CN105586327B - A kind of source of people antalzyme protein purification process - Google Patents
A kind of source of people antalzyme protein purification process Download PDFInfo
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Abstract
The invention belongs to protein purification arts, specifically, the present invention relates to a kind of methods for purifying source of people antalzyme protein.The invention further relates to the applications of the production method.The present invention provides a kind of source of people antalzyme protein purification process, which includes: the pre-treatment of fermentation liquid；Bestarose MMC Diamond capture；Bestdex G-25 buffer exchange；Destination protein is concentrated.The present invention establishes the new process that compound cation exchange medium isolates and purifies source of people lysozyme in fermentation liquid, and step is few, at low cost, can obtain higher albumen input-output ratio, provides an efficient production technology for the industrialized production of human lysozyme protein.
Description
Technical field
The invention belongs to protein purification arts, specifically, the present invention relates to a kind of sides for purifying source of people antalzyme protein
Method.The invention further relates to the applications of the production method.
Background technique
With the development of biotechnology, the method for obtaining albumen is more and more, and classical protein separation process is logical
Often the following steps are included: (1) is crushed biological tissue, protein is extracted with buffer appropriate；It (2) will be thin with centrifugal process
Sub-cellular particles (such as nucleus, mitochondria, microsome or ribosomes and the cell fragment removal of born of the same parents；(3) using salting out method or
Organic solvent method precipitates related protein component；(4) further separate various protein using chromatography or electrophoresis；
(5) if with if possible, by crystallization of protein or freeze-dried powder is made.
Following problems need to be considered by obtaining source of people albumen with this method: first, people is tissue-derived few, and destination protein content
Good tissue-derived just less of high, active high dissolubility stability；Second, the step of each isolating and purifying, can lose certain
The albumen and protein active of amount, but separating step is very little cannot to reach certain purity again；Third, the step each isolated and purified
Reagent used in rapid and relevant parameter are all different the scope of application of different albumen；4th, different albumen it is accurate
Qualitative, quantitative usually requires specific method.
With biochemical development, the artificial synthesized of peptide has become possibility, however this method is suitable only for synthesis and contains
The albumen for having the little albumen of more than ten of amino acid residue, moreover being synthesized in chemical apparatuses whether with naturally occurring albumen have one
The conformation of cause needs further to study with function.
Currently, can also be in a variety of expression bodies such as Escherichia coli, yeast, insect and mammal with technique for gene engineering
High efficient expression in system, but the albuminiferous method of this life relates equally to the problem of isolating and purifying.
Lysozyme is the polypeptide para-immunity antimicrobial molecule for decomposing mucopolysaccharide.It has heat-resisting, acidproof physicochemical property, solvable
Yu Shui, ethyl alcohol, grease type solvent achieve the effect that crack gram-positive bacterium by lytic cell wall.Lysozyme is killed
Bacterium capability study shows: the lysozyme of denier can kill micrococcus lysodeikticus, gamboge coccus, bacillus megaterium, rod-like stem
The natures common bacteria such as bacterium, Bacillus acidi lactici, micrococcus, sarcina, staphylococcus.Lysozyme currently on the market all comes
It is foreign protein for human body derived from other kind animal bodies, there is very strong antigenicity, side effect is big.
Natural human lysozyme is primarily present in the secretion and organ such as human milk, saliva, tears, placenta, due to source
It is limited and is not easy to extract, so expensive.Currently, scientific research personnel has utilized chemical synthesis or has made from human tissue
The approach such as standby cDNA obtain human lysozyme gene, some to be expressed in the systems such as fungi, bacterium, reaction of animals device,
But yield is very limited, is not able to satisfy it in the extensive use of the industries such as food, medicine, herding.
