CN108570491B - Method for detecting stability of microbial transglutaminase - Google Patents
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Abstract
The invention discloses a method for detecting the stability of microbial transglutaminase (MTGase), which comprises the steps of adjusting the pH value of an MTGase solution to 5.0-6.0, and then carrying out cation exchange chromatography; the pH value of the elution buffer solution is 8.0-9.0, the elution method is linear elution, two isomers (MTGase I1 and MTGase I2) of the MTGase are obtained by separation through pH gradient elution of the equilibrium buffer solution and the elution buffer solution, and the stability of the MTGase can be measured according to the ratio of MTGase I2/MTGase I1. The invention also discloses a separation method of MTGase isomerides. The method is simple, rapid and high in sensitivity, provides a more convenient, rapid and reliable detection method for the production quality control link of the MTGase, and simultaneously provides technical guarantee for the control of the later application stability of the MTGase.
Description
Technical Field
The invention relates to detection of enzyme product stability, in particular to a method for detecting MTGase stability, and belongs to the field of enzymology properties and separation and purification.
Background
Glutamine transaminase (TGase for short, EC2.3.2.13) is a transferase for catalyzing acyl transfer, and can catalyze the formation of epsilon- (gamma-glutaminyl) lysine covalent bonds in and among protein molecules, thereby catalyzing the cross-linking of the protein molecules, and further changing the properties and functions of the protein. Glutamine transaminases are widely found in plants, animals and microorganisms. And microbial transglutaminase (MTGase) has the characteristics of non-calcium ion dependence, low substrate specificity, low cost and the like relative to animal and plant TGase, so that the MTGase is widely applied to various fields, such as food processing aspects of meat products, aquatic products, dairy products, vegetable proteins and the like, and mainly changes the mechanical property, the water holding capacity, the mouthfeel and other properties of food. In recent years, MTGase has been widely used in textile and biomedical fields, for example, MTGase is widely used for binding proteins or polypeptides with small molecules, polymers, chemical materials, DNA and other proteins. Many researchers have utilized the cross-linking properties of MTGase to perform site-specific modifications and the like on therapeutic drug proteins. In the textile industry, MTGase is mainly used for the treatment of wool fabrics and also used for the antique treatment of jean clothes and the like; the wool contains glutamine and lysine residues, and the addition of TGase can reduce fiber damage and fading during processing.
Because the chemical nature of MTGase is protein, the MTGase is easily influenced by various factors in the processes of production, sale and application to reduce the enzyme activity, so that the improvement of the stability of the MTGase in industry is a major problem to be solved, and strict control needs to be realized in each production link. The scholars are dedicated to screening of excellent strains, so that strains with better production stability are obtained; scholars strive to optimize the strain fermentation process, so that a stable fermentation process is obtained, and the influence of unstable factors on the stability of MTGase in the fermentation process is reduced; researchers are dedicated to the optimization of the process after fermentation, different stabilizers are added in the treatment after fermentation to protect the MTGase, and researches show that the stability of the MTGase can be improved by adding substances such as proper sugar, salt ions, amino acid and the like, so that the influence of the treatment after fermentation on the MTGase is reduced; meanwhile, researchers have also been working on the long-term stable preservation of MTGase, such as lowering the preservation temperature, adjusting the liquid MTGase product at a suitable pH, adding a protective agent to increase the stability of MTGase, and extending the shelf life (patent publication No. CN 104024406A, CN 105462950A, CN 1684593).
Although many researchers have made many studies on the stability of MTGase, there are still many problems in practical production and application, such as: (1) due to the instability of the fermentation process, the stability of MTGase obtained by fermentation of each batch has certain difference; (2) MTGase is protein, and even if the operation link is strictly controlled in the processes of fermentation post-treatment and transportation, the stability of the MTGase is still influenced by external unstable factors; (3) the pharmaceutical field has strict requirements on the purity, stability and the like of the MTGase, the fine purification process of the MTGase may influence the stability of the MTGase, and a strict and accurate stability identification method is required to screen the MTGase meeting the requirements; (4) although the MTGase powder preparation is produced on a large scale at present, the biological enzyme preparation and various industrial fields are developed to liquid dosage forms, and the stability of MTGase in a liquid state is more unstable than that in a solid state, so that the identification of the stability of the liquid MTGase becomes a more concerned problem.
It is reported in the literature that due to differences in folding and assembly of proteins, different isomers of the same protein, especially antibodies used in the biomedical field, exist, and different isomer antibody proteins have obvious property differences, so that the isomers of the protein can be separated. Through our experiments, MTGase also has isomers, and the isomers of MTGase can be separated by performing pH gradient elution by a cation exchange chromatography method. The detection of enzymatic properties shows that the MTGase I1 and the MTGase I2 have obvious differences in temperature stability, pH stability, storage stability and the like.
