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Heat-resisting lysozyme, and preparation method and applications thereof

A lysozyme and heat-resistant technology, applied in the field of bioengineering, can solve the problems of the target product lysozyme, such as unsatisfactory purity and yield, imperfect production process, and damaged vitality.

Active Publication Date: 2021-05-18
江苏海枫达生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently the most widely used is egg white lysozyme, which is rich in egg white, has significant antibacterial function, is easy to extract, and has low production cost. However, egg white lysozyme has poor tolerance to heat stability and denaturants. It is used as feed As an additive, there is a high-temperature treatment step in the feed pelleting process, because egg white lysozyme cannot tolerate high temperature and its vitality is damaged, which seriously limits its application
In recent years, although microbial fermentation technology has gradually appeared on the market to produce recombinant human lysozyme, its tolerance to heat stability and denaturing agents has been enhanced, and its unit activity is higher than that of egg white lysozyme. Its heat resistance and related antibacterial properties need to be further improved; in addition, the existing industrial production process of lysozyme is not perfect, and its related process conditions need to be further optimized, and the separation and concentration technology of the target protein in the fermentation broth also exists Certain problems, which lead to the purity and yield of the target product lysozyme are all unsatisfactory

Method used

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  • Heat-resisting lysozyme, and preparation method and applications thereof
  • Heat-resisting lysozyme, and preparation method and applications thereof
  • Heat-resisting lysozyme, and preparation method and applications thereof

Examples

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Effect test

Embodiment 1

[0033] The construction of embodiment 1-Pichia pastoris HFD-01

[0034] In this example, Pichia pastoris GS115 was used as the starting strain, and Pichia pastoris HFD-01 was constructed by physical mutagenesis and temperature gradient acclimation. The specific steps include:

[0035] (1) UV mutagenesis + plasma mutagenesis

[0036] Inoculate Pichia pastoris GS115 on a YPD plate, culture at 28°C for 3 days, wash the bacteria with sterile water, make a suspension, and dilute to 1×10 6 cells / mL, alternately irradiate 15 times with UV lamp-incandescent lamp, among which: UV lamp (40W) is irradiated for 60s at a distance of about 20cm, and incandescent lamp (40W) is irradiated for 2min at a distance of about 20cm. After the irradiation is completed, add the bacterial liquid to the YPD liquid In the culture medium, plasma was irradiated at normal pressure and room temperature for 80s. The plasma mutagenesis conditions were: 110W, a distance of about 2mm, and a helium flow rate of ...

Embodiment 2

[0041] Embodiment 2-The construction of the recombinant bacterial strain expressing thermostable lysozyme

[0042] In this example, Pichia pastoris HFD-01 constructed in Example 1 is used to construct a recombinant strain expressing thermostable lysozyme, which specifically includes steps:

[0043] Synthesize the human lysozyme (HLM) gene fragment, preferably the human lysozyme gene with good heat resistance and stress resistance, and add enzyme cutting sites Xho I and Not I to its two ends, for example, for heat-resistant human lysozyme The gene of the enzyme is modified so that its amino acid site has the following two mutations: R41H (to improve the heat resistance of human lysozyme) and A92L (to provide tolerance to deforming agents); the above-mentioned gene fragments are inserted by double enzyme digestion In the plasmid pPicZαA, the expression vector pPicZαA-HLM was constructed; the expression vector pPicZαA-HLM was monoclonally amplified by Escherichia coli DH5α compet...

Embodiment 3

[0045] Example 3 - Industrial production of lysozyme using recombinant Pichia pastoris strains

[0046] This example uses the recombinant Pichia strain constructed and mutated by Pichia pastoris HFD-01 in Example 2 to carry out heat-resistant lysozyme ((genetic modification of human lysozyme, amino acid site mutation: R41H and A92L) The industrialized production of above-mentioned recombinant Pichia pastoris strain adopts the conventional operation in this field to be prepared into seed liquid standby, and the production technological process of heat-resistant lysozyme is as follows figure 1 As shown, it includes the following steps:

[0047] (1) Seed solution preparation: prepare medium, sterilize and cool down, divide the medium into sterile eggplant bottles to make a slope. After the eggplant bottle is cultured to confirm its sterility, pick the seed liquid and draw a line evenly on the slant medium. After the scribing is completed, place the slant in the incubator for cul...

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Abstract

The invention relates to heat-resisting lysozyme. The lysozyme is prepared by introducing human lysozyme genes into pichia yeast for expression; and the pichia yeast is HFD-01 and assigned the accession number CCTCC NO: M2021148. A preparation method of the heat-resisting lysozyme is also related. The preparation method includes adopting physical mutagenesis and temperature gradient acclimation to construct the pichia yeast HFD-01, introducing the heat-stable human lysozyme genes into the pichia yeast HFD-01 to construct recombinant pichia yeast strains and using the recombinant strains to perform fermentation and cultivation so as to obtain the heat-resisting lysozyme. Applications of the heat-resisting lysozyme in preparation of animal feed are related as well. Thus, the pichia yeast HFD-01 with good tolerance, heat resistance and fast growth velocity can be constructed; through the introduction of the heat-stable human lysozyme genes into the pichia yeast HFD-01, the strains that normally and rapidly grow and can efficiently and stably express lysozyme under high temperature can be obtained; and industrial fermentation conditions are optimized, so that the yield and purity of the lysozyme can be enhanced, and energy consumption can be reduced.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a heat-resistant lysozyme and its preparation method and application. Background technique [0002] Lysozyme, also known as muramidase or N-acetylmuramide glycanohydrlase, is an alkaline enzyme that can hydrolyze mucopolysaccharides in pathogenic bacteria. Destroy the β-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the cell wall, decompose the insoluble mucopolysaccharides in the cell wall into soluble glycopeptides, cause the cell wall to rupture, and the contents escape to dissolve the bacteria . Lysozyme can also directly combine with negatively charged viral proteins, and form double salts with DNA, RNA, and apoproteins to inactivate the virus. Therefore, lysozyme has antibacterial, anti-inflammatory, antiviral and other effects. [0003] Currently the most widely used is egg white lysozyme, which is rich in egg white, has significant a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/36C12N15/81A23K20/189A23K50/75A23K10/30A23K20/147A23K10/37A23K10/22A23K10/18A23K20/174A23K20/20
CPCA23K10/18A23K10/22A23K10/30A23K10/37A23K20/147A23K20/174A23K20/189A23K20/20A23K50/75C12N9/2462C12N15/815C12Y302/01017Y02P60/87
Inventor 陈绍帮陆荣亮李久银
Owner 江苏海枫达生物科技有限公司