Currently, the foundation of separation purifying technique is generally all raw to commercial scale by laboratory research, intermediate experiment production
The amplification process of producing line.Pichia pastoris meeting chromogenesis substance during expressing recombinant human lysozyme, makes fermentation liquid in Huang
Green, while can also generate the impurity such as foreign protein, nucleic acid, it is therefore desirable to purification process is carried out to fermentation liquid, could be used for subsequent
Research.At present about the isolation and purification method of lysozyme, mainly there are crystallisation, ion-exchange and affinity chromatography.Crystallisation
It is that lysozyme is precipitated with crystalline state mainly according to solution condition in most traditional method for preparing lysozyme, this method operation
Simplicity, cost is very low, but yield is relatively low, is difficult to obtain high purity product；There is processing in the isolation technics based on affine
Measure smaller, the expensive drawback of affinity media；Ion-exchange is most common method of purification, and this method is to utilize solution
In between various charged particles and ion-exchanger binding force difference to isolate destination protein, it is easy, efficiently, can be automatic
Change continuous operation, but existing problem is, fermentation liquid is minimal medium, and ion concentration is very high, is needed before loading to fermentation liquid
It is diluted to reduce ionic strength, otherwise chromatographic column filler is rigidly small, and is easily blocked, and the trouble for causing regeneration to use is made
At separating rate and inefficiency.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of people's bacteriolyzes easy to operate, low in cost, product purity is high
Enzyme purification method, process cycle is short, high income to reach, and is easy to Linear Amplifer to production-scale purpose.
In order to solve the above-mentioned technical problem, the present invention establishes compound cation exchange medium and isolates and purifies in fermentation liquid
The new process of source of people lysozyme: fermentation liquid through centrifugation, after filtration treatment without dilution can directly upper column purification, purify mesh through a step
Albumen i.e. to reach electrophoresis pure.Albumen is captured using compound cation exchange column Bestarose MMC Diamond first, so
Buffer exchange is carried out with solvent resistant column Bestdex G-25 afterwards, it is finally dense with cation exchange column SP Bestarose FF
Contracting destination protein, final protein concentration is up to 27mg/mL；Each step yield is respectively 92.67%, 91.9% and 94.68%, always
Protein yield is 80.63%, about 15 times of purification；It is simple spike that HPLC, which detects finished product, and purity is greater than 99%.
Source of people antalzyme protein purification process of the invention the following steps are included:
The pre-treatment of fermentation liquid；
Bestarose MMC Diamond capture；
Bestdex G-25 buffer exchange；
Destination protein is concentrated.
The pre-treatment of fermentation liquid mainly includes the solid impurity removed containing in human lysozyme solution, adjustment pH value etc..
In the present invention, the pH containing human lysozyme solution is adjusted to neutral meta-acid, the pH value with subsequent chromatographic column buffer
Match.Meanwhile utilizing the little particle impurity in filter membrane removal solution.
When removing impurity, first solution can be centrifuged, collect supernatant, then handle supernatant using hollow fiber ultrafiltration membrane
Liquid further removes the finely ground particle substance in sample.
When adjusting pH value, by the fermentation liquid tune pH to 6.5 of the lysozyme containing recombination human source, then 12000rpm, is centrifuged
15min collects supernatant.Then, Bestarose MMC Diamond buffer solution B (1.5M NaCl, 20mM PB, pH is added
6.5) adjusting sample conductance keeps it consistent (about 43ms/cm) with buffer solution A (0.4M NaCl, 20mM PB, pH 6.5) conductance, i.e.,
As former state for Bestarose MMC Diamond column loading.
In the present invention, the fermentation liquid of human lysozyme solution can be directly used containing human lysozyme solution.
Bestarose MMC Diamond capture needs the physicochemical property according to target product, selects suitable chromatostrip
Part.
Source of people lysozyme is basic protein, and isoelectric point is higher (pI ≈ 10), optimal pH ≈ 6.5, at room temperature in neutral salt
There is higher natural activity in solution.Therefore select the pH of buffer close to neutrality in purification process, it is pure in separation to avoid sample
It is denaturalized during changing.And source of people lysozyme is positively charged under pH neutrallty condition, therefore sun is generally selected in traditional handicraft
Purification media of the Ion Exchange Medium (such as SP, CM) as human lysozyme.
5 column volumes of equilibration buffer, until the pH of chromatographic column effluent liquid, conductance are consistent with equilibration buffer, it will be upper
Sample cleans 3~5 column volumes until baseline with equilibration buffer after end of the sample with 30mL/min flow velocity loading after stating processing
Stablize, i.e., all unbonded substances are all flushed splitter.Then destination protein is eluted with elution buffer, according to
UV280 eluting peak collects sample.
In one embodiment of the present of invention, the chromatographic column of use and the condition of buffer are as follows:
Chromatographic column: BXK 50/30, Vt=300mL.
Equilibration buffer A:0.4M NaCl, 20mM PB, pH 6.5.
Elution buffer B:1.5M NaCl, 20mM PB, pH 6.5.
Since the elution samples volume of Bestarose MMC Diamond column is larger, protein concentration is lower, needs to sample
Product, which carry out concentration, can be only achieved requirement.
