CN109182287A

CN109182287A - A kind of extracting method with catalytic activity transaminase

Info

Publication number: CN109182287A
Application number: CN201810935519.7A
Authority: CN
Inventors: 叶质强; 周拥军; 冯露; 鄂松; 王心洁
Original assignee: HUBEI HONGYUAN PHARMACEUTICAL TECHNOLOGY Co Ltd
Current assignee: HUBEI HONGYUAN PHARMACEUTICAL TECHNOLOGY Co Ltd
Priority date: 2018-08-16
Filing date: 2018-08-16
Publication date: 2019-01-11

Abstract

The present invention proposes a kind of extracting method of transaminase with catalytic activity, remove smudge cells body by the somatic cells centrifugation containing transaminase, washing and adding ammonium sulfate after being crushed, obtain supernatant solution, again into supernatant solution be added ammonium sulfate staticly settled with isolated transaminase crude product, finally into transaminase crude product be added mixed solution be stirred, be centrifuged and go out to obtain finished product transaminase after supernatant；The present invention proposes the active transaminase gone out in somatic cells by ammonium sulfate precipitation precipitation and separation, the mixed solution configured by purified water, polyethylene glycol and dimethyl sulfoxide can wash off most ammonium sulfate in transaminase crude product of saltouing, the mixed solution will not dissolve transaminase simultaneously, extraction efficiency is high, and it is able to maintain the catalytic activity of transaminase, a large amount of waste water will not be generated in the process, it is environmentally friendly and low in cost suitable for large-scale production.

Description

A kind of extracting method with catalytic activity transaminase

Technical field

The invention belongs to biopharmaceutical technology more particularly to a kind of extraction sides of the transaminase with catalytic activity Method.

Background technique

Transaminase is the class of enzymes of transamination between catalytic amino acid and ketone acid, is prevalent in animal, plant tissue In microorganism, in cardiac muscle, brain, liver, Shen Deng animal tissue and mung bean sprouts, it participates in amino acid in biocatalytic reaction A series of decomposition and synthesis, the a-amino acid that it is catalyzed certain monoamino-acid are transferred on the ketone group of another 2-ketoacid, are generated corresponding Amino acid, while there is certain selectivity, the especially application in asymmetric syntheses, embody the efficient, single-minded of it and Stability, and catalytic condition is mild, more save the cost.

The number of patent application that State Intellectual Property Office announced on May 13rd, 2014 is 201410198961.8, name Claim: a method of continuously extracting glutamine transaminage from fermentation liquid, this patent proposes the bacterium containing enzyme obtained by fermentation Body, by Ceramic Composite retaining molecular weight be 6000 albumen, control 4-10 DEG C of temperature, then by solvent deposition, concentration, very Sky is dry plus auxiliary material protects the methods of enzyme activity to obtain purer transaminase, which continuously extracts glutamine from fermentation liquid The method of transaminase has the following problems: 1. membrane separation process equipment investment in actual fabrication process is larger, and ceramic membrane needs anti- Multiple cleaning activation, can generate a large amount of waste water, cause higher cost；2. using part enzyme can be made in solvent deposition and concentration process Loss of activity, treatment process is longer, and practicability is narrow, is not suitable for being prepared on a large scale.

The number of patent application that State Intellectual Property Office announced on November 23rd, 2009 is 200910154642.6, name Claim: a kind of glutamine transaminage preparation method, this patent propose to contain by the way that centrifugation after fermentation liquid break process is obtained supernatant There is transaminase, obtains crude enzyme liquid using micro-filtration membrane sterilization and removing impurity, using ultrafiltration membrane treatment, retain certain molecular weight Albumen to get transaminase product is arrived, which has the following problems: by micro-filtration and super To purify transaminase, preparation cost is higher, and equipment investment amount is big, is not suitable for equally being mass produced for filter.

Enzyme easily loses catalytic activity to external environment sensitive, so its extracting method is key, most of enzyme preparations are needed High-purity is wanted, therefore generally uses ultrafiltration or chromatography, and for catalysis enzyme, it is desirable that relative purity can be lower, general to use Salt precipitation obtains crude protein, needs to carry out chromatography or dialysis desalting later, obviously has considerable restraint for large-scale production, especially It is that wastewater flow rate that is big to the investment of desalter and generating is larger, the lower product investment of added value is just uneconomical.Therefore, this hair A kind of bright extracting method for proposing transaminase with catalytic activity so as to solve the deficiencies in the prior art place.

Summary of the invention

In view of the above-mentioned problems, the present invention proposes that ammonium sulfate precipitation precipitation and separation goes out the active transaminase in somatic cells, lead to Cross the mixed solution that purified water, polyethylene glycol and dimethyl sulfoxide configure can wash off it is most in transaminase crude product of saltouing Ammonium sulfate, while the mixed solution will not dissolve transaminase, extraction efficiency is high, and is able to maintain the catalytic activity of transaminase, process In will not generate a large amount of waste water, it is environmentally friendly and low in cost be suitable for large-scale production.

