Escherichia coli engineering bacteria for expressing recombinant sucrose phosphorylase

A sucrose phosphorylase, Escherichia coli technology, applied in recombinant DNA technology, enzymes, bacteria and other directions, can solve the problems of complex process, high cost, low extraction rate, etc., and achieve high purification efficiency, low cost and high specificity. Effect

Inactive Publication Date: 2011-09-07
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the complex process, low extraction rate and high cost of natural SPase extracted directly from wild fungi, finding

Method used

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  • Escherichia coli engineering bacteria for expressing recombinant sucrose phosphorylase
  • Escherichia coli engineering bacteria for expressing recombinant sucrose phosphorylase
  • Escherichia coli engineering bacteria for expressing recombinant sucrose phosphorylase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Construction of Escherichia coli sp1515 genetic engineering bacteria.

[0051] 1. Primer design

[0052] According to the SPase gene sequence (GenBank Accession NO.D90314.1) of Leuconostoc mesenteroides ATCC12291, a pair of primers for amplifying the SPase gene were designed with Primerpremier 5, namely the upstream primer P1 and the downstream primer P2:

[0053] Upstream primer P1: 5'-GC TCTAGA CGCAATCGTTACAGTTATTACTAGC-3';

[0054] Downstream primer P2: 5'-CCC AAGCTT GTTCTGAGTCAAATTATCACTGCTC-3'.

[0055] Xba I and Hind III restriction enzyme sites (underlined) were introduced at the 5' ends of P1 and P2, respectively, and protective bases were added.

[0056] 2. PCR amplification of Leuconostoc enteromenis SPase fragment

[0057] (1) Genomic DNA extraction of Leuconostoc enterolis

[0058] a) Streak and activate Leuconostoc mesenteroides ATCC 12291 stored in glycerol cryopreservation tubes, inoculate a single colony in 5ml MRS medium and culture ov...

Embodiment 2

[0093] Example 2 Expression of Escherichia coli sp1515 genetic engineering bacteria

[0094] 1. IPTG induces the expression of Escherichia coli sp1515 genetically engineered bacteria

[0095] (1) Pick a single colony of the recombinant bacteria and culture it overnight in LB medium, and use the bacterial sample containing the empty vector and E.coli BL21(DE3) as controls;

[0096] (2) The next day, the experimental group and the control group were inoculated into fresh 50mL LB medium according to the volume ratio of 3% inoculum, and cultured at 37°C with shaking at 200r / min;

[0097] (3) Wait until the bacterial solution grows to OD 600 At 0.6-0.8, add the inducer IPTG to a final concentration of 0.5mmol / L, and induce at 30°C for 6 hours;

[0098] (4) The bacteria liquid was centrifuged (4° C., 8000 r / min, 15 min) to collect the bacteria.

[0099] 2. SDS-PAGE electrophoresis detection of expression products

[0100] (1) Wash the expression product collected by the above ce...

Embodiment 3

[0109] The influence of embodiment 3 different induction temperature, different IPTG concentration and different induction time on SPase recombinant protein expression

[0110] The expression conditions of genetically engineered bacteria were optimized through further research on induction temperature, IPTG induction concentration and induction time.

[0111] 1. The influence of different induction temperature

[0112] (1) Pick a single colony of recombinant bacteria and culture it overnight in LB medium;

[0113] (2) The next day, the seed solution was inoculated into fresh 50mL LB medium according to the volume ratio of 3% inoculum, and cultured with shaking at 37°C and 200r / min;

[0114] (3) Wait until the bacterial solution grows to OD 600 At 0.6-0.8, IPTG was added to a final concentration of 0.1 mmol, and then induced at 20°C, 25°C, 30°C, and 35°C for 10 hours. The result is as Figure 5 shown.

[0115] 2. Effect of different IPTG concentrations

[0116] (1) (2) ar...

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Abstract

The invention belongs to the field of biotechnology, disclosing escherichia coli engineering bacteria for expressing recombinant sucrose phosphorylase. Sucrose phosphorylase SPase genes which are derived from leuconostoc mesenteroides ATCC 12291 are introduced into escherichia coli. The invention also discloses a construction method of the escherichia coli engineering bacteria for expressing the recombinant sucrose phosphorylase and application thereof. The escherichia coli engineering bacteria for expressing the recombinant sucrose phosphorylase have the characteristics of convenience of operation, stability for expression, high efficiency, low cost, easiness of purification and the like when used for producing the SPase on a scale.

Description

technical field [0001] The invention relates to the technical field of design biology, in particular to a kind of Escherichia coli engineering bacterium highly expressing recombinant SPase gene. Background technique [0002] Sucrose phosphorylase (EC 2.4.1.7, Sucrose Phosphorylase, SPase), a glucosyltransferase, is a specific enzyme that catalyzes the transfer of glucosidic bonds and belongs to the glycosyl hydrolase 13 family. The enzyme mainly catalyzes two types of reactions: one is to transfer the glucose group in 1-phosphate glucose to the acceptor, such as D-fructose as the acceptor, which can generate sucrose under the catalysis of the enzyme; the other is to transfer the glucose group in sucrose The glucose group is transferred to the receptor, which includes inorganic phosphoric acid, water, substances containing phenolic hydroxyl group, alcoholic hydroxyl group and carboxyl group. If phosphoric acid is used as the receptor, 1-phosphate glucose and D-fructose can be...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/52C12N9/00C12R1/19
Inventor 姜岷隋姗姗马江锋侯顾伟奚永兰陈可泉韦萍欧阳平凯
Owner NANJING UNIV OF TECH
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