CN101245358A - Method for producing succinic acid with biotransformation - Google Patents
Method for producing succinic acid with biotransformation Download PDFInfo
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- CN101245358A CN101245358A CNA2007100104477A CN200710010447A CN101245358A CN 101245358 A CN101245358 A CN 101245358A CN A2007100104477 A CNA2007100104477 A CN A2007100104477A CN 200710010447 A CN200710010447 A CN 200710010447A CN 101245358 A CN101245358 A CN 101245358A
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- inulin
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Abstract
The invention relates to a microbial method for producing succinic acid, which is the method for producing succinic acid by biological conversion; the inulin plants are taken as the raw materials, the inulin-rich organs of the inulin plants are inoculated with microbes after the pretreatment by sterilization for carrying out the biological conversion reaction and producing the succinic acid. The production cost is low, the sources of the raw materials are wide, the process is simple, the technical line is mature and the industrial implementation can be realized.
Description
Technical field
The present invention relates to microbial method and produce succsinic acid, it is a raw material with the inulin plant, is processed into the utilizable fermentation raw material of microorganism by means such as pre-treatment, utilizes microbial method to produce the technology of succsinic acid again.
Background technology
Succsinic acid is one of intermediate product of tricarboxylic acid cycle, also is one of metabolic end product of many anaerobions simultaneously.Simultaneously, succsinic acid is again a kind of broad-spectrum important foundation industrial chemicals.At field of food, succsinic acid can be applied to foodstuff additive and seasonings; At field of medicaments, it can be used as the auxiliary material of pharmaceutical products; At chemical field, it can be used as whipping agent, sanitising agent and ion chelating agent etc.; In petrochemical industry, be used as precursor compound and produce important chemical products such as butyleneglycol, tetrahydrofuran (THF), g-butyrolactone.At present, the succsinic acid product mainly by petroleum chemicals by chemical method production because raw materials for production are non-renewable petroleum resources, cause production cost directly to be subjected to the influence of international oil price, and cause problem of environmental pollution easily.Therefore utilize microorganism, transform carbohydrate resource abundant in the plant and produce the focus that succsinic acid becomes concern.
Occurring in nature has many microorganisms can under anaerobic utilize sugared source fermentation such as glucose, fructose, seminose to produce a spot of succsinic acid.People such as Guettler filter out the mutant strain of the high yield succsinic acid of anti-8g/L fluoro acetate from anaerobism bacterial strain Actinobacillussuccmogen, be fermenting substrate with glucose, and the concentration of the output of succsinic acid in fermentor tank reaches 110g/L.The U.S. used C.-Chem AG in 2002, by to colibacillary pyruvate formate-lyase gene (pfl), the inactivation of lactate dehydrogenase gene (ldh) and three genes of glucose phosphotransferase gene (pstG) constructs a strain engineering bacteria AFP111, this project bacterium grows in the aerobic stage for nutrition source fast by utilizing corn steeping water, enters anaerobic stage transforming glucose then and produces succsinic acid.
Yet cheap elementary biomass material is one of effective way that reduces fermentation costs.Cellulosic material is with low cost, cellulose resource is very abundant on the earth, yet the cost-effective cellulose biomass raw material that utilizes is one of international difficult problem at present, the method of generally taking is to adopt diluted acid to handle cellulosic material, and then by expressing cellulase, and can utilize the microbial catalyst of glucose and wood sugar, realize biorefinery.Though this method is effective, because the use of acid has inevitably brought environmental problem again in the Mierocrystalline cellulose treating processes.W-Gum be simple and easy to most the glucosyl group raw material, microorganism use also very easily.But, if but do not meet the national conditions of China as the raw material of biorefinery technology with W-Gum.Because China's cultivated area is limited, China produces 1.2 hundred million tons of corns per year, output is half of the not enough U.S. also, per capita less than 100 kilograms, and culture to consume and account for 70-80%, the corn that is used for industry accounts for about 10% of corn ultimate production, and in only more than 1,000 ten thousand tons of every year, therefore domestic W-Gum does not satisfy the development of large-scale bio-based industry far away.
