CN107475222A - The heat-resisting human lysozyme of genetic engineering transformation - Google Patents
The heat-resisting human lysozyme of genetic engineering transformation Download PDFInfo
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- CN107475222A CN107475222A CN201710935533.2A CN201710935533A CN107475222A CN 107475222 A CN107475222 A CN 107475222A CN 201710935533 A CN201710935533 A CN 201710935533A CN 107475222 A CN107475222 A CN 107475222A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2462—Lysozyme (3.2.1.17)
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B4/00—General methods for preserving meat, sausages, fish or fish products
- A23B4/10—Coating with a protective layer; Compositions or apparatus therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B4/00—General methods for preserving meat, sausages, fish or fish products
- A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
- A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
- A23B4/20—Organic compounds; Microorganisms; Enzymes
- A23B4/22—Microorganisms; Enzymes; Antibiotics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
- A23B7/154—Organic compounds; Microorganisms; Enzymes
- A23B7/155—Microorganisms; Enzymes; Antibiotics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/1203—Addition of, or treatment with, enzymes or microorganisms other than lactobacteriaceae
- A23C9/1216—Other enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K40/00—Shaping or working-up of animal feeding-stuffs
- A23K40/10—Shaping or working-up of animal feeding-stuffs by agglomeration; by granulation, e.g. making powders
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3571—Microorganisms; Enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01017—Lysozyme (3.2.1.17)
Abstract
The invention discloses a kind of heat-resisting human lysozyme of genetic engineering transformation；Include the mutation of two amino acid sites of R41H and A92L:Wherein R41H can improve the heat resistance of human lysozyme, and A92L improves the tolerance level to denaturant, and the heat resistance of raising human lysozyme that not only can be by a larger margin after R41H and A92L combinations, and can improve tolerance level of the human lysozyme to denaturant；By the application the invention also discloses the heat-resisting human lysozyme that a kind of genetic engineering is transformed in the preparation of high temperature granulation type feed, comparatively the heat resistance of the heat-resisting human lysozyme of described genetic engineering transformation is higher than in general enzyme, in the high temperature granulation process of 80 85 DEG C of temperature, the activity at least preceding 8 30min is able to maintain that more than 50%；The invention also discloses a kind of application of the heat-resisting human lysozyme in food fresh keeping of genetic engineering transformation, the antistaling agent using heat-resisting human lysozyme as main active, for the fresh-keeping of aquatic products or meat products, more than one times of shelf-life of low-temperature meat product can be extended.
Description
Technical field
The present invention relates to bioengineering field, it is more particularly related to a kind of resistance to thermal man of genetic engineering transformation
Lysozyme.
Background technology
Lysozyme from human body is a kind of as caused by human body, bacillus, staphylococcus aureus etc. is removed from office blue
Positive bacteria has the enzyme of antibacterial action, is made up of 130 amino acid, zoogenous lysozyme structurally and functionally have it is stronger
Conservative, the mechanism of action are all that can destroy the primary structure of bacteria cell wall：N-Acetyl-D-glucosamine and -acetylmuramic acid
Between β-Isosorbide-5-Nitrae glycosidic bond, and then kill bacterium.Because the cell membrane primary structure of Gram-positive pathogenic bacterium is by N- acetyl Portugal
Grapes glucosamine and -acetylmuramic acid composition, and gram-negative bacteria cell wall is gone back outside the cell membrane of both material compositions
There is the structure of one layer of lipopolysaccharides, therefore lysozyme has very strong killing action to gram-positive bacteria, for Gram-negative bacteria
Also there is stronger inhibitory action.
Lysozyme most widely used at present is egg white lysozyme, and its rich content in egg white, bacteria resistance function is notable, also
With being easy to extract, the advantages such as production cost is low.But the heat endurance of egg white lysozyme and poor to the tolerance of denaturant,
And in the production processes such as feed granulation, often there is the step of high temperature granulation, so egg white lysozyme is as feed addictive
High temperature is granulated or the application of the technique such as spray drying is limited by very large etc. being related in production process.And egg is molten
The units activity of bacterium enzyme only has 1/3rd of human lysozyme.
Although human lysozyme is relatively preferable for the tolerance of high temperature and denaturant relative to other lysozymes, still
The harsh techniques such as feed addition granulation can not be fully met, the high temperature that more than 80 degrees Celsius of granulation process generally use is warm herein
Under degree, most protein and enzyme can all inactivate because of high-temperature denatured.Comparatively the heat resistance of lysozyme is better than one
As enzyme, prior document report human lysozyme can be resistant to 70-75 DEG C of high temperature within a certain period of time, but this is to many temperature
Industry post processing higher than 80 DEG C is obviously not enough, therefore build the new restructuring lysozyme for screening more heat-resisting and resistance to denaturant
With important application value and wide market prospects.
