CN102250977A - Method for preparing L-isoleucine by using immobilized cells - Google Patents
Method for preparing L-isoleucine by using immobilized cells Download PDFInfo
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- CN102250977A CN102250977A CN2011101767657A CN201110176765A CN102250977A CN 102250977 A CN102250977 A CN 102250977A CN 2011101767657 A CN2011101767657 A CN 2011101767657A CN 201110176765 A CN201110176765 A CN 201110176765A CN 102250977 A CN102250977 A CN 102250977A
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Abstract
The invention discloses a method for preparing L-isoleucine by using immobilized cells, which comprises the following steps: 1) preparing a culture medium; 2) activating strain; 3) performing amplification culture of the strain and collecting bacterial cells; 4) immobilizing bacterial cells; 5) converting L-isoleucine; and 6) separating and purifying isoleucine. When the method for preparing L-isoleucine by using the immobilized cells, which is provided by the invention, is used, the drawbacks of a microorganism fermentation process are overcome, the process flow is simplified, the utilization rate of the substrate is improved, the conversion rate of the L-isoleucine substrate is improved, and the yield of the product is improved.
Description
Technical field
The present invention relates to the amino acid whose preparation method in a kind of cell engineering field, especially a kind of method of utilizing immobilized cell to prepare the L-Isoleucine.
Background technology
The L-Isoleucine belongs to neutral amino acids, be referred to as branched-chain amino acid with L-Xie Ansuan, L-leucine, a kind of as in the amino acid starting material medicine, be mainly used in aminoacids complex transfusion, three branched-chain amino acids transfusion amino acids oral-liquor etc., be used for the treatment of hepatopathy, hepatic coma, weak disease such as weak is one of important amino acid starting material medicine, simultaneously also be widely used in industries such as food and feed, its market outlook are very wide.At present domestic production L-Isoleucine all adopts microbe fermentation method, but ubiquity separate purify thoroughly, product yield is low and the problem of aspect such as unstable product quality.And the method that relevant immobilized cell enzyme process prepares the L-Isoleucine is not appeared in the newspapers.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method of utilizing immobilized cell to prepare the L-Isoleucine, can solve the shortcoming of microbe fermentation method, simplify technological process, improve the utilization ratio of substrate, substrate L-Isoleucine transformation efficiency height, product yield height.
For solving the problems of the technologies described above, the technical solution adopted in the present invention is: a kind of method of utilizing immobilized cell to prepare the L-Isoleucine, and this method may further comprise the steps:
1) preparation of substratum
The liquid seeds activation medium: use glucose 5g, NaCl5g, extractum carnis 5g and peptone 10g, water is settled to 1000ml, 121 ℃ down sterilization to make the pH value after 30 minutes be 6.8～7.2 liquid seeds activation medium,
Shake a bottle enlarged culturing base: use glucose 50g, (NH
4)
2SO
420g, corn steep liquor 15g, KH
2PO
41g, MgSO
40.5g, lightweight CaCO
36g, water is settled to 1000ml, 121 ℃ down sterilization to make the pH value after 30 minutes be 6.8～7.2 bottle enlarged culturing base that shakes,
Fermentation transforms nutrient solution: with glucose 120～150g, (NH
4)
2SO
420g, corn steep liquor 15～20g, KH
2PO
41g, MgSO
40.5g, lightweight CaCO
36g, water is settled to 1000ml, 112 ℃ down sterilization to make pH value after 30 minutes be 6.8～7.2 fermentation conversion nutrient solution;
2) actication of culture: will make 10 with the 5ml stroke-physiological saline solution through the brevibacterium flavum of freeze-drying preservation
8Individual/ml10
9Behind the bacteria suspension of individual/ml, insert in the liquid seeds activation medium at 30 ℃～31 ℃ and cultivate 20～24 bacterial classifications after making activation after little down;
3) collection of strain expanded culture and somatic cells: with step 2) bacterial classification inoculation that makes is to shaking in bottle enlarged culturing base, shaking bottle liquid amount of enlarged culturing base is 50～100mL/250mL triangular flask, the bacterial classification inoculation amount is 5%～10%, shaking culture is 22～24 hours in the constant-temperature shaking culture device of 150～200 rev/mins of 30 ℃～31 ℃, rotating speed, then with the centrifugal collection somatic cells of fermented liquid;
4) somatic cells immobilization: the somatic cells collected in step 3) ratio and the embedding medium in 1: 10～1: 5 mixed, splash into and make the immobilized bacterium somatocyte in the forming agent behind the curing molding;
5) conversion of L-Isoleucine: with the immobilized bacterium somatocyte that makes in step 4) dress post, the conversion nutrient solution that will the ferment gel column of flowing through carries out bio-transformation, changes into the L-Isoleucine, collects effusive conversion fluid;
6) separation and purification of L-Isoleucine: after the conversion fluid collected in the step 5) removed impurity with activated carbon decolorizing, be evaporated to 30%～40% of original volume, under 10 ℃～15 ℃, leave standstill and educated crystalline substance in 3～5 hours, promptly make L-Isoleucine crystal crude product.
