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Production method for sucrose phosphorylase and application of production method

A sucrose phosphorylase and a technology for producing sucrose, applied in the biological field, can solve the problems of low specific enzyme activity of sucrose phosphorylase, difficulty in meeting industrial applications, poor soluble expression effect, etc., and achieve high yield

Active Publication Date: 2020-01-07
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complex metabolic regulation mechanism in wild-type strains that can produce sucrose phosphorylase, the yield of sucrose phosphorylase produced by this method is not high, and it is difficult to meet the requirements of industrial applications
[0005] Recently, many researchers have tried to use genetic engineering principles to construct recombinant strains that can produce sucrose phosphorylase to increase the production of sucrose phosphorylase. However, the use of these recombinant strains to ferment and produce sucrose phosphorylase still has poor soluble expression. And the disadvantage that the sucrose phosphorylase produced is lower than the enzyme activity

Method used

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  • Production method for sucrose phosphorylase and application of production method
  • Production method for sucrose phosphorylase and application of production method
  • Production method for sucrose phosphorylase and application of production method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Construction of different recombinant bacteria that can produce sucrose phosphorylase

[0062] Specific steps are as follows:

[0063] Chemically synthesize the gene (the nucleotide sequence of the gene is shown in SEQ ID NO.1) of the sucrose phosphorylase encoding aminoacid sequence as shown in SEQ ID NO.2; The gene obtained and pET-20b (+) plasmid pass Ligate after double enzyme digestion (Nco I and Xho I), transform the ligation product into Escherichia coli E.coli BL21(DE3), spread the transformed product on LB solid medium, culture at 37°C for 8-10h, and place on LB solid medium The transformants were picked from above, cultured in LB liquid medium, cultured at 37°C for 10 hours, and the plasmid was extracted. The plasmid was sequenced, and the recombinant plasmid pET20b-SPase and recombinant Escherichia coli pET20b-SPase / E.coli were obtained if the sequencing was correct. BL21.

[0064] The recombinant plasmid pET20b-SPase and plasmid pG-KJE8 (contain...

Embodiment 2

[0068] Embodiment 2: the production of sucrose phosphorylase

[0069] Specific steps are as follows:

[0070] The recombinant Escherichia coli pET20b-SPase / E.coli BL21 obtained in Example 1, the recombinant Escherichia coli pET20b-SPase-pG-KJE8 / E.coli BL21, the recombinant Escherichia coli pET20b-SPase-pKJE7 / E.coli BL21, the recombinant Escherichia coli Bacillus pET20b-SPase-pG-Tf2 / E.coli BL21 and recombinant Escherichia coli pET20b-SPase-pGro7 / E.coli BL21 were spread on LB solid medium and cultured at 37°C for 8-10 hours to obtain a single colony; Put the colony into the LB liquid medium, cultivate at 37°C for 12-14h, and obtain the seed liquid; insert the seed liquid into the LB liquid medium according to the inoculum amount of 1% (v / v), and cultivate at 37°C, 180rpm to OD 600 After reaching 0.8-0.9, add L-arabinose with a final concentration of 0.5g / L, tetracycline with a final concentration of 10ng / mL, and IPTG with a final concentration of 0.5mM in the culture medium, an...

Embodiment 3

[0074] Example 3: Effects of Arabinose and Tetracycline Concentrations on Sucrose Phosphorylase Yield and Specific Enzyme Activity

[0075] Specific steps are as follows:

[0076] Spread the recombinant Escherichia coli pET20b-SPase-pG-KJE8 / E.coli BL21 obtained in Example 1 on LB solid medium and culture at 37°C for 8-10 hours to obtain a single colony; pick a single colony and insert it into LB liquid culture cultured at 37°C for 12-14 hours to obtain seed liquid; the seed liquid was inserted into LB liquid medium according to the inoculation amount of 1% (v / v), and cultivated to OD at 37°C and 180rpm 600 After the concentration is 0.8 to 0.9, add L-arabinose at the final concentrations of 0.25g / L, 0.5g / L, 1.0g / L, and 2.0g / L to the culture medium, and the final concentrations are 5ng / mL and 10ng / mL, respectively. Tetracycline / mL and IPTG with a final concentration of 0.5mM were induced for 12 hours at 16°C and 180rpm to obtain a fermentation broth; the fermentation broth was c...

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Abstract

The invention discloses a production method for sucrose phosphorylase and an application of the production method, and belongs to the field of biotechnology. The invention provides the production method for sucrose phosphorylase. The method produces the sucrose phosphorylase with high yield and high specific enzyme activity. By the method, recombinant escherichia coli is expressed in a fermentation medium containing L-arabinose, tetracycline and IPTG for 12 hours by induction, allowing enzyme activity of the sucrose phosphorylase in a supernatant of cell disruption liquid up to 32.43U / mL, andspecific enzyme activity of the produced sucrose phosphorylase up to 18.5 U / mg.

Description

technical field [0001] The invention relates to a method for producing sucrose phosphorylase and its application, belonging to the field of biotechnology. Background technique [0002] Sucrose phosphorylase (SPase, EC 2.4.1.7) belongs to the GH13 family and mainly exists in Bifidobacterium longum, Leuconostoc mesenteroides and Pseudomonas saccharophila. [0003] Sucrose phosphorylase is active on various substrates and can catalyze various substrates to generate corresponding products. Therefore, it has a wide range of applications in industries such as food, cosmetics and medicine. At present, the application of sucrose phosphorylase mainly includes three aspects: (1) use sucrose as a donor, and use rhamnose, xylose, fructose and galactose as acceptors to catalyze the corresponding oligosaccharides. Oligosaccharides will have one more glucose group, which can be used as an additive in food and medicine; (2) catalyze certain unstable substances to synthesize their stable de...

Claims

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Application Information

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