CN102199632A - Method for preparing pyruvic acid by converting DL-lactic acid - Google Patents
Method for preparing pyruvic acid by converting DL-lactic acid Download PDFInfo
- Publication number
- CN102199632A CN102199632A CN201110092393XA CN201110092393A CN102199632A CN 102199632 A CN102199632 A CN 102199632A CN 201110092393X A CN201110092393X A CN 201110092393XA CN 201110092393 A CN201110092393 A CN 201110092393A CN 102199632 A CN102199632 A CN 102199632A
- Authority
- CN
- China
- Prior art keywords
- lactic acid
- sodium
- pyruvic acid
- pseudomonas fluorescens
- hydroxypropionate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of pyruvic acid preparation method, and relates to a method for preparing pyruvic acid through a process of converting DL-lactic acid by using lactic acid oxidase (LOD) and catalase (CAT) in Pseudomonas fluorescens. The invention provides a method for preparing pyruvic acid through a process of converting DL-lactic acid by using lactic acid oxidase (LOD) and catalase (CAT) in microbe bacterial strain (ATCC 948)-Pseudomonas fluorescens crude enzymes liquor. According to the invention, the conversion rate of the products can be raised and the products stability can be maintained.
Description
Technical field
The invention belongs to the preparation method's of pyruvic acid technical field, relate to a kind of be used to the autofluorescence pseudomonas (
Pseudomonas fluorescens) in Lactate Oxidase (LOD) and catalase (CAT) transform the method that DL-lactic acid prepares pyruvic acid.
Background technology
Pyruvic acid (Pyruvate) is a kind of important medicine, Chemicals, and it not only has a very important role in the bio-energy metabolism, and can be used as the precursor of multiple useful compound.The method of traditional chemosynthesis pyruvic acid is to be raw material with tartrate, obtain pyruvic acid by reacting down with sal enixum high temperature, also need to introduce sodium cyanide and acetyl halide synthesis of acetyl prussiate in this reaction process, the one-step hydrolysis acetyl prussiate of going forward side by side, this raw materials technology cost is higher, and productive rate is also lower.
It is also often lower that fermentation method is produced the pyruvic acid transformation efficiency, this is because pyruvic acid is in most active Centroid in carbohydrate metabolism, in cell, be easy to be converted into other compounds, therefore be difficult to accumulation, and pyruvic acid is as a kind of composition of tunning mixture, suitable lower concentration often, separating and extracting pyruvic acid from the fermented liquid of complexity generally is difficult to carry out, and expense is also expensive.
The Production by Enzymes pyruvic acid is the recent research technology that adopt more, and serum lactic dehydrogenase can make lactic acid (Lactate) step generate pyruvic acid, and this enzymic catalytic reaction is as follows:
Lactate?+?NAD+?←→Pyruvate?+?NADH?+?H
+
This reaction is reversible reaction, and molecular balance tends to lactic acid on one side; Coenzyme NAD+be micromolecular compound is difficult to fixingly, be difficult for regeneration, and cost an arm and a leg, these effects limit the application of serum lactic dehydrogenase.
Lactate Oxidase is as a kind of flavoprotein, be as cofactor with FMN or FAD, the form of its transfer transport is different from classical electron transport chain, but directly make substrate with oxygen, FMN or FAD and zymoprotein are in conjunction with very firm, entire reaction course does not need free external source coenzyme to participate in, and this has just solved the problem of regenerating coenzyme.The catalytic biochemical reaction formula of Lactate Oxidase is in the bacterial cell:
CH
3CH
2OHCOOH(lactic acid)+O
2 
CH
3The COCOOH(pyruvic acid)+H
2O
2
The hydrogen peroxide that this reaction generates continues not have enzymic catalytic reaction with pyruvic acid and generates acetate and carbonic acid gas.
Summary of the invention
Purpose of the present invention be exactly provide at the defective of above-mentioned existence a kind of be used to the autofluorescence pseudomonas (
Pseudomonas fluorescens) in Lactate Oxidase (LOD) and catalase (CAT) transform the method that DL-lactic acid prepares pyruvic acid.
The present invention adopt from Pseudomonas fluorescens (
Pseudomonas fluorescens) in Lactate Oxidase (LOD) and catalase (CAT) transform the method that DL-lactic acid prepares pyruvic acid, utilize Lactate Oxidase (LOD) in microorganism strains (ATCC948)-Pseudomonas fluorescens crude enzyme liquid and catalase (CAT) to transform DL-lactic acid and prepare pyruvic acid.