Embedding medium in the step 4) is that concentration is 1～5% sodium alginate soln.
Forming agent in the step 4) is that concentration is 1～5% calcium chloride solution, and be 2～3 hours set time, and solidification value is 30 ℃～40 ℃.
It is 400～800mL/ hour that fermentation in the step 5) transforms the flow through flow velocity of gel column of nutrient solution, and the bio-transformation temperature is 30～31 ℃.
The conversion fluid of collecting in the step 5) is upper prop repeatedly, and the substrate that transforms in the nutrient solution up to fermentation is reformed completely into the L-Isoleucine.
A kind of method of utilizing immobilized cell to prepare the L-Isoleucine provided by the invention, owing to adopt the method for immobilized cell, overcome microbe fermentation method that present production L-Isoleucine all adopts exist separate purify thoroughly, product yield is low and shortcoming such as unstable product quality, can collect the preparation that conversion fluid carries out the L-Isoleucine easily, technological process is simplified, improved the utilization ratio of substrate, substrate L-Isoleucine transformation efficiency is more than 90%, the product yield is higher than 70%, and helps the automatic control in the commercial process.
Embodiment
Embodiment one
A kind of method of utilizing immobilized cell to prepare the L-Isoleucine, this method may further comprise the steps:
1) preparation of substratum
The liquid seeds activation medium: use glucose 5g, NaCl5g, extractum carnis 5g and peptone 10g, water is fixed to 1000ml, 121 ℃ down sterilization to make the pH value after 30 minutes be 6.8～7.2 liquid seeds activation medium,
Shake a bottle enlarged culturing base: use glucose 50g, (NH
4)
2SO
420g, corn steep liquor 15g, KH
2PO
41g, MgSO
40.5g, lightweight CaCO
36g, water is settled to 1000ml, 121 ℃ down sterilization to make the pH value after 30 minutes be 6.8～7.2 bottle enlarged culturing base that shakes,
Fermentation transforms nutrient solution: use glucose 120g, (NH
4)
2SO
420g, corn steep liquor 15g, KH
2PO
41g, MgSO
40.5g, lightweight CaCO
36g, water is settled to 1000ml, 112 ℃ down sterilization to make pH value after 30 minutes be 6.8～7.2 fermentation conversion nutrient solution;
2) actication of culture: will make 10 with the 5ml stroke-physiological saline solution through the brevibacterium flavum of freeze-drying preservation
8Behind the bacteria suspension of individual/ml, insert the bacterial classification after 30 ℃ cultivation made activation after 20 hours down in the liquid seeds activation medium;
3) collection of strain expanded culture and somatic cells: with step 2) bacterial classification inoculation that makes is to shaking in bottle enlarged culturing base, shaking bottle liquid amount of enlarged culturing base is the 50mL/250mL triangular flask, the bacterial classification inoculation amount is 5%, and shaking culture is 24 hours in the constant-temperature shaking culture device of 150 rev/mins of 31 ℃, rotating speed;
4) somatic cells immobilization: is that 5% sodium alginate soln mixes with the somatic cells collected in the step 3) in 1: 5 ratio and concentration, splashes into concentration and be in 5% the calcium chloride solution to be cured, and be 3 hours set time, and solidification value is 30 ℃.
