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Method for producing extracellular pullulanase by applying auto-induction culture medium and dual-temperature control strategy

A technology of self-induction medium and pullulanase, which is applied in the field of microbial fermentation and production of pullulanase, which can solve the problems of low enzyme production ability of wild strains and no self-induction medium for pullulanase fermentation production.

Active Publication Date: 2012-09-19
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2001, Tang Baoying and others isolated a strain of pullulanase-producing Bacillus from the soil, which has good enzymatic properties (75°C, pH 4.6), but the enzyme-producing ability of wild strains is not good. High, only 1.6 U / mL, after mutation breeding, the enzyme production level was finally increased to 8.8 U / mL
However, at present, the self-induction medium is mainly used for the expression of isotope-labeled recombinant proteins analyzed by nuclear magnetic resonance, and there is no report on the use of the self-induction medium for the fermentation production of recombinant pullulanase
Moreover, there are few reports on the combined strategy of self-induction culture and dual temperature regulation for recombinant pullulanase fermentation production.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Autoinduction medium: α-lactose 10 g / L, glucose 0.5 g / L, glycerol 5 g / L, KH 2 PO 4 6.8 g / L, MgSO 4 0.24 g / L, peptone 10 g / L, Na 2 HPO 4 12H 2 O 17.9 g / L, Na 2 SO 4 0.71 g / L, NH 4 Cl 2.67 g / L, trace element solution 200 μL / L.

[0052] recombinant Escherichia coli E. coli The BL21 (DE3) / pET28a(+)-pul A strain was streaked on LB solid medium containing 50 μg / mL kanamycin and cultured overnight in a 37°C incubator. Pick a single colony from the plate and inoculate it into a 250 mL Erlenmeyer flask filled with 50 mL LB medium (50 μg / mL kanamycin), place it in a shaker at 37 °C and 200 rpm, and culture until OD 600Reach around 2.0~3.0. The seed culture solution was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of the above-mentioned autoinduction medium at an inoculum size of 5%, and cultured at 37°C for 60 hours (at this time OD 600 reach about 15), the end of fermentation.

[0053] Through the recombinant bacteria culture and fermentation enzym...

Embodiment 2

[0055] Autoinduction medium: α-lactose 20 g / L, glucose 1 g / L, glycerol 5 g / L, KH 2 PO 4 6.8 g / L, MgSO 4 0.24 g / L, peptone 10 g / L, Na 2 HPO 4 12H 2 O 17.9 g / L, Na 2 SO 4 0.71 g / L, NH 4 Cl 2.67 g / L, trace element solution 200 μL / L.

[0056] recombinant Escherichia coli E. coli The BL21 (DE3) / pET28a(+)-pul A strain was streaked on LB solid medium containing 50 μg / mL kanamycin and cultured overnight in a 37°C incubator. Pick a single colony from the plate and inoculate it into a 250 mL Erlenmeyer flask filled with 50 mL LB medium (50 μg / mL kanamycin), place it in a shaker at 37 °C and 200 rpm, and culture until OD 600 Reach around 2.0~3.0. The seed culture solution was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of the above-mentioned autoinduction medium at an inoculum size of 5%, and cultured at 25°C for 60 hours (at this time OD 600 reach about 15), the end of fermentation.

[0057] Through the recombinant bacteria culture and fermentation enzyme...

Embodiment 3

[0059] Autoinduction medium: α-lactose 20 g / L, glucose 1 g / L, glycerol 5 g / L, KH 2 PO 4 6.8 g / L, MgSO 4 0.24 g / L, peptone 10 g / L, Na 2 HPO 4 12H 2 O 17.9 g / L, Na 2 SO 4 0.71 g / L, NH 4 Cl 2.67 g / L, trace element solution 200 μL / L.

[0060] recombinant Escherichia coli E. coli The BL21 (DE3) / pET28a(+)-pul A strain was streaked on LB solid medium containing 50 μg / mL kanamycin and cultured overnight in a 37°C incubator. Pick a single colony from the plate and inoculate it into a 250 mL Erlenmeyer flask filled with 50 mL of LB medium (50 μg / mL kanamycin), place it in a shaker at 37 °C and 200 rpm, and culture until OD 600 Reach around 2.0~3.0. The seed culture solution was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of the above-mentioned autoinduction medium at an inoculation amount of 5%, and cultured in a shaker at 37°C and 200 rpm for 4 hours, and then the temperature of the shaker was Lower the temperature to 25°C and continue to incubate for 56 ...

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Abstract

The invention provides a method for producing extracellular pullulanase by applying an auto-induction culture medium and a dual-temperature control strategy, belonging to the technical field of pullulanase production through microbial fermentation. The method has the following beneficial effects: pullulanase coding genes from Klebsiella variicola CCTCC M2012108 are inserted into an expression vector pET28a(+) to construct recombinant plasmids and E.coli is converted to obtain a recombination strain E.coli BL21(DE3) / pET28a(+)-pulA containing the target pullulanase gene; the auto-induction culture medium is utilized to culture the recombinant E.coli BL21(DE3) / pET28a(+)-pulA and ferment the recombinant E.coli BL21(DE3) / pET28a(+)-pulA to generate enzyme by adopting the dual-temperature control mode of firstly culturing at 37 DEG C for 2-4 hours and then continuing culture at 25 DEG C for 48-72 hours; and after adopting the optimized fermentation conditions of the auto-induction culturemedium and dual-temperature, the extracellular pullulanase activity can reach 60-70U / mL. The method provides an effective strategy for producing extracellular pullulanase with recombinant E.coli and has great significance in the production process for developing novel recombinant pullulanase in future and application value of pullulanase.

Description

technical field [0001] The invention discloses a method for producing extracellular pullulanase by applying self-inducing medium and dual temperature regulation strategy, which belongs to the technical field of microbial fermentation and production of pullulanase. Background technique [0002] Pullulanase (EC 3.2.1.41) is a debranching enzyme that specifically decomposes α-1,6-glycosidic bonds in pullulan, starch, amylopectin and corresponding branched oligosaccharides. Compared with other pullulan hydrolases, pullulanase specifically hydrolyzes α-1,6-glucosidic bonds in pullulan to generate end products mainly composed of maltotriose. This property determines that it has great value in improving the effect of amylase on starch, increasing the utilization rate of starch, reducing grain consumption, improving product quality and developing new products. It is widely used in starch processing industry, food, detergent, textile etc. have important uses and good market prospect...

Claims

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Application Information

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