CN102228823B - Modified beer waste yeast adsorbent, preparation method and application thereof - Google Patents
Modified beer waste yeast adsorbent, preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biosorbent preparation and application, and specifically discloses a modified beer waste yeast adsorbent, a preparation method and an application thereof. The modified beer waste yeast adsorbent provided by the present invention is a novel adsorbent which is prepared from a cross-linked beer waste yeast modified through citric acid, and provides good absorbability for lysozyme such that the modified beer waste yeast adsorbent can be applicable for extracting the lysozyme.
Description
Technical field
The present invention relates to preparation and the applied technical field of biological adsorption agent, be specifically related to a kind of modified beer waste yeast adsorbent and its preparation method and application.
Background technology
Beer waste yeast is the accessory substance of fermentation industry, be rich in-OH ,-NH
2,-the functional groups such as COOH.As a kind of adsorbent cheaply, often be used to adsorb the harmful substance in waste water.Through the various functional groups of its surface grafting that are everlasting, increase active sites and count to strengthen its adsorption capacity.Much research is reported, utilizes the group on beer waste yeast surface, carries out derivative reaction, carrys out the chelating adsorbing metal ions.But the research the beer waste yeast through modification for adsorbing lysozyme, there is not yet report.
Lysozyme is a kind of enzyme be in daily use, mainly be present in egg, people's the body fluid of tears, saliva and other animals, it can destroy-acetylmuramic acid and the β between NAG-1 in cell membrane, 4 glycosidic bonds, make the insoluble glutinous polysaccharide of cell membrane resolve into the solubility glycopeptide, cause the cell wall rupture Dissolve things inside to be overflowed and make bacterolysis.Because there is this character, lysozyme is applied (R.Islam usually used as anticorrisive agent, J.Kite, A.S.Baker, A.Ching Jr., M.R.Islam, African J.Biotechnology.Affinity purification of hen egg lysozyme using sephadex.5 (2006) 1902.1515-1520).Usually extract lysozyme from egg, wherein the content of lysozyme is 0.34% (G.Zhang et al.Tris (hydroxymethyl) aminomethane-modified magnetic microspheres for rapid affinity purificationof lysozyme.Talanta 83 (2011) 1515-1520).Because the foreign protein coexisted in egg is more, therefrom separate, Purification of Lysozyme is cumbersome.At present, the separation of relevant lysozyme, purifying mainly contains following these methods: film separates (R.Ghosh, S.S.Silva, Z.F.Cui, Lysozyme separation by hollow-fibre ultrafiltration, 19-24 Biochem.Eng.J.6 (2000) 19-24), ion-exchange (E.Lichan, S.Nakai, J.Sim, D.B.Bragg, K.V.Lo, Lysozyme Separation from Egg White by Cation Exchange Column Chromatography, FoodSci.51 (1986) 1032-1036.C.Guerin-Dubiard, M.Pasco, A.Hietanen, A.Q.del Bosque, F.Nau, T.Croguennec, Hen egg white fractionation by ion-exchange chromatography.J.Chromatogr.A 1090 (2005) 58-67), crystallization (Y.C.Cheng, R.F.Lobo, S.I.Sandler, A.M.Lenhoff, Kinetics and equilibria of lysozyme precipitation and crystallization in concentrated ammonium sulfate solutions.Biotechnology and Bioengineering.
