CN109852591B - Method for extracting lipoxygenase from soybean whey wastewater by reverse pH gradient - Google Patents
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Abstract
The invention discloses a method for extracting lipoxygenase from soybean whey wastewater by reverse pH gradient, belonging to the technical field of food processing wastewater utilization. The method comprises the steps of pretreating soybean whey wastewater, extracting lipoxygenase by a reverse pH gradient method, removing impure protein at a pH value far away from the isoelectric point of the lipoxygenase, separating out the lipoxygenase at the isoelectric point of the lipoxygenase, and performing ultrafiltration and gel chromatography to obtain the refined lipoxygenase. The invention utilizes the soybean whey wastewater to obtain lipoxygenase with high industrial value, and the lipoxygenase has high extraction rate, high purity and good enzyme activity. The method is convenient to operate, safe, efficient and low in cost, can be used for developing and utilizing lipoxygenase in industrial soybean whey wastewater to realize resource recycling, and has remarkable economic and social benefits.
Description
Technical Field
The invention relates to the technical field of food processing wastewater utilization, in particular to a method for extracting lipoxygenase from soybean whey wastewater by reversing pH gradient.
Background
The soybean is an important crop with rich protein content, and the soybean protein isolate processed by taking the soybean protein isolate as a raw material is a refined soybean protein product with high protein purity and deep processing performance, can be used as an intermediate raw material to be added into food production, and has wide application.
The soybean protein isolate can be processed by methods such as an alkali-soluble acid precipitation method, an ultrafiltration concentration method, a reverse micelle extraction method, a reverse phase high performance liquid chromatography method and the like, wherein the alkali-soluble acid precipitation method is a mature and widely applied process method for producing the soybean protein isolate at present. However, when the soy protein isolate is produced by using the alkali-dissolving and acid-precipitating process, a large amount of soy whey wastewater is produced, and the wastewater contains abundant substances such as protein, saccharides and the like, so that resource waste and environmental pollution are easily caused.
The soybean protein isolate is widely applied to the production of the food industry, the annual demand is about 60 ten thousand tons, but about 40 tons of wastewater is generated for producing 1 ton of SPI, and the generation amount of the wastewater is large. At present, the main treatment method for the soybean protein isolate processing wastewater is to directly carry out multi-stage biological treatment on the soybean protein isolate processing wastewater, and the COD and the BOD in the wastewater are reduced by using an anaerobic method and an aerobic method so as to reach the national wastewater discharge standard, the treatment cost is very high, and a large amount of active substances contained in the wastewater are not reasonably utilized. Therefore, the high-added-value products in the soybean whey wastewater are recycled by adopting the modern separation technology, so that the effective resources can be recycled, the sewage is purified, and better economic benefits and social benefits are generated.
In recent years, in order to reduce wastewater discharge and environmental pollution, many researchers have been dedicated to the separation and extraction of functional substances from soybean whey wastewater, for example, jiang et al have utilized two-stage foam separation technology to recover soybean whey protein from soybean whey wastewater; the Chinese medicine Lingxu chrysanthemum and the like extract oligosaccharide in the soybean whey wastewater by using a membrane technology and form a soybean oligosaccharide product. However, no research report is found on the separation and extraction of lipoxygenase from soybean whey wastewater.
Lipoxygenase (LOX), also known as lipoxygenase, belongs to oxidoreductase, is a non-heme iron-containing protein, can specifically catalyze polyunsaturated fatty acids with cis, cis-4-ene structures, and forms a hydro-oxidation derivative with conjugated double bonds through intramolecular oxygenation. Lipoxygenase (LOX) can cause the processed products of fruits and vegetables to generate poor flavor, and the color, the fragrance and the taste of oil and fat and oil-containing food are deteriorated in the storage and processing processes. However, lipoxygenase (LOX) is used as a green food additive in wheat flour, and can oxidize the pigment in flour, fade it and whiten the flour product. Lipoxygenase (LOX) can be applied to disease resistance, insect resistance and injury reaction of plants, and the relation between the Lipoxygenase (LOX) and the disease resistance of the plants is reported on crops such as wheat, tobacco, rice, cotton and the like. Lipoxygenase (LOX) is industrially useful for the industrial production of dyes, paints, detergents, etc., and also as an intermediate for the synthesis of pharmaceuticals.
Although LOX has wide application in the fields of food, chemical industry and the like, LOX is directly extracted from raw materials such as soybeans and the like, the cost is too high, and no enzyme preparation which is industrially produced exists; the recombinant LOX is efficiently expressed by the genetic engineering bacteria, and the safety and other problems exist, so that the large-scale production and application of the recombinant LOX are difficult to break through. It can be seen that finding a method for obtaining lipoxygenase suitable for industrial applications is a problem that needs to be solved urgently at present.
