CN101418288B - Method for refining egg-white lysozyme by circulation hyperfiltration-salting out crystal separation - Google Patents
Method for refining egg-white lysozyme by circulation hyperfiltration-salting out crystal separation Download PDFInfo
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- CN101418288B CN101418288B CN2008102195047A CN200810219504A CN101418288B CN 101418288 B CN101418288 B CN 101418288B CN 2008102195047 A CN2008102195047 A CN 2008102195047A CN 200810219504 A CN200810219504 A CN 200810219504A CN 101418288 B CN101418288 B CN 101418288B
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Abstract
The invention provides a method for refining egg-white lysozyme by the application of cycling ultrafiltration and salting-out crystallization separation. The method comprises the following steps: firstly, performing pretreatments of dilution, micro-filtration, ultrafiltration and the like on egg white to obtain a raw material liquid of egg white lysozyme; secondly, allowing the raw material liquid to circularly flow through membrane modules in a tangential flow ultra-filter so that an egg-white lysozyme solution is continuously separated and concentrated; thirdly, further concentrating the egg-white lysozyme solution through the ultrafiltration and the salting-out crystallization; and fourthly, performing salting-out crystallization on the egg-white lysozyme solution in a crystallizing tank. With the method, the crystal particle size of the obtained lysozyme is larger than 0.1 millimeter, the particle size is uniform and the appearance is complete, and the lysozyme has a typical crystal X-ray diffraction peak; besides, the specific activity can reach more than 20, 000 U/mg, the enzyme reclamation rate can reach more than 70 percent, and the purification factor reaches 25.1.
Description
Technical field
The present invention relates to a kind of separation purification method of hen egg white lysozyme, be specifically related to the method for a kind of loop ultrafiltration-salting-out crystallization separation and purification hen egg white lysozyme.
Background technology
Hen egg white lysozyme is a kind of typical protein, existing application widely on medical treatment, food and biotechnology at present.Along with the appearance of various new bioseparation technologies, the method that is used for the N,O-Diacetylmuramidase separation and purification is also more and more many, from initial direct crystallization method, the precipitator method to affinity chromatography, ion exchange method, ultra-filtration membrane separation etc.At present, hen egg white lysozyme production method commonly used is an ion exchange method, but the yield of hen egg white lysozyme and enzyme are alive not ideal.The ultra-filtration membrane isolation technique is a kind of non-heating and separating technology, has no phase transformation, energy consumption is low and separation efficiency is high characteristics, has great superiority for the separating bio macromole.In addition, ultra-filtration membrane separate also have easy to operate, compact construction, maintenance cost is lower, the characteristics that are easy to automatization.And the separation of adopting the ultra-filtration membrane isolation technique to carry out hen egg white lysozyme concentrates, high and few destruction of yield.But ultra-filtration membrane separates the separation that is applied to hen egg white lysozyme must overcome problems such as concentration polarization, flux decay.Crystallization is a very important chemical unit operation, can obtain the very high crystal of purity from the considerable solution of impurity, and simultaneously used equipment is simple, easy to operate.For many materials, a unit operation the most cheap of pure product is produced in crystallization often from impure mixture or impure solution, also be method most economical in the suitability for industrialized production.But salting-out crystallization method production hen egg white lysozyme crystallization auxiliary consumption is big, crystal size is little, foreign matter content is higher, be difficult to reach problems such as purity and crystalline form requirement.
Loop ultrafiltration-salting-out crystallization method has been coupled, and ultra-filtration membrane separates and two kinds of processes of crystallization, under the situation that crystallization auxiliary exists, make the degree of supersaturation of solute maintain a higher level, and make membrane flux can maintain a comparatively ideal level, the degree of supersaturation of solute is progressively improved, more help the crystalline growth, and improved the rate of recovery of solute in mother liquor, also can recycle simultaneously crystallization auxiliary.The method that loop ultrafiltration-salting-out crystallization separation and purification hen egg white lysozyme is not arranged at present as yet.
