A kind of Escherichia coli producing sucrose phosphorylase

A technology of Escherichia coli and glucose phosphate, applied in the field of microorganisms, can solve the problems of low expression level, low yield of sucrose phosphorylase, difficulty in meeting the requirements of industrial applications, etc.

Active Publication Date: 2021-03-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, sucrose phosphorylase is mainly distributed in bacterial microorganisms, and a small amount is distributed in plant cells. The enzyme is mainly obtained through biological fermentation. The complex metabolic regulation mechanism in wild-type strains makes the yield of sucrose phosphorylase low. Type strains ferment and produce sucrose phosphorylase, which is difficult to meet the requirements of industrial applications
At present, the heterologous expression of sucrose phosphorylase in Escherichia coli and Bacillus subtilis has been reported in the literature, but there are problems such as low expression level and low enzyme activity

Method used

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  • A kind of Escherichia coli producing sucrose phosphorylase
  • A kind of Escherichia coli producing sucrose phosphorylase

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Effect test

Embodiment 1

[0023] The construction of embodiment 1 Escherichia coli SPL02 genetically engineered bacteria

[0024] (1) Gene optimization: Sucrosephosphorylase (GenBank Accession NO.D90314) derived from Leuconostoc mesenteroides ATCC 12291 was analyzed and optimized to obtain the gene sequence shown in SEQ ID NO.1, and two restriction sites NcoI and XhoI were added to The two ends of the optimized sucrose phosphorylase are synthesized to obtain the SPase gene.

[0025] (2) Construction of genetically engineered bacteria: the synthetic SPase gene and pET-28a vector were double-digested with restriction endonucleases NcoI and XhoI, and the products after digestion were connected with Solution I, and then the recombinant vector pET-28a-SPase Transformed into Escherichia coli BL21 (DE3) for expression.

Embodiment 2

[0026] The preparation of embodiment 2 recombinant sucrose phosphorylase

[0027] Inoculate a single colony of the recombinant strain in the LB liquid medium containing Kana, cultivate overnight at 37°C, 200r / min, and insert it into the TB liquid medium containing Kana at 37°C, 200r / min Min culture to bacterial density OD 600 After reaching 0.6, add IPTG inducer and induce at 25°C, 200r / min for 24h, then collect the bacterial liquid. Centrifuge the bacterial liquid in a low-temperature refrigerated centrifuge at 7000 r / min at 4°C for 15 minutes to collect the bacterial cells. Bacteria use 50mmol / L K 2 HPO4 / KH 2 Bacteria were collected after washing twice with PO4 buffer (pH6.5). Add the collected wet bacteria to 50mmol / L K 2 HPO4 / KH 2 PO4 buffer (pH 6.5) buffer solution was used to make a bacterial suspension, placed on ice and fixed, and then the bacterial cells were disrupted by ultrasonic waves. Ultrasonic breaker working time: 2s, intermittent time: 4s, total time: ...

Embodiment 3

[0029] The enzyme activity assay of embodiment 3 recombinant sucrose phosphorylase

[0030]In phosphate buffer, sucrose phosphorylase can catalyze sucrose and inorganic phosphoric acid to generate glucose-1-phosphate and D-fructose. The sucrose hydrolysis reaction can be carried out first, and then the content of the generated D-fructose can be detected by DNS to determine the sucrose phosphate [Choi H.C.,Seo D.H.,Jung J.H.,et al.Developmemt of new assay for sucrosephosphorylase and its application to the characterization of Bifidobacterium longumSJ32 sucrosephosphorylase[J].Food Science and Biotechnology,2011,20(2):513-51 .

[0031] The enzyme activity determination method refers to [Wu Jing, Wu Dan, Zhu Jie, et al. A recombinant Bacillus subtilis expressing sucrose phosphorylase derived from L. mesenteroides. Chinese invention patent application, application number: 201710637427.6, publication number: CN107236696A]. The assay steps include: 500 μL of 5% sucrose solution, 50...

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Abstract

The invention discloses an Escherichia coli producing sucrose phosphorylase, which belongs to the technical field of microorganisms. In the present invention, the nucleotide sequence shown in SEQ ID NO.1 is constructed by using pET-28a as a carrier to construct the recombinant plasmid pET-28a-SPase, which is transformed into Escherichia coli BL21 (DE3), and the recombinant Escherichia coli is constructed and fermented to produce recombinant sucrose phosphorylase, and mutagenized the constructed recombinant bacteria by ultraviolet mutagenesis, and screened out Escherichia coli strains with high production of sucrose phosphorylase. The enzyme activity is 206.91U / mg, and its stable enzyme production can lay the foundation for further theoretical research and practical production application, and the practical application is of great significance.

Description

technical field [0001] The invention relates to an Escherichia coli producing sucrose phosphorylase, belonging to the technical field of microorganisms. Background technique [0002] Sucrose phosphorylase (EC 2.4.1.7, Sucrose phosphorylase, hereinafter referred to as SPase) can mainly catalyze two types of reactions: one is to transfer phosphorylated glucose as a donor to different substances, such as D-fructose as an acceptor Another catalytic method is to transfer the glucose group obtained by decomposing sucrose by sucrose phosphorylase to different types of acceptors, such as inorganic phosphoric acid, substances containing phenolic hydroxyl groups, alcoholic hydroxyl groups and carboxyl groups, and catalyze the synthesis of various glycosides . [0003] Sucrose phosphorylase is mainly distributed in bacteria. According to literature reports, the enzyme mainly exists in Leuconostoc mesenteroides, Stococcus mutans, Pseudomonassaccharophila, Bifidobacterium longum, Bifid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/10C12P19/02C12P19/44C12R1/19
CPCC12N1/20C12N9/1051C12P19/02C12P19/44C12Y204/01007C12N1/205C12R2001/19
Inventor 陈献忠李晓玉沈微
Owner JIANGNAN UNIV
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