CN107475222B

CN107475222B - Genetically engineered thermostable human lysozyme

Info

Publication number: CN107475222B
Application number: CN201710935533.2A
Authority: CN
Inventors: 杨波; 金大勇; 杨建�; 郭春雨; 赵阳; 杨光
Original assignee: Jinfusai Beijing Biotechnology Co ltd; Nantong University
Current assignee: Jinfusai Beijing Biotechnology Co ltd; Nantong University
Priority date: 2017-10-10
Filing date: 2017-10-10
Publication date: 2020-04-03
Anticipated expiration: 2037-10-10
Also published as: CN107475222A

Abstract

The invention discloses a genetically engineered heat-resistant human lysozyme; it includes mutations in two amino acid sites of R41H and A92L: wherein R41H can improve the heat resistance of human lysozyme, and A92L can improve the tolerance to denaturants , and the combination of R41H and A92L can not only greatly improve the heat resistance of human lysozyme, but also improve the tolerance of human lysozyme to denaturants; the invention also discloses a genetically engineered heat-resistant The application of human lysozyme in the preparation of high-temperature granulated feed, the heat resistance of the genetically engineered heat-resistant human lysozyme is relatively higher than that of ordinary enzymes, and the high-temperature granulation at a temperature of 80-85 ° C is used. During the process, the activity in at least the first 8-30min can be maintained at more than 50%; the invention also discloses the application of a genetically engineered heat-resistant human lysozyme in food preservation, and the heat-resistant human lysozyme is mainly used. The preservative of active ingredients, used for the preservation of aquatic products or meat products, can prolong the shelf life of low-temperature meat products by more than one time.

Description

Genetically engineered heat-resistant human lysozyme

Technical Field

The invention relates to the field of bioengineering, in particular to genetically engineered heat-resistant human lysozyme.

Background

The lysozyme from human body is an enzyme which is produced by human body and has antibacterial action on gram-positive bacteria such as bacillus, staphylococcus aureus, etc. and is composed of 130 amino acids, the original lysozyme of animal has stronger conservative property in structure and function, and the action mechanism is the main structure which can destroy the bacterial cell wall, namely β -1,4 glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid, so as to kill bacteria.

The lysozyme which is most widely applied at present is egg white lysozyme which has the advantages of rich content in egg white, obvious bacteriostatic function, easy extraction, low production cost and the like. However, the heat stability and the tolerance to denaturants of egg white lysozyme are poor, and a high-temperature granulation step is often required in the production process of feed granulation and the like, so that the application of the egg white lysozyme as a feed additive and the like in the production process of high-temperature granulation or spray drying and other processes is greatly limited. And the unit activity of the egg white lysozyme is only one third of that of human lysozyme.

Although human lysozyme has relatively better tolerance to high temperature and denaturant than other lysozyme, the lysozyme still cannot completely meet the strict process of feed addition granulation and the like, and the granulation process usually adopts high temperature above 80 ℃, and most of proteins and enzymes are inactivated due to high-temperature denaturation. The heat resistance of the lysozyme is relatively better than that of common enzymes, and the prior literature reports that human lysozyme can resist the high temperature of 70-75 ℃ within a certain time, but the heat resistance is obviously insufficient for industrial post-treatment of a plurality of temperatures higher than 80 ℃, so that the construction and screening of the novel recombinant lysozyme with higher heat resistance and denaturant resistance have important application value and wide market prospect.

Disclosure of Invention

An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.

Another purpose of the invention is to provide a heat-resistant human lysozyme, the heat resistance and the ability of resisting a denaturant of which are obviously improved compared with the wild type, and the amino acid sequence of the heat-resistant human lysozyme is shown as SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3;

the invention also aims to provide a gene HLM for coding the heat-resistant human lysozyme, wherein the nucleotide sequence of the gene HLM is shown as SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6;

still another object of the present invention is to provide an application of heat-resistant human lysozyme, which has various excellent effects, especially as a feed additive, in a high temperature granulation process at 80 ℃ or higher, in the breeding industry; the preservative is used for preserving food, and has high-efficiency antibacterial performance and high safety to human bodies.

To achieve these objects and other advantages in accordance with the present invention, there is provided a genetically engineered heat-resistant human lysozyme, wherein the amino acid sequence of the heat-resistant human lysozyme is represented by SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.

The gene HLM for coding the genetically engineered heat-resistant human lysozyme has a nucleotide sequence shown in SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6.

A eukaryotic expression vector pPicZ α A-HLM containing the gene HLM of the heat-resistant human lysozyme.