volume 94(2006) _ 177-188.C.J.Coen, J.M.Prausnitz, H.W.Blanch, Protein salting-out:Phase equilibria in two-protein systems Biotechnology and Bioengineering
volume 53, Issue6,pages 567-574,20 March 1997.) and affinity precipitation (T.Goto, T.Ohkuri, S.Shioi, Y.Abe, T.Imoto, T.Ueda, Crystal structures of K33 mutant hen lysozymes with enhanced activities.Journal of Biochemistry Volume 144, Issue 5, November 2008, Pages 619-623.).At present, with the method separation of absorption and the research of protein purification, report is arranged more.Mainly by static, ion-exchange, hydrogen bond and affine absorption, come selective absorption or specific binding target protein (T.Kawai, K.Saito, W.Lee, Protein binding to polymer brush, based on ion-exchange, hydrophobic and affinity interactions, J.Chromatogr.B 790 (2003) 131-142.).The research of this respect also has much, such as: A.X.Lu etc. by introducing Fe (III) absorption lysozyme (A.X.Lu et al.Preparation of Fe (III)-immobilized collagen fiber for lysozyme adsorption.Colloids and Surfaces A:Physicochem.Eng.Aspects 301 (2007) 85-93.) on collagenous fibres, the selection dye ligands such as C.Garcia-Diego adsorb lysozyme (C.Garcia-Diego, J.Cuellar.Preparation and characterization of a dye-ligand adsorbent for lysozyme adsorption.Chemical Engineering Journal 143 (2008) 337-348.), Z.Lei etc. utilize carbosphere absorption lysozyme (Z.Lei et al.Adsorption of lysozyme on spherical mesoporous carbons (SMCs) replicated from colloidal silica arrays by chemical vapor deposition.Journal of Colloid and Interface Science 339 (2009) 439-445.), D.Aktas Uygun and G.Zhang etc. adsorb lysozyme etc. (D.Aktas Uygun et al.Magnetic hydrophobic affinity nanobeads for lysozyme separation.Materials Science and Engineering C 29 (2009) 2165-2173.G.Zhang et.al.Tris (hydroxymethyl) aminomethane-modified magnetic microspheres for rapid.affinity purification of lysozyme.Talanta 83,2011.) by magnetic microsphere.Yet the equal more complicated of these methods, cost are higher.So, be necessary to study a kind of simply, separate fast, the method for Purification of Lysozyme.
Summary of the invention
For the deficiencies in the prior art, the object of the present invention is to provide a kind of modified beer waste yeast adsorbent and its preparation method and application.
Modified beer waste yeast adsorbent provided by the present invention is a kind of new adsorbent made with the crosslinked beer waste yeast of citric acid modification (crosslinking agent is glutaraldehyde), this adsorbent has good adsorption capacity to lysozyme, can be applied to extract lysozyme.
In order to realize above-mentioned technical purpose, the present invention has taked following technical measures:
A kind of modified beer waste yeast adsorbent, the step of its preparation method is as follows:
(1) pretreatment of beer waste yeast:
Take beer waste yeast and be placed in conical flask, the water that adds 8-12 times of weight, take out standing 20-40min after 20-30 ℃ of water-bath vibration 0.5-1.5h, abandon the upper strata turbid solution, it is colourless that water continues beer waste yeast is washed till to supernatant, then after using absolute ethanol washing 2-4 time, standing, then suction filtration makes Separation of Solid and Liquid, and it is dry that the gained solid is placed in 60 ℃ of baking ovens, standby;
(2) preparation of crosslinked beer waste yeast:
Take through the pretreated beer waste yeast of step (1) and join in the glutaraldehyde solution of 2wt%, the consumption of glutaraldehyde solution is the every g beer waste yeast of 30-35mL, collect product after 30-40 ℃ of shaking table oscillating reactions 10-12h, product first washes with water three times, use again absolute ethanol washing three times, to remove the unreacted glutaraldehyde in product surface, finally be placed in 60 ℃ of oven dryings, obtain yellow crosslinked beer waste yeast (also claiming the unmodified yeast), standby;
(3), modify the preparation of beer waste yeast:
Take crosslinked beer waste yeast prepared by step (2), amount according to the crosslinked beer waste yeast of the every g of 10-13mL adds N, dinethylformamide (DMF), then add and the citric acid of the quality such as crosslinked beer waste yeast and the sodium dihydrogen phosphate (NaH of 0.5 times of crosslinked beer waste yeast quality
2pO
4as catalyst), be placed in three-neck flask 130-140 ℃ of reaction 1.5-2.5h, and cooling for reflux, utilize the water produced in water knockout drum minute dereaction process; After reaction finishes, suction filtration carries out Separation of Solid and Liquid, and it is colourless washing solid product to filtrate with water, use again absolute ethanol washing 2-4 time, finally be placed in 60 ℃ of oven dryings, must modify beer waste yeast (also claiming to modify yeast), i.e. modified beer waste yeast adsorbent of the present invention.Reaction equation is as follows:
Advantage and the beneficial effect of the inventive method are as follows:
Prepare new adsorbent with the citric acid modification beer waste yeast, not only raw material beer waste yeast wide material sources, cheap and easy to get, can take full advantage of a large amount of waste yeasts that brewing industry produces.And, after it is carried out to simple modification, regeneration, and synthetic method is simple, needed reaction reagent kind, consumption are less, do not need harsh experiment condition, and cost is relatively low.Adsorbent after modification, in the process to the lysozyme test for nominal samples, adsorption conditions is easier to reach, and the adsorbent consumption is few, energy of adsorption comparatively fast reaches balance, and adsorption capacity is higher.Applicable acidity and temperature range are larger, can in wider acidity and temperature range, use.