At present, no method is available for completely avoiding the difference of MTGase stability, so that the method for detecting the MTGase stability in a rapid, accurate and high-sensitivity mode is very important to find, and the stability of the MTGase produced in the fermentation process, the post-fermentation treatment link and the fine purification link can be detected, on one hand, the fermentation process is monitored, on the other hand, the stability of the ex-factory sample can be determined, and thus, a client can be guided to use the MTGase more well. Customers in different fields can also use the method to detect the stability of the MTGase purchased by the customers, so as to select the MTGase suitable for the customers. During the production of MTGase in a factory, only simple functional detection is carried out on the MTGase. In the prior art, a rapid, accurate and high-sensitivity MTGase detection method is not available, and the stability of MTGase can be determined.
Disclosure of Invention
The invention aims to provide a method for detecting the stability of MTGase, which is simple, quick and high in sensitivity by combining an ion exchange separation method. Therefore, the stability of the MTGase is monitored and detected in real time, and a stricter MTGase stability detection method is established.
The invention provides a method for detecting the stability of microbial transglutaminase (MTGase), which comprises the following steps:
(1) preparation of MTGase solution
Dissolving MTGase in a buffer solution, adjusting the pH value of the solution to be between 5.0 and 6.0, and centrifugally collecting to obtain MTGase supernatant;
in the invention, the buffer solution is selected from phosphate buffer solution, Tris-HCl buffer solution and acetate buffer solution; preferably, the buffer is a phosphate buffer.
In the invention, the concentration of the buffer solution is 15-50 mM; preferably, 15-30 mM; further preferably, it is 20 mM.
In the invention, the pH value of the MTGase is between 5.0 and 6.0. The pH condition of the MTGase is decisive for the determination of its stability, and is strictly controlled within the pH range of 5.0-6.0.
In the invention, the MTGase terminal enzyme activity concentration is 3.0-30U/mL; preferably, the concentration is 3.0-10U/mL; further preferably, 3.0-5.0U/mL; further preferably 3.0U/mL or 5.0U/mL.
In the invention, the centrifugation condition can be conventional centrifugation condition, including but not limited to 8000r/mim centrifugation for 10-15 min; preferably, 8000r/mim are centrifuged for 10 min.
In the invention, the temperature of the centrifugation is 4-20 ℃; preferably, it is 4 ℃.
In the present invention, the MTGase may be, but is not limited to, MTGase produced by Streptomyces mobaraensis.
In the present invention, the MTGase can be present in the form of an untreated fermentation broth, or in the form of a commercial MTGase (e.g., any Streptomyces mobaraensis-derived MTGase commercially available, or a high-purity Streptomyces mobaraensis-derived MTGase for use in the medical field after fine purification).
In the present invention, when the MTGase is present in the form of an untreated microbial fermentation broth, the fermentation broth may be centrifuged first, the supernatant collected, then dissolved in a buffer solution, the pH of the solution adjusted to 5.0-6.0, and the supernatant collected by centrifugation. Or, pre-cooled absolute ethyl alcohol is firstly used for treating fermentation liquor, after standing, the fermentation liquor is centrifuged and collected to obtain crude extract of MTGase (or, the crude extract is freeze-dried to obtain MTGase of solid preparation), then the MTGase is dissolved in buffer solution, the pH value of the solution is adjusted to be between 5.0 and 6.0, and supernatant is centrifuged and collected.
Wherein the volume ratio of the absolute ethyl alcohol to the fermentation liquor is 1: 1.
Wherein the standing time is 0.5-1.5h, preferably 1 h.
Wherein, the centrifugation condition in each step can be but is not limited to 8000r/mim centrifugation for 10-15 min; preferably, 8000r/mim are centrifuged for 10 min.
(2) Ion exchange chromatography
And (2) carrying out ion exchange chromatography on the MTGase supernatant prepared in the step (1).
In the invention, the ion exchange chromatography is cation exchange chromatography; for example, the type of cation exchange chromatography purification column used is Hitrap SP Sepharose Fast Flow (1 mL).
In the invention, the equilibrium buffer solution adopted in the ion exchange chromatography is selected from phosphate buffer solution, Tris-HCl buffer solution and acetate buffer solution; preferably, the buffer is a phosphate buffer.
In the invention, the concentration of the equilibrium buffer solution adopted in the ion exchange chromatography is 15-50 mM; the pH is 5.0-6.0.
In the invention, the elution buffer solution adopted in the ion exchange chromatography is selected from phosphate buffer solution, Tris-HCl buffer solution and acetate buffer solution; preferably, the buffer is a phosphate buffer.
In the invention, the concentration of the elution buffer solution adopted in the ion exchange chromatography is 15-50 mM; the pH is 8.0-9.0.
In the invention, the flow rates of the equilibrium buffer solution, the sample and the elution buffer solution are 0.5-2.0 mL/min; preferably, it is 1 mL/min.
In the present invention, the equilibration buffer, elution buffer and enzyme product are all sterilized by filtration through a filter membrane, for example, a 0.22 μm filter membrane.
In the invention, the separation elution method is to collect elution peaks in a linear elution mode; the volume percentage of the elution buffer solution adopted in the ion exchange chromatography is changed to 0-100 percent; that is, the linear elution refers to a uniform increase in the volume percentage of elution buffer from 0% to 100% over time.
In the present invention, the volume of the elution buffer is 20 to 70 column volumes.
