CN112813055B - Heat-resistant lysozyme and preparation method and application thereof - Google Patents
Heat-resistant lysozyme and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a heat-resistant lysozyme which is prepared by introducing a humanized lysozyme gene into pichia pastoris for expression, wherein the pichia pastoris is HFD-01, and the preservation number is CCTCC NO: M2021148; the invention also relates to a preparation method of the heat-resistant lysozyme, which comprises the following steps: constructing pichia pastoris HFD-01 by adopting physical mutagenesis and temperature gradient domestication, introducing a thermostable humanized lysozyme gene into the pichia pastoris HFD-01 to construct a recombinant pichia pastoris strain, and performing fermentation culture by adopting the recombinant strain to obtain heat-resistant lysozyme; the invention also relates to application of the heat-resistant lysozyme in preparation of animal feed. The invention constructs pichia pastoris HFD-01 with good tolerance, heat resistance and high growth speed, and simultaneously introduces the heat stable human lysozyme gene into the pichia pastoris HFD-01 to obtain the bacterial strain which can normally and rapidly grow at a high temperature and can efficiently and stably express the lysozyme.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to heat-resistant lysozyme and a preparation method and application thereof.
Background
Lysozyme (also called muramidase) or N-acetylmuramidase (N-acetylmuramidase glycohydrolase) is an alkaline enzyme capable of hydrolyzing mucopolysaccharide in pathogenic bacteria, and mainly breaks beta-1, 4 glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine in cell walls to decompose insoluble mucopolysaccharide of the cell walls into soluble glycopeptides, so that the ruptured contents of the cell walls escape to dissolve the bacteria. Lysozyme can also be directly combined with virus protein with negative charge, and forms double salt with DNA, RNA and apoprotein to inactivate virus. Therefore, the lysozyme has the functions of antibiosis, antiphlogosis, antivirus, and the like.
The egg white lysozyme is widely applied at present, the egg white lysozyme has rich content in egg white, remarkable bacteriostatic function, easy extraction and low production cost, but the egg white lysozyme has poor heat stability and poor tolerance of a denaturant, when the egg white lysozyme is used as a feed additive, a high-temperature treatment step is carried out in the feed granulation process, and the egg white lysozyme has poor high-temperature resistance and impaired activity, so that the application is severely limited. In recent years, although the recombinant human lysozyme produced by using a microbial fermentation technology gradually appears in the market, the heat stability and the tolerance of a denaturant of the recombinant human lysozyme are enhanced to a certain extent, and the unit activity of the recombinant human lysozyme is higher than that of egg white lysozyme, the heat resistance, the related antibacterial activity and the like of the recombinant human lysozyme still need to be further improved; in addition, the existing lysozyme industrial production process is not complete, the relevant process conditions need to be further optimized, and the separation and concentration technology of the target protein in the fermentation liquor has certain problems, so that the purity and the yield of the target product lysozyme are not ideal.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to construct a pichia pastoris strain with excellent performance, introduce a humanized lysozyme gene with better heat resistance and stress resistance into a eukaryotic cell pichia pastoris by utilizing a biotechnology to express, and optimize a lysozyme production process and a product concentration and purification process to obtain high-purity heat-resistant lysozyme with higher yield.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a heat-resistant lysozyme which is prepared by introducing a human lysozyme gene into Pichia pastoris for expression, wherein the Pichia pastoris is HFD-01, is classified and named as Pichia pastoris (Pichia pastoris), has the preservation number of M2021148, the preservation date of 2021 year, 1 month and 25 days, the preservation unit is China Center for Type Culture Collection (CCTCC), and the preservation unit address is China, Wuhan university.
Further, the activity loss rate of the lysozyme at the temperature of 100-120 ℃ is not higher than 15%, and the activity loss rate of the lysozyme at the temperature of 80-100 ℃ is not higher than 10%.
The second aspect of the present invention provides a method for preparing the heat-resistant lysozyme of the first aspect of the present invention, comprising the steps of:
step A, constructing pichia pastoris HFD-01, wherein the preservation number is CCTCC NO: M2021148;
b, introducing a thermostable humanized lysozyme gene into the Pichia pastoris HFD-01 constructed in the step A to construct a recombinant Pichia pastoris strain;
and C, adopting the recombinant pichia pastoris strain constructed in the step B to perform fermentation culture, and concentrating and purifying a product to obtain the heat-resistant lysozyme.
