Method for producing trehalose synthase from integrated recombinant bacillus subtilis and manufacturing trehalose

A technology of Bacillus subtilis and trehalose synthase, applied in the biological field, can solve the problems of restricting the high-efficiency expression of foreign genes, no discovery of trehalose synthase, and the use of antibiotics

Active Publication Date: 2013-07-24
南宁中诺生物工程有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

2. There is no obvious codon preference, and the expression product is not easy to form inclusion body
Replicating plasmids are usually not very stable in Bacillus subtilis, which limits efficient expression of foreign genes
At the same time, there is also the problem of the need to use antibiotics in the production process
At present, there is no research report on the expression and production of trehalose synthase by using the integrated Bacillus subtilis expression system, and then using the obtained trehalose synthase to produce trehalose

Method used

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  • Method for producing trehalose synthase from integrated recombinant bacillus subtilis and manufacturing trehalose
  • Method for producing trehalose synthase from integrated recombinant bacillus subtilis and manufacturing trehalose
  • Method for producing trehalose synthase from integrated recombinant bacillus subtilis and manufacturing trehalose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] This example will include the overlapping promoter P43 promoter derived from Bacillus subtilis (Bacillus subtilis), the signal peptide DNA fragment of the α-acetolactate decarboxylase gene derived from Bacillus brevis (Bacillus brevis) and the DNA fragment derived from Corynebacterium glutamicum ( Corynebacterium glutamicum) trehalose synthase gene monocistronic trehalose synthase expression element was cloned into the integrated plasmid pMLK83.

[0027] 1. Construction of recombinant plasmid pMLK83-P43

[0028] According to the promoter P43 sequence annotated in Genbank, the upstream primer was designed as 5'attgctggacgcttatggac 3' and the downstream primer was 5'cgggatccattcctctcttacctataat 3'. PCR reaction system 100ul: DNA template (Bacillus subtilis 1A751 total DNA) 1ul (about 20ng), 5×PrimeSTAR Buffer 20ul, 10pmol / ul dNTP 2ul, 10pmol / ul forward and reverse primers 2ul each, 2.5U / ul PrimeSTAR HS DNA polymerase 1ul, add ddH 2 0 to 100ul. PCR reaction program: 94°...

Embodiment 2

[0072] This example will include the overlapping promoter P43 promoter from Bacillus subtilis, the β-amylase gene signal peptide DNA fragment from Clostridium thermosulfurogenes and the curved high-temperature single The monocistronic trehalose synthase expression element of the trehalose synthase gene of Thermomonospora curvata was cloned into the integration plasmid pMLK83.

[0073] The following DNA fragments are artificially synthesized:

[0074] 1 GGATCCATGA TTGGAGCTTT TAAAAGGTTG GGTCAAAAAT TGTTTTTGAC

[0075] 51 ATTGTTAACG GCATCATTAA TTTTTGCATC TTCTATAGTA ACTGCTAATG

[0076] 101 CAGTGCAGAT GACCGGGGAC CCCATCCCCG ACACCTTCAC CCACGAAAAG

[0077] 151 CCGCGCGACC CCTACTGGTA CAAGCACGCG GTCTTCTACG AGGTGCTGGT

[0078] 201 GCGCGGGTTC AGCGACTCCA ACGACGACGG CACCGGAGAC CTGCGCGGCC

[0079] 251 TCATCAACCG GCTGGACTAT CTGCAGTGGC TGGGCATCGA CTGCATCTGG

[0080] 301 CTGCTGCCGA TCTACCAGTC GCCGCTGCGG GACGGCGGCT ACGACATCAG

[0081] 351 CGACTACACC AAGATCCTGC CGGAGTTCGG CGATCTGGG...

Embodiment 3

[0115] This example will include the promoter derived from the maltose amylase gene amyM from Bacillus lincheniformis, the signal peptide DNA fragment from the α-acetolactate decarboxylase gene from Bacillus brevis and the DNA fragment from the aquatic The monocistronic trehalose synthase expression element of the trehalose synthase gene of Thermus aquaticus was cloned into the integration plasmid pMLK83.

[0116] 1. Construction of recombinant plasmid pMLK83-amyM

[0117] According to the promoter amyM sequence annotated in Genbank, the upstream primer was designed as 5'cccaagcttctgtacacttgcgtcctcca 3' and the downstream primer was 5'cgggatcctctcctcccctttcaatgtg 3'. PCR reaction system 100ul: DNA template (Bacillus licheniformis ATCC 14580 total DNA) 1ul (about 20ng), 5×PrimeSTAR Buffer 20ul, 10pmol / ul dNTP 2ul, 10pmol / ul forward and reverse primers 2ul each, 2.5U / ul PrimeSTAR HS DNA polymerase 1ul, add ddH 2 0 to 100ul. PCR reaction program: 94°C 5min; 94°C 30s, 60°C 30s,...

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Abstract

The invention discloses a method for producing trehalose synthase by taking integrated recombinant bacillus subtilis as a strain and producing trehalose from the trehalose synthase. The method specifically comprises the following steps of: integrating a trehalose synthase expression element in a bacillus subtilis chromosome to construct integrated recombinant bacillus subtilis, and fermenting in a nutrient culture medium to produce the trehalose synthase by taking the recombinant bacillus subtilis as a strain, wherein via simple separation, the trehalose synthase in the fermentation liquor can be directly used for manufacturing trehalose. The method disclosed by the invention has the advantages that the trehalose synthase is produced by virtue of a food safety expression system, the exogenous gene contained in the strain with integrated expression can be used for stable passage and expression, and the expressed trehalose synthase is secreted to the outside of cells, has no antimicrobial activity, and achieves the requirements of food applications for enzymic preparation, thus being beneficial to further manufacturing for trehalose.

Description

technical field [0001] The invention belongs to the field of biotechnology, specifically a method for producing trehalose synthase and trehalose by means of an integrated recombinant bacillus subtilis, and a method for using the produced trehalose synthase to produce trehalose. Background technique [0002] Trehalose is composed of two molecules of glucose connected by α,α-1,1-glucosidic bonds, and is a natural disaccharide that exists widely in nature. It is very stable in nature and has protective effects on a variety of biologically active substances. In recent years, studies have found that trehalose can be widely used in the preservation of umami and texture improvement of various foods. With the progress of society and the improvement of people's living standards, people pay more and more attention to the quality and taste of food, which increases people's demand for trehalose. Therefore, the large-scale, cheap and efficient production of trehalose has great economic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N1/21C12N9/90C12P19/24C12P19/12C12R1/125C12R1/10C12R1/145C12R1/08C12R1/15C12R1/01
Inventor 黄日波李晓明罗兆飞韦航韦宇拓蒙健宗廖东庆韦玉琴李丛卢运琨
Owner 南宁中诺生物工程有限责任公司
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