In the present invention, the pH change is increased from the initial 5.0-6.0 to 8.0-9.0, and the pH condition is determined for the stability determination and is strictly controlled within the pH change range.
In the present invention, the total protein amount of the sample is 5-20mg, and the total enzyme activity is 100-300U.
(3) Determination of the ratio of MTGase I2/MTGase I1
In the cation exchange chromatography process, the pH value of the enzyme solution obtained by elution is continuously increased along with the continuous increase of the proportion of the elution buffer solution, two isomers (MTGase I1 and MTGase I2) of MTGase can be obtained by separation through pH gradient elution separation, the peak integral area is respectively calculated according to the elution peak, and the peak area ratio of the two isomers is calculated.
In the invention, the corresponding pH range of the isomer MTGase I1 is 5.0-6.5.
In the invention, the corresponding pH range of the isomer MTGase I2 is 6.6-8.0.
In the invention, the stability of the MTGase is judged according to the ratio of MTGase I2/MTGase I1. The ratio of MTGase I2/MTGase I1 is inversely related to the stability of MTGase.
For example, if the value of MTGase I2/MTGase I1 is less than 1.5, the preservation rate of MTGase is more than 80% when the MTGase is preserved for 5 days at 37 ℃ and pH of 7.0;
if the value of MTGase I2/MTGase I1 is higher than 2.0, the preservation rate of MTGase is less than 60 percent when the MTGase is preserved for 5 days at the pH of 7.0 and the temperature of 37 ℃;
if the value of MTGase I2/MTGase I1 is not less than 1.5 and not more than 2.0, the preservation rate of MTGase is not less than 60% and not more than 80% when the MTGase is preserved for 5 days at the pH of 7.0 and the temperature of 37 ℃.
In one embodiment of the present invention, the method for detecting the stability of MTGase specifically operates as follows:
(1) preparation of MTGase solution
(i) Fermenting Streptomyces mobaraensis strain, centrifuging, collecting supernatant, and adjusting pH of fermentation liquid to 5.0-6.0 with phosphate buffer solution; or
(ii) Fermenting Streptomyces mobaraensis strain, centrifuging to collect supernatant, treating with precooled absolute ethanol at a ratio of 1:1, standing for 1h, centrifuging at 8000r/mim for 15min, collecting precipitate to obtain crude extract of MTGase, dissolving with 15-50mM phosphate buffer solution with pH of 5.0-6.0, centrifuging at 8000r/mim for 15min, and collecting supernatant; or
(iii) Dissolving MTGase from commercially available Streptomyces mobaraensis with 15-50mM phosphate buffer solution with pH of 5.0-6.0, centrifuging at 8000r/mim for 15min, and collecting supernatant; or
(iv) After fine purification, the MTGase with high purity and used for the medical field and derived from the streptomyces mobaraensis is dissolved by a phosphate buffer solution with 15-50mMpH5.0-6.0, and centrifuged at 8000r/mim for 15min to collect supernatant.
(2) Cation exchange chromatography
And (2) performing cation exchange chromatography on the MTGase supernatant obtained in the step (1) at room temperature by using an AKTAavantan 25 purification system, wherein the type of the selected purification column is Hitrap SP Sepharose Fast Flow (1mL), the selected equilibration buffer solution is 20mM phosphate buffer solution with pH5.5, the selected elution buffer solution is 20mM phosphate buffer solution with pH9.0, and the equilibration buffer solution, the elution buffer solution and the sample are filtered and sterilized by using a filter membrane with the diameter of 0.22 mu m.
The separation and elution method is linear elution, 0-100% of elution buffer solution, the elution volume is 20-70 column volumes, and the flow rate is 1 mL/min. The pH change increased from the initial 5.5 to 9.0. The total protein amount of the sample loading is 5-20mg, and the total enzyme activity is 100-300U.
With the increasing of the proportion of the elution buffer solution and the increasing of the pH, two isomers (MTGase I1 and MTGase I2) of MTGase can be obtained by separation according to the change of the pH, the peak integral area is calculated according to the elution peak respectively, and the peak area ratio of the two isomers is calculated.
(3) Determination of the ratio of MTGase I2/MTGase I1
The stability of the MTGase is judged according to the proportion of MTGase I2/MTGase I1, the values of MTGase I2/MTGase I1 and the stability of the MTGase are in negative correlation, and the MTGase with required specific stability can be determined according to the actual needs and the values of MTGase I2/MTGase I1. Both the temperature stability and the pH stability of MTGase I1 were higher than MTGase I2, as shown in fig. 7 and 8. Therefore, the higher the MTGase I2 content in MTGase, the lower the MTGase stability.
The invention also provides a separation method of MTGase isomerides, which comprises the following steps:
(1) preparation of MTGase solution
Dissolving MTGase in buffer solution, adjusting the pH value of the solution to be between 5.0 and 6.0, centrifuging and collecting to obtain MTGase supernatant.
In the invention, the equilibrium buffer solution adopted in the ion exchange chromatography is selected from phosphate buffer solution, Tris-HCl buffer solution and acetate buffer solution; the concentration of the buffer solution is 15-50mM, and preferably, the buffer solution is phosphate buffer solution.
In the invention, the pH value of the MTGase is 5.0-6.0.