The application of the heat-resistant human lysozyme in feed preparation is characterized in that the addition amount of the heat-resistant human lysozyme in the feed is 1-10 ten thousand IU/kg.

An application of the heat-resistant human lysozyme in the preparation of high-temperature granulated feeds.

Preferably, the temperature in the high-temperature granulation process is 80-85 ℃, the time in the high-temperature granulation process is 8-30min, and the activity of the heat-resistant human lysozyme in the granulated feed is more than 50 percent after the high-temperature granulation process.

An application of the heat-resistant human lysozyme in food preservation.

Preferably, the heat-resistant human lysozyme is added into the milk product before pasteurization, and the addition amount of the heat-resistant human lysozyme is 1-3% of the mass percentage of the milk product.

Preferably, the heat-resistant human lysozyme is added into the fresh fruits and vegetables before packaging, and the addition amount of the heat-resistant human lysozyme is 0.1-0.3% of the mass percentage of the fresh fruits and vegetables;

or adding the heat-resistant human lysozyme into the fruits and vegetables before heating and cooking, wherein the addition amount of the heat-resistant human lysozyme is 1-3% of the mass percentage of the fruits and vegetables.

The preservative prepared by using the heat-resistant human lysozyme is used for preserving aquatic products or meat products by adopting a spraying method or an immersion method, and the mass percentage concentration of the heat-resistant human lysozyme in the preservative is 1-3%.

The invention at least comprises the following beneficial effects:

the invention obtains a heat-resistant human lysozyme with heat resistance and denaturant resistance obviously improved compared with wild type through a genetic engineering modification method; the screening results in two site mutations of human lysozyme, R41H and A92L. R41H can improve the heat resistance of human lysozyme, A92L improves the tolerance to denaturant, and R41H and A92L are combined to ensure that the heat resistance of human lysozyme is substantially improved and the tolerance of human lysozyme to denaturant can be improved; the invention has positive effect on maintaining the activity of human lysozyme in high-temperature industrial environment or high-temperature granulation as a feed additive; the heat-resistant human lysozyme is added into the feed, so that the propagation of harmful bacteria in the feed can be effectively inhibited, and the feed can be prevented from deteriorating in the transportation and storage processes; the heat resistance of the heat-resistant human lysozyme modified by the genetic engineering is relatively higher than that of a common enzyme, and the activity can be maintained above 50% in at least the first 8-30min in the high-temperature granulation process at the temperature of 80-85 ℃; the heat-resistant human lysozyme can effectively prolong the storage time of the dairy product and can protect the human body after drinking; the preservative prepared by the heat-resistant human lysozyme is used for preserving aquatic products or meat products, and can prolong the shelf life of low-temperature meat products by more than one time; the heat-resistant human lysozyme modified by the genetic engineering not only has stronger killing and inhibiting effects on pathogenic bacteria, but also has the advantage of broad-spectrum antibiosis, and is suitable for corrosion prevention of various foods due to high bacteriostasis efficiency and high safety on human bodies.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a plasmid map of pPicZ α A correctly carrying HLM gene in the genetic engineering method of the present invention;

FIG. 2 is a schematic diagram showing the relative conserved tertiary structure of lysozyme in accordance with the present invention;

FIG. 3 is a schematic representation of the SDS-PAGE electrophoresis Coomassie blue staining of the purified thermostable human lysozyme according to the present invention;

FIG. 4 is a diagram showing the comparison result of the heat resistance temperature T50 of each mutant human lysozyme and wild lysozyme of the present invention.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

Terms to which this invention relates unless defined 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. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described.

The term "signal peptide" means a short peptide (usually 16-30 amino acids long) located at the N-terminus of newly synthesized secreted proteins, including those secreted from cells that are ultimately localized extracellularly or within certain organelles (endoplasmic reticulum, Golgi body or soma). After the target protein is transported to an organelle or a cell, the signal peptide is specifically cut off by enzyme, so that mature protein is obtained.

The term "PCR" means Polymerase Chain Reaction (Polymerase Chain Reaction), abbreviated PCR. The Polymerase Chain Reaction (PCR) is a method for in vitro enzymatic synthesis of specific DNA fragment, and several reactions of high-temp. denaturation, low-temp. annealing (renaturation) and proper-temp. extension are formed into a cycle, and cyclically implemented so as to make the target DNA quickly amplified, and said invented product possesses the characteristics of strong specificity, high sensitivity, simple operation and saving time. It can be used not only for basic research of gene separation, cloning and nucleic acid sequence analysis, but also for diagnosis of diseases or any places with DNA and RNA. Polymerase Chain Reaction (PCR) is also called cell-free molecular cloning or specific DNA sequence in vitro primer-directed enzymatic amplification technology.

