CN112760309B - Method for multi-stage purification of nuclease P1 - Google Patents
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Abstract
The invention discloses a method for multi-stage purification of nuclease P1, which is to carry out multi-stage purification on nuclease P1 crude enzyme solution through ultrafiltration membrane ultrafiltration, heat inactivation and resin impurity removal. The method can efficiently separate and purify the nuclease P1, and the enzyme recovery rate of the nuclease P1 reaches 82.6 percent; and the cost is low, and the method is suitable for large-scale industrial production. Meanwhile, the purity of the nuclease P1 obtained by the method is greatly improved, the specific activity is 7.3 times of that of the crude enzyme solution, and the impurities are few, so the method can be used for the food industry.
Description
Technical Field
The invention relates to the field of biology, and in particular relates to a method for multi-stage purification of nuclease P1.
Background
With the development of the biotechnology industry and the pharmaceutical industry, research and development of nucleotides and derivatives thereof have been hot and have gradually formed another major industry following amino acids. The nucleotide is a biochemical substance with high added value, has wide application, and supports the development of some larger industries, such as high-end dairy industry, pharmaceutical industry and the like. The milk powder is used as a functional additive in the dairy industry, and the milk powder is added to enable the milk powder to be closer to breast milk, so that the capability of resisting bacillary dysentery of infants can be effectively enhanced, and the occurrence of diarrhea is reduced; in the field of medicine, nucleotides are used as pharmaceutical intermediates as raw materials for producing many nucleotide derivatives, and are used for synthesizing hundreds of compounds such as CDP, UDP, GTP, CDP-choline, SAM, cGMP, CTP, UTP, cAMP, UDP-acetylglucosamine, GDP-mannose, GDP-glucose, UDP-glucose, and the like. The nucleotide medicine has unique curative effect in the aspects of resisting cancer and virus, treating cardiovascular diseases, diabetes, interference induction and the like, and has good application effect and huge application market.
The enzyme required in preparing nucleotide by an enzymatic hydrolysis method is nuclease Pl, which is phosphodiesterase capable of hydrolyzing RNA to obtain four 5' -nucleotides, the impurities of the enzymatic hydrolysis product are few, the subsequent separation process is simple, and the method is generally adopted for producing the nucleotide abroad. However, crude nuclease P1 contains a large amount of miscellaneous enzymes, and has a large influence on the efficiency of enzymatic hydrolysis, and it is generally necessary to purify it before use.
The existing methods for separating the nuclease P1, such as an ammonium sulfate salting-out method, a centrifugal spray drying method and the like, have the problems of low sample recovery rate, expensive separation materials or equipment, small sample treatment capacity and low separation purity by a single method; and nuclease P1 obtained by the ammonium sulfate salting-out method contains more impurities, has low titer, and cannot be directly applied to the food industry.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the technical problem of providing a method for multi-stage purification of nuclease P1 aiming at the defects of the prior art.
In order to solve the technical problems, the invention discloses a method for multi-stage purification of nuclease P1, which comprises the steps of carrying out ultrafiltration on nuclease P1 crude enzyme liquid by an ultrafiltration membrane, carrying out heat inactivation and carrying out resin impurity removal to carry out multi-stage purification.
Preferably, as shown in figure 1, crude nuclease P1 enzyme solution is sequentially subjected to ultrafiltration by an ultrafiltration membrane, heat inactivation and resin impurity removal for multi-stage purification.
Specifically, the method comprises the following steps:
(1) performing ultrafiltration on nuclease P1 crude enzyme liquid by an ultrafiltration membrane, and collecting trapped fluid;
(2) carrying out heat inactivation on trapped fluid obtained in the step (1), carrying out solid-liquid separation, and collecting filtrate;
(3) and (3) loading the filtrate obtained in the step (2) into resin for removing impurities, and collecting effluent, namely the nuclease P1 solution after multi-stage purification.
In the step (1), the nuclease P1 crude enzyme solution is prepared by fermentation of penicillium citrinum, and contains other proteases besides nuclease P1, such as: alkaline phosphatase, amylase, etc., with impurities accounting for about 30% of the total protein content.
Wherein the Penicillium citrinum (Penicillium citrinum) is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 2014.