5) conversion of L-Isoleucine: with the immobilized bacterium somatocyte dress post that makes in the step 4), to ferment and transform the nutrient solution gel column of flowing through and carry out bio-transformation, be converted into the L-Isoleucine, it is 400mL/ hour that fermentation transforms the flow through flow velocity of gel column of nutrient solution, the bio-transformation temperature is 30 ℃, collects effusive conversion fluid;
6) separation and purification of L-Isoleucine: the conversion fluid of collecting in the step 5) is heated to 95 ℃ kept 10 minutes, then 4500 rev/mins centrifugal 10 minutes, transfer the gac of pH to 4.5 adding 2% to keep 25 minutes for 60 ℃, carry out vacuum filtration again, be evaporated to 30% of original volume, transfer pH to 6.0, adding concentration by 1: 1 volume ratio is 95% alcohol, under 10 ℃, leave standstill and educated crystalline substance in 3 hours, vacuum filtration can obtain L-Isoleucine crystal crude product, and this crystallization can be made with extra care by recrystallization.
Can be with the conversion fluid collected in step 5) upper prop repeatedly, the substrate that transforms in the nutrient solution up to fermentation is reformed completely into the L-Isoleucine.
Embodiment two
A kind of method of utilizing immobilized cell to prepare the L-Isoleucine, this method may further comprise the steps:
1) preparation of substratum
The liquid seeds activation medium: use glucose 5g, NaCl5g, extractum carnis 5g and peptone 10g, water is fixed to 1000ml, 121 ℃ down sterilization to make the pH value after 30 minutes be 6.8～7.2 liquid seeds activation medium,
Shake a bottle enlarged culturing base: use glucose 50g, (NH
4)
2SO
420g, corn steep liquor 15g, KH
2PO
41g, MgSO
40.5g, lightweight CaCO
36g, water is settled to 1000ml, 121 ℃ down sterilization to make the pH value after 30 minutes be 6.8～7.2 bottle enlarged culturing base that shakes,
Fermentation transforms nutrient solution: use glucose 150g, (NH
4)
2SO
420g, corn steep liquor 20g, KH
2PO
41g, MgSO
40.5g, lightweight CaCO
36g, water is settled to 1000ml, 112 ℃ down sterilization to make pH value after 30 minutes be 6.8～7.2 fermentation conversion nutrient solution;
2) actication of culture: will make 10 with the 5ml stroke-physiological saline solution through the brevibacterium flavum of freeze-drying preservation
9Behind the bacteria suspension of individual/ml, insert the bacterial classification after 31 ℃ cultivation made activation after 24 hours down in the liquid seeds activation medium;
3) collection of strain expanded culture and somatic cells: with step 2) bacterial classification inoculation that makes is to shaking in bottle enlarged culturing base, shaking bottle liquid amount of enlarged culturing base is the 100mL/250mL triangular flask, the bacterial classification inoculation amount is 8%, shaking culture is 22 hours in the constant-temperature shaking culture device of 200 rev/mins of 30 ℃, rotating speed, then with the centrifugal collection somatic cells of fermented liquid;
4) somatic cells immobilization: is that 3% sodium alginate soln mixes with the somatic cells collected in the step 3) in 1: 8 ratio and concentration, splashes into concentration and be in 3% the calcium chloride solution to be cured, and be 2 hours set time, and solidification value is 40 ℃.
5) conversion of L-Isoleucine: with the immobilized bacterium somatocyte dress post that makes in the step 4), to ferment and transform the nutrient solution gel column of flowing through and carry out bio-transformation, be converted into the L-Isoleucine, it is 500mL/ hour that fermentation transforms the flow through flow velocity of gel column of nutrient solution, the bio-transformation temperature is 31 ℃, collects effusive conversion fluid;
6) separation and purification of L-Isoleucine: the conversion fluid of collecting in the step 5) is heated to 95 ℃ kept 10 minutes, then 4500 rev/mins centrifugal 10 minutes, transfer the gac of pH to 4.5 adding 2% to keep 25 minutes for 60 ℃, carry out vacuum filtration again, be evaporated to 35% of original volume, transfer pH to 6.0, adding concentration by 1: 1 volume ratio is 95% alcohol, under 15 ℃, leave standstill and educated crystalline substance in 5 hours, vacuum filtration can obtain L-Isoleucine crystal crude product, and this crystallization can be made with extra care by recrystallization.
