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Method for synthesizing sucrose-6-phosphate from recombinant high-temperature-resistant sucrose phosphate synthase

A technology of phosphate synthase and high temperature resistance, which is applied in the field of enzyme engineering and can solve the problems of difficulty in the preparation and purification of sucrose-6-phosphate

Active Publication Date: 2020-03-17
NORTHEAST NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently, sucrose-6-phosphate has not been commercialized worldwide due to the difficulty in its preparation and purification

Method used

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  • Method for synthesizing sucrose-6-phosphate from recombinant high-temperature-resistant sucrose phosphate synthase
  • Method for synthesizing sucrose-6-phosphate from recombinant high-temperature-resistant sucrose phosphate synthase
  • Method for synthesizing sucrose-6-phosphate from recombinant high-temperature-resistant sucrose phosphate synthase

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Embodiment 1

[0059] The preparation of embodiment 1 escherichia coli BL21 (DE3) competent cell

[0060] Shake 4ml of Escherichia coli BL21(DE3) to the exponential growth phase; centrifuge at 10000g for 2min to collect the bacteria; add 1ml of 0.1M calcium chloride to resuspend the cells; centrifuge at 10000g for 2min to collect the bacteria; add 1ml of 0.1M calcium chloride and resuspend Suspend bacteria in ice bath for 45 minutes; heat shock at 42°C for 2 minutes; add LB medium preheated at 37°C and place on a shaker for 1.5 hours; collect bacteria by centrifugation, and spread bacteria on agarose gel containing kanamycin LB board.

Embodiment 2

[0061] Example 2 Cloning of high temperature resistant sucrose phosphate synthase gene, construction of overexpression plasmid, transformation of Escherichia coli BL21 (DE3)

[0062] Insert the gene of high temperature resistant sucrose phosphate synthase (the uniprot database number is tll1590) into the plasmid pET28a, the restriction endonuclease sites are NdeI and XhoI, the end of the cDNA of high temperature resistant sucrose phosphate synthase is added with stop codon TAA, The obtained plasmid was named pET28a_tll1590; 0.1-10 μg of plasmid pET28a_tll1590 was transferred per 0.1 ml of Escherichia coli BL21 (DE3).

Embodiment 3

[0063] Example 3 Induced expression and affinity chromatography purification of high temperature resistant sucrose phosphate synthase

[0064] (1) Pick several Escherichia coli colonies, place them in 10ml LB liquid medium, place them in a shaker for culture, the temperature of the shaker is 37°C, and the speed of the shaker is 200rpm, cultivate overnight; expand to 1L LB liquid medium cultured in a shaker, the temperature of the shaker is 37°C, the speed of the shaker is 200rpm, and cultivated to the exponential growth phase; add 0.1-10mM IPTG to induce protein expression, the induction temperature is 37°C, and the induction time is 16h; the next day Bacteria were collected by centrifugation at 5000g;

[0065] (2) Discard the supernatant, add 50ml bacterial lysate (50mM Tris-HCl, pH 8.0, 150mM NaCl and 20mM imidazole), fully suspend the bacteria, ultrasonically lyse the bacteria for 20min, power 30%, turn on for 3 seconds, turn off 3 seconds;

[0066] (3) Centrifuge at 1200...

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Abstract

The invention discloses a method for synthesizing sucrose-6-phosphate from recombinant high-temperature-resistant sucrose phosphate synthase and belongs to the field of enzyme engineering. The methodcomprises the following steps: cloning a gene of a high-temperature-resistant sucrose phosphate synthase, constructing overexpression plasmids, transforming the overexpression plasmids into Escherichia coli BL21 (DE3), inducing expression of the high-temperature-resistant sucrose phosphate synthase, and performing affinity chromatography purification to obtain a recombinant high-temperature-resistant sucrose phosphate synthase, making the recombinant high-temperature-resistant sucrose phosphate synthase with the concentration of 0.01-10 [mu]g / ml, fructose-6-phosphate with the concentration of0.1-20 mM and uridine diphosphate glucose with the concentration of 0.1-20 mM react at 70 DEG C for 1-24h to obtain sucrose-6-phosphate. The optimal reaction temperature of the recombinant high-temperature-resistant sucrose phosphate synthase is 70 DEG C, the tolerable temperature reaches 100 DEG C, the enzyme activity can be kept at 100 DEG C, generation of sucrose-6-phosphate is catalyzed, and the recombinant high-temperature-resistant sucrose phosphate synthase is suitable for industrial production of sucrose-6-phosphate.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering, and in particular relates to a method for synthesizing sucrose-6-phosphate by using recombinant high-temperature resistant sucrose phosphate synthase. Background technique [0002] Sucrose-phosphate synthase (EC 2.4.1.14) (Sucrose-phosphate synthase, SPS) belongs to GT-B type glycosyltransferase. SPS exists in living organisms such as plants and cyanobacteria. SPS is a key enzyme for the synthesis of sucrose in plants and cyanobacteria, and it is also the rate-limiting enzyme for the synthesis of sucrose. Plants and cyanobacteria can further use sucrose phosphate phosphorylase (SPP) to hydrolyze the phosphate radical on sucrose-6-phosphate to generate sucrose. [0003] At present, due to the difficulty in the preparation and purification of sucrose-6-phosphate, sucrose-6-phosphate has not been commercialized worldwide. Contents of the invention [0004] The purpose of the present ...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12P19/12C12R1/19
CPCC12N9/1066C12N15/70C12P19/12C12Y204/01014
Inventor 苏纪勇李昱颖姚圆
Owner NORTHEAST NORMAL UNIVERSITY
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