Conversion DL-lactic acid of the present invention prepares the method for pyruvic acid, and its detailed step is:
(1) bacterial classification is selected: select for use microorganism strains (ATCC948)-Pseudomonas fluorescens (
Psendomonasfluorescens), employing is that the substratum of sole carbon source is cultivated with lactic acid, this bacterial strain has high Lactate Oxidase vigor and activity of catalase;
(2) slant culture: pseudomonas fluorescens strain is inoculated in the agarose that contains 1.5-2.0% and is added with on the solid inclined-plane minimum medium of 1.0-2.0%DL-Sodium.alpha.-hydroxypropionate, cultivated 20-30 hour for 25-35 ℃;
(3) first order seed is cultivated: with the bacterial strain of step (2) cultivation, aseptic condition encircles in 50～100mL with inoculation articulating 1～2 down and contains in the liquid-based basal culture medium (LLM) of 1.0-2.0%DL-Sodium.alpha.-hydroxypropionate, under the 25-35 ℃ of condition, on shaking table shaking culture 20-30 hour, make first order seed;
(4) enlarged culturing: with 3-5% (volume ratio) inoculum size, connect first order seed and contain among the LLM of 1.0-2.0%DL-Sodium.alpha.-hydroxypropionate, under the 25-35 ℃ of condition, on shaking table shaking culture 20-30 hour, make secondary seed in 500 mL;
(5) fermentor cultivation: with 3-5% (volume ratio) inoculum size, connecing secondary seed contains among the LLM of 0.1-0.5% glucose, 1.0-2.0%DL-Sodium.alpha.-hydroxypropionate in 2L, under the 25-35 ℃ of condition, cultivate feed supplement in 6-8 hour, feed supplement liquid is the sodium lactate solution of 15-25%, and feed supplement speed is 0.2-0.3 mL/min, cultivates to stop fermentation culture in 10-15 hour, at this moment Lactate Oxidase enzyme work reaches the highest, during detect the pyruvic acid growing amount with capillary electrophoresis (CE) method;
(6) collect thalline: the fermented liquid 6000-8000 that gets step (4)
gCentrifugal 10-15 minute, collect bacterial sediment, and wash 2～3 times with the potassium phosphate buffer of pH7.4,33mM; Thalline is dissolved in the potassium phosphate buffer of pH7.4,33mM again, makes the concentration of thalline reach every liter 100～200 gram wet cell;
(7) ultrasonic treatment cell: 5-10 minute (pulse 5 seconds, 5 seconds recurrent intervals) of ultrasonic disruption somatic cells, centrifugal then 30-60 minute (30,000-35,000
g), get supernatant and promptly get the Pseudomonas fluorescens crude enzyme liquid, 4 ℃ of storages, standby;
(8) transformation experiment: the Pseudomonas fluorescens crude enzyme liquid in the step (7) is mixed with the DL-Sodium.alpha.-hydroxypropionate, make the final concentration of DL-Sodium.alpha.-hydroxypropionate in the mixture reach 5.54-1,080mM, adding final concentration is the sodium ethylene diamine tetracetate (EDTA) of 0-1.0 mM, the crude enzyme liquid protein concentration reaches 70-300 mg/L, 37-42 ℃, under the pH7.2 condition, 150-200 rev/min vibrated 5-160 hour; The sampling in 10 minutes-15 hours of every interval detects base consumption amount and product growing amount;
(9) the sample removal zymoprotein of saltouing: adding saturation ratio in the sampling sample is the ammonium sulfate precipitation zymoprotein of 50-60%, 6000-8000
g Centrifugal 10 minutes, obtain supernatant liquor and be sample;
(10) sample detection: after treating that step (9) has been separated, get 1～5 μ L sample feeding, utilize capillary electrophoresis (CE) to measure the content of substrate lactic acid and product pyruvic acid, calculate transformation efficiency.
Liquid-based basal culture medium (LLM) prescription consists of (being weight percentage): dipotassium hydrogen phosphate 0.05%, and sodium-chlor 0.05%, sal epsom 0.05%, ferrous sulfate 0.001%, yeast extract paste 0.1%, surplus is a water.
