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A kind of purification method of malt limit dextrinase

A technology of limit dextrinase and purification method, applied in the field of food biology, can solve the problems of low limit dextrinase activity, poor thermal stability, etc., and achieve the effects of good reproducibility, high purification multiple, and wide application prospects.

Inactive Publication Date: 2011-12-07
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] For a long time, due to the low activity of limit dextrinase and poor thermal stability in malt, it has not been paid attention to

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The extraction of the first step limit dextrinase: add the 0.1mol / L acetic acid-sodium acetate buffer solution that the pH value that contains 20mmol / L L-cysteine ​​hydrochloride is 5.5 to the malt powder, make solid-liquid ratio 1:5g / mL, placed in a water bath constant temperature oscillator with a rotation speed of 100r / min, extracted for 20 hours at a temperature of 35°C, and centrifuged in a centrifuge with a rotation speed of 4000r / min for 12 minutes to obtain the malt limit Dextrinase Crude Extract I.

[0024] The second step of ammonium sulfate step-by-step precipitation: add ammonium sulfate with a saturation of 30% to the crude extract of malt limit dextrinase I to carry out a salting out, and add ammonium sulfate to the supernatant obtained by centrifugation. The saturation is 75%, after the second salting out for 5 hours, centrifuge to obtain malt limit dextrin crude enzyme solution II;

[0025] The third step of ion exchange chromatography: the volume of th...

Embodiment 2

[0029] The extraction of the first step limit dextrinase: add the 0.1mol / L acetic acid-sodium acetate buffer solution that contains the pH value of 5.4 of 22mmol / L L-cysteine ​​hydrochloride to malt powder, make solid-liquid ratio 1:5.37 g / mL, placed in a water bath constant temperature oscillator with a rotation speed of 100r / min, extracted for 17.5h at a temperature of 37.5°C, and centrifuged for 15min in a centrifuge with a rotation speed of 3000r / min to obtain malt Extreme dextrinase crude extract Ⅰ.

[0030] The second step of ammonium sulfate step-by-step precipitation: adding ammonium sulfate with a saturation of 35% to the crude extract of malt limit dextrinase I for a salting out, and adding ammonium sulfate to the supernatant obtained by centrifugation to Saturation is 80%, secondary salting out for 10 hours, and centrifugation to obtain maltolimited dextrinase crude enzyme solution II;

[0031] The third step of ion exchange chromatography: the volume of the anion ...

Embodiment 3

[0035] The first step is ammonium sulfate step-by-step precipitation: add ammonium sulfate with a saturation of 35% to the purchased crude extract of malt limit dextrinase I for a salting out, and add sulfuric acid to the supernatant obtained by centrifugation ammonium to a saturation of 85%, and after secondary salting out for 18 hours, the crude extract of malt limit dextrinase II was obtained;

[0036]The second step of ion-exchange chromatography: the volume of the anion-exchange resin bed is 100 mL, equilibrated with 20 mmol / L Tris-HCl buffer solution with a pH value of 7.0, and then 5.0 g of malt limit dextrinase crude extract II is dissolved in 20 mL of distilled water, Load the sample, and elute with the gradient of the above buffer and the buffer containing 0 mol / L NaCl (i.e. clear water) at a flow rate of 3mL / min, collect the eluted fraction corresponding to the protein absorption peak, perform enzyme activity determination, and collect Solution I containing a limite...

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Abstract

The invention discloses a purification method of malt limit dextrinase. The method includes: 1) Ammonium sulfate step-by-step precipitation: add ammonium sulfate with a saturation of 25-35% to the enzyme crude extract Ⅰ to remove impurity proteins, and then add ammonium sulfate to the supernatant to a saturation of 75-35%. 85%, salting out to get the crude enzyme solution II; 2) Ion exchange chromatography: After the filler is pretreated and packed, equilibrate with 15~20mmol / LTris-HCl buffer solution with a pH value of 7.0, load the sample, and then use 0~ Gradient elution with 1mol / L NaCl buffer solution; 3) Gel filtration chromatography: After the sample separated by ion exchange chromatography column is further purified by gel filtration chromatography column, it is freeze-dried to obtain a highly purified limit dextrinase product . The method has the advantages of simple process, safety, low cost, high product purity and good purification effect, and is an effective and reliable method for purifying limit dextrinase.

Description

technical field [0001] The invention relates to the refining of malt limit dextrinase in the field of food biotechnology, in particular to a purification method of malt limit dextrinase. Background technique [0002] Limit dextrinase (pullulan 6-glucan hydrolase), also known as α-dextrin 6-glucan hydrolase or pullulanase (Pullulan 6-glucan hydrolase), belongs to starch hydrolase . It can specifically catalyze the α-1,6 glycosidic bonds in the branch points of pullulan, pullulan, α-limit dextrin, β-limit dextrin, etc. Its main function is to hydrolyze the α-1,6 glycosidic bonds that cannot be hydrolyzed by α-amylase and β-amylase, so as to accelerate the degradation rate of starch, change the composition of the final product, and increase the hydrolysis rate of starch. [0003] As a unique enzyme that degrades α-1,6 glycosidic bonds, limit dextrinase plays an important role in food, medicine, daily chemical, textile and other industries by improving starch and other product...

Claims

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Application Information

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IPC IPC(8): C12N9/24
Inventor 胡飞冯倩倩
Owner SOUTH CHINA UNIV OF TECH
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