The present invention carries out sample concentration using SP Bestarose FF column, therefore needs to buffer sample before it is concentrated
Liquid displacement, makes its conductance be less than 10ms/cm (Bestarose MMC Diamond column elution samples conductance is about 135ms/cm).
In one embodiment of the present of invention, the item of chromatographic column and buffer that when Bestdex G-25 buffer exchange uses
Part is as follows:
Chromatographic column: BXK 200/500, Vt=5L.
Equilibration buffer: 20mM NaAC (sodium acetate, CH3COONa), 4.75 pH.
2 column volumes of equilibration buffer, until the pH of chromatographic column effluent liquid, conductance are consistent with equilibration buffer, it will
Bestarose MMC Diamond elution samples should be less than 1.5L with 200mL/min flow velocity loading, loading volume.End of the sample
After continue to be cleaned with equilibration buffer, according to the peak UV280 collect sample.
Destination protein is concentrated using SP Bestarose FF in the present invention.
3~5 column volumes of equilibration buffer, until the pH of chromatographic column effluent liquid, conductance are consistent with equilibration buffer, it will
It is straight that the sample of transposed buffer with 20mL/min flow velocity loading, after end of the sample with equilibration buffer cleans 3~5 column volumes
To baseline stability, i.e., all unbonded substances are all flushed splitter.Then destination protein, root are eluted with elution buffer
Sample is collected according to UV280 eluting peak.
In a preference of the invention, using the chromatographic column and buffer of SP Bestarose FF concentration destination protein
Condition it is as follows:
Chromatographic column: BXK 50/30, Vt=280ml.
Equilibration buffer: 20mM NaAC, pH 4.75.
Elution buffer: 0.15M NaCl, 20mM PB (phosphate buffer), pH 7.5.
Innovation of the invention is as follows:
1. establishing the new process that compound cation exchange medium isolates and purifies source of people lysozyme in fermentation liquid: fermentation liquid
Through centrifugation, after filtration treatment without dilution can directly upper column purification, it is pure to reach electrophoresis through step purifying destination protein.It adopts first
Albumen is captured with compound cation exchange column Bestarose MMC Diamond, then uses solvent resistant column Bestdex G-
25 carry out buffer exchange, destination protein finally are concentrated with cation exchange column SP Bestarose FF, final protein concentration can
Up to 27mg/mL；Each step yield is respectively 92.67%, 91.9% and 94.68%, and total protein yield is 80.63%, purifying times
About 15 times of number；It is simple spike that HPLC, which detects finished product, and purity is greater than 99%.
2. zymologic property research finds that the enzyme Michaelis constant Km is 0.0233g/mL, optimal pH 6.0, in slant acidity condition
Under (pH=5.8~6.4) stability it is preferable, 37 DEG C be incubated for 1h after still keep 80% or more vigor；45 DEG C of optimum temperature, not
Synthermal lower heat resistance is different, and after 30 DEG C~60 DEG C incubation 1h, enzyme activity decline 20%, after 70 DEG C of incubations 1h, enzyme activity declines
70%, but after 90 DEG C of high temperature incubation 1h, enzyme activity still has 60%.Influence of the different metal ions and additive to enzyme activity is:
Na+、Mn2+、Fe2+、Mg2+、K+And Ca2+Ion promotes the activity of h-LYZ to some extent；Zn2+、Ba2+、Cu2+、Co2+Inhibit h-
The activity of LYZ, other than EDTA additive can promote activity, remaining additive inhibits it.
3. using Odontothrips loti detection h-LYZ to nine plants of bacteriostasis for trying bacterium, while being examined with micro broth dilution method
Survey MIC and MBC.The result shows that micrococcus luteus is most sensitive to h-LYZ, bacteriostatic diameter reaches 47.8mm；Staphylococcus aureus
Bacterium and pasteurella multocida are more sensitive, and inhibition zone diameter is respectively 14.6mm and 25.9mm；To P. aeruginosa, pathogenic
Escherichia coli, staphylococcus epidermis also produce weaker bacteriostasis, diameter 12.3mm, 9.2mm, 8.0mm；To white
Candida albicans also functions to certain inhibiting effect, bacteriostatic diameter 10.5mm.Bacillus cercus and enterococcus faecalis act on not it
It is sensitive.When fighting micrococcus luteus, pathogenic escherichia coli and pasteurella multocida, in extremely low mass concentration (MIC
< 32 μ g/mL) can they be generated with growth inhibition effect, while finding to anti-Staphylococcus aureus and P. aeruginosa
When bacterium, MBC is better than certain brand product.