Summary of the invention
The objective of the invention is to defective, the method for a kind of loop ultrafiltration-salting-out crystallization separation and purification hen egg white lysozyme is provided at prior art
The object of the invention is achieved through the following technical solutions:
The method of a kind of loop ultrafiltration-salting-out crystallization separation and purification hen egg white lysozyme may further comprise the steps:
(1) pre-treatment of egg white obtains the hen egg white lysozyme stock liquid
With Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution egg white is diluted 6-12 doubly, use high pressure homogenizer under 18-28MPa behind the homogeneous, use 0.22 μ m or 0.45 μ m micro-filtrate membrane filtration to remove solid impurity, be the ultrafiltration membrance filter of 20-40kD with molecular weight cut-off then, filtrate is the hen egg white lysozyme stock liquid;
(2) separation of hen egg white lysozyme stock liquid and concentrated
20-50 ℃, working pressure be under the 0.10-0.45MPa in tangential flow ultra-filtration unit continuous loop ultrafiltration hen egg white lysozyme stock liquid, to the lysozyme content of hen egg white lysozyme stock liquid be 1-4% (w/v);
(3) the integrated hen egg white lysozyme stock liquid that further concentrates of ultrafiltration and salting-out crystallization
Adding NaCl to NaCl concentration then in the hen egg white lysozyme stock liquid is 1-4% (w/v), in tangential flow ultra-filtration unit, continuing ultrafiltration and concentration lysozyme content to the hen egg white lysozyme stock liquid under 20-50 ℃, working pressure 0.10-0.45MPa is 3-6% (w/v);
(4) hen egg white lysozyme salting-out crystallization in crystallizer
The trapped fluid of tangential flow ultra-filtration unit is entered crystallizer, adding the concentration of NaCl to NaCl in the hen egg white lysozyme stock liquid is 3-6% (w/v), Tc is 25-35 ℃, stir, N,O-Diacetylmuramidase nucleation and growing the grain to crystal in crystallizer are no longer grown up, crystal in the centrifugation magma, vacuum-drying can obtain N,O-Diacetylmuramidase crystal of the present invention.
The pH of the described Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution of step (1) is 5.0-8.0, and concentration is 0.1-0.3mol/L concentration, in phosphate radical.
The described tangential flow ultra-filtration unit of step (2) uses the ultrafiltration membrance filter hen egg white lysozyme stock liquid of molecular weight cut-off as 3-10kD.
The speed of the described stirring of step (4) is 100-300r/min.
The inventive method organically combines the separation and purification process that is used for hen egg white lysozyme with ultra-filtration technique and salting-out crystallization technology, selective permeation by filter membrane separates solute with continuous Circulation with impurity, and constantly be concentrated into saturated, salting-out crystallization under the effect of crystallization auxiliary.
The present invention compared with prior art has following advantage and beneficial effect:
(1) the present invention has fully utilized salting-out crystallization method and ultrafiltration process advantage separately preferably.The advantage of salting-out crystallization method is that energy consumption is low, can improve the rate of recovery of solute from mother liquor, can carry out at low temperatures, keeps the activity of hen egg white lysozyme effectively; And ultrafiltration can the realization of holding back by ultra-filtration membrane reach purifying to concentrating of solute equally under lower temperature, and easy to operate.
(2) the present invention overcomes the hen egg white lysozyme salting-out crystallization and separates separately technological deficiency with ultra-filtration membrane.The defective of salting-out crystallization is that the crystallization auxiliary consumption is big, and crystal size is little, and foreign matter content is higher, is difficult to reach purity and crystalline form requirement; And ultra-filtration technique still can't overcome the concentration polarization phenomenon in the operational process, and along with the raising of treatment solution concentration, viscosity, the decay of flux is comparatively obvious, and, must there be energy consumption big or influence active follow-up drying process after concentrating.Integrated based on salting-out crystallization and two kinds of processes of ultrafiltration, promote at polymer film surface under the situation of the heterogeneous nucleation of protein, reduce the crystallization auxiliary add-on, loop ultrafiltration makes that the degree of supersaturation of solute is progressively improved, more favourable crystalline growth, and further improve the rate of recovery of solute from mother liquor.
(3) method of loop ultrafiltration of the present invention-salting-out crystallization separation and purification hen egg white lysozyme has shortened the Production Flow Chart of N,O-Diacetylmuramidase, has kept lysozyme activity, has improved production efficiency, and has reduced the quantity discharged of waste liquid.
(4) adopt method of the present invention, the N,O-Diacetylmuramidase crystal particle diameter that is obtained is greater than 0.1mm, and epigranular, complete shape and appearance have typical crystal X-ray diffraction peak; Can reach 20 than vigor, more than the 000U/mg, the enzyme rate of recovery can reach more than 70%, and purification of factor reaches more than 25.
Description of drawings
Fig. 1 is the process flow sheet of loop ultrafiltration of the present invention-salting-out crystallization separation and purification hen egg white lysozyme;
Fig. 2 is that the embodiment of the invention obtains hen egg white lysozyme X-ray diffraction in crystals collection of illustrative plates;
Fig. 3 is that the embodiment of the invention obtains hen egg white lysozyme crystalline light micrograph;
Fig. 4 is that the embodiment of the invention obtains hen egg white lysozyme crystalline scanning electron photomicrograph;
Among Fig. 1 in the membrane module 1 molecular weight cut-off of ultra-filtration membrane be 20-40kD, the molecular weight cut-off of ultra-filtration membrane is 3-10kD in the membrane module 2.