The reaction formula that the common catalyzed conversion DL-lactic acid of Lactate Oxidase (LOD) and catalase (CAT) prepares pyruvic acid is as follows:
The present invention has following characteristics:
(1) Lactate Oxidase in the pseudomonas fluorescens strain enzyme liquid has stronger specificity to lactic acid, can not only catalysis L type lactic acid, and can act on D type lactic acid;
(2) enzymatic optimal reactive temperature is 39-40 ℃;
(3) it is unaffected substantially to place 30 min enzyme activities at 35 ℃;
In the time of (4) 50 ℃, the transformation period that enzyme is lived also surpasses 30 min;
(5) enzymatic optimal pH is pH7.2;
(6) pH from 6.0 to 8.5, the having good stability of enzyme;
(7) Michaelis constant K
mWith maximum enzyme speed of response r
MaxBe respectively 9.53 mM and 2.46 mMminmg zymoproteins, but when concentration of substrate surpassed 16.6 mM, the substrate that enzyme reaction rate occurred suppressed.
Beneficial effect of the present invention is: conversion DL-lactic acid of the present invention prepares the method for pyruvic acid, set about from Lactate Oxidase and catalase, adopt with lactic acid be the substratum of sole carbon source cultivate microorganism strains-Pseudomonas fluorescens with high Lactate Oxidase vigor and activity of catalase (
Psendomonas fluorescens), after cultivating a large amount of thalline of acquisition, its intracellular enzyme of separation and Extraction, form biological catalyst efficiently, with lactic acid is substrate, carries out bio-transformation, obtains the product pyruvic acid, and catalase can prevent that but hydrogen peroxide from continuing not have the generation of enzymic catalytic reaction with pyruvic acid, thereby has not only improved product yield but also kept product stability.
Specific as follows:
(1) Lactate Oxidase of bacterial strain crude enzyme liquid is stronger to the conversion capability of substrate DL-lactic acid, and 70 mg/L crude enzyme liquid zymoproteins, 3 h can transform the DL-lactic acid of 92% 5.54 mM.Crude enzyme liquid can not only catalysis L type lactic acid, and can act on D type lactic acid, and it is very valuable that this enzymatic conversion method at pyruvic acid is used.
(2) sodium ethylene diamine tetracetate of lower concentration (EDTA) transforms the necessary metal ion of disadvantageous other pyruvic acid metabolic enzyme catalysis to pyruvic acid in can this Pseudomonas fluorescens crude enzyme liquid of complexing, thereby the activity that suppresses these enzymes, increase product stability, improve enzymatic conversion efficient.
(3) catalase that has of enzyme liquid itself is higher, enough with the hydrogen peroxide of the oxidase catalyzed generation of decomposing lactic acid, has avoided the influence of hydrogen peroxide to the product pyruvic acid, thereby has increased the stability that generates pyruvic acid.
Description of drawings
It is the conversion process of the DL-lactic acid of 5.54 mM to initial concentration that Fig. 1 illustrates Pseudomonas fluorescens enzyme liquid zymoprotein (70 mg/L), is the change curve of substrate DL-lactic acid and product pyruvic acid;
It is the conversion process of the DL-lactic acid of 5.54 mM to initial concentration that Fig. 2 illustrates Pseudomonas fluorescens enzyme liquid zymoprotein (70 mg/L), is conversion rate curve (product pyruvic acid concentration is to the ratio of initial substrate DL-lactic acid concn);
It is the conversion process of the DL-lactic acid of 116 mM to initial concentration that Fig. 3 illustrates Pseudomonas fluorescens enzyme liquid zymoprotein (300 mg/L), is the change curve of substrate DL-lactic acid and product pyruvic acid;
It is the conversion process of the DL-lactic acid of 116 mM to initial concentration that Fig. 4 illustrates Pseudomonas fluorescens enzyme liquid zymoprotein (300 mg/L), is conversion rate curve (product pyruvic acid concentration is to the ratio of initial substrate DL-lactic acid concn);
It is the conversion process of the DL-lactic acid of 1,080 mM to initial concentration that Fig. 5 illustrates Pseudomonas fluorescens enzyme liquid zymoprotein (300 mg/L), is the change curve of substrate DL-lactic acid and product pyruvic acid;
It is the conversion process of the DL-lactic acid of 1,080 mM to initial concentration that Fig. 6 illustrates Pseudomonas fluorescens crude enzyme liquid zymoprotein (300 mg/L), is conversion rate curve (product pyruvic acid concentration is to the ratio of initial substrate DL-lactic acid concn);
Fig. 7 illustrates and does not add EDTA and add EDTA and Pseudomonas fluorescens enzyme liquid is transformed the result that influences of pyruvic acid that DL-lactic acid generates; The pyruvic acid concentration that does not add the generation of EDTA Pseudomonas fluorescens enzyme liquid conversion DL-lactic acid reduces the (solid line among the figure rapidly, (a) represent pyruvic acid concentration, (c) represent the DL-lactic acid concn), and after adding the EDTA of 1 mmol/L, the pyruvic acid that transforms the generation of DL-lactic acid does not but have the too big variation (dotted line among the figure in a long time, (b) represent pyruvic acid concentration, (d) represent the DL-lactic acid concn).This result shows that the EDTA of lower concentration can increase in the enzyme-catalyzed reaction, the stability of the pyruvic acid that enzymatic conversion lactic acid generates.
Embodiment
Below in conjunction with specific embodiment technical scheme of the present invention is described in detail.
Embodiment 1: Pseudomonas fluorescens crude enzyme liquid zymoprotein (70 mg/L) is the conversion of the DL-lactic acid of 5.54 mM to initial concentration.
(1) slant culture: pseudomonas fluorescens strain (ATCC948) is inoculated on the solid inclined-plane minimum medium that contains 2.0% agarose and be added with the 1.0%DL-Sodium.alpha.-hydroxypropionate, cultivated 22 hours for 30 ℃;
(2) first order seed is cultivated: with the bacterial strain of step (1) cultivation, aseptic condition encircles in 50～100mL with inoculation articulating 1～2 down and contains in the liquid-based basal culture medium (LLM) of 1.0%DL-Sodium.alpha.-hydroxypropionate, under 30 ℃ of conditions, shaking culture is 24 hours on shaking table, makes first order seed;
(3) enlarged culturing: with 5% (volume ratio) inoculum size, connect first order seed and contain among the LLM of 1.0%DL-Sodium.alpha.-hydroxypropionate in 500 mL, under 30 ℃ of conditions, shaking culture is 24 hours on shaking table, makes secondary seed;
(4) fermentor cultivation: with 5% (volume ratio) inoculum size, connecing secondary seed contains among the LLM of 0.2% glucose, 1.0%DL-Sodium.alpha.-hydroxypropionate in 2L, under 30 ℃ of conditions, cultivate feed supplement in 7 hours, feed supplement liquid is 20% sodium lactate solution, and feed supplement speed is 0.24 mL/min, cultivates to stop fermentation culture in 12.5 hours, at this moment Lactate Oxidase enzyme work reaches the highest, during detect the pyruvic acid growing amount with capillary electrophoresis (CE);
(5) collect thalline: get centrifugal 10 minutes of 6000 rev/mins of the fermented liquids of step (4), collect bacterial sediment, and wash 2～3 times with the potassium phosphate buffer of pH7.4,33mM; Thalline is dissolved in the potassium phosphate buffer of pH7.4,33mM again, makes the concentration of thalline reach every liter 100～200 gram wet cell;
(6) ultrasonic treatment cell: in 5 minutes (pulse 5 seconds, 5 seconds recurrent intervals) of ultrasonic disruption somatic cells, centrifugal then 30 minutes (30,000 rev/mins) are got supernatant liquor and promptly got the Pseudomonas fluorescens crude enzyme liquid, and are 4 ℃ of storages, standby;
(7) transformation experiment: the Pseudomonas fluorescens crude enzyme liquid in the step (6) is mixed with DL-Sodium.alpha.-hydroxypropionate, make that the final concentration of DL-Sodium.alpha.-hydroxypropionate reaches 5.54mM in the mixture, the crude enzyme liquid protein concentration reaches 70 mg/L, 39 ℃, under the pH7.2 condition, 200 rev/mins vibrated 5 hours; The sampling in 10～30 minutes of every interval detects base consumption amount and product growing amount;
(8) the sample removal zymoprotein of saltouing: add saturation ratio in the sampling sample and be 50% ammonium sulfate precipitation zymoprotein, 6000 rev/mins centrifugal 10 minutes, obtain supernatant liquor and be sample;
(9) sample detection: after treating that step (8) has been separated, get 1～5 μ L sample feeding, utilize capillary electrophoresis (CE) to measure the content of substrate lactic acid and product pyruvic acid, calculate transformation efficiency.
The DL-lactic acid that 70 mg/L crude enzyme liquid zymoproteins, 3 h can transform 92% 5.54 mM generates 5.10 mM pyruvic acid.
Embodiment 2: Pseudomonas fluorescens crude enzyme liquid zymoprotein (300 mg/L) is the conversion of the DL-lactic acid of 116 mM to initial concentration.