The formulation of 4.H-LYZ quality standard, project include: purity, property, identification, inspection, enzyme activity determination, storage, answer
With etc..
Detailed description of the invention
Fig. 1 separation purifying technique process.
Fig. 2 Bestarose MMC Diamond thin layer chromatography figure.
Fig. 3 sample collection.
Fig. 4 sample crosses Bestarose MMC Diamond column each component electrophoretogram.
Wherein, Fig. 4 A, M: albumen marker；1: positive control；2: fermented supernatant fluid；3: fermentation liquid pH is adjusted to 6.5；4: stream
It wears at 500mL；5: flowing through at 1000mL；6: flowing through at 2000mL；7: flowing through at 3000mL.Fig. 4 B, M: albumen marker；1: sun
Property control；2: cleaning；3: 0~400mL of elution；4: 800~1200mL of elution；5: 1200~1600mL of elution.
Fig. 5 HPLC purity detecting.
Fig. 6 human lysozyme freeze-dried powder.
The bis- counting backward techniques of Fig. 7 survey the Michaelis constant of h-LYZ.
Fig. 8 difference pH is on the active influence of h-LYZ.
The pH stability of Fig. 9 H-LYZ.
Figure 10 different temperatures is on the active influence of h-LYZ.
The thermal stability of Figure 11 H-LYZ.
Figure 12 different metal ions are on the active influence of h-LYZ.
Figure 13 different additive is on the active influence of h-LYZ.
Specific embodiment
The material and method that the present invention uses are as follows.
One, material
1.1 bacterial strain
Bacillus cercus (Bacillus cereus), pasteurella multocida (Pasteurlla multocida), table
Skin staphylococcus (StapHylococcus epidermidis), staphylococcus aureus (StapHylococcus aureus),
It is enterococcus faecalis (Enterococcus faecalis), pseudomonas aeruginosa (Pseudomonas aeruginosa), pathogenic big
Intestines Escherichia (Escherichia coli), Candida albicans (Monilia albican), micrococcus luteus
(Micrococcus luteus) is studied by Microbiological Lab, Fudan University and Huashan Hospital Affiliated To Fudan Univ antibiotic
It is provided.
1.2 main agents
Three, experimental method
H-LYZ's isolates and purifies in 3.1 fermentation liquids
1) pre-treatment of fermentation liquid: the fermentation liquid of the lysozyme containing recombination human source 1M NaOH tune pH to 6.5, then
12000rpm is centrifuged 15min, collects supernatant.Supernatant is handled using hollow fiber ultrafiltration membrane, is further removed in sample
Finely ground particle substance.It is eventually adding Bestarose MMC Diamond buffer solution B (1.5M NaCl, 20mM PB, pH 6.5) adjusting
Sample conductance keeps it consistent (about 43ms/cm) with buffer solution A (0.4M NaCl, 20mM PB, pH 6.5) conductance, as
Bestarose MMC Diamond column loading is as former state.
2) Bestarose MMC Diamond is captured: according to the physicochemical property of target product, selecting suitable chromatostrip
Part.Source of people lysozyme is basic protein, and isoelectric point is higher (pI ≈ 10), optimal pH ≈ 6.5, at room temperature in neutral salt solution
There is higher natural activity.Therefore select the pH of buffer close to neutrality in purification process, process is being isolated and purified to avoid sample
It is middle to be denaturalized.And source of people lysozyme is positively charged under pH neutrallty condition, therefore generally cation is selected to hand in traditional handicraft
Change purification media of the medium (such as SP, CM) as human lysozyme.
Chromatographic column: BXK 50/30, Vt=300mL.BXK, explosion-proof control box (cabinet).
Equilibration buffer A:0.4M NaCl, 20mM PB, pH 6.5.PB, phosphate buffer.
Elution buffer B:1.5M NaCl, 20mM PB, pH 6.5.
5 column volumes of equilibration buffer, until the pH of chromatographic column effluent liquid, conductance are consistent with equilibration buffer, it will be upper
Sample cleans 3~5 column volumes until baseline with equilibration buffer after end of the sample with 30mL/min flow velocity loading after stating processing
Stablize, i.e., all unbonded substances are all flushed splitter.Then destination protein is eluted with elution buffer, according to
UV280 eluting peak collects sample.