In earlier research, researchers find that a large amount of lipoxygenase is contained in the SPI processing wastewater, china is the biggest SPI producing country in the world and can generate a large amount of SPI processing wastewater every year, and therefore, the separation and extraction of the lipoxygenase in the soybean protein isolate processing wastewater and the resource utilization of the SPI processing wastewater have great economic and social values and very important practical significance.
Disclosure of Invention
In order to make up the defects of the prior art, the invention provides a method for extracting lipoxygenase from soybean whey wastewater by reversing pH gradient.
The technical scheme of the invention is as follows:
a method for extracting lipoxygenase from soybean whey wastewater by reverse pH gradient comprises the following steps:
1) Pretreatment of soybean whey wastewater
Centrifuging the soybean whey wastewater to remove large-particle impurities, and keeping a supernatant as a pretreatment solution of the soybean whey wastewater;
2) Removing foreign protein in pretreatment liquid of soybean whey wastewater
Adjusting the pH value of the soybean whey wastewater pretreatment liquid to 7.8 to 8.2, and centrifuging to remove precipitates;
3) Extraction of lipoxygenase
Adjusting the pH value of the supernatant obtained by centrifuging in the step 2) to 5.7-6.0, centrifuging to remove the supernatant, and dissolving the obtained precipitate with distilled water to obtain the lipoxygenase crude enzyme solution.
The method comprises the steps of pretreating soybean whey wastewater, extracting lipoxygenase by a reverse pH gradient method, removing impure protein at a pH value far away from the isoelectric point of the lipoxygenase, and then separating out the lipoxygenase at the isoelectric point of the lipoxygenase.
Preferably, the method for extracting lipoxygenase from soybean whey wastewater by reverse pH gradient further comprises the step 4): the lipoxygenase crude enzyme liquid is primarily purified by using an ultrafiltration concentration method.
Preferably, the method for extracting lipoxygenase from soybean whey wastewater by reverse pH gradient further comprises the step 5): and (4) further purifying the enzyme solution obtained in the step 4) by using gel filtration chromatography to obtain pure lipoxygenase.
Preferably, in the step 4), the lipoxygenase crude enzyme solution is subjected to ultrafiltration concentration by 8 to 10 times under the pressure of 200 to 300KPa by using a modified polyethersulfone ultrafiltration membrane with the size of 30 KDa.
Preferably, in the step 4), ultrafiltration concentration is carried out by using an ultrafiltration system in a two-stage filtration mode
Preferably, in the step 5), gel filtration chromatography is carried out by using a Sephadex G-75 gel column of 2-70KDa, balancing 2-3 column volumes by using 0.02mol/L phosphate buffer solution with pH7.8, loading after balancing, and eluting by using 0.02mol/L phosphate buffer solution with pH7.8 as eluent; the flow rate of the eluate was 0.5mL/min.
Preferably, in the step 2), the pH of the pretreatment solution of the soybean whey wastewater is adjusted to 8.0.
Preferably, in step 3), the pH of the supernatant is adjusted to 6.0.
Preferably, in step 2) and step 3), the pH of the system is adjusted by using a sodium hydroxide solution.
Preferably, in the step 1), the centrifugal separation is carried out at the rotating speed of 3000-15000r/min for 8-20min.
The invention has the beneficial effects that:
1. the invention provides a method for extracting lipoxygenase from soybean whey wastewater by reversing pH gradient, which comprises the steps of pretreating the soybean whey wastewater, adjusting the pH to be far away from the isoelectric point of the lipoxygenase, removing impurities, adjusting the pH to the isoelectric point of the lipoxygenase, obtaining the lipoxygenase with high enzyme activity and high enzyme activity recovery rate, wherein the enzyme activity recovery rate is 73.54%, and the specific enzyme activity is 10661.32U/mg.
2. The lipoxygenase obtained by isoelectric precipitation is further subjected to ultrafiltration concentration and gel filtration chromatography purification to obtain the lipoxygenase with very high purity, the final LOX purity is 90.43%, the specific enzyme activity reaches 25546.08U/mg, the enzyme activity recovery rate is 40.29%, and the purification multiple is increased by 25.44 times.
3. The invention utilizes the soybean whey wastewater to obtain lipoxygenase with high industrial value, and the lipoxygenase has high extraction rate, high purity and good enzyme activity.
4. The method is convenient to operate, safe, efficient and low in cost, can be used for developing and utilizing lipoxygenase in industrial soybean whey wastewater to realize resource recycling, and has remarkable economic and social benefits.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a graph of a protein content determination standard;
FIG. 2 is a graph of the precipitation rate and specific enzyme activity of Lipoxygenase (LOX) at various pH values;
FIG. 3 is a pH gradient precipitated protein electrophoretogram;
FIG. 4 is a LOX specific activity plot in the pellet and supernatant at various PEG6000 concentrations;
FIG. 5 is a graph showing the effect of sodium polyacrylate addition on lipoxygenase specific enzyme activity;
FIG. 6 is a graph showing the influence of the cut-off molecular weight of the ultrafiltration membrane on the LOX enzyme activity recovery rate and the ultrafiltration time;
FIG. 7 is a graph showing the effect of ultrafiltration operating pressure on LOX enzyme activity recovery;
FIG. 8 is a graph showing the effect of ultrafiltration concentration factor on LOX unit enzyme activity and recovery rate;
FIG. 9 is a graph showing the effect of the ultrafiltration on the LOX unit enzyme activity and recovery rate;
FIG. 10 is a Sephadex G-75 chromatogram;
FIG. 11 shows the result of SDS-PAGE analysis of the LOX purification process.
Detailed Description
1. The method for measuring the enzyme activity of the lipoxygenase in the embodiment comprises the following steps:
definition of enzyme activity: at 25 ℃ and pH9.0, the enzyme adding amount of 1umol product catalyzed and generated by a reaction system for 1min at 234nm by using linoleic acid as a substrate is defined as one enzyme activity unit.
Preparation of a substrate: dispersing 0.25mL of Tween 20 in 10mL of 0.2mol/L borate buffer solution with the pH value of 9.0, continuously shaking and dropwise adding 0.27mL of linoleic acid to uniformly mix, then dropwise adding a NaOH solution with a certain concentration into the system to clarify the system, adjusting the pH value of the system to 9.0, finally diluting to 500mL with 0.2mol/L borate buffer solution with the pH value of 9.0 to be used as a substrate solution, and storing at 4 ℃ for later use.
And (3) activity determination: adding 0.2mL of sample solution into 1.5mL of substrate solution, uniformly mixing, reacting at the constant temperature of 25 ℃ for 3min, adding 5mL of absolute ethyl alcohol to stop the reaction, adding 5mL of distilled water to clarify the reaction system, and measuring the absorbance at 234 nm. The blank sample is 1.5mL of substrate solution, 5mL of absolute ethanol is added, 0.2mL of sample solution is added, and after the temperature is kept at 25 ℃ for 3min, 5mL of distilled water is added.
The above enzyme activity measurement methods are referred to the following two documents:
1. the influence of Wangbuyi.ultra high pressure on the properties of soybean lipoxygenase, trophic suppressive factors and proteins [ D ]. Jiangnan university, 2008.
2、 Catod L, Halmos A L, Small D M. Measurement of lipoxygenase in Australian white wheat flour: the effect of lipoxygenase on the quality properties of white salted noodles[J]. Journal of the Science of Food and Agriculture, 2006, 86(11): 1670-1678. DOI: 10.1002/jsfa.2539.
2. The protein content measurement method in the present embodiment:
measuring the protein content in the sample by using a Bradford method, taking 6 clean test tubes and marking the tube number, wherein 1.0ml of distilled water is added into one test tube to be blank, 5 test tubes are respectively added with bovine serum albumin standard solution with different volume concentrations of 0.1mg/ml, and water is supplemented to 1.0ml. Then 5.0ml Coomassie brilliant blue G-250 reagent is added into each test tube, shaken evenly and placed for 5min, and the light absorption value is measured at 595nm of an ultraviolet-visible spectrophotometer. A standard curve was plotted with the A595 absorbance as the ordinate and the concentration of bovine serum albumin (mg/mL) as the abscissa, as shown in FIG. 1.
3. SDS-PAGE analysis experiment in this embodiment
SDS-PAGE analysis of LOX sample solutions by reference to the Laemmli method, sample 2
The loading buffer was dissolved and heated in a boiling water bath for 3min and centrifuged at 8000r/min for 3min. The concentration of the separation gel is 15 percent, the concentration of the concentrated gel is 5 percent, the electrode buffer solution is a Tris-Gly buffer solution with the pH value of 8.3, the separation gel buffer solution is a Tris-HCl buffer solution with the pH value of 8.8 of 1.5mol/L, the concentrated gel buffer solution is a Tris-HCl buffer solution with the pH value of 6.8 of 1.0mol/L, and the electrophoresis is carried out for 120min at room temperature. After electrophoresis, the gel is dyed for 8 hours by Coomassie brilliant blue R-250 stain, decolored for 24 hours and then photographed for analysis. And analyzing the protein precipitation rate and purity according to the principle that the protein content in the sample is linearly related to the grayscale value of the SDS-PAGE gel electrophoresis band, collecting an electrophoresis Image by using a gel imaging system, analyzing the grayscale value of each electrophoresis band by using Image J software, and calculating the protein Precipitation Rate (PR) and the Purity (PU) in each sample according to the formulas (1) and (2).
Wherein PG pro Representing the gray value of the protein to be detected in the sediment; EG pro Representing the gray value of the protein to be detected in the soybean whey wastewater; np and Ne represent precipitation and dilution times of soybean whey wastewater, respectively; SG (steam generator) pro Representing the gray value of the protein to be detected in the sample; SG all Representing the total grey value of all proteins in the pellet.
4. Lipoxygenase study by precipitation along pH gradient
The isoelectric point of LOX is between pH5.7-6.0, so 6 parts of each wastewater treatment solution with 1 part as a control group and the other 5 parts as an experimental group are respectively adjusted to pH5.0, pH6.0, pH7.0, pH8.0 and pH9.0 by 1mol/L NaOH, kept stand at 4 ℃ for 5h and then centrifuged in a centrifuge for 10min, supernatant and precipitate are collected, wherein the precipitate is dissolved by a certain amount of distilled water and adjusted to be neutral, centrifuged for 10min, supernatant is taken and the enzyme activity and protein content of LOX are measured, an analysis experiment is carried out by SDS-PAGE, and the precipitation rate of LOX in the precipitate is measured, and the result is shown in figure 2.
As can be seen from fig. 2, when the pH of the soybean whey wastewater is adjusted to 6.0, the specific enzyme activity and the precipitation rate of LOX in the precipitate are both higher than those in the precipitates under other pH conditions, and the specific enzyme activity in the supernatant is lower than those in the supernatants under other pH conditions, because the isoelectric point of LOX is located near pH6.0, when the pH of the solution is 6.0, the specific enzyme activity of LOX in the precipitate is 2816.56U/mg, the purification fold is increased by 2.80 times, and the LOX precipitation rate reaches 76.82%. And when the pH of the wastewater treatment liquid is adjusted to 8.0, the specific enzyme activity and the precipitation rate of LOX in the precipitate are lower than those of LOX under other pH conditions, and the specific enzyme activity of LOX in the supernatant is higher than that of the supernatant under other pH conditions, because the wastewater treatment liquid can precipitate more alkaline protein under the condition of pH8.0, and LOX basically remains in the supernatant, when the pH of the solution is 8.0, the specific enzyme activity of LOX in the supernatant is higher, and when the pH of the solution is 6.0, the specific enzyme activity and the precipitation rate of LOX in the precipitate are higher.
According to the experimental result, the pH value of the soybean whey wastewater is adjusted to be far away from the isoelectric point of LOX to remove the impure protein with the isoelectric point at the pH value, and then the supernatant obtained by centrifugation is adjusted to the pH value of the isoelectric point of LOX.
Example 1
A method for extracting lipoxygenase from soybean whey wastewater by reverse pH gradient comprises the following steps:
1) Pretreatment of soybean whey wastewater
Taking 1000mL of soybean whey wastewater, centrifuging at 12000r/min for 10min, and reserving supernatant as a pretreatment solution of the soybean whey wastewater for experiments;
2) Removing foreign protein in pretreatment liquid of soybean whey wastewater
Adjusting the pH value of the soybean whey wastewater pretreatment liquid to 8.0 by using a sodium hydroxide solution, and centrifuging to remove precipitates;
3) Extraction of lipoxygenase
Adjusting the pH value of the supernatant obtained by centrifuging in the step 2) to 6.0 by using a sodium hydroxide solution, centrifuging to remove the supernatant, dissolving the obtained precipitate with distilled water, adjusting the pH to be neutral to obtain a lipoxygenase crude enzyme solution, measuring the enzyme activity and the protein content in the crude enzyme solution, and analyzing and measuring the lipoxygenase precipitation rate and purity by combining SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), wherein the results are shown in table 1 and figure 3.
TABLE 1 LOX parameter table for reversed pH gradient precipitation
According to the results of the studies on the LOX precipitation of the soybean whey wastewater at each pH, it was found that the LOX precipitation effect of the soybean whey wastewater was excellent at pH6.0, and the foreign proteins in the soybean whey wastewater were removed well at pH 8.0. Therefore, in this experiment, the pH of the soybean whey wastewater was adjusted to 8.0 to remove a part of the foreign proteins, and then the supernatant was adjusted to pH6.0 to precipitate the objective protein, and the results are shown in FIG. 3 and Table 2. As can be seen from the figure, the band 6 in FIG. 3 contains significantly less hetero-proteins, and the molecular weights of the proteins in the band are significantly different, which is beneficial to further separation and purification. As can be seen from Table 1, the purification multiple of LOX in the supernatant with the pH value of 8.0 is 3.10, and the enzyme activity recovery rate reaches 97.32%, which indicates that the process has a certain purification effect on LOX and can better maintain the activity of LOX enzyme. When the pH value is adjusted to 6.0, the purification multiple of LOX in the precipitate is improved by 10.62 times, the specific enzyme activity is up to 10661.32U/mg, the enzyme activity recovery rate and purification rate of LOX are 73.54 percent and 39.55 percent respectively, and the reverse pH gradient precipitation process has a good precipitation effect on LOX.
Comparative example 1
Method for precipitating lipoxygenase by PEG6000
Reference: yuwuying, zhao Jinmei, xiao Qing Hu, et al research on precipitation of porcine trypsin by polyethylene glycol [ J ]. Proceedings of Siwa university (Nature science edition), 2011, 30 (5): 96-99.
