CN103436503A - Purification method of L-carnitine dehydrogenase - Google Patents
Purification method of L-carnitine dehydrogenase Download PDFInfo
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- CN103436503A CN103436503A CN2013103786666A CN201310378666A CN103436503A CN 103436503 A CN103436503 A CN 103436503A CN 2013103786666 A CN2013103786666 A CN 2013103786666A CN 201310378666 A CN201310378666 A CN 201310378666A CN 103436503 A CN103436503 A CN 103436503A
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Abstract
The invention discloses a purification method of L-carnitine dehydrogenase. The purification method comprises the following steps of: preparation of the crude enzyme liquid of L-carnitine dehydrogenase, fractional salting out by using ammonium sulfate and DEAE-cellulose ion-exchange column chromatography. After the treatment of the purification method provided by the invention, the L-carnitine dehydrogenase is purified preliminarily; the purification multiple of the L-carnitine dehydrogenase is 104.7, the enzyme activity of the L-carnitine dehydrogenase is 7.49 U/ml and the specific activity is 2.848 U/mg.
Description
Technical field
The invention belongs to biochemical field, particularly, the present invention relates to the extraction and purification method of VBT desaturase.
Background technology
VBT (L-carnitine), another name L-BETAIN, vitamins B t, levocarnitine etc., chemistry L-beta-hydroxy-gamma by name-Trimethylamine 99 butyric acid, be essential a kind of biostearin compound in human body.Since Engel in 1973 reports first case mankind's CN deficiency diseases (CD), the Physiology and biochemistry of relevant VBT, pathology and the clinical report of CD day by day increase, and become an important topic of clinical medicine and trophology.The various diseases that VBT is caused by CD as pharmacological agent has been obtained good effect, and the improvement and bring new ideas of its production method is significant for the scale operation of VBT.Produce at present in the method for VBT, enzyme transforming process has that environmental friendliness, cost are low, the Product Safety high, is more attractive method.
In enzyme transforming process, VBT desaturase (EC1.1.1.108) is a needed important enzyme, so its character using and promoting extremely important the method.Although have a small amount of document, this fermentoid is reported, in the enzyme of having reported at these, great majority all exist enzymes and live and the not high problem of specificity.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of extraction and purification method of VBT desaturase.The enzyme of VBT desaturase product prepared by the method 7.49U/ml alive, specific activity reaches 2.848U/mg.
The present invention solves the problems of the technologies described above adopted technical scheme: the extraction and purification method of VBT desaturase comprises the following steps:
(1) acquisition of VBT desaturase crude enzyme liquid:
1. strain fermentation:
The bacterial strain activation: 30 ℃ of cultivation 20-30h of L-3 strains bacterial strain access slant medium that the strain that Ochrobactrum is belonged to can be produced the VBT desaturase (EC1.1.1.108) of advantageous property are activated;
One-level is cultivated: again the bacterial strain after activation is connect to a ring to liquid nutrient medium, culture condition is: dress liquid 20mL in the 100mL Erlenmeyer flask, and 30 ℃, shaking table revolution 180rpm, cultivate 24 hours;
Secondary is cultivated: then with 1% inoculum size, bacterium liquid is joined in liquid nutrient medium, with one-level, cultivating under same culture conditions and cultivating 24h;
Three grades of cultivations, the solution culture fermentation 27h of dress 10L in the 15L fermentor tank, the condition of fermentation: 30 ℃ of leavening temperatures, stirring velocity 180rpm, pH6.5;
2. the concentration of fermented liquid
The centrifugal 30min of fermented liquid 8000rpm after fermentation ends is collected to thalline, and use 50mmol/L, the PB damping fluid washed cell of pH7.0 once; After having washed, the thalline of gained is suspended in 500mL PB damping fluid, with high pressure homogenizer, carries out broken wall, the centrifugal 30min of 8000rpm removes cell wall fragments afterwards, obtains crude enzyme liquid, preserves under 4 ℃;
(2) purifying of VBT desaturase
1. grade ammonium sulfate salting-out
Slowly add the ammonium sulfate powder in described crude enzyme liquid, make saturation ratio reach respectively 35%, 45%, 55%, 65%, constantly regulate pH makes it be stabilized in 7.0 simultaneously, saltout and spend the night, in each concentration by crude enzyme liquid with the centrifugal 30min of 8000rpm, ratio with 1:2 in precipitation adds 50mmol/L, surveying enzyme after the PB damping fluid of pH7.0 lives and protein content, the part that specific activity is higher collects the dialysis tubing of packing into, use 0.01mol/L, under the Tris-HCl damping fluid of pH8.0 and 4 ℃, dialysis, changed one time dialyzate every 12 hours, changes three times; After dialysis finishes, 8000rpm gets supernatant with the purifying for next step in centrifugal 30 minutes;
2. DEAE-cellulose ion exchange chromatography
DEAE-cellulose chromatography column (5.5 * 50cm) is first used 0.01mol/L, the Tris-HCl damping fluid balance of pH8.0, this ion exchange column on enzyme liquid after dialysing again, the 0.01mol/L that is 0 to 1mol/L by NaCl concentration, the Tris-HCl damping fluid of pH8.0 carries out gradient elution, flow velocity is 1mL/min, and 15min collects a pipe; Measure each tubulin content and measure and respectively manage enzyme work by Folin-phenol method; By specific activity, higher several pipes are collected and are merged.
The composition of described slant medium is: the 1.0%L-carnitine, and 1.0% yeast extract paste, 1.0% glucose, 0.2% 3 water dipotassium hydrogen phosphate, 0.05% potassium primary phosphate,, 0.05% magnesium sulfate heptahydrate, 1.0% (v/v) liquid microelement, 1.5% agar powder, pH7.0.
The composition of described liquid nutrient medium is: the 0.5%L-carnitine, and 0.1% yeast extract paste, 0.2% glucose, 0.2% 3 water dipotassium hydrogen phosphate, 0.05% potassium primary phosphate,, 0.05% magnesium sulfate heptahydrate, 1.0% (v/v) liquid microelement, 1.5% agar powder, pH6.5.
The present invention compared with prior art, has following advantage:
(1) VBT desaturase of the present invention purification after oversalting and DEAE-Cellulose ion exchange chromatography reaches 104.7 times, enzyme 7.49U/ml alive, and specific activity reaches 2.848U/mg.
(2) technique of the present invention is simple, easy to operate, and production cost is low.
The accompanying drawing explanation
The protein content typical curve that Fig. 1 is the Folin-phenol method in embodiment 2.
Fig. 2 is that the enzyme of respectively managing in embodiment 2 is lived and protein content.
Embodiment
In order to understand better content of the present invention, below in conjunction with specific embodiment, be described further.Should be understood that these embodiment, only for the present invention is further described, limit the scope of the invention and be not used in.Should be understood that in addition after having read content of the present invention, the person skilled in art makes some nonessential change or adjustment to the present invention, still belongs to protection scope of the present invention.
Embodiment
Experimental technique
1. the mensuration of thalli growth amount
The bacterial growth amount is determined in the absorbancy at 600nm place by measuring fermented liquid, adjusts the extension rate of bacterium liquid, makes absorbancy be less than 1.000.
2.L-carnitin dehydrogenases enzyme activity determination
3.6mL after 37 ℃ of preheatings is containing the Tris-HCl(50mM of 10mM VBT and 1mM NAD; PH9.0) add the 0.4mL testing sample in damping fluid, measure at once the light absorption value at 340nm place after fully mixing, every a reading of 30 seconds records, adjust the extension rate of testing sample, the per minute absorbancy is changed between 0.060-0.100.It is the enzyme unit that lives that enzyme work is defined as enzyme amount that 37 ℃ of per minutes generate the NADH of 1 μ mol.
3 determination of protein concentration
3.1 spectrophotometry
By the absorption value at spectrophotometric determination 280nm place, and calculate according to formula 1OD280=1.67mg/mL.
3.2Folin-phenol method
Folin-phenol method reagent: 1, (1) 4% sodium carbonate (2) 0.2M sodium hydroxide (3) 1% copper sulfate (4) 2% Seignette salts.(1) and (2) equal-volume mix.(3) and (4) equal-volume mix.Then be mixed into to reagent first with 50:1 two nights.Used the same day.2,1M Folin-phenol reagent second.