The content of the invention
It is an object of the invention to solve at least the above, and provide the advantages of at least will be described later.
It is a further object to provide a kind of heat-resisting human lysozyme, its heat resistance and resistance to denaturant ability ratio are wild
Type significantly improves, the amino acid sequence such as SEQ ID NO of the heat-resisting human lysozyme:1、SEQ ID NO:2 or SEQ ID NO:3
It is shown；
It is a still further object of the present invention to provide a kind of gene HLM, the gene HLM for encoding the heat-resisting human lysozyme
Nucleotide sequence such as SEQ ID NO:4、SEQ ID NO:5 or SEQ ID NO:Shown in 6；
It is a still further object of the present invention to provide a kind of application of heat-resisting human lysozyme, the heat-resisting human lysozyme is applied to
Aquaculture, there is many-sided excellent beneficial effect, especially as can in high temperature granulation process of the feed addictive more than 80 DEG C
Keep higher activity；For food fresh keeping, not only with efficient bacteriostasis property but also property safe to the human body height.
In order to realize according to object of the present invention and further advantage, there is provided a kind of resistance to thermal man of genetic engineering transformation
Lysozyme, wherein, the amino acid sequence such as SEQ ID NO of the heat-resisting human lysozyme:1、SEQ ID NO:2 or SEQ ID NO:
Shown in 3.
A kind of gene HLM of the heat-resisting human lysozyme of genetic engineering transformation described in coding, wherein, encode the resistance to thermal man
The gene HLM of lysozyme nucleotide sequence such as SEQ ID NO:4、SEQ ID NO:5 or SEQ ID NO:Shown in 6.
A kind of carrier for expression of eukaryon pPicZ α A-HLM of gene HLM containing described heat-resisting human lysozyme.
A kind of application of described heat-resisting human lysozyme in feed preparation, wherein, the heat-resisting human lysozyme is in feed
In addition be the IU/kg of 1-10 ten thousand.
A kind of application of described heat-resisting human lysozyme in the preparation of high temperature granulation type feed.
Preferably, the temperature of high temperature granulation process is 80-85 DEG C, and time of high temperature granulation process is 8-30min, high temperature
After granulation process, the activity of heat-resisting human lysozyme is more than 50% in pellet.
A kind of application of described heat-resisting human lysozyme in food fresh keeping.
Preferably, the heat-resisting human lysozyme, the heat-resisting human lysozyme are added into dairy produce before pasteurization
Addition for the dairy produce weight/mass percentage composition 1%-3%.
Preferably, the heat-resisting human lysozyme is added into fresh fruit of vegetables before packing, the heat-resisting human lysozyme
Addition is the 0.1%-0.3% of the weight/mass percentage composition of the fresh fruit of vegetables；
Or, the heat-resisting human lysozyme is added into the fruits and vegetables before heating and cooking, the addition of the heat-resisting human lysozyme
For the 1%-3% of the weight/mass percentage composition of the fruits and vegetables.
The antistaling agent that a kind of heat-resisting human lysozyme described in application prepares, wherein, the antistaling agent uses spray-on process
Or infusion process is used for fresh-keeping, the quality hundred used of the heat-resisting human lysozyme in the antistaling agent of aquatic products or meat products
It is 1%-3% to divide specific concentration.
The present invention comprises at least following beneficial effect：
The method that the present invention is transformed by genetic engineering obtains a kind of heat resistance and resistance to denaturant ability ratio wild type is obvious
The heat-resisting human lysozyme improved；Screening has obtained the mutation in two sites of human lysozyme:R41H and A92L.R41H can improve people
The heat resistance of lysozyme, A92L improves the tolerance level to denaturant, and not only makes one bacteriolyze after R41H and A92L combinations
The heat resistance of enzyme has obtained substantial raising, and can improve tolerance level of the human lysozyme to denaturant；The present invention for
Human lysozyme is used for the industrial environment of high temperature, or positive work is maintained as active in the high temperature granulation of feed addictive
With；Heat-resisting human lysozyme of the present invention is added in feed, can effectively suppress the breeding of harmful bacteria in feed, energy
It is rotten to prevent that feed from occurring during shipping storage；The heat-resisting human lysozyme of genetic engineering of the present invention transformation it is heat-resisting
Comparatively property is higher than in general enzyme, in the high temperature granulation process of 80-85 DEG C of temperature, the work at least preceding 8-30min
Performance enough maintains more than 50%；Described heat-resisting human lysozyme can effectively extend the holding time of dairy produce, and drink
After protective effect can be produced to human body；The antistaling agent prepared using described heat-resisting human lysozyme, for aquatic products or
Meat products it is fresh-keeping, more than one times of shelf-life of low-temperature meat product can be extended；Genetic engineering of the present invention is transformed resistance to
Thermal man's lysozyme not only has stronger killing and inhibitory action to pathogenic bacteria, while has the advantages of broad-spectrum antiseptic, due to suppression
The high efficiency of bacterium and to the safe of human body, is suitable for the anti-corrosion of various food.