In the invention, preferably, the MTGase terminal enzyme activity concentration is 3.0-10U; preferably, it is about 3.0-5.0U/mL.
In the invention, the centrifugation condition can be but is not limited to 8000r/mim centrifugation for 10-15 min; preferably, 8000r/mim are centrifuged for 10 min.
In the invention, the temperature of the centrifugation is 4-20 ℃; preferably, it is 4 ℃.
In the invention, the MTGase is produced by Streptomyces mobaraensis.
In the present invention, the MTGase can be present in the form of an untreated fermentation broth, or can be a commercial MTGase (e.g., any Streptomyces mobaraensis-derived MTGase commercially available, or a highly purified Streptomyces mobaraensis-derived MTGase for use in the medical field).
In the present invention, when the MTGase is present in the form of an untreated microbial fermentation broth, the fermentation broth may be centrifuged first, the supernatant collected, then dissolved in a buffer solution, the pH of the solution adjusted to 5.0-6.0, and the supernatant collected by centrifugation. Or, pre-cooled absolute ethyl alcohol is firstly used for treating fermentation liquor, after standing, the fermentation liquor is centrifuged and collected to obtain crude extract of MTGase (or, the crude extract is freeze-dried to obtain MTGase of solid preparation), then the MTGase is dissolved in buffer solution, the pH value of the solution is adjusted to be between 5.0 and 6.0, and supernatant is centrifuged and collected. The final pH of MTGase directly affects the separation of its isomers, therefore, the pH of MTGase cannot be higher than 6.0, and the pH cannot be lower than 5 for the protection of MTGase, because the pH of MTGase is stable in the range of 5.0-9.0.
Wherein the volume ratio of the absolute ethyl alcohol to the fermentation liquor is 1: 1.
Wherein the standing time is 0.5-1.5h, preferably 1 h.
Wherein, the centrifugation condition in each step can be but is not limited to 8000r/mim centrifugation for 10-15 min; preferably, 8000r/mim are centrifuged for 10 min.
(2) Ion exchange chromatography
And (2) carrying out ion exchange chromatography on the MTGase supernatant prepared in the step (1).
In the present invention, the ion exchange chromatography is cation exchange chromatography, and the type of the cation exchange chromatography purification column can be selected as Hitrap SP Sepharose Fast Flow (1 mL).
In the invention, the equilibrium buffer solution adopted in the ion exchange chromatography is selected from phosphate, Tris-HCl buffer solution and acetate buffer solution; the concentration of the buffer solution is 15-50 mM; preferably, the buffer is a phosphate buffer, and the pH is 5.0-6.0.
In the invention, the elution buffer solution adopted in the ion exchange chromatography is selected from phosphate, Tris-HCl buffer solution and acetate buffer solution; the concentration of the buffer solution is 15-50mM, and the pH value is 8.0-9.0. The volume percentage change of the elution buffer is 0-100%.
In the invention, the flow rates of the equilibrium buffer solution, the sample and the elution buffer solution are 0.5-2.0 mL/min; preferably, it is 1 mL/min.
In the present invention, the equilibration buffer, elution buffer and enzyme product are all sterilized by filtration through a filter membrane, for example, a 0.22 μm filter membrane.
In the invention, the separation elution method is to collect elution peaks in a linear elution mode; the linear elution refers to a uniform increase in the volume percentage of elution buffer from 0% to 100% over time.
In the present invention, the volume of the elution buffer is 20 to 70 column volumes.
In the present invention, the pH change is increased from the initial 5.0-6.0 to 8.0-9.0, and the pH condition is determined for the stability determination and is strictly controlled within the pH change range.
In the present invention, the total protein amount of the sample is 5-20mg, and the total enzyme activity is 100-300U.
(3) Separation of MTGase isomers MTGase I1, MTGase I2
The pH value of the enzyme solution obtained by elution is increased along with the increasing of the proportion of the elution buffer solution, and when the pH value is in the range of 5.0-6.5, the MTGase isomer MTGase I1 is obtained by collection; the MTGase I2 was collected as an isomer of MTGase when the pH was in the range of 6.6-8.0.
The invention also provides MTGase I1 and/or MTGase I2 which are isomers of MTGase separated by the separation method of the MTGase isomers, namely MTGase Isomer 1 and/or MTGase Isomer 2, wherein the molecular weight of the MTGase Isomer 1 and/or MTGase Isomer 2 is 37.816KDa, the molecular weight of the MTGase Isomer is the same as that of MTGase, and four sequences at the N tail end are Asp-Ser-Asp-Glu. Wherein the thermal stability and the pH stability of the MTGase I1 under the same conditions are both obviously higher than that of MTGase I2.
The invention also provides application of the MTGase isomer MTGase I1 or MTGase I2 in catalyzing acyl transfer.
The MTGase stability identification method obtained by the invention is rapid, accurate and high in sensitivity. The rapid screening of the MTGase is convenient, and more standard technical guarantee is provided for the MTGase in the fields of food, medicine, textile and the like.
