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Freeze-drying process of recombinant protease K

A protease and freeze-drying technology, which is applied in the freeze-drying process of recombinant proteinase K, can solve the problems of easy moisture absorption, unfavorable long-term storage, and poor reconstitution and solubility of freeze-dried powder, and achieve short freeze-drying time, easy-to-dissolve shelf life, low cost effect

Active Publication Date: 2022-04-05
合肥巅峰生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There are not many reports on the freeze-drying process of proteinase K. The re-enactment of the early freeze-drying process found that the freeze-drying time is long, the reconstitution and solubility are poor, and the freeze-dried powder is easy to absorb moisture, which is not conducive to long-term storage; in summary, the research and development of a A freeze-drying process with simple operation, low cost, short freeze-drying time, easy dissolution of the product and long shelf life, and its application to the industrialized freeze-drying of recombinant proteinase K is particularly important, and is urgently needed by those skilled in the art. key technical issues

Method used

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  • Freeze-drying process of recombinant protease K

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Comparison scheme
Effect test

Embodiment 1

[0023] The present embodiment provides a kind of lyophilization process of recombinant proteinase K, comprising the following steps:

[0024] S1. Use affinity chromatography to purify, put the solution into a dialysis bag, add peg20000 to the dialysis bag, concentrate, and detect, control the activity of the solution to 4000u / ml. ;

[0025] S2. Add equal proportions of protease freeze-dried storage solution; the protease freeze-dried storage solution includes: 3w / v% of sucrose, 2.5w / v% of trehalose, 0.3w / v% of mannitol, and 0.1w / v% of sorbitol , tert-butanol 0.7v / v%, Tween-80 0.05v / v% and PVP 0.1w / v%;

[0026] S3. Freeze-drying procedure: first, add 300mm*400mm*10mm protease solution to a 300mm*400mm tray, freeze at -30°C, and keep it for 2 hours for pre-freezing. After pre-freezing, start vacuuming and open Heating program, the temperature rising curve of the lyophilization is: keep at -20°C for 1h, keep at -10°C for 6h, keep at 0°C for 4h, keep at 5°C for 2h, keep at 10°C ...

Embodiment 2

[0028] The present embodiment provides a kind of lyophilization process of recombinant proteinase K, comprising the following steps:

[0029] S1. Use affinity chromatography to purify, put the solution into a dialysis bag, add peg20000 to the dialysis bag, concentrate, and detect, control the activity of the solution to 4000u / ml. ;

[0030] S2. Add an equal proportion of protease freeze-dried storage solution; the protease freeze-dried storage solution includes: sucrose 1w / v%, trehalose 1w / v%, mannitol 0.1w / v%, sorbitol 0.04w / v%, tert-butanol 0.5v / v%, Tween-80 0.01v / v% and PVP 0.05w / v%;

[0031] S3. Freeze-drying procedure: First, add 300mm*400mm*10mm protease solution to the tray of 300mm*400mm, freeze at -35°C, and keep it for 1.5 hours for pre-freezing. After pre-freezing, turn on the vacuum pump and open the The heating program, the temperature rising curve of the lyophilization is: keep at -25°C for 0.5h, keep at -15°C for 5h, keep at -5°C for 3h, keep at 0°C for 1h, ke...

Embodiment 3

[0033] The present embodiment provides a kind of lyophilization process of recombinant proteinase K, comprising the following steps:

[0034] S1. Use affinity chromatography to purify, put the solution into a dialysis bag, add peg20000 to the dialysis bag, concentrate, and detect, control the activity of the solution to 4000u / ml. ;

[0035] S2. Add an equal proportion of protease freeze-dried storage solution; the protease freeze-dried storage solution includes: sucrose 5w / v%, trehalose 4w / v%, mannitol 0.5w / v%, sorbitol 0.2w / v%, tert-butanol 1v / v%, Tween-80 0.1v / v% and PVP 0.2w / v%;

[0036] S3. Freeze-drying procedure: First, add 300mm*400mm*10mm protease solution to the tray of 300mm*400mm, freeze at -25°C, and keep it for 2.5 hours for pre-freezing. After pre-freezing, turn on the vacuum and open the The heating program, the temperature rising curve of the lyophilization is: keep at -15°C for 1.5h, keep at -5°C for 7h, keep at 5°C for 5h, keep at 10°C for 3h, keep at 15°C ...

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Abstract

The invention provides a freeze-drying process of recombinant protease K. The freeze-drying process comprises the following steps: S1, purifying a protease k solution, and concentrating the protease k solution; s2, adding a protease freeze-drying preservation solution in equal proportion; s3, performing a freeze-drying procedure: firstly adding a protease solution for pre-freezing, and then performing freeze-drying in a vacuum state, wherein a freeze-drying temperature curve is as follows: keeping for 0.5-1.5 hours at-25 DEG C to-15 DEG C, keeping for 5-7 hours at-15 DEG C to-5 DEG C, keeping for 3-5 hours at-5 DEG C to 5 DEG C, keeping for 1-3 hours at 0-10 DEG C, keeping for 1-3 hours at 5-15 DEG C, and keeping for 7-9 hours at 10-20 DEG C; and then freeze-drying is finished. The freeze-drying process of the recombinant protease K provided by the invention is simple to operate, low in cost and short in freeze-drying time, and the product is easy to dissolve and long in shelf life.

Description

technical field [0001] The invention relates to the technical field of enzyme freeze-drying, in particular to a freeze-drying process of recombinant proteinase K. Background technique [0002] Proteinase K was first discovered in 1974 in the extract of Candida albicans (tritirachium albumimber), because this enzyme can digest natural keratin (Keratin) rich in disulfide bonds, it can use keratin as the only carbon source and Nitrogen source growth, proteinase K is also named after this. [0003] Proteinase K is a serine protease with proteolytic activity and broad-spectrum cutting ability to natural proteins, preferring to cut aliphatic amino acids and aromatic amino acids, especially the carboxy-terminal peptide bond of alanine. [0004] Proteinase K can maintain activity in a wide range of environments, and it is also active in pH 4-12.5, high-salt buffer and 70°C environment, and can react with denaturants and detergents (such as urea, SDS, guanidine hydrochloride, Guani...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/60C12N9/96
CPCY02P60/85
Inventor 罗强梁良丁海平
Owner 合肥巅峰生物科技有限公司
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