Further advantage, target and the feature of the present invention embodies part by following explanation, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Brief description of the drawings
Fig. 1 is the correct pPicZ α A plasmid maps for carrying HLM genes in genetic engineering remodeling method of the present invention；
Fig. 2 is the relatively conservative tertiary structure schematic diagram of lysozyme of the present invention；
Fig. 3 is that the SDS-PAGE electrophoresis coomassie brilliant blue staining results of the heat-resisting human lysozyme of purifying of the present invention are shown
It is intended to；
Fig. 4 is the heat-resisting warm-natured of each the mutant human lysozyme and wild type lysozyme of genetic engineering of the present invention transformation
Spend T50 comparative result schematic diagram.
Embodiment
The present invention is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art with reference to specification text
Word can be implemented according to this.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein are not precluded from one or more
The presence or addition of individual other elements or its combination.
Unless otherwise defined, otherwise all technologies used herein and scientific terminology all have the term that the present invention relates to
Have and generally understand identical implication with those skilled in the art.Although in the practice or test of the present invention
In any method, apparatus similar or equivalent with described herein and material can be used, but will now be described method for optimizing, device and
Material.
Term " signal peptide " means a short peptide (usual 16-30 amino acid longs), positioned at the secretory protein newly synthesized
N- ends, these protein be finally positioned within extracellular or some organelles including those (endoplasmic reticulum, golgiosome or
Body) the albumen from intracellular secretory.Signal peptide by destination protein transporte to cells device or it is extracellular after, will be specific
Digestion removes, so as to obtain the protein of maturation.
Term " PCR " means PCR (Polymerase Chain Reaction), abbreviation PCR.Polymerase
Chain reaction (PCR) is a kind of method of external enzyme' s catalysis specific DNA fragment, by high-temperature denatured, process annealing (renaturation) and
A few step reaction composition a cycles such as thermophilic extension, circulation carry out, target DNA is expanded rapidly, have high specificity, spirit
The features such as sensitivity is high, easy to operate, time saving.It cannot be only used for the basic research such as Gene Isolation, clone and nucleic acid sequence analysis,
It can be additionally used in the diagnosis of disease or any place for having DNA, RNA.Polymerase chain reaction (Polymerase Chain
Reaction, abbreviation PCR) it is also known as cell-free molecular cloning or the external primer orientation enzymatic amplification technique of specific DNA sequences.
Term " primer " means a bit of single stranded DNA or RNA, no except nonspecific limitation as the starting point of DNA replication dna
Then the term covers primer artificial synthesized in DNA replication dna biological in nature and PCR (PCR) (usually
DNA primer).It is that new nucleotides can be only added to have because of archaeal dna polymerase in synthesizing in DNA why to need primer
DNA on.Except nonspecific limitation, otherwise sense primer is the replication origin as the end of DNA profiling 3 ' in DNA replication dna
Primer, anti-sense primer be in DNA replication dna, as DNA profiling 5 ' hold replication origin primer.
Term " buffer solution " means a kind of when toward when adding a certain amount of bronsted lowry acids and bases bronsted lowry in some solution, having and hinder pH value of solution change
The solution of change effect.
Term " NAD+" mean oxidized nicotinamide adenine dinucleotide, oxidized coenzyme I, it is a kind of electric with transmitting
The small organic molecule of son (more accurate for, be to transmit hydrogen ion) function, it is the coenzyme of dehydrogenase.
Term " coenzyme " means can be by chemical group from an enzyme for one kind necessary to the activation plays of certain enzyme
The organic molecule being transferred on another enzyme, such as some B family vitamins.
As Figure 1-Figure 4, the present invention provides a kind of heat-resisting human lysozyme of genetic engineering transformation, wherein, it is described heat-resisting
The amino acid sequence of human lysozyme such as SEQ ID NO:1、SEQ ID NO:2 or SEQ ID NO:Shown in 3.