In order to further optimize the preparation method, the technical measures adopted by the invention also comprise the following steps:
further, in the above preparation method, the constructing step of pichia pastoris HFD-01 comprises: the original strain is subjected to mutagenesis by adopting ultraviolet lamp-incandescent lamp alternative irradiation, and then is subjected to mutagenesis by adopting normal-pressure room-temperature plasma irradiation; and (3) domesticating and screening the strains obtained by mutagenesis by adopting a temperature gradient to obtain the pichia pastoris HFD-01 with high tolerance and high temperature resistance.
Further, the construction steps of the pichia pastoris HFD-01 specifically comprise: inoculating Pichia pastoris GS115 to a YPD plate, culturing at 28-30 ℃ for 2-3 days, washing the thallus with sterile water to prepare a suspension, and diluting to 1 × 106And (2) irradiating the cells per mL by adopting an ultraviolet lamp-incandescent lamp for 15-20 times alternately, wherein: irradiating with ultraviolet lamp (40W) for 60-120sAnd (3) irradiating the YPD liquid culture medium for 1-3min by an incandescent lamp (40W) at a distance of about 20-25 cm away from about 20-25 cm, adding the bacterial liquid into the YPD liquid culture medium after irradiation is finished, and performing plasma irradiation for 40-100 s at normal pressure and room temperature, wherein the plasma mutagenesis conditions are as follows: 100-120W, the distance is about 1.5-3 mm, the helium gas flow rate is 8-15L/min, after mutagenesis is finished, the bacterial liquid is added into an YPD liquid culture medium, and the YPD liquid culture medium is cultured for 48 hours at the temperature of 28-30 ℃ to obtain a bacterial strain with good activity and tolerance; inoculating the strain obtained by mutagenesis into YPD medium, culturing at 28-30 deg.C for 2-3d, and measuring OD600And (if the culture medium is more than 1, the culture medium is suitable for growth), transferring the bacterial liquid into a fresh culture medium in sequence, and culturing at 30 ℃, 32 ℃, 35 ℃ and 38 ℃ until the pichia pastoris which can be stably produced at 38 ℃ and has the highest growth speed is obtained, is named as pichia pastoris HFD-01, and is preserved, and the preservation number is CCTCC NO: M2021148.
Further, the construction steps of the recombinant pichia pastoris strain comprise: synthesizing a human-derived lysozyme gene fragment, and adding enzyme cutting sites at both ends of the human-derived lysozyme gene fragment; inserting the gene fragment into a plasmid through double enzyme digestion, constructing a plasmid expression vector and performing monoclonal amplification; and transforming the expression vector into the pichia pastoris HFD-01, and performing mutation by alternately irradiating with an ultraviolet lamp and an incandescent lamp to obtain the recombinant pichia pastoris strain capable of efficiently expressing the heat-resistant lysozyme.
Further, the construction steps of the recombinant pichia pastoris strain specifically comprise: synthesizing a Human Lysozyme (HLM) gene segment, preferably a human lysozyme gene with better heat resistance and stress resistance, and adding enzyme cutting sites Xho I and Not I at two ends of the human lysozyme gene segment; the gene fragment is inserted into a plasmid pPicZ alpha A through double enzyme digestion to construct an expression vector pPicZ alpha A-HLM; carrying out monoclonal amplification of an expression vector pPicZ alpha A-HLM by using escherichia coli DH5 alpha competent cells; electrically converting an expression vector pPicZ alpha A-HLM into pichia pastoris HFD-01, inoculating the recombinant strain to a YPD plate, culturing at 30-35 ℃ for 2-3 days, washing the strain with sterile water to prepare a suspension, and diluting to 1 × 106And (2) irradiating the cells per mL by adopting an ultraviolet lamp-incandescent lamp for 4-8 times alternately, wherein: irradiating for 30-60 s under the ultraviolet lamp (40W) at a distance of about 20-25 cm, irradiating for 1-2 min under the incandescent lamp (40W) at a distance of about 20-25 cm, and finishing irradiationAnd (3) coating the bacterial liquid on a flat plate, and culturing for 48h at 30-35 ℃ to obtain the recombinant pichia pastoris strain capable of stably and efficiently expressing the heat-resistant lysozyme.