The invention has the beneficial effects that: (1) the MTGase isomer is discovered and provided for the first time by using an ion exchange chromatography method according to a pH gradient elution method, fills the theoretical blank in the MTGase research field, and is important for understanding the properties of MTGase; (2) the proportion of MTGase I1 and MTGase I2 is combined with the stability of MTGase to obtain the relation between the proportion of MTGase I1 and MTGase I2 and the stability of MTGase, a new idea for identifying the stability of MTGase is provided, and the blank in the stability identification technology of MTGase is filled; (3) the method is simple and convenient, has high sensitivity, and can better guide actual production and application; (4) the MTGase is widely applied to the field of medicine in recent years, the medicine field has extremely strict requirements on the quality of the MTGase, and the method has high accuracy and high sensitivity, and can strictly screen and control the MTGase used in the medicine field.
Drawings
FIG. 1MTGase ion exchange chromatography chromatogram.
FIG. 2 is an electrophoretogram showing the results of MTGase ion exchange chromatography.
FIG. 3 shows the enzyme activity preservation rate of two different batches of enzyme powder in liquid preservation at 37 ℃.
FIG. 4 shows enzyme activity preservation rates of ten different batches of enzyme powder in liquid preservation at 37 ℃.
FIG. 5 shows the separation of MTGase isomers at different starting pH.
FIG. 6 shows the separation of MTGase isomers under different buffer conditions.
FIG. 7 thermal stability results for MTGase I1 and MTGase I2.
FIG. 8MTGase I1 and MTGase I2pH stability results.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.
Example 1 separation of MTGase isomers
(1) The MTGase enzyme powder was dissolved in 20mM phosphate buffer pH5.5 at a unit enzyme activity of about 3.0U/mL and a conductance of 2.0 mS/cm.
(2) And (2) carrying out cation exchange chromatography on the MTGase obtained in the step (1) by using an AKTAavantan 25 purification system at room temperature, wherein the type of the selected purification column is Hitrap SP Sepharose Fast Flow (1mL), the selected equilibration buffer solution is 20mM phosphate buffer solution with pH5.5, the selected elution buffer solution is 20mM phosphate buffer solution with pH9.0, and the equilibration buffer solution, the elution buffer solution and the sample are filtered and sterilized by using a filter membrane with the diameter of 0.22 mu m.
(3) The separation and elution method is linear elution, 0-100% of elution buffer solution, the elution volume is 30 column volumes, and the flow rate is 1 mL/min; the pH of the eluted enzyme solution was increased from the initial pH of 5.5 to 9.0; the total enzyme activity of the sample loading was about 150U.
Along with the increasing of the proportion of the elution buffer solution, the pH value of the enzyme solution obtained by elution is increased continuously, and two isomers of MTGase can be separated according to the change of the pH value: MTGase I1 and MTGase I2. As shown in FIG. 1, as the pH increased, the protein from the column was eluted by the elution buffer, and MTG I1 and MTG I2 chromatographic peaks appeared at pH5.5-6.5 and pH6.6-8.0, respectively. The electrophoresis results of the two isomers are shown in FIG. 2, and it can be found that MTG I1 and MTG I2 both have MTGase target bands at about 38 kDa. It can be shown that under the same conditions, MTG I1 and MTG I2 have different charges, and therefore, two isomers of MTGase can be separated with increasing pH.
Example 2 storage stability of MTGase from two different production batches
(1) Selecting 2MTGase solid preparation products with different production batches, wherein the labels are 20131025 batches and 20140717 batches respectively, and the MTGase is derived from Streptomyces mobaraensis strains.
(2) Respectively dissolving 2 batches of MTGase solid preparations in 20mM phosphate buffer solution with pH7.0, fully stirring uniformly, centrifuging at 8000r/min and 4 ℃ for 10min, and collecting supernatant.
(3) Filtering with 0.22 μm sterile filter membrane, sterilizing, storing in sterile freezing tube, and storing in 37 deg.C incubator.
(4) And detecting the enzyme activity of the MTGase at the same time point on the same day of preservation, day 1, day 2, day 3, day 4, day 5 and day 6 respectively, and calculating the enzyme activity preservation rates of two batches of MTGase.
(5) As shown in FIG. 3, it can be seen that MTGase of different batches obtained by fermentation of the same strain was greatly different when it was preserved at pH7.037 ℃. 20131025 batches had significantly higher stability than 20140717 batches; under the condition of pH7.0, the preservation rate of the enzyme activity when the 20131025 batch of MTGase is preserved for 6 days is about 94 percent, while the preservation rate of the enzyme activity when the 20140717 batch of MTGase is preserved for 6 days is only 18 percent; the enzyme activity preservation rate of the MTGase of 20131025 batch is kept stable basically within 6 days, and is more than 90%, while the enzyme activity preservation rate of the MTGase of 20140717 batch is gradually reduced along with the increase of time.
EXAMPLE 3 determination of the MTGase I2/MTGase I1 ratio of two different production batches of MTGase enzyme powder
(1) The enzyme powders of the MTGase from 20131025 and 20140717 batches were dissolved in 30mM phosphate buffer pH5.5 at a unit enzyme activity of about 5.0U/mL and a conductance of 2.7 mS/cm.