Add 0,0.20,0.40,0.60,0.80 in test tube, 1.00mL bovine serum albumin (500 μ g/mL), add the not enough 1mL of water, parallelly does two parts, respectively adds according to the order of sequence 5mL reagent first, shakes up, room temperature is placed 10 minutes, then adds successively 0.5mL reagent second, shakes up, and 30 ℃ are incubated 30 minutes.Then colorimetric estimation (OD on spectrophotometer
500).X-coordinate is protein content, and ordinate zou is optical density value drawing standard curve.Testing sample as above reacts and measures, and the reference standard curve, draw protein content.
3.3Lineweaver-Burk graphing method
The 1/s reciprocal of enzymatic reaction concentration of substrate of take is X-coordinate, and the 1/v reciprocal of enzyme ' s reaction speeding is that ordinate zou is made a rectilinear, and vertical axis intercept is-1/v
max, slope is Km/v
max, the transverse axis intercept is-1/Km.
The strain first Ochrobactrum belonged to can be produced 30 ℃ of cultivation 20-30h of L-3 strains bacterial strain access slant medium of the VBT desaturase (EC1.1.1.108) of advantageous property, again the bacterial strain after activation is connect to a ring to liquid nutrient medium, culture condition is: dress liquid 20mL in the 100mL Erlenmeyer flask, 30 ℃, shaking table revolution 180rpm, cultivate 24 hours.Then with 1% inoculum size, bacterium liquid is joined respectively containing 0,0.05%, 0.1%, 0.2%, in the liquid nutrient medium of 0.5%, 1.0% yeast extract paste, under the same terms, cultivate 24h.Measure respectively the increment of thalline in each substratum.With ultrasonic cell-break crusher machine somatic cells, power 200W, work 4 seconds, stops 4 seconds, circulates 99 times.By centrifugal 30 minutes of the bacterium liquid 8000rpm after fragmentation, getting supernatant was that crude enzyme liquid is surveyed the VBT dehydrogenase activity.
Measure respectively enzyme (in Table 1) and the increment (in Table 2) alive of each substratum.
The VBT dehydrogenase activity that under the different yeast extract paste concentration of table 1, L-3 produces
The different yeast extract paste concentration of table 2 is on L-3 increment and enzyme impact alive
From table 1,2, can find out, consider the increment of thalline and the enzyme of crude enzyme liquid and live, the substratum that yeast extract paste concentration is 0.1% is the optimal medium of producing the VBT desaturase.
1. according to following one-tenth assignment system substratum, slant medium: 1.0%L-carnitine, 1.0% yeast extract paste, 1.0% glucose, 0.2% 3 water dipotassium hydrogen phosphate, 0.05% potassium primary phosphate, 0.05% magnesium sulfate heptahydrate, 1.0% (v/v) liquid microelement, 1.5% agar powder, pH7.0.
Liquid nutrient medium: the 0.5%L-carnitine, 0.1% yeast extract paste, 0.2% glucose, 0.2% 3 water dipotassium hydrogen phosphate, 0.05% potassium primary phosphate,, 0.05% magnesium sulfate heptahydrate, 1.0% (v/v) liquid microelement, 1.5% agar powder, pH6.5.
2. bacterial strain activation: 30 ℃ of cultivation 20-30h of L-3 strains bacterial strain access slant medium that the strain that Ochrobactrum is belonged to can be produced the VBT desaturase (EC1.1.1.108) of advantageous property are activated;
3. one-level is cultivated: again the bacterial strain after activation is connect to a ring to liquid nutrient medium, culture condition is: dress liquid 20mL in the 100mL Erlenmeyer flask, and 30 ℃, shaking table revolution 180rpm, cultivate 24 hours;
4. secondary is cultivated: then with 1% inoculum size, bacterium liquid is joined in liquid nutrient medium, with described one-level, cultivating under identical culture condition and cultivating 24h;
5. three grades of cultivations, the solution culture fermentation 27h of dress 10L in the fermentor tank of 15L, the condition of fermentation: 30 ℃ of leavening temperatures, stirring velocity 180rpm, pH6.5;
6. the concentration of fermented liquid: the centrifugal 30min of fermented liquid 8000rpm after fermentation ends is collected to obtain to the 74.24g thalline, by 1:2(w/v) add 50mmol/L, the PB damping fluid of pH7.0, carry out broken wall with high pressure homogenizer, the centrifugal 30min of 8000rpm removes cell wall fragments afterwards, obtains crude enzyme liquid 670mL.Measure the crude enzyme liquid enzyme and live, and use the spectrophotometry protein content.
