CN102586353A - Non-dependent production method of gamma-polyglutamic acid from glutamic acid - Google Patents
Non-dependent production method of gamma-polyglutamic acid from glutamic acid Download PDFInfo
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Abstract
The invention discloses and provides a non-dependent production method of gamma-polyglutamic acid from glutamic acid. The gamma-polyglutamic acid produced by the prior art is low in concentration and high in cost. According to the non-dependent production method disclosed by the invention, an organic acid or a mixture of the organic acid and a nitrogen source is added into a culture medium by flowing during the process of culturing aerobic bacillus sp. to induce thalli to transform a saccharine material and the organic acid into the gamma-polyglutamic acid, and a precursor L-glutamic acid does not need to be additionally added into the culture medium. The fermentation method for producing the gamma-polyglutamic acid, disclosed by the invention, is short in period and easy to operate, can be used for industrial large-scale fermentation and has great application value.
Description
Technical field
The invention belongs to the bio-science technical field, 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, cultivate, continue in the fermenting process to stir, stream adds organic acid in fermention medium; Under the fed-batch fermentation condition, cultivate bacillus micro-organism, make it produce gamma-polyglutamic acid-.Wherein organic acid concentration maintains between 5～20g/L, and bacillus micro-organism is selected from 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 organic acid that stream adds in fermention medium is selected from any one in Hydrocerol A, isocitric acid, succsinic acid, α-Tong Wuersuan, fumaric acid, oxysuccinic acid or the oxaloacetic acid, or above-mentioned any one organic acid salt; Machine acid is to add fermention medium with the mode that constant speed stream adds or intermittent flow adds in the fed-batch fermentation process.Stream adds organic acid and also can flow simultaneously and add nitrogenous source, carbon source and inorganic salt in fermention medium, makes nitrogen concentration maintain 1～10g/L, and wherein nitrogenous source is selected from any one in ammonium chloride, Semen Maydis powder, ammonium sulfate, peptone Carnis Bovis seu Bubali cream or the yeast extract paste
In the method for the present invention, after organic acid added in the substratum, its effect was as γ-indirect precursor of PGA synthetic, in the fermenting process, was absorbed into by thalline and was endogenous L-glutamic acid, increased the output of polyglutamic acid.
Other compositions in the fermention medium except that organic 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 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 organic acid, has cost advantage.
Use method of the present invention to produce polyglutamic acid; Adding organic acid through stream in fermention medium maintains in certain concentration range it; Avoided toxicity and the growth-inhibiting effect of organic 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 more than the 38g/.
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 Hydrocerol A batch fermentation conditional curve figure in the 10L fermentor tank.(among the figure ▲ represent remaining glucose concn, ● expression γ-PGA concentration, ■ representes dry cell weight, ◆ represent remaining citric acid concentration)
Fig. 2 is subtilis (Bacillus subtilis) C10 fed-batch fermentation conditional curve figure (constant speed stream adding citric acid solution) in the 10L fermentor tank.(among the figure ▲ represent remaining glucose concn, ● expression γ-PGA concentration, ■ representes dry cell weight, ◆ the expression citric acid concentration,---expression constant speed stream adding citric acid)
Fig. 3 is subtilis (Bacillus subtilis) C10 fed-batch fermentation conditional curve figure (intermittent flow adding citric acid and ammonium chloride solution) in the 10L fermentor tank.(among the figure ▲ and represent remaining glucose concn, zero expression γ-PGA concentration, representes dried cell weight, ◆ expression citric acid concentration, ↓ expression stream adding citric acid and ammonium chloride mixing solutions.)
Fig. 4 is the comparison diagram as a result of batch fermentation and two kinds of fed-batch fermentations.(fed-batch fermentation 1 expression constant speed stream adding citric acid; Fed-batch fermentation 2 expression intermittent flow adding citric acid and ammonium chloride mixing solutionss.)
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.
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 10g/l, Hydrocerol A 20g/l, KH2PO4 0.5g/l, MgSO47H2O 0.5g/l, FeCl3 0.05g/l, MnSO4H2O0.1g/l, CaCl2 0.11g/l, pH 6.5; When citric acid concentration was lower than 5g/L in the fermention medium, beginning constant speed stream added the sodium citrate soln that concentration is 250g/L, kept that citric acid concentration is about 5～10g/L in the substratum.γ in the substratum-PGA concentration stops stream and adds when no longer increasing.
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.Fermentation is the result show, final consumption of glucose 46g/L consumes Hydrocerol A 31.1g/L, and the output of γ-PGA is 34.5g/L, and the fermenting process curve is seen Fig. 2.
Step 1) is identical with step 1) among the embodiment 1.
Step 2) with embodiment 1 in step 2) identical.
Step 3) among step 3) and the embodiment 1 is except following difference; All the other technical characterictics are identical; Difference is when citric acid concentration is lower than 5g/L in the fermention medium, the mixing solutions of beginning intermittent flow adding citric acid and ammonium chloride, and concentration is respectively 250g/L and 48g/l; Keep that citric acid concentration is about 5～20g/L in the substratum, ammonium chloride concentration is 1-10g/L.γ in the substratum-PGA concentration stops stream and adds when no longer increasing.
Step 4) is identical with step 4) among the embodiment 1.
Fermentation is the result show, final consumption of glucose 57g/L consumes Hydrocerol A 29.8g/L, and the output of γ-PGA is 38.2g/L, and the fermenting process curve is seen Fig. 3.
Comparative Examples 1
Step 1) is identical with the step 1) of embodiment 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 10g/l, Hydrocerol A 20g/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 the step 4) of embodiment 1.
