Method for separating and purifying alpha-amylase by virtue of two aqueous phase extraction

A separation and purification, two-phase technology, applied in the field of separation and purification of α-amylase, can solve the problems of small sample processing volume, α-amylase with many impurities, and low titer, and achieve low cost, high-efficiency separation and purification, and impurity little effect

Inactive Publication Date: 2011-10-19
HEILONGJIANG UNIV
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  • Application Information

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Problems solved by technology

[0004] The present invention aims to solve the problems of low sample recovery rate, expensive separation materials or equipment, small amount of sample processing, and low separation purity in a single method in current separation methods such as ammonium sulfate salting-out method and centrifugal spray drying method. The α-amylase obtained by the separation method of ammonium sulfate salting-out method contains more impurities, has low titer, and cannot be directly applied to the defects in the food industry, and a method for separating and purifying α-amylase by two-phase extraction is provided

Method used

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specific Embodiment approach 1

[0013] Specific embodiment one: In this embodiment, two-phase extraction and separation of α-amylase is carried out according to the following steps:

[0014] 1. Prepare fungal α-amylase crude enzyme solution;

[0015] 2. Preparation of PEG / (NH 4 ) 2 SO 4 Aqueous two phase, PEG / (NH 4 ) 2 SO 4 The aqueous two phase consists of 34% polyethylene glycol 1500 (PEG1500), 24% (NH 4 ) 2 SO 4 , 20% NaCl and the balance of deionized water;

[0016] 3. According to the fungal α-amylase crude enzyme solution and PEG / (NH 4 ) 2 SO 4 The two-phase aqueous phase was uniformly mixed at a weight ratio of 1:1, and then adjusted to a pH value of 6.5, then allowed to stand at room temperature for 2 minutes, and the PEG / (NH 4 ) 2 SO 4 The upper liquid phase in the two aqueous phases;

[0017] 4. The collected PEG / (NH 4 ) 2 SO 4 The upper liquid phase of the two-phase aqueous phase and the mass concentration of 35% (NH 4 ) 2 SO 4 The aqueous solution constitutes a two-phase puri...

specific Embodiment approach 2

[0021] Specific embodiment two: the difference between this embodiment and specific embodiment one is: use Aspergillus oryzae 2197, Aspergillus oryzae ZLA16, Aspergillus oryzae ZLB35, Aspergillus oryzae ZLC06, Aspergillus oryzae ZLD14, Aspergillus oryzae ZLE09 or Aspergillus oryzae ZLF13 fermentation in step one Crude enzyme solution of fungal alpha-amylase. Other steps and parameters are the same as those in Embodiment 1.

specific Embodiment approach 3

[0022] Embodiment 3: The difference between this embodiment and Embodiment 1 or 2 is that in Step 4, the lower phase liquid of the two-phase purification system is taken, put into a dialysis bag and concentrated by dialysis with PEG6000. Other steps and parameters are the same as those in Embodiment 1 or 2.

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Abstract

The invention discloses a method for separating and purifying alpha-amylase by virtue of two aqueous phase extraction, and relates to a method for separating and purifying alpha-amylase. The method can be used for solving the problems of low sample recovery rate, expensive separation material or equipment, small sample processing amount and low separation purity of single method in the existing amylase separating method such as ammonium sulfate salting out method, centrifugal spray drying method and the like, as well as the defects that the alpha-amylase obtained by the ammonium sulfate salting out method contains more impurities, has low potency and cannot be directly applied to the food industry. The method for separating and purifying alpha-amylase by virtue of two-aqueous phase extraction comprises the following steps: (1) preparing a coarse enzyme liquid of fungus alpha-amylase; (2) preparing two aqueous phases of PEG (polyethylene glycol)/(NH4)2SO4; (3) extracting; and (4) purifying. The method disclosed by the invention can be used for separating and purifying alpha-amylase.

Description

technical field [0001] The invention relates to a method for separating and purifying alpha-amylase. Background technique [0002] Fungal α-amylase is an α-amylase produced by the fermentation of Aspergillus microorganisms. Different from bacterial α-amylase, the optimum temperature of fungal α-amylase is about 55°C, and it will be inactivated when it exceeds 60°C; the products of its hydrolyzed starch are mainly high content of maltose, some oligosaccharides and a small amount of glucose. The optimum temperature for bacterial α-amylase is high (medium-temperature α-amylase is 70-80°C, high-temperature resistant α-amylase is 95-105°C), and the main product of hydrolyzing starch is dextrin. Therefore, bacterial α-amylase can only be used in the fermentation industry, while fungal α-amylase is widely used in the production of starch syrup, oligosaccharides, beer, baked goods, flour products, etc., and has a very broad market prospect. [0003] At present, the ammonium sulfa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/30C12R1/69
Inventor 葛菁萍宋刚凌宏志董海丽平文祥
Owner HEILONGJIANG UNIV
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