CN102586354A - Glutamic acid-independent production method of gamma-polyglutamic acid - Google Patents
Glutamic acid-independent production method of gamma-polyglutamic acid Download PDFInfo
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Abstract
The invention discloses a glutamic acid-independence production method of gamma-polyglutamic acid. Gamma-polyglutamic acid produced by the prior art is low in concentration and high in cost. According to the invention, in a process of culturing aerobic Bacillus sp., an organic acid or a mixture of the organic acid and a nitrogen source is added to a culture medium in a flowing manner, bacteria are promoted to convert the sugar raw materials and organic acid into gamma-polyglutamic acid, and a precursor L-glutamic acid is not needed to be added to the culture medium. The fermentation process for producing gamma-poly glutamic acid is short in period and easy to operate, can be used for plant-scale fermentation, and has good application value.
Description
Technical field
The invention belongs to the working method of polyglutamic acid, be specifically related to a kind of novel method of L-glutamic acid dependent/non-dependent production of gamma-polyglutamic acid-.
Background technology
Gamma-polyglutamic acid-(Poly-γ-glutamatic acid, γ-PGA), be a kind of water-soluble and biodegradable polyamino acid by multiple microorganisms.Polyglutamic acid is a kind of new type natural polymer to human body and environment toxicological harmless; Its particular structure characteristic is given height water-absorbent, good penetration property, biocompatibility and biodegradability; Being widely used in many fields such as agricultural, medicine, food, makeup, is the Green Chemistry product that a kind of generally acknowledged utmost point has development potentiality.The production of gamma-polyglutamic acid-mainly utilizes some bacterial strains of bacillus, direct fermentation production.Be polymer based with the petroleum derivation article relatively, the gamma-polyglutamic acid-compound can be synthetic through renewable raw materials, has sizable market potential.
γ-PGA working method is divided into two types: one type is in fermention medium, to add L-L-glutamic acid as γ-PGA synthetic precursor; Absorb the L-glutamic acid in the substratum by mikrobe; And utilize its synthetase series that the polymerization of monomer L-glutamic acid is produced γ-PGA; Be L-glutamic acid dependency compound method, the described γ of patent CN1644677A-PGA working method for example; Other one type is in fermention medium, not add L-L-glutamic acid; There is mikrobe directly the saccharine material in the substratum to be converted into endogenous L-glutamic acid; Synthetase series polymerization by self produces γ-PGA again, and this is called de novo synthesis, i.e. L-glutamic acid dependent/non-dependent compound method; Be also referred to as de novo synthesis, for example the described γ of patent CN 1932007A-PGA working method.
In two kinds of working methods, the gamma-polyglutamic acid-production level receives the influence of medium component and culture condition to a great extent.The optimal medium composition of production γ-PGA has been confirmed in many researchs.Normally used substratum comprises carbon source, nitrogenous source and inorganic salts, and L-L-glutamic acid dependency compound method need add the precursor of a large amount of external source L-glutamic acid as polyglutamic acid.In general suitable carbon source is glucose normally, glycerine, and SANMALT-S, Hydrocerol A, fructose, sucrose, starch etc., suitable nitrogenous source is ammonium chloride normally, ammonium sulfate, peptone, yeast extract paste etc.
Existing lot of documents has been reported the method that L-glutamic acid dependency compound method is produced polyglutamic acid, comprises the optimization of nutrient media components, and batch fermentation and stream add fermentation process, the extraction and separation method of γ-PGA etc.The mode that stream adds fermentation comprises: stream adds carbohydrate, and to be the cell growth with polymkeric substance synthesize provides energy (referring to patent: CN1556859A); Stream adds the L-glutamate precursor increases polyglutamic acid output.In the method for present fermentative prodn polyglutamic acid, need to add L-glutamic acid in the substratum and caused high raw materials cost, become one of main bottleneck of suitability for industrialized production and application as precursor.And do not add the fermenting process of L-glutamic acid, and can only produce the polyglutamic acid of lower concentration, low excessively production efficiency is not suitable for the production of industrially scalable.Therefore, through developing new fermentation method for producing, substitute L-L-glutamic acid with comparatively cheap substrate, the gamma-polyglutamic acid-of production high density becomes the new demand in market.
Summary of the invention
The objective of the invention is deficiency, a kind of L-glutamic acid dependent/non-dependent working method of gamma-polyglutamic acid-is provided to prior art, can be with comparatively cheap cost, the gamma-polyglutamic acid-of production high density.
