CN1276087C - Process for biologically synthesizing gamma-amino butyric acid - Google Patents
Process for biologically synthesizing gamma-amino butyric acid Download PDFInfo
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- CN1276087C CN1276087C CN 200510049187 CN200510049187A CN1276087C CN 1276087 C CN1276087 C CN 1276087C CN 200510049187 CN200510049187 CN 200510049187 CN 200510049187 A CN200510049187 A CN 200510049187A CN 1276087 C CN1276087 C CN 1276087C
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Abstract
The present invention discloses a method for biologically synthesizing gamma-amino butyric acid. The present invention is characterized in that after lactobacillus brevis whose the preservation number is CGMCC NO. 1306 is activated by an agar slant culture medium, the actobacillus brevis is inoculated to a GYP seed culture medium or an MRS seed culture medium; after the lactobacillus brevis is cultured for 10 to 30 hours, the lactobacillus brevis whose the inoculum size is from 0.5% to 5% is inoculated to a GYP fermentation culture medium or an MRS fermentation culture medium; at the temperature of 25 DEG C to 35 DEG C, after the lactobacillus brevis is cultured for 48h to 120h without moving, fermentation liquor containing mycelia can be obtained; the mycelia are collected in a centrifugal separating mode; the centrifugated mycelia are washed by deionized water which is sterilized; 0.25 to 2g of wet mycelia are taken and are suspended in 15 to 50mL of a citric acid-disodium hydrogen phosphate buffering system, wherein the content of L-sodium glutamate is from 5mM to 60mM, and the reaction time is from 1 to 10 hours; after the reaction solution is centrifugated, solution containing the gamma-amino butyric acid is obtained. The method has the advantages of low production cost, moderate reaction condition, little environment pollution, simple method, easy operation, high productive efficiency, high product purity, and the lightening of subsequent separation and purification processes.
Description
Technical field
The present invention relates to a kind of method of biosynthesizing γ-An Jidingsuan.
Background technology
γ-An Jidingsuan (being called for short GABA) is a kind of nonprotein amino acid with important physiologically active, is the important neurotransmitter of central nervous system.The lot of documents report, GABA has hypotensive and cholesterol, reduces neuronal activity, prevent that neurocyte is overheated, not only having and calming the nerves and resist the depressed effect that removes, and can also cerebral function improvement and long-term memory, increase growth hormone secretion, prevent obesity, the sharp kidney of strong liver, improve function such as climacteric syndrome.Along with the research that deepens continuously to the GABA physiologically active, the physiological function of the γ-An Jidingsuan that current research is found is more and more.GABA also has been subjected to more concern more and more, though GABA extensively exists in vivo, no matter in the animal and plant, content is but all very low, separates very difficultly, need synthesize preparation to it.Synthesis preparation method for it mainly contains chemical synthesis and biosynthetic means.Compare with chemical synthesis process, the synthetic GABA of biological process utilizes biological intravital L-Glutamic decarboxylase as catalyzer, is substrate with L-Sodium Glutamate (L-MSG), with L-Sodium Glutamate (L-MSG) α-decarboxylation, produces γ-An Jidingsuan and CO
2, major advantage is the synthesis condition gentleness, does not need expensive starting material, and environmental pollution is little, and energy consumption is little.One of key that adopts the synthetic GABA of biological process is exactly to filter out the strain excellent with high glutamic acid decarboxylase activity.Bibliographical information is arranged, the biosynthesizing of GABA adopts intestinal bacteria as producing bacterial strain usually, but have the hidden danger of food safety and sanitation aspect with intestinal bacteria as producing bacterial strain, this laboratory screening has the lactobacillus strain of the synthetic GABA ability of high-performance bio to a strain.This paper has reported the method for a kind of biosynthesizing GABA.
Summary of the invention
The method that the purpose of this invention is to provide a kind of biosynthesizing γ-An Jidingsuan.
Deposit number is: the short lactobacillus of CGMCC NO.1306 (Lactobacillus brevis), after the agar slant culture-medium activation, transfer in GYP seed culture medium or MRS seed culture medium, cultivate after 10～30 hours, inoculum size with 0.5%～5% is inoculated in GYP or the MRS fermention medium, leave standstill under 25 ℃～35 ℃ and cultivate 48h～120h, promptly get the fermented liquid of mycetome, the thalline centrifugation is collected; Thalline after centrifugal is again with the deionized water wash of the bacterium of going out, get 0.25～2g wet thallus, be suspended in citric acid-Sodium phosphate dibasic buffer system of 15～50mL, L-Sodium Glutamate content is 5mM～60mM, reacted the centrifugal solution that contains γ-An Jidingsuan that promptly gets of reaction solution 1～10 hour.
Deposit number is: the short lactobacillus of CGMCC NO.1306 (Lactobacillus brevis), and lactic acid producing when fermentation, microscopic examination is paired or become chain to occur, do not move, no spore, bacterium colony is little, smooth surface; Belong to gram positive bacterial strain, amphimicrobian.
