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Method for improving sucrose phosphorylase expression efficiency by molecular chaperone co-expression

A technology of sucrose phosphorylase and efficiency, applied in the field of molecular chaperone co-expression to improve the expression efficiency of sucrose phosphorylase, to reduce the formation of inclusion bodies, improve enzyme activity, and promote the effect of increasing the soluble expression level and activity of SPase

Active Publication Date: 2019-03-19
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, there has been no report that the co-expression of molecular chaperones improves the expression efficiency of SPase

Method used

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  • Method for improving sucrose phosphorylase expression efficiency by molecular chaperone co-expression
  • Method for improving sucrose phosphorylase expression efficiency by molecular chaperone co-expression
  • Method for improving sucrose phosphorylase expression efficiency by molecular chaperone co-expression

Examples

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Embodiment 1

[0050] Example 1 Construction method of molecular chaperone co-expression system

[0051] 1. Cloning of the sucrose phosphorylase gene

[0052] The sucrose phosphorylase gene was synthesized by a chemical total synthesis method, and its nucleotide sequence is shown in SEQ ID NO.2.

[0053] 2. Construction of recombinant plasmid pET-20b-SPase

[0054] First, pET-20b was double-digested with NcoI and XhoI, and then the synthetic gene fragment was inserted between the two restriction sites of NcoI and XhoI of pET-20b.

[0055] 3. CaCl 2 Preparation of E. coli Competent Cells and Thermal Transformation Steps

[0056] Streak the preserved E.coli BL21 (DE3) glycerol strain on an LB solid plate without an anti-plate, and place the plate upside down in a 37°C incubator for about 12 hours to isolate a single colony. Pick a single colony with neat edges and a moderate size in fresh medium, and culture it in a shaker at 37°C and 180rpm until OD 600 Stop the culture when it is 0.3, p...

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Abstract

The invention discloses a method for improving the sucrose phosphorylase expression efficiency by molecular chaperone co-expression, and belongs to the technical field of bioengineering and enzyme engineering. According to the method, pGro7 expressed molecular chaperone protein can be used for effectively reducing formation of an inclusion body by co-expression of recombinant plasmid pET-20b-SPasewith pGro7, and improvement of SPase soluble expression level and vitality can be promoted. By optimizing the expression condition and using a molecular chaperone co-expression system, the intracellular enzyme activity of SPase can be 24.33U / mL, and the specific enzyme activity can be 6.58U / mg.

Description

technical field [0001] The invention relates to a method for co-expressing molecular chaperones to improve the expression efficiency of sucrose phosphorylase, belonging to the technical fields of bioengineering and enzyme engineering. Background technique [0002] Sucrose phosphorylase (SPase, EC 2.4.1.7) belongs to the GH13 family and is mainly found in Bifidobacterium longum, Leuconostoc mesenteroides, and Pseudomonas saccharophila. Because of its broad substrate specificity, it has been widely used in food, cosmetics and pharmaceutical industries, mainly including three aspects: (1) with sucrose as donor, rhamnose, xylose, fructose and galactose as Acceptors, catalyzed to obtain corresponding oligosaccharides, which will have one more glucose group; (2) substances that can catalyze certain unstable substances to synthesize their stable derivatives, such as sucrose phosphorylase in sucrose Catalyzes the 2-O-α-glucosylation of L-ascorbic acid with high efficiency and excel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12R1/19
CPCC12N9/1051C12N15/70C12Y204/01007
Inventor 韩瑞枝姚栋倪晔肖静王克芬
Owner JIANGNAN UNIV
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