Respectively adding 50 percent PEG6000 solutions with different masses into a certain amount of soybean whey wastewater pretreatment solution at 4 ℃ to ensure that the mass fractions of PEG6000 in the solution are respectively 5 percent, 10 percent, 15 percent, 20 percent, 25 percent and 30 percent, uniformly stirring, standing for 6 hours at 4 ℃, centrifuging for 10 minutes at 12000r/min after the PEG6000 is fully combined with protein, dissolving the obtained precipitate by using 0.2mol/L of borate buffer solution with the pH value of 8.0, determining the LOX enzyme activity and the protein content in the solution, carrying out experimental analysis by using SDS-PAGE, and determining the precipitation rate of LOX in the precipitate.
The results of LOX study in the wastewater from the separation of soybean whey by PEG6000 precipitation are shown in FIG. 4. As can be seen from fig. 4, the different concentrations of PEG6000 have different effects on LOX precipitation, when the concentration of PEG6000 is below 10%, the specific enzyme activity of LOX in the precipitate is higher than the specific enzyme activity of supernatant and soybean whey wastewater, and when the concentration of PEG6000 is 10%, the LOX purification times and precipitation rate are the highest, respectively 1.70 and 32.83%, which indicates that the PEG6000 separation and precipitation technology has a certain precipitation effect on LOX in soybean whey wastewater. When the concentration of PEG6000 is higher than 10%, the specific enzyme activity of LOX in the precipitate is lower than that of LOX in the supernatant, the LOX precipitation rate is in a descending trend, and the specific enzyme activity of LOX in the supernatant is not obviously improved, so that when the concentration of PEG6000 is higher than 10%, the effect of the PEG6000 on the LOX precipitation in the soybean whey wastewater is poor. Moreover, studies have shown that: the PEG molecule has extensibility in aqueous solution, and the PEG in protein solution is difficult to remove, so that the PEG-protein conjugate is difficult to purify, and the yield of the target product is low.
Therefore, the separation effect of the isoelectric point precipitation technology on the LOX in the soybean whey wastewater can be obtained by integrating the LOX precipitation rate and the purification multiple.
Comparative example 2
Reference: technology research on extracting lysozyme from egg shells [ J ] food and fermentation technology, 2005, 41 (2): 22-25.
Respectively taking 6 parts of 20mL of soybean whey wastewater, sequentially adding 2mL,3mL,4mL,5mL and 6mL of 0.5% sodium polyacrylate, uniformly mixing, standingAnd centrifuging and separating precipitates and supernatant after flocculating and settling for 24 hours. Adding a small amount of distilled water to the LOX-sodium polyacrylate precipitate, and adding 0.5mol/L Na dropwise 2 CO 3 Dissolving the precipitate, adding 5% CaCl 2 And (3) dissociating the LOX and the sodium polyacrylate until no precipitate is separated out, finally, centrifuging and separating in a centrifugal machine, and analyzing the LOX precipitation rate by combining SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) after the obtained supernatant is used for enzyme activity determination and protein content determination.
The sodium polyacrylate contains a large amount of carboxyl, can be combined with protein with opposite ions through electrostatic action to form a polymer polyelectrolyte-protein composite precipitate, and has low consumption, and can be dissolved by water again, so that the protein activity is well maintained. Fig. 5 is a result of studying LOX precipitation in soybean whey wastewater using 0.5% sodium polyacrylate solution, and it can be seen from fig. 5 that the LOX specific activities obtained in the sodium polyacrylate-LOX complex precipitation are both higher than the LOX specific activities in the supernatant, wherein the LOX purification times and the precipitation rates are as high as 1.89 and 40.82%, respectively, and thus it can be seen that sodium polyacrylate has a certain precipitation effect on LOX in soybean whey wastewater, but the extraction effect of the precipitation method is not ideal.
Example 2
A method for extracting lipoxygenase from soybean whey wastewater by reverse pH gradient comprises the following steps:
1) Pretreatment of soybean whey wastewater
Taking 1000mL of soybean whey wastewater, centrifuging at 12000r/min for 10min, and reserving supernatant as a pretreatment solution of the soybean whey wastewater for experiments;
2) Removing foreign protein in pretreatment liquid of soybean whey wastewater
Adjusting the pH value of the soybean whey wastewater pretreatment liquid to 8.0 by using a sodium hydroxide solution, and centrifuging to remove precipitates;
3) Extraction of lipoxygenase
Adjusting the pH value of the supernatant obtained by centrifuging in the step 2) to 6.0 by using a sodium hydroxide solution, centrifuging to remove the supernatant, dissolving the obtained precipitate by using distilled water, and adjusting the pH value to be neutral to obtain a lipoxygenase crude enzyme solution;
4) Purifying the lipoxygenase crude enzyme liquid by using an ultrafiltration concentration method
And (3) carrying out ultrafiltration concentration on the lipoxygenase crude enzyme liquid by 10 times under the pressure of 200KPa by using an ultrafiltration system and a modified polyether sulfone ultrafiltration membrane with the size of 30KDa and selecting a two-stage ultrafiltration mode.