The term "primer" means a short piece of single-stranded DNA or RNA that serves as a point of initiation of DNA replication, and unless specifically limited, encompasses DNA replication in nature and primers (typically DNA primers) artificially synthesized in the Polymerase Chain Reaction (PCR). Primers are required because DNA polymerases can only add new nucleotides to an existing DNA strand during DNA synthesis. Unless specifically limited, the upstream primer is a primer that serves as a replication origin at the 3 'end of the DNA template at the time of DNA replication, and the downstream primer is a primer that serves as a replication origin at the 5' end of the DNA template at the time of DNA replication.

The term "buffer" means a type of solution that has the effect of retarding the pH change of a solution when certain amounts of acid and base are added to the solution.

The term "NAD⁺"means oxidized nicotinamide adenine dinucleotide, oxidized coenzyme I, is a small molecule organic substance having a function of transferring electrons (more precisely, transferring hydrogen ions), and is a coenzyme of dehydrogenase.

The term "coenzyme" means a class of small organic molecules, such as some B vitamins, necessary for the activity of a particular enzyme to function, which can transfer chemical groups from one enzyme to another.

As shown in figures 1-4, the invention provides a heat-resistant human lysozyme modified by genetic engineering, wherein the amino acid sequence of the heat-resistant human lysozyme is shown as SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.

In the scheme, the genetically engineered heat-resistant human lysozyme comprises mutations of two amino acid sites, namely R41H and A92L, wherein R41H can improve the heat resistance of the human lysozyme, A92L improves the tolerance to a denaturing agent, and R41H and A92L can simultaneously improve the heat resistance of the human lysozyme and the tolerance to the denaturing agent after being combined.

The following will specifically describe the genetic engineering process of the heat-resistant human lysozyme of the present invention by means of specific examples.

Example 1

Constructing a human lysozyme eukaryotic expression vector containing R41H and A92L pre-mutation sites and having no redundant amino acid at the N-terminal, namely a plasmid pPicZ α A-HLM.

The lysozyme is a protein coded by higher animals, and the expression in escherichia coli or bacillus subtilis may have the problems of low activity, incorrect folding, insoluble inclusion body formation and the like, so a eukaryotic expression system-Pichia pastoris is preferably adopted, the Pichia pastoris can be used for carrying out post-translational modification such as glycosylation and phosphorylation on the expressed protein, and the expressed animal protein has higher activity than that expressed by a prokaryotic system, and the Pichia pastoris can secrete the protein to the outside by adding signal peptides such as α -factor and the like to the N-end of the target protein, so that the phenomenon that the concentration is too high due to the fact that the target protein is expressed in a large amount in a short time, the proteins are not correctly folded and are mutually cross-linked and aggregated to form an inclusion body can be avoided, and the process flow of post-extraction and purification is greatly simplified.

In the expression system of Pichia pastoris, the most classical and efficient secretion expression system is to add α -factor signal peptide to the N-terminal of the target protein to secrete the target protein to the outside, and Kex2 signal peptidase can specifically recognize and cleave off α -factor signal peptide to obtain the mature protein, however, according to the report of Shuichio Goda et al, since the first amino acid of human lysozyme is basic amino acid Lys, it can form an ionic bond with Glu-Ala-Glu-Ala acidic amino acid at the C-terminal of α -factor signal peptide, thereby preventing the correct cleavage of these four amino acids, this makes the final product carry 4 extra amino acids Glu-Ala-Glu-Ala compared with normal human lysozyme after the human lysozyme using α -factor signal peptide is expressed in Pichia pastoris, and form an ionic bond with the N-terminal tail of lysozyme itself, thereby preventing the function thereof and making the protein easily form amyloid precipitate, thereby greatly reducing the activity of lysozyme.

In order to solve the problem, the sequence and the cloning method of the α -factor signal peptide are optimized, the DNA sequence of the Glu-Ala-Glu-Ala amino acid at the C end of the α -factor signal peptide is removed, and the human lysozyme without any redundant amino acid at the N-end can be obtained after Kex2 protease is cut, so that the activity of the human lysozyme expressed by pichia pastoris is greatly increased.

In order to clone the HLM protein gene into a pichia pastoris plasmid pPicZ α A, a PCR amplification method is adopted, and the HLM gene is amplified from a pUC57 cloning vector by using primers HLM-F and HLM-R, wherein the nucleotide sequences of the primers HLM-F and HLM-R are shown as SEQ ID NO:7 and SEQ ID NO: 8.

(1) And (3) PCR reaction system:

(2) PCR reaction procedure:

the amplification reaction is carried out using any commercially available PCR instrument, such as the Thermo Scientific ArkTik Thermocycler PCR instrument. After the reaction was completed, the PCR product was purified using a PCR purification kit manufactured by Gen-Foci Biotech (Ann Arbor, MI, USA) according to the manufacturer's instructions.

The pichia pastoris expression vector pPicZ α a was subjected to NdeI and XhoI double digestion, the digested product was subjected to heat denaturation inactivation, run through 0.7% agarose electrophoresis, and the large fragment band of the digested linearized pPicZ α a vector was excised with a surgical blade under an ultraviolet lamp, and the digested pPicZ α a vector was recovered from the agarose gel using the gel recovery kit of Gen-Foci (Ann Arbor, MI, USA) according to the manufacturer's instructions for cloning ligation of the gene of interest, and the gel recovery step prevented self-ligation of the vector.

By adopting a lightning cloning kit (Jinfossai (Beijing) biotechnology) and a homologous recombination method, a gene DNA fragment for expressing HLM obtained by PCR amplification is connected with a pPicZ α A vector subjected to XhoI/NotI enzyme digestion to obtain the clone of the eukaryotic expression coding human lysozyme HLM gene.

The purified PCR product and the linearized pPicZ α A vector were mixed in a ratio of 1:1 to 10:1, preferably 3:1 (e.g., 90 ng: 30 ng), and 2 Xlightning cloning mix (King Fusai (Beijing) Biotech) containing the lightning clonase, ligation buffer and NAD in a total reaction volume of 1/2 was added⁺After the reaction is completed, 1 mu of the ligation product is taken to transform Escherichia coli DH5 α competent cells, and is spread on a low-salt high-pH LB (1% peptone, 0.5% yeast extract, 0.5% NaCl, pH 7.5) resistant plate containing 25ug/ml bleomycin for overnight culture, a single colony is picked up, a plasmid is cultured and extracted, and a pPicZ α A plasmid positive clone which correctly carries the HLM gene is screened by colony PCR and gene sequencing, wherein the plasmid is pPicZ α A-HLM, and the map of the plasmid pPicZ α A-HLM is shown in FIG. 1.

Example 2

Primers were designed for two different mutation sites of R41H and a92L, respectively, and R41H and a92L mutations were introduced into plasmid pPicZ α a-HLM by PCR reaction.

The lysozyme structure of different species is relatively conserved, as shown in figure 2, through the analysis of lysozyme sequence and the comparison of heat tolerance of different species, two amino acids of human lysozyme are selected to be mutated R41H and A92L, and through the site-directed mutagenesis method, two different mutations R41H and A92L are introduced into a plasmid pPicZ α A-HLM.

The constructed plasmid pPicZ α A-HLM is used as a template, and PCR reaction is carried out by using primers R41H-F and R41H-R to introduce R41H mutation, wherein the nucleotide sequences of the primers R41H-F and R41H-R are shown as SEQ ID NO. 9 and SEQ ID NO. 10.

The experimental procedure for introducing the R41H mutation was as follows:

the point mutation PCR reaction was carried out using a point mutation kit manufactured by Gen-Foci Biotech (Ann Arbor, MI, USA) company according to the manufacturer's instructions.

(1) And (3) PCR reaction system:

(2) PCR reaction procedure:

after the PCR reaction was completed, 0.5. mu.l of restriction enzyme DpnI (New England Biolabs, Ipswich, MA, USA) was directly added to the PCR reaction tube, mixed well and reacted at 37 ℃ for 1 hour, then 1. mu.l of reaction product was taken to directly transform E.coli DH5 α competent cells, and spread on ampicillin-containing resistant LB plate for overnight culture.

PCR reactions were performed to introduce the A92L mutation using primers A92L-F and A92L-R, the nucleotide sequences of which are shown in SEQ ID NO:11 and SEQ ID NO:12, A92L-F and A92L-R. The experimental procedure for introducing the a92L mutation can be referred to the experimental procedure for introducing the R41H mutation.

