CN102199632B - Method for preparing pyruvic acid by converting DL-lactic acid - Google Patents
Method for preparing pyruvic acid by converting DL-lactic acid Download PDFInfo
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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 the method for catalase (CAT) preparing pyruvic acid by converting DL-lactic 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 as raw material take tartrate, by with sal enixum high temperature under reaction obtain pyruvic acid, also need to introduce sodium cyanide and acetyl halide synthesis of acetyl prussiate in this reaction process, the one-step hydrolysis acetyl cyanide compound 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, is difficult for regeneration, and expensive, these effects limit the lactic dehydrogenase application of enzymes.
Lactate Oxidase is as a kind of flavoprotein, 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, the external source coenzyme that whole reaction process does not need to dissociate participates in, and this has just solved the problem of regenerating coenzyme.The biochemical reaction formula of Lactate Oxidase catalysis 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 to carry out generating acetic acid and carbonic acid gas without enzymic catalytic reaction with pyruvic acid.
Summary of the invention
Purpose of the present invention be exactly provide for the defective of above-mentioned existence a kind of be used to the autofluorescence pseudomonas (
Pseudomonas fluorescens) in Lactate Oxidase (LOD) and the method for catalase (CAT) preparing pyruvic acid by converting DL-lactic acid.
The present invention adopt from Pseudomonas fluorescens (
Pseudomonas fluorescens) in Lactate Oxidase (LOD) and the method for catalase (CAT) preparing pyruvic acid by converting DL-lactic acid, utilize Lactate Oxidase (LOD) and catalase (CAT) preparing pyruvic acid by converting DL-lactic acid in microorganism strains (ATCC948)-Pseudomonas fluorescens crude enzyme liquid.
The method of preparing pyruvic acid by converting DL-lactic acid of the present invention, its detailed step is:
(1) bacterial classification is selected: select microorganism strains (ATCC948)-Pseudomonas fluorescens (
Psendomonas fluorescens), adopt the substratum take lactic acid as sole carbon source to cultivate, 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, encircle in 50～100mL and contain in the liquid-based basal culture medium (LLM) of 1.0-2.0%DL-Sodium.alpha.-hydroxypropionate with inoculation articulating 1～2 under the aseptic condition, 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 in 500 mL, under the 25-35 ℃ of condition, on shaking table shaking culture 20-30 hour, make secondary seed;
(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: ultrasonic disruption somatic cells 5-10 minute (pulse 5 seconds, 5 seconds recurrent intervals), then centrifugal 30-60 minute (30,000-35,000
g), get supernatant and namely get the Pseudomonas fluorescens crude enzyme liquid, 4 ℃ of storages, for subsequent use;
(8) transformation experiment: the Pseudomonas fluorescens crude enzyme liquid in the step (7) is mixed with DL-LACTIC ACID sodium, make the final concentration of DL-LACTIC ACID sodium 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; Every interval sampling in 10 minutes-15 hours detection substrate consumption 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 water.
