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Nanofiber biological membrane immobilized bi-enzyme system and trehalose catalytic synthesis method thereof

A technology of nanofibers and biofilms, applied in the biological field, can solve the problems of low immobilization efficiency of single-enzyme catalytic systems, lack of competitive advantages in substrates, and low enzyme utilization efficiency, so as to improve immobilization efficiency and utilization efficiency, The effect of reducing the difficulty of separating and purifying trehalose and improving the conversion efficiency

Inactive Publication Date: 2016-08-10
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The technical problem to be solved by the present invention is to solve the technical route and efficiency problem of trehalose synthesis in the existing synthetic system; Hydrolase (MTHase) enzyme utilization efficiency is low; and single enzyme catalytic system (trehalose synthase TreS) immobilization efficiency is not high, the substrate does not have a competitive advantage, and the problem of high separation cost

Method used

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  • Nanofiber biological membrane immobilized bi-enzyme system and trehalose catalytic synthesis method thereof
  • Nanofiber biological membrane immobilized bi-enzyme system and trehalose catalytic synthesis method thereof
  • Nanofiber biological membrane immobilized bi-enzyme system and trehalose catalytic synthesis method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0050] This example illustrates the construction of engineering bacteria co-expressing trehalose synthase gene TreS and amyloid CsgA-SpyTag fusion gene

[0051] The gene sequence of the fusion protein CsgA-SpyTag is based on the CsgA sequence published by NCBI (NCBI Reference Sequence: NC_000913.3) and the reported literature (Nguyen P Q, Botyanszki Z, Tay P K R, et al. Programmable biofilm-based materials from engineered curli In nanofibres[J]. Nature communications, 2014, 5.), SpyTag (polypeptide sequence AHIVMVDAYKPTK) is fused and connected by a flexible linker of 6 amino acid residues GSGGSG.

[0052] Specifically, the constructed strains E. coli BL21(DE3)(pET22b-treS)(Jiang L, Lin M, Zhang Y, et al. Identification and characterization of a novel trehalose synthase gene derived from saline-alkali soil metagenomes[J]. PloS one, 2013, 8(10 ): e77437.) inoculated into LB liquid medium (peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L) at 37°C, 200 rpm for overn...

Embodiment 2

[0064] This example illustrates the construction of engineering bacteria expressing β-amylase-SpyCatcher fusion gene BA-SC.

[0065] The fusion protein BA-SpyCatcher gene sequence is based on the NCBI published β-amylase BA sequence (GenBank: AJ250858.1) and the published SpyCatcher gene sequence (GenBank: JQ478411.1) through the flexible linker fusion of 6 amino acid residues GSGGSG The BA sequence of β-amylase is obtained by whole gene synthesis method.

[0066] According to the β-amylase gene sequence and the SpyCatcher gene sequence, the upstream and downstream primers F1 and R1 (amplification of the β-amylase gene BA), F2 and R2 (amplification of the polypeptide tag SpyCatcher) were designed and synthesized.

[0067] F1:5'-CG GGATCC ATGTTCATTTTGAGTC-3'

[0068] R1: 5'-ACTGCCACCGCCACCGCTACCGCCACCGCCCCAATTTATCTGTATAA-3'

[0069] F2: 5'-ACTGCCACCGCCACCGCTACCGCCACCGCCCCAATTTATCTGTATAA-3'

[0070] R2: 5'-CC CTCGAG TTAAATATGAGCGTCACCTTTAGTT-3'

[0071] Among them, BamH ...

Embodiment 3

[0077] This example illustrates the effect of creating functional nanofibrous biofilms on the surface of trehalose synthase-containing cells

[0078] strains from the plate E. coli BL21(DE3) (PETDuet-TreS-CsgA-ST) was inoculated into 50ml LB medium containing ampicillin resistance, cultured overnight at 37°C, 200rpm / min for 12h; Resistance 50 mL autoinduction medium (glycerol 5g / L, Na2HPO4 6g / L, K2HPO4 3g / L, NH4Cl 1g / L, NaCl 0.5g / L, MgSO4-7H2O 0.5g / L, soybean peptone 10g / L ), 30°C, 180r / min shaking culture to induce expression for 10-12 h, collect the fermentation broth, wash twice with pH 7.0 PBS buffer and resuspend, the enzyme activity of trehalose synthase cells was determined to be 8000U / mL. The Escherichia coli containing trehalose synthase was bound and adsorbed by Congo red solution, and it was observed that Congo red was obviously adsorbed on the surface of the bacterial cells. At the same time, the Escherichia coli that could produce CsgA amyloid fibers was analyz...

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Abstract

The invention provides a nanofiber biological membrane immobilized bi-enzyme system and a trehalose catalytic synthesis method thereof. A starchiness nanofiber biological membrane is arranged on the surface of an escherichia coli cell generating high-temperature-resistant trehalose synthase through fermentation, polypeptide tag SpyTag-SpyCatcher capable of gene coding is used for specificity covalent binding and efficient immobilization recombination of beta-amylase, a trehalose synthase-beta amylase bi-enzyme catalytic system is constructed autonomously, and finally extracellular and intracellular two-step catalysis is conducted continuously. The immobilization efficiency of beta-amylase can reach 50-62%, maltose is generated by 20-25wt% of soluble starch under catalysis of beta-amylase on the extracellular biological membrane, enters the cell and reacts with trehalose synthase to generate trehalose, and the conversion rate of starch can reach 45-55% after the immobilized cell is reused for 10-16 times.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for efficiently preparing trehalose from cheap starchy raw materials catalyzed by Escherichia coli by means of genetic engineering and enzyme engineering. Background technique [0002] Trehalose is widely distributed in bacteria, algae, yeast, lower plants, insects and other invertebrates, and is one of the main oligosaccharides recently developed in the world. Trehalose can effectively maintain the stability of intracellular plasma membrane and protein when cells are in stressful environments such as starvation, dryness, high temperature, freezing, high osmotic pressure and toxic reagents, and can protect genetically engineered enzymes, various viruses, vaccines, Exciting results have been achieved for antibodies, protein factors, nucleic acids, etc. This unique biological function makes trehalose have broad application prospects in food, cosmetics, molecular biology, medici...

Claims

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Application Information

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IPC IPC(8): C12N11/18C12N1/21C12N9/26C12N9/10C12P19/12C12R1/19
CPCC12N9/1051C12N9/2425C12N11/18C12P19/12C12Y204/01245C12Y302/01002Y02P20/50
Inventor 江凌宋小刚黄和唐苏苏
Owner NANJING UNIV OF TECH
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