The technical scheme that the inventive method adopted comprises the steps:
Bacillus micro-organism is inoculated in the fermenting container that contains fermention medium, under aerobic conditions, cultivates, continue in the fermenting process to stir, in fermention medium, add oxalic acid, make it produce gamma-polyglutamic acid-.Oxalic acid adopts fed-batch mode to join in the fermention medium, under the fed-batch fermentation condition, cultivates bacillus micro-organism, makes it produce gamma-polyglutamic acid-; When concentration of oxalic acid is lower than 1g/L, begin stream and add oxalic acid solution, make it be maintained to 1-12g/L.Wherein bacillus micro-organism is selected from any one in subtilis, bacillus amyloliquefaciens, Bacillus licheniformis or the bacillus megaterium with gamma-polyglutamic acid-synthesis capability, preferred subtilis C10 (Bacillus subtilis C10) (preserving number CGMCC No.4924) or subtilis ZJU7 (Bacillus subtilis zju-7) (preserving number CGMCC No.1250) bacterial strain.The fermention medium that uses in present method does not comprise L-glutamic acid.Oxalic acid can be replaced by oxalate.Machine acid is to add fermention medium with the mode that intermittent flow adds in the fed-batch fermentation process.Stream also can flow when adding oxalic acid and add nitrogenous source in fermention medium, makes nitrogen concentration maintain 1～10g/L, and wherein nitrogenous source is selected from ammonium chloride, any one in ammonium sulfate, peptone or the yeast extract paste.
In the method for the present invention, oxalic acid adds in the substratum, and its effect is as γ-PGA synthetic stimulant; Stimulate somatic cells to produce the γ-PGA of high density; It is synthetic that the precursor L-glutamic acid of γ-PGA comes from thalline self, and in the fermenting process, oxalic acid is by the thalline absorption and be decomposed into carbonic acid gas.Therefore, this method also provides a kind of new purposes for oxalic acid or oxalate simultaneously.
Other compositions in the fermention medium except that oxalic acid comprise existing nitrogenous source, carbon source and the inorganic salt that are used to produce gamma-polyglutamic acid-.As use glucose, sucrose, fructose, glycerine, starch as carbon source; Use ammonium chloride, ammonium sulfate, peptone, Carnis Bovis seu Bubali cream, yeast extract paste, Semen Maydis powder etc. as nitrogenous source; Also comprise various inorganic salt such as molysite that thalli growth is required, magnesium salts, sylvite, calcium salt, phosphoric acid salt.
The present invention's said " oxalic acid adopts fed-batch mode to join in the fermention medium; under the fed-batch fermentation condition, cultivate bacillus micro-organism; make it produce gamma-polyglutamic acid-" is meant during the fermentation; Constantly in substratum, add oxalic acid, the concentration of oxalic acid is maintained in certain scope, promptly constantly oxalic acid is carried out feed supplement." mode that intermittent flow adds " is adding off and on when being meant adding oxalic acid or is not to add with constant speed.
The present invention compares with prior art has following beneficial effect:
The inventive method makes mikrobe continue to produce the high density gamma-polyglutamic acid-, and has avoided adding expensive L-glutamic acid in the employed substratum, and synthetic needed substrate is cheap saccharine material and oxalic acid, has cost advantage.
Use method of the present invention to produce polyglutamic acid; Adding oxalic acid through stream in fermention medium maintains in certain concentration range it; Avoided toxicity and the growth-inhibiting effect of oxalic acid on the one hand for somatic cells; Impel the constantly synthetic γ-PGA of thalline on the other hand, significantly increase γ-PGA output, its concentration can reach 37.5g/L.
The method cycle of the present invention is short, and easy handling can be used for plant-scale production.
Description of drawings
Fig. 1 is that subtilis (Bacillus subtilis) C10 adds oxalic acid batch fermentation conditional curve figure in the 10L fermentor tank.
Fig. 2 is subtilis (Bacillus subtilis) C10 fed-batch fermentation conditional curve figure (intermittent flow adds oxalic acid solution) in the 10L fermentor tank.
The comparison diagram as a result of Fig. 3 batch fermentation and fed-batch fermentation.
Embodiment
Below in conjunction with Figure of description and embodiment the present invention is described further, but is not used for limiting scope of the present invention.
The L-glutamic acid dependent/non-dependent working method of gamma-polyglutamic acid-may further comprise the steps:
The activation of step 1) bacterial strain: C10 rules on flat board with subtilis (Bacillus subtilis), 37 ℃, cultivates 12h.Plate culture medium consists of: peptone 10g/L, and Carnis Bovis seu Bubali cream 5g/L, sodium-chlor 5g/L, glucose 10g/L, agar 15g/L, pH 7.
