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Tagatose-producing bacillus subtilis genetic engineering bacteria 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 low utilization rate of enzyme recycling, difficulty in recycling, Escherichia coli unfavorable for industrial production of food preparations, etc.

Active Publication Date: 2021-04-06
天津怡和生物科技有限责任公司
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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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  • Tagatose-producing bacillus subtilis genetic engineering bacteria and method for preparing tagatose
  • Tagatose-producing bacillus subtilis genetic engineering bacteria and method for preparing tagatose
  • Tagatose-producing bacillus subtilis genetic engineering bacteria 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 homologous fragment upstream of the gene and the 500 bp downstream homologous fragment 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 supercompet...

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 a tagatose-producing Bacillus subtilis genetically engineered bacterium and a method for preparing tagatose. The genetically engineered bacterium comprises constructing a single-expression or co-expression thermostable α-glucan phosphorylase, thermostable glucose phosphate Mutase, thermostable glucose phosphate isomerase, thermostable 6‑phosphate tagatose epimerase, and thermostable 6‑phosphate tagatose phosphatase. Starch can be effectively converted into tagatose by using the genetically engineered bacteria. Compared with the existing methods for producing tagatose, the method of the present invention has the advantages of being suitable for whole cell recycling, high safety performance, high yield, simple production process, low cost and easy large-scale preparation.

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 Patents(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 天津怡和生物科技有限责任公司
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