CN102653736A - Fermenