Multienzyme complex as well as construction method and application thereof

A technique for building methods, complexes, and applications in chemical instruments and methods, microorganism-based methods, biochemical equipment and methods, etc.

Active Publication Date: 2021-06-01
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on the assembly of backboneless multi-enzyme complexes using SpyTag / SpyCatcher as functional modules is still in its infancy

Method used

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  • Multienzyme complex as well as construction method and application thereof
  • Multienzyme complex as well as construction method and application thereof
  • Multienzyme complex as well as construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0055] Embodiment 1: Construction of engineering bacteria of CvFAP-linker-SpyTag and TLL-linker-SpyCatcher

[0056] All construction processes are based on the basic principle of polymerase chain reaction (PCR).

[0057]In order to construct pET28a-CvFAP-linker-SpyTag (the selected linker is a flexible peptide segment (EAAAK) 2 That is, EAAAK EAAAK), using the seamless cloning method, because the SpyTag gene fragment is very short (only 39bp), so this gene fragment was designed in the primer by insertion mutation, using pET28a-CvFAP as the template, and CvFAP-linker -SpyTag-F / R is used as a primer, and the plasmid pET28a-CvFAP-linker-SpyTag is obtained by PCR amplification. After the PCR product is purified, it is transformed, and after the positive plasmid is obtained, it is sequenced and verified. The accurately sequenced plasmid is stored for future use.

[0058] In order to construct pET28a-TLL-linker-SpyCatcher (here the linker is (GGGGS) 2, That is, GGGGSGGGGS), becaus...

Embodiment 2

[0065] Example 2: Fusion protein expression, purification and characterization

[0066] (1) Transformation and expression of recombinant plasmids: the obtained fusion protein recombinant plasmids pET28a-CvFAP-linker-SpyTag and pET28a-TLL-linker-SpyCatcher were introduced into E.coli BL21(DE3) by heat shock transformation method, and the positive clones obtained After amplified culture, IPTG (final concentrations of 0.5 mM and 0.1 mM, respectively) was added to induce expression, and cultured at 17° C. and 20° C. for 20 h, respectively. After the fermentation process is completed, the TLL-linker-SpyCatcher crude enzyme is purified by affinity chromatography to obtain an enzyme protein with a purity higher than 95%. After the eluted protein is collected, it is concentrated and subpackaged and quickly frozen with liquid nitrogen.- Store at 20°C for later use; in addition, because the catalytic activity of the crude CvFAP enzyme is higher than that of the pure enzyme, the CvFAP-li...

Embodiment 3

[0069] Example 3: Construction of multi-enzyme complex in vitro and optimization of assembly conditions

[0070] (1) Construction of TLL-CvFAP multi-enzyme complex in vitro: mix CvFAP-linker-SpyTag crude enzyme and TLL-linker-SpyCatcher pure enzyme extracellularly in 20mM, pH 7.4 PBS at a molar ratio of 1:1 of the target protein, each The final concentration of the enzyme was 20 μM / L, and stood at 30°C for 12 hours; after the assembly, samples were taken for SDS-PAGE protein electrophoresis, and 30 μL soybean oil was used as the substrate (the density of soybean oil was 0.91 g / mL, and its average molecular weight was 900g / moL), 30°C, 1000rpm, under blue light irradiation, take part of the assembly mixture to catalyze the conversion of soybean oil; after the reaction, fully extract with 1mL ethyl acetate, centrifuge at 10000rpm, room temperature for 4min, take a clear sample of the upper layer for high-efficiency gas chromatography analysis , to detect the formation of the prod...

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Abstract

The invention relates to a TLL-CvFAP multienzyme complex as well as a construction method and application thereof, the construction method comprises the following steps: firstly constructing a TLL-linker-SpyCatcher recombinant protein engineering bacterium and a CvFAP-linker-SpyTag recombinant protein engineering bacterium, then performing expression to obtain a CvFAP-linker-SpyTag protein and a TLL-linker-SpyCatcher protein, mixing the two proteins outside a cell, and performing self-assembly to obtain the TLL-CvFAP multienzyme complex. The TLL-CvFAP multi-enzyme complex is used as a catalyst in the production of alkane (alkene) hydrocarbon from grease, and the catalytic efficiency of cascade reaction and the yield of alkane (alkene) hydrocarbon are improved.

Description

technical field [0001] The invention belongs to the field of biological enzyme catalysis, and in particular relates to a new multi-enzyme complex of photodecarboxylase (CvFAP)-lipase and its construction method and application. Background technique [0002] The reduction of petroleum energy and the continuous deterioration of the environment have accelerated the research process of sustainable development. Developing technologies for producing biofuels from renewable biomass resources and replacing petroleum fuels with biofuels has become an important task of scientific research by relevant researchers in various countries. Among the renewable biomass resources, fats and oils represented by waste catering oil and non-edible oil have received extensive attention in recent years. Fatty acid, as the main component of oil hydrolysis products, only needs to remove one carboxyl group to form various alkanes (alkenes), which is a nearly perfect match with petroleum components. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/70C12N1/21C07K19/00C12P5/00C12R1/19
CPCC12N9/20C12N9/88C12N15/70C12P5/00C12Y401/01C12Y301/01003
Inventor 杨博张细珍马云建王永华吴斌仲宣儒蓝东明
Owner SOUTH CHINA UNIV OF TECH
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