Wherein the enzyme activity of the nuclease P1 in the crude nuclease liquid is 5000-7000U/mL.
Wherein the specific activity of the nuclease P1 in the crude nuclease liquid is 800-1000U/g.
Preferably, the crude nuclease P1 enzyme solution is subjected to solid-liquid separation, the filtrate is collected, and then the filtrate is subjected to multi-stage purification.
In the step (1), the ultrafiltration membrane is a polyether sulfone membrane.
In the step (1), the molecular weight of the ultrafiltration membrane is 1-30KDa, preferably 2-20KDa, and more preferably 5-15 KDa.
In the step (1), the ultrafiltration comprises the following steps:
(i) performing solid-liquid separation on a nuclease P1 crude enzyme solution to obtain a filtrate, and performing ultrafiltration on the filtrate in an ultrafiltration membrane until a retention solution is 1/4 of the volume of the filtrate;
(ii) (ii) continuing to add ultrafiltration solvent to the retentate to its original volume, and then ultrafiltering to a retentate volume that is 1/4 times the volume of the filtrate from step (i);
(iii) (iii) repeating step (ii) and continuing the cycle 2-5 times.
In steps (i) and (ii), the temperature of the ultrafiltration is 20-40 ℃.
In step (ii), the ultrafiltration solvent is water.
In the step (2), the temperature for heat inactivation is 70-100 ℃, and preferably 85-90 ℃.
In the step (2), the time is 1-60min, preferably 20-30 min.
In the step (3), the resin is macroporous resin which takes polystyrene as an inert framework and takes sulfonic group as a functional group.
In the step (3), the sampling amount of the impurity removal is 0.5-2 BV.
In the step (3), the sampling rate of the impurity removal is 0.1-0.5 BV/h.
The method for purifying the nuclease P1 in multiple stages can effectively improve the specific activity and yield of the nuclease P1.
Wherein, the specific activity of the nuclease P1 is improved by more than 4.7 times and reaches 7.3 times.
Wherein, the total yield of the nuclease P1 reaches 82.6 percent.
Has the advantages that: compared with the prior art, the invention has the following advantages:
1. the method can efficiently separate and purify the nuclease P1, and the enzyme recovery rate of the nuclease P1 reaches 82.6 percent; and the cost is low, and the method is suitable for large-scale industrial production.
2. The nuclease P1 obtained by the method has greatly improved purity, 7.3 times of specific activity of crude enzyme liquid and less impurities, so the method can be used in food industry.
Drawings
The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
FIG. 1 is a process flow diagram of the present invention.
Detailed Description
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
In the following examples, the ultrafiltration membrane is a polyethersulfone membrane with a molecular weight of 1-30 KDa.
The resin in the following examples is a macroporous resin with polystyrene as an inert skeleton and sulfonic acid group as a functional group.
In the invention, the definition of the enzyme activity, the definition of the specific activity and the definition of a protein determination method of the nuclease P1 are as follows:
definition of the enzymatic activity of nuclease P1: 1.9mL of a substrate solution (containing 1% by mass of RNA; 0.2M of pH5.2 in acetic acid buffer and 0.0005M of ZnSO)4) After 10min in a constant-temperature water bath at 70 ℃, 0.1mL of nuclease P1 is added, after 15min of heat preservation at 70 ℃, 2.0mL of nucleic acid precipitator (0.25% ammonium molybdate-2.5% perchloric acid in mass ratio) is added, after 20min of ice water bath, centrifugation is carried out, supernatant is taken and diluted by certain times by distilled water, the determination value is between 0.2 and 0.6, and the light absorption value at 260nm is A260. The control was made by adding the precipitant first, and the other operations were the same as before. Under the above conditions, 1 unit of enzyme activity was defined as the difference in absorbance at 260nm between the amounts of nucleotides produced per minute and 1.0.
Definition of specific activity: the protein concentration of the nuclease P1 in terms of enzyme activity ratio is the specific activity of the nuclease P1, and the representative is the purity of the nuclease P1.