Can be with the conversion fluid collected in step 5) upper prop repeatedly, the substrate that transforms in the nutrient solution up to fermentation is reformed completely into the L-Isoleucine.
Embodiment three
A kind of method of utilizing immobilized cell to prepare the L-Isoleucine, this method may further comprise the steps:
1) preparation of substratum:
The liquid seeds activation medium: use glucose 5g, NaCl5g, extractum carnis 5g and peptone 10g, water is settled to 1000ml, 121 ℃ down sterilization to make the pH value after 30 minutes be 6.8～7.2 liquid seeds activation medium,
Shake a bottle enlarged culturing base: use glucose 50g, (NH
4)
2SO
420g, corn steep liquor 15g, KH
2PO
41g, MgSO
40.5g, lightweight CaCO
36g, water is settled to 1000ml, 121 ℃ down sterilization to make the pH value after 30 minutes be 6.8～7.2 bottle enlarged culturing base that shakes,
Fermentation transforms nutrient solution: use glucose 140g, (NH
4)
2SO
420g, corn steep liquor 18g, KH
2PO
41g, MgSO
40.5g, lightweight CaCO
36g, water is settled to 1000ml, 112 ℃ down sterilization to make pH value after 30 minutes be 6.8～7.2 fermentation conversion nutrient solution;
2) actication of culture: will make 10 with the 5ml stroke-physiological saline solution through the brevibacterium flavum of freeze-drying preservation
8Behind the bacteria suspension of individual/ml, insert the bacterial classification after 31 ℃ cultivation made activation after 23 hours down in the liquid seeds activation medium;
3) collection of strain expanded culture and somatic cells: with step 2) bacterial classification inoculation that makes is to shaking in bottle enlarged culturing base, shaking bottle liquid amount of enlarged culturing base is the 80mL/250mL triangular flask, the bacterial classification inoculation amount is 10%, shaking culture is 23 hours in the constant-temperature shaking culture device of 175 rev/mins of 30 ℃, rotating speed, then with the centrifugal collection somatic cells of fermented liquid;
4) somatic cells immobilization: is that 1% sodium alginate soln mixes with the somatic cells collected in the step 3) in 1: 10 ratio and concentration, splashes into concentration and be in 1% the calcium chloride solution to be cured, and be 2.5 hours set time, and solidification value is 35 ℃.
5) conversion of L-Isoleucine: with the immobilized bacterium somatocyte dress post that makes in the step 4), to ferment and transform the nutrient solution gel column of flowing through and carry out bio-transformation, be converted into the L-Isoleucine, it is 800mL/ hour that fermentation transforms the flow through flow velocity of gel column of nutrient solution, the bio-transformation temperature is 30 ℃, collects effusive conversion fluid;
6) separation and purification of L-Isoleucine: the conversion fluid of collecting in the step 5) is heated to 95 ℃ kept 10 minutes, then 4500 rev/mins centrifugal 10 minutes, transfer the gac of pH to 4.5 adding 2% to keep 25 minutes for 60 ℃, carry out vacuum filtration again, be evaporated to 40% of original volume, transfer pH to 6.0, adding concentration by 1: 1 volume ratio is 95% alcohol, under 12 ℃, leave standstill and educated crystalline substance in 4 hours, vacuum filtration can obtain L-Isoleucine crystal crude product, and this crystallization can be made with extra care by recrystallization.
Can be with the conversion fluid collected in step 5) upper prop repeatedly, the substrate that transforms in the nutrient solution up to fermentation is reformed completely into the L-Isoleucine.