(1) slant culture: pseudomonas fluorescens strain is inoculated on the solid inclined-plane minimum medium that contains 1.5% agarose and be added with the 2.0%DL-Sodium.alpha.-hydroxypropionate, cultivated 29 hours for 30 ℃;
(2) first order seed is cultivated: with the bacterial strain of step (1) cultivation, aseptic condition encircles in 50～100mL with inoculation articulating 1～2 down and contains in the liquid-based basal culture medium (LLM) of 2.0%DL-Sodium.alpha.-hydroxypropionate, under 30 ℃ of conditions, shaking culture is 24 hours on shaking table, makes first order seed;
(3) enlarged culturing: with 3% (volume ratio) inoculum size, connect first order seed and contain among the LLM of 2.0%DL-Sodium.alpha.-hydroxypropionate in 500 mL, under 30 ℃ of conditions, shaking culture is 20 hours on shaking table, makes secondary seed;
(4) fermentor cultivation: with 5% (volume ratio) inoculum size, connecing secondary seed contains among the LLM of 0.5% glucose, 2.0%DL-Sodium.alpha.-hydroxypropionate in 2L, under 30 ℃ of conditions, cultivate feed supplement in 7 hours, feed supplement liquid is 25% sodium lactate solution, and feed supplement speed is 0.2 mL/min, cultivates to stop fermentation culture in 15 hours, at this moment Lactate Oxidase enzyme work reaches the highest, during detect the pyruvic acid growing amount with capillary electrophoresis (CE);
(5) collect thalline: get centrifugal 15 minutes of 8000 rev/mins of the fermented liquids of step (4), collect bacterial sediment, and wash 2～3 times with the potassium phosphate buffer of pH7.4,33mM; Thalline is dissolved in the potassium phosphate buffer of pH7.4,33mM again, makes the concentration of thalline reach every liter 100～200 gram wet cell;
(6) ultrasonic treatment cell: in 5 minutes (pulse 5 seconds, 5 seconds recurrent intervals) of ultrasonic disruption somatic cells, centrifugal then 60 minutes (30,000 rev/mins) are got supernatant liquor and promptly got the Pseudomonas fluorescens crude enzyme liquid, and are 4 ℃ of storages, standby;
(7) transformation experiment: the Pseudomonas fluorescens crude enzyme liquid in the step (6) is mixed with DL-Sodium.alpha.-hydroxypropionate, make that the final concentration of DL-Sodium.alpha.-hydroxypropionate reaches 116mM in the mixture, the crude enzyme liquid protein concentration reaches 300 mg/L, adding final concentration is the sodium ethylene diamine tetracetate (EDTA) of 1.0 mM, 39 ℃, under the pH7.2 condition, 200 rev/mins vibrated 75 hours; The sampling in 5～20 hours of every interval detects base consumption amount and product growing amount.
(8) the sample removal zymoprotein of saltouing: add saturation ratio in the sampling sample and be 50% ammonium sulfate precipitation zymoprotein, 6000 rev/mins centrifugal 10 minutes, obtain supernatant liquor and be sample;
(9) sample detection: after treating that step (8) has been separated, get 1～5 μ L sample feeding, utilize capillary electrophoresis (CE) to measure the content of substrate lactic acid and product pyruvic acid, calculate transformation efficiency.66% 116 mM DL-lactic acid can be converted into pyruvic acid in crude enzyme liquid 18 h of 300 mg/L.
Embodiment 3: Pseudomonas fluorescens crude enzyme liquid zymoprotein (300 mg/L) is the conversion of the DL-lactic acid of 1,080 mM to initial concentration.