A kind of gene HLM of the heat-resisting human lysozyme of genetic engineering transformation described in coding, wherein, encode the resistance to thermal man
The gene HLM of lysozyme nucleotide sequence such as SEQ ID NO:4、SEQ ID NO:5 or SEQ ID NO:Shown in 6.
In such scheme, the heat-resisting human lysozyme of described genetic engineering transformation includes the prominent of two amino acid sites
Become, respectively R41H and A92L, wherein, R41H can improve the heat resistance of human lysozyme, and A92L is improved to the resistance to of denaturant
Spent, and the heat resistance of human lysozyme and the tolerance level to denaturant can be improved simultaneously after R41H and A92L combinations.
Have below by specific embodiment to the genetic engineering transformation process of heat-resisting human lysozyme of the present invention
Body explanation.
Embodiment 1
Human lysozyme carrier for expression of eukaryon of the N- ends without additional amino acid that preceding site is mutated containing R41H and A92L is built,
That is plasmid pPicZ α A-HLM.
Lysozyme is the albumen of higher mammal coding, expresses being possible to meeting inside Escherichia coli or bacillus subtilis
Active low, the problems such as can not correctly folding and form insoluble inclusion body, it is advantageous to red using eukaryotic expression system-finish
Family name yeast Pichia pastoris.Repaiied after the translations such as pichia can be glycosylated to the albumen of expression, phosphorylation
Decorations so that the animal protein of expression is higher than the activity that prokaryotic system is expressed.And pichia can be by purpose egg
White N- ends are plus the signal peptide such as α-factor, by protein secretion to extracellular, on the one hand can avoid due in the short time largely
Excessive concentration caused by express express target protein, albumen has little time correct fold and the aggregation that is cross-linked with each other forms showing for inclusion body
As on the other hand greatly simplifying the technological process of rear extraction purification.
In pichia expression system, most classical and efficient secreting, expressing system is by the N- in destination protein
End add α-factor signal peptides, destination protein is secreted into it is extracellular, and Kex2 signal peptidases can with it is specific identification and cut
α-factor signal peptides are sliced off, so as to obtain the albumen of maturation.And according to Shuichiro Goda etc. report, due to people's bacteriolyze
First amino acid of enzyme is basic amino acid Lys, can be with the Glu-Ala-Glu-Ala acidity ammonia of α-factor signal peptide C-terminals
Base acid forms ionic bond, so as to hinder the correct excision of this four amino acid.This allows for the people using α-factor signal peptides
After lysozyme is expressed in pichia, final product can take 4 unnecessary amino acid compared with normal human lysozyme
Glu-Ala-Glu-Ala, and ionic bond is formed with lysozyme N- ends tail itself, the performance of its function is hindered, and make albumen
Amylaceous precipitation is easily formed, so as to substantially reduce the activity of lysozyme.
In order to solve this problem, we optimize the sequence and cloning process of α-factor signal peptides, by coding for alpha-
The DNA sequence dna of the Glu-Ala-Glu-Ala amino acid of factor signal peptide C-terminals removes, and can be obtained after the cutting of Kex2 protease
To human lysozyme of the N- ends without any additional amino acid, so as to considerably increase the human lysozyme for using pichia to express
Activity.
The codon that the gene of coding human lysozyme (human lysozyme, HLM) be directed to pichia is excellent
Change, by DNA sequence dna by complete sequence gene chemical synthesis, and be connected to cloned plasmids pUC57 EcoRV sites.In order to by HLM albumen
Gene cloning is into pichia plasmid pPicZ α A, the method that is expanded using PCR, using primer HLM-F and HLM-R by HLM
Gene amplifies from pUC57 cloning vectors to be come, the nucleotide sequence such as SEQ ID NO of the primer HLM-F and HLM-R:7 Hes
SEQ ID NO:Shown in 8.
(1) PCR reaction systems：
(2) PCR response procedures：
Entered using arbitrary commercial PCR instrument, such as Thermo Scientific ArkTik Thermocycler PCR instruments
Row amplified reaction.After reaction terminates, the PCR produced using Gen-Foci Biotech (Ann Arbor, MI, USA) company is pure
Change kit, PCR primer is purified according to the explanation of manufacturer.
Pichia expression vector pPicZ α A are subjected to NdeI and XhoI double digestions, digestion products are passed through into thermal denaturation
After inactivation, 0.7% agarose electrophoresis is run, and cuts the pPicZ α of the linearisation of digestion with knife blade under uviol lamp
A carrier large fragment bands, using Gen-Foci (Ann Arbor, MI, USA) glue reclaim kit, according to the explanation of manufacturer
The pPicZ α A carriers of digestion are reclaimed from Ago-Gel, the clone for target gene connects, and glue reclaim step can be to prevent
Only carrier connects certainly.
Using lightning Cloning Kit (Jin Fusai (Beijing) biotechnology), by the method for homologous recombination, PCR is expanded
The expression HLM of acquisition gene DNA fragment is connected with the pPicZ α A carriers of XhoI/NotI digestions, obtains eukaryotic expression coding
The clone of human lysozyme HLM genes.
By the PCR primer purified and the pPicZ α A carriers of linearisation according to 1:1 to 10:1 ratio, preferably 3:1 ratio
Example mixing is (for example, 90ng：30ng), 2x lightnings clone mix (Jin Fusai (Beijing) biological sections of reaction cumulative volume 1/2 are added
Skill), lightning clone enzyme, coupled reaction buffer solution and NAD are included in mix+Coenzyme, 50 DEG C, 15-60 minutes, preferably 30 minutes
It is attached.After the completion of reaction, 1 μ connection products are taken to convert bacillus coli DH 5 alpha competent cell, and be coated in containing 25ug/ml
Mistake on the high pH of less salt of bleomycin LB (1% peptone, 0.5% yeast extract, 0.5%NaCl, pH7.5) resistance plate
Night cultivates.Single bacterium colony is chosen, cultivates and extracts plasmid, correct carrying HLM genes are screened by bacterium colony PCR and gene sequencing
PPicZ α A plasmid positives are cloned, and the plasmid is pPicZ α A-HLM, collection of illustrative plates such as Fig. 1 institutes of the plasmid pPicZ α A-HLM
Show.
Embodiment 2
Respectively for mutational site design primers different R41H and A92L two, and by PCR reactions by R41H and
A92L mutation are imported in plasmid pPicZ α A-HLM.
Bacteriolyze enzymatic structure between different plant species is all relatively conservative, as shown in Figure 2.By to different plant species bacteriolyze enzyme sequence
Analysis and heat resistance comparison, have selected human lysozyme two amino acid carry out be mutated R41H and A92L；It is prominent by pinpointing
The method of change, two different mutation R41H and A92L are imported in plasmid pPicZ α A-HLM.
Using the plasmid pPicZ α A-HLM of structure as masterplate, enter performing PCR using primer R41H-F and R41H-R and react,
To introduce R41H mutation, the nucleotide sequence such as SEQ ID NO of the primer R41H-F and R41H-R:9 and SEQ ID NO:10
It is shown.
Experimental procedure for introducing R41H mutation is as follows：
The point mutation kit produced using Gen-Foci Biotech (Ann Arbor, MI, USA) company, according to production
The explanation of business carries out point mutation PCR reactions.
(1) PCR reaction systems：
(2) PCR response procedures：
After PCR reactions terminate, 0.5 μ l restriction enzyme DpnI (New are directly added in PCR reaction tubes
England Biolabs, Ipswich, MA, USA), reacted 1 hour at 37 DEG C after mixing, then take 1 μ l reaction products directly to turn
Change bacillus coli DH 5 alpha competent cell, and be coated on the resistance LB plates containing ampicillin and be incubated overnight.Choose single bacterium colony,
Cultivate and extract plasmid, be sequenced with Sanger methods to determine correctly to clone.
Using primer A92L-F and A92L-R enter performing PCR react, with introduce A92L mutation, the primer A92L-F and
A92L-R nucleotide sequence such as SEQ ID NO:11 and SEQ ID NO:Shown in 12.For introducing the experimental procedure of A92L mutation
It can refer to the experimental procedure for introducing R41H mutation.
Embodiment 3
The plasmid pPicZ α A-HLM for importing two mutational sites are transferred to pichia bacterial strain with electric shocking method, and screened
Positive colony.(pichia is eucaryote, can carry out posttranslational modification to the foreign protein of expression, helps to carry molten
The units activity of bacterium enzyme and help form correct space structure.)
The pPicZ α A-HLM plasmids for expressing wild type or human lysozyme containing specific point mutation are confirmed through sequencing
Afterwards, extracted with a large amount of extracts kits of plasmid, then carry out linearization for enzyme restriction with SacI restriction enzymes, it is purified
Afterwards, pichia GS115 competent cells are changed with electric shocking method, is sieved on YPD+200ug/ml bleomycin antibiotic flat boards
The positive colony for selecting HLM gene orders to be incorporated into GS115 Yeast genomes.Then after each positive colony being drawn into single bacterium colony,
It is coated onto respectively on YPD+400 to 4000ug/ml bleomycin antibiotic flat boards, the high clone of the insertion of screening multicopy, expression quantity
(according to Invitrogen research, the expression quantity of destination protein is directly proportional with the copy number of insertion).
