Eureka AIR delivers breakthrough ideas for toughest innovation challenges, trusted by R&D personnel around the world.

Starch induction type recombinant bacillus subtilis as well as preparation method and application thereof

A technology of Bacillus subtilis and amylase, applied in the fields of genetic engineering and enzyme engineering, can solve the problem of high production cost

Active Publication Date: 2015-11-11
QILU UNIV OF TECH
View PDF1 Cites 9 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Although the double-enzyme method has many advantages above, there are still shortcomings: the two enzymes used in the double-enzyme method conversion need to be fermented and purified separately to obtain, so the production cost of the enzyme is significantly higher than that of the single-enzyme method
However, the existing promoters of Bacillus subtilis have many problems in terms of quantity, expression level and regulation mode.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Starch induction type recombinant bacillus subtilis as well as preparation method and application thereof
  • Starch induction type recombinant bacillus subtilis as well as preparation method and application thereof
  • Starch induction type recombinant bacillus subtilis as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] The α-amylase promoter PamyQ derived from Bacillus amyloliquefaciens (B.Amyloliquefaciens) was constructed into the shuttle plasmid PHT43 to replace the original Pgrac promoter;

[0119] Proceed as follows:

[0120] (i) using the shuttle plasmid PHT43 as a template to perform PCR amplification to obtain a PHT fragment;

[0121] Described PCR primer sequence is as follows:

[0122] PHT-up: 5'-ACTCAAACATCAAAATCTTACAAA-3'

[0123] PHT-down: 5'-CTTTTCGTATGTGCGGGGCGTGATAAGATAAAAAAATTTTTCACGCTTACATCAT-3'

[0124] The PCR amplification system is 50 μl:

[0125] 2×TaqPCR MasterMix 25 μl, primer PHT-up (10 μmol / L) 2.5 μl, primer PHT-down (10 μmol / L) 2.5 μl, template 2.5 μl, use ddH 2 O make up 50 μl;

[0126] The PCR amplification procedure is as follows:

[0127] Pre-denaturation at 95°C for 5min; denaturation at 95°C for 30sec, annealing at 51°C for 30sec, extension at 72°C for 40sec, 30 cycles; extension at 72°C for 10min, storage at -20°C;

[0128] (ii) extracting the...

Embodiment 2

[0173] The maltooligosaccharide-based trehalose synthase gene derived from Arthrobacternicotinovorans was fused with the maltooligosaccharide-based trehalose hydrolase gene, and the fusion expression gene was constructed into the shuttle plasmid PHT43.

[0174] (viii) Using Arthrobacternicotinovorans genomic DNA as a template, respectively design primers F1 and R1 (amplification of maltooligosaccharide-based trehalose synthase TreY), F2 and R2 (amplification of maltooligosaccharide-based trehalose hydrolase TreZ);

[0175] F1: 5'-GGATCCGTGTTGACACCGAAATCGACCTACC-3'

[0176] R15'-CCTCGGGGGTGAACGTGC-3'

[0177] F2:5'-ATGAGTTCGCCATTCGAGGT-3'

[0178] R2: 5'-GACGTCGTCGAGCAGGTGGATGGAGG-3'

[0179] Using the Arthrobacternicotinovorans genome as a template and F1 and R1 as primers, the product TreY was obtained through the PCR process;

[0180] The PCR system is 50 μl:

[0181] 2×HiFi-PCRmaster25μl; Upstream primer F12.5μl; Downstream primer R12.5μl; Template 2.5μl; ddH 2 O 17.5 ...

Embodiment 3

[0200] Transformation of the recombinant shuttle plasmid PamyQ-PHT43-TreY-TreZ in Bacillus subtilis WB800n;

[0201] A single colony of Bacillus subtilis WB800n was picked and inoculated in TBY medium (1% tryptone, 0.5% yeast extract, 1% NaCl), and cultivated overnight in a 37°C incubator.

[0202] Use 100mL LBSP medium (tryptone 10g / L, yeast extract 5g / L, NaCl 1g / L, glucose 250mmol / L, K 2 HPO 4 / KH 2 PO 4 50mmol / L, pH7.2) to dilute 2mL of the overnight culture, cultivate at 37°C and 220rpm until the OD value is 1.0; place the bacterial solution on ice for 10min; centrifuge at 10000rpm for 5min in a cold centrifuge tube at 4°C to enrich cells;

[0203] SHMG (sucrose 250mmol, Hepes1mmol, MgCl 2 0.5 mmol, glycerol 10%) to wash the enriched cells three times, and finally dissolve in 3 mL of ice-bathed SHMG; aliquot competent cells in 100 μl each, and store at -80°C.

[0204] Take a tube of competent cells and quickly place them in 37°C water until they dissolve; take 1-10 μ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to starch induction type recombinant bacillus subtilis as well as a preparation method and application thereof. The starch induction type recombinant bacillus subtilis contains a recombinant vector, wherein the recombinant vector is characterized in that in front of a BamHI restriction enzyme cutting site of a PHT43 plasmid, a Pgrac promoter is replaced with an alpha-amylase promoter PamyQ by performing for three times a mode of overlapping PCR continuously, and then an MTSase-MTHase fusion enzyme gene is inserted behind the BamHI restriction enzyme cutting site. When the used alpha-amylase promoter and an amylase signal peptide are combined with the expression gene, i.e., the MTSase-MTHase fusion enzyme gene, the expression effect of the starch induction type is better than that of other induction types.

Description

technical field [0001] The present invention relates to a starch-inducible recombinant Bacillus subtilis and its preparation method and application, in particular to a starch-inducible recombinant Bacillus subtilis producing maltooligosaccharide-based trehalose synthase-maltooligosaccharide-based trehalose hydrolase fusion enzyme The invention relates to a method for manufacturing trehalose, belonging to the technical fields of genetic engineering and enzyme engineering. Background technique [0002] Trehalose is a ubiquitous non-reducing disaccharide linked by glucose through α-1,1 glycosidic bonds, among which α,α-1,1-trehalose has been isolated and found to be widely present in plants, in animals and microorganisms. Natural trehalose has a good and non-specific protective effect on organisms or biological macromolecules, enabling organisms with trehalose to survive harsh environments such as starvation, dryness, low temperature, high temperature, and radiation. With the...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N15/63C12N15/66C12N1/21C12N15/75C12N9/90C12R1/125
Inventor 王腾飞王瑞明刘强
Owner QILU UNIV OF TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com