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Lysozyme mutant with improved specific activity

A lysozyme and specific activity technology, applied in the field of bioengineering, can solve the problems of less preparation, poor thermal stability, inability to meet the requirements, and achieve the effects of rapid growth, low production cost and high safety.

Active Publication Date: 2020-05-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the lysozyme sold on the market is mainly extracted from egg whites and animal organs. This kind of lysozyme has poor thermal stability and its activity is only half of that of human lysozyme.
However, human lysozyme is limited by factors such as raw material sources, purification and refining costs, etc., and the amount of preparation is small, which cannot meet the needs of various fields

Method used

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  • Lysozyme mutant with improved specific activity
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Construction of lysozyme expression vector pPIC9K-m5HLM fused to pentapeptide

[0028] Lysozyme exists in large quantities in nature and has a significant bactericidal effect. In order to further expand the antibacterial spectrum and antibacterial function of lysozyme, it is necessary to carry out molecular modification of lysozyme to develop a new type of lysozyme that is more suitable for modern production needs. The research on egg white lysozyme showed that the hydrophobic short peptide was connected to the C-terminus of egg white lysozyme through recombinant technology, and the bactericidal activity of the modified lysozyme was found to be significantly enhanced against Escherichia coli. Both human lysozyme and egg white lysozyme belong to c-type lysozyme, so the C-terminus of human lysozyme is also hydrophobically modified to improve its antibacterial effect. Three hydrophobic short peptides of different lengths were selected for C-terminal fusion, namel...

Embodiment 2

[0030] Example 2 Construction of recombinant Pichia pastoris genetic engineering strain KM71-pPIC9K-m5HLM expressing m5HLM lysozyme

[0031] The plasmid pPIC9K-m5HLM was digested with SacI and linearized, then electrotransformed into Pichia pastoris KM71, and coated with a YPD-resistant plate (peptone 20g L -1 , yeast extract 10g·L -1, glucose 20g·L -1 , 20g·L -1 Agar powder, G418 1000μg·mL -1 ) were cultured at 30°C for 48 hours, and a single colony was picked from the plate to obtain the recombinant strain KM71-pPIC9K-m5HLM. Inoculate a single colony in a 250mL shake flask containing 25mL of antibiotic-free YPD medium, culture it at 30°C and 200 rpm for 48 hours, collect the bacteria by centrifugation, and add it to 25mL of YP medium (peptone 20g L -1 , yeast extract 10g·L -1 ), cultured at 28°C and 200 rpm for 72 hours, supplemented with methanol at a final concentration of 1% (v / v) every 12 hours, and the fermentation supernatant obtained by centrifugation after induc...

Embodiment 3

[0032] Example 3 Fermentation of recombinant bacterial strain KM71-pPIC9K-m5HLM

[0033] The recombinant strain KM71-pPIC9K-m5HLM was expanded in fermenter respectively. Draw 100 μL of the bacterial liquid in the glycerol tube and inoculate it into 100 mL of YPD medium and cultivate it for about 18 hours as the seed culture liquid, and then inoculate the seed culture liquid into the 1L basal salt fermentation medium (K 2 SO 4 18.2g·L -1 , MgSO 4 ·7H 2 O 14.9g·L -1 , CaSO 4 2H 2 O 0.93g·L -1 , KOH 4.13g·L -1 , 85%H 3 PO 4 26.7mL·L -1 , glycerol 30g·L -1 , PTM 1 4.35mL·L -1 ) for fermentation.

[0034] The 3L tank culture is divided into two stages: the first stage is the glycerol phase culture, at this time, 50% glycerol is added as the carbon source, the temperature is 30°C, the pH is 5.5, the dissolved oxygen is more than 20%, and the culture is to OD 600 When it is approximately equal to 100, it begins to enter the second stage of methanol induction phase;...

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Abstract

The invention discloses a lysozyme mutant with improved specific activity, belonging to the technical field of bioengineering. The invention discloses the human lysozyme mutant which is obtained by adding hydrophobic oligopeptide Val-Ile-Pro-Leu-Phe to the C end of human lysozyme through genetic engineering. The specific activity of the human lysozyme mutant is improved by 126%. The invention alsodiscloses a recombinant plasmid containing a mutant human lysozyme gene, and a recombinant genetically-engineered pichia pastoris strain which is obtained by transforming pichia pastoris with the recombinant plasmid and capable of efficiently expressing the human lysozyme mutant. The obtained human lysozyme mutant has the characteristics of high specific activity and simple preparation, has potential clinical application value, and also has wide application in feed and food industries.

Description

technical field [0001] The invention relates to a lysozyme mutant with improved specific activity, belonging to the technical field of bioengineering. Background technique [0002] Lysozyme, also known as muramidase or N-acetyl murein hydrolase, can hydrolyze the β-1, 4 glycosidic bonds in bacterial cell walls, destroy the structure of cell wall peptidoglycan, and thus protect host cells from bacterial infection. Human lysozyme belongs to c-type lysozyme, which consists of 130 amino acids and has a relative molecular weight of 14700. Human lysozyme also has anti-virus, immune-enhancing and anti-tumor effects. As a natural protein, lysozyme can be digested and absorbed as a nutrient in the gastrointestinal tract. It is non-toxic to humans and animals, and will not remain in the body. It is a highly safe drug, feed and food additive. . In animal husbandry, lysozyme can be used as feed preservative and fungicide. In the food industry, lysozyme can be added to food as an ant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/36C12N15/56C12N15/81C12N1/21A23L3/3571A23L27/30C12R1/84
CPCC12N9/2462C12Y302/01017C12N15/815A23L3/3571A23L27/31A23V2002/00A23V2200/10A23V2250/54A23V2250/24
Inventor 吴丹郑璞陈鹏程张莉芝
Owner JIANGNAN UNIV
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