Embodiment 4
Heat-resisting human lysozyme secreting, expressing in pichia is induced, and the post processing for carrying out heat-resisting human lysozyme is pure
Change.
Expression of the vaccine protein driven by methanol induction AOX1 promoters in pichia.Using the side of fermentation
Method carries out HLM Yeast expression.Using BMGY culture mediums, 30 DEG C of fermented and cultureds, start stream plus methanol after glucose exhausts
Human lysozyme induced expression is carried out to final concentration 1%.A methanol, the after fermentation knot of induction 60 hours were added every 18 hours
Beam, carry out the post processing purifying of human lysozyme.
The vaccine protein of pichia expression is secreted into extracellular, it is possible to directly centrifuges or filters off and be degerming
Body, take supernatant, be concentrated by ultrafiltration to 1/5 volume, with pH7.4 PBS phosphate buffers carry out the displacement of ultrafiltration solvent and
Desalination, rear directly upper prop purifying.Using the ion-exchange chromatography medias of Bio-Rex 70 (Bio-rad, Herculus,
Carlifornia, USA), carry out first step cation exchange chromatography.Second step weak cation exchange resin CM
Sepharose removes the other impurities of the overwhelming majority, finally obtains purity and reaches more than 99%, N-terminal is without additional amino acid
Human lysozyme.Albumen after purification runs SDS-PAGE glue, with the result of coomassie brilliant blue staining (swimming lane as shown in Figure 3：1st, albumen
Matter molecular weight standard；2nd, wild type lysozyme；3rd, R41H saltant types；4th, A92L saltant types；5th, R41H/A92L double-mutants).
Embodiment 5
Detect the heat resistance of the human lysozyme after R41H and A92L mutation and the tolerance to denaturant.
The activity of lysozyme after wild type or mutation is detected using standard method.In Optimal pH (6.5 or 7.0)
Under, in 10 minutes incubation times, the temperature range used is 30 to 90 DEG C.Thermostabilization is determined by measuring the half-life period of enzyme
Property.The human lysozyme of all purifying is dilute in reaction buffer (0.1M potassium phosphate buffer, pH 7.0)
Release to 10 μ g/ml, and be incubated the different time, measure activity of residual enzyme at the standard conditions.
Determine their heat resistance (temperature of activity residue 50%, T50)：Wild type lysozyme and mutant enzyme are slow in reaction
10 μ g/ml are diluted in fliud flushing (pH6.5), are then heated 30 minutes, in the range of 50 DEG C to 90 DEG C, sample point interval 5
℃.After heating, human lysozyme enzyme is immediately placed on 10 minutes on ice, then the people using determination method described above measurement residual is molten
Bacterium enzymatic activity.
Human lysozyme determination of activity：
1st, 800ul Micrococcus micrococcus luteus cell suspending liquids are loaded onto in a cuvette as blank, another ratio
One, color ware is used to compare, and each sample is such.
2nd, by cuvette equalized temperature to 25 DEG C.
3rd, the light absorbs A450 in wavelength 450nm is monitored until constant using the spectrophotometer of appropriate temperature control.
4th, 30ul reaction buffers are added into blank cuvette, 30ul human lysozymes solution adds into control sample pond
30ul samples are to remaining test tube.
5th, mix immediately, and record the decrement of 5 minutes of A450 light absorbs.
The time (T1/2) of remaining 1/2 enzyme activity after the wild type of table 4 and the heat treatment of saltant type lysozyme
As a result as shown in table 4 and Fig. 4, compared with wild type lysozyme, R41H mutant can be by the heat-resisting warm-natured of lysozyme
Degree T50 brings up to 76 DEG C for 74 DEG C from highest, and the heat resisting temperature T50 of lysozyme can be brought up to 78 for 74 DEG C by A92L mutant from highest
DEG C, and the T50 of lysozyme can be brought up to 81 DEG C for 74 DEG C by R41H and A92L double-mutants from highest, it was found from experimental result,
The heat resistance of human lysozyme effectively can be brought up to more than 80 DEG C by R41H and A92L double-mutants；Remaining 1/2 enzyme after processing
Time T1/2 living is also significantly improved, and needs high-temperature process work for lysozyme is applied into the feeds such as high temperature granulation and other
Industry agricultural is using condition of providing convenience.
A kind of application of described heat-resisting human lysozyme in feed preparation, wherein, the heat-resisting human lysozyme is in feed
In addition be the IU/kg of 1-10 ten thousand.
