Preparation method of nuclease P1

A nuclease and ribonucleic acid technology, applied in the field of preparation of nuclease P1, can solve the problems of low enzyme activity and long time consumption, and achieve the effects of improving fermentation enzyme activity, free of cost, and shortening fermentation time.

Active Publication Date: 2022-01-28
NANJING UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The production of nuclease P1 at home and abroad is mainly through liquid submerged fermentation of Penicillium citrinum, and the existing method takes a long time and has low enzyme activity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The ribonucleic acid powder was dissolved into 4% concentration (mass ratio) with deionized water, and the pH was adjusted to 5.0 with NaOH, and the temperature was raised to 70° C. After stabilizing for 30 min, nuclease P1 powder was added (nuclease P1: ribonucleic acid=1:100 , the enzyme activity of nuclease P1 is 100,000 U / g), and the enzymolysis is carried out under stirring at 100 rpm, and the enzymolysis ends after 3 hours.

[0034] Cool the enzymolysis solution to 20°C, use a roll-type polysulfone ultrafiltration membrane with a molecular weight of 3000 Daltons to ultrafilter the enzymolysis solution to 1 / 10 of the original volume, add water to the original volume, and then ultrafilter to 1 / 10 of the original volume 10. Repeat adding water and ultrafiltration again. The obtained ultrafiltration membrane retentate is the fermentation accelerator 1.

Embodiment 2

[0036] The ribonucleic acid powder was dissolved into 4% concentration (mass ratio) with deionized water, and the pH was adjusted to 5.0 with NaOH, and the temperature was raised to 70° C. After stabilizing for 30 min, nuclease P1 powder was added (nuclease P1: ribonucleic acid=1:100 , the enzyme activity of nuclease P1 is 100,000 U / g), and the enzymolysis is carried out under stirring at 100 rpm, and the enzymolysis ends after 3 hours.

[0037] Cool the enzymolysis solution to 20°C, use a roll-type polysulfone ultrafiltration membrane with a molecular weight of 6000 Daltons to ultrafilter the enzymolysis solution to 1 / 10 of the original volume, add water to the original volume, and then ultrafilter to 1 / 10 of the original volume 10. Repeat adding water and ultrafiltration again. The obtained ultrafiltration membrane retentate is the fermentation accelerator 2.

Embodiment 3

[0039] The ribonucleic acid powder was dissolved into 4% concentration (mass ratio) with deionized water, and the pH was adjusted to 5.0 with NaOH, and the temperature was raised to 70° C. After stabilizing for 30 min, nuclease P1 powder was added (nuclease P1: ribonucleic acid=1:100 , the enzyme activity of nuclease P1 is 100,000 U / g), and the enzymolysis is carried out under stirring at 100 rpm, and the enzymolysis ends after 3 hours.

[0040] Cool the enzymolysis solution to 20°C, use a roll-type polysulfone ultrafiltration membrane with a molecular weight of 10,000 Daltons to ultrafilter the enzymolysis solution to 1 / 10 of the original volume, add water to the original volume, and then ultrafilter to 1 / 10 of the original volume 10. Repeat adding water and ultrafiltration again. The obtained ultrafiltration membrane retentate is the fermentation accelerator 3.

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Abstract

The invention relates to the technical field of biology, and particularly discloses a preparation method of nuclease P1. Penicillium citrinum is fermented in a fermentation culture medium containing a fermentation accelerant to prepare a fermentation liquor containing nuclease P1. A preparation method of the fermentation accelerant comprises the following steps: S1, carrying out enzymolysis on ribonucleic acid, so as to obtain enzymatic hydrolysate; and S2, carrying out ultrafiltration on the enzymatic hydrolysate obtained in step S1 through an ultrafiltration membrane to obtain a retentate which is the fermentation accelerant. The fermentation accelerant is added for fermentation, the single-batch fermentation time is shortened to 18 h from 55 h, and the enzyme activity is improved to 7300-8200 U / mL from 2500 U / mL. According to the method, the fermentation time can be remarkably shortened, the fermentation enzyme activity can be remarkably improved, the addition amount of the used fermentation accelerant is small, meanwhile, the used fermentation accelerant is waste in the nucleotide production process, and the method is free of cost, easy to operate and remarkable in effect.

Description

technical field [0001] The invention relates to the field of microbial fermentation, in particular to a preparation method 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, UD...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12N9/22C12N1/38C12N1/14C12R1/80
CPCC12N9/22C12N1/38C12N1/14C12Y301/30001Y02A50/30
Inventor陈晓春张磊汤亦文滕红兵蔡家栋
OwnerNANJING UNIV OF TECH