(2) And (2) carrying out cation exchange chromatography on the MTGase obtained in the step (1) by using an AKTAavantan 25 purification system at room temperature, wherein the type of the selected purification column is Hitrap SP Sepharose Fast Flow (1mL), the selected equilibration buffer solution is 30mM phosphate buffer solution with pH5.5, the selected elution buffer solution is 30mM phosphate buffer solution with pH8.5, and the equilibration buffer solution, the elution buffer solution and the sample are filtered and sterilized by using a filter membrane with the diameter of 0.22 mu m.
(3) The separation and elution method is linear elution, 0-100% of elution buffer solution, the elution volume is 30 column volumes, and the flow rate is 1 mL/min; the pH of the enzyme solution obtained by elution is increased from the initial 5.5 to 8.5; the total enzyme activity of the sample loading was about 250U.
(4) With the increasing of the proportion of the elution buffer solution, the pH value of the enzyme solution obtained by elution is increased, two isomers (MTGase I1 and MTGase I2) of MTGase can be separated according to the change of the pH value, the peak integral area is calculated according to the elution peak respectively, and the ratio result of the two isomers is shown in the following table 1.
TABLE 1 ratio of MTGase I2/MTGase I1 for two different production batches of MTGase enzyme powder
EXAMPLE 4 Studies of the storage stability of Ten different production batches of MTGase
(1) Selecting 10 MTGase solid preparation products with different production batches, wherein the numbers are 140733, 140735, 140736, 140737, 140739, 140803, 140804, 140807 and 140834 respectively, and the MTGase is derived from streptomyces mobaraensis strains.
(2) Respectively dissolving 10 batches of MTGase solid preparations in 20mM phosphate buffer solution with pH7.0, fully stirring uniformly, centrifuging at 8000r/min and 4 ℃ for 10min, and collecting supernatant.
(3) Filtering with 0.22 μm sterile filter membrane, sterilizing, storing in sterile freezing tube, and storing in 37 deg.C incubator.
(4) And detecting the enzyme activity of the MTGase at the same time point on the same day of preservation, the same day of preservation 5 and the same day of preservation 30, and calculating the enzyme activity preservation rates of the MTGase in two batches.
(5) As shown in FIG. 4, it can be seen that MTGase of different batches obtained by fermentation of the same strain was greatly different when it was stored at 37 ℃. Specific results of enzyme activity are shown in Table 2, and the stability of the MTGase batches of 140733, 140803, 140804, 140833 and 140834 is obviously higher than that of the enzyme powder batches of 140735, 140736, 140737, 140739 and 140807 (P is less than 0.05). When the enzyme is stored for 5 days, the enzyme activity preservation rates of the first five batches of enzyme powder are more than 80%, and the enzyme activity preservation rates of the last five batches of enzyme powder are all lower than 60%; when the enzyme powder is preserved for 30 days, the enzyme activity preservation rates of 140733, 140803, 140804, 140833 and 140834 batches of the enzyme powder are all over 25 percent except 140804 batches, and the enzyme activity preservation rates of 140735, 140736, 140737, 140739 and 140807 batches of the enzyme powder are almost 0.
TABLE 2 preservation results of MTGase enzyme powder at 37 deg.C for ten different production batches
EXAMPLE 5 determination of MTGase I2/MTGase I1 ratios of ten different production batches of MTGase enzyme powder
(1) 140733, 140803, 140804, 140833, 140834, 140735, 140736, 140737, 140739 and 140807 ten different batches of MTGase enzyme powder are dissolved in 20mM phosphate buffer solution with pH5.3, the unit enzyme activity is about 3.0U/mL, and the electric conductance is 2.0 mS/cm.
(2) And (2) carrying out cation exchange chromatography on the MTGase obtained in the step (1) by using an AKTAavantan 25 purification system at room temperature, wherein the type of the selected purification column is Hitrap SP Sepharose Fast Flow (1mL), the selected equilibration buffer solution is 20mM phosphate buffer solution with pH5.3, the selected elution buffer solution is 20mM phosphate buffer solution with pH9.0, and the equilibration buffer solution, the elution buffer solution and the sample are filtered and sterilized by using a filter membrane with the diameter of 0.22 mu m.
(3) The separation and elution method is linear elution, 0-100% of elution buffer solution, the elution volume is 30 column volumes, and the flow rate is 1 mL/min; the pH of the eluted enzyme solution was increased from the initial pH of 5.3 to 9.0; the total enzyme activity of the sample loading was about 150U.
(4) Along with the increasing of the proportion of the elution buffer solution, the pH value of the enzyme solution obtained by elution is increased continuously, two isomers (MTGase I1 and MTGase I2) of MTGase can be obtained by separation according to the change of the pH value, the peak integral area is calculated according to the elution peak respectively, and the proportion result of the two isomers is shown in the following table 3; the ratio of MTGase I2/MTGase I1 of the first five batches of enzyme powder is between 0.9 and 1.5, and the ratio of MTGase I2/MTGase I1 of the last five batches of enzyme powder is between 2.1 and 3.1 and is more than 2.0.