(1) slant culture: pseudomonas fluorescens strain is inoculated on the solid inclined-plane minimum medium that contains 2.0% agarose and be added with the 1.0%DL-Sodium.alpha.-hydroxypropionate, cultivated 20 hours for 30 ℃;
(2) first order seed is cultivated: with the bacterial strain of step (1) cultivation, aseptic condition encircles in 50～100mL with inoculation articulating 1～2 down and contains in the liquid-based basal culture medium (LLM) of 1.0%DL-Sodium.alpha.-hydroxypropionate, under 30 ℃ of conditions, shaking culture is 24 hours on shaking table, makes first order seed;
(3) enlarged culturing: with 5% (volume ratio) inoculum size, connect first order seed and contain among the LLM of 1.0%DL-Sodium.alpha.-hydroxypropionate in 500 mL, under 30 ℃ of conditions, shaking culture is 24 hours on shaking table, makes secondary seed;
(4) fermentor cultivation: with 5% (volume ratio) inoculum size, connecing secondary seed contains among the LLM of 0.2% glucose, 1.0%DL-Sodium.alpha.-hydroxypropionate in 2L, under 30 ℃ of conditions, cultivate feed supplement in 7 hours, feed supplement liquid is 20% sodium lactate solution, and feed supplement speed is 0.3 mL/min, cultivates to stop fermentation culture in 12.5 hours, at this moment Lactate Oxidase enzyme work reaches the highest, during detect the pyruvic acid growing amount with capillary electrophoresis (CE);
(5) collect thalline: get centrifugal 10 minutes of 6000 rev/mins of the fermented liquids of step (4), collect bacterial sediment, and wash 2～3 times with the potassium phosphate buffer of pH7.4,33mM; Thalline is dissolved in the potassium phosphate buffer of pH7.4,33mM again, makes the concentration of thalline reach every liter 100～200 gram wet cell;
(6) ultrasonic treatment cell: in 5 minutes (pulse 5 seconds, 5 seconds recurrent intervals) of ultrasonic disruption somatic cells, centrifugal then 30 minutes (30,000 rev/mins) are got supernatant liquor and promptly got the Pseudomonas fluorescens crude enzyme liquid, and are 4 ℃ of storages, standby;
(7) transformation experiment: the Pseudomonas fluorescens crude enzyme liquid in the step (6) is mixed with DL-Sodium.alpha.-hydroxypropionate, make that the final concentration of DL-Sodium.alpha.-hydroxypropionate reaches 1 in the mixture, 080mM, the crude enzyme liquid protein concentration reaches 300 mg/L, adding final concentration is the sodium ethylene diamine tetracetate (EDTA) of 0.5 mM, 39 ℃, under the pH7.2 condition, 200 rev/mins vibrated 160 hours; The sampling in 15～24 hours of every interval detects base consumption amount and product growing amount.
(8) the sample removal zymoprotein of saltouing: add saturation ratio in the sampling sample and be 50% ammonium sulfate precipitation zymoprotein, 6000 rev/mins centrifugal 10 minutes, obtain supernatant liquor and be sample;
(9) sample detection: after treating that step (8) has been separated, get 1～5 μ L sample feeding, utilize capillary electrophoresis (CE) to measure the content of substrate lactic acid and product pyruvic acid, calculate transformation efficiency.41% 1,080 mM DL-lactic acid can be converted into pyruvic acid in crude enzyme liquid 114 h of 300 mg/L.
Claims (3)
- One kind be used to the autofluorescence pseudomonas ( Pseudomonas fluorescensATCC948) Lactate Oxidase in (LOD) and catalase (CAT) transform the method that DL-lactic acid prepares pyruvic acid, it is characterized in that: utilize Lactate Oxidase (LOD) and catalase (CAT) conversion DL-lactic acid in microorganism strains (ATCC948)-Pseudomonas fluorescens crude enzyme liquid to prepare pyruvic acid.
- 2. conversion DL-lactic acid as claimed in claim 1 prepares the method for pyruvic acid, and its detail operations step is:(1) bacterial classification is selected: select for use microorganism strains (ATCC948)-Pseudomonas fluorescens ( Psendomonas fluorescens), employing is that the substratum of sole carbon source is cultivated with lactic acid, this bacterial strain has high Lactate Oxidase vigor and activity of catalase;(2) slant culture: pseudomonas fluorescens strain is inoculated in the agarose that contains 1.5-2.0% and is added with on the solid inclined-plane minimum medium of 1.0-2.0%DL-Sodium.alpha.-hydroxypropionate, cultivated 20-30 hour for 25-35 ℃;(3) first order seed is cultivated: with the bacterial strain of step (2) cultivation, aseptic condition encircles in 50～100mL with inoculation articulating 1～2 down and contains in the liquid-based basal culture medium (LLM) of 1.0-2.0%DL-Sodium.alpha.