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Method for producing isomaltulose by whole cell transformation

A technology of isomaltulose and host cells, applied in the field of genetic engineering, can solve the problems of weak production intensity, low sucrose conversion rate, low yield and the like, and achieve the effect of high yield and high production intensity

Active Publication Date: 2019-06-25
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Therefore, there is an urgent need to find a recombinant bacterium that can produce sucrose isomerase with high specific activity and high sucrose conversion rate to solve the problem of low conversion rate, weak production intensity and low yield of sucrose in enzymatic biosynthesis. Defects

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Construction of Bacillus subtilis engineered bacteria that can express sucrose isomerase derived from Pantoea dispersa

[0054] Specific steps are as follows:

[0055] (1) Obtain the nucleotide sequence of sucrose isomerase (containing signal peptide) derived from Pantoea dispersa UQ68J (GeneID: AY223549.1) from GENBANK (shown in SEQ ID NO. 2) and obtain this sequence by artificial synthesis ; This sequence and the expression plasmid pMA5 are digested with restriction enzymes Nde I and Mlu I and then connected to obtain the recombinant plasmid pMA5-sim-1; the recombinant plasmid pMA5-sim-1 is transformed into E. coli JM109 To obtain recombinant E.coli JM109 / pMA5-sim-1;

[0056] (2) Obtain the nucleotide sequence of sucrose isomerase (containing signal peptide) derived from Pantoea dispersa UQ68J (GeneID: AY223549.1) from GENBANK (shown in SEQ ID NO. 2), and remove this nucleotide sequence The partial sequence corresponding to the signal peptide on the above and co...

Embodiment 2

[0060] Example 2: Construction of Bacillus subtilis engineered bacteria that can express genes encoding sucrose isomerase from other sources

[0061] Specific steps are as follows:

[0062] (1) Obtain the nucleotide sequence of sucrose isomerase derived from Erwinia rhapontici NX-5 (shown in SEQ ID NO.3) and the nucleoside of sucrose isomerase derived from Enterobacter sp.FMB-1 from GENBANK The acid sequence (shown in SEQ ID NO. 4) and the nucleotide sequence of the sucrose isomerase derived from Serratia plymuthica (shown in SEQ ID NO. 5) were artificially synthesized to obtain these sequences; these sequences were respectively restricted The expression plasmid pMA5 digested with sex endonuclease Nde I and Mlu I was ligated to obtain recombinant plasmids pMA5-sim-3, pMA5-sim-4 and pMA5-sim-5; the recombinant plasmids pMA5-sim-3, pMA5 -sim-4 and pMA5-sim-5 were transformed into E.coli JM109, respectively, to obtain recombinant E.coli JM109 / pMA5-sim-3, E.coli JM109 / pMA5-sim-4 and E...

Embodiment 3

[0065] Example 3: Expression of sucrose isomerase from different sources

[0066] Specific steps are as follows:

[0067] (1) Transform the plasmid pMA5 into Bacillus subtilis (Bacillus subtilis) 168 for expression, and obtain the recombinant strain BS168 / pMA5 as a blank control;

[0068] (2) Pick the recombinant bacteria BS168 / pMA5-sim-1, recombinant bacteria BS168 / pMA5-sim-2 obtained in Example 1, and the recombinant bacteria BS168 / pMA5-sim-3 and recombinant bacteria BS168 / pMA5 obtained in Example 2. Single colonies of -sim-4 and recombinant strain BS168 / pMA5-sim-5 were respectively inoculated into 10 mL of LB medium, and cultured at 37°C and 180 rpm for 12 hours to obtain seed liquid;

[0069] (3) The seed solution was inoculated into 50mL fermentation medium with 1% inoculum, cultured at 25℃ and 180rpm for 24h to obtain recombinant bacteria BS168 / pMA5-sim-1 and recombinant bacteria BS168 / pMA5-sim respectively -2. The culture solution of recombinant bacteria BS168 / pMA5-sim-3, reco...

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Abstract

The invention discloses a method for producing isomaltulose by whole cell transformation, and belongs to the technical field of genetic engineering. The method includes utilizing whole cell transformation of bacillus subtilis engineering bacteria of a gene capable of expressing and encoding sucrose isomerase (nucleotide sequence as shown in SEQ ID NO. 1) to produce isomaltulose. Conversion of theisomaltulose by the method has the advantages of high conversion rate, production intensity and high yield. By means of conversion and production of the isomaltulose for 9h in the method, 500g / L sucrose in a reaction system can be converted into 443g / L isomaltulose, the conversion rate of the sucrose is as high as 94%, the yield of the isomaltulose is as high as 88.6%, and the space-time yield ofthe isomaltulose is as high as 49.2g / L h.

Description

Technical field [0001] The invention relates to a method for producing isomaltulose by using whole cell transformation, and belongs to the technical field of genetic engineering. Background technique [0002] Isomaltulose (α-D-glucopyranosyl-1,6-D-fructose), also known as palatinose, is an isomer of sucrose, with similar physical properties to sucrose Nature and taste, but different from sucrose, its sweetness is only half that of sucrose, and it is not cariogenic; and after isomaltulose is eaten by the human body, the speed of releasing monosaccharides in the human blood is very slow. Stimulate the secretion of insulin, especially suitable for diabetics and obese people; in addition, isomaltulose, as a reducing functional disaccharide, is currently the only sweetener with no dosage limit. Therefore, isomaltulose is a natural New and new functional sugars are widely used in the food industry. [0003] At present, the main methods of producing isomaltulose are as follows: one is m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/61C12N15/75C12N15/77C12N9/90C12P19/12
Inventor 饶志明胡孟凯刘菲张显杨套伟徐美娟
Owner JIANGNAN UNIV
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