CN108118044A - A kind of method of separating-purifying egg white lysozyme - Google Patents

A kind of method of separating-purifying egg white lysozyme Download PDF

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CN108118044A
CN108118044A CN201810106401.3A CN201810106401A CN108118044A CN 108118044 A CN108118044 A CN 108118044A CN 201810106401 A CN201810106401 A CN 201810106401A CN 108118044 A CN108118044 A CN 108118044A
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resin
egg white
lysozyme
eluent
interception
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刘冬梅
黄燕燕
周钦育
黄泳尧
林钟培
林梓茵
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South China University of Technology SCUT
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    • 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)
    • 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)

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Abstract

The invention discloses a kind of methods with base exchange method combination hyperfiltration technique separating-purifying egg white lysozyme, include the following steps：(1) egg white pre-processes：Screen filtration is crossed after egg white stirring；(2) resin pre-processes；(3) adsorb：Pretreated resin, standing adsorption after stirring are added in after adjusting egg white filtrate pH value；(3) elute：It is stirred and eluted with NaCl solution, filter out eluent, repetition elutes and merges eluent；(4) ultrafiltration：By eluent ultra-filtration centrifuge tube ultrafiltration removing protein, desalination, ultrafiltrate is obtained；(5) it is dry：To get to the lysozyme after ultrafiltrate is dried.Gained lysozyme measures high purity 96% by SDS PAGE electrophoresis, and Rate activity is more than 20000U/mg.Present invention process is simple, and cost is relatively low, and the lysozyme purity prepared is high, activity is high.

Description

A kind of method of separating-purifying egg white lysozyme
Technical field
The invention belongs to biological technical field, more particularly to a kind of method of separating-purifying lysozyme.
Background technology
Chen Yan et al. in《Biology magazine》It is mentioned in " progress of lysozyme " that the 2nd phase of volume 26 in 2009 delivers Lysozyme is a kind of alkaline protein, be widely present in the egg white of birds, poultry and the tear of mammal, saliva, blood plasma, In milk, placenta and body fluid, histocyte, but the content in egg white is most abundant (account for egg white 3.5%).It can be selected Property hydrolyze in peptide glycan-Isosorbide-5-Nitrae glycosidic bond, while do not destroy other tissues, and itself is nontoxic, thus be a kind of natural The good fungicide of security performance, preservative, can be applied to the anti-corrosion of the industries such as food, medicine and daily use chemicals.
At present, the preparation method of lysozyme mainly has crystallisation, ion-exchange, ultrafiltration, polyelectrolyte precipitation method, parent With chromatography etc..Tao Feng clouds et al. in《Beijing Union University's journal (natural science edition)》What the 3rd phase of volume 20 in 2006 delivered It is referred in " progress of antalzyme crystallization " using NaCl as precipitating reagent, MgCl2 solution is eluent, and sodium acetate solution is slow The Static Membrane crystallisation of fliud flushing.Using this method, the easy to operate but production cycle is long, and raw material availability is low, is not suitable for industry metaplasia Production.
Wang Wei armies et al. in《Agricultural University Of South China's journal》03 phase in 1996 deliver " deaminated regenerated chitin is affine Deaminated regenerated chitin is taken for the first time in a column chromatography Purification of Lysozyme " text, with new column chromatography extraction purification bacteriolyze Enzyme.The advantages of affinity chromatography can quickly realize the separation of purpose thing and other impurities is fully demonstrated, but this method amplification is tired Difficulty, the rate of recovery is low, and the blocking of chromatographic column is more easily caused when viscosity is big, therefore is not suitable for industrialized production.
Remaining sea sweet smell et al. in《Chinese poultry resource》24 phases in 2008 deliver " base exchange method extraction egg white lysozyme is ground Study carefully " in cation exchange resin processes extract egg white lysozyme, the ammonium sulfate eluent cost of use is higher, the lysozyme of preparation Enzyme purity and specific activity of enzyme it is not high enough.
