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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
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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

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  • 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

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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...

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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

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Application Information

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IPC IPC(8): C12N15/63C12N15/66C12N1/21C12N15/75C12N9/90C12R1/125
Inventor 王腾飞王瑞明刘强
Owner QILU UNIV OF TECH
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