Embodiment
The present invention will be further described below in conjunction with embodiment and accompanying drawing 1, but embodiments of the present invention are not limited thereto.
Embodiment 1
According to technical process shown in Figure 1, utilize the step of loop ultrafiltration-salting-out crystallization separation and purification hen egg white lysozyme as follows:
(1) use pH=5.0, concentration (in phosphate radical) be Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution of 0.2mol/L with 6 times of egg white dilutions, under 28MPa behind the homogeneous, crossing after the 0.22 μ m microfiltration membrane with molecular weight cut-off is the ultrafiltration membrance filter of 40kD, gets filtrate;
(2) with molecular weight cut-off being the ultra-filtration membrane bag of 3kD, is 4% (w/v) at the continuous loop ultrafiltration hen egg white lysozyme of tangential flow ultra-filtration unit stock liquid to lysozyme content under 20 ℃, working pressure 0.45MPa;
(3) in hen egg white lysozyme solution, add NaCl to its concentration be 1% (w/v), be the ultra-filtration membrane bag of 3kD with molecular weight cut-off, continuation ultrafiltration and concentration to the N,O-Diacetylmuramidase in the solution is 6% (w/v) under 20 ℃, working pressure 0.45MPa; The average film flux is 0.25mL/ (cm with this understanding
2Min);
(4) trapped fluid in the tangential flow ultra-filtration unit is entered crystallizer, add NaCl to its concentration in hen egg white lysozyme solution be 3% (w/v), Tc is 35 ℃, stirring velocity is 100r/min, N,O-Diacetylmuramidase nucleation and growing the grain to crystal in crystallizer are no longer grown up, crystal in the centrifugation magma, vacuum-drying, can obtain the average crystalline particle diameter is the N,O-Diacetylmuramidase crystal of 0.1mm, obtain N,O-Diacetylmuramidase X-ray diffraction in crystals collection of illustrative plates as shown in Figure 2, have typical crystal X-ray diffraction peak.
Embodiment 2
According to technical process shown in Figure 1, utilize the step of loop ultrafiltration-salting-out crystallization separation and purification hen egg white lysozyme as follows:
(1) use pH=7.0, concentration (in phosphate radical) be Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution of 0.1mol/L with 12 times of egg white dilutions, under 18MPa behind the homogeneous, crossing after the 0.45 μ m microfiltration membrane with molecular weight cut-off is the ultrafiltration membrance filter of 30kD, gets filtered solution;
(2) with molecular weight cut-off being the ultra-filtration membrane bag of 5kD, is 1% (w/v) at the continuous loop ultrafiltration hen egg white lysozyme of tangential flow ultra-filtration unit stock liquid to lysozyme content under 30 ℃, working pressure 0.10MPa;
(3) in hen egg white lysozyme solution, add NaCl to its concentration be 3% (w/v), be the ultra-filtration membrane bag of 5kD with molecular weight cut-off, continuation ultrafiltration and concentration to the N,O-Diacetylmuramidase in the solution is 3% (w/v) under 30 ℃, working pressure 0.20MPa; The average film flux is 0.21mL/ (cm with this understanding
2Min)
(4) trapped fluid in the tangential flow ultra-filtration unit is entered crystallizer, add NaCl to its concentration in hen egg white lysozyme solution be 4% (w/v), Tc is 25 ℃, stirring velocity is 300r/min, N,O-Diacetylmuramidase nucleation and growing the grain to crystal in crystallizer are no longer grown up, crystal in the centrifugation magma, vacuum-drying, can obtain average mean crystal size is the N,O-Diacetylmuramidase crystal of 0.15mm.To obtain N,O-Diacetylmuramidase crystalline purification effect as shown in table 1.
