Strain for producing D-psicose 3-epimerase and application thereof

A technology of epimerase and psicose, applied in the field of bioengineering, can solve problems such as difficult separation, pollution, and low amount of existence

Pending Publication Date: 2022-04-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] As a rare sugar, D-psicose exists in a very small amount in nature, and it is not realistic to extract it directly
At the same time, chemical synthesis will generate by-products, causing separation difficulties, greatly increasing production costs, and complex reaction processes will produce excessive pollution

Method used

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  • Strain for producing D-psicose 3-epimerase and application thereof
  • Strain for producing D-psicose 3-epimerase and application thereof
  • Strain for producing D-psicose 3-epimerase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Construction of recombinant Bacillus subtilis containing different promoters

[0054] The specific operation steps are as follows:

[0055] (1) Acquisition of D-psicose 3-epimerase gene: the artificially synthesized nucleotide sequence is shown in SEQ ID NO.1.

[0056] (2) Acquisition of different promoter genes: the promoters used are hag, ylbP, hagP43, ylbPylbP, haghag, amyE, aprE, gsiB, HpaII, nprE, sigX, P43hag, ylbPhag, hagylbP, ylbPP43, P43ylbP, P43P43; The nucleotide sequences are respectively shown in SEQ ID NO.2 to SEQ ID NO.18.

[0057] Among the single promoters, except for HpaII, which uses the PMA5 plasmid as a template for cloning, other single promoters can be amplified by PCR using B.Subtilis168 as a template, and double promoters can be amplified by fusion PCR using a vector containing a single promoter as a template. increase.

[0058] (3) The gene of D-psicose 3-epimerase and the gene of the promoter are fused by fusion PCR, and then the...

Embodiment 2

[0060] Example 2: Fermentative production of D-psicose 3-epimerase by recombinant Bacillus subtilis containing a promoter

[0061] Specific steps are as follows:

[0062] (1) Add the recombinant Bacillus subtilis prepared in Example 1 to the LB liquid medium, and carry out the shaker culture at 37° C. and 200 rpm for 12 hours to prepare the seed solution;

[0063] (2) Inoculate the prepared seed liquid into the fermentation medium according to the inoculum amount of 3% (v / v), and carry out shaker culture at 37°C and 200 rpm to prepare the fermentation liquid;

[0064] The enzyme activity of D-psicose 3-epimerase in the fermentation broth was detected respectively, and the results are shown in Table 1 and figure 1 shown.

[0065] Table 1: Enzyme activity of D-psicose 3-epimerase prepared by different recombinant Bacillus subtilis

[0066] Recombinant bacteria Fermentation enzyme activity (U / mL) B. subtilis WB800 / pP43NMK-P43-dpe 15.05 B. subtilis WB800 / ...

Embodiment 3

[0069] Example 3: Construction of recombinant Bacillus subtilis containing different RBS

[0070] Specific steps are as follows:

[0071] Using the plasmid pP43NMK-hag-dpe as a template and using RBS-F / RBS-R as primers, one-step PCR was performed to mutate the RBS sequence on the hag promoter to construct linearized plasmids containing different RBS sequences;

[0072] Among them, the primers are as follows:

[0073] RBS-F: tgccttaacaacatattcrrggaggrrcaaaacaatgaagcatggta

[0074] RBS-R: taccatgcttcattgttttgyycctccyygaatatgttgttaaggca

[0075] The constructed linearized plasmid was transformed into Escherichia coli DH5α competent cells; the transformed Escherichia coli DH5α competent cells were coated with LB solid medium (containing 100 μg·mL -1 ampicillin), cultured upside down at 37°C for 12 hours; a certain number of positive transformants were picked, plasmids were extracted, and sequenced for verification. After the verification was correct, the recombinant plasmid pP4...

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Abstract

The invention discloses a strain for producing D-psicose 3-epimerase and application of the strain, and belongs to the technical field of biological engineering. The invention provides a method for screening a promoter and improving the expression quantity of D-psicose 3-epimerase through RBS optimization of the promoter. According to the recombinant bacillus subtilis constructed by using the vectors pP43NMK-hag and pP43NMK-hag-RBS4 provided by the invention, the enzyme activity of the target gene D-psicose 3-epimerase is improved, and the enzyme activity after modification is respectively 1.30 times and 1.69 times of the enzyme activity of the original vector. The invention also provides an antibiotic-free vector and an antibiotic-free recombinant bacillus subtilis strain, and the highest fermentation enzyme activity of a shake flask is 24.72 U/mL by adopting the antibiotic-free strain B.subtilis 1A751-dal-/pP43NMK-hag-RBS4-dpe-dal provided by the invention.

Description

technical field [0001] The invention relates to a bacterial strain producing D-psicose 3-epimerase and application thereof, belonging to the technical field of bioengineering. Background technique [0002] D-allulose (D-allulose) is the epimer of D-fructose at the C-3 position. It can be used as a low-calorie sweetener and can generate a pleasant taste through the Maillard reaction. It can improve the gelation of food; it can regulate biological functions such as lowering blood sugar and blood lipid levels, reducing fat accumulation, and scavenging reactive oxygen species (ROS). [0003] As early as 2014, the laws and regulations of the US Food and Drug Administration listed D-psicose as generally safe, allowing it to be added to food, dietary supplements and pharmaceutical preparations. Therefore, D-psicose has broad application prospects in fields such as medicine and food. [0004] As a rare sugar, D-psicose exists in a very small amount in nature, and it is unrealistic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N15/67C12N1/21C12N15/61C12N9/90C12P19/24C12P19/02C12R1/125
CPCC12N9/90C12N15/67C12P19/24C12P19/02C12R2001/125C12N15/74C12N15/75C12N15/70C12N9/88C12N9/16
Inventor 张涛胡梦莹江波李梦丽
Owner JIANGNAN UNIV
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