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Aspergillus niger mutant strain with high yield of glucoamylase and application thereof

A technology of Aspergillus niger strain and saccharification enzyme, which is applied in the directions of glycosylase, mutant preparation, enzyme, etc., can solve the problems of up-regulation, large gap, and low saccharification enzyme activity, and achieves strong specificity, efficient decomposition, and improved The effect of catalytic efficiency

Active Publication Date: 2022-01-11
SHANDONG LONGKETE ENZYME PREPARATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The main problem currently existing is that the glucoamylase activity of the production strain is not high, the catalytic efficiency of the enzyme is not high, and there are more α-glucosidases produced by fermentation, which is far behind foreign countries. In addition, the price of raw materials is gradually rising, and the saccharification The price of enzyme products has also increased along with the trend, limiting the application in other industries and affecting the transformation and upgrading of these industries

Method used

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  • Aspergillus niger mutant strain with high yield of glucoamylase and application thereof
  • Aspergillus niger mutant strain with high yield of glucoamylase and application thereof
  • Aspergillus niger mutant strain with high yield of glucoamylase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Mutagenesis and screening of the strain of Example 1

[0034] 1.1 Preparation of mutagen and antidote

[0035] Mutagen: in a biological safety cabinet, draw 0.5 mL of diethyl sulfate into a 50 mL centrifuge tube, add 2 mL of absolute ethanol, fully dissolve the diethyl sulfate, and then add sterile water to make the volume to 20 mL, put in Locked storage in a dedicated refrigerator.

[0036] Antidote: Use sterile water to prepare 25% sodium thiosulfate and store it in a brown bottle.

[0037] 1.2 Preparation of mutagenic strains

[0038] Solid slant activated strain: Take the cryopreserved glycerol tube seeds of Aspergillus niger origin strain CGMCC No. 10788, spread it on the seed slant medium, and cultivate at 34°C for 5 days.

[0039] Seed slant medium formula: sodium nitrate 0.43%, dipotassium hydrogen phosphate 0.2%, magnesium sulfate 0.06%, potassium chloride 0.05%, ferrous sulfate 0.01%, sucrose 2.1%, agar powder 1.5%, the rest is water, pH is natural .

[0...

Embodiment 2

[0049] Example 2 Production of saccharification enzyme by liquid fermentation

[0050] Solid slant activated strain: Take the frozen glycerol tube seeds of Aspergillus niger mutant strain GA-9216, spread them on the seed slant medium, and cultivate at 35°C for 5 days.

[0051] Seed slant medium formula: sodium nitrate 0.2%, dipotassium hydrogen phosphate 0.2%, magnesium sulfate 0.06%, potassium chloride 0.03%, ferrous sulfate 0.01%, sucrose 2.1%, agar powder 2.5%, the rest is water, pH is natural .

[0052] Liquid seed culture: on the above-mentioned seed sloping medium, pick about 2cm*2cm of bacterial grass, insert into 200mL liquid seed medium (use 500mL conical flask), 35 ° C, shaker rotation speed 300 rev / min, cultivate to The solid content of bacteria in the seed solution was 40%.

[0053] Liquid seed medium: sodium nitrate 0.5%, dipotassium hydrogen phosphate 0.2%, magnesium sulfate 0.16%, potassium chloride 0.03%, ferrous sulfate 0.09%, sucrose 4.8%, the rest is water...

Embodiment 3

[0063] Embodiment 3 Enzyme activity assay method adopted in the present invention

[0064] 3.1 Determination method of glucoamylase

[0065] 1) Definition of enzyme activity: 1g solid enzyme powder (or 1ml liquid enzyme), under certain conditions (if not specified, the conditions are: 40°C, pH value 4.6), 1h decomposes soluble starch to produce 1mg glucose, which is 1 Enzyme activity unit, expressed in U / g (U / mL).

[0066] 2) Principle

[0067] Saccharification enzymes have the function of catalyzing the hydrolysis of starch, which can start from the non-reducing end of starch molecules and decompose α-1,4-glucosidic bonds to generate glucose. Glucose molecules contain aldehyde groups, which can be oxidized by sodium hypoiodate. After acidification of excess sodium hypoiodate, iodine is precipitated, and then titrated with sodium thiosulfate standard solution to calculate the enzyme activity.

[0068] 3) Reagents and solutions

[0069] ①Acetic acid-sodium acetate buffer so...

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Abstract

The invention provides an Aspergillus niger GA-9216 with high yield of glucoamylase, and the preservation number of the Aspergillus niger GA-9216 is CGMCC No.23220. The activity of the saccharifying enzyme produced by the strain GA-9216 can be up to 190500U / mL, the optimum action temperature is 80 DEG C, the optimum action pH is 6.0, the enzymatic property has high integrating degree with the liquefaction process of the current related industries such as starch sugar and fermentation, in the industries, before the saccharifying enzyme is added, the temperature of liquefied starch slurry is greater than 75 DEG C, the pH is about 5.5, and when the saccharifying enzyme produced by the strain is added, cooling and pH regulation are not needed, and the cost is saved. The enzyme activity of [alpha]-glucosidase in fermentation liquor of the strain is remarkably reduced, so that when glucoamylase produced by the strain is used for producing glucose, the catalytic efficiency is greatly improved, and the DE value reaches 98% within 1 hour; and the saccharifying enzyme is high in specificity and can efficiently decompose [alpha]-1, 4-glucosidic bonds, and the content of glucose in the product is as high as 94%.

Description

Technical field: [0001] The invention belongs to the technical field of fermentation engineering, in particular to an Aspergillus niger mutant strain with high saccharification enzyme production and an industrial fermentation technology. Background technique: [0002] Glucoamylase is one of the enzyme preparations with the largest output and the widest application range in my country. Glucoamylase EC3.2.1.3 is the abbreviation of glucoamylase (abbreviated as GA or G), which is secreted by microorganisms and is the main enzyme for hydrolyzing starch. The non-reducing end hydrolysis of α-1,4 glycosidic bonds releases individual β-D-glucose one by one. It has been widely used in starch processing, brewing and fermentation industry, textile industry, bread industry, pharmaceutical industry, feed industry and other industries. [0003] Aspergillus, a common filamentous fungus, is widely used in the production of glucoamylase due to its strong protein secretion ability and good ...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N9/34C12N15/01C12P19/20C12P19/02C12R1/685
CPCC12N1/14C12N9/2428C12N15/01C12P19/20C12P19/02C12Y302/01003Y02E50/10
Inventor 钱娟娟佟新伟郭庆文王克芬王兴吉刘文龙
Owner SHANDONG LONGKETE ENZYME PREPARATION