The present invention adopts short lactobacillus as bacterial classification, thalli growth reaches stationary phase, collecting thalline, adopt whole-cell catalytic, is substrate with L-Sodium Glutamate (L-MSG), with citric acid-Sodium phosphate dibasic is buffer system, do not have other medium components such as other carbon nitrogen source, utilize the lactic acid thalline to contain the high reactivity L-Glutamic decarboxylase, L-Sodium Glutamate (L-MSG) α-decarboxylation, produce γ-An Jidingsuan, GABA output reaches 1～6g/L.The advantage of present method is that production cost is low, reaction conditions is gentle, environmental pollution is little, method is simple, easy handling, and the cycle is short, production efficiency is high, and product purity is higher, has alleviated the later separation purifying process.
Embodiment
A. bacterial strain
This laboratory screening.
B. substratum
1000mL agar slant storage medium: glucose 10g, yeast extract paste 10g, peptone 5g, sodium acetate 2g, MgSO
47H
2O 20mg, MnSO
44H
2O 1mg, NaCl 1mg, FeSO
47H
2O 1mg, agar 15g, pH 6.8.
1000mL GYP seed culture medium: glucose 10g, yeast extract paste 10g, peptone 5g, sodium acetate 2g, MgSO
47H
2O 20mg, MnSO
44H
2O 1mg, NaCl 1mg, FeSO
47H
2O 1mg, pH 6.6-7.0.
1000mL GYP fermention medium: glucose 10g, yeast extract paste 10g, peptone 5g, sodium acetate 2g, MgSO
47H
2O 20mg, MnSO
44H
2O 1mg, NaCl 1mg, FeSO
47H
2O 1mg, a hydration L-Sodium Glutamate 10g, pH 6.6-7.0.
1000mL MRS seed culture medium: extractum carnis 10g, yeast extract paste 5g, peptone 10g, K
2HpO
42g, ammonium citrate 2g, glucose 20g, tween-80 1mL, sodium-acetate 5g, inorganic salt solution 5mL (MgSO
47H
2O, 11.5%; MnSO
42H
2O, 2.4%), pH 6.6.
1000mL MRS fermention medium: extractum carnis 10g, yeast extract paste 5g, peptone 10g, K
2HPO
42g, ammonium citrate 2g, glucose 20g, tween-80 1mL, sodium-acetate 5g, inorganic salt solution 5mL (MgSO
47H
2O, 11.5%; MnSO
42H
2O, 2.4%), a hydration L-Sodium Glutamate 10g, pH 6.6.
C. preparation technology
This bacterial strain is after the agar slant culture-medium activation, transfer in GYP seed culture medium or MRS seed culture medium, cultivate after 10～30 hours, inoculum size inoculation GYP or MRS fermention medium with 0.5%～5%, under 30 ℃, leave standstill and cultivate 48h～120h, promptly get the fermented liquid of mycetome, thalline is by centrifugal (8000r/min, 5min) collection.Thalline after centrifugal is again with the deionized water wash of the bacterium of going out, get 0.25～2g wet thallus, be suspended in citric acid-Sodium phosphate dibasic buffer system of 25mL, L-Sodium Glutamate content is 5mM-100mM, reacted the centrifugal solution that contains γ-An Jidingsuan that promptly gets of reaction solution 1～10 hour.
This bacterial strain is after the agar slant culture-medium activation, transfer in GYP seed culture medium or MRS seed culture medium, cultivate after 10～30 hours, inoculum size inoculation GYP or MRS fermention medium with 0.5%～5%, under 30 ℃, leave standstill and cultivate 48h～120h, promptly get the fermented liquid of mycetome, thalline is by centrifugal (8000r/min, 5min) collection.Thalline after centrifugal is again with the deionized water wash of the bacterium of going out, get 0.25～2g wet thallus, be suspended in citric acid-Sodium phosphate dibasic buffer system of 25mL, L-Sodium Glutamate content is 5mM-100mM, reacted the centrifugal solution that contains γ-An Jidingsuan that promptly gets of reaction solution 1～10 hour.
Embodiment 1
This bacterial strain is after the agar slant culture-medium activation, transfer in the GYP seed culture medium, incubation time is 24 hours, seed culture fluid is inoculated in the GYP fermention medium with 1% inoculum size, adds a hydration L-Sodium Glutamate of 1% in the fermention medium, the liquid amount of fermention medium is 200mL/500mL, leave standstill and cultivated 60 hours, promptly get the fermented liquid of mycetome, thalline is by centrifugal (8000r/min, 5min) collection.Thalline after centrifugal is got the 0.5g wet thallus again with the deionized water wash of the bacterium of going out, is suspended in citric acid-Sodium phosphate dibasic buffer system of 25mL, L-Sodium Glutamate content is 10mM, reacted 1 hour, reaction solution is centrifugal to be got through efficient liquid phase chromatographic analysis, the about 1g/L of alpha-aminobutyric acid content.
