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Genetically engineered bacteria of high-yield malto-oligosaccharide-based trehalose-hydrolyzing enzyme and application of genetically engineered bacteria

A trehalose hydrolase and malto-oligosaccharide-based technology, which is applied in the fields of genetic engineering and fermentation engineering, can solve the problems of low enzyme activity, low soluble expression, unfavorable industrial application and the like, and achieve the effect of improving soluble expression

Active Publication Date: 2016-09-28
LIYANG WEIXIN CHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Double-enzyme production of trehalose takes starch as the substrate, and the conversion rate is as high as 80%, which has the advantage of low cost, but the soluble expression of the maltooligosaccharide-based trehalose hydrolase gene in the host bacteria is very low, resulting in more inclusions Body, low enzyme activity, not conducive to its industrial application

Method used

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  • Genetically engineered bacteria of high-yield malto-oligosaccharide-based trehalose-hydrolyzing enzyme and application of genetically engineered bacteria
  • Genetically engineered bacteria of high-yield malto-oligosaccharide-based trehalose-hydrolyzing enzyme and application of genetically engineered bacteria
  • Genetically engineered bacteria of high-yield malto-oligosaccharide-based trehalose-hydrolyzing enzyme and application of genetically engineered bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Construction of pET-32a(+)-treZ / E.coli Origami(DE3)

[0035] PCR amplified maltooligosaccharide-based trehalose hydrolase gene treZ (nucleotide sequence is SEQ ID NO.1), and the obtained treZ gene fragment was digested and purified with EcoR I and Hind III vector pET-32a ( +), ligated with T4DNAligase at 4°C for 16h. The ligation product was transformed into the cloning host E.coli JM109 and coated with LB solid medium (containing 100 μg·mL -1 ampicillin), cultivated in a 37°C incubator for 8 hours, picked a single colony, and passed through LB liquid medium (containing 100 μg·mL -1 ampicillin) at 37°C for 8 hours, the plasmids were extracted from the cells, verified by enzyme digestion, and then the DNA sequence of the verified recombinant plasmids was determined to obtain the recombinant plasmid pET-32a(+)-treZ. The pET-32a(+) vector contains a thioredoxin (TrxA) fusion tag. Thioredoxin (thioredoxin A, TrxA) is a small molecule protein with a conserved se...

Embodiment 2

[0040] Example 2 Shake flask fermentation to produce enzyme

[0041] 1. The recombinant pET-24a(+)-treZ / E.coliBL21(DE3) and pET-32a(+)-treZ constructed with pET-24a(+) as the vector and E.coli BL21(DE3) as the host / E.coli Origami(DE3), use a pipette gun to draw 10 μl of the bacterial solution and inoculate it into 10 mL of LB liquid medium (adding a final concentration of 30 μg·mL -1 Kanamycin or 100 μg·mL -1 Ampicillin), placed in a constant temperature shaker at 37°C, 200r·min -1 , cultivated for 8-10h, as the seed solution.

[0042] Inject the cultivated seed solution into 40 mL TB medium with a 4% inoculum size (adding a final concentration of 30 μg·mL -1 Kanamycin or 100 μg·mL -1 Ampicillin), placed in a constant temperature shaker at 37°C, with a rotation speed of 200r min -1 , cultivated for 2h; add the final concentration of 0.05-0.4mmol L -1 IPTG, 25°C, speed 200r·min- 1 , cultivated for 24-48h. Centrifuge the fermentation broth to remove the supernatant, col...

Embodiment 3

[0051] Example 3 Effects of Different Induction Times on the Fermentation and Enzyme Production of Recombinant Bacteria

[0052] 1. Seed culture: Inoculate the pET-32a(+)-treZ / E.coli Origami(DE3) strain stored in a glycerol tube at -80°C into the seed medium, and cultivate it on a constant temperature shaker at a temperature of 37°C and a rotation speed of 200rpm , cultivated for 8h.

[0053] 2. Enzyme production by fermentation:

[0054] The seed solution was added to the fermentation medium with an inoculum of 8%. The dissolved oxygen was maintained at 30% by controlling the stirring speed and ventilation rate, the temperature was controlled at 37° C., and the pH was controlled at 7.0 by feeding 25% (v / v) ammonia water. After the initial glycerin is consumed and the dissolved oxygen rises to 80-100%, the batch fermentation culture ends. With specific growth rate μ=0.2h -1 The feed medium was added exponentially. Select in bacterial concentration OD 600 When reaching 30...

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Abstract

The invention discloses genetically engineered bacteria of a high-yield malto-oligosaccharide-based trehalose-hydrolyzing enzyme and application of the genetically engineered bacteria and belongs to the technical field of genetic engineering and fermentation engineering. The genetically engineered bacteria pET-32a(+)-treZ / E.coli Origami(DE3) of the high-yield malto-oligosaccharide-based trehalose-hydrolyzing enzyme is constructed by taking E.coli Origami(DE3) as a host. The strain is used as a production strain and is fermented in a fermentation tank to generate the enzyme; and the activity of the enzyme can reach 204.U.mL<-1> and is about 4.9 times as much as that of shaking bottle fermentation. The production cost of the malto-oligosaccharide-based trehalose-hydrolyzing enzyme is reduced and a foundation is laid for industrial production of trehalose.

Description

technical field [0001] The invention relates to a genetically engineered bacterium with high yield of maltoligosaccharide-based trehalose hydrolase and application thereof, belonging to the technical fields of genetic engineering and fermentation engineering. Background technique [0002] Trehalose (Trehalose) is composed of two glucopyranose linked by 1,1-glycosidic bonds. It is a stable non-reducing disaccharide. It has three optical isomers, namely αα type and αβ type. and ββ-type. [0003] Trehalose is a safe non-reducing disaccharide that widely exists in nature. It has special biological functions such as moisture retention, anti-freeze and anti-drying properties, and thermal-acid stability. It has non-specific protective effects on biological macromolecules , so it has great application potential in medicine, agriculture, cosmetics, food and other industries. Since the 1980s, countries have successively carried out studies on the physiology, biochemistry and molecul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P19/14C12P19/12C12R1/19
CPCC12N9/2408C12P19/12C12P19/14C12Y302/01141
Inventor 吴敬宿玲恰吴世雄
Owner LIYANG WEIXIN CHEM
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