-hydroxypropionate, under the 25-35 ℃ of condition, on shaking table shaking culture 20-30 hour, make first order seed;(4) enlarged culturing: with 3-5% (volume ratio) inoculum size, connect first order seed and contain among the LLM of 1.0-2.0%DL-Sodium.alpha.-hydroxypropionate, under the 25-35 ℃ of condition, on shaking table shaking culture 20-30 hour, make secondary seed in 500 mL;(5) fermentor cultivation: with 3-5% (volume ratio) inoculum size, connecing secondary seed contains among the LLM of 0.1-0.5% glucose, 1.0-2.0%DL-Sodium.alpha.-hydroxypropionate in 2L, under the 25-35 ℃ of condition, cultivate feed supplement in 6-8 hour, feed supplement liquid is the sodium lactate solution of 15-25%, and feed supplement speed is 0.2-0.3 mL/min, cultivates to stop fermentation culture in 10-15 hour, at this moment Lactate Oxidase enzyme work reaches the highest, during detect the pyruvic acid growing amount with capillary electrophoresis (CE) method;(6) collect thalline: get fermented liquid 6000-8000 rev/min of step (4) centrifugal 10-15 minute, collect bacterial sediment, and wash 2～3 times with the potassium phosphate buffer of pH7.4,33mM; Thalline is dissolved in the potassium phosphate buffer of pH7.4,33mM again, makes the concentration of thalline reach every liter 100～200 gram wet cell;(7) ultrasonic treatment cell: in 5-10 minute (pulse 5 seconds, 5 seconds recurrent intervals) of ultrasonic disruption somatic cells, centrifugal then 30-60 minute (30,000-35,000 rev/min) are got supernatant liquor and promptly got the Pseudomonas fluorescens crude enzyme liquid, and are 4 ℃ of storages, standby;(8) transformation experiment: the Pseudomonas fluorescens crude enzyme liquid in the step (7) is mixed with the DL-Sodium.alpha.-hydroxypropionate, make the final concentration of DL-Sodium.alpha.-hydroxypropionate in the mixture reach 5.54-1,080mM, adding final concentration is the sodium ethylene diamine tetracetate (EDTA) of 0-1.0 mM, the crude enzyme liquid protein concentration reaches 70-300 mg/L, 37-42 ℃, under the pH7.2 condition, 150-200 rev/min vibrated 5-160 hour; The sampling in 10 minutes-15 hours of every interval detects base consumption amount and product growing amount;(9) the sample removal zymoprotein of saltouing: adding saturation ratio in the sampling sample is the ammonium sulfate precipitation zymoprotein of 50-60%, 6000-8000 rev/min centrifugal 10 minutes, obtain supernatant liquor and be sample;(10) sample detection: after treating that step (9) has been separated, get 1～5 μ L sample feeding, utilize capillary electrophoresis (CE) to measure the content of substrate lactic acid and product pyruvic acid, calculate transformation efficiency.
- 3. conversion DL-lactic acid as claimed in claim 1 prepares the method for pyruvic acid, it is characterized in that: liquid-based basal culture medium (LLM) consists of following component by weight: dipotassium hydrogen phosphate 0.05%, sodium-chlor 0.05%, sal epsom 0.05%, ferrous sulfate 0.001%, yeast extract paste 0.1%, surplus are water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110092393 CN102199632B (en) | 2011-04-13 | 2011-04-13 | Method for preparing pyruvic acid by converting DL-lactic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110092393 CN102199632B (en) | 2011-04-13 | 2011-04-13 | Method for preparing pyruvic acid by converting DL-lactic acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102199632A true CN102199632A (en) | 2011-09-28 |
| CN102199632B CN102199632B (en) | 2013-04-10 |
Family
ID=44660616
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110092393 Expired - Fee Related CN102199632B (en) | 2011-04-13 | 2011-04-13 | Method for preparing pyruvic acid by converting DL-lactic acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102199632B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102284570A (en) * | 2011-07-14 | 2011-12-21 | 张家港市明华机械制造有限公司 | Single-oil cylinder small-circle bent pipe device |
| WO2017035253A1 (en) | 2015-08-24 | 2017-03-02 | The Regents Of The University Of California | Methods and compositions for protein purification and enzyme reaction |
| CN107735027A (en) * | 2015-06-15 | 2018-02-23 | 雅培糖尿病护理股份有限公司 | Stable lactic acid responsive enzyme, electrode and sensor and preparation and use their method |
| CN108841878A (en) * | 2018-06-19 | 2018-11-20 | 四川同晟生物医药有限公司 | A method of coexpression Pfansteihl oxidizing ferment and catalase coupling production Sodium Pyruvate |