Embodiment 2
This bacterial strain is after the agar slant culture-medium activation, transfer in the GYP seed culture medium, incubation time is 24 hours, seed culture fluid is inoculated in the sub-GYP fermention medium with 1% inoculum size, added a hydration L-Sodium Glutamate of 1% in the fermention medium, the liquid amount of fermention medium is 200mL/500mL, leave standstill and cultivated 60 hours, promptly get the fermented liquid of mycetome, thalline is by centrifugal (8000r/min, 5min) collection.Thalline after centrifugal is got the 0.5g wet thallus again with the deionized water wash of the bacterium of going out, is suspended in citric acid-Sodium phosphate dibasic buffer system of 25mL, L-Sodium Glutamate content is 20mM, reacted 2 hours, reaction solution is centrifugal to be got through efficient liquid phase chromatographic analysis, the about 2g/L of alpha-aminobutyric acid content.
Embodiment 3
This bacterial strain is after the agar slant culture-medium activation, transfer in the GYP seed culture medium, incubation time is 24 hours, seed culture fluid is inoculated in the GYP fermention medium with 1% inoculum size, adds a hydration L-Sodium Glutamate of 1% in the fermention medium, the liquid amount of fermention medium is 200mL/500mL, leave standstill and cultivated 60 hours, promptly get the fermented liquid of mycetome, thalline is by centrifugal (8000r/min, 5min) collection.Thalline after centrifugal is got the 0.5g wet thallus again with the deionized water wash of the bacterium of going out, is suspended in citric acid-Sodium phosphate dibasic buffer system of 25mL, L-Sodium Glutamate content is 40mM, reacted 3 hours, reaction solution is centrifugal to be got through efficient liquid phase chromatographic analysis, the about 4g/L of alpha-aminobutyric acid content.
Embodiment 4
This bacterial strain is after the agar slant culture-medium activation, transfer in the GYP seed culture medium, incubation time is 24 hours, seed culture fluid is inoculated in the GYP fermention medium with 1% inoculum size, adds a hydration L-Sodium Glutamate of 1% in the fermention medium, the liquid amount of fermention medium is 200mL/500mL, leave standstill and cultivated 60 hours, promptly get the fermented liquid of mycetome, thalline is by centrifugal (8000r/min, 5min) collection.Thalline after centrifugal is got the 0.5g wet thallus again with the deionized water wash of the bacterium of going out, is suspended in citric acid-Sodium phosphate dibasic buffer system of 25mL, L-Sodium Glutamate content is 60mM, reacted 5 hours, reaction solution is centrifugal to be got through efficient liquid phase chromatographic analysis, the about 6g/L of alpha-aminobutyric acid content.
Claims (1)
1. the method for a biosynthesizing γ-An Jidingsuan, it is characterized in that, deposit number is: the short lactobacillus of CGMCCNO.1306 (Lactobacillus brevis), after the agar slant culture-medium activation, transfer in GYP seed culture medium or MRS seed culture medium, cultivate after 10～30 hours, inoculum size with 0.5%～5% is inoculated in GYP or the MRS fermention medium, leave standstill under 25 ℃～35 ℃ and cultivate 48h～120h, promptly get the fermented liquid of mycetome, the thalline centrifugation is collected; Thalline after centrifugal is again with the deionized water wash of the bacterium of going out, get 0.25～2g wet thallus, be suspended in citric acid-Sodium phosphate dibasic buffer system of 15～50mL, L-Sodium Glutamate content is 5mM～60mM, reacted the centrifugal solution that contains γ-An Jidingsuan that promptly gets of reaction solution 1～10 hour.
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Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011512144A (en) * | 2008-02-21 | 2011-04-21 | ビーエーエスエフ ソシエタス・ヨーロピア | Method for producing γ-aminobutyric acid |
| CN101724587B (en) * | 2009-09-10 | 2011-10-12 | 浙江师范大学 | Lactobacillus brevis L2 bacterial strain of high yield gamma-aminobutyrique and screening method and applications thereof |
| CN102174449B (en) * | 2011-03-04 | 2012-08-22 | 天津科技大学 | Method for producing high-yield gamma-propalanine and application thereof |
| CN102260720A (en) * | 2011-07-20 | 2011-11-30 | 贵州大学 | Method for producing gamma-aminobutyric acid by using Lactobacillus fructivorans |
| CN102559552B (en) * | 2012-01-09 | 2014-06-11 | 天津科技大学 | Production method and application of high-yield gamma-aminobutyric acid |
| CN102796779B (en) * | 2012-08-24 | 2013-11-20 | 南通励成生物工程有限公司 | Biological method for preparing gamma-aminobutyric acid |
| CN103966274B (en) * | 2013-02-05 | 2017-03-15 | 中国科学院海洋研究所 | A kind of method that gamma aminobutyric acid is produced as raw materials through biotransformation with aquatic products and its processing fent |
| CN108300742A (en) * | 2017-07-26 | 2018-07-20 | 南通励成生物工程有限公司 | A kind of method that current adding substrate enzyme prepares γ-aminobutyric acid |
| CN113061061A (en) * | 2021-02-19 | 2021-07-02 | 安徽丰乐农化有限责任公司 | Preparation method of chemical fertilizer rich in gamma-aminobutyric acid |
| CN114058653B (en) * | 2021-12-29 | 2024-02-09 | 南通励成生物工程有限公司 | Method for preparing gamma-aminobutyric acid by biological synthesis method |
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