It is indicated above it is domestic to the isolating and purifying of lysozyme, extract preparation method and once used crystallisation, ion exchange layer Analysis method, affinity chromatography etc., but have certain drawback.Even current industrial production mainly using base exchange method, passes through Resin cation absorption, high eluting salt, desalting and dewatering and atomization drying and etc. obtain lysozyme, but due to lysozyme eluent Amount of solution is big, salt content is high, commonly uses ultrafiltration apparatus burden weight, ultrafiltration membrane because of the vulnerable to pollution that works long hours, the service life of film It is greatly affected；The conditions such as the selected process for separation and purification of above-mentioned technology, eluent, ultrafiltration centrifugation opportunity are not suitable for, The of high cost of separating-purifying is caused, the lysozyme purity after separation is not high, and extraction process falls behind so that the problems such as enzyme activity loss is big.
The content of the invention
The purpose of the present invention is making up the deficiencies in the prior art, a kind of base exchange method combination hyperfiltration technique point is provided Method from purification egg white lysozyme.A kind of Rate activity is provided up to 20000U/mg, the lysozyme of high purity 96%.The present invention It is simple for process, without complex device, the lysozyme of high-purity and high bioactivity can be obtained, existing lysozyme is overcome to prepare work The shortcomings that skill, can be mass-produced.
The purpose of the present invention is achieved through the following technical solutions：
A kind of method of base exchange method combination hyperfiltration technique separating and purifying protein lysozyme, includes the following steps：
(1) egg white pre-processes：After egg white is stirred evenly, cross 80~120 mesh filter screens and obtain egg white filtrate；
(2) resin pre-processes：Resin is taken to be washed with distilled water to without apparent impurity and is filtered dry, is first impregnated with hydrochloric acid solution Afterwards, then with distilled water flushing Resin A is obtained；Resin A obtains resin B after sodium hydroxide solution immersion with distilled water flushing；Wherein, Resin is selected from 724 Macroporous weak acid cation exchange resins, 732 type cation exchange resins, D152 macropore Subacidity cations One kind in exchanger resin；
(3) adsorb：Resin B obtained by adding in step (2) into egg white filtrate, stirs and evenly mixs rear standing adsorption, supernatant discarding Liquid is washed with distilled water, and obtains resin L；
(4) elute：NaCl solution is added in into resin L obtained by step (3), stirring elution filters out eluent, repeats to elute Merge gained eluent, reservation filters out the resin after eluent；
(5) ultrafiltration：Eluent obtained by step (4) is first placed in the ultra-filtration centrifuge tube that interception is 50KD, centrifuges and take Subnatant；Subnatant is placed in the ultra-filtration centrifuge tube that interception is 3KD afterwards, centrifuges and takes upper strata trapped fluid to get to described Lysozyme.
Preferably, the volume ratio of egg white filtrate and resin B is 10 in step (3)：2~4, it is 6 to control egg white filtrate pH value ~10.
Preferably, the volume ratio of egg white filtrate and resin B is 10 in step (3)：2.4~2.5.
Preferably, egg white filtrate pH value is 9.0~9.5 in step (3).
Preferably, the concentration of NaCl solution described in step (4) is 0.5~2.5mol/L, the frequency of the stirring elution For 60~100r/min, the time is 20~100min.
Preferably, step (4) described NaCl solution is 1~1.5moL/L, and stirring elution time is 60~80min, NaCl The dosage of solution is 0.5~1 times of step (1) the egg white filtrate volume.
Preferably, interception described in step (5) is that the rotating speed of 50KD centrifuge tubes is 4000~6000g, centrifugation time 5 ~15min；The interception is that the rotating speed of 3KD centrifuge tubes is 6000~7000g, and centrifugation time is 50~70min.
Preferably, step (5) described interception is that the rotating speed of 50KD centrifuge tubes is 5000g, centrifugation time 10min；Institute It is 6500g, centrifugation time 60min to state the rotating speed that interception is 3KD centrifuge tubes.
Preferably, resin described in step (2) pre-process, by resin be soaked in 2~4 times of resin volumes 0.8~ 1.2mol/L hydrochloric acid solutions, soaking time are 10~12h, are 5.5~6.5 with distilled water flushing to efflux pH value, obtain resin A；Resin A is soaked in 0.8~1.2mol/L sodium hydroxide solutions of 2~4 times of resin volumes again, soaking time is 10~12h, It is 8.5~9.5 with distilled water flushing to efflux pH value, obtains resin B.
Preferably, step (4) resin filtered out after eluent continues step (2) as Resin A and prepares resin B。
Compared with prior art, the present invention having the following advantages that and advantageous effect：
(1) present invention is eluted using NaCl solution instead of traditional ammonium sulfate, not only reduces cost, but also Add the security of lysozyme product so that product more suits food and medicine industry.