Step 2) preparation of seed: activatory subtilis (Bacillus subtilis) C10 bacterial classification picking is inserted liquid seed culture medium, and liquid amount is a 250ml triangular flask 20ml substratum, 37 ℃, 200rpm (rev/min), shaking culture 12h.
Above-mentioned seed culture medium is formed: peptone 10g/L, and Carnis Bovis seu Bubali cream 5g/L, sodium-chlor 5g/L, glucose 10g/L, pH 7.
The step 3) shake flask fermentation: the seed liquor for preparing is inserted fermention medium in 5% ratio, and liquid amount is a 250ml triangular flask 30ml substratum, 37 ℃, and 200rpm, shaking culture 36h.
Above-mentioned fermention medium is formed: glucose 120g/L, and ammonium chloride 18g/L, KH2PO4 5g/L, MgSO47H2O 0.5g/L, FeCl3 0.05g/L, MnSO4H2O 0.1g/L, CaCl20.11g/L, pH 7.Except that said components, add in oxalic acid, succsinic acid, oxysuccinic acid, Hydrocerol A, oxaloacetic acid, isocitric acid, α-2 ' ketoisocaproic or the fumaric acid any one in this fermention medium, addition is 10g/L.
Step 4) product separation and Extraction: the centrifugal thalline of removing in the fermented liquid after the fermentation ends, add 4 times of volumes methanol in the supernatant, place 1h for 4 ℃, centrifugal collecting precipitation obtains white γ-PGA after the lyophilize.Table 1 is seen in the influence of different sorts oxalic acid cell growth and γ-PGA output.
Experimental result shows, adds 10g/L oxalic acid, produces the γ-PGA of maximum concentration, and γ-PGA output reaches 25.3g/L.
The influence of table 1 oxalic acid cell growth and γ-PGA output
Fermention medium in the step 3) is formed: glucose 80g/L, and ammonium chloride 18g/L, KH2PO4 0.5g/L, MgSO47H2O 0.5g/L, FeCl30.05g/L, MnSO4H2O 0.1g/L, CaCl20.11g/L, pH 7.Except that said components, add oxalic acid in this fermention medium in addition, get 6 kinds of additions (respectively in 6 experimental tank), addition is respectively 0g/L, 5g/L, 10g/L, 12g/L, 15g/L, 20g/L.All the other steps characteristic are all identical with embodiment 1.
Table 2 is seen in the influence of oxalic acid addition cell growth and γ-PGA output.
Experimental result shows that along with the increase that adds concentration of oxalic acid, the γ of generation-PGA concentration increases; Finally under the condition that adds 12g/L oxalic acid; Produce the γ-PGA of maximum concentration, reach 27.1g/L, when concentration of oxalic acid surpasses 15g/L severe inhibition the growth and γ-PGA generation of thalline.
Table 3 oxalic acid addition is to the influence of polyglutamic acid output
The activation of step 1) bacterial strain: zju-7 rules on flat board with subtilis (Bacillus subtilis), 37 ℃, cultivates 12h.Plate culture medium consists of: peptone 10g/L, and Carnis Bovis seu Bubali cream 5g/L, sodium-chlor 5g/L, agar 15g/L, pH 7.
Step 2) preparation of seed: activatory subtilis (Bacillus subtilis) zju-7 bacterial classification picking is inserted liquid seed culture medium, and liquid amount is a 250ml triangular flask 20ml substratum, 37 ℃, and 200rpm, shaking culture 12h.
Above-mentioned seed culture medium is formed: peptone 10g/L, Carnis Bovis seu Bubali cream 5g/L, sodium-chlor 5g/L, pH7.
The step 3) shake flask fermentation: the seed liquor for preparing is inserted fermention medium in 5% ratio, and liquid amount is a 250ml triangular flask 30ml substratum, 37 ℃, and 200rpm, shaking culture 4h.
Above-mentioned fermention medium is formed: glucose 100g/L, and peptone 40g/L, oxalic acid 10g/L, KH2PO4 0.5g/L, MgSO47H2O 2g/L, NaCl2 5g/L, CaCl2 1g/L, pH 7.
Step 4) is identical with the step 4) of embodiment 1.
Experimental result shows that this method can make the γ-PGA that subtilis (Bacillus subtilis) zju-7 is produced high density, and output reaches 29.5g/L.