A kind of extracting method of the transaminase with catalytic activity, includes the following steps:

Step 1: passing through fermented and cultured and induces the somatic cells containing transaminase, using centrifugation, washing and broken obtains Transaminase mixed liquor；

Step 2: the ammonium sulfate that concentration is 6% -30%, control temperature are added in the transaminase mixed liquor into above-mentioned step 1 Degree is lower than 10 DEG C, then is centrifuged and removes smudge cells body, obtains supernatant solution；

Step 3: ammonium sulfate is added in the supernatant solution into above-mentioned step 2 and is staticly settled, is centrifuged, is obtained after precipitating To transaminase crude product；

Step 4: being added mixed solution in the transaminase crude product into above-mentioned step 3, be stirred, be centrifuged after stirring, Supernatant is removed after centrifugation again, carries out vacuum freeze drying preservation after obtaining finished product transaminase solid.

Further improvement lies in that: the somatic cells in the step 1 are as follows: one plant of production glutamine transaminage transgenosis bacterium Strain accesses seed culture medium with 4% inoculum concentration, and at 8 DEG C, the somatic cells obtained after 48h are cultivated under the conditions of 210r/min.

Further improvement lies in that: it is described Step 1: Step 2: the centrifugation in step 3 and step 4 be density gradient from Heart method, it is described Step 1: Step 2: step 3 and step 4 are completed at a temperature of 10 DEG C.

Further improvement lies in that: first somatic cells are put into centrifuge in the step 1, control centrifuge in from Heart acceleration is 8000G, centrifugation time 5min, then is removed supernatant, obtains bacterium mud.

Further improvement lies in that: first by one plant of production glutamine transaminage transgenic strain with 4% in the step 1 Inoculum concentration accesses seed culture medium, cultivates at 8 DEG C, under the conditions of 210r/min and obtains a kind of turning ammonia with catalytic activity for 24 hours afterwards Enzyme somatic cells, then somatic cells are put into centrifuge, controlling the centrifugal acceleration in centrifuge is 8000G, centrifugation time For 5min, then be removed supernatant, obtain bacterium mud, then be added into bacterium mud phosphate buffer solution that 2 times of volume pH values are 7.4 into Row agitator treating 1 time, then the bacterium mud after washing is imported in centrifuge and is centrifuged, controls the centrifugal acceleration in centrifuge For 8000G, the deionized water for then adding 5 times of amounts is stirred uniformly, obtains suspension, finally use suspension The high pressure of 1200psi obtains bacterial cell disruption liquid at a temperature of broken recycle 2 times lower than 10 DEG C.

Further improvement lies in that: the ammonium sulfate that concentration is 6% -30% is first added in the step 2, is stirred 20-30min, control temperature are lower than 10 DEG C, then import in centrifuge be centrifuged and remove smudge cells body, control in centrifuge Centrifugal acceleration is 8000G, obtains supernatant solution.

Further improvement lies in that: first supernatant solution is poured into container in the step 3, is slowly added to ammonium sulfate solids, It being stirred until solid slowly dissolves, the concentration for controlling ammonium sulfate in container is 50%-70%, it is then allowed to stand precipitating 2 hours, then Supernatant supernatant solution after precipitating is imported in centrifuge and is centrifuged, controlling the centrifugal acceleration in centrifuge is 12000G, Then supernatant is removed, transaminase crude product is obtained.

Further improvement lies in that: the mixed solution in the step 4 is that purified water, polyethylene glycol and dimethyl sulfoxide are pressed It is mixed according to ratio 3:4:3-2:5:3, is subsequently cooled to 10 DEG C hereinafter, manufactured mixed solution, the mixed solution pH value For 6-9.

Further improvement lies in that: mixed solution first is added into transaminase crude product in the step 4, is stirred 30min keeps temperature to be lower than 5 DEG C, then is centrifuged by centrifuge, and controlling the centrifugal acceleration in centrifuge is 12000G, Supernatant is removed after centrifugation, is obtained transaminase solid, then transaminase solid is put into vacuum freeze drier and is dried, is obtained into Finished product transaminase is finally stored under -20 DEG C of environment by product transaminase.

Further improvement lies in that: the mixed solution is reusable after concentration, precipitation and filtering.