(2) before elution step is arranged in ultrafiltration step by the present invention, ultrafiltration membrane can be blocked to avoid sticky egg white solution, So as to extend the service life of ultrafiltration membrane, while improve the purity of ultrafiltered product.
(3) present invention base exchange method combination hyperfiltration technique separating-purifying egg white lysozyme, can both obtain high-purity The lysozyme product of high activity is spent, while reduces ultrafiltration pressure again, reduces operating cost.
(4) present invention process is simple, with short production cycle, mild condition, at low cost without special installation, can expand life Production.
Description of the drawings
Fitting A (resin demand) and D (elution time) response surface design interaction of Fig. 1 embodiment 1Box-Behnken Optimal Experimentals Function influence figure.
Fig. 2 embodiment 1Box-Behnken Optimal Experimentals are fitted A (resin demand) and D (elution time) contour map.
Fig. 3 embodiment 1Box-Behnken Optimal Experimentals fitting B (eluate concentration) and D (elution time) response surface design is handed over Interaction influences figure.
Fig. 4 embodiment 1Box-Behnken Optimal Experimentals are fitted B (eluate concentration) and D (elution time) contour map.
Fig. 5 embodiment 1Box-Behnken Optimal Experimentals are fitted A (resin demand) and C (pH value) response surface design reciprocation Influence figure.
Fig. 6 embodiment 1Box-Behnken Optimal Experimentals are fitted A (resin demand) and C (pH value) contour map.
The PAGE gel electrophoretogram of 3 eluent of Fig. 7 embodiments.
The PAGE gel electrophoretogram of 3 ultrafiltrate of Fig. 8 embodiments.
The gel imaging figure of 3 ultrafiltrate of Fig. 9 embodiments.
Figure 10 process flow charts.
Specific embodiment
To be best understood from the present invention, the present invention is described in further details with reference to embodiment, but the present invention claims The scope of protection is not limited to the scope represented by embodiment.
First, base exchange method single factor test optimizes
1. egg white resin ratio optimizes
It is 10 to control egg white and resin volume ratio respectively：2、10：2.5、10：3、10：3.5、10：4.As it can be seen from table 1 When egg white resin ratio is 10：When 2.5, the Rate activity highest of gained lysozyme, ratio at this time separates lysozyme suitable for extraction.When When amount of resin is further added by, Rate activity is gradually reduced, and illustrates that excessive resin is unfavorable for extracting lysozyme from egg white.
The different egg white resins of table 1 compare the influence of bacteriolyze specific activity of enzyme
Project/sequence number 1 2 3 4 5
Egg white resin ratio 10:2 10:2.5 10:3 10:3.5 10:4
Enzyme content/(mg/ml) 1.789 2.050 2.094 2.137 2.094
Rate activity/(U/mg) 11178.25 14997.78 13492.07 12749.49 12178.69
2. egg white filtrate pH value optimizes
It is 6,7,8,9,10 to adjust egg filtrate pH value respectively.From table 2 it can be seen that when egg white filtrate pH value is 9, gained The Rate activity highest of lysozyme, pH value at this time separate lysozyme suitable for extraction.When pH value is too high or too low, the Rate activity of enzyme It will be lower.
Influence of the different egg white filtrate pH value of table 2 to bacteriolyze specific activity of enzyme
Project/sequence number 1 2 3 4 5
pH 6 7 8 9 10
Enzyme content/(mg/ml) 1.920 2.094 2.268 2.181 2.094
Rate activity/(U/mg) 10027.37 12298.08 13117.87 16048.71 13372.67
3.NaCl solution concentrations optimize
It is 0.5mol/L, 1.0mol/L, 1.5mol/L, 2.0mol/L, 2.5mol/L to control eluent NaCl concentration respectively. From table 3 it can be seen that when NaCl concentration is 1mol/L, the Rate activity highest of gained lysozyme, eluate concentration at this time is fitted Preferably extract separation lysozyme.When NaCl concentration is higher than 1mol/L, the higher NaCl solution elution of concentration is added in, specific activity of enzyme is anti- And decline.
Influence of the different NaCl concentrations of table 3 to bacteriolyze specific activity of enzyme
Project/sequence number 1 2 3 4 5
NaCl concentration/(mol/l) 0.5 1 1.5 2 2.5
Enzyme content/(mg/ml) 1.615 2.137 2.485 2.094 2.007
Rate activity/(U/mg) 9287.28 15205.81 12472.37 11223.49 12956.05
4. elution time optimizes
It is 20min, 40min, 60min, 80min, 100min to control elution time respectively.From table 4, it can be seen that when elution When time is 60min, the Rate activity highest of gained lysozyme, elution time at this time separates lysozyme suitable for extraction.Work as elution During overlong time, specific activity of enzyme declines instead.
Influence of the different elution times of table 4 to bacteriolyze specific activity of enzyme
Project/sequence number 1 2 3 4 5
Elution time/min 20 40 60 80 100
Enzyme content/(mg/ml) 1.398 1.789 2.137 2.181 2.224
Rate activity/(U/mg) 10017.76 12016.62 18130.00 14099.94 13599.30
2nd, base exchange method condition optimizing designs
Base exchange method condition optimizing is carried out using Design Expert8.0.6 softwares.Carrying out single factor test point above During analysis, egg white resin ratio, egg white filtrate pH value, NaCl concentration, elution time these four single factor tests is selected to be analyzed, found When egg white resin ratio is 10：2.5, egg white filtrate pH value is 9, concentration of sodium chloride solution 1mol/L, elution time 60min When, the Rate activity for obtaining lysozyme is that Rate activity is highest in respective single factor test Optimal Experimental group.Therefore these are selected Part carries out Box-Behnken Optimal Experimentals, experimental result is as shown in table 5 as benchmark.
Table 5Box-Behnken experimental result tables
It is analyzed using the center combination design experimental data of Box-Benhken, then utilizes Design Expert 8.0.6 software is analyzed, and experiment analysis results are as shown in table 6.
Table 6Box-Behnken experimental analysis tables
As shown in Table 6, the p=0.0014 ＜ 0.05 of model of fit, coefficient of determination R2=0.9208, illustrate model and reality Situation fitting is good.Learn bacteriolyze specific activity of enzyme R1 to resin demand in the every 100ml egg white filtrates of independent variable according to above analysis (A), NaCl concentration (B), pH value (C), the multiple regression equation of elution time (D)：
R1=20866.66-1432.26A+194.18B+486.75C+539.58D-961.62AB-12 52.19AC- 2929.88AD-21.46BC-2724.86BD+90.11CD-6079.24A2-5995.65B2-3410.58C2-3903.162
The linear relationship that the equation expresses between the Rate activity of lysozyme and 4 independents variable is significant, i.e., this side Method is reliable.The quadratic term coefficient ratio of regression equation is larger, and the interaction coefficient of AD, BD are very big in interaction term coefficient, specification tree Interaction between fat dosage and elution time, NaCl concentration and elution time is very big, and the interaction coefficient of AB, AC are larger, say Interaction is larger between bright resin demand and NaCl concentration, resin demand and pH value, and the interaction coefficient of BC, CD are smaller, Illustrate that the interaction between NaCl concentration and pH value, pH value and elution time is small.
In order to examine the validity of equation, variance analysis is carried out to the mathematical model of the Rate activity of the lysozyme of measure, and The partial regression coefficient of each factor is detected.The regression coefficient of A is more significant in first order, p=0.0806, illustrates egg white resin The bacteriolyze specific activity of enzyme for comparing extraction has more significant effect.The partial regression coefficient of A, B reach the pole level of signifiance, C, D in quadratic term And more significant level.Interaction item AD, BD regression coefficient is notable compared with other factors, illustrates resin demand and elution time, NaCl Interaction item between concentration and elution time influences bacteriolyze specific activity of enzyme more notable.Degree of fitting (coefficient of determination) R2= 0.9208, illustrate that the equation is good to experimental fit degree.
Each condition of highest bacteriolyze specific activity of enzyme in order to obtain, absorption and elution needs suitable cross selection.Therefore, Response surface design figure is fitted according to the Box-Behnken Optimal Experimentals of Fig. 1~Fig. 6 and variance analysis is learnt, to bacteriolyze specific activity of enzyme Influence size order is AD ＞ BD ＞ AC>AB>CD>BC.
Seek quadratic regression equation single order local derviation, when response R1 (bacteriolyze specific activity of enzyme) be maximum when each factor level For resin demand (A) in every 100ml egg white filtrate be 24.20ml, NaCl concentration (B) is 1.00mol/L, egg white filtrate pH value (C) be 9.10, elution time (D) is 62.61min.Theoretical prediction bacteriolyze specific activity of enzyme is 21041.1U/mg at this time.
Parallel verified experiment is carried out to each optimal conditions of base exchange method obtained, is repeated five times, it is actual such as following table It obtains lysozyme and is averaged Rate activity as 20509.58U/mg.
Table 7 screens bacteriolyze specific activity of enzyme measurement result after optimum condition
Group 1 2 3 4 5 Average
Enzyme content/(mg/ml) 3.138 2.964 2.964 2.877 2.964 2.982
Rate activity/(U/mg) 21030.64 21084.93 21759.65 18768.48 19904.18 20509.58
3rd, Ultrafiltration Purifying condition optimizing designs
The eluent obtained by optimum condition that base exchange method condition optimizing above is designed is placed in interception and is In the ultra-filtration centrifuge tube of 50KD, in 4000~6000g, 5~15min is centrifuged, except foreigh protein removing, discards centrifuge tube upper liquid.Again Subnatant is placed in the ultra-filtration centrifuge tube that interception is 3KD, in 6500g, centrifuges 60min, retain upper strata trapped fluid.Take part Upper strata trapped fluid carries out enzyme content and Rate activity measures, as a result as shown in table 8 below.
8 eluent centrefuge experiment result of table
50KD centrifuge tube rotating speeds (g) 50KD centrifugation times (min) Enzyme content (mg/mL) Rate activity (U/mg)
0 0 2.982 20509.58
4000 5 2.877 19116.05
4000 10 3.007 19616.18
4000 15 3.095 18256.67
5000 5 3.138 19596.73
5000 10 3.225 23718.61
5000 15 3.095 22618.89
6000 5 3.182 21685.86
6000 10 3.486 21511.98
6000 15 3.617 19906.07
Compare the combination of each centrifugal rotational speed of 50KD ultra-filtration centrifuge tubes and centrifugation time.With the rise of centrifugal rotational speed, enzyme Content gradually rises, and bacteriolyze specific activity of enzyme is then first to rise to reduce afterwards, this shows that too low centrifugal rotational speed causes lysozyme can Upper strata can be partly trapped within, though and excessively high centrifugal rotational speed can be by lysozyme all through lower floor, simultaneously to the miscellaneous egg in part It cannot effectively remove in vain, not be trapped equally or lysozyme is damaged during high speed centrifugation, enzyme activity is caused to decline.With That centrifugation time extends, Rate activity increased, but centrifugation time is not suitable for long, long centrifugation time can make efficiency It reduces, is unfavorable for mass producing.
Known by table 8, be that the condition that 50KD ultra-filtration centrifuge tubes should be taken is under 5000g when bacteriolyze specific activity of enzyme is maximum Centrifuge 10min.
Embodiment 1：Base exchange method purification lysozyme research
(1) egg white pre-processes：After egg white is stirred 35min with blender, filtered with 100 mesh filter screens.
(2) resin pre-processes：724 resins of 150ml are taken, 5 times is washed with distilled water to without apparent impurity, is filtered dry, adds in 3 The 1moL/L hydrochloric acid solutions of times resin volume, impregnate 10h, and abandoning supernatant must be set with distilled water flushing to eluate pH for 6.0 Fat A；The 1moL/LNaOH solution of 4 times of resin volumes is added in into Resin A again, impregnates 12h, abandoning supernatant is rushed with distilled water It is 9.0 to be washed till eluate pH, and it is spare to obtain resin B with distilled water immersion.
(3) adsorb：Gained egg white pH value is 9.10 in regulating step (1), and tree obtained by step (2) is added in into egg white filtrate The volume ratio of fat B, egg white and resin B is 10：2.4, it adds in after resin B and 30min is stirred with 100r/min, standing adsorption 5h is abandoned Resin L is washed with distilled water to obtain after removing supernatant.
(4) elute：The NaCl solution of 1moL/L is added in into resin L obtained by step (3), the dosage of NaCl solution is step (1) 0.75 times of gained egg white filtrate volume stirs elution 62.6min with 75r/min, filters out eluent, repeat elution three times, Merge eluent.