Further, the fermentation culture step of the recombinant pichia pastoris strain comprises the following steps: preparing a seed solution by using the recombinant pichia pastoris strain to perform primary and secondary seed culture, transferring the cultured seed solution into a fermentation tank to perform fermentation culture, concentrating and purifying a fermentation broth, and performing spray drying, sieving and packaging to obtain a lysozyme finished product; furthermore, the culture conditions of the fermentation culture of the recombinant pichia pastoris strain in each step are controlled as follows: preparing a seed solution: culturing at 25-35 ℃ for 40-60 h; first-order seed culture: 25 to 35 ℃, a rotation speed of 100 to 280rpm, and an air flow of 10 to 25m3H, the tank pressure is 0.03-0.06 Mpa, and the culture is carried out for 20-30 h; secondary seed culture: 25 to 35 ℃, a rotation speed of 80 to 200rpm, and an air flow of 80 to 200m3H, the tank pressure is 0.03-0.06 Mpa, and the culture is carried out for 10-20 h; fermentation culture: 25 to 35 ℃, a rotation speed of 50 to 130rpm, and an air flow of 500 to 2000m3H, the pressure in the tank is 0.03-0.06 Mpa, and the culture time is 100-130 h.
Further, the concentration and purification step comprises concentration, separation and purification by adopting a separation and concentration membrane with a proper pore size and a resin exchange method; further, filtration was carried out using a hollow fiber membrane of 10 ten thousand molecular weight, and purification was carried out using a CM-Sepharose FF cation exchange resin column.
A third aspect of the invention provides the use of a thermostable lysozyme according to any one of the first aspects of the invention in the preparation of an animal feed.
Further, in the application, the lysozyme comprises water-soluble lysozyme and dressing type lysozyme. The water-soluble lysozyme is mainly used for feeding young animals which can not eat feed or are inconvenient to eat feed in a mode of drinking water or preparing a spray so as to prevent and treat intestinal unhealthy problems; the mixing type is designed for animals which can eat feed, and is directly added into the feed to be uniformly mixed for feeding or added into the feed for granulation.
Further, in the application, when the water-soluble lysozyme is adopted, the addition amount of the water-soluble lysozyme is 300g-500g/T water; when the material mixing type lysozyme is adopted, the addition amount of the material mixing type lysozyme is 100-300 g/T feed.
Further, in the application, the lysozyme can replace antibiotics to be used as a feed additive.
Further, in the application, the animal feed is chicken feed, preferably broiler feed.
Further, the chicken feed also comprises the following components: 45-65 parts of corn, 12-20 parts of crude protein, 3-8 parts of bran, 5-15 parts of soybean meal, 0.2-2 parts of fish meal, 0.5-3 parts of wheat middling, 2-6 parts of corn protein powder, 1-5 parts of yeast powder, 0.5-1.5 parts of mineral premix, 0.1-0.5 part of vitamin and 1-3 parts of traditional Chinese medicine premix; wherein, the traditional Chinese medicine premix comprises: 1-5 parts of folium isatidis, 1-5 parts of astragalus membranaceus, 1-5 parts of radix isatidis, 1-5 parts of tenebrio molitor, 1-5 parts of pine needle, 1-5 parts of dandelion, 1-5 parts of garlic leaves and 1-5 parts of hawthorn. In the chicken feed, the content of flavor lactic acid bacteria can be effectively increased by adding lysozyme, the quality of chicken is improved, the metabolism of the chicken can be improved by adding the traditional Chinese medicine premix, various diseases (rickets, pharyngitis, respiratory system diseases and the like) can be prevented, the fatality rate of the chicken is reduced to a certain extent, the feed intake can be improved to a certain extent by the feed, and the utilization rate of the chicken feed is effectively improved.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
the invention constructs the pichia pastoris HFD-01 with good tolerance, heat resistance and higher growth speed, it can realize the rapid normal growth of the strain at a higher temperature, and simultaneously introduce the heat-stable human lysozyme gene into the constructed pichia pastoris HFD-01 and carry out mutagenesis on the gene, so that the strain can express the heat-resistant lysozyme efficiently and stably while growing normally and rapidly at a higher temperature, the measures can reduce the energy consumption cost for maintaining lower fermentation temperature by adopting cooling water in the industry, are favorable for realizing the industrial production of the heat-resistant lysozyme, and the heat-resistant lysozyme is granulated at high temperature, the loss rate of the enzyme activity can be controlled within 10 percent, the loss rate of the lysozyme is effectively reduced, the lysozyme prepared by fermentation has excellent antibacterial property, the antibiotic can be replaced to a certain extent, and the method is green and environment-friendly, so that the application of the antibiotic in feed additives and high-temperature use environments is widely increased; in addition, the invention optimizes the industrial fermentation conditions according to the characteristics of the constructed fermentation strains, has simple process flow, reduces energy consumption to a certain extent, and obtains the lysozyme with high production efficiency, high purity and no environmental pollution, thereby being suitable for large-scale culture.