Step 1) is identical with embodiment 1 step 1).
Step 2) preparation of seed: activatory subtilis (Bacillus subtilis) C10 bacterial classification picking is inserted liquid seed culture medium, and liquid amount is a 1000ml triangular flask 250ml substratum, 37 ℃, and 200rpm, shaking culture 12h.
Above-mentioned seed culture medium is formed: peptone 10g/L, and Carnis Bovis seu Bubali cream 5g/L, sodium-chlor 5g/L, glucose 10g/L, pH 7.
Step 3) inserts the 10L fermentor tank with the seed liquor for preparing in 5% ratio, and the fermention medium liquid amount is 6L, and fermentation parameter is controlled to be: 37 ℃ of leavening temperatures, stir 400rpm, pH6.5, air flow 0.4L/h, fermentation 34h.
Above-mentioned fermention medium is formed: glucose 80g/l, and ammonium chloride 18g/l, oxalic acid 10g/L, KH2PO4 0.5g/l, MgSO47H2O 0.5g/l, FeCl3 0.05g/l, MnSO4H2O0.1g/l, CaCl20.11g/l, pH 6.5.
Step 4) is identical with embodiment 1 step 4).
Fermentation is the result show, final consumption of glucose 37g/L consumes oxalic acid 7g/L, and the output of γ-PGA is 17.3g/L, and the fermenting process curve is seen Fig. 1.
Step 1) is identical with step 1) among the embodiment 4.
Step 2) with embodiment 4 in step 2) identical.
Step 3) inserts the 10L fermentor tank with the seed liquor for preparing in 5% ratio, and the fermention medium liquid amount is 6L, and fermentation parameter is controlled to be: 37 ℃ of leavening temperatures, stir 400rpm, pH6.5, air flow 0.4L/h.When fermention medium mesoxalic acid concentration is lower than 1g/L, begin to flow and add the sodium oxalate solution that concentration is 50g/L, keeping substratum mesoxalic acid concentration is about 1～10g/L.γ in the substratum-PGA concentration stops stream and adds when no longer increasing.
Above-mentioned fermention medium is formed: glucose 80g/l, and ammonium chloride 18g/l, oxalic acid 10g/l, KH2PO4 0.5g/l, MgSO47H2O 0.5g/l, FeCl3 0.05g/l, MnSO4H2O0.1g/l, CaCl2 0.11g/l, pH 6.5.
Step 4) is identical with step 4) among the embodiment 4.
Fermentation is the result show, final consumption of glucose 70g/L consumes oxalic acid 16g/L, and the output of γ-PGA is 36.3g/L, and the fermenting process curve is seen Fig. 2.
Step 1) is identical with step 1) among the embodiment 4.
Step 2) preparation of first order seed: activatory subtilis (Bacillus subtilis) C10 bacterial classification picking is inserted liquid seed culture medium, and liquid amount is a 1000ml triangular flask 250ml substratum, 37 ℃, and 200rpm, shaking culture 12h.
The preparation of secondary seed: with the secondary seed jar of 500ml primary seed solution access 15L, the seeding tank liquid amount is 6L, mixing speed 600rpm, and 37 ℃, incubation time is 5 hours.
Above-mentioned one-level, secondary seed medium are formed: peptone 10g/L, and Carnis Bovis seu Bubali cream 5g/L, sodium-chlor 5g/L, glucose 10g/L, pH 7.
Step 3) inserts the 100L fermentor tank with the secondary seed solution for preparing in 5% ratio, and the fermention medium liquid amount is 60L, and fermentation parameter is controlled to be: 37 ℃ of leavening temperatures, stir 400rpm, pH6.5, air flow 0.4L/h.When fermention medium mesoxalic acid concentration was lower than 1g/L, the beginning intermittent flow added the sodium oxalate solution that concentration is 50g/L, and keeping substratum mesoxalic acid concentration is about 1～10g/L.
γ in the substratum-PGA concentration stops stream and adds when no longer increasing.
Above-mentioned fermention medium is formed: glucose 80g/l, and ammonium chloride 18g/l, oxalic acid 10g/l, KH2PO4 1.5g/l, MgSO47H2O 0.5g/l, FeCl3 0.05g/l, MnSO4H2O0.1g/l, CaCl20.11g/l, pH 6.5.
Step 4) is identical with step 4) among the embodiment 4.
Fermentation is the result show, final consumption of glucose 76g/L consumes oxalic acid 17.9g/L, and the output of γ-PGA is 37.5g/L.