3) Bestdex G-25 buffer exchange: due to Bestarose MMC Diamond column elution samples volume compared with
Greatly, protein concentration is lower, and needing to carry out sample concentration can be only achieved requirement.SP Bestarose FF column is used in experiment
Sample concentration is carried out, therefore needs to carry out buffer exchange to sample before it is concentrated, its conductance is made to be less than 10ms/cm (Bestarose
MMC Diamond column elution samples conductance is about 135ms/cm).
Chromatographic column: BXK 200/500, Vt=5L.
Equilibration buffer: 20mM NaAC, pH 4.75.
2 column volumes of equilibration buffer, until the pH of chromatographic column effluent liquid, conductance are consistent with equilibration buffer, it will
Bestarose MMC Diamond elution samples should be less than 1.5L with 200mL/min flow velocity loading, loading volume.End of the sample
After continue to be cleaned with equilibration buffer, according to the peak UV280 collect sample.
4) destination protein: 3~5 column volumes of equilibration buffer is concentrated in SP Bestarose FF, until chromatographic column effluent
The pH of liquid, conductance are consistent with equilibration buffer, by the sample of transposed buffer with 20mL/min flow velocity loading, after end of the sample
3~5 column volumes are cleaned until baseline stability with equilibration buffer, i.e., all unbonded substances are all flushed splitter.
Then destination protein is eluted with elution buffer, sample is collected according to UV280 eluting peak.
Chromatographic column: BXK 50/30, Vt=280ml
Equilibration buffer: 20mM NaAC, pH 4.75.
Elution buffer: 0.15M NaCl, 20mM PB, pH 7.5.
Protein concentration detection: it is measured using " Folin-Phenol " method.It is said referring to Lowry method determination of protein concentration kit
Bright book.
Purity of protein detection: it is identified using SDS-PAGE electrophoresis and reverse phase C8HPLC.
The freeze-drying of 3.2 h-LYZ after purification
The physicochemical property research of 3.3 recombination h-LYZ
3.3.1 the Km value measurement of enzyme
It using micrococcus lysodeikticus as the substrate of enzyme, prepares different concentration gradients (0.1,0.2,0.3,0.4,0.5g/L), surveys
Determine enzyme activity, is mapped with double counting backward techniques.
3.3.2pH to the influence of enzyme activity
A series of 0.1M phosphate buffer of different pH value (pH=5.8~8.0) is chosen, at 25 DEG C, measurement is after purification
Liquid of protease vigor at various ph values the enzyme activity measured under remaining each pH value is converted with highest energy value for 100
It maps at opposite enzyme activity.
3.3.3pH stability
Liquid of protease after purification is placed in 37 DEG C, incubates in the 0.1M phosphate buffer of different pH value (pH=5.8~8.0)
It educates 1 hour.Then at 25 DEG C, enzyme activity is surveyed under conditions of optimum pH.Enzyme activity before incubation is 100, the enzyme measured after incubation
Vigor is converted into opposite enzyme activity and maps.
3.3.4 influence of the temperature to enzyme activity
In the buffer of optimum pH, choose different temperatures (15 DEG C~65 DEG C), the liquid of protease measured after purification exists
Vigor under different temperatures, with highest energy value for 100, the enzyme activity that remaining is measured at each temperature is converted into opposite enzyme activity and makees
Figure.
3.3.5 thermal stability
In the buffer of optimum pH, under different temperatures (30 DEG C~100 DEG C), it is incubated for 1 hour.Then at 25 DEG C, most
Enzyme activity is surveyed under conditions of suitable pH value.Enzyme activity is 100 before being incubated for, and the enzyme activity that remaining is measured at each temperature is converted into relatively
Enzyme activity mapping.
3.3.6 influence of the metal ion to enzyme activity
Under optimum pH and optimum temperature, it is separately added into the metal ion (Cu of 0.01M2+、Mg2+、Na+、Ca2+、Ba2+、
Zn2+、K+、Fe2+、Co2+、Mn2+), measure the influence to enzyme activity.The enzyme activity measured under metal ion is not added as 100, incite somebody to action
The enzyme activity measured under the conditions of remaining is converted into opposite enzyme activity and maps.
3.3.7 influence of the additive to enzyme activity
Under optimum pH and optimum temperature, it is separately added into additive (glucose, EDTA, probiotics, the cream of 10%w/v
Sugar and trehalose), mixture is being kept the temperature 1 hour at 25 DEG C, is measuring enzyme activity.It is with the enzyme activity that not doping measures
100, the enzyme activity measured under the conditions of remaining is converted into opposite enzyme activity and maps.