Example 3

The plasmid pPicZ α A-HLM introduced into the two mutation sites is transferred into a Pichia pastoris strain by an electric shock method, and positive clones are screened (the Pichia pastoris is a eukaryote, can perform post-translational modification on an expressed exogenous protein, and is beneficial to improving unit activity of lysozyme and forming a correct space structure.)

The pPicZ α A-HLM plasmid of the human lysozyme expressing the wild type or containing specific point mutation is subjected to sequencing confirmation, extracted by a plasmid mass extraction kit, then enzyme digestion linearization is carried out by SacI restriction enzyme, after purification, Pichia pastoris GS115 competent cells are converted by an electric shock method, positive clones with HLM gene sequences integrated into GS115 yeast genomes are screened on a YPD +200ug/ml bleomycin antibiotic plate, then each positive clone is screened after being screened, and is respectively coated on a YPD + 400-4000 ug/ml bleomycin antibiotic plate, and the clones with multiple copies and high expression amount are screened (according to research of Invitrogen, the expression amount of target protein is in direct proportion to the copy number of the insert).

Example 4

Inducing the heat-resistant human lysozyme to secrete and express in the pichia pastoris, and carrying out post-treatment purification on the heat-resistant human lysozyme.

The expression of the vaccine protein driven by AOX1 promoter in Pichia pastoris is induced by methanol. Yeast expression of HLM was performed using a fermentative approach. Human lysozyme-induced expression was performed using BMGY medium, fermentation at 30 ℃ and starting with methanol feed to a final concentration of 1% after glucose depletion. Methanol is supplemented every 18 hours, fermentation is finished after 60 hours of induction, and post-treatment purification of human lysozyme is carried out.

The vaccine protein expressed by pichia pastoris is secreted to the extracellular, so the bacteria can be directly removed by centrifugation or filtration, the supernatant is taken, after ultrafiltration and concentration to 1/5 volume, the PBS phosphate buffer solution with pH7.4 is used for carrying out ultrafiltration solvent replacement and desalination, and then the purified solution is directly loaded on a column. A first step of ion exchange chromatography purification was performed using Bio-Rex 70 ion exchange chromatography media (Bio-rad, Herculus, Carlifornia, USA). And in the second step, removing most other impurities by using weak cation exchange resin CMSepharose, and finally obtaining the human lysozyme with the purity of more than 99 percent and no redundant amino acid at the N end. The purified protein was run on SDS-PAGE gel, and the results of staining with Coomassie Brilliant blue are shown in FIG. 3 (lane: 1, protein molecular weight standard; 2, wild-type lysozyme; 3, R41H mutant; 4, A92L mutant; 5, R41H/A92L double mutant).

Example 5

The heat resistance and the ability to tolerate denaturing agents of human lysozyme were examined after R41H and A92L mutations.

The activity of wild-type or post-mutation lysozyme was tested using standard methods. At optimal pH (6.5 or 7.0) a temperature range of 30 to 90 ℃ was used over a 10 minute incubation time. Thermostability was determined by measuring the half-life of the enzyme. All purified human lysozyme were diluted to 10. mu.g/ml in reaction buffer (0.1M potassium phosphotebuffer, pH 7.0) and incubated for various periods of time, and the residual enzyme activity was measured under standard conditions.

Determination of their heat resistance (temperature of 50% remaining of activity, T50): the wild-type lysozyme and the mutant enzyme were diluted to 10. mu.g/ml in the reaction buffer (pH 6.5), and then heated for 30 minutes with sampling points spaced 5 ℃ apart in the range of 50 ℃ to 90 ℃. After heating, the human lysozyme was immediately placed on ice for 10 minutes, and then the residual human lysozyme activity was measured using the assay described above.

Human lysozyme activity assay:

1. 800 ul of Micrococcus cell suspension was loaded into one cuvette as a blank, and another cuvette was used for control, one for each sample.

2. The cuvette temperature was equilibrated to 25 ℃.

3. The light absorption at a wavelength of 450nm a450 was monitored until constant using a suitably temperature-controlled spectrophotometer.

4. 30ul of reaction buffer was added to the blank tube, 30ul of human lysozyme solution was added to the control cell, and 30ul of sample was added to the remaining tubes.