Drawings
FIG. 1 is a schematic view of a process for producing heat-resistant lysozyme in accordance with an embodiment of the present invention;
FIG. 2 is a scanning electron microscope image of in vitro antibacterial test using lysozyme in accordance with an embodiment of the present invention; wherein A, B, C, D respectively represents the scanning electron microscope images of the bacteria after lysozyme acts for 0min, 10min, 30min and 1 h;
FIG. 3 is a diagram of the actual antibacterial effect of in vitro antibacterial test using lysozyme in one embodiment of the present invention.
The pichia pastoris HFD-01 constructed by the invention is preserved, and the preservation information is as follows: the yeast is classified and named as Pichia pastoris (Pichia pastoris), the preservation number is CCTCC NO: M2021148, the preservation date is 2021 year, 1 month and 25 days, the preservation unit is China Center for Type Culture Collection (CCTCC), and the preservation unit address is China, Wuhan university and Wuhan university.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The experimental methods of the following examples, which are not specified under specific conditions, are generally determined according to national standards. The experimental materials not shown in the following examples are all commercially available materials. If there is no corresponding national standard, it is carried out according to the usual international standards, to the conventional conditions or to the conditions recommended by the manufacturer. Unless otherwise indicated, all parts are parts by weight and all percentages are percentages by mass. Unless defined or indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention.
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict. The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting.
Example 1 construction of Pichia pastoris HFD-01
In the embodiment, pichia pastoris GS115 is used as an initial strain, and pichia pastoris HFD-01 is constructed by physical mutagenesis and temperature gradient domestication, and the specific steps comprise:
(1) ultraviolet mutagenesis + plasma mutagenesis
Inoculating Pichia pastoris GS115 to YPD plate, culturing at 28 deg.C for 3 days, washing thallus with sterile water to obtain suspension, and diluting to 1 × 106Per mL, alternately irradiating 15 times with an ultraviolet lamp-incandescent lamp, wherein: irradiating with an ultraviolet lamp (40W) for 60s at a distance of about 20cm, irradiating with an incandescent lamp (40W) for 2min at a distance of about 20cm, adding the bacterial liquid into YPD liquid culture medium after irradiation, and performing plasma irradiation at normal pressure and room temperature for 80s under the plasma mutagenesis conditions: 110W, the distance is about 2mm, the helium gas flow rate is 10L/min, after mutagenesis is completed, bacterial liquid is added into a YPD liquid culture medium to be cultured for 48h at the temperature of 28 ℃, the growth condition of bacterial colonies is checked every 6h to select bacterial strains with good activity, good tolerance and accelerated growth speed, the diameter of the bacterial colonies and the growth speed of the bacterial colonies are observed in each round of screening process to select the bacterial strains with the highest growth speed, and therefore next round of screening is carried out.
Respectively inoculating the strain obtained by screening by ultraviolet mutagenesis and plasma mutagenesis and the original strain GS115 into YPD culture medium, culturing at 28 deg.C under shaking at 200r/min for 3d, and measuring OD every 6h600The influence of the mutagenesis experiment on the production condition of the pichia pastoris is observed, and the experimental result can obtain that compared with the original strain, the growth of the mutagenesis strain isThe speed is increased by about 20%, and the concentration of the bacterial liquid is also increased by about 25%, which shows that the mutagenesis effectively promotes the growth of pichia pastoris.
(2) Temperature gradient acclimation
Inoculating the strain obtained by screening by ultraviolet mutagenesis and plasma mutagenesis to YPD medium, culturing at 28 deg.C for 2-3d, and determining OD with original strain GS115 as control600(more than 1 is considered to be suitable for growth), transferring the bacterial liquid to a fresh culture medium in sequence, culturing at 30 ℃, 32 ℃, 35 ℃ and 38 ℃, and measuring the OD of the bacterial liquid every 6 hours600Until obtaining the pichia pastoris which can be stably produced at 38 ℃ and has the fastest growth speed, carrying out 3-cycle domestication at 30 ℃, 2-cycle domestication at 32 ℃, 5-cycle domestication at 35 ℃ and 15-cycle domestication at 38 ℃, and finally screening to obtain the mutation-domesticated strain, wherein the growth speed of the mutation-domesticated strain at 38 ℃ exceeds the growth speed of the original strain at 28 ℃, and is about 1.12 times.
