Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Protease K freeze-drying protective agent and preparation method thereof

A freeze-drying protective agent and protease technology, applied in biochemical equipment and methods, freeze-drying transportation, peptide/protein components, etc., can solve the problems of cumbersome preparation process, achieve good solubility, good appearance, and improve storage stability Effect

Pending Publication Date: 2021-11-26
WUHAN HANHAI NEW ENZYMES BIOLOGICAL TECH CO LTD
View PDF5 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some enzyme lyoprotectants disclosed in the prior art usually require more than 10 formulations to achieve a relatively stable protective effect, and the deployment process is cumbersome

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Protease K freeze-drying protective agent and preparation method thereof
  • Protease K freeze-drying protective agent and preparation method thereof
  • Protease K freeze-drying protective agent and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] The separation and purification of embodiment 1 proteinase K

[0088]Extracting proteinase K: using yeast secretion expression or other expression systems to prepare proteinase K, the proteinase K supernatant in the lower tank of fermentation (the supernatant of proteinase K-containing fermentation products after centrifugation to remove microbial cells) can be obtained in the following ways Purified enzyme solution.

[0089] 1. Coarse separation:

[0090] (a) Salting-out method: using ammonium sulfate to fractionally precipitate proteins. First, prepare a saturated ammonium sulfate solution (usually the saturation is 33% to 50%), slowly add an equal volume of saturated ammonium sulfate solution to the protein supernatant with a protein concentration of 20 mg / mL while stirring, and place the solution at 4 ℃, stir with a magnetic stirrer for 6 hours or overnight to fully precipitate the protein; centrifuge the protein solution at 12,000g at 4°C for 20min, discard the s...

Embodiment 2

[0096] The preparation method of embodiment 2 freeze-dried powder:

[0097] The purified enzyme solution prepared in Example 1 is mixed alone or with a protective agent, and poured into a clean and sterile glass plate with a glass bottom diameter of 14 cm to ensure that the liquid level is no more than 1 cm to ensure the best sublimation effect (liquid level too high, too much liquid volume will affect the sublimation efficiency), and then freeze-dried on the plate layer of the freeze dryer.

[0098] The freeze dryer program is set as follows;

[0099] a. Pre-freezing stage: set the layer temperature at -45°C for 2.5 hours; annealing temperature at -25°C for 2 hours; then lower the temperature to -45°C for 3 hours; return temperature to 1°C / min;

[0100] b. Primary sublimation: set the vacuum value below 20pa, rise from -45°C to -18°C in step a for 22 hours; in the second stage, the temperature of the sheet layer rises to -15°C, and the duration is 12h; in the third stage, th...

Embodiment 3

[0103] Example 3 Screening of lyoprotectant components and preparation of lyophilized enzyme powder

[0104] In 50mM pH7.0 HEPES buffer, sucrose, trehalose, maltose, inositol, glycine, L-serine, α-alanine, PEG6000, PEG8000, BSA and glycerol were used as lyoprotectants respectively, and the specific activity Proteinase K with a concentration of 48U / mg and a concentration of 60mg / mL was mixed with an equal volume of 30mg / mL sucrose, 30mg / mL trehalose, 30mg / mL maltose, 30mg / mL inositol, 30mg / mL glycine, 30mg / mL L-serine, 30mg / mL α-alanine, 3mg / ml PEG6000, 3mg / ml PEG8000, 30mg / ml BSA, 20% glycerol were mixed, and 1‰ volume of 1M CaCl was added 2 Solution, so that the final concentration of proteinase K in the solution is 30mg / mL, the final concentration of buffer solution HEPES (pH 7.0) is 50mM, Ca 2+ The concentration is 5mM. The freeze-dried enzyme powder was prepared according to the steps in Example 2, and the enzyme activity of the freeze-dried enzyme powder and the proteas...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
buffering capacityaaaaaaaaaa
Login to View More

Abstract

The invention discloses a freeze-drying protective agent for protease K and a preparation method, and belongs to the technical field of biology. According to the invention, Ca2<+> and one or more of cane sugar, trehalose, maltose, glycine, L-serine and alpha-alanine are used as freeze-drying protective agent components, the freeze-drying survival rate of protease K in the vacuum freeze-drying process can be effectively improved, and the storage stability of protease K freeze-dried powder is improved. The residual activity of the protease K freeze-dried powder prepared by applying the freeze-drying protective agent disclosed by the invention can be maintained at 90% or above after accelerated incubation for 14 days at the temperature of 42 DEG C; after the protease K freeze-dried powder is stored in a refrigerator at the temperature of minus 20 DEG C for 1 year, the enzyme activity of the protease K freeze-dried powder is maintained at 90% or above; the enzyme powder can still ensure good solubility at the concentration of 60 mg / mL; the redissolution time in water does not exceed 10 seconds; and the specific activity of a protein is greater than 40 U / mg.

Description

technical field [0001] The invention relates to a freeze-drying protective agent for proteinase K and a preparation method thereof, belonging to the field of biotechnology. Background technique [0002] Proteinase K, a serine protease related to subtilisin, is a major class of proteases produced by Tritirachium album Limber. Because the microorganisms that can synthesize this kind of protease can grow in the environment with keratin (Kerantin) as the only carbon and nitrogen source, it is called proteinase K. [0003] Proteinase K has high-efficiency enzymatic activity and broad substrate specificity. It can preferentially decompose ester bonds and peptide bonds adjacent to the C-terminals of hydrophobic amino acids, sulfur-containing amino acids, and aromatic amino acids. It is often used to degrade proteins to produce short peptides. It has the typical catalytic triad Asp39-His69-Ser224 characteristic of serine proteases and has two Ca around the active center 2+ The bin...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/48A61K9/19A61K47/26A61K47/18A61K47/10A61K47/42
CPCA61K38/482C12Y304/21014A61K9/19A61K47/26A61K47/183A61K47/10A61K47/42
Inventor 罗漫杰施婧妮雷宵宫安王梁鄢文琪
Owner WUHAN HANHAI NEW ENZYMES BIOLOGICAL TECH CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com