Table 1
Embodiment 3
According to technical process shown in Figure 1, utilize the step of loop ultrafiltration-salting-out crystallization separation and purification hen egg white lysozyme as follows:
(1) use pH=8.0, concentration (in phosphate radical) be Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution of 0.3mol/L with 8 times of egg white dilutions, under 24MPa behind the homogeneous, crossing after the 0.22 μ m microfiltration membrane with molecular weight cut-off is the ultrafiltration membrance filter of 20kD, gets filtered solution;
(2) with molecular weight cut-off being the ultra-filtration membrane bag of 10kD, is 2% (w/v) at the continuous loop ultrafiltration hen egg white lysozyme of tangential flow ultra-filtration unit stock liquid to lysozyme content under 50 ℃, working pressure 0.2MPa;
(3) in hen egg white lysozyme solution, add NaCl to its concentration be 4% (w/v), be the ultra-filtration membrane bag of 10kD with molecular weight cut-off, continuation ultrafiltration and concentration to the N,O-Diacetylmuramidase in the solution is 4% (w/v) under 50 ℃, working pressure 0.3MPa; The average film flux is 0.27mL/ (cm with this understanding
2Min)
(4) trapped fluid in the tangential flow ultra-filtration unit is entered crystallizer, add NaCl to its concentration in hen egg white lysozyme solution be 6% (w/v), Tc is 30 ℃, stirring velocity is 200r/min, N,O-Diacetylmuramidase nucleation and growing the grain to crystal in crystallizer are no longer grown up, crystal in the centrifugation magma, vacuum-drying, can obtain average mean crystal size is the N,O-Diacetylmuramidase crystal of 0.2mm, the light micrograph of N,O-Diacetylmuramidase crystalline pattern such as Fig. 3 that obtains and the electron scanning micrograph of Fig. 4, visible crystals epigranular, complete shape and appearance.
Claims (4)
1. the method for loop ultrafiltration-salting-out crystallization separation and purification hen egg white lysozyme is characterized in that may further comprise the steps:
(1) pre-treatment of egg white obtains the hen egg white lysozyme stock liquid
With Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution egg white is diluted 6-12 doubly, use high pressure homogenizer under 18-28MPa behind the homogeneous, use 0.22 μ m or 0.45 μ m micro-filtrate membrane filtration to remove solid impurity, be the ultrafiltration membrance filter of 20-40kD with molecular weight cut-off then, filtrate is the hen egg white lysozyme stock liquid;
(2) separation of hen egg white lysozyme stock liquid and concentrated
20-50 ℃, working pressure be under the 0.10-0.45MPa in tangential flow ultra-filtration unit continuous loop ultrafiltration hen egg white lysozyme stock liquid, to the lysozyme content of hen egg white lysozyme stock liquid be 1-4% (w/v);
(3) the integrated hen egg white lysozyme stock liquid that further concentrates of ultrafiltration and salting-out crystallization
Adding NaCl to NaCl concentration then in the hen egg white lysozyme stock liquid is 1-4% (w/v), in tangential flow ultra-filtration unit, continuing ultrafiltration and concentration lysozyme content to the hen egg white lysozyme stock liquid under 20-50 ℃, working pressure 0.10-0.45MPa is 3-6% (w/v);
(4) hen egg white lysozyme salting-out crystallization in crystallizer
The trapped fluid of tangential flow ultra-filtration unit is entered crystallizer, adding the concentration of NaCl to NaCl in the hen egg white lysozyme stock liquid is 3-6% (w/v), Tc is 25-35 ℃, stir, N,O-Diacetylmuramidase nucleation and growing the grain to crystal in crystallizer are no longer grown up, crystal in the centrifugation magma, vacuum-drying can obtain N,O-Diacetylmuramidase crystal of the present invention.
2. method according to claim 1 is characterized in that: the pH of the described Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution of step (1) is 5.0-8.0, and concentration is 0.1-0.3mol/L, in phosphate radical.
3. method according to claim 1 is characterized in that: the described tangential flow ultra-filtration unit of step (2) uses the ultrafiltration membrance filter hen egg white lysozyme stock liquid of molecular weight cut-off as 3-10kD.
4. method according to claim 1 is characterized in that: the speed of the described stirring of step (4) is 100-300r/min.
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| CN2008102195047A CN101418288B (en) | 2008-11-28 | 2008-11-28 | Method for refining egg-white lysozyme by circulation hyperfiltration-salting out crystal separation |
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| CN2008102195047A CN101418288B (en) | 2008-11-28 | 2008-11-28 | Method for refining egg-white lysozyme by circulation hyperfiltration-salting out crystal separation |
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| CN107446905B (en) * | 2017-08-24 | 2020-04-24 | 陕西慧康生物科技有限责任公司 | Method for purifying recombinant human lysozyme |
| CN110606867A (en) * | 2019-09-24 | 2019-12-24 | 中国科学院力学研究所 | Method for obtaining high-purity protein crystal |
| CN110746483A (en) * | 2019-10-28 | 2020-02-04 | 上海奥浦迈生物科技有限公司 | Method for controlling generation of particles in ultrafiltration and diafiltration process |
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