Summary of the invention
The method that the purpose of this invention is to provide a kind of producing succinic acid with biotransformation; Its production cost is low, and raw material sources are extensive, and technology is simple, mature technical route, but industrialized implementation.
For achieving the above object, the technical solution used in the present invention is:
A kind of method of producing succinic acid with biotransformation is a raw material with the inulin plant, and the inulin plant is rich in the organ pre-treatment of inulin after sterilization inserts microorganism, carries out bioconversion reaction, produces succsinic acid; The succsinic acid that produces can be that the organic acid form exists, and also can be that the form with organic acid salt exists
Inulin is meant a series of fructose polymers, and end has a glucosyl residue; The inulin plant is meant a class plant of being rich in inulin in organs such as piece root, stem tuber, is rich in the plant of inulin as jerusalem artichoke, witloof and/or Garden Dahlia etc.; The described organ that is rich in inulin is meant piece root or the stem tuber of inulin plant;
Described pre-treatment be meant with the inulin plant be rich in inulin organ its by physics, chemical or biological method is processed into the raw material that jerusalem artichoke sheet, jerusalem artichoke powder, inulin etc. are convenient to following process; Or further above-mentioned raw materials is passed through chemical method: as the dilute acid hydrolysis method, or biological process: as utilize inulinase that it is degraded into the sacchariferous raw material of the utilizable richness of microorganism.
Bioconversion reaction comprises the anaerobically fermenting production succsinic acid of microorganism, also comprises that the microorganism aerobic bioreactor transforms the production succsinic acid simultaneously; Described microorganism is meant that a class can utilize monose such as glucose, fructose, wood sugar, seminose, or oligosaccharides such as sucrose, maltose, Nutriflora P, or polysaccharide carbon source such as inulin, the microorganism commonly used that produces succsinic acid.As: wild-type microorganisms Anaerobiospirillumsucciniciproducens, Actinobacillus succinogenes or Mannheimiasucciniciproducens, or by genetic engineering and genetic engineering modified microorganism E.coli, Saccharomyces cereviciae, Actinobacillus succinogenes, Mannheimiasucciniciproducens, 4naerobiospirillum succiniciproducens, Zymomonasmobilis or lactic bacterium strains.
Detailed process can be,
1) stem tuber of inulin plant is processed into jerusalem artichoke powder and/or inulin according to a conventional method after, make the solution of mass concentration 2～20%.
2) in every mass per liter concentration is 2～20% jerusalem artichoke powder or inulin solution, the ratio preparation inulin or the jerusalem artichoke powder enzyme digestion reaction liquid that add the inulinase of 1000U～100000U unit, the work of inulinase enzyme is defined as the per minute hydrolysis substrate and produces the needed enzyme amount of 1 micromole's fructose, transfer pH=4～6,50～65 ℃ of reaction 2～60h, enzyme reaction is carried out fully, becomes the utilizable raw material that is rich in monose of microorganism, reaction solution is transferred to neutrality, sterilization;
What 3) can utilize that the microorganism of inulin inserts sterilization is in the substratum of main carbon source with jerusalem artichoke powder or inulin, and jerusalem artichoke powder or inulin mass concentration are 2～20%, and 20～60 ℃ of culture temperature are cultivated 40～300h, succsinic acid.
4) microorganism commonly used that can utilize the monose carbon source to produce succsinic acid is inserted in the inulin enzymolysis solution of sterilization, makes that sugared concentration reaches 2%～20% in the substratum, and culture temperature is 20～60 ℃, cultivates 40～300h, succsinic acid.