Combining examples 2, 3, 4, and 5, it was found that the value of MTGase I2/MTGase I1 was less than 1.5 when the preservation rate was more than 80% at pH7.037 ℃ for 5 days, and the value of MTGase I2/MTGase I1 was more than 2.0 when the preservation rate was less than 60% at pH7.037 ℃ for 5 days.
TABLE 3 ratios of MTGase I2/MTGase I1 for ten different production batches of MTGase enzyme powder
Example 6 separation of MTGase isomers at different starting pH conditions
(1) 20140717 batches of MTGase enzyme powder were dissolved in 20mM phosphate buffer pH5.0, pH6.0, pH6.5 at a unit enzyme activity of about 5.0U/mL and a conductance of 2 mS/cm.
(2) At room temperature, the MTGase obtained in step (1) is subjected to cation exchange chromatography using AKTAavantan 25 purification system, the type of purification column used is Hitrap SP Sepharose Fast Flow (1mL), the equilibration buffer is 20mM phosphate buffer with pH5.0, pH6.0 and pH6.5, the elution buffer is 20mM phosphate buffer with pH9.0, and the equilibration buffer, the elution buffer and the sample are all sterilized by filtration with 0.22 μm filter membrane.
(3) The separation and elution method is linear elution, 0-100% of elution buffer solution, the elution volume is 30 column volumes, and the flow rate is 1 mL/min; the pH of the enzyme solution obtained by elution is increased to 9.0 from the initial pH of 5.0, 6.0 and 6.5 respectively; the total enzyme activity of the sample loading is 250U.
(4) With the increasing of the proportion of the elution buffer solution, the pH value of the enzyme solution obtained by elution is increased, two isomers (MTGase I1 and MTGase I2) of MTGase can be separated according to the change of the pH value, the peak integral area is calculated according to the elution peak respectively, and the proportion results of the two isomers are shown in the following table 4 and fig. 5. From Table 4 and FIG. 5, it was found that the MTGase isomers could not be separated at the initial pH of 6.5, because MTGase I1 was not substantially bound to the cation exchange column at pH6.5, and a small amount of MTGase I1 was not separated from MTGase I1 and MTGase I2 at the same elution conditions as pH5.0-6.0 even if bound to the column at pH6.5, because of the high initial pH, and the two peaks overlapped, and only one absorption peak was observed. The results show that the ratio of MTGase I2/MTGase I1 varies from 2.1 to 2.2 under the starting condition of pH5.0 to 6.0, and in example 3, at pH5.5, the ratio of MTGase I2/MTGase I1 of 20140717 batches of enzyme powder is 2.1 and is greater than or equal to 2.0, which is consistent with the conclusion of example 5, therefore, pH5.0 to 6.0 does not affect the judgment result of stability.
TABLE 4 ratio of MTGase I2/MTGase I1 in MTGase at different starting pH conditions
Example 7 separation of MTGase isomers at different elution volumes
(1) 20140717 batches of MTGase enzyme powder were dissolved in 20mM phosphate buffer pH5.2 at a unit enzyme activity of about 5.0U/mL and a conductance of 2 mS/cm.
(2) And (2) carrying out cation exchange chromatography on the MTGase obtained in the step (1) by using an AKTAavantan 25 purification system at room temperature, wherein the type of the selected purification column is Hitrap SP Sepharose Fast Flow (1mL), the selected equilibration buffer solution is 20mM phosphate buffer solution with pH5.2, the selected elution buffer solution is 20mM phosphate buffer solution with pH8.0, and the equilibration buffer solution, the elution buffer solution and the sample are filtered and sterilized by using a filter membrane with the diameter of 0.22 mu m.
(3) The separation and elution method is linear elution, 0-100% of elution buffer solution has the elution volumes of 20, 30, 50 and 70 column volumes respectively, and the flow rate is 1 mL/min; the pH of the eluted enzyme solution was increased from the initial pH of 5.2 to 8.0; the total enzyme activity of the sample loading is 250U.
(4) As the ratio of the elution buffer solution and the pH value are increased, two isomers of MTGase (MTGase I1 and MTGase I2) can be separated according to the change of the pH value, the peak integral area is calculated according to the elution peak respectively, and the ratio results of the two isomers are shown in the following table 5. The results show that under the condition of the elution volume of 20-70CV, the ratio of MTGase I2/MTGase I1 changes between 2.1 and 2.3, and in example 3, at pH5.5, the ratio of MTGase I2/MTGase I1 of 20140717 batches of enzyme powder is 2.1, and is more than or equal to 2.0, which is consistent with the conclusion of example 5, therefore, the judgment result that the elution volume of 20-70CV does not influence the stability is not influenced.
TABLE 5 ratio of MTGase I2/MTGase I1 in MTGase at different elution volumes
EXAMPLE 8 separation of MTGase isomers with different buffers
(1) 20131025 batches of MTGase enzyme powder were dissolved in 50mM phosphate, Tris-HCl, acetate buffer pH5.7, respectively, and the unit enzyme activity was about 5.0U/mL.