The protein determination method comprises the following steps: coomassie Brilliant blue, which binds to hydrophobic domains of proteins with high sensitivity. Coomassie brilliant blue G250 in phosphoric acid showed a red-brown color with a maximum absorption peak at 465 nm. The blue color of the absorption peak changes to 595nm when it binds to proteins to form complexes, and the high extinction effect of the Coomassie brilliant blue G250-protein complex results in high sensitivity of protein quantitation. In a certain range, after the Coomassie brilliant blue G250-protein complex is colored, the absorbance and the protein content are in a linear relation at 595nm, so that the method can be used for measuring the protein concentration.
In the following examples, the crude nuclease P1 was prepared as follows:
(1) the strain is as follows: penicillium citrinum (Penicillium citrinum) with preservation number of CGMCC No.2014 is described in Chinese patent CN 101067116A.
(2) Fermentation medium: 20g/L glucose, 2g/L peptone, 0.5g/L potassium dihydrogen phosphate, 0.5g/L dipotassium hydrogen phosphate, 0.2g/L magnesium sulfate and 0.2g/L calcium chloride.
(3) Scraping the strain in bottle into sterile water, inoculating 2 bottles into fermentation medium in 100L fermentation tank, fermenting at pH6 and 32 deg.C for 30 hr.
(4) After the fermentation is finished, filtering to obtain clear liquid, wherein the obtained filtrate is the nuclease P1 crude enzyme.
The crude enzyme solutions in the following examples were also prepared according to this method, only with some parameters being different.
Example 1
Purification of nuclease P1 was carried out as follows:
firstly, fermenting mould to prepare nuclease P1 crude enzyme solution, wherein the enzyme activity is 5600U/mL, and the specific activity is 852U/g.
Secondly, carrying out solid-liquid separation on the crude enzyme liquid obtained in the step one, collecting filtrate, and carrying out ultrafiltration on the filtrate by using an ultrafiltration membrane with the molecular weight of 3 KD; specifically, the method comprises the following steps:
(i) carrying out ultrafiltration on the filtrate in an ultrafiltration membrane at the temperature of 30 ℃ until the trapped fluid is 1/4 of the volume of the filtrate, and collecting the trapped fluid;
(ii) (ii) continuing replenishing water into the trapped fluid to the volume of the filtrate in the step (i), performing ultrafiltration to 1/4 with the trapped fluid being the initial volume, and collecting the trapped fluid;
(iii) (iii) repeating the step (ii), ending the circulation for 2 times, and collecting the trapped fluid.
And thirdly, transferring the trapped fluid obtained in the second step into a reaction kettle, heating for 15min at 80 ℃ under a stirring state, and carrying out solid-liquid separation to obtain a clear liquid.
Pumping the clear liquid obtained in the step three into a chromatographic column loaded with ion exchange resin, wherein the sample loading amount is 0.8BV, the sample loading flow rate is 0.25BV/h, and collecting effluent, namely the nuclease P1 solution obtained after purification.
The recovery rate of the nuclease P1 in the embodiment is 81.4%, and the specific activity is 4.7 times of that of the crude enzyme.
Example 2
Purification of nuclease P1 was carried out as follows:
firstly, preparing nuclease P1 crude enzyme liquid by fermenting mould, wherein the enzyme activity is 5900U/mL, and the specific activity is 863U/g.
Secondly, carrying out solid-liquid separation on the crude enzyme liquid obtained in the step one, collecting filtrate, and carrying out ultrafiltration on the filtrate by using an ultrafiltration membrane with the molecular weight of 6 KD; specifically, the method comprises the following steps:
(i) carrying out ultrafiltration on the filtrate in an ultrafiltration membrane at the temperature of 32 ℃, and collecting the retentate until the retentate is 1/4 of the volume of the filtrate;
(ii) (ii) continuing replenishing water into the trapped fluid to the volume of the filtrate in the step (i), performing ultrafiltration to 1/4 with the trapped fluid being the initial volume, and collecting the trapped fluid;
(iii) (iii) repeating the step (ii), ending the cycle for 3 times, and collecting the trapped fluid.
And thirdly, transferring the trapped fluid obtained in the second step into a reaction kettle, heating for 20min at 85 ℃ under the stirring state, and carrying out solid-liquid separation to obtain clear liquid.