The elution process of having adsorbed the adsorbent after the lysozyme is simple, and eluant, eluent is exactly that common NaCl solution, elution efficiency is high.The adsorption capacity of the beer waste yeast after citric acid modification is 10.6 times of unmodified, illustrates that modifying beer waste yeast has obviously improved its adsorption capacity to lysozyme.Adsorbent is reused four times, and adsorption rate still can keep 94.6% of first adsorption rate, illustrates that the regenerability of adsorbent of the present invention is good, can reuse.
When adsorbent of the present invention is applied to the actual sample egg, can fast and effeciently from egg, extract lysozyme, adsorption capacity is high.And good vigor recovery rate and very high purification are arranged.It is good that the biologically active of the lysozyme after enrichment keeps.And the results in electrophoresis also confirms, modifying yeast has purification preferably to the lysozyme in egg.
The accompanying drawing explanation
Fig. 1. be the unmodified yeast of embodiment 1 preparation and the infrared spectrogram of modifying yeast;
Fig. 1 shows, the unmodified yeast is at 1726cm
-1there is no obvious peak, show that the unmodified yeast surface does not have carboxyl.Modify yeast at 1726cm
-1and 1167cm
-1for in ester group-stretching vibration peak (Mao of C=O and C-O, J., Won, S.W., Vijayaraghavan, K., Yun, Y.-S., 2009.Surface modification of Corynebacterium glutamicum for enhanced Reactive Red 4 biosorption.Bioresour.Technol.100,1463-1466).3302cm
-1for O-H and N-H stretching vibration peak, the peak of modification bacterium obviously broadens, show the introducing due to citric acid, make the surface of beer waste yeast contain more O-H (Deng, S., Ting, Y.P., 2005.Characterization of PEI-modified biomass and biosorption of Cu (II), Pb (II) and Ni (II) .Water Res.39,2167-2177.).These results show, citric acid has been grafted to the surface of beer waste yeast, on its surface, has introduced a large amount of carboxyls.
Fig. 2. be the unmodified yeast of embodiment 1 preparation and the xps energy spectrum figure of modification yeast;
Fig. 3. be the unmodified yeast of embodiment 1 preparation and the O1s figure of modification yeast;
The result of Fig. 2 and Fig. 3 shows that larger variation has occurred the ratio of modifying rear each element of yeast surface, and especially the ratio of oxygen element improves very large.The ratio that table 1 has been listed the unmodified yeast and modified each element of yeast, the ratio of oxygen element brings up to 33.88% by original 24.36%.Reason is that the hydroxyl of the carboxyl of citric acid and yeast surface esterification has occurred has been connected more citric acid residue on the surface of beer waste yeast, make yeast surface-COOH content improves greatly, the ratio of oxygen element is corresponding increase also.Show that modification that citric acid passes through the esterification success arrived the surface of yeast, this result is consistent with infrared parsing.
Table 1 unmodified yeast and each element ratio of modification yeast are relatively
Fig. 4. be the unmodified yeast of embodiment 1 preparation and the acidity curve map of modification yeast absorption lysozyme;
Fig. 5. be the unmodified yeast of embodiment 1 preparation and the time plot of modification yeast absorption lysozyme;
Fig. 6. be the unmodified yeast of embodiment 1 preparation and the temperature profile of modification yeast absorption lysozyme;
Fig. 7. be the unmodified yeast of embodiment 1 preparation and the adsorption isotherm of modification yeast absorption lysozyme;
Can know the unmodified yeast and modify yeast experiment adsorption capacity (maximal absorptive capacity) from figure and be respectively 89.9mgg
-1, 954.7mgg
-1, the modification yeast is 10.6 times of unmodified yeast.