Embodiment 7
Step 1) is identical with step 1) among the embodiment 4.
Step 2) with embodiment 4 in step 2) identical.
Step 3) inserts the 10L fermentor tank with the seed liquor for preparing in 5% ratio, and the fermention medium liquid amount is 6L, and fermentation parameter is controlled to be: 37 ℃ of leavening temperatures, stir 400rpm, pH6.5, air flow 0.4L/h.When fermention medium mesoxalic acid concentration is lower than 1g/L, begin to flow the mixing solutions (sodium oxalate concentration is 50g/L, and ammonium chloride concentration is 50g/L) that adds sodium oxalate and ammonium chloride, keeping substratum mesoxalic acid and ammonium chloride concentration is about 1～10g/L.γ in the substratum-PGA concentration stops stream and adds when no longer increasing.
Above-mentioned fermention medium is formed: glucose 80g/l, and ammonium chloride 10g/l, oxalic acid 10g/l, KH2PO4 0.5g/l, MgSO47H2O 0.5g/l, FeCl3 0.05g/l, MnSO4H2O0.1g/l, CaCl20.11g/l, pH 6.5.
Step 4) is identical with step 4) among the embodiment 4.
Fermentation is the result show, final consumption of glucose 82g/L consumes oxalic acid 19.6g/L, and the output of γ-PGA is 42g/L.
Embodiment 7 is that the mode that adopts stream to add oxalic acid and ammonium chloride simultaneously produces γ-PGA.
Claims (8)
1. the L-glutamic acid dependent/non-dependent working method of gamma-polyglutamic acid-is characterized in that its process is following: bacillus micro-organism is inoculated in the fermenting container that contains fermention medium, under aerobic conditions, cultivates, continue in the fermenting process to stir; In fermention medium, add oxalic acid, make it produce gamma-polyglutamic acid-.
2. the L-glutamic acid dependent/non-dependent working method of gamma-polyglutamic acid-according to claim 1; It is characterized in that; Described oxalic acid adopts fed-batch mode to join in the fermention medium, under the fed-batch fermentation condition, cultivates bacillus micro-organism, makes it produce gamma-polyglutamic acid-.
3. the L-glutamic acid dependent/non-dependent working method of gamma-polyglutamic acid-according to claim 2 is characterized in that, when concentration of oxalic acid is lower than 1g/L, begins stream and adds oxalic acid solution, makes it be maintained to 1-12g/L.
4. the L-glutamic acid dependent/non-dependent working method of gamma-polyglutamic acid-according to claim 3; It is characterized in that described bacillus micro-organism is selected from any one in subtilis, bacillus amyloliquefaciens, Bacillus licheniformis or the bacillus megaterium.
5. the L-glutamic acid dependent/non-dependent working method of gamma-polyglutamic acid-according to claim 4; It is characterized in that described subtilis is selected subtilis C10 (Bacillus subtilis C10) (preserving number CGMCC No.4924) or subtilis ZJU7 (Bacillus subtilis zju-7) (preserving number CGMCC No.1250) bacterial strain for use.
6. according to the L-glutamic acid dependent/non-dependent working method of the said gamma-polyglutamic acid-of claim 5, it is characterized in that stream also flows when adding oxalic acid and adds nitrogenous source in fermention medium, makes nitrogen concentration maintain 1～10g/L.
7. according to the L-glutamic acid dependent/non-dependent working method of the said gamma-polyglutamic acid-of claim 6, it is characterized in that selected nitrogenous source is selected from any one in ammonium chloride, ammonium sulfate, Semen Maydis powder, Carnis Bovis seu Bubali cream, peptone or the yeast extract paste.
8. according to the L-glutamic acid dependent/non-dependent working method of the described gamma-polyglutamic acid-of the arbitrary claim of claim 1-7, it is characterized in that said oxalic acid is replaced by oxalate.
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| CN109182404A (en) * | 2018-10-15 | 2019-01-11 | 天津科技大学 | A method of utilizing stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments |
| CN114875085A (en) * | 2022-05-09 | 2022-08-09 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Liquid fermentation method for increasing yield of gamma-polyglutamic acid |
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| CN114875085A (en) * | 2022-05-09 | 2022-08-09 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Liquid fermentation method for increasing yield of gamma-polyglutamic acid |
| CN114875085B (en) * | 2022-05-09 | 2024-02-02 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Liquid fermentation method for improving gamma-polyglutamic acid yield |
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Application publication date: 20120718 |