In order to further improve the purity of the LOX extracting solution, an ultrafiltration system and a modified polyether sulfone ultrafiltration membrane are used for carrying out ultrafiltration concentration optimization experiments on the LOX extracting solution, so that the optimal ultrafiltration condition is explored for industrial production of LOX.
The conditions are obtained by using an ultrafiltration system and a modified polyethersulfone ultrafiltration membrane to carry out an ultrafiltration concentration optimization experiment. In order to ensure that LOX is completely intercepted and obtain higher enzyme activity recovery rate, an ultrafiltration membrane package with the molecular weight cutoff of LOX being one third to one sixth is selected according to the molecular weight of LOX and the selection principle of the ultrafiltration membrane package, so that the influence of factors such as the molecular weight cutoff of the ultrafiltration membrane, operation pressure, an ultrafiltration mode, concentration multiple and the like on the ultrafiltration process is researched.
2.1 Optimization of ultrafiltration membrane cut-off molecular weight
The influence of ultrafiltration membranes with different molecular weight cut-off on the recovery rate of enzyme activity of LOX is different, and ultrafiltration concentration treatment is carried out on extract obtained by isoelectric point fractional precipitation by respectively selecting ultrafiltration membranes with molecular weight cut-off of 10KDa, 30KDa and 50KDa according to the molecular weight of LOX and the selection principle of an ultrafiltration membrane package and integrating standby kits of a tangential flow membrane package. In the ultrafiltration process, the operation pressure is 200KPa, the precipitation extract is concentrated 6 times by adopting a primary ultrafiltration mode, so as to explore the influence of different cut-off molecular weights of the ultrafiltration membrane on the LOX enzyme activity recovery rate and the ultrafiltration time, and the result is shown in figure 6.
As can be seen from FIG. 6, as the molecular weight cut-off of the ultrafiltration membrane increases, the recovery rate of the enzyme activity decreases in turn, and the time for the ultrafiltration concentration at the same fold decreases in turn. When the molecular weight cut-off of the ultrafiltration membrane is 10KDa, the enzyme activity recovery rate can reach 70.84 percent at most, but the used ultrafiltration time is the most. When the molecular weight of the ultrafiltration membrane is 30KDa, the recovery rate of enzyme activity is 69.51%, the used time is 196min, and the time is greatly reduced compared with that of a 10KDa ultrafiltration membrane. And the LOX enzyme activity recovery rate is lowest when the molecular weight cut-off of the ultrafiltration membrane is 50KDa, and the used time is also least. The reason is that the size of the molecular weight cut-off of the ultrafiltration membrane can influence the membrane flux of the ultrafiltration membrane, and the membrane flux is an important index for representing the efficiency of the ultrafiltration membrane, namely, the larger the molecular weight cut-off of the ultrafiltration membrane is, the higher the membrane flux is, the shorter the ultrafiltration time is, and the higher the ultrafiltration efficiency is. However, in order to ensure complete interception of LOX and obtain higher recovery rate of enzyme activity, the molecular weight intercepted by the ultrafiltration membrane in the ultrafiltration process should be selected to be one third to one sixth of the molecular weight of LOX, so that the molecular weight intercepted by the ultrafiltration membrane is lower than the molecular weight of the molecules to be intercepted and higher than the molecular weight of the molecules to pass through. Therefore, the ultrafiltration membrane with the molecular weight cutoff of 50KDa has a poor LOX interception effect, while the ultrafiltration membrane with the molecular weight cutoff of 10KDa has the best LOX interception effect, but the used time is long, and in industrial production, not only the LOX enzyme activity recovery rate is considered, but also the ultrafiltration time is considered, so that the LOX enzyme activity recovery rate and the ultrafiltration time are comprehensively considered, and the ultrafiltration membrane with the molecular weight cutoff of 30KDa is selected for ultrafiltration treatment.
2.2 Optimization of ultrafiltration operating pressure
Besides most whey proteins, the soybean whey wastewater also contains small molecular polysaccharides such as soybean oligosaccharides, so that the viscosity of the soybean whey wastewater is increased along with the increase of ultrafiltration multiple, and certain loss of LOX is generated by operating pressure, so that the enzyme activity recovery rate of LOX can be better maintained by determining the appropriate operating pressure. The concentration multiple is 6 times, the molecular weight cut-off of the ultrafiltration membrane is 30KDa, so that the proper operation pressure and time in the ultrafiltration concentration process can be researched, and the research result is shown in figure 7.
As can be seen from fig. 7, as the operation pressure increases, the recovery rates of enzyme activity and the ultrafiltration time both decrease sequentially, and when the operation pressure is 100KPa and 200KPa respectively, the recovery rates of LOX enzyme activity are 69.51% and 68.92%, respectively, and the used ultrafiltration time is 196min and 161min, respectively, thus it can be seen that the difference between the recovery rates of LOX enzyme activity at the two operation pressures is almost the same, but the difference between the used ultrafiltration times is much. When the operation pressure is 300KPa, the LOX enzyme activity recovery rate and the ultrafiltration time are respectively 62.08 percent and 144min, and the LOX enzyme activity recovery rate and the used ultrafiltration time are both smaller when the pressure is larger. This is because the operating pressure is too high during ultrafiltration, the ultrafiltration membrane surface is prone to generate concentration polarization phenomenon, and even a gel layer may be formed, so that some components in the material are deposited in the membrane pores, which causes the membrane pores to be reduced or blocked, and the membrane is prone to rupture due to too high operating pressure. Therefore, in the ultrafiltration process, in order to improve the membrane separation efficiency and reduce the pollution of the ultrafiltration membrane, the operation pressure is generally controlled below 300KPa, so the operation pressure is 200KPa.
2.3 Optimization of concentration factor
The influence of different ultrafiltration factors on the LOX unit enzyme activity and the enzyme activity recovery rate is different, and the influence result is shown in FIG. 8.
As can be seen from FIG. 8, as the concentration factor of ultrafiltration increases, the LOX unit enzyme activity increases sequentially, and the recovery rate of enzyme activity decreases gradually. When the concentration is 10 times, the unit enzyme activity and the recovery rate of the enzyme activity of LOX are 17946.88U/mL and 66.23% respectively, and the concentrated solution after ultrafiltration needs to be further purified, so that the larger the ultrafiltration concentration multiple is, the smaller the volume of the obtained concentrated solution is, and the workload of the subsequent purification process is greatly reduced.
2.4 optimization of the Ultrafiltration mode
According to the optimized experimental results, in the ultrafiltration process, the operation pressure is 200KPa, an ultrafiltration membrane of 30KDa is selected to carry out 10-time concentration on the extract obtained by isoelectric point fractional precipitation, and the selection of the ultrafiltration mode can also have certain influence on the recovery rate of LOX enzyme activity and unit enzyme activity.
The isoelectric point fractional precipitation extract was concentrated by single-stage ultrafiltration and two-stage ultrafiltration to determine a suitable ultrafiltration, the results are shown in fig. 9.
As can be seen from fig. 9, the LOX unit enzyme activity and recovery rates obtained by the first-stage ultrafiltration are 17946.88U/mL and 66.23% respectively, while the two-stage ultrafiltration divides the precipitation extract into two equal parts, concentrates the two parts by 3 times respectively, mixes the two parts together, and concentrates the two parts by 4 times, and finally the LOX unit enzyme activity recovery rates and unit enzyme activities are 19713.092U/mL and 71.08% respectively. And selecting a two-stage ultrafiltration mode for sample treatment according to the final results of the two ultrafiltration modes.
Under the condition that the operation pressure is 200KPa, an extracting solution obtained by isoelectric point fractional precipitation is concentrated by 10 times by adopting an ultrafiltration membrane with the molecular weight cutoff of 30KDa and a two-stage ultrafiltration mode, the unit enzyme activity of the obtained concentrated solution is 19713.092U/mL, the purification multiple is improved by 16.79 times, and the LOX purity and the enzyme activity recovery rate respectively reach 51.85 percent and 71.08 percent, so that the extracting solution obtained by isoelectric point fractional precipitation is further optimized by utilizing an ultrafiltration system, and the purity of LOX in the extracting solution can be greatly improved.
Example 3
A method for extracting lipoxygenase from soybean whey wastewater by reverse pH gradient comprises the following steps:
1) Pretreatment of soybean whey wastewater
Taking 1000mL of soybean whey wastewater, centrifuging at 12000r/min for 10min, and reserving supernatant as a pretreatment solution of the soybean whey wastewater for experiments;
2) Removing foreign protein in pretreatment liquid of soybean whey wastewater
Adjusting the pH value of the soybean whey wastewater pretreatment liquid to 8.0 by using a sodium hydroxide solution, and centrifuging to remove precipitates;
3) Extraction of lipoxygenase
Adjusting the pH value of the supernatant obtained by centrifuging in the step 2) to 6.0 by using a sodium hydroxide solution, centrifuging to remove the supernatant, dissolving the obtained precipitate with distilled water, and adjusting the pH to be neutral to obtain a lipoxygenase crude enzyme solution;
4) Primarily purifying the lipoxygenase crude enzyme liquid by using an ultrafiltration concentration method
And (3) performing ultrafiltration concentration on the lipoxygenase crude enzyme solution by 10 times at the pressure of 200KPa by using an ultrafiltration system and a modified polyethersulfone ultrafiltration membrane with the size of 30KDa in a two-stage ultrafiltration mode.
5) Further purifying the enzyme solution obtained in the step 4) by using a gel filtration chromatography method to obtain pure lipoxygenase; gel filtration chromatography uses Sephadex G-75 gel column of 2-70KDa, uses phosphate buffer solution with pH7.8 and 0.02mol/L to balance 2 to 3 column volumes, and samples are loaded after the balance is finished, and uses phosphate buffer solution with pH7.8 and 0.02mol/L as eluent to elute; the flow rate of the eluent is 0.5mL/min; automatically collecting samples, combining peak parts, and measuring LOX enzyme activity and protein content in the solution.