The In Vitro Bacteriostatic research of 3.4 recombination h-LYZ
3.4.1 the preparation of solution
Source of people antalzyme protein after the source of people lysozyme mark product (certain brand company) of purchase and this experiment purifying freeze-drying is dry
Powder, is diluted to 1.0g/L with ultrapure water respectively, and -20 DEG C of preservations are spare.
3.4.2 the activation culture of test strain
It is false that oese picks them separately micrococcus luteus (M.luteus), staphylococcus epidermis (S.epidermidis), verdigris
Monad (P.aeruginosa), Candida albicans (Monilia albican), Enteropathogenic Escherichia coli (E.coli), wax
Shape bacillus (B.cereus), staphylococcus aureus (S.aureus), pasteurella multocida (P.multocida), press
Continuous method of scoring is inoculated in MH plate, 37 DEG C of 24~36h of culture.It chooses single bacterium colony and is seeded to new MH plate, continue to cultivate, it is standby
With.Wherein 2% calf serum need to be added in activation culture pasteurella multocida in the medium.Enterococcus faecalis
(E.faecalis) it is activated on MRS plate, method is same as above.
3.4.3 the bacteriostatic activity of Odontothrips loti detection h-LYZ
Agar is added in beef-protein medium, 121 DEG C of high pressure sterilization 20min are cooled to 60 DEG C or so, use suction pipe
Accurate 15mL culture medium of drawing is added in sterile petri dish (lower layer), to its solidification.Separately take the bacterium solution (bacterium of 100 μ L test strains
Concentration 1.5 × 105CFU/mL it) is mixed well with the 10mL culture medium (being cooled to 50 DEG C or so), is added to the culture medium solidified
Sterile Oxford cup, is then gently put into culture dish with aseptic nipper by upper (upper layer), to its solidification.Then add in Oxford cup
Enter 50 μ L of enzyme solution to be measured, 37 DEG C of cultures for 24 hours, measure antibacterial circle diameter.
According to same method, the antibacterial circle diameter of certain brand source of people lysozyme of purchase is measured, as positive control；With nothing
Bacterium physiological saline is as negative control.
3.4.3 the MIC and MBC of micro broth dilution method detection h-LYZ
Using 96 orifice plates, using micro-broth dilution method h-LYZ 9 kinds are detected with the MIC and MBC of bacterial strain, and with certain
Brand source of people lysozyme makees standard control.
Sterile working, by (128,64,32,16,8,4,2,1,0.5,0.25, the 0.125 μ g/ of various concentration after doubling dilution
ML h-LYZ solution) is added separately in 96 hole polystyrene plates of sterilizing, and the 1st to the 11st hole adds medical fluid, every 10 μ L of hole；12nd
H-LYZ solution is not added in hole, as growth control.Diluting bacterium to be measured to concentration with MH fluid nutrient medium is 0.5 Maxwell than turbid, i.e.,
1.5×108CFU/mL, after MH meat soup 1:1000 dilution, to every 100 μ L of Kong Zhongjia.37 DEG C of cultures 18~for 24 hours.With same sample prescription
Method measures the MIC and MBC of certain brand source of people lysozyme, in this, as control.Experiment is parallel in triplicate.
MIC determines: occurring that disperse is muddy, to have precipitating be bacterial growth index for bottom in 96 orifice plates, the visible bacterium of naked eyes
The minimum quality concentration of growth is the minimal inhibitory concentration MIC of h-LYZ.
MBC determines: first measuring MIC, then will successively 100 μ L of culture solution be sucked out in the hole for having no bacterial growth, is applied to general
In logical agar plate, 37 DEG C of cultures 18~count bacterium colony afterwards for 24 hours, the culture hole mass concentration of clump count < 5 is h- on plate
The minimum bactericidal concentration MBC of LYZ.
H-LYZ's isolates and purifies result in 1 fermentation liquid of embodiment
After the fermentation liquid of the type lysozyme of c- containing recombination human source such as is centrifuged, is filtered at the pre-treatments, by Bestarose MMC
The purifying of one step of Diamond column, it is pure (Fig. 4) that source of people lysozyme product can reach electrophoresis.Bestarose MMC Diamond elution
Sample is concentrated, final sample concentration is about 27mg/mL after buffer exchange using SP Bestarose FF column.It is pure
During change, the yield of each step h-LYZ is respectively 92.67%, 91.9% and 94.68% (table 3.1), and destination protein is always received
Rate is 80.63%, and purification is up to 15 times.