| CN108949656A (en) * | 2018-04-19 | 2018-12-07 | 江南大学 | A kind of engineering bacteria and its application in production pyruvic acid |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1451756A (en) * | 2003-05-15 | 2003-10-29 | 山东大学 | Process for preparing pyruvic acid using lactic acid oxidase or whole cell transformed lactic acid contg. said oxidase |
-
2011
- 2011-04-13 CN CN 201110092393 patent/CN102199632B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1451756A (en) * | 2003-05-15 | 2003-10-29 | 山东大学 | Process for preparing pyruvic acid using lactic acid oxidase or whole cell transformed lactic acid contg. said oxidase |
Non-Patent Citations (2)
| Title |
|---|
| 许平等: "乳酸氧化酶转化乳酸产丙酮酸", 《应用与环境生物学报》 * |
| 谷劲松等: "乳酸氧化酶研究进展", 《中国生物工程杂志》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102284570A (en) * | 2011-07-14 | 2011-12-21 | 张家港市明华机械制造有限公司 | Single-oil cylinder small-circle bent pipe device |
| CN107735027A (en) * | 2015-06-15 | 2018-02-23 | 雅培糖尿病护理股份有限公司 | Stable lactic acid responsive enzyme, electrode and sensor and preparation and use their method |
| WO2017035253A1 (en) | 2015-08-24 | 2017-03-02 | The Regents Of The University Of California | Methods and compositions for protein purification and enzyme reaction |
| EP3341112A4 (en) * | 2015-08-24 | 2019-06-12 | The Regents of the University of California | Methods and compositions for protein purification and enzyme reaction |
| US11300567B2 (en) | 2015-08-24 | 2022-04-12 | The Regents Of The University Of California | Methods and compositions for protein purification and enzyme reaction |
| CN108949656A (en) * | 2018-04-19 | 2018-12-07 | 江南大学 | A kind of engineering bacteria and its application in production pyruvic acid |
| CN108949656B (en) * | 2018-04-19 | 2021-03-26 | 江南大学 | Engineering bacterium and application thereof in production of pyruvic acid |
| CN108841878A (en) * | 2018-06-19 | 2018-11-20 | 四川同晟生物医药有限公司 | A method of coexpression Pfansteihl oxidizing ferment and catalase coupling production Sodium Pyruvate |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102199632B (en) | 2013-04-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101643429B1 (en) | Production of butanediol by anaerobic microbial fermentation | |
| Levin et al. | Hydrogen production by Clostridium thermocellum 27405 from cellulosic biomass substrates | |
| Troshina et al. | Production of H2 by the unicellular cyanobacterium Gloeocapsa alpicola CALU 743 during fermentation | |
| CN109439701B (en) | Method for preparing ergothioneine by biosynthesis and fermentation medium | |
| Sinha et al. | Biohydrogen production from various feedstocks by Bacillus firmus NMBL-03 | |
| Madigan et al. | Growth of a photosynthetic bacterium anaerobically in darkness, supported by “oxidant-dependent” sugar fermentation | |
| CN102199632B (en) | Method for preparing pyruvic acid by converting DL-lactic acid | |
| US10378028B2 (en) | System for hydrogen production under limited aerobic conditions | |
| CN103397005B (en) | Production method of glucose oxidase | |
| CN100338221C (en) | Preparation of lactic acid from a pentose-containing substrate | |
| Dipasquale et al. | Introducing capnophilic lactic fermentation in a combined dark-photo fermentation process: a route to unparalleled H 2 yields | |
| Clark et al. | Levels of enzymes involved in the synthesis of acetate from CO2 in Clostridium thermoautotrophicum | |
| Bai et al. | Effects of substrate components on hydrogen fermentation of multiple substrates | |
| CN112063532B (en) | Geotrichum linum and application thereof in preparation of (S) -1- (2-trifluoromethylphenyl) ethanol | |
| CN102146415A (en) | Gene knockout bacterium of gluconobacter oxydans and preparation method thereof | |
| Tekucheva et al. | Combined biological hydrogen-producing systems: a review | |
| CN102586343A (en) | Biocatalysis method for preparing pyruvic acid from L-alanine | |
| CN101641434B (en) | Novel microorganism | |
| CN101497871B (en) | Alcohol fermentation anaerobic high temperature bacterium culture medium, preparation and use thereof | |
| US20130210071A1 (en) | Consolidated bioprocessing method using thermophilic microorganisms | |
| CN101054597A (en) | Method for producing cystine by microorganism transformation | |
| CN109486720B (en) | Bacillus subtilis for producing L-xylulose and application thereof | |
| CN113502306A (en) | Method for producing sclareolide by catalyzing sclareol | |
| CN1280418C (en) | Process for preparing pyruvic acid using lactic acid oxidase or whole cell transformed lactic acid contg. said oxidase | |
| CN110885774A (en) | Method for optimizing glutamic acid fermentation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130410 Termination date: 20190413 |