Pumping the clear liquid obtained in the step three into a chromatographic column loaded with ion exchange resin, wherein the sample loading amount is 1.2BV, the sample loading flow rate is 0.28BV, and collecting effluent, namely obtaining nuclease P1 solution after purification.
The recovery rate of the nuclease P1 in the embodiment is 79.4%, and the specific activity is 5.2 times of that of the crude enzyme.
Example 3
Purification of nuclease P1 was carried out as follows:
firstly, preparing crude nuclease P1 enzyme solution by fermenting mould, wherein the enzyme activity is 6200U/mL, and the specific activity is 912U/g.
Secondly, carrying out solid-liquid separation on the crude enzyme liquid obtained in the step one, collecting filtrate, and carrying out ultrafiltration on the filtrate by using an ultrafiltration membrane with the molecular weight of 12 KD; specifically, the method comprises the following steps:
(i) carrying out ultrafiltration on the filtrate in an ultrafiltration membrane at 33 ℃ until the retention solution is 1/4 of the volume of the filtrate, and collecting the retention solution;
(ii) (ii) continuing replenishing water into the trapped fluid to the volume of the filtrate in the step (i), performing ultrafiltration to 1/4 with the trapped fluid being the initial volume, and collecting the trapped fluid;
(iii) (iii) repeating the step (ii), ending the cycle for 4 times, and collecting the trapped fluid.
And thirdly, transferring the trapped fluid obtained in the second step into a reaction kettle, heating for 25min at 85 ℃ under the stirring state, and carrying out solid-liquid separation to obtain clear liquid.
Pumping the clear liquid obtained in the step three into a chromatographic column loaded with ion exchange resin, wherein the sample loading amount is 1BV, the sample loading flow rate is 0.24BV, and collecting effluent, namely the nuclease P1 solution after purification.
The recovery rate of the nuclease P1 in the embodiment is 82.6%, and the specific activity is 7.3 times of that of the crude enzyme.
Example 4
Purification of nuclease P1 was carried out as follows:
firstly, fermenting mould to prepare nuclease P1 crude enzyme liquid, wherein the enzyme activity is 6400U/mL, and the specific activity is 903U/g.
Secondly, carrying out solid-liquid separation on the crude enzyme liquid obtained in the step one, collecting filtrate, and carrying out ultrafiltration on the filtrate by using an ultrafiltration membrane with the molecular weight of 10 KD; specifically, the method comprises the following steps:
(i) carrying out ultrafiltration on the filtrate in an ultrafiltration membrane at 25 ℃ until the retention solution is 1/4 of the volume of the filtrate, and collecting the retention solution;
(ii) (ii) continuing replenishing water into the trapped fluid to the volume of the filtrate in the step (i), performing ultrafiltration to 1/4 with the trapped fluid being the initial volume, and collecting the trapped fluid;
(iii) (iii) repeating the step (ii), ending the cycle for 3 times, and collecting the trapped fluid.
And thirdly, transferring the trapped fluid obtained in the second step into a reaction kettle, heating for 30min at 90 ℃ under a stirring state, and carrying out solid-liquid separation to obtain a clear liquid.
Pumping the clear liquid obtained in the step three into a chromatographic column loaded with ion exchange resin, wherein the sample loading amount is 1BV, the sample loading flow rate is 0.20BV, and collecting effluent, namely the nuclease P1 solution after purification.
The recovery rate of the nuclease P1 in the embodiment is 72.7%, and the specific activity is 6.0 times of that of the crude enzyme.
Example 5
Purification of nuclease P1 was carried out as follows:
firstly, preparing nuclease P1 crude enzyme liquid by fermenting mould, wherein the enzyme activity is 5800U/mL, and the specific activity is 846U/g.
Secondly, carrying out solid-liquid separation on the crude enzyme liquid obtained in the step one, collecting filtrate, and carrying out ultrafiltration on the filtrate by using an ultrafiltration membrane with the molecular weight of 20 KD; specifically, the method comprises the following steps:
(i) carrying out ultrafiltration on the filtrate in an ultrafiltration membrane at the temperature of 35 ℃ until the trapped fluid is 1/4 of the volume of the filtrate, and collecting the trapped fluid;
(ii) (ii) continuing replenishing water into the trapped fluid to the volume of the filtrate in the step (i), performing ultrafiltration to 1/4 with the trapped fluid being the initial volume, and collecting the trapped fluid;
(iii) (iii) repeating the step (ii), ending the cycle for 3 times, and collecting the trapped fluid.