The invention has the benefit that

1. the present invention goes out the active transaminase in somatic cells by ammonium sulfate precipitation precipitation and separation, pass through purified water, poly- second two The mixed solution that pure and mild dimethyl sulfoxide configures can wash off most ammonium sulfate in transaminase crude product of saltouing, while this is mixed Transaminase will not be dissolved by closing solution, and extraction efficiency is high, and is able to maintain the catalytic activity of transaminase, will not be generated in the process a large amount of Waste water, it is environmentally friendly and low in cost suitable for large-scale production；

2. the mixed solution in the present invention can be recycled after concentration, precipitation and filtering and be recycled, method is practical, and ring It protects economical；

3. equipment used in transaminase extractive technique of the present invention is less, while without using desalter, substantially reducing Equipment input cost, suitable for the production of different scales, versatility is high.

Detailed description of the invention

Fig. 1 is that the finished product transaminase that the embodiment of the present invention extracts quantitatively is catalyzed HPLC testing result figure；

Fig. 2 is the transaminase and standard enzyme proteins gel electrophoresis comparison diagram at extraction of the embodiment of the present invention；

Fig. 3 is that standard apotransminase is quantitatively catalyzed HPLC testing result figure.

Specific embodiment

In order to realize invention technological means, reach purpose and effect is easy to understand, below with reference to specific implementation Mode, the present invention is further explained.

Embodiment

Step 1: glutamine transaminage transgenic strain first is produced by one plant, seed culture medium is accessed with 4% inoculum concentration, 8 DEG C, it is cultivated under the conditions of 210r/min and obtains a kind of transaminase somatic cells with catalytic activity afterwards for 24 hours, then somatic cells are put Enter in centrifuge, the centrifugal acceleration controlled in centrifuge is 8000G, centrifugation time 5min, then is removed supernatant, is obtained Bacterium mud, then the phosphate buffer solution that 2 times of volume pH values are 7.4 is added into bacterium mud and is followed by stirring and washing 1 time, after then washing Bacterium mud import centrifuge in be centrifuged, control centrifuge in centrifugal acceleration be 8000G, then add 5 times amount Deionized water is stirred uniformly, obtains suspension, and suspension is finally used to the high pressure of 1200psi, is being lower than 10 DEG C of temperature Lower broken recycle 2 times of degree obtains bacterial cell disruption liquid；

Step 2: the ammonium sulfate that concentration is 20% is first added, is stirred 25min, controlled at 8 DEG C, then imports centrifugation Centrifugation is carried out in machine and removes smudge cells body, and the centrifugal acceleration controlled in centrifuge is 8000G, obtains supernatant solution；

Step 3: first pouring into the supernatant solution in above-mentioned steps two in container, be slowly added to ammonium sulfate solids, is stirred straight It is slowly dissolved to solid, the concentration for controlling ammonium sulfate in container is 60%, is then allowed to stand precipitating 2 hours, then by the supernatant after precipitating Supernatant solution is imported in centrifuge and is centrifuged, and the centrifugal acceleration controlled in centrifuge is 12000G, is then removed supernatant, is obtained To transaminase crude product；

Step 4: first by purified water, polyethylene glycol and dimethyl sulfoxide, proportionally 3:4:3 is mixed, and is subsequently cooled to 5 DEG C, modulation pH value is 7, manufactured mixed solution, then mixed solution is added into transaminase crude product, is stirred 30min, is kept Temperature is 2 DEG C, then is centrifuged by centrifuge, and controlling the centrifugal acceleration in centrifuge is 12000G, after centrifugation in removal Clearly, transaminase solid is obtained, then transaminase solid is put into vacuum freeze drier and is dried, obtains finished product transaminase, most Finished product transaminase is stored under -20 DEG C of environment afterwards.

The finished product transaminase and standard apotransminase gel electrophoresis comparison extracted to the present embodiment obtains Fig. 2, leads to It crosses finished product transaminase that embodiment is extracted and standard apotransminase carries out quantitative catalysis HPLC detection and obtains Fig. 1 and Fig. 3.

It is obtained by Fig. 1, the present embodiment extracts the product that finished product transaminase is quantitatively catalyzed HPLC: substrate=9.6:1；Pass through Fig. 3 show that standard apotransminase is quantitatively catalyzed the product of HPLC: substrate=11.85:1.

It is obtained by Fig. 1-3, the finished product transaminase and standard enzyme protein active that the present embodiment extracting method is extracted compare, low In its 10% -20%, be normal range (NR), enzymatic activity is effective.

The present invention goes out the active transaminase in somatic cells by ammonium sulfate precipitation precipitation and separation, passes through purified water, poly- second The mixed solution that two pure and mild dimethyl sulfoxides configure can wash off most ammonium sulfate in transaminase crude product of saltouing, and simultaneously should Mixed solution will not dissolve transaminase, and extraction efficiency is high, and is able to maintain the catalytic activity of transaminase, will not generate in the process a large amount of Waste water, it is environmentally friendly and low in cost be suitable for large-scale production；

Mixed solution in the present invention can be recycled after concentration, precipitation and filtering to be recycled, and method is practical, and environmental protection It is economical；

Equipment used in transaminase extractive technique of the present invention is less, while without using desalter, greatly reducing Equipment input cost, suitable for the production of different scales, versatility is high.