The yield of h-LYZ in 3.1 purification process of table
PAGE gel electrophoresis and HPLC carry out Purity as a result, reverse phase C8 chromatographic column uses second cyanogen gradient, as a result
For simple spike (Fig. 5), PAGE gel electrophoresis is greater than 99% through laser gray scale scanning instrument measurement purity.To the source of people after concentration
(Fig. 6) is lyophilized in bacteriolyze enzyme sample, and the h-LYZ activity after freeze-drying is 120,000U/mg, every bottle of content 100mg.
Embodiment 2Km value measurement result
It reacts first rate and micrococcus luteus concentration of substrate to map according to the bis- counting backward techniques of Lineweaver-Burk, as a result sees Fig. 7.
Straight line is extended ,-(1/Km)=- 43 is obtained, according to KmThe definition of value, when enzymatic reaction reaches maximum speed VmBottom when half
Object concentration, obtains Km=0.0233g/mL.
The optimum pH of 3 enzyme of embodiment
The activity of source of people lysozyme is significantly affected when the pH value of phosphoric acid buffer system is greater than 7.0, enzyme activity substantially reduces, pH
When value is between 5.8~6.4, enzyme activity is relatively stable；When pH is 6.0, enzymatic activity is up to 93000U/mg.
Embodiment 4pH stability
From fig. 9, it can be seen that the acid resistance of this product is stronger, pH between 5.8~6.4,37 DEG C be incubated for one hour after still
The vigor that remain 80% or more illustrates that stability of the enzyme within the scope of slant acidity is preferable.
The optimum temperature of 4 enzyme of embodiment
Under the conditions of 15 DEG C~65 DEG C, source of people lysozyme enzyme activity first increases with the raising of temperature to be reduced afterwards, the people at 45 DEG C
The enzyme activity of source lysozyme reaches peak value, and active 102000U/mg illustrates that 45 DEG C are the enzyme optimum temperatures.
5 thermal stability of embodiment
As shown in figure 11, heat resistance is different at different temperature for the source of people lysozyme, and 30 DEG C~60 DEG C are incubated for one
After hour, enzyme activity downward trend is unobvious, enzyme activity decline 20% or so；Under the conditions of 70 DEG C, source of people antalzyme activity only deposits 30%；
But after 90 DEG C are incubated for 1 hour, enzyme activity still has 60%, after 100 DEG C are incubated for 1 hour, still there is 20% original enzyme activity, experimental result
Illustrate that this engineering source of people lysozyme has good heat resistance.
Influence of 6 metal ion of embodiment to enzyme activity
As shown in figure 12, a series of different types of metal ion solutions are prepared, when final concentration of 0.01mol/L measures enzyme
Vigor, the results showed that, Na+、Mn2+、Fe2+、Mg2+、K+、Ca2+Ion promotes the activity of h-LYZ in various degree, promotes degree: Na+
> Mg2+> Fe2+> K+> Mn2+> Ca2+；Zn2+、Ba2+、Cu2+、Co2+Inhibit the activity of h-LYZ, inhibition level: Cu2+> Zn2+
> Ba2+> Co2+。
Influence of 7 additive of embodiment to enzyme activity
Prepare different additive solutions, when final concentration of 0.01mol/L measures enzyme activity, as a result as shown in figure 13, removes
EDTA can promote h-LYZ activity outer, remaining generates inhibiting effect to its activity.
8 present invention of embodiment and current lysozyme purification technics comparing
Isolation and purification method and its advantage and disadvantage for relating generally to source of people lysozyme at present are as shown in the table:
The present invention establishes the new process that source of people lysozyme in fermentation liquid is isolated and purified with compound cation exchange medium.
Fermentation liquid through centrifugation, after filtration treatment without dilution can directly upper column purification, it is pure to reach electrophoresis through step purifying destination protein,
It is simple spike that HPLC, which detects finished product,.Entire process cycle is short, high income, is easy to Linear Amplifer to production scale.
The source of people c- type antalzyme protein of the invention of embodiment 9 is compared with certain brand source of people lysozyme
The expression quantity of the source of people c- type antalzyme protein of this experiment expression is up to 1.65g/L, than living up to 160,
000U/mg, product purity > 99%.The yield of albumen and the purity of product are highest.
