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Bacillus subtilis genetically-engineered bacteria for producing tagatose and method for preparing tagatose

A technology of Bacillus subtilis and genetically engineered bacteria, applied in the field of bioengineering, can solve the problems of cumbersome purification steps, difficult recycling, and low utilization rate of enzymes

Active Publication Date: 2021-02-09
天工生物科技(天津)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Aiming at the problems existing in the existing method for preparing tagatose catalyzed by multiple enzymes, such as Escherichia coli is not conducive to the industrial production of food preparations, the purification steps are cumbersome, the recovery and utilization rate of the enzyme is low and the recycling is difficult, and the low concentration of the substrate starch Feeding problem, the main purpose of the present invention is to provide a kind of method that utilizes Bacillus subtilis whole cell to catalyze high-concentration starch to prepare and produce high-concentration tagatose

Method used

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  • Bacillus subtilis genetically-engineered bacteria for producing tagatose and method for preparing tagatose
  • Bacillus subtilis genetically-engineered bacteria for producing tagatose and method for preparing tagatose
  • Bacillus subtilis genetically-engineered bacteria for producing tagatose and method for preparing tagatose

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1: Construction of Bacillus subtilis recombinant strain SCK8

[0055] (1) Construction of recombinant integration vector pSS-upp-FR

[0056] According to the KEGG database derived from Bacillus subtilis Bacillus subtilis 168 uracil phosphoribosyltransferase encoding gene upp Gene sequence (NCBI-ProteinID: NP_391570), designed primers, and obtained by PCR amplification upp The 500 bp upstream homologous fragment and the 500 bp downstream homologous fragment of the gene are joined by simple cloning (You, C., Zhang, X. Z., & Zhang, Y. H. (2012). Simple cloning via direct transformation of PCR product (DNA Multimer) to Escherichia coli and Bacillus subtilis . Appl. Environ. Microbiol., 78 (5), 1593-1595. doi:10.1128 / AEM.07105-11) was constructed into the integration vector pSS to obtain the recombinant integration vector pSS-upp-FR.

[0057] (2) Construction of Bacillus subtilis recombinant strain SCK8

[0058] Preparation of Bacillus subtilis ...

Embodiment 2

[0062] Example 2 Construction of Bacillus subtilis recombinant strain SCK8-ST1

[0063] (1) Construction of recombinant integration vector pSS-amyE-FR

[0064] According to the KEGG database derived from Bacillus subtilis Bacillus subtilis 168 genes encoding α-amylase amyG Gene sequence (NCBI-ProteinID: NP_388186), designed primers, and obtained by PCR amplification amyGThe 500 bp upstream homologous fragment and the 500 bp downstream homologous fragment of the gene are joined by simple cloning (You, C., Zhang, X. Z., & Zhang, Y. H. (2012). Simple cloning via direct transformation of PCR product (DNA Multimer) to Escherichia coli and Bacillus subtilis . Appl. Environ. Microbiol., 78 (5), 1593-1595. doi:10.1128 / AEM.07105-11) was constructed into the integration vector pSS to obtain the recombinant integration vector pSS-amyE-FR.

[0065] (2) Construction of Bacillus subtilis recombinant strain SCK8-ST1

[0066] Prepare Bacillus subtilis strain SCK8 supercompete...

Embodiment 3

[0070] Example 3 Construction of Bacillus subtilis recombinant strain SCK8-ST2

[0071] (1) Construction of recombinant integration vector pSS-spoIIAC-FR

[0072] According to the KEGG database derived from Bacillus subtilis Bacillus subtilis 168 sporulation RNA polymerase σ F factor coding gene spoIIAC Gene sequence (NCBI-ProteinID: NP_390226), designed primers, and obtained sporulation by PCR amplification spoIIAC The 500 bp upstream homologous fragment and the 500 bp downstream homologous fragment of the gene are joined by simple cloning (You, C., Zhang, X. Z., & Zhang, Y. H. (2012). Simplecloning via direct transformation of PCR product (DNA Multimer) to Escherichia coli and Bacillus subtilis . Appl. Environ. Microbiol., 78 (5), 1593-1595. doi:10.1128 / AEM.07105-11) was constructed into the integration vector pSS to obtain the recombinant integration vector pSS- spoIIAC -FR.

[0073] (2) Construction of Bacillus subtilis recombinant strain SCK8-ST2

[00...

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Abstract

The invention discloses bacillus subtilis genetically-engineered bacteria for producing tagatose and a method for preparing the tagatose. The genetically-engineered bacteria are used for constructingheat-resistant alpha-glucan phosphorylase, heat-resistant phosphoglucomutase, heat-resistant glucose phosphate isomerase, heat-resistant 6-tagatose phosphate epimerase and heat-resistant 6-tagatose phosphate phosphatase which are independently expressed or co-expressed. By utilizing the genetically-engineered bacteria, starch can be effectively converted into the tagatose. Compared with the existing method for producing the tagatose, the method disclosed by the invention has the advantages of suitability for whole-cell recycling, high safety, high yield, simple production process, low cost, easiness in large-scale preparation and the like.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a tagatose-producing genetically engineered bacterium and a method for preparing tagatose. Background technique [0002] Tagatose is a rare monosaccharide that occurs naturally and is the ketose form of galactose, the epimer of fructose. The sweetness of tagatose is similar to that of sucrose, and its calories are only one-third of that of sucrose, so it is called a low-calorie sweetener. Natural tagatose is mainly found in dairy products such as yogurt and milk powder. Tagatose offers a very fresh and pure sweetness, with a taste profile similar to fructose. Studies have shown that tagatose has important physiological functional properties such as low calorie, low glycemic index, anti-caries, anti-oxidation, prebiotics, improvement of intestinal function, immune regulation, and drug precursors, and can be widely used in food, beverages, medicine, It has great economic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12N11/14C12P19/02C12R1/125
CPCC12N9/1051C12N9/90C12N9/92C12N9/16C12N15/75C12P19/02C12Y504/02002C12Y503/01009C12Y501/03C12P19/24C12Y204/01001C12N9/1205C12Y207/01101C12N15/52C12R2001/125C12N11/00C12Y301/03
Inventor 马延和石婷李运杰韩平平李元
Owner 天工生物科技(天津)有限公司
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