The reaction formula of Lactate Oxidase (LOD) and catalase (CAT) co-catalysis preparing pyruvic acid by converting DL-lactic 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 substantially unaffected 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, Enzymic stability is good;
(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: the method for preparing pyruvic acid by converting DL-lactic acid of the present invention, set about from Lactate Oxidase and catalase, the microorganism strains-Pseudomonas fluorescens of adopting substratum take lactic acid as sole carbon source to cultivate to have 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 efficient biological catalyst, take lactic acid as substrate, carry out bio-transformation, obtain the product pyruvic acid, and catalase can prevent that but hydrogen peroxide continuation and pyruvic acid from carrying out the generation without enzymic catalytic reaction, 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 hydrogen peroxide to product acetone effect of 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 concentration);
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 concentration);
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 concentration);
Fig. 7 illustrates and does not add EDTA and add the acetone effect of acid result of EDTA to the generation of Pseudomonas fluorescens enzyme liquid conversion DL-LACTIC ACID; The pyruvic acid concentration that does not add the generation of EDTA Pseudomonas fluorescens enzyme liquid conversion DL-LACTIC ACID reduces rapidly (the solid line among the figure, (a) represent pyruvic acid concentration, (c) represent DL-LACTIC ACID concentration), and after adding the EDTA of 1 mmol/L, transform the pyruvic acid of DL-LACTIC ACID generation in a long time but without the too large variation (dotted line among the figure, (b) represent pyruvic acid concentration, (d) represent DL-LACTIC ACID concentration).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, encircle in 50～100mL and contain in the liquid-based basal culture medium (LLM) of 1.0%DL-Sodium.alpha.-hydroxypropionate with inoculation articulating 1～2 under the aseptic condition, 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, then centrifugal 30 minutes (30,000 rev/mins) are got supernatant liquor and namely got the Pseudomonas fluorescens crude enzyme liquid, and are 4 ℃ of storages, for subsequent use;
(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; Every interval sampling in 10～30 minutes detection substrate consumption 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, encircle in 50～100mL and contain in the liquid-based basal culture medium (LLM) of 2.0%DL-Sodium.alpha.-hydroxypropionate with inoculation articulating 1～2 under the aseptic condition, 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, then centrifugal 60 minutes (30,000 rev/mins) are got supernatant liquor and namely got the Pseudomonas fluorescens crude enzyme liquid, and are 4 ℃ of storages, for subsequent use;
(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; Every interval sampling in 5～20 hours detection substrate consumption 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 ACIDs 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, encircle in 50～100mL and contain in the liquid-based basal culture medium (LLM) of 1.0%DL-Sodium.alpha.-hydroxypropionate with inoculation articulating 1～2 under the aseptic condition, 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, then centrifugal 30 minutes (30,000 rev/mins) are got supernatant liquor and namely got the Pseudomonas fluorescens crude enzyme liquid, and are 4 ℃ of storages, for subsequent use;
(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; Every interval sampling in 15～24 hours detection substrate consumption 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 (1)
1. the method for a preparing pyruvic acid by converting DL-lactic acid, its detailed operation steps is:
(1) bacterial classification is selected: select Pseudomonas fluorescens (
Psendomonas fluorescens) ATCC948, adopt the substratum take lactic acid as sole carbon source to cultivate, 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, encircle in 50～100mL and contain in the liquid-based basal culture medium of 1.0-2.0%DL-Sodium.alpha.-hydroxypropionate with inoculation articulating 1～2 under the aseptic condition, under the 25-35 ℃ of condition, on shaking table shaking culture 20-30 hour, make first order seed; Described liquid-based basal culture medium consists of following component by weight: 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 water;
(4) enlarged culturing: with volume ratio 3-5% inoculum size, connect first order seed and contain in the liquid-based basal culture medium of 1.0-2.0%DL-Sodium.alpha.-hydroxypropionate in 500 mL, under the 25-35 ℃ of condition, on shaking table shaking culture 20-30 hour, make secondary seed;
(5) fermentor cultivation: with volume ratio 3-5% inoculum size, connecing secondary seed contains in the liquid-based basal culture medium 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;
(6) collect thalline: get step (4) fermented liquid 6000-8000 rev/min 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: ultrasonic disruption somatic cells 5-10 minute, pulse 5 seconds, 5 seconds recurrent intervals, then centrifugal 30-60 minute, rotating speed is 30,000-35,000 rev/min, get supernatant liquor and namely get the Pseudomonas fluorescens crude enzyme liquid, 4 ℃ of storages, for subsequent use;
(8) transformation experiment: the Pseudomonas fluorescens crude enzyme liquid in the step (7) is mixed with DL-LACTIC ACID sodium, make the final concentration of DL-LACTIC ACID sodium in the mixture reach 5.54-1,080mM, adding final concentration is the sodium ethylene diamine tetracetate 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; Every interval sampling in 10 minutes-15 hours detection substrate consumption 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 the content of Capillary Electrophoresis substrate lactic acid and product pyruvic acid, calculate transformation efficiency.
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| 谷劲松等.乳酸氧化酶研究进展.《中国生物工程杂志》.2003,第23卷(第5期),36-41. |
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