Fig. 8. with modifying yeast absorption lysozyme, with langmuir adsorption isotherm simulation curve, (the langmuir isotherm equations is the unmodified yeast prepared for embodiment 1: C
e/ q
e=1/bq
m+ C
e/ q
m, q
eadsorbance during for molecular balance, q
mfor maximal absorptive capacity, C
ethe concentration of Lysozyme in Solution during for adsorption equilibrium, b is constant);
Fig. 9. with modifying yeast absorption lysozyme, with Freundlich adsorption isotherm simulation curve, (the Freundlich isotherm equations is the unmodified yeast prepared for embodiment 1: lnq
e=lnK+lnC
e/ n, q
eadsorbance during for molecular balance, q
mfor maximal absorptive capacity, C
ethe concentration of Lysozyme in Solution during for adsorption equilibrium, K and n are empiricals);
The Langmuir that table 2 is Fig. 8 and Fig. 9 and Freundlich adsorption isotherm analog parameter
From table, the gained linearly dependent coefficient can be found out, the process of unmodified yeast and modification yeast absorption lysozyme more meets Langmuir adsorption isotherm pattern, the absorption that shows lysozyme is to take the monolayer chemisorbed as main, and unmodified yeast and modification yeast are respectively 82.17mgg to the theoretical maximum adsorption capacity of lysozyme
-1and 909.09mgg
-1, more identical with the experiment value in Fig. 7.
Electrophoretogram (a. standard protein (Marker) of modification yeast Purification of Lysozyme from egg that Figure 10 is embodiment 1 preparation; B. pure lysozyme standard specimen; C. original egg white; D. the solution after adsorbing; E. the lysozyme eluted).As can be seen from the figure, the supernatant after the absorption lysozyme is the 4th swimming lane d, almost there is no lysozyme, illustrates that the beer waste yeast of modifying is more complete to the absorption of lysozyme.The 5th swimming lane e is the lysozyme eluted after adsorbent absorption, and purity is higher, and the content of foreign protein is less.Illustrate and modify yeast separation, enrichment lysozyme from egg quickly and efficiently.
The specific embodiment
The beer waste yeast the present invention relates to is the discarded object that general Brewage industry produces, and in the present invention, the actual beer waste yeast used is taken from Hubei brewery.
Embodiment 1:
A kind of modified beer waste yeast adsorbent, the step of its preparation method is as follows:
1. the pretreatment of beer waste yeast: take the 15.0g beer waste yeast in the 250mL conical flask, add 150mL distilled water, take out standing 30min after 25 ℃ of water-bath vibration 1h, abandon the upper strata turbid solution, it is colourless that water continues beer waste yeast is washed till to supernatant, then after using absolute ethanol washing 3 times, standing, then suction filtration makes Separation of Solid and Liquid, and it is dry that the gained solid is placed in 60 ℃ of baking ovens, standby;
2. the preparation of crosslinked beer waste yeast: by 1.5g, the beer waste yeast pretreated through step 1 joins in the glutaraldehyde solution of 50mL 2%wt, collect product after 35 ℃ of shaking table oscillating reactions 12h, product first washes with water three times, use again absolute ethanol washing three times, to remove the unreacted glutaraldehyde in product surface, finally be placed in 60 ℃ of oven dryings, obtain yellow crosslinked beer waste yeast (also claiming the unmodified yeast), standby;
3. modify the preparation of beer waste yeast: the crosslinked beer waste yeast that takes the preparation of 2g step 2 joins in 25mL DMF (DMF), then adds 2g citric acid and 1g sodium dihydrogen phosphate (NaH
2pO
4), be placed in 140 ℃ of three-neck flasks reaction 2h, and cooling for reflux, utilize the water produced in water knockout drum minute dereaction process; After reaction finishes, suction filtration carries out Separation of Solid and Liquid, and it is colourless washing solid product to filtrate with water, use again absolute ethanol washing 3 times, finally be placed in 60 ℃ of oven dryings, must modify beer waste yeast (also claiming to modify yeast), i.e. modified beer waste yeast adsorbent of the present invention.