The purifying of embodiment 3L-carnitin dehydrogenases
1. grade ammonium sulfate salting-out
Slowly add the ammonium sulfate powder in the crude enzyme liquid of embodiment 2 preparations, make saturation ratio reach respectively 35%, 45%, 55%, 65%, constantly regulate pH makes it be stabilized in 7.0 simultaneously, saltout and spend the night, in each concentration by crude enzyme liquid with the centrifugal 30min of 8000rpm, ratio with 1:2 in precipitation adds 50mmol/L, surveys enzyme after the PB damping fluid of pH7.0 and lives and protein content.The results are shown in Table 3:
Table 3 grade ammonium sulfate salting-out result
As can be seen from Table 3, VBT desaturase sedimentation effect between ammonium sulfate saturation ratio 35%-45% is best, and the specific activity that precipitation records after again dissolving is the highest.When being 35% and 55%, the ammonium sulfate saturation ratio can precipitate a large amount of impurity albumen.In order to extract as much as possible the VBT desaturase, again carry out salt fractionation after 35% and 55% precipitation is dissolved again, but do not obtain obvious sediment 45% the time, therefore give up this part enzyme.45% precipitation and 65% precipitation are merged, measure enzyme and live and protein concentration, total enzyme is lived as 696.51U, and albumen is 3791.74mg, specific activity 0.184U/mg.By the enzyme liquid dialysis tubing of packing into, use 0.01mol/L, dialysis under the Tris-HCl damping fluid of pH8.0 and 4 ℃, changed one time dialyzate every 12 hours, change three times.The rear 8000rpm of dialysis end gets supernatant in centrifugal 30 minutes and carries out the DEAE-cellulose ion exchange chromatography.
2. DEAE-cellulose ion exchange chromatography
DEAE-cellulose chromatography column (5.5 * 50cm) is first used 0.01mol/L, the Tris-HCl damping fluid balance of pH8.0, this ion exchange column on enzyme liquid after dialysing again, the 0.01mol/L that is 0 to 1mol/L by NaCl concentration, the Tris-HCl damping fluid of pH8.0 carries out gradient elution, flow velocity is 1mL/min, and 15min collects a pipe.Measure each tubulin content and measure and respectively manage enzyme work by Folin-phenol method.The protein content typical curve of Folin-phenol method is shown in Fig. 1.Each is managed, and enzyme is lived and protein content is shown in Fig. 2.
From No. 39 to No. 45, Guan Jun has enzyme work as can be seen from Figure 2, and their enzyme work, protein concentration are in Table 4.
Showing No. 439-45 pipe enzyme lives and the determination of protein concentration result
According to shown in table 4, by specific activity, 39,40,41 and No. 42 four higher pipes merge, the enzyme liquid enzyme obtained is lived as 7.49U/mL, protein concentration 2.63mg/mL, specific activity 2.8U/mg.
Each stage purification interpretation of result of embodiment 3L-carnitin dehydrogenases
The VBT desaturase is through grade ammonium sulfate salting-out, dialysis and the preliminary purification of DEAE-cellulose ion exchange chromatography, and analytical results is in Table 5.
Table 5L-carnitin dehydrogenases preliminary purification result
As mentioned above, just can realize preferably the present invention.