Fermentation is the result show, final consumption of glucose 26g/L consumes Hydrocerol A 20g/L, and the output of γ-PGA is 27.7g/L, and the fermenting process curve is seen Fig. 1.
Comparative Examples 1 is that the mode of disposable adding organic acid fermentation (batch fermentation) in subtilis (Bacillus subtilis) C10 produces γ-PGA; Embodiment 1 carries out constant speed stream to add under the mode of organic acid (fed-batch fermentation 1) and produce γ-PGA; Embodiment 2 carries out intermittent flow to add under the mode of organic acid (fed-batch fermentation 2) and produce γ-PGA; Comparative Examples 1 compares with the fermentation result of embodiment 1 and 2, can find that output that two kinds of streams add fermentation (fed-batch fermentation) is apparently higher than batch fermentation.(referring to Fig. 4)
Step 1) is identical with step 1) among the embodiment 1.
Step 2) with embodiment 1 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 succsinic acid concentration was lower than 5g/L in the fermention medium, the beginning intermittent flow added the sodium succinate solution that concentration is 50g/L, keeps that succsinic acid concentration is about 5～10g/L in the substratum.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, succsinic acid 20g/l, KH2PO4 1.5g/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 1.
Fermentation is the result show, final consumption of glucose 28g/L consumes succsinic acid 25.4g/L, and the output of γ-PGA is 24.1g/L.
Comparative Examples 2
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 organic acid in this fermention medium, like in succsinic acid, oxysuccinic acid, Hydrocerol A, oxaloacetic acid, isocitric acid, α-Tong Wuersuan or the fumaric acid any one, 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 organic acid cell growth and γ-PGA output.
Experimental result shows, adds the 10g/L α-Tong Wuersuan, produces the γ-PGA of maximum concentration, and γ-PGA output reaches 15.4g/L.
The influence of table 1 organic acid cell growth and γ-PGA output
Comparative Examples 2 is that the various organic acids of disposable adding (batch fermentation) produce γ-PGA in subtilis (Bacillus subtilis) C10.
Comparative Examples 2 compares with the fermentation result of embodiment 3, can find that the output of the disposable adding of any organic acid (batch fermentation) does not all have stream to add the output height of fermentation (fed-batch fermentation).
Claims (8)
1. the L-glutamic acid dependent/non-dependent working method of a 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; Stream adds organic acid in fermention medium, under the fed-batch fermentation condition, cultivates bacillus micro-organism, makes it produce gamma-polyglutamic acid-.
2. the L-glutamic acid dependent/non-dependent working method of a kind of gamma-polyglutamic acid-according to claim 1 is characterized in that, when organic acid concentration is lower than 5g/L, begins stream and adds organic acid solution, makes it be maintained to 5-20g/L.
3. the L-glutamic acid dependent/non-dependent working method of a kind of gamma-polyglutamic acid-according to claim 1; It is characterized in that; The described organic acid that stream adds in fermention medium is selected from any one in Hydrocerol A, isocitric acid, succsinic acid, α-Tong Wuersuan, fumaric acid, oxysuccinic acid or the oxaloacetic acid, perhaps above-mentioned any one organic acid salt.
4. the L-glutamic acid dependent/non-dependent working method of a kind of gamma-polyglutamic acid-according to claim 1; 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 a kind 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. the L-glutamic acid dependent/non-dependent working method of a kind of gamma-polyglutamic acid-according to claim 2 is characterized in that, described organic acid is to add fermention medium with the mode that constant speed stream adds or intermittent flow adds.
7. according to the L-glutamic acid dependent/non-dependent working method of the said a kind of gamma-polyglutamic acid-of claim 2, it is characterized in that, in fermention medium stream add organic acid simultaneously also stream add nitrogenous source, carbon source and inorganic salt, make nitrogen concentration maintain 1～10g/L.
8. according to the L-glutamic acid dependent/non-dependent working method of the said a kind of gamma-polyglutamic acid-of claim 7, it is characterized in that described 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.
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| CN106479907A (en) * | 2015-09-02 | 2017-03-08 | 上海师范大学 | A kind of culture medium improving composite bacteria fermentation Biomass and its using method |
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| CN113122590A (en) * | 2021-06-03 | 2021-07-16 | 淮北师范大学 | Epsilon-polylysine biosynthesis method based on citric acid fermentation waste |
| 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 |
| WO2024084510A1 (en) * | 2022-10-20 | 2024-04-25 | Council Of Scientific And Industrial Research An Indian Registered Body Incorporated Under The Regn. Of Soc. Act (Act Xxi Of 1860) | Glutamic acid independent process for synthesis of poly gamma glutamic acid |
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| CN106479907A (en) * | 2015-09-02 | 2017-03-08 | 上海师范大学 | A kind of culture medium improving composite bacteria fermentation Biomass and its using method |
| CN109415751A (en) * | 2016-05-26 | 2019-03-01 | 密执安大学评议会 | Composition and method for microbial co culture |
| CN109415751B (en) * | 2016-05-26 | 2023-02-28 | 密执安大学评议会 | Compositions and methods for microbial co-culture |
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| CN110747240A (en) * | 2019-12-01 | 2020-02-04 | 内蒙古阜丰生物科技有限公司 | Fermentation process of polyglutamic acid |
| CN113122590A (en) * | 2021-06-03 | 2021-07-16 | 淮北师范大学 | Epsilon-polylysine biosynthesis method based on citric acid fermentation waste |
| 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 |
| WO2024084510A1 (en) * | 2022-10-20 | 2024-04-25 | Council Of Scientific And Industrial Research An Indian Registered Body Incorporated Under The Regn. Of Soc. Act (Act Xxi Of 1860) | Glutamic acid independent process for synthesis of poly gamma glutamic acid |
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Application publication date: 20120718 |