It is prepared that the adsorbent of using in following examples is embodiment 1.
Embodiment 2: adsorbent prepared by the inventive method is applied to adsorb lysozyme
Determining of adsorption conditions:
1. determining of absorption acidity: take respectively the crosslinked beer waste yeast of 0.0200g and modified beer waste yeast adsorbent (all parallel take many parts), be placed in respectively the 50mL conical flask, add 10.00mL 0.2mgmL
-1the lysozyme mark liquid of different acidity (pH=3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.4,8.0), take out supernatant, mensuration after shaking table absorption 12h under 25 ℃ under 120rpm, calculate the adsorption rate of adsorbent to lysozyme, its adsorber acid line of writing music, be shown in Fig. 4;
For modified beer waste yeast, along with the increase of pH, the adsorption rate of lysozyme increases gradually, and when pH4.0, adsorption rate reaches maximum, then increases pH, and adsorption rate is almost constant; In order to make lysozyme keep higher activity, select the experiment pH of pH7.4 as following examples.For crosslinked beer waste yeast, pH<4 o'clock, lysozyme does not adsorb, and along with the increase of pH, adsorption rate increases.Under same pH, with modified beer waste yeast, to compare, the adsorption rate of crosslinked beer waste yeast is all the time lower than modified beer waste yeast.
2. determining of adsorption time: take respectively the crosslinked beer waste yeast of 0.0200g and modified beer waste yeast adsorbent (all parallel take many parts), be placed in respectively the 50mL conical flask, all add the 0.2mgmL of 10.00mL with buffer solution (the 0.05M Tris-hydrochloric acid system) configuration of pH7.4
-1lysozyme mark liquid, shaking table absorption under 120rpm under 25 ℃, take out supernatant and measure after different time, calculate the adsorption rate of adsorbent to lysozyme, draws its adsorption time curve, sees Fig. 5;
Reach balance after modified beer waste yeast absorption 40min, absorption fully.Crosslinked beer waste yeast is along with the increase of adsorption time, and adsorption rate has slowly to be increased, but changes little.The adsorption process that this modified beer waste yeast is described can reach balance fast.The adsorption time of following examples is decided to be 40min.
3. determining of adsorption temp: take respectively the crosslinked beer waste yeast of 0.0200g and modified beer waste yeast adsorbent (all parallel take many parts), be placed in respectively the 50mL conical flask, all add the 0.2mgmL of 10.00mL with buffer solution (the 0.05M Tris-hydrochloric acid system) configuration of pH7.4
-1lysozyme mark liquid, put into the shaking bath of different temperatures, takes out supernatant after absorption 40min and measure under 120rpm, calculates the adsorption rate of adsorbent to lysozyme, draws its temperature curve, sees Fig. 6;
In the time of 20～45 ℃, temperature is little on the impact of adsorption rate, and adsorption rate, all more than 80%, has a maximum at 25 ℃, therefore selects 25 ℃ as the optimal adsorption temperature.
4. the impact of lysozyme initial concentration on adsorbance: take respectively the crosslinked beer waste yeast of 0.0200g and modified beer waste yeast adsorbent (all parallel take many parts), be placed in respectively the 50mL conical flask, add respectively 0.0 of the buffer solution (0.05M Tris-hydrochloric acid system) of pH7.4 configuration for 10.00mL, 0.2,0.4,0.5,0.7,1.0,2.0,3.0mgmL
-1lysozyme mark liquid takes out supernatant after 120rpm absorption 40min under 25 ℃ and measures in shaking bath, calculates the adsorbance of adsorbent to lysozyme, draws its adsorption isotherm, sees Fig. 7;
Embodiment 3: the regenerability test of modified beer waste yeast adsorbent
1. get 0.0200g and modify yeast in the 50.0mL conical flask, add the 0.2mgmL of 10.00mL with buffer solution (the 0.05M Tris-hydrochloric acid system) configuration of pH7.4
-1lysozyme mark liquid, put into 25 ℃ of shaking baths, shaking table absorption 40min under 120rpm, standing after, take out supernatant, measure the adsorption rate (be now the adsorption rate of use) for the first time of lysozyme.After remaining adsorbent natural air drying, add 10.00mL 1.0MNaCl solution, put into 25 ℃ of shaking baths, shaking table wash-out 40min under 120rpm, standing after, take out supernatant, repeat above-mentioned absorption-wash-out after remaining adsorbent natural air drying, adsorbent is used altogether four times, and the adsorption capacity while using for the 4th time still keeps 94.6% (in Table 4) of using first, the regenerability that this adsorbent is described is good, can reuse.