The present invention can avoid the industrial biotechnology industry occurring and people and animals strive the situation that grain is striven ground, utilize jerusalem artichoke, witloof, Garden Dahlia etc. are rich in the plant of inulin, particularly those can be saline and alkaline, the inulin plant that famine is grown with being coated with, as: jerusalem artichoke, by its pre-treatment is become the jerusalem artichoke sheet, the jerusalem artichoke powder, inulin or these raw materials are through further processing, handle or the inulinase processing by diluted acid, formation can be for the fermentation substrate of microorganism utilization, by Anaerobiospirillum succiniciproducens, Actinobacillussuccinogenes, Mannheimia succiniciproducens, E.coli, Saccharomycescerevisiae, Zymomonas mobilis, microorganisms such as milk-acid bacteria, biotransformation method is produced the technology of succsinic acid.
The plant that occurring in nature exists a big class to be rich in inulin, and inulin not only can directly utilize for certain micro-organisms, can also be easy to the sugar that microorganism utilizes by being hydrolyzed into fructose, glucose etc.Simultaneously, the jerusalem artichoke in this class plant etc., also having can be in the saltings, the growth of desert ground, pest-resistant disease-resistant characteristics.Therefore, utilize the inulin plant of this class significant to China's developing industry biotechnology.
Embodiment
The making of embodiment 1 jerusalem artichoke powder
The jerusalem artichoke stem tuber is dried, be ground into powder, make the jerusalem artichoke powder through pulverizer.
The making of embodiment 2 inulin
The jerusalem artichoke stem tuber is cleaned, immerse 1min in 90 ℃ the 5%NaOH solution, pulling decortication out and cleaning the jerusalem artichoke stem tuber of to peel, heat through the 4000W microwave oven, every heating 2kg needs 8min, the jerusalem artichoke stem tuber of heating is put into juice extractor squeeze the juice, and expressed juice is through Plate Filtration, pass through cation exchange resin column the moon, ion exchange resin column again, the clarified liq (pH5.0～8.0) of inulin is rich in formation, through the concentrated spray drying, makes inulin.
Embodiment 3 inulinase enzymolysis inulins or jerusalem artichoke powder
In every mass per liter concentration is 2% jerusalem artichoke powder solution, the ratio preparation inulin or the jerusalem artichoke powder enzyme digestion reaction liquid that add the inulinase of 1000U unit, the work of inulinase enzyme is defined as the per minute hydrolysis substrate and produces the needed enzyme amount of 1 micromole's fructose, transfer pH=4.5,60 ℃ of reaction 26h, enzyme reaction is carried out fully, glucose quality concentration is 0.18% in the solution, the fructose mass concentration is 1.60%, and reaction solution is transferred to neutrality, sterilization;
Embodiment 4 inulinase enzymolysis inulins or jerusalem artichoke powder
In every mass per liter concentration is 2% jerusalem artichoke powder solution, the ratio preparation inulin or the jerusalem artichoke powder enzyme digestion reaction liquid that add the inulinase of 20000U unit, the work of inulinase enzyme is defined as the per minute hydrolysis substrate and produces the needed enzyme amount of 1 micromole's fructose, transfer pH=5.0,55 ℃ of reaction 5h, enzyme reaction is carried out fully, glucose quality concentration is 0.08% in the solution, the fructose mass concentration is 1.58%, and reaction solution is transferred to neutrality, sterilization;
Embodiment 5 inulinase enzymolysis inulins or jerusalem artichoke powder
In every mass per liter concentration is 5% jerusalem artichoke powder solution, the ratio preparation inulin or the jerusalem artichoke powder enzyme digestion reaction liquid that add the inulinase of 20000U unit, the work of inulinase enzyme is defined as the per minute hydrolysis substrate and produces the needed enzyme amount of 1 micromole's fructose, transfer pH=4.8,65 ℃ of reaction 8h, enzyme reaction is carried out fully, glucose quality concentration is 0.29% in the solution, the fructose mass concentration is 4.38%, and reaction solution is transferred to neutrality, sterilization;