Following table is the comparison result of certain brand source of people lysozyme and this product available on the market.
Claims (2)
1. a kind of source of people antalzyme protein purification process, which is characterized in that the source of people antalzyme protein purification process includes following
Step:
1) pre-treatment of fermentation liquid will contain recombined human including the solid impurity and adjustment pH value in removal human lysozyme solution
The fermentation liquid tune pH to 6.5 of source lysozyme；When removing impurity, first solution is centrifuged, collects supernatant, it is then super using doughnut
Filter membrane handles supernatant, further removes the finely ground particle substance in sample；
2) Bestarose MMC Diamond is captured, and Bestarose MMC Diamond elution buffer B is added, and adjusts sample
Conductance keeps it consistent with equilibration buffer A conductance, reaches 43ms/cm, and as Bestarose MMC Diamond column loading is former
Sample, Bestarose MMC Diamond capture need the physicochemical property according to target product, select suitable chromatographic condition, use
Purification media of the cation exchange medium as human lysozyme, the chromatographic column of use and the condition of buffer are as follows:
Chromatographic column: BXK 50/30, Vt=300mL；
Equilibration buffer A:0.4M NaCl, 20mM phosphate buffer, pH6.5；
Elution buffer B:1.5M NaCl, 20mM phosphate buffer, pH6.5；
Equilibration buffer A balances 5 column volumes, until the pH of chromatographic column effluent liquid, conductance are consistent with equilibration buffer, by above-mentioned place
Loading after reason as former state with 30mL/min flow velocity loading, after end of the sample with equilibration buffer A clean 3~5 column volumes until
Baseline stability, i.e., all unbonded substances are all flushed splitter, then elute destination protein, root with elution buffer B
Sample is collected according to UV280 eluting peak；
3) Bestdex G-25 buffer exchange carries out buffer exchange to sample before concentration, its conductance is made to be less than 10ms/cm,
The chromatographic column of use and the condition of buffer are as follows:
Chromatographic column: BXK 200/500, Vt=5L；
Equilibration buffer: 20mM NaAC, pH4.75；
2 column volumes of equilibration buffer, until the pH of chromatographic column effluent liquid, conductance are consistent with equilibration buffer, it will
Bestarose MMC Diamond elution samples are less than 1.5L, end of the sample with 200mL/min flow velocity loading, loading volume
After continue to be cleaned with equilibration buffer, according to the peak UV280 collect sample；
4) be concentrated destination protein, using SP Bestarose FF column carry out sample concentration, using SP Bestarose FF column into
The step of row sample concentration includes: 3~5 column volumes of equilibration buffer, until the pH of chromatographic column effluent liquid, conductance and balance
Buffer is consistent, by the sample of transposed buffer with 20mL/min flow velocity loading, cleans 3 with equilibration buffer after end of the sample
~5 column volumes are all flushed splitter, are then washed with elution buffer until baseline stability, i.e., all unbonded substances
De- destination protein collects sample according to UV280 eluting peak, and the chromatographic column of use and the condition of buffer are as follows:
Chromatographic column: BXK 50/30, Vt=280mL；
Equilibration buffer: 20mM sodium acetate, pH4.75；
Elution buffer: 0.15M NaCl, 20mM phosphate buffer, pH7.5.
2. source of people antalzyme protein purification process according to claim 1, which is characterized in that straight containing human lysozyme solution
Connect the fermentation liquid using human lysozyme solution.
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| CN109295034A (en) * | 2017-07-25 | 2019-02-01 | 上海复华兴生物技术有限公司 | A kind of source of people antalzyme protein LYC8 and its production technology and application |
| CN109295036A (en) * | 2017-07-25 | 2019-02-01 | 上海复华兴生物技术有限公司 | A kind of recombinant human lysozyme industrialized purification process |
| CN109295035A (en) * | 2017-07-25 | 2019-02-01 | 上海复华兴生物技术有限公司 | A kind of chromatography method of recombination human source lysozyme |
| CN107446905B (en) * | 2017-08-24 | 2020-04-24 | 陕西慧康生物科技有限责任公司 | Method for purifying recombinant human lysozyme |
| CN115094051B (en) * | 2022-04-08 | 2023-09-22 | 福建福大百特生物科技有限公司 | Recombinant lysozyme from bemyces fumosoroseus as well as preparation method and application thereof |
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