The crude LOX enzyme solution was further purified by gel filtration chromatography, and the elution results are shown in FIG. 10. As can be seen from FIG. 10, 5 protein peaks were obtained by Sephadex G-75 gel chromatography, and the samples collected from each peak were subjected to enzyme activity measurement, and the samples having LOX enzyme activity were combined and concentrated.
To further verify the purification results of LOX in the soybean whey wastewater, SDS-PAGE analysis was performed on samples at each purification stage, and the results are shown in table 2 and fig. 11. From FIG. 11, it can be seen that the soybean whey wastewater contains more protein bands, the molecular weight is more concentrated in 10-100kDa, the protein band in lane 3 is reduced, and more impure proteins are precipitated in lane 4, thereby removing a part of the impure proteins. The lipoxygenase band is significantly reduced in lane 5, while a small fraction of the hetero-protein is precipitated in lane 6 in addition to LOX, thus requiring further purification by concentration through a 30KDa ultrafiltration membrane and gel filtration chromatography. As can be seen from the figure, the LOX extract shows a single protein band after further purification, which indicates that the purification process obtains LOX with higher purity, thereby achieving the purpose of separating and purifying LOX in the soybean whey wastewater.
TABLE 2 LOX purification results in Soy whey wastewater
After dissolving the precipitate with the pH value of 6.0, concentrating the precipitate by a 30KDa ultrafiltration membrane and purifying the precipitate by SephadexG-75 gel chromatography, the final LOX purity of the LOX is 90.43 percent, the specific enzyme activity reaches 25546.08U/mg, the enzyme activity recovery rate is 40.29 percent, the purification multiple is improved by 25.44 times, and according to the analysis result of SDS-PAGE gel electrophoresis, the impure protein bands in the chromatographic solution are obviously reduced, and the purification effect is more obvious. Therefore, the purification scheme can be used for developing and utilizing LOX in the soybean whey wastewater so as to achieve the aim of recycling bioactive substances in the soybean whey wastewater.
Claims (4)
1. A method for extracting lipoxygenase from soybean whey wastewater by reverse pH gradient is characterized by comprising the following steps:
1) Pretreatment of soybean whey wastewater
Centrifuging the soybean whey wastewater to remove large-particle impurities, and reserving supernatant as a pretreatment solution of the soybean whey wastewater;
2) Removing foreign protein in pretreatment liquid of soybean whey wastewater
Adjusting the pH value of the soybean whey wastewater pretreatment liquid to 7.8 to 8.2, and centrifuging to remove precipitates;
3) Extraction of lipoxygenase
Regulating the pH value of the supernatant obtained by centrifuging in the step 2) to 5.7-6.0, centrifuging to remove the supernatant, and dissolving the obtained precipitate with distilled water to obtain a lipoxygenase crude enzyme solution;
further comprises a step 4), primarily purifying the lipoxygenase crude enzyme liquid by using an ultrafiltration concentration method;
further comprising the step 5), further purifying the enzyme solution obtained in the step 4) by using gel filtration chromatography to obtain pure lipoxygenase; in the step 5), the gel filtration chromatography utilizes a Sephadex G-75 gel column of 2-70KDa, 2-3 column volumes are balanced by phosphate buffer solution with pH7.8 and 0.02mol/L, and after the balance is finished, the gel is loaded and eluted by taking the phosphate buffer solution with pH7.8 and 0.02mol/L as eluent; the flow rate of the eluent is 0.5mL/min;
in the step 4), carrying out ultrafiltration concentration on the lipoxygenase crude enzyme liquid by 8 to 10 times under the pressure of 200 to 300KPa by using a modified polyethersulfone ultrafiltration membrane with the size of 30 KDa;
in the step 2), adjusting the pH value of the soybean whey wastewater pretreatment liquid to 8.0;
in step 3), the pH of the supernatant was adjusted to 6.0.
2. The method for extracting lipoxygenase from soybean whey waste water by reverse pH gradient as claimed in claim 1, wherein: and 4), performing ultrafiltration concentration by using an ultrafiltration system in a two-stage filtration mode.
3. The method for extracting lipoxygenase from soybean whey waste water by reverse pH gradient as claimed in claim 1, wherein: in the step 2) and the step 3), the pH value of the system is adjusted by using a sodium hydroxide solution.
4. The method for extracting lipoxygenase from soybean whey waste water by reverse pH gradient as claimed in claim 1, wherein: and in the step 1), centrifuging at the rotating speed of 3000-15000r/min for 8-20min.
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| 大豆肽的理化性质及其对脂肪氧合酶活性的影响;荣建华;《食品工业科技》;20020831;第23卷(第8期);19-21 * |
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