Embodiment 6 inulinase enzymolysis inulins or jerusalem artichoke powder
In every mass per liter concentration is 10% jerusalem artichoke powder solution, the ratio preparation inulin or the jerusalem artichoke powder enzyme digestion reaction liquid that add the inulinase of 100000U unit, the work of inulinase enzyme is defined as the per minute hydrolysis substrate and produces the needed enzyme amount of 1 micromole's fructose, transfer pH=5.5,50 ℃ of reaction 5h, enzyme reaction is carried out fully, glucose quality concentration is 0.24% in the solution, the fructose mass concentration is 9.12%, and reaction solution is transferred to neutrality, sterilization;
Embodiment 7 inulinase enzymolysis inulins or jerusalem artichoke powder
In every mass per liter concentration is 20% jerusalem artichoke powder or inulin solution, the ratio preparation inulin or the jerusalem artichoke powder enzyme digestion reaction liquid that add the inulinase of 100000U unit, the work of inulinase enzyme is defined as the per minute hydrolysis substrate and produces the needed enzyme amount of 1 micromole's fructose, transfer pH=4.0,60 ℃ of reaction 11h, enzyme reaction is carried out fully, glucose quality concentration is 1.41% in the solution, the fructose mass concentration is 17.06%, and reaction solution is transferred to neutrality, sterilization;
It is the raw material production succsinic acid with the inulin that application examples 1 is utilized intestinal bacteria
Get the 10ml crude enzyme liquid, unit of activity 380U/ml, the inulin solution (pH=4) of adding 1L mass concentration 15%, 65 ℃ of reaction 30h detect through HPLC, and enzyme reaction is carried out fully, glucose quality concentration 1.6% in the product, fructose mass concentration 13.2%.Reaction solution is transferred to neutrality, 121 ℃ of sterilization 20min.
With the intestinal bacteria of genetic engineering modified mistake (preserving number: KCTC 0506BP) in the LB substratum 30 ℃ cultivate 14h, centrifugal acquisition thalline inserts among the 100mlLB and cultivates in (500ml Erlenmeyer flask), culture temperature is 30 ℃, is cultured to OD
600Absorbance reaches at 0.6 o'clock, inserts in the 5L fermentor tank that contains 1 liter of LB substratum and cultivates, and culture temperature is 30 ℃, and nutrient solution pH is controlled at 6.0 (by 1M NaOH, 1M HCl regulates), and logical sterile air stir culture, to OD
600Value is 8.5.
Then,, add the inulin enzymolysis solution 0.5L of sterilization, make that saccharic amount concentration reaches 5% in the substratum, stop oxygen supply simultaneously and feed aseptic CO by the fermentor tank feeding-system
2, culture temperature is 30 ℃, cultivates 80h, gets the 42g succsinic acid.
Use HPLC-UV system and Hypersil BDS C18 reverse-phase chromatographic column to detect the content of succsinic acid, use HPLC-pulse ampere detector and anion-exchange column (HAMILTON RCX-10) to detect glucose, fructose concentration.
It is the raw material production succsinic acid with the inulin that application examples 2 is utilized intestinal bacteria
Get the 10ml crude enzyme liquid, unit of activity 5000U/ml, the inulin solution (pH=6) of adding 0.5L mass concentration 10%, 55 ℃ of reaction 5h detect through HPLC, and enzyme reaction is carried out fully, glucose quality concentration 1.1% in the product, fructose mass concentration 8.4%.Reaction solution is transferred to neutrality, 121 ℃ of sterilization 20min.
With the intestinal bacteria of genetic engineering modified mistake (preserving number: KCTC 0506BP) in the LB substratum 30 ℃ cultivate 14h, centrifugal acquisition thalline inserts among the 100mlLB and cultivates in (500ml Erlenmeyer flask), culture temperature is 30 ℃, is cultured to OD
600Absorbance reaches at 0.8 o'clock, inserts in the 5L fermentor tank that contains 1 liter of LB substratum and cultivates, and culture temperature is 30 ℃, and nutrient solution pH is controlled at 6.0 (by 1M NaOH, 1M HCl regulates), and logical sterile air stir culture, to OD
600Value is 6.8.