And thirdly, transferring the trapped fluid obtained in the second step into a reaction kettle, heating for 45min at 95 ℃ under a stirring state, and carrying out solid-liquid separation to obtain a clear liquid.
Pumping the clear liquid obtained in the step three into a chromatographic column loaded with ion exchange resin, wherein the sample loading amount is 1BV, the sample loading flow rate is 0.3BV, and collecting effluent, namely the nuclease P1 solution after purification.
The recovery rate of the nuclease P1 in the embodiment is 53.8%, and the specific activity is 5.3 times of that of the crude enzyme.
Comparative example 1
Purification of nuclease P1 was carried out as follows:
firstly, preparing crude nuclease P1 enzyme solution by fermenting mould, wherein the enzyme activity is 6200U/mL, and the specific activity is 912U/g.
Secondly, carrying out solid-liquid separation on the crude enzyme liquid obtained in the step one, collecting filtrate, and carrying out ultrafiltration on the filtrate by using an ultrafiltration membrane with the molecular weight of 12 KD; specifically, the method comprises the following steps:
(i) carrying out ultrafiltration on the filtrate in an ultrafiltration membrane at 33 ℃ until the retention solution is 1/4 of the volume of the filtrate, and collecting the retention solution;
(ii) (ii) continuing replenishing water into the trapped fluid to the volume of the filtrate in the step (i), performing ultrafiltration to 1/4 with the trapped fluid being the initial volume, and collecting the trapped fluid;
(iii) (iii) repeating the step (ii), ending the cycle for 4 times, and collecting the trapped fluid.
The recovery rate of the nuclease P1 in the embodiment is 94.6%, and the specific activity is 2.3 times of that of the crude enzyme.
Comparative example 2
Purification of nuclease P1 was carried out as follows:
firstly, preparing crude nuclease P1 enzyme solution by fermenting mould, wherein the enzyme activity is 6200U/mL, and the specific activity is 912U/g.
And secondly, carrying out solid-liquid separation on the crude enzyme liquid obtained in the step one, transferring the clear liquid into a reaction kettle, heating at 85 ℃ for 25min under a stirring state, and carrying out solid-liquid separation to obtain the clear liquid.
The recovery rate of the nuclease P1 in the embodiment is 92.3%, and the specific activity is 1.7 times of that of the crude enzyme.
Comparative example 3
Purification of nuclease P1 was carried out as follows:
firstly, preparing crude nuclease P1 enzyme solution by fermenting mould, wherein the enzyme activity is 6200U/mL, and the specific activity is 912U/g.
And secondly, performing solid-liquid separation on the crude enzyme liquid obtained in the step one, pumping clear liquid into a chromatographic column loaded with ion exchange resin, wherein the sample loading amount is 1BV, the sample loading flow rate is 0.24BV, and collecting effluent, namely the nuclease P1 solution obtained after purification.
The recovery rate of the nuclease P1 in the embodiment is 93.8%, and the specific activity is 1.8 times of that of the crude enzyme.
Comparative example 4
Purification of nuclease P1 was carried out as follows:
firstly, preparing crude nuclease P1 enzyme solution by fermenting mould, wherein the enzyme activity is 6200U/mL, and the specific activity is 912U/g.
Secondly, carrying out solid-liquid separation on the crude enzyme liquid obtained in the step one, collecting filtrate, and carrying out ultrafiltration on the filtrate by using an ultrafiltration membrane with the molecular weight of 12 KD; specifically, the method comprises the following steps:
(i) performing ultrafiltration on the filtrate in an ultrafiltration membrane at 33 ℃, performing ultrafiltration until the trapped fluid is 1/4 of the volume of the filtrate, and collecting the trapped fluid;
(ii) (ii) continuing replenishing water into the trapped fluid to the volume of the filtrate in the step (i), performing ultrafiltration to 1/4 with the trapped fluid being the initial volume, and collecting the trapped fluid;
(iii) (iii) repeating the step (ii), ending the cycle for 4 times, and collecting the trapped fluid.
