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Pullulanase, pullulanase producing strain and application of the pullulanase

A pullulanase and strain technology, applied in the field of biological enzymes, can solve the problems of high price, no screening of pullulanase-producing strains, and limited application.

Active Publication Date: 2012-07-25
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Pullulanase has greatly improved the utilization rate of starch resources, but the pullulanase-producing strains isolated at home and abroad have low enzyme activity (generally 2~5 U / mL), and expensive, which limits its application in industry
These enzyme-producing strains are mainly derived from Bacillus (Bacillus) and Klebsiella (Klebsiella), and have not been screened from Aeromonas by experimental methods pullulanase producing strain

Method used

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  • Pullulanase, pullulanase producing strain and application of the pullulanase
  • Pullulanase, pullulanase producing strain and application of the pullulanase
  • Pullulanase, pullulanase producing strain and application of the pullulanase

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1: Screening, isolation and identification of pullulanase-producing strains

[0031] Take 10 g of soil sample and put it into a conical flask filled with 90 mL of sterile water and glass beads, shake it for 30 min to fully disperse the sample, draw 100 μL of culture solution and dilute it, and spread it on the mixture with pullulan as the only Plate enriched medium with carbon source was cultured in a 30°C incubator for 48 h. Two single colonies of different shapes were picked from the enrichment medium and inoculated on the strain isolation medium, and the picked single colonies were streaked repeatedly. Subsequently, the colonies purified by streaking on the plate were inoculated into the enzyme-producing seed medium, and cultured with shaking at 30 °C for 24 h to obtain a bacterial suspension. The bacterial suspension was inserted into the enzyme-producing fermentation medium with 4% inoculum, and cultured with shaking at 30 °C for 48 h; the supernatant wa...

Embodiment 2

[0033] Example 2: Aeromonas sp . As fermentation condition optimization

[0034] The pullulanase of the As strain is an inducible enzyme and needs to be expressed under substrate-inducing conditions. Therefore, when optimizing the conditions for the production of pullulanase by the As strain, soluble starch was used as the enzyme-producing inducer. After the As strain was isolated and purified, a single colony was picked and inoculated in 25 mL of seed medium, and cultured with shaking at 30 °C for 16–18 h; then 2 mL was transferred into 50 mL of fermentation medium, and incubated for 30 ℃. Cultivate with shaking at ℃ for 48 h, and take the supernatant to measure the enzyme activity. Adjust the pH value of the fermentation medium (pH 6.5 and pH 7.5) and fermentation temperature (30 ℃ and 37 ℃), measure the activity of pullulanase in the fermentation broth every 12 h, and establish the conditions for high-yield pullulanase of strain As .

Embodiment 3

[0035] Example 3: Aeromonas sp . Preliminary Purification of Pullulanase from As Fermentation Supernatant

[0036] Aeromonas sp . After the As strain was fermented for 48 h, 20 μL of the fermentation supernatant was used to detect protein expression bands by 12% SDS-PAGE. Take 30 mL of fermentation supernatant, concentrate it to 1 mL by ultrafiltration with Amicon ultrafiltration tube (molecular weight 30 KDa), the buffer solution is 20 mM Tris-HCl (pH 8.0), take 20 μL for SDS-PAGE detection, and detect the first Specific activity of pullulanase after one ultrafiltration (U / mg protein). For the second ultrafiltration purification, Amicon ultrafiltration tubes with a molecular weight cut-off of 50 KDa and 20 mM Tris-HCl (pH 8.0) were used. The final volume after ultrafiltration was 800 μL, and 20 μL was taken out for SDS-PAGE detection (such as Figure 4 ), while measuring the specific activity of pullulanase.

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Abstract

The invention belongs to the technical field of biological enzymes, and in particular relates to a pullulanase, a pullulanase producing strain and application of the pullulanase. The pullulanase is derived from Aeromonas sp. As in bacteria. The pullulanase produced by the strain is pullulanase type I, and can specifically hydrolyze alpha-1,6 glycosidic bond in pullulan or starch. The pullulanase is expressed under an induction condition in which 1.5% soluble starch is used as a substrate. Under a condition of medium pH of 7.5 and culture temperature of 30 DEG C, shake-flask fermentation is carried out for 48h to produce the pullulanase at a level up to 24.8U / mL. For the pullulanase produced by the strain, the optimum reaction temperature is 60 DEG C, the optimum pH is 6.0, and the relative enzyme activity in the pH range of 6.0-9.0 is greater than 90 percent. After treatment for 50min at 60 DEG C, the residual activity of the pullulanase reaches about 50 percent. The pullulanase type I provided by the invention can be applied to industries such as starch processing, environment protection, foods, medical care and the like.

Description

technical field [0001] The invention belongs to the technical field of biological enzymes, and in particular relates to screening and identification of a novel pullulanase and enzyme-producing strains thereof. The invention also provides the production method, enzymatic characteristics and application of the pullulanase. Background technique [0002] Starch is a product of photosynthesis in green plants and consists of amylose and amylopectin. Among them, amylose is mainly connected by α-1,4 glucose glycosidic bonds, while amylopectin also contains branched chains, and the branches are connected by α-1,6 glucose glycosidic bonds. Starch is one of the most widely used industrial raw materials at present, such as the preparation of glucose syrup, high maltose syrup and so on. However, since starch contains about 4% to 5% of α-1,6 glycosidic bonds, the use of α-amylase (hydrolyzing α-1,4 glycosidic bonds) cannot completely hydrolyze starch, which affects the improvement of st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/44C12R1/01
Inventor 吕红冯碧薇王儒恺周峻岗
Owner FUDAN UNIV
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