(2) And (2) carrying out cation exchange chromatography on the MTGase obtained in the step (1) by using an AKTAavantan 25 purification system at room temperature, wherein the types of the selected purification columns are Hitrap SP Sepharose Fast Flow (1mL), the types of the selected equilibrium buffer solutions are 20mM phosphate, Tris-HCl and acetate buffer solutions with pH5.7, the types of the selected elution buffer solutions are 20mM phosphate, Tris-HCl and acetate buffer solutions with pH8.5, and the equilibrium buffer solutions, the elution buffer solutions and the samples are filtered and sterilized by using 0.22 mu m filter membranes.
(3) The separation and elution method is linear elution, 0-100% of elution buffer solution, the elution volumes are respectively 30 column volumes, and the flow rate is 1 mL/min; the pH of the eluted enzyme solution was increased from the initial pH of 5.7 to 8.5; the total enzyme activity of the sample loading is 250U.
(4) With the increasing of the proportion of the elution buffer and the increasing of the pH, two isomers of MTGase (MTGase I1 and MTGase I2) can be separated according to the change of the pH, as shown in FIG. 6, the peak integral areas are respectively calculated according to the elution peaks, and the proportion results of the two isomers are shown in the following Table 6. The results show that the ratio of MTGase I2/MTGase I1 varies between 1.1 and 1.3 under different buffer conditions, and in example 3, at pH5.5, the ratio of MTGase I2/MTGase I1 of 20131025 batches of enzyme powder is 0.9 and is less than or equal to 1.5, which is consistent with the conclusion of example 5, therefore, different buffers do not influence the judgment result of stability.
TABLE 6 ratio of MTGase I2/MTGase I1 in MTGase under different buffer conditions
The present invention is not limited to the above embodiments, and variations and advantages that can be realized by those skilled in the art are included in the present invention without departing from the spirit and scope of the inventive concept, and the scope of the present invention is defined by the appended claims.
Claims (7)
1. A method for detecting the stability of MTGase, which is characterized by comprising the following steps:
(1) preparation of MTGase solution
Dissolving MTGase in a buffer solution, adjusting the pH value of the solution to 5.0-6.0, and centrifugally collecting to obtain MTGase supernatant; the MTGase is produced by Streptomyces mobaraensis;
(2) ion exchange chromatography
Performing cation exchange chromatography on the MTGase supernatant prepared in the step (1);
(3) determination of the ratio of MTGase I2/MTGase I1
In the ion exchange chromatography process, the pH of the MTGase obtained by elution is continuously increased along with the continuous increase of the proportion of an elution buffer solution, two isomers of the MTGase, namely MTGase I1 and MTGase I2, can be obtained by separation according to the change of the pH, the peak integral area is respectively calculated according to the elution peak, and the peak area ratio of the two isomers is calculated;
the corresponding pH range of the isomer MTGase I1 is 5.0-6.5; the corresponding pH range of the isomer MTGase I2 is 6.6-8.0;
the stability of the MTGase is judged according to the ratio of MTGase I2/MTGase I1, and the lower the ratio of MTGase I2/MTGase I1, the higher the stability of the MTGase.
2. The method of claim 1, wherein in step (1), the buffer is selected from the group consisting of phosphate buffer, Tris-HCl buffer, and acetate buffer.
3. The method according to claim 1, wherein in the step (1), when the MTGase is present in the form of an untreated microbial fermentation broth, the fermentation broth is centrifuged, the supernatant is collected, and then dissolved in a buffer solution, the pH of the solution is adjusted to 5.0 to 6.0, and the supernatant is collected by centrifugation; or, pre-cooled absolute ethyl alcohol is firstly used for treating fermentation liquor, after standing, the fermentation liquor is centrifuged and collected to obtain the crude extract of MTGase, then the crude extract is dissolved in buffer solution, the pH value of the solution is adjusted to be 5.0-6.0, and the supernatant is centrifuged and collected.
4. The method of claim 1, wherein in step (2), the cation exchange chromatography is performed using an equilibration buffer selected from phosphate buffer, Tris-HCl buffer, and acetate buffer, and the pH is 5.0-6.0.
5. The method of claim 1, wherein in step (2), the elution buffer used in the cation exchange chromatography is selected from the group consisting of phosphate buffer, Tris-HCl buffer, and acetate buffer, and has a pH of 8.0 to 9.0.
6. The method as claimed in claim 1, wherein in step (2), the total protein amount of the supernatant of the sample MTGase is 5-20mg and the total enzyme activity is 100-300U.
7. The method of claim 1 wherein if the value of MTGase I2/MTGase I1 is less than 1.5, the MTGase has a retention of greater than 80% when stored at 37 ℃ for 5 days at pH 7.0;
if the value of MTGase I2/MTGase I1 is higher than 2.0, the preservation rate of MTGase is less than 60 percent when the MTGase is preserved for 5 days at the temperature of 37 ℃ and the pH value of 7.0;
if the value of MTGase I2/MTGase I1 is not less than 1.5 and not more than 2.0, the preservation rate of MTGase is not less than 60% and not more than 80% when the MTGase is preserved for 5 days at the pH of 7.0 and the temperature of 37 ℃.
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| CN105754892A (en) * | 2016-02-01 | 2016-07-13 | 华东师范大学 | Separation and purification method of microbial transglutaminase |
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