Escherichia coli for producing sucrose phosphorylase

A technology of Escherichia coli and glucose phosphate, applied in the field of microorganisms, can solve the problems of low expression, difficult to meet the requirements of industrial applications, and low enzyme activity.

Active Publication Date: 2019-11-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 strain...

Method used

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  • Escherichia coli for producing sucrose phosphorylase
  • Escherichia coli for producing sucrose phosphorylase

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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 escherichia coli for producing sucrose phosphorylase and belongs to the technical field of microorganisms. A nucleotide sequence shown as SEQ ID NO.1 is used for constructing arecombinant plasmid pET-28a-SPase with pET-28a as a vector, the recombinant plasmid is transformed into escherichia coli BL21(DE3), and recombinant escherichia coli is constructed. Recombinant sucrose phosphorylase is produced by fermentation, the recombinant escherichia coli strain with high yield of sucrose phosphorylase is screened out by adopting an ultraviolet mutagenesis method to mutagenize the constructed recombinant escherichia coli, the enzyme activity of a supernatant on the broken walls in fermentation cells is 819.16 U/mL, the specific enzyme activity is 206.91 U/mg, the stable enzyme production can lay a foundation for further theoretical research and practical application, and practical application is significant.

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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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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