Experiments prove that the mutagenic-domesticated strain can normally and rapidly grow at 30-38 ℃, has greater advantages compared with the original strain GS115, has the optimal growth rate at 30-35 ℃, can hardly grow at 40 ℃, and can still grow although the growth rate of the mutagenic-domesticated strain is reduced; based on fermentation culture energy consumption of pichia pastoris, selecting 30-35 ℃ as the culture temperature. The mutant strain with excellent performance obtained by physical mutagenesis and temperature gradient domestication screening is named as pichia pastoris HFD-01, and is preserved with the preservation number of CCTCC NO: M2021148.
Example 2 construction of recombinant strains expressing thermostable Lysozyme
In this example, the pichia pastoris HFD-01 constructed in example 1 is used to construct a recombinant strain expressing thermostable lysozyme, which specifically includes the steps of:
synthesizing Human Lysozyme (HLM) gene fragment, preferably human lysozyme gene with good heat resistance and stress resistance, adding enzyme cutting sites Xho I and Not I at two ends of the human lysozyme gene fragment, for example, modifying the gene of heat-resistant human lysozyme to make the amino acid sites have the following two mutations of R41H (improving the heat resistance of human lysozyme) and R41H (improving the heat resistance of human lysozyme)A92L (providing resistance to deforming agents); the gene fragment is inserted into a plasmid pPicZ alpha A through double enzyme digestion to construct an expression vector pPicZ alpha A-HLM; carrying out monoclonal amplification of an expression vector pPicZ alpha A-HLM by using escherichia coli DH5 alpha competent cells; electrically transforming an expression vector pPicZ alpha A-HLM into Pichia pastoris HFD-01, wherein the construction and use methods of all the biological materials adopt conventional operation methods in the field; inoculating the above recombinant strain to YPD plate, culturing at 32 deg.C for 3d, washing with sterile water to obtain suspension, and diluting to 1 × 106Per mL, alternately irradiating 6 times with an ultraviolet lamp-incandescent lamp, wherein: irradiating with an ultraviolet lamp (40W) for 30s at a distance of about 22cm, irradiating with an incandescent lamp (40W) for 1min at a distance of about 22cm, coating the bacterial liquid on a flat plate after irradiation, culturing at 32 ℃ for 48h, taking a recombinant strain constructed by the original strain GS115 and a recombinant strain constructed by pichia pastoris HFD-01 and not subjected to mutagenesis as controls, and centrifuging after culture to obtain a supernatant liquid for measuring the content of lysozyme so as to obtain the recombinant pichia pastoris strain which can stably and efficiently express heat-resistant lysozyme at 32 ℃ and has a high growth speed.
Experiments prove that the recombinant pichia pastoris strain constructed and mutagenized by pichia pastoris HFD-01 can normally and quickly grow at 32 ℃, the growth speed of the recombinant pichia pastoris strain is superior to that of the recombinant strain constructed by the original strain GS115 (about 15 percent higher), the growth speed of the recombinant pichia pastoris strain is superior to that of the recombinant strain constructed by the pichia pastoris HFD-01 and not mutagenized (about 8 percent higher), and the expressed lysozym content of the recombinant pichia pastoris strain is also superior to that of the recombinant strain constructed by the original strain GS115 (about 12 percent higher) and that of the recombinant strain constructed by the pichia pastoris HFD-01 and not mutagenized (about 10 percent higher).
Example 3 Industrial production of lysozyme Using recombinant Pichia Strain
In this example, the recombinant Pichia pastoris strain constructed and mutagenized by Pichia pastoris HFD-01 of example 2 was used for industrial production of heat-resistant lysozyme (gene modification of human lysozyme, amino acid site mutation: R41H and A92L), the recombinant Pichia pastoris strain was prepared into seed liquid for later use by conventional operations in the art, and the production process of heat-resistant lysozyme is shown in FIG. 1 and includes the following steps:
(1) preparing a seed solution: preparing a culture medium, sterilizing, cooling, and packaging the culture medium in sterile eggplant bottles to form inclined planes. After the eggplant bottle is cultured and the sterility is confirmed, picking the seed solution, uniformly streaking in a slant culture medium, placing the slant in an incubator for culture after streaking is finished, controlling the temperature to be 25-35 ℃ and the culture time to be 40-60 h, and eluting with sterile water to prepare the seed solution after the eggplant bottle culture meets the requirements, and inoculating the seed solution into a first-stage tank for continuous culture.