Claims (3)
1.L-the extraction and purification method of carnitin dehydrogenases, is characterized in that, comprises the following steps:
(1) acquisition of VBT desaturase crude enzyme liquid:
1. strain fermentation:
The bacterial strain activation: 30 ℃ of cultivation 20-30h of L-3 strains bacterial strain access slant medium that the strain that Ochrobactrum is belonged to can be produced the VBT desaturase (EC1.1.1.108) of advantageous property are activated;
One-level is cultivated: again the bacterial strain after activation is connect to a ring to liquid nutrient medium, culture condition is: dress liquid 20mL in the 100mL Erlenmeyer flask, and 30 ℃, shaking table revolution 180rpm, cultivate 24 hours;
Secondary is cultivated: then with 1% inoculum size, bacterium liquid is joined in liquid nutrient medium, with one-level, cultivating under same culture conditions and cultivating 24h;
Three grades of cultivations, the solution culture fermentation 27h of dress 10L in the 15L fermentor tank, the condition of fermentation: 30 ℃ of leavening temperatures, stirring velocity 180rpm, pH6.5;
2. the concentration of fermented liquid
The centrifugal 30min of fermented liquid 8000rpm after fermentation ends is collected to thalline, and use 50mmol/L, the PB damping fluid washed cell of pH7.0 once; After having washed, the thalline of gained is suspended in the 500mLPB damping fluid, with high pressure homogenizer, carries out broken wall, the centrifugal 30min of 8000rpm removes cell wall fragments afterwards, obtains crude enzyme liquid, preserves under 4 ℃;
(2) purifying of VBT desaturase
1. grade ammonium sulfate salting-out
Slowly add the ammonium sulfate powder in described crude enzyme liquid, make saturation ratio reach respectively 35%, 45%, 55%, 65%, constantly regulate pH makes it be stabilized in 7.0 simultaneously, saltout and spend the night, in each concentration by crude enzyme liquid with the centrifugal 30min of 8000rpm, ratio with 1:2 in precipitation adds 50mmol/L, surveying enzyme after the PB damping fluid of pH7.0 lives and protein content, the part that specific activity is higher collects the dialysis tubing of packing into, use 0.01mol/L, under the Tris-HCl damping fluid of pH8.0 and 4 ℃, dialysis, changed one time dialyzate every 12 hours, changes three times; After dialysis finishes, 8000rpm gets supernatant with the purifying for next step in centrifugal 30 minutes;
2. DEAE-cellulose ion exchange chromatography
DEAE-cellulose chromatography column (5.5 * 50cm) is first used 0.01mol/L, the Tris-HCl damping fluid balance of pH8.0, this ion exchange column on enzyme liquid after dialysing again, the 0.01mol/L that is 0 to 1mol/L by NaCl concentration, the Tris-HCl damping fluid of pH8.0 carries out gradient elution, flow velocity is 1mL/min, and 15min collects a pipe; Measure each tubulin content and measure and respectively manage enzyme work by Folin-phenol method; By specific activity, higher several pipes are collected and are merged.
2. the extraction and purification method of VBT desaturase according to claim 1, it is characterized in that, the composition of described slant medium is: 1.0%L-carnitine, 1.0% yeast extract paste, 1.0% glucose, 0.2% 3 water dipotassium hydrogen phosphate, 0.05% potassium primary phosphate, 0.05% magnesium sulfate heptahydrate, 1.0% (v/v) liquid microelement, 1.5% agar powder, pH7.0.
3. the extraction and purification method of VBT desaturase according to claim 1, it is characterized in that, the composition of described liquid nutrient medium is: 0.5%L-carnitine, 0.1% yeast extract paste, 0.2% glucose, 0.2% 3 water dipotassium hydrogen phosphate, 0.05% potassium primary phosphate, 0.05% magnesium sulfate heptahydrate, 1.0% (v/v) liquid microelement, 1.5% agar powder, pH6.5.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1771323A (en) * | 2003-04-07 | 2006-05-10 | 巴斯福股份公司 | L-carnitin dehydrogenases, their derivatives and method for producing substituted (s) alkanols |
| CN101514984A (en) * | 2008-02-22 | 2009-08-26 | 上海蓝怡科技有限公司 | Reagent for determining serum free carnitine |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1771323A (en) * | 2003-04-07 | 2006-05-10 | 巴斯福股份公司 | L-carnitin dehydrogenases, their derivatives and method for producing substituted (s) alkanols |
| CN101514984A (en) * | 2008-02-22 | 2009-08-26 | 上海蓝怡科技有限公司 | Reagent for determining serum free carnitine |
Non-Patent Citations (1)
| Title |
|---|
| 王磊: "Ochrobactrum L-3的L-肉碱脱氢酶研究", 《中国优秀硕士学位论文全文数据库（电子期刊） 医药卫生科技辑》, 15 January 2008 (2008-01-15), pages 28 - 39 * |
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Application publication date: 20131211 |