A method for multistage purification of nuclease p1

A technology of nuclease and nuclease crude enzyme, which is applied in the biological field, can solve the problems of small sample processing volume, low titer, and low sample recovery rate, and achieve the effects of efficient separation and purification, low cost, and improved purity

Active Publication Date: 2022-05-17
NANJING TECH UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the nuclease P1 crude enzyme contains more miscellaneous enzymes, which has a greater impact on the efficiency of enzymatic hydrolysis, and generally needs to be purified before use.
[0004] At present, the methods of separating nuclease P1 such as ammonium sulfate salting-out method and centrifugal spray drying method have the problems of low sample recovery, expensive separation materials or equipment, small sample processing capacity, and low separation purity by a single method; The nuclease P1 obtained by the method separation method contains more impurities, and the titer is low, and cannot be directly applied in the food industry

Method used

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  • A method for multistage purification of nuclease p1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Purify nuclease P1 as follows:

[0053] 1. Prepare nuclease P1 crude enzyme solution by fermenting mold, the enzyme activity is 5600U / mL, and the specific activity is 852U / g.

[0054] 2. Separating the solid-liquid of the crude enzyme liquid obtained in step 1, collecting the filtrate, and ultrafiltering it with an ultrafiltration membrane with a molecular weight of 3KD; specifically, comprising the following steps:

[0055] (i) put the filtrate in an ultrafiltration membrane, and perform ultrafiltration at 30° C., and ultrafilter until the retentate is 1 / 4 of the volume of the filtrate, and collect the retentate;

[0056](ii) continue replenishing water to the volume of the filtrate described in step (i) in the retentate, then ultrafilter until the retentate is 1 / 4 of the initial volume, and collect the retentate;

[0057] (iii) Step (ii) is repeated, and the cycle ends for 2 times, and the retentate is collected.

[0058] 3. Transfer the retained liquid obtained in ...

Embodiment 2

[0062] Purify nuclease P1 as follows:

[0063] 1. Prepare nuclease P1 crude enzyme solution by fermenting mold, the enzyme activity is 5900U / mL, and the specific activity is 863U / g.

[0064] 2. Separating the solid-liquid of the crude enzyme liquid obtained in step 1, collecting the filtrate, and ultrafiltering it with an ultrafiltration membrane with a molecular weight of 6KD; specifically, comprising the following steps:

[0065] (i) Put the filtrate in the ultrafiltration membrane, at 32°C, perform ultrafiltration, ultrafiltration until the retentate is 1 / 4 of the volume of the filtrate, and collect the retentate;

[0066] (ii) continue replenishing water to the volume of the filtrate described in step (i) in the retentate, then ultrafilter until the retentate is 1 / 4 of the initial volume, and collect the retentate;

[0067] (iii) Step (ii) is repeated, the cycle is completed for 3 times, and the retentate is collected.

[0068] 3. Transfer the retained liquid obtained in...

Embodiment 3

[0072] Purify nuclease P1 as follows:

[0073] 1. Prepare nuclease P1 crude enzyme solution by fermenting mold, the enzyme activity is 6200U / mL, and the specific activity is 912U / g.

[0074] 2. Separating the solid-liquid of the crude enzyme liquid obtained in step 1, collecting the filtrate, and ultrafiltering it with an ultrafiltration membrane with a molecular weight of 12KD; specifically, comprising the following steps:

[0075] (i) put the filtrate in an ultrafiltration membrane, perform ultrafiltration at 33° C., and ultrafilter until the retentate is 1 / 4 of the volume of the filtrate, and collect the retentate;

[0076] (ii) continue replenishing water to the volume of the filtrate described in step (i) in the retentate, then ultrafilter until the retentate is 1 / 4 of the initial volume, and collect the retentate;

[0077] (iii) Step (ii) is repeated, the cycle is completed 4 times, and the retentate is collected.

[0078] 3. Transfer the retained liquid obtained in st...

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Abstract

The invention discloses a method for multistage purification of nuclease P1, that is, performing multistage purification of nuclease P1 crude enzyme solution through ultrafiltration membrane ultrafiltration, heat inactivation and resin impurity removal. The method of the invention can efficiently separate and purify the nuclease P1, and the enzyme recovery rate of the nuclease P1 reaches 82.6 percent; and the method has low cost and is applicable to large-scale industrial production. At the same time, the purity of the nuclease P1 obtained by the method of the invention is greatly improved, the specific activity is 7.3 times that of the crude enzyme solution, and there are few impurities, so it can be used in the food industry.

Description

technical field [0001] The invention relates to the field of biology, in particular to a method for multistage purification of nuclease P1. Background technique [0002] With the development of biotechnology industry and pharmaceutical industry, the research and development of nucleotides and their derivatives has become a hot spot, and gradually formed another major industry after amino acids. Nucleotides are a class of biochemical substances with high added value. They have a wide range of uses and support the development of some large industries, such as high-end dairy and pharmaceutical industries. As a functional additive in the dairy industry, its addition can make milk powder closer to breast milk, effectively enhance the ability of infants and young children to resist bacillary dysentery, and reduce the occurrence of diarrhea; Raw materials for the production of glycoside derivatives, used for the synthesis of CDP, UDP, GTP, CDP-choline, SAM, cGMP, CTP, UTP, cAMP, U...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/22C12R1/80
CPCC12N9/22Y02P20/10
Inventor 陈晓春张磊汤亦文李梓阳蔡家栋
Owner NANJING TECH UNIV
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