In such scheme, heat-resisting human lysozyme of the present invention is added in feed, can effectively suppress to raise
The breeding of harmful bacteria in material, it can prevent feed from occurring during shipping storage rotten, the heat-resisting human lysozyme is in feed
Addition be defined so that significant anti-inflammation effect can be played, do not limit its addition one and be set to the IU/kg of 1-10 ten thousand.
A kind of application of heat-resisting human lysozyme as claimed in claim 1 in the preparation of high temperature granulation type feed.
In one preferred scheme, the temperature of high temperature granulation process is 80-85 DEG C, and the time of high temperature granulation process is 8-
30min, after high temperature granulation process, the activity of heat-resisting human lysozyme is more than 50% in pellet.
In such scheme, the high temperature that more than 80 degree Celsius of the granulation process generally use of feed is at this temperature, big absolutely
Most protein and enzyme can all inactivate because of high-temperature denatured.The heat-resisting human lysozyme of genetic engineering transformation of the present invention
Comparatively heat resistance is higher than in general enzyme, at least preceding 8-30min being up in temperature in 80-85 DEG C of high temperature granulation process
Interior activity is able to maintain that more than 50%.
A kind of application of described heat-resisting human lysozyme in food fresh keeping.
In one preferred scheme, the heat-resisting human lysozyme, the resistance to thermal man are added into dairy produce before pasteurization
The addition of lysozyme is the 1%-3% of the weight/mass percentage composition of the dairy produce.
In such scheme, anti-corrosion of the heat-resisting human lysozyme of the present invention particularly suitable for pasteurize milk, in milk
Appropriate human lysozyme is added in product, can effectively extend the holding time of dairy produce, and can be to human body after drinking
Produce protective effect.In addition, human lysozyme is a kind of nospecific immunity factor, it is special to have to corruptibility microorganism in enteron aisle
Killing action.Human lysozyme can directly or indirectly promote the propagation of Bifidobacterium in enteron aisle in vivo；Stomach can be promoted
Enteral milk casein forms fine curdled milk, extends in the enteron aisle residence time, is advantageous to digest and assimilate；Enteric bacteria bacterium can be promoted
The normalization of group；It can also strengthen the internal defense factors such as properdin, r- globulin, to strengthen the resistance to infection
Power.In addition, added in cheese processing a certain amount of lysozyme can prevent the later stage of middle and later periods cheese from bubbling, flavor be deteriorated and
The quality of cheese base fluid in cheese ageing process is not influenceed, simultaneously, moreover it is possible to bacteriostasis is played, it is unlikely to cause butyric acid to ferment, this
Be other preservatives it is incomparable.
In one preferred scheme, the heat-resisting human lysozyme is added into fresh fruit of vegetables before packing, the resistance to thermal man is molten
The addition of bacterium enzyme is the 0.1%-0.3% of the weight/mass percentage composition of the fresh fruit of vegetables；
Or, the heat-resisting human lysozyme is added into the fruits and vegetables before heating and cooking, the addition of the heat-resisting human lysozyme
For the 1%-3% of the weight/mass percentage composition of the fruits and vegetables.
In such scheme, a certain amount of heat-resisting human lysozyme of the present invention is added into fresh fruit of vegetables, is typically added
Dosage is 0.1%-0.3%, is then vacuum-packed again, can play good fresh-keeping effect；Or added into fruits and vegetables
The 1%-3% heat-resisting human lysozyme, then carries out heating and cooking, can play good antiseptic effect again.
The antistaling agent that a kind of heat-resisting human lysozyme described in application prepares, wherein, the antistaling agent uses spray-on process
Or infusion process is used for fresh-keeping, the quality hundred used of the heat-resisting human lysozyme in the antistaling agent of aquatic products or meat products
It is 1%-3% to divide specific concentration.
In such scheme, added using spray-on process or infusion process in aquatic products and meat products molten containing the resistance to thermal man
Aquatic products and meat products can be played fresh-keeping effect by bacterium enzyme antistaling agent, and the antistaling agent is molten with resistance to thermal man of the present invention
Bacterium enzyme is main active component, and the mass percent concentration used of the heat-resisting human lysozyme in the antistaling agent is
1%-3%, more than one times of shelf-life of low-temperature meat product can be extended.
Although embodiment of the present invention is disclosed as above, it is not restricted in specification and embodiment listed
With it can be applied to various suitable the field of the invention completely, can be easily for those skilled in the art
Other modification is realized, therefore under the universal limited without departing substantially from claim and equivalency range, it is of the invention and unlimited
In specific details and shown here as the legend with description.