Then,, add the inulin enzymolysis solution 2L of sterilization, make that saccharic amount concentration reaches 10% in the substratum, stop oxygen supply simultaneously and feed aseptic CO by the fermentor tank feeding-system
2, culture temperature is 40 ℃, cultivates 60h, gets the 56g succsinic acid.
Use HPLC-UV system and Hypersil BDS C18 reverse-phase chromatographic column to detect the content of succsinic acid, use HPLC-pulse ampere detector and anion-exchange column (HAMILTON RCX-10) to detect glucose, fructose concentration.
It is the raw material production succsinic acid with the jerusalem artichoke powder that application examples 3 is utilized intestinal bacteria
Get the 10ml crude enzyme liquid, unit of activity 4900U/ml, the jerusalem artichoke powder solution (pH=5) of adding 2L mass concentration 5%, 60 ℃ of reaction 3.5h detect through HPLC, and enzyme reaction is carried out fully, glucose quality concentration 0.3% in the product, fructose mass concentration 4.6%.Reaction solution is transferred to neutrality, 121 ℃ of sterilization 20min.
With the intestinal bacteria of genetic engineering modified mistake (preserving number: KCTC 0506BP) in the LB substratum 40 ℃ cultivate 12h, centrifugal acquisition thalline inserts among the 100mlLB and cultivates in (500ml Erlenmeyer flask), culture temperature is 40 ℃, is cultured to OD
600Absorbance reaches at 0.3 o'clock, inserts in the 5L fermentor tank that contains 1 liter of LB substratum and cultivates, and culture temperature is 40 ℃, and nutrient solution pH is controlled at 7.0 (by 1M NaOH, 1M HCl regulates), and logical sterile air stir culture, to OD
600Value is 10.
Then,, add the jerusalem artichoke powder enzymolysis solution 1L of sterilization, make that saccharic amount concentration reaches 6% in the substratum, stop oxygen supply simultaneously and feed aseptic CO by the fermentor tank feeding-system
2, culture temperature is 32 ℃, cultivates 40h, gets the 36g succsinic acid.
Use HPLC-UV system and Hypersil BDS C18 reverse-phase chromatographic column to detect the content of succsinic acid, use HPLC-pulse ampere detector and anion-exchange column (HAMILTON RCX-10) to detect glucose, fructose concentration.
It is the raw material production succsinic acid with the jerusalem artichoke powder that application examples 3 is utilized intestinal bacteria
Get the 10ml crude enzyme liquid, unit of activity 8000U/ml, the jerusalem artichoke powder solution (pH=4) of adding 1.5L mass concentration 20%, 50 ℃ of reaction 7h detect through HPLC, and enzyme reaction is carried out fully, glucose quality concentration 1.2% in the product, fructose mass concentration 17.7%.Reaction solution is transferred to neutrality, 121 ℃ of sterilization 20min.
With the intestinal bacteria of genetic engineering modified mistake (preserving number: KCTC 0506BP) in the LB substratum 40 ℃ cultivate 12h, centrifugal acquisition thalline inserts among the 100mlLB and cultivates in (500ml Erlenmeyer flask), culture temperature is 40 ℃, is cultured to OD
600Absorbance reaches at 0.53 o'clock, inserts in the 5L fermentor tank that contains 1 liter of LB substratum and cultivates, and culture temperature is 40 ℃, and nutrient solution pH is controlled at 6.5 (by 1M NaOH, 1M HCl regulates), and logical sterile air stir culture, to OD
600Value is 12.5.
Then,, add the jerusalem artichoke powder enzymolysis solution 1.5L of sterilization, make that saccharic amount concentration reaches 8% in the substratum, stop oxygen supply simultaneously and feed aseptic CO by the fermentor tank feeding-system
2, culture temperature is 36 ℃, cultivates 50h, gets the 38g succsinic acid.