5. Mix immediately and record the 5 minute decrease in a450 light absorption.

The time of 1/2 enzyme activity remained after heat treatment of wild type and mutant lysozyme (T1/2)

As shown in the table and FIG. 4, compared with wild lysozyme, the R41H mutant can increase the heat resistance temperature T50 of lysozyme from 74 ℃ maximum to 76 ℃, the A92L mutant can increase the heat resistance temperature T50 of lysozyme from 74 ℃ maximum to 78 ℃, while the R41H and A92L double mutants can increase the T50 of lysozyme from 74 ℃ maximum to 81 ℃, and the experimental results show that the R41H and A92L double mutants can effectively increase the heat resistance of human lysozyme to more than 80 ℃; the time T1/2 of the enzyme activity of the 1/2 remained after the treatment is obviously prolonged, and convenience is provided for applying the lysozyme to feeds such as high-temperature pelleting and other industrial agricultural applications requiring high-temperature treatment.

In the scheme, the heat-resistant human lysozyme is added into the feed, so that the propagation of harmful bacteria in the feed can be effectively inhibited, the feed can be prevented from deteriorating in the transportation and storage processes, the addition amount of the heat-resistant human lysozyme in the feed is based on the effect of playing a remarkable antibacterial and anti-inflammatory effect, and is not limited to be 1-10 ten thousand IU/kg.

Use of the heat-resistant human lysozyme of claim 1 in the preparation of a high temperature pelleted feed.

In a preferred scheme, the temperature in the high-temperature granulation process is 80-85 ℃, the time in the high-temperature granulation process is 8-30min, and the activity of the heat-resistant human lysozyme in the granulated feed is more than 50 percent after the high-temperature granulation process.

In the above scheme, the feed granulation process usually adopts a high temperature of more than 80 ℃, and at the temperature, most of proteins and enzymes are inactivated due to high temperature denaturation. The heat resistance of the heat-resistant human lysozyme modified by the genetic engineering is relatively higher than that of the common enzyme, and the activity of the heat-resistant human lysozyme can be maintained above 50% at least within the first 8-30min in the high-temperature granulation process at the temperature of 80-85 ℃.

An application of the heat-resistant human lysozyme in food preservation.

In a preferable scheme, the heat-resistant human lysozyme is added into a milk product before pasteurization, and the addition amount of the heat-resistant human lysozyme is 1-3% of the mass percentage of the milk product.

In the scheme, the heat-resistant human lysozyme is particularly suitable for preserving pasteurized milk, and a proper amount of human lysozyme is added into a milk product, so that the preservation time of the milk product can be effectively prolonged, and the heat-resistant human lysozyme can protect a human body after being drunk. In addition, human lysozyme is a nonspecific immune factor and has a special killing effect on putrefactive microorganisms in the intestinal tract. The human lysozyme can directly or indirectly promote the proliferation of the bifidobacteria in the intestinal tract in vivo; can promote the lactocasein in the stomach and intestine to form fine curd, prolong the retention time in the intestinal tract and facilitate the digestion and absorption; can promote normalization of intestinal bacteria flora; it can also enhance the in vivo defense factors such as serum bactericidal protein, gamma-globulin, etc., to enhance resistance to infection. In addition, a certain amount of lysozyme is added in the cheese processing, so that the late foaming and the flavor deterioration of cheese in the middle and later stages can be prevented, the quality of cheese base liquid in the cheese aging process is not influenced, and meanwhile, the lysozyme can play a role in inhibiting bacteria, so that the butyric acid fermentation is not caused, which is incomparable with other preservatives.

In a preferable scheme, the heat-resistant human lysozyme is added into fresh fruits and vegetables before packaging, and the addition amount of the heat-resistant human lysozyme is 0.1-0.3% of the mass percentage of the fresh fruits and vegetables;

In the scheme, a certain amount of the heat-resistant human lysozyme is added into fresh fruits and vegetables, the addition amount is generally 0.1-0.3%, and then vacuum packaging is carried out, so that a good fresh-keeping effect can be achieved; or 1% -3% of the heat-resistant human lysozyme is added into the fruits and vegetables, and then the fruits and vegetables are heated and boiled, so that a good anti-corrosion effect can be achieved.

In the scheme, the preservative containing the heat-resistant human lysozyme is added into the aquatic products and the meat products by adopting a spraying method or an immersion method, so that the preservative can play a role in preserving the aquatic products and the meat products, the preservative takes the heat-resistant human lysozyme as a main active ingredient, the mass percentage concentration of the heat-resistant human lysozyme in the preservative is 1-3%, and the quality guarantee period of the low-temperature meat products can be prolonged by more than one time.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

<110> university of Nantong, Cibotai (Beijing) Biotechnology Ltd

<120> genetically engineered heat-resistant human lysozyme

<160>8

<210>1

<211>130

<212>PRT

<213> Artificial sequence

<220>

<221>MUTAGEN

<222>(41,92)