<110>Nantong University;Jin Fusai（Beijing）Bio tech ltd
<120>The heat-resisting human lysozyme of genetic engineering transformation
<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 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 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 (10)
1. a kind of heat-resisting human lysozyme of genetic engineering transformation, wherein, the amino acid sequence such as SEQ of the heat-resisting human lysozyme
ID NO:1、SEQ ID NO:2 or SEQ ID NO:Shown in 3.
2. a kind of gene HLM for the heat-resisting human lysozyme for encoding genetic engineering transformation as claimed in claim 1, wherein, coding
The gene HLM of heat-resisting human lysozyme nucleotide sequence such as SEQ ID NO:4、SEQ ID NO:5 or SEQ ID NO:6 institutes
Show.
A kind of 3. carrier for expression of eukaryon pPicZ α A- of the gene HLM containing heat-resisting human lysozyme as claimed in claim 2
HLM。
4. a kind of application of heat-resisting human lysozyme as claimed in claim 1 in feed preparation, wherein, the bacteriolyze of resistance to thermal man
Addition of the enzyme in feed is the IU/kg of 1-10 ten thousand.
A kind of 5. application of heat-resisting human lysozyme as claimed in claim 1 in the preparation of high temperature granulation type feed.
6. application of the heat-resisting human lysozyme as claimed in claim 5 in the preparation of high temperature granulation type feed, wherein, high temperature is granulated
The temperature of process is 80-85 DEG C, and time of high temperature granulation process is 8-30min, heat-resisting in pellet after high temperature granulation process
The activity of human lysozyme is more than 50%.
A kind of 7. application of the heat-resisting human lysozyme in food fresh keeping as described in right will go 1.
8. application of the heat-resisting human lysozyme in food fresh keeping as claimed in claim 7, wherein, to dairy before pasteurization
The heat-resisting human lysozyme is added in product, the addition of the heat-resisting human lysozyme is the weight/mass percentage composition of the dairy produce
1%-3%.
9. application of the heat-resisting human lysozyme in food fresh keeping as claimed in claim 7, wherein, before packing to fresh fruit of vegetables
The middle addition heat-resisting human lysozyme, the addition of the heat-resisting human lysozyme is the weight/mass percentage composition of the fresh fruit of vegetables
0.1%-0.3%；
Or, the heat-resisting human lysozyme is added into the fruits and vegetables before heating and cooking, the addition of the heat-resisting human lysozyme is institute
State the 1%-3% of the weight/mass percentage composition of fruits and vegetables.
10. a kind of antistaling agent for applying heat-resisting human lysozyme as claimed in claim 1 to prepare, wherein, the antistaling agent
The fresh-keeping of aquatic products or meat products is used for using spray-on process or infusion process, the heat-resisting human lysozyme in the antistaling agent makes
Mass percent concentration is 1%-3%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108165567A (en) * | 2017-12-19 | 2018-06-15 | 佛山科学技术学院 | The synthesis of human lysozyme gene and its construction method of expression product |
| CN112813055A (en) * | 2021-03-26 | 2021-05-18 | 江苏海枫达生物科技有限公司 | Heat-resistant lysozyme and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01120286A (en) * | 1987-11-02 | 1989-05-12 | Agency Of Ind Science & Technol | Modified human lysozyme, gene coding the same, and production of modified human lysozyme |
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2017
- 2017-10-10 CN CN201710935533.2A patent/CN107475222B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01120286A (en) * | 1987-11-02 | 1989-05-12 | Agency Of Ind Science & Technol | Modified human lysozyme, gene coding the same, and production of modified human lysozyme |
Non-Patent Citations (3)
| Title |
|---|
| MICHIRO MURAKI: "Alteration of the substrate specificity of human lysozyme by site-specific intermolecular cross-linking", 《FEBS LETTERS》 * |
| RYOTA KUROKI等: "Design and creation of a Ca2" binding site in human lysozyme to enhance structural stability", 《PROC.NATI.ACAD.SCI.》 * |
| 王怀蓬等: "溶菌酶在饲料领域的研究进展", 《饲料研究》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108165567A (en) * | 2017-12-19 | 2018-06-15 | 佛山科学技术学院 | The synthesis of human lysozyme gene and its construction method of expression product |
| CN112813055A (en) * | 2021-03-26 | 2021-05-18 | 江苏海枫达生物科技有限公司 | Heat-resistant lysozyme and preparation method and application thereof |
| CN112813055B (en) * | 2021-03-26 | 2021-10-08 | 江苏海枫达生物科技有限公司 | Heat-resistant lysozyme and preparation method and application thereof |
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