And thirdly, transferring the trapped fluid obtained in the second step into a reaction kettle, heating for 25min at 85 ℃ under the stirring state, and carrying out solid-liquid separation to obtain clear liquid.
The recovery rate of the nuclease P1 in the embodiment is 88.2%, and the specific activity is 3.9 times of that of the crude enzyme.
Comparative example 5
Purification of nuclease P1 was carried out as follows:
firstly, preparing crude nuclease P1 enzyme solution by fermenting mould, wherein the enzyme activity is 6200U/mL, and the specific activity is 912U/g.
Secondly, carrying out solid-liquid separation on the crude enzyme liquid obtained in the step one, collecting filtrate, and carrying out ultrafiltration on the filtrate by using an ultrafiltration membrane with the molecular weight of 12 KD; specifically, the method comprises the following steps:
(i) carrying out ultrafiltration on the filtrate in an ultrafiltration membrane at 33 ℃ until the retention solution is 1/4 of the volume of the filtrate, and collecting the retention solution;
(ii) (ii) continuing replenishing water into the trapped fluid to the volume of the filtrate in the step (i), performing ultrafiltration to 1/4 with the trapped fluid being the initial volume, and collecting the trapped fluid;
(iii) (iii) repeating the step (ii), ending the cycle for 4 times, and collecting the trapped fluid.
Pumping the trapped fluid obtained in the step two into a chromatographic column loaded with ion exchange resin, wherein the sample loading amount is 1BV, the sample loading flow rate is 0.24BV, and collecting effluent, namely the nuclease P1 solution after purification.
The recovery rate of the nuclease P1 in the embodiment is 88.2%, and the specific activity is 4.2 times of that of the crude enzyme.
Comparative example 6
Purification of nuclease P1 was carried out as follows:
firstly, preparing crude nuclease P1 enzyme solution by fermenting mould, wherein the enzyme activity is 6200U/mL, and the specific activity is 912U/g.
And secondly, carrying out solid-liquid separation on the crude enzyme liquid obtained in the step one, transferring the obtained enzyme liquid into a reaction kettle, heating at 85 ℃ for 25min under a stirring state, and carrying out solid-liquid separation to obtain a clear liquid.
And thirdly, pumping the clear liquid obtained in the third step into a chromatographic column loaded with ion exchange resin, wherein the sample loading amount is 1BV, the sample loading flow rate is 0.24BV, and collecting effluent, namely the nuclease P1 solution after purification.
The recovery rate of the nuclease P1 in the embodiment is 87.5%, and the specific activity is 3.1 times of that of the crude enzyme.
The invention provides a method for multi-stage purification of nuclease P1, and a plurality of methods and ways for implementing the method, and the above description is only a preferred embodiment of the invention, and it should be noted that, for those skilled in the art, a plurality of modifications and embellishments can be made without departing from the principle of the invention, and these modifications and embellishments should also be regarded as the protection scope of the invention. All the components not specified in the present embodiment can be realized by the prior art.
Claims (3)
1. A method for multi-stage purification of nuclease P1 is characterized in that crude nuclease P1 is subjected to ultrafiltration by an ultrafiltration membrane, heat inactivation and resin impurity removal to carry out multi-stage purification;
wherein, the nuclease P1 crude enzyme solution is prepared by fermenting penicillium citrinum; wherein, said Penicillium citrinum (A) and (B)Penicillium citrinum) Has been preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 2014;
the ultrafiltration membrane is a polyether sulfone membrane, and the molecular weight of the ultrafiltration membrane is 12 kD;
wherein the heat inactivation is heating at 85 ℃ for 25 min;
wherein the resin is macroporous resin which takes polystyrene as an inert framework and takes sulfonic group as a functional group;
wherein the sampling amount of the resin for impurity removal is 1BV, and the sampling rate of the impurity removal is 0.24 BV/h.
2. The method as claimed in claim 1, wherein the nuclease activity of the nuclease P1 in the crude nuclease solution is 5000-7000U/mL.
3. The method as claimed in claim 1, wherein the specific activity of nuclease P1 in the crude nuclease solution is 800-1000U/g.
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