Use HPLC-UV system and Hypersil BDS C18 reverse-phase chromatographic column to detect the content of succsinic acid, use HPLC-pulse ampere detector and anion-exchange column (HAMILTON RCX-10) to detect glucose, fructose concentration.
Claims (6)
1. the method for a producing succinic acid with biotransformation, it is characterized in that: with the inulin plant is raw material, and the inulin plant is rich in the organ pre-treatment of inulin after sterilization inserts microorganism, carries out bioconversion reaction, produces succsinic acid.
2. method according to claim 1 is characterized in that: inulin is meant a series of fructose polymers, and end has a glucosyl residue; Described inulin plant is meant that jerusalem artichoke, witloof and/or Garden Dahlia be rich in the plant of inulin; The described organ that is rich in inulin is meant piece root or the stem tuber of inulin plant.
3. method according to claim 1 is characterized in that: described pre-treatment be meant with the inulin plant be rich in inulin organ its by physics, chemical or biological method processing is in flakes, powder, inulin be convenient to the raw material of following process,
Or further above-mentioned raw materials is degraded into the sacchariferous raw material of the utilizable richness of microorganism by chemical method or biological process with it.
4. method according to claim 1 is characterized in that: described microorganism is meant the microorganism commonly used that a class can utilize monose, oligosaccharides or polysaccharide carbon source to produce succsinic acid.
5. method according to claim 4, it is characterized in that: described microorganism is meant wild-type microorganisms Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes or Mannheimia succiniciproducens, or by genetic engineering and genetic engineering modified microorganism E.coli, Saccharomyces cereviciae, Actinobacillus succinogenes, Mannheimiasucciniciproducens, Anaerobiospirillum succiniciproducens, Zymomonasmobilis or lactic bacterium strains.
6. method according to claim 1, it is characterized in that: detailed process can be,
1) stem tuber of inulin plant is processed into jerusalem artichoke powder and/or inulin according to a conventional method after, make the solution of mass concentration 2～20%.
2) in every mass per liter concentration is 2～20% jerusalem artichoke powder or inulin solution, the ratio preparation inulin or the jerusalem artichoke powder enzyme digestion reaction liquid that add the inulinase of 1000U～100000U unit, the work of inulinase enzyme is defined as the per minute hydrolysis substrate and produces the needed enzyme amount of 1 micromole's fructose, transfer pH=4～6,50～65 ℃ of reaction 2～60h, enzyme reaction is carried out fully, becomes the utilizable raw material that is rich in monose of microorganism, reaction solution is transferred to neutrality, sterilization;
What 3) can utilize that the microorganism of inulin inserts sterilization is in the substratum of main carbon source with jerusalem artichoke powder or inulin, and jerusalem artichoke powder or inulin mass concentration are 2～20%, and 20～60 ℃ of culture temperature are cultivated 40～300h, succsinic acid.
4) microorganism commonly used that can utilize the monose carbon source to produce succsinic acid is inserted in the inulin enzymolysis solution of sterilization, makes that sugared concentration reaches 2%～20% in the substratum, and culture temperature is 20～60 ℃, cultivates 40～300h, succsinic acid.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101532036B (en) * | 2008-03-11 | 2012-03-14 | 浙江杭州鑫富药业股份有限公司 | Method for producing butane diacid by fermenting Jerusalem artichoke raw material |
| CN102618569A (en) * | 2012-03-15 | 2012-08-01 | 南京工业大学 | Construction of butanol producing genetic engineering bacteria, strain and application thereof |
-
2007
- 2007-02-15 CN CNA2007100104477A patent/CN101245358A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101532036B (en) * | 2008-03-11 | 2012-03-14 | 浙江杭州鑫富药业股份有限公司 | Method for producing butane diacid by fermenting Jerusalem artichoke raw material |
| CN102618569A (en) * | 2012-03-15 | 2012-08-01 | 南京工业大学 | Construction of butanol producing genetic engineering bacteria, strain and application thereof |
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