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Engineering escherichia coli capable of efficient soluble expression of 4-alpha-glycosyltransferase

A technology of glycosyltransferase and Escherichia coli, which is applied in the field of genetic engineering and microbial engineering, can solve the problem of not realizing the high-efficiency expression of 4-α-glycosyltransferase

Active Publication Date: 2018-11-23
NANJING FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] However, the expression of 4-α-glycosyltransferase in the genetically engineered bacteria constructed in the above research is still at a very low level, and they have not realized the high-efficiency expression of 4-α-glycosyltransferase. Therefore, how to realize 4-α-glycosyltransferase - The industrial production and high-efficiency production of α-glycosyltransferase still needs further research

Method used

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  • Engineering escherichia coli capable of efficient soluble expression of 4-alpha-glycosyltransferase
  • Engineering escherichia coli capable of efficient soluble expression of 4-alpha-glycosyltransferase
  • Engineering escherichia coli capable of efficient soluble expression of 4-alpha-glycosyltransferase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Construction of recombinant strain E.coli BL21(DE3) / pET24-4GT

[0040] 1. Codon optimization and synthesis of 4-α-glycosyltransferase coding gene

[0041] The amino acid sequence of 4-α-glycosyltransferase can be obtained from NCBI database, and the sequence number is WP_004067291.1.

[0042] According to the codon preference of Escherichia coli, the codon-optimized 4-α-glycosyltransferase coding nucleotide (DNA) sequence was artificially designed and synthesized, and after sequencing verification, the target gene fragment was obtained (synthesized at both ends) Nde I and Xho I restriction sites were added to facilitate gene cloning).

[0043] The amino acid sequence of 4-α-glycosyltransferase is shown in SEQ ID NO.1, and its optimized nucleotide sequence is shown in SEQ ID NO.2.

[0044] 2. Construction of recombinant plasmids

[0045] The synthesized gene fragment was double-digested with Nde I and Xho I, and after gel recovery, it was ligated with the p...

Embodiment 2

[0055] Example 2: Construction of recombinant strain E.coli BL21(DE3) / pET24-4GT / pGro7

[0056] 1. Construction of recombinant strains containing different molecular chaperone plasmids

[0057] Five chaperone plasmids pG-KJE8, pGro7, pKJE7, pG-Tf2, and pTf16 (purchased from Takara Company) were introduced into competent cells E.coli BL21(DE3), and they were respectively constructed to contain pG-KJE8, pGro7, and pKJE7 , pG-Tf2, pTf16 plasmids of five E.coli BL21 (DE3) host bacteria, respectively named as E.coli BL21 (DE3) / pG-KJE8, E.coliBL21 (DE3) / pGro7, E.coli BL21 (DE3 ) / pKJE7, E.coli BL21(DE3) / pG-Tf2, E.coliBL21(DE3) / pTf16.

[0058] Then these five kinds of host bacteria were prepared into competent cells according to the preparation method of Escherichia coli competent cells; then the recombinant plasmid pET24a-4GT was introduced into E.coli BL21(DE3) competent cells containing five molecular chaperones Among them, E.coli BL21(DE3) / pET24-4GT / pG-KJE8, E.coli BL21(DE3) / ...

Embodiment 3

[0063] Example 3: Construction of recombinant strain E.coli BL21(DE3) / pETduet-GroES-GroEL-4GT

[0064] 1. Construction of recombinant plasmid pETduet-4GT

[0065] The pET24-4GT was double-digested with Nde I and Xho I, and after gel recovery, it was ligated with the purified pETduet-1 vector (purchased from Novagen) that was digested with the same two enzymes and gel recovered.

[0066] The ligation product was transformed into E. coli E.coli JM109 competent cells, and the transformed competent cells were spread on LB-agar plates containing 100 μg / mL ampicillin resistance, and cultured at 37°C overnight; pick the transformants and transfer them to LB medium medium, shake culture at 37°C for 8 hours, and extract the plasmid; the recombinant plasmid was verified by enzyme digestion and gene sequencing, confirming that the recombinant vector pETduet-4GT was correct.

[0067] The pETduet-1 vector contains two T7 promoters, 4GT is located downstream of one of the T7 promoters, and...

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Abstract

The invention discloses an engineering escherichia coli capable of efficient soluble expression of 4-alpha-glycosyltransferase and belongs to the technical field of genetic engineering and microbial engineering. The engineering escherichia coli capable of efficient soluble expression of 4-alpha-glycosyltransferase in the invention takes pETduet-1 carrier or pET24a(+) carrier as an expression vector and takes E.coli BL21(DE3) strain as a strain of expression host while expressing 4-alpha-glycosyltransferase originating from thermococcus litoralis and stress and chaperon GroES-GroEL; the engineering escherichia coli capable of efficient soluble expression of 4-alpha-glycosyltransferase of the invention is cultured through fermentation for 24 h to produce a fermentation broth containing enzymes with an activity of as high as 82.2 U / mL.

Description

technical field [0001] The invention relates to an Escherichia coli engineering bacterium capable of efficiently and solublely expressing 4-alpha-glycosyltransferase, belonging to the technical fields of genetic engineering and microbial engineering. Background technique [0002] 5-α-Glycosyltransferase is a multifunctional enzyme belonging to the family of amylolytic enzymes. Since 4-α-glycosyltransferase has unique catalytic properties, it can catalyze four reactions of cyclization, disproportionation, hydrolysis and coupling, so it has a wide range of uses in the production of modified starch and macrocyclodextrin. [0003] For example, in terms of starch modification, Cho K.H. et al. used 4-α-glycosyltransferase derived from Thermus hydrophyta to treat rice starch. The results showed that after the enzyme treatment, the retrogradation phenomenon of starch was greatly improved. Inhibition, HaV.Do et al. used 4-α-glycosyltransferase from Thermus aquaticus to prepare starc...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P19/04C12R1/19
CPCC12N9/1048C12N15/70C12P19/04C12Y204/00
Inventor 段绪果张心怡沈镇炎周烽华苏二正
Owner NANJING FORESTRY UNIV
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