Claims (5)
1. method of utilizing immobilized cell to prepare the L-Isoleucine is characterized in that this method may further comprise the steps:
1) preparation of substratum
The liquid seeds activation medium: use glucose 5g, NaCl5g, extractum carnis 5g and peptone 10g, water is settled to 1000ml, 121 ℃ down sterilization to make the pH value after 30 minutes be 6.8～7.2 liquid seeds activation medium,
Shake a bottle enlarged culturing base: use glucose 50g, (NH
4)
2SO
420g, corn steep liquor 15g, KH
2PO
41g, MgSO
40.5g, lightweight CaCO
36g, water is settled to 1000ml, 121 ℃ down sterilization to make the pH value after 30 minutes be 6.8～7.2 bottle enlarged culturing base that shakes,
Fermentation transforms nutrient solution: with glucose 120～150g, (NH
4)
2SO
420g, corn steep liquor 15～20g, KH
2PO
41g, MgSO
40.5g, lightweight CaCO
36g, water is settled to 1000ml, 112 ℃ down sterilization to make pH value after 30 minutes be 6.8～7.2 fermentation conversion nutrient solution;
2) actication of culture: will make 10 with the 5ml stroke-physiological saline solution through the brevibacterium flavum of freeze-drying preservation
8Individual/ml～10
9Behind the bacteria suspension of individual/ml, insert the bacterial classification after 30 ℃～31 ℃ cultivation made activation after 20～24 hours down in the liquid seeds activation medium;
3) collection of strain expanded culture and somatic cells: with step 2) bacterial classification inoculation that makes is to shaking in bottle enlarged culturing base, shaking bottle liquid amount of enlarged culturing base is 50～100mL/250mL triangular flask, the bacterial classification inoculation amount is 5%～10%, shaking culture is 22～24 hours in the constant-temperature shaking culture device of 150～200 rev/mins of 30 ℃～31 ℃, rotating speed, then with the centrifugal collection somatic cells of fermented liquid;
4) somatic cells immobilization: the somatic cells collected in step 3) ratio and the embedding medium in 1: 10～1: 5 mixed, splash into and make the immobilized bacterium somatocyte in the forming agent behind the curing molding;
5) conversion of L-Isoleucine: with the immobilized bacterium somatocyte that makes in step 4) dress post, the conversion nutrient solution that will the ferment gel column of flowing through carries out bio-transformation, changes into the L-Isoleucine, collects effusive conversion fluid;
6) separation and purification of L-Isoleucine: after the conversion fluid collected in the step 5) removed impurity with activated carbon decolorizing, be evaporated to 30%～40% of original volume, under 10 ℃～15 ℃, leave standstill and educated crystalline substance in 3～5 hours, promptly make L-Isoleucine crystal crude product.
2. a kind of method of utilizing immobilized cell to prepare the L-Isoleucine according to claim 1 is characterized in that: the embedding medium in the step 4) is that concentration is 1～5% sodium alginate soln.
3. a kind of method of utilizing immobilized cell to prepare the L-Isoleucine according to claim 1, it is levied and is: the forming agent in the step 4) is that concentration is 1～5% calcium chloride solution, and be 2～3 hours set time, and solidification value is 30 ℃～40 ℃.
4. a kind of method of utilizing immobilized cell to prepare the L-Isoleucine according to claim 1 is characterized in that: it is 400～800mL/ hour that fermentation in the step 5) transforms the flow through flow velocity of gel column of nutrient solution, and the bio-transformation temperature is 30～31 ℃.
5. a kind of method of utilizing immobilized cell to prepare the L-Isoleucine according to claim 1, it is characterized in that: the conversion fluid of collecting in the step 5) is upper prop repeatedly, and the substrate that transforms in the nutrient solution up to fermentation is reformed completely into the L-Isoleucine.
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Cited By (2)
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| CN102505027A (en) * | 2011-12-27 | 2012-06-20 | 开原市天慕生物科技有限公司 | Isoleucine fermenting process |
| CN108004278A (en) * | 2017-11-23 | 2018-05-08 | 山东福瑞达生物科技有限公司 | A kind of method of glutamate-producing strain viable capsule fermenting and producing gamma-polyglutamic acid |
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| CN108004278A (en) * | 2017-11-23 | 2018-05-08 | 山东福瑞达生物科技有限公司 | A kind of method of glutamate-producing strain viable capsule fermenting and producing gamma-polyglutamic acid |
| CN108004278B (en) * | 2017-11-23 | 2020-06-23 | 山东福瑞达生物科技有限公司 | Method for producing gamma-polyglutamic acid by fermenting glutamic acid producing bacterium and live bacterium capsules |
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Application publication date: 20111123 |