Embodiment 2：Ultrafiltration further purifies lysozyme
The eluent of the gained of embodiment 1 institute is placed in the ultra-filtration centrifuge tube that interception is 50KD, in 5000g, is centrifuged 10min except foreigh protein removing, obtains centrifuge tube subnatant, then subnatant is placed in the ultra-filtration centrifuge tube that interception is 3KD, in 6500g centrifuges 60min, retains upper strata trapped fluid.
Embodiment 3：PAGE gel electrophoresis and gel imaging Experimental Research lysozyme purity
The ultrafiltrate of 2 gained of the eluent of 1 gained of embodiment and embodiment is probed into for following electrophoresis experiment；
By the separately sampled progress PAGE gel electrophoresis experiment of the ultrafiltrate of the eluent of embodiment 1 and embodiment 2, electricity Result of swimming is as shown in Figure 7 and Figure 8.Fig. 7 is eluent electrophoresis result figure, and 5 swimming lanes are washed for what is obtained in parallel condition embodiment 1 De- liquid；Fig. 8 is ultrafiltrate electrophoresis result figure, and 10 swimming lanes are the ultrafiltrate obtained in parallel condition embodiment 2；As seen from the figure, Eluent and ultrafiltrate can see apparent band in 14.4KDa series, meet egg white lysozyme relative molecular weight.And eluent It can be seen that foreign protein removal effect is preferable after ultrafiltration, the lysozyme purity of extraction is high.
Further to estimate the purity of lysozyme, gained ultrafiltrate, electrophoresis are made SDS-PAGE and coagulate in another Example 2 Glue carries out Image Acquisition, as shown in Figure 9 with gel imager to gel.Swimming lane 7 is low molecular weight protein standard items in Fig. 9, Remaining swimming lane is that ultrafiltrate is made in embodiment 2, image is analyzed with Image Lab 5.2.1 softwares, the data obtained As shown in table 9.
9 gel imaging data results of table
Swimming lane No. BandNo. Molecular weight (KDa) RelativeFront Volume(Int) Band% Lane%
1 1 15.1 0.661 2,509,010 100.0 34.1
2 1 15.1 0.661 2,387,280 100.0 35.7
3 1 14.8 0.667 2,310,858 100.0 28.1
4 1 14.6 0.672 2,325,078 100.0 34.5
5 1 14.4 0.676 2,288,792 100.0 25.0
6 1 14.4 0.679 2,127,116 100.0 20.5
7 1 97.4 0.130 245,616 9.0 4.5
7 2 66.2 0.184 383,250 14.1 7.1
7 3 43.0 0.263 559,356 20.5 10.3
7 4 31.0 0.385 361,872 13.3 6.7
7 5 20.1 0.567 553,140 20.3 10.2
7 6 14.4 0.676 619,080 22.7 11.4
8 1 14.5 0.675 1,916,610 100.0 32.7
9 1 14.4 0.679 2,228,768 100.0 33.1
10 1 14.4 0.678 2,355,315 100.0 31.1
11 1 14.4 0.679 2,258,568 100.0 31.6
12 1 14.4 0.695 2,489,419 100.0 31.4
As shown in Table 9, protein molecular weight distribution in 14.4~15.1KDa, meets lysozyme in ultrafiltrate Distribution.To in addition to swimming lane 7 all swimming lane Volume values be averaged as 2290619, the Article 6 band of Protein standards (lysozyme band) content is 0.125ug/ul, and applied sample amount 5ul, Volume value are 619080, and experiment measures albumen in ultrafiltrate Matter content be 2.40ug/ul, applied sample amount 10ul (sample-loading buffers：Sample=4：1), Volume values are 2290619, therefore are estimated Lysozyme purity be 0.125 × 2290619 × 5 × 100%/(2.40 × 619080)=96.36%, the Rate activity of the lysozyme For 23718.61U/mg.

