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Genetic recombination escherichia coli and method for efficiently producing pullulanase

A technology of recombinant Escherichia coli and pullulanase, applied in the direction of microorganism-based methods, biochemical equipment and methods, genetic engineering, etc., can solve the problems of low extracellular enzyme activity and rising production cost of pullulanase

Inactive Publication Date: 2013-03-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In summary, in the microbial fermentation production process of pullulanase, whether it is wild bacteria or engineered bacteria, the main problem is that the activity of extracellular enzymes is low, which causes the production cost of pullulanase to rise, and the industrialization Obstacles to production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] LB medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0. When needed, add kanamycin (50 μg / mL) before use, and add 15 g / L agar powder to the solid medium.

[0054] pick recombinant Escherichia coli E. coli BL21(DE3) / pET28a(+)-PelB- pul A single colony of positive clones was inoculated in 3 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured overnight at 37 °C with shaking at 200 rpm. Take 1 mL of the culture medium and transfer it to 50 mL of LB liquid medium containing 50 μg / mL kanamycin, and culture at 37°C and 200 rpm until OD 600 About 0.6. The inducer IPTG was added to the culture to a final concentration of 0.8 mmol / L, and the induction culture was carried out at a culture temperature of 30°C for 12 hours. After the cultivation, the fermentation broth was centrifuged at 10,000 rpm for 10 min to obtain the fermentation supernatant for the determination of the extracellular enzyme activity of the strain. Using LB medium and IPTG i...

Embodiment 2

[0056]LB medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0. When needed, add kanamycin (50 μg / mL) before use, and add 15 g / L agar powder to the solid medium.

[0057] pick recombinant Escherichia coli E. coli BL21(DE3) / pET28a(+)-PelB- pul A single colony of positive clones was inoculated in 3 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured overnight at 37 °C with shaking at 200 rpm. Take 1 mL of the culture medium and transfer it to 50 mL of LB liquid medium containing 50 μg / mL kanamycin, and culture at 37°C and 200 rpm until OD 600 About 0.6. The inducer IPTG was added to the culture to a final concentration of 0.4 mmol / L, and the induction culture was carried out at a culture temperature of 30°C for 12 hours. After the cultivation, the fermentation broth was centrifuged at 10,000 rpm for 10 min to obtain the fermentation supernatant for the determination of the extracellular enzyme activity of the strain. Using LB medium and IPTG in...

Embodiment 3

[0059] Autoinduction medium (g / L): β-lactose 10, anhydrous glucose 0.5, glycerol 5, KH 2 PO 4 6.8, MgSO 4 0.24, tryptone 10, yeast extract 5, Na 2 HPO 4 7.1, Na 2 SO 4 0.71, NH 4 Cl 2.67, trace element solution 400 μL / L, pH 7.5~8.0. Trace element solution (g / L): FeCl 3 8.125, CaCl 2 2.22, MnCl 2 2.52, ZnSO 4 1.61, CoCl 2 0.26, CuCl 2 0.27, NiCl 2 0.26, Na 2 MoO 4 0.41, Na 2 SeO 3 0.346,H 3 BO 3 0.124, HCl 2.19.

[0060] Recombinant bacteria E. coli BL21(DE3) / pET28a(+)-PelB- pul The bacterial solution was inoculated in 50 mL of autoinduction medium containing 50 mg / L kanamycin, and cultured at 37°C and 200 rpm for 3 hours with shaking, then added glycine with a final concentration of 6 g / L, and the temperature was changed to 20°C for 60 hours . After the cultivation, the fermentation broth was centrifuged at 10,000 rpm for 10 minutes to obtain the fermentation supernatant for the determination of the extracellular enzyme activity of the st...

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Abstract

The invention discloses a genetic recombination escherichia coli and a method for efficiently producing pullulanase, belonging to the technical field of microbial fermentation. The method comprises the following steps of: connecting a signal peptide PelB of a plasmid pET20b(+) and bacillus naganoensis CCTCC NO:M2012388 pullulanase genes, and commonly inserting the signal peptide and the bacillus naganoensis into an expression vector pET28a(+) to construct a recombination strain E.coliBL21(DE3) / pET28a(+)-PelB-pul with the signal peptide and target genes; and using the strategies of auto-induction culturing and adding glycine to control the cell permeability in the fermentation and production of extracellular pullulanase, using an auto-induction culture medium with lactose in a concentration of 10g / L, shaking and culturing for about 2 hours under the conditions of a temperature of 37 DEG C and a speed of 200rpm, then adding the glycine with a final concentration of 6g / L and transferring to culturing for 70 hours at a temperature of 20 DEG C. The enzyme activity of the extracellular pullulanase reaches 505U / mL and is enhanced by 1260 times to the enzyme activity of the original strains of the wild fungus. The application of the invention is of great significance.

Description

technical field [0001] The invention relates to a genetically recombined Escherichia coli and a method for efficiently producing pullulanase, which belong to the technical field of microbial fermentation and production of pullulanase. Background technique [0002] Pullulanase (pullulanase, EC.3.2.1.41) is a kind of debranching enzyme that can specifically hydrolyze the α-1,6-glycosidic bond in pullulan, amylopectin and its limit dextrin. In the process of starch saccharification, pullulanase and glucoamylase work together, pullulanase cuts amylopectin branch points, and glucoamylase only needs to cut linear oligosaccharides, which can significantly improve the hydrolysis efficiency of starch. The use of pullulanase can not only reduce the amount of glucoamylase, but also increase the DE value to more than 97%, and improve the quality and purity of the hydrolyzed product glucose or maltose. [0003] At present, a variety of pullulanase-producing microorganisms have been foun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/56C12N15/70C12N1/21C12N9/44C12R1/07C12R1/19
Inventor 聂尧徐岩严伟
Owner JIANGNAN UNIV
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