(2) First order seed culture
2.1) preparation of the culture Medium and Sterilization
Weighing materials such as 1-3% of peptone, 1-3% of glucose, 1-3% of yeast powder and the like according to a formula of a culture medium, putting the materials into a first-stage seeding tank to a constant volume to a measured volume, sterilizing the materials by using steam, and quickly cooling the sterilized materials to 25-35 ℃ for inoculation.
2.2) inoculation
And inoculating the seed liquid after the temperature of the first-stage seed tank is reduced to the process range.
2.3) Process control
The fermentation process is controlled at 25-35 deg.C, rotation speed of 100-280 rpm, and aeration rate of 10-25 m3H, the pressure in the tank is 0.03-0.06 MPa, and no mixed bacteria are detected under the microscope. Sampling and detecting physical and chemical indexes and sterility in the fermentation process, culturing the seed liquid for 20-30 h, performing microscopic examination on the seed liquid without sundry bacteria, and transferring the seed liquid into a secondary seed tank for continuous fermentation.
(3) Second stage seed culture
3.1) preparation of the culture Medium and Sterilization
Weighing 1-3% of glycerol, 1-2% of magnesium sulfate, 1-2% of potassium sulfate, 0.1-0.5% of potassium hydroxide and other materials according to a formula of a culture medium, putting the materials into a secondary seeding tank to fix the volume to a measured volume, sterilizing the materials by using steam, and quickly cooling the sterilized materials to 25-35 ℃ to wait for inoculation.
3.2) inoculation
And after the first-stage seed liquid meets the requirements, the first-stage seed liquid is inoculated into a second-stage seed tank for continuous culture.
3.3) Process control
The culture process is controlled at 25-35 deg.C, rotation speed of 80-200 rpm, and ventilation amount of 80-200 m3H, pot pressure (0.03)About 0.06) Mpa, and no mixed bacteria by microscopic examination. Sampling and detecting physical and chemical indexes and sterility in the fermentation process, culturing in seed liquid for 10-20 h, performing microscopic examination, and transferring into a fermentation tank.
(4) Fermentation culture
4.1) preparation of the culture Medium and Sterilization
Weighing 1-3% of glycerol, 1-2% of magnesium sulfate, 1-2% of potassium sulfate, 0.1-0.5% of calcium carbonate and other materials according to a formula of a culture medium, putting the materials into a fermentation tank to a constant volume to a measured volume, sterilizing the materials by using steam, and quickly cooling the materials to the temperature of (25-35) DEG C after the sterilization is finished.
4.2) inoculation
And inoculating the second-stage seed liquid into a fermentation tank after meeting the requirement.
4.3) Process control
The fermentation process is controlled at 25-35 deg.C, 50-130 rpm, and 500-2000 m ventilation3H, the pressure of a tank is (0.03-0.06) Mpa, no mixed bacteria are detected by microscopic examination, and 5-15% of methanol, 20% of ammonia water and the like are supplemented in the culture process.
4.4) Can-out Condition
And (4) after the culture time of the fermentation liquor is 100-130 h, conveying the fermentation liquor to a fermentation liquor storage tank when no foreign bacteria exist in microscopic examination.
(5) Concentrating, separating and purifying fermentation liquor
Adjusting the pH value of the fermentation liquor to 6.0-7.0, performing plate-and-frame filtration on the fermentation liquor to collect supernatant, wherein the supernatant adopts a hollow fiber membrane with the molecular weight of 10 ten thousand, and collecting concentrated liquor. Purifying with CM-Sepharose FF cation exchange resin chromatographic column, eluting with Tris-HCl, collecting active peak solution, and dialyzing to remove ions to obtain purified fermentation liquid.
(6) Pretreatment of fermentation liquor
Adding 2-10% of carrier into the purified fermentation liquor, stirring for 15-30 min, and then carrying out spray drying operation.
(7) Spray drying
The air inlet temperature (130-180) DEG C and the air outlet temperature (65-95) DEG C need to be controlled during spraying, and the air inlet and outlet temperatures need to be adjusted if the spraying powder is poor in flowability, adheres to a tower and the like.
(8) Sieving and packaging
Controlling the granularity of the spray powder: all pass through an analysis sieve with the aperture of 1.19mm, and more than 90 percent pass through an analysis sieve with the aperture of 0.59 mm.
Packaging specification: 500g, 1000g, 10kg, 15kg, 20kg, 25 kg.