<223>Human lysozyme R41H A92L mutant protein sequence

<400>1

Lys Val Phe Glu Arg Cys Glu Leu Ala Arg Thr Leu Lys Arg Leu Gly

1 5 1015

Met Asp Gly Tyr Arg Gly Ile Ser Leu Ala Asn Trp Met Cys Leu Ala

20 25 30

Lys Trp Glu Ser Gly Tyr Asn Thr His Ala Thr Asn Tyr Asn Ala Gly

35 40 45

Asp Arg Ser Thr Asp Tyr Gly Ile Phe Gln Ile Asn Ser Arg Tyr Trp

50 55 60

Cys Asn Asp Gly Lys Thr Pro Gly Ala Val Asn Ala Cys His Leu Ser

65 70 75 80

Cys Ser Ala Leu Leu Gln Asp Asn Ile Ala Asp Leu Val Ala Cys Ala

85 90 95

Lys Arg Val Val Arg Asp Pro Gln Gly Ile Arg Ala Trp Val Ala Trp

100 105 110

Arg Asn Arg Cys Gln Asn Arg Asp Val Arg Gln Tyr Val Gln Gly Cys

115 120 125

Gly Val

130

<210>2

<211>130

<212>PRT

<213> Artificial sequence

<220>

<221>MUTAGEN

<222>(41)

<223>Human lysozyme R41H mutant protein sequence

<400>2

Lys Val Phe Glu Arg Cys Glu Leu Ala Arg Thr Leu Lys Arg Leu Gly

1 5 10 15

Met Asp Gly Tyr Arg Gly Ile Ser Leu Ala Asn Trp Met Cys Leu Ala

20 25 30

Lys Trp Glu Ser Gly Tyr Asn Thr His Ala Thr Asn Tyr Asn Ala Gly

35 40 45

Asp Arg Ser Thr Asp Tyr Gly Ile Phe Gln Ile Asn Ser Arg Tyr Trp

50 55 60

Cys Asn Asp Gly Lys Thr Pro Gly Ala Val Asn Ala Cys His Leu Ser

65 70 75 80

Cys Ser Ala Leu Leu Gln Asp Asn Ile Ala Asp Ala Val Ala Cys Ala

85 90 95

Lys Arg Val Val Arg Asp Pro Gln Gly Ile Arg Ala Trp Val Ala Trp

100 105 110

Arg Asn Arg Cys Gln Asn Arg Asp Val Arg Gln Tyr Val Gln Gly Cys

115 120 125

Gly Val

130

<210>3

<211>130

<212>PRT

<213> Artificial sequence

<220>

<221>MUTAGEN

<222>(92)

<223>Human lysozyme A92L mutant protein sequence

<400>3

Lys Val Phe Glu Arg Cys Glu Leu Ala Arg Thr Leu Lys Arg Leu Gly

1 5 10 15

Met Asp Gly Tyr Arg Gly Ile Ser Leu Ala Asn Trp Met Cys Leu Ala

20 25 30

Lys Trp Glu Ser Gly Tyr Asn Thr Arg Ala Thr Asn Tyr Asn Ala Gly

35 40 45

Asp Arg Ser Thr Asp Tyr Gly Ile Phe Gln Ile Asn Ser Arg Tyr Trp

50 55 60

Cys Asn Asp Gly Lys Thr Pro Gly Ala Val Asn Ala Cys His Leu Ser

65 70 75 80

Cys Ser Ala Leu Leu Gln Asp Asn Ile Ala Asp Leu Val Ala Cys Ala

85 90 95

Lys Arg Val Val Arg Asp Pro Gln Gly Ile Arg Ala Trp Val Ala Trp

100 105 110

Arg Asn Arg Cys Gln Asn Arg Asp Val Arg Gln Tyr Val Gln Gly Cys

115 120 125

Gly Val

130

<210>4

<211>130

<212>DNA

<213> Artificial sequence

<220>

<221>MUTAGEN

<222>(124，125，126，277，278，279)

<223>human lysozyme gene R41H A92L mutant DNA sequence

<400>4

atgaaggttt tcgaaaggtg cgaattggct agaactttga agagattggg aatggacggt 60

tacagaggca tttctttggc aaattggatg tgcttggcta agtgggaatc aggttacaac 120

actcatgcta ctaattataa cgccggcgat agatctaccg attacggcat tttccagatc 180

aactccagat attggtgcaa cgacggtaaa acaccaggag cagttaacgc ttgtcatttg 240

tcttgttccg ccttgttgca agataatatt gccgatttgg ttgcttgcgc taaaagagtt 300

gttagagatc cacaaggaat tagagcttgg gttgcttgga gaaataggtg ccaaaataga 360

gacgttagac aatacgttca aggttgcgga gtttaa 396

<210>5

<211>396

<212>DNA

<213> Artificial sequence

<220>

<221>MUTAGEN

<222>(124，125，126)