The result of table 4 adsorbent reactivation performance test
Embodiment 4:
Adsorbent of the present invention is processed for actual sample, and step is as follows:
1. (0.05M) PBS that is 6.50 by pH value dilution egg white to 5 times volume, fully the egg white solution after agitation and dilution, fully mix egg white and buffer solution.Then adjust the pH value to 4.60 of dilution with 0.05M phosphoric acid, heating water bath to 100 ℃, and insulation 20min allows ovalbumin fully precipitate.The centrifugal 10min of cooling rear 10000r/min, get supernatant stand-by;
2. take 0.0200g modified beer waste yeast adsorbent in the 50mL conical flask, the supernatant that adds 10.00mL step 1 to obtain, put into 25 ℃ of shaking baths, under 120rpm, reacts 40min.Then take out, standing, get supernatant.Survey the vigor of lysozyme.
3. in above-mentioned steps 1, take in the modified beer waste yeast adsorbent of supernatant, add 10.00mL1.0M NaCl solution, put into 25 ℃ of shaking baths, shaking table wash-out 40min under 120rpm, measure the enzyme activity of eluent, the separation and purification result that table 3 is lysozyme in egg.
Table 3
Extract and separate lysozyme (parallel three times) from egg, the results are shown in Table 3.Before purifying, the ratio vigor of egg is 168Umg
-1, after extraction, in the adsorbent eluent, the ratio vigor of lysozyme is 4605Umg
-1, the single step purification multiple is 27.5, the recovery rate of lysozyme total activity is 71.3%.
Ratio vigor before ratio vigor/purifying after purification=purifying
Total activity before total activity/purifying after enzyme activity recovery rate=purifying.
Claims (1)
1. the application of modified beer waste yeast adsorbent in the absorption lysozyme, preparation method's step of described modified beer waste yeast adsorbent is as follows:
(1) pretreatment of beer waste yeast:
Take beer waste yeast and be placed in conical flask, the water that adds 8-12 times of weight, take out standing 20-40min after 20-30 ℃ of water-bath vibration 0.5-1.5h, abandon the upper strata turbid solution, it is colourless that water continues beer waste yeast is washed till to supernatant, then after using absolute ethanol washing 2-4 time, standing, then suction filtration makes Separation of Solid and Liquid, and it is dry that the gained solid is placed in 60 ℃ of baking ovens, standby;
(2) preparation of crosslinked beer waste yeast:
Take through the pretreated beer waste yeast of step (1) and join in the glutaraldehyde solution of 2wt%, the consumption of glutaraldehyde solution is the every g beer waste yeast of 30-35mL, collect product after 30-40 ℃ of shaking table oscillating reactions 10-12h, product first washes with water three times, use again absolute ethanol washing three times, to remove the unreacted glutaraldehyde in product surface, finally be placed in 60 ℃ of oven dryings, obtain yellow crosslinked beer waste yeast, standby;
(3), modify the preparation of beer waste yeast:
Take crosslinked beer waste yeast prepared by step (2), amount according to the crosslinked beer waste yeast of the every g of 10-13mL adds N, dinethylformamide, add again and the citric acid of the quality such as crosslinked beer waste yeast and the sodium dihydrogen phosphate of 0.5 times of crosslinked beer waste yeast quality, be placed in three-neck flask 130-140 ℃ of reaction 1.5-2.5h, and cooling for reflux, utilize the water produced in water knockout drum minute dereaction process; After reaction finishes, suction filtration carries out Separation of Solid and Liquid, and it is colourless washing solid product to filtrate with water, then uses absolute ethanol washing 2-4 time, finally is placed in 60 ℃ of oven dryings, obtains.
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| 陈聪等.辛巴蓝F-3GA修饰的啤酒废酵母菌亲和吸附溶菌酶.《分析化学(FENXI HUAXUE)研究简报》.2011, |
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