In the production process of the process, the pollution control measures for product cross production specified in the production process pollution and cross pollution management regulations are strictly executed, and the specific measures for preventing cross pollution comprise:
1) during cross production, materials of different products are placed in different areas, and marks are made;
2) when the same operator feeds materials, the material feeding of one product is finished, then the material feeding of the other product is carried out, and when the operation is carried out by multiple operators, the operation is carried out on the premise that the materials of the respective products are obviously distinguished;
3) when different products are produced by using the same equipment in a cross mode, the equipment must be thoroughly cleaned according to the cleaning requirement, the natural color of the equipment can be seen after cleaning, the part directly contacted with the material has no obvious residue, and residue detection is carried out if necessary;
4) when different products are produced in a cross mode, related operation rules must be carefully read, and loss caused by process execution errors is prevented.
The culture medium and other formulas and the used reagents adopted in the production process are all conventional components in the field.
In the production process, the optimal control temperature in the fermentation process is 32 ℃, the growth rate of the recombinant strain is higher, wherein compared with 30 ℃, the expression quantity of the heat-resistant lysozyme is improved by about 4.3 percent, the energy consumption is reduced by about 14.7 percent, and the purity of the lysozyme is improved (the purity is about 97.5 percent) by adopting the process and the mutant recombinant strain.
Example 4 Performance validation of Heat-resistant Lysozyme produced by fermentation of a recombinant Pichia Strain
In this example, the antibacterial property, heat resistance and the like of the heat-resistant lysozyme prepared in example 3 were verified, and the examples specifically include:
(1) verification of antibacterial Properties
1% agarose in Tris-HCl buffer pH 7.210 mM after autoclavingCooling to 40 deg.C, adding bacteria to 10 deg.C6Pouring the plate, punching the plate by a puncher, adding lysozyme solutions (0-100mg/ml) with different concentrations, incubating at 37 ℃ for 4-6 hours, autoclaving LB nutrient agar, cooling to 40 ℃, covering the agarose plate, and irradiating the plate under an ultraviolet lamp for 20 minutes. Incubating in an incubator at 37 ℃ overnight, and observing the growth condition of bacteria between agarose and the nutrient agar layer.
The experimental results are as follows: as shown in FIG. 2, the MRSE strain is used for carrying out an experiment, and compared with lysozyme (i.e. lysozyme acts for 0min, see part A of FIG. 2), partial bacterial cell walls can be observed to be discontinuous under an electron microscope after lysozyme acts for 10min (see part B of FIG. 2), and a small amount of vacuoles appear; after 30min of action (see part C of figure 2), the number of bacteria with discontinuous cell walls is increased under an electron microscope, the degree of vacuole is increased when the time is 10min, and the forms of part of bacteria are irregularly changed; after 1h of action (see part D of figure 2), a large number of bacteria cell walls are discontinuous under an electron microscope, and vacuole-like changes of different degrees of a large number of bacteria can be seen; as shown in FIG. 3, the MRSA strain is used for the experiment, and the lysozyme has good antibacterial effect on the MRSA strain according to the actual antibacterial effect graph.
Experiments prove that the minimum inhibitory concentration MIC range of the lysozyme is 0.125 to more than 4000U/mL, and the lysozyme is particularly effective to MRSA and MRSE strains and drug-resistant bacteria such as clarithromycin, roxithromycin and the like. With clarithromycin, roxithromycin in a ratio of 1: 1, combination use: the synergistic effect is obviously more than 2-16 times, and the combination and the single use have significant difference (P is less than 0.05).
(2) Verification of Heat resistance
The lysozyme is respectively put in a drying oven and is acted for 30min at 80 ℃, 100 ℃ and 120 ℃. Respectively measuring the initial state and the enzyme activity after 30min of action, and calculating the activity loss rate, wherein the experimental results are shown in the following table:
| item | Untreated control | Acting at 80 deg.C for 30min | Acting at 100 deg.C for 30min | Acting at 120 deg.C for 30min |
| Vitality, ten thousand U/g | 4.50 | 4.15 | 4.07 | 3.91 |
| Rate of activity loss% | - | 7.78 | 9.56 | 13.11 |
As can be seen from the table above, the lysozyme expressed by the recombinant pichia pastoris strain constructed by pichia pastoris HFD-01 still has higher activity after being acted for 30min at the temperature of 80-120 ℃, and if the lysozyme is used for feed granulation, the temperature of the common feed granulation is 80-90 ℃, and the granulation time is less than 30min, so the loss rate of the activity of the lysozyme is controlled within 10 percent, the unnecessary waste is greatly reduced, and excellent conditions are provided for the high-temperature treatment process of the lysozyme.