<223>human lysozyme gene R41H mutant DNA sequence

<220>

<221>CDS

<222>(1)...(396)

<400>5

atgaaggttt tcgaaaggtg cgaattggct agaactttga agagattggg aatggacggt 60

tacagaggca tttctttggc aaattggatg tgcttggcta agtgggaatc aggttacaac 120

actcatgcta ctaattataa cgccggcgat agatctaccg attacggcat tttccagatc 180

aactccagat attggtgcaa cgacggtaaa acaccaggag cagttaacgc ttgtcatttg 240

tcttgttccg ccttgttgca agataatatt gccgatgcag ttgcttgcgc taaaagagtt 300

gttagagatc cacaaggaat tagagcttgg gttgcttgga gaaataggtg ccaaaataga 360

gacgttagac aatacgttca aggttgcgga gtttaa 396

<210>6

<211>396

<212>DNA

<213> Artificial sequence

<220>

<221>MUTAGEN

<222>(277，278，279)

<223>human lysozyme gene A92L mutant DNA sequence

<220>

<221>CDS

<222>(1)...(396)

<400>6

atgaaggttt tcgaaaggtg cgaattggct agaactttga agagattggg aatggacggt 60

tacagaggca tttctttggc aaattggatg tgcttggcta agtgggaatc aggttacaac 120

actcatgcta ctaattataa cgccggcgat agatctaccg attacggcat tttccagatc 180

aactccagat attggtgcaa cgacggtaaa acaccaggag cagttaacgc ttgtcatttg 240

tcttgttccg ccttgttgca agataatatt gccgatttgg ttgcttgcgc taaaagagtt 300

gttagagatc cacaaggaat tagagcttgg gttgcttgga gaaataggtg ccaaaataga 360

gacgttagac aatacgttca aggttgcgga gtttaa 396

<210>7

<211>46

<212>DNA

<213> Artificial sequence

<220>

<400>7

gaagaagggg tatctctcga gaaaagaaaa gtgtttgaac gctgcg 46

<210>8

<211>36

<212>DNA

<213> Artificial sequence

<220>

<400>8

aagagaggct gaagctgaag gttttcgaaa ggtgcg 36

<210>9

<211>33

<212>DNA

<213> Artificial sequence

<220>

<400>9

ctgggctata acacccatgc gaccaactat aac 33

<210>10

<211>33

<212>DNA

<213> Artificial sequence

<220>

<400>10

gttatagttg gtcgcatggg tgttatagcc cag 33

<210>11

<211>34

<212>DNA

<213> Artificial sequence

<220>

<400>11

gataacattg cggatttagt ggcgtgcgcg aaac 34

<210>12

<211>34

<212>DNA

<213> Artificial sequence

<220>

<400>12

gtttcgcgca cgccactaaa tccgcaatgt tatc 34

Claims

1. A genetically engineered heat-resistant human lysozyme is disclosed, wherein the amino acid sequence of the heat-resistant human lysozyme is shown as SEQ ID NO. 1.

2. A gene HLM of heat-resistant human lysozyme coded by genetic engineering is characterized in that the nucleotide sequence of the gene HLM coding the heat-resistant human lysozyme is shown in SEQ ID NO. 4.

3. A eukaryotic expression vector pPicZ α A-HLM comprising a gene HLM of heat-resistant human lysozyme of claim 2.

4. Use of the thermostable lysozyme according to claim 1 for the preparation of a high temperature pelleted feed, characterized in that the temperature of the high temperature pelleting process is 85 ℃.

5. The use of the heat-resistant human lysozyme of claim 1 in food preservation, wherein the heat-resistant human lysozyme is added to a dairy product before pasteurization, and the addition amount of the heat-resistant human lysozyme is 1-3% of the mass percentage of the dairy product.

6. The application of the heat-resistant human lysozyme in food preservation according to claim 1, characterized in that the heat-resistant human lysozyme is added into fruits and vegetables before heating and boiling, and the addition amount of the heat-resistant human lysozyme is 1% -3% of the mass percentage of the fruits and vegetables.