Claims (10)

  1. A kind of 1. method of separating-purifying egg white lysozyme, which is characterized in that comprise the following steps：
    (1) egg white pre-processes：After egg white is stirred evenly, cross 80~120 mesh filter screens and obtain egg white filtrate；
    (2) resin pre-processes：Resin is taken to be washed with distilled water to without apparent impurity and is filtered dry, after first being impregnated with hydrochloric acid solution, then Resin A is obtained with distilled water flushing；Resin A obtains resin B after sodium hydroxide solution immersion with distilled water flushing；Wherein, resin selects Tree is exchanged from 724 Macroporous weak acid cation exchange resins, 732 type cation exchange resins, D152 macropores Subacidity cation One kind in fat；
    (3) adsorb：Resin B obtained by adding in step (2) into egg white filtrate, stirs and evenly mixs rear standing adsorption, and abandoning supernatant is used Water washing is distilled, obtains resin L；
    (4) elute：NaCl solution is added in into resin L obtained by step (3), stirring elution filters out eluent, repeats elution and merges Gained eluent, reservation filter out the resin after eluent；
    (5) ultrafiltration：Eluent obtained by step (4) is first placed in the ultra-filtration centrifuge tube that interception is 50KD, centrifuges and remove layer Liquid；Subnatant is placed in the ultra-filtration centrifuge tube that interception is 3KD afterwards, centrifuges and takes upper strata trapped fluid to get to the bacteriolyze Enzyme.
  2. 2. according to the method described in claim 1, it is characterized in that, the volume ratio of egg white filtrate and resin B is in step (3) 10：2~4, it is 6~10 to control egg white filtrate pH value.
  3. 3. according to the method described in claim 2, it is characterized in that, the volume ratio of egg white filtrate and resin B is in step (3) 10：2.4~2.5.
  4. 4. according to claims 1 to 3 any one of them method, which is characterized in that egg white filtrate pH value is 9.0 in step (3) ~9.5.
  5. 5. according to claims 1 to 3 any one of them method, which is characterized in that the concentration of NaCl solution described in step (4) For 0.5~2.5mol/L, the frequency of the stirring elution is 60~100r/min, and the time is 20~100min.
  6. 6. according to the method described in claim 5, it is characterized in that, step (4) described NaCl solution be 1~1.5moL/L, stir Elution time is mixed as 60~80min, the dosage of NaCl solution is 0.5~1 times of step (1) the egg white filtrate volume.
  7. 7. according to claims 1 to 3 any one of them method, which is characterized in that interception described in step (5) for 50KD from The rotating speed of heart pipe is 4000~6000g, and centrifugation time is 5~15min；The interception is that the rotating speed of 3KD centrifuge tubes is 6000 ~7000g, centrifugation time are 50~70min.
  8. 8. the method according to the description of claim 7 is characterized in that step (5) described interception is the rotating speed of 50KD centrifuge tubes For 5000g, centrifugation time 10min；The interception is that the rotating speed of 3KD centrifuge tubes is 6500g, centrifugation time 60min.
  9. 9. according to claims 1 to 3 any one of them method, which is characterized in that resin described in step (2) pre-processes, will Resin is soaked in 0.8~1.2mol/L hydrochloric acid solutions of 2~4 times of resin volumes, and soaking time is 10~12h, is rushed with distilled water It is 5.5~6.5 to be washed till efflux pH value, obtains Resin A；Resin A is soaked in 0.8~1.2mol/L of 2~4 times of resin volumes again Sodium hydroxide solution, soaking time are 10~12h, are 8.5~9.5 with distilled water flushing to efflux pH value, obtain resin B.
  10. 10. according to claims 1 to 3 any one of them method, which is characterized in that after filtering out eluent described in step (4) Resin continues step (2) as Resin A and prepares resin B.
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CN110066778A (en) * 2019-05-09 2019-07-30 江南大学 A kind of combined separation method of lysozyme from egg white and ovotransferrins
CN113475619A (en) * 2021-07-13 2021-10-08 江南大学 Method for efficiently fermenting egg white and preparing original-flavor dried egg white

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CN109136201A (en) * 2018-07-15 2019-01-04 爱必信（上海）生物科技有限公司 A kind of purification technique of enzyme biochemical reagents
CN110066778A (en) * 2019-05-09 2019-07-30 江南大学 A kind of combined separation method of lysozyme from egg white and ovotransferrins
CN110066778B (en) * 2019-05-09 2021-03-02 江南大学 Combined separation method of lysozyme and ovotransferrin in egg white
CN113475619A (en) * 2021-07-13 2021-10-08 江南大学 Method for efficiently fermenting egg white and preparing original-flavor dried egg white
CN113475619B (en) * 2021-07-13 2022-07-22 江南大学 Method for efficiently fermenting egg white and preparing original-flavor dried egg white

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