Example 5 application of Heat-resistant Lysozyme produced by fermentation of recombinant Pichia Strain
The heat-resistant lysozyme prepared in the embodiment 3 is applied to preparation of broiler feed, and the broiler feed comprises the following components: 60 parts of corn, 15 parts of crude protein, 6 parts of bran, 8 parts of soybean meal, 0.2-2 parts of fish meal, 2.5 parts of wheat middling, 3 parts of corn protein powder, 2.5 parts of yeast powder, 1 part of mineral premix, 0.3 part of vitamin, 2 parts of traditional Chinese medicine premix (specifically comprising 2 parts of folium isatidis, 3 parts of radix astragali, 5 parts of radix isatidis, 1 part of yellow mealworm, 1 part of pine needle, 2 parts of dandelion, 3 parts of garlic leaves and 3 parts of hawthorn), and heat-resistant lysozyme; wherein the addition amount of the heat-resistant lysozyme is 200g/T feed. The broiler chicken is fed for 40 days by adopting the feeds of the feed experimental group (containing lysozyme and Chinese medicine premix), the control group 1 (not added with lysozyme and containing Chinese medicine premix), the control group 2 (containing lysozyme and not added with Chinese medicine premix) and the control group 3 (not added with lysozyme and Chinese medicine premix), each group adopts 40 broiler chickens, the average weight difference among the groups is not significant, all data are counted after the feeding is finished, and the experimental results are shown in the following table:
| daily gain | Meat ratio of materials | Mortality rate | |
| Feed group | 25.32±0.62 | 1.52±0.31 | 2.5％ |
| Control group 1 | 22.51±0.39 | 1.31±0.42 | 5.0 |
| Control group | |||
| 2 | 24.27±0.53 | 1.46±0.25 | 5.0％ |
| Control group 3 | 21.51±0.92 | 1.23±0.38 | 7.5％ |
According to the experimental results shown in the table, the feed added with lysozyme and the lysozyme and traditional Chinese medicine premix can obviously improve the daily weight gain of the broilers, reduce the feed conversion ratio and reduce the death rate to a certain extent, which indicates that the broilers eating the feed containing lysozyme have better antibacterial and antiviral abilities and enhanced resistance. By verification, when the feed containing lysozyme is eaten, the spleen index can be obviously improved, but the influence on the thymus index and the bursal disease index is small (P is more than 0.05); can obviously improve the lymphocyte transformation rate (P is less than 0.05) and the number of white blood cells; the content of the flavor lactobacillus can be effectively increased, and the quality of chicken is improved; in addition, the heat-resistant lysozyme can also be used as a feed additive of daily ration of broiler chickens to completely replace antibiotics, and the optimal dosage of the heat-resistant lysozyme is 300g-500g/T water for 1 week.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.
Claims (5)
1. The application of the heat-resistant lysozyme is characterized in that the heat-resistant lysozyme is prepared by introducing a genetically modified humanized lysozyme gene into pichia pastoris for expression, wherein the modification of the genetically modified humanized lysozyme gene enables the encoded amino acid R41 to be mutated into H, A92 and mutated into L, the pichia pastoris is HFD-01, and the preservation number is CCTCC NO: M2021148.
2. The application of claim 1, wherein the heat-resistant lysozyme has a activity loss rate of not higher than 15% at a temperature of 100-120 ℃ and a activity loss rate of not higher than 10% at a temperature of 80-100 ℃.
3. The application of the heat-resistant lysozyme in preparation of animal feed containing heat-resistant lysozyme is characterized in that the heat-resistant lysozyme is prepared by introducing a genetically modified human lysozyme gene into pichia pastoris for expression, wherein the modification of the human lysozyme gene enables the encoded amino acid R41 to be mutated into H, A92 and mutated into L, the pichia pastoris is HFD-01, and the preservation number is CCTCC NO: M2021148.
4. The use according to claim 3, wherein the heat resistant lysozyme comprises water soluble lysozyme and dressing lysozyme.
5. The use according to claim 4, wherein when the water soluble lysozyme is used, the amount of the lysozyme is 300-500 g/T water; when the material mixing type lysozyme is adopted, the addition amount of the material mixing type lysozyme is 100-300 g/T feed.
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