-3Y-31Y, AKR-3Y-189Q, three-dimensional representation gene AKR-3Y-232W, and the two mutation sites. AKR points based on different mutants, under microwave radiation, to help with the crosslinking agent diacetylene AKR two mutants, crosslinked morphology o...

Embodiment 3

[0050] Obtained by different methods annular assembly

[0051] Concentration on the introduction of functions and mechanisms of protein covalently assembled in AKR-114-189, can be easily assembled into a rod, and a linear strip. Using different concentrations of enzyme formed SEM AKR-114-189 assembly, TEM and CLSM as FIG. Figure 4 Shown, respectively, when using six different concentrations (5.02mg · mL -1 , 16.04mg · mL -1 , 32.16mg · mL -1 , 40.20mg · mL -1 , 60.13mg · mL -1 ) Of the enzyme protein AKR-114-189 covalent crosslinking assembly, with increasing protein concentration, are also more likely mutant AKR form a ring ( Figure 4 A, 4B, 4C, 4D). When considering the increased protein concentration, increased chance of collision between molecules, a greater chance of cyclization, resulting in formation of a protein loop. Thus, depending on the enzyme protein was added to an unnatural amino acid azide specific covalent binding can regulate and control the behavior of the prote...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Sizeaaaaaaaaaa
Login to View More

Abstract

The invention relates to a method for preparing a stable zymoprotein ring through a precise regulation and control assembly. The method comprises the following steps that by taking aldo-keto reductase as model protein, 4-azido-L-phenylalanine is inserted at a fixed point to obtain two mutants of the aldo-keto reductase; then, under the action of microwaves, a diacetylene crossing agent induces covalent assembly of the target enzyme protein of a cell lysis solution; and the morphology of the zymoprotein assembly is regulated and controlled by controlling the protein concentration, the mutation site and the dosage of the cross-linking agent in the cell lysis supernatant, and the cyclic cross-linked zymoprotein is obtained. According to the method disclosed by the invention, the cyclic assembly of zymoprotein is formed only by using the cell lysis solution by utilizing the biological orthogonal chemical specific connection between non-natural amino acid molecules in the protein and cyclooctyl diacetylene. the zymoprotein ring obtained by the invention shows strong catalytic activity, thermal stability and digestion stability in simulated gastric juice and intestinal juice, so that the application of the zymoprotein ring in catalysis and drug delivery materials becomes possible.

Description

Technical field [0001] The present invention relates to a method for assembling the ring enzyme protein, particularly to a process for preparing stabilized enzyme protein assembling precision regulation loop. Background technique [0002] Nature presents a remarkable complexity of assembly system, these systems composed of various materials, in particular all kinds of biological macromolecules, such as proteins. This inspired the ambitions scientists to explore and control of biological macromolecules assembly process in the lab to use as nano-materials, bio-catalyst mixed, host tissue engineering and drug delivery agents. At the molecular level, from the lower to the nanostructures assemble into an organized system will have new features. Protein is a member of the living body extremely versatile, since they have a complex topology and wide range of functions in nature. In addition, the inherent bioavailability, less toxicity, biodegradability, non-antigenic, high nutritional va...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N9/96C12N9/04C12N15/70
CPCC12N9/96C12N9/0006C12N15/70
Inventor 王安明章鹏飞张静
Owner HANGZHOU NORMAL UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com