The basic principles, main features and advantages of the invention have been shown and described above.The technical staff of the industry should Understand, the present invention is not limited to the above embodiments, and the above embodiments and description only describe originals of the invention Reason, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes and improvements It all fall within the protetion scope of the claimed invention.The claimed scope of the invention is by appended claims and its equivalent circle It is fixed.

Claims

1. a kind of extracting method of the transaminase with catalytic activity, characterized by the following steps:

2. a kind of extracting method of transaminase with catalytic activity according to claim 1, it is characterised in that: the step Somatic cells in rapid one are as follows: one plant produces glutamine transaminage transgenic strain and accesses seed culture medium with 4% inoculum concentration, At 8 DEG C, the somatic cells obtained after 48h are cultivated under the conditions of 210r/min.

3. a kind of extracting method of transaminase with catalytic activity according to claim 1, it is characterised in that: the step Rapid one, described Step 1: Step 2: step 3 Step 2: the centrifugation in step 3 and step 4 is density-gradient centrifugation method It is completed at a temperature of 10 DEG C with step 4.

4. a kind of extracting method of transaminase with catalytic activity according to claim 1, it is characterised in that: the step First somatic cells are put into centrifuge in rapid one, the centrifugal acceleration controlled in centrifuge is 8000G, and centrifugation time is 5min, then it is removed supernatant, obtain bacterium mud.

5. a kind of extracting method of transaminase with catalytic activity according to claim 1, it is characterised in that: the step Glutamine transaminage transgenic strain first is produced by one plant in rapid one, and seed culture medium is accessed with 4% inoculum concentration, at 8 DEG C, It is cultivated under the conditions of 210r/min and obtains a kind of transaminase somatic cells with catalytic activity afterwards for 24 hours, then somatic cells are put into In centrifuge, the centrifugal acceleration controlled in centrifuge is 8000G, centrifugation time 5min, then is removed supernatant, obtains bacterium Mud, then the phosphate buffer solution that 2 times of volume pH values are 7.4 is added into bacterium mud and is followed by stirring and washing 1 time, it then will be after washing Bacterium mud is imported in centrifuge and is centrifuged, and controlling the centrifugal acceleration in centrifuge is 8000G, then adds going for 5 times of amounts Ionized water is stirred uniformly, obtains suspension, and suspension is finally used to the high pressure of 1200psi, is being lower than 10 DEG C of temperature Lower broken recycle 2 times obtains bacterial cell disruption liquid.

6. a kind of extracting method of transaminase with catalytic activity according to claim 1, it is characterised in that: the step The ammonium sulfate that concentration is 6% -30% is first added in rapid two, is stirred 20-30min, control temperature is lower than 10 DEG C, then leads Enter to carry out centrifugation in centrifuge and remove smudge cells body, the centrifugal acceleration in control centrifuge is 8000G, and it is molten to obtain supernatant Liquid.

7. a kind of extracting method of transaminase with catalytic activity according to claim 1, it is characterised in that: the step First supernatant solution is poured into container in rapid three, is slowly added to ammonium sulfate solids, be stirred until solid slowly dissolve, control In container the concentration of ammonium sulfate be 50%-70%, be then allowed to stand precipitating 2 hours, then by the supernatant supernatant solution after precipitating import from It is centrifuged in scheming, the centrifugal acceleration controlled in centrifuge is 12000G, then removes supernatant, obtains transaminase crude product.

8. a kind of extracting method of transaminase with catalytic activity according to claim 1, it is characterised in that: the step Mixed solution in rapid four is that proportionally 3:4:3-2:5:3 is mixed for purified water, polyethylene glycol and dimethyl sulfoxide, then 10 DEG C are cooled to hereinafter, manufactured mixed solution, the mixed solution pH value are 6-9.

9. a kind of extracting method of transaminase with catalytic activity according to claim 1, it is characterised in that: the step Mixed solution first is added into transaminase crude product in rapid four, is stirred 30min, keeps temperature to be lower than 5 DEG C, then pass through centrifuge It is centrifuged, the centrifugal acceleration controlled in centrifuge is 12000G, removes supernatant after centrifugation, obtains transaminase solid, then will Transaminase solid is put into vacuum freeze drier drying process, obtains finished product transaminase, finished product transaminase is finally stored in -20 Under DEG C environment.

10. a kind of extracting method of transaminase with catalytic activity according to claim 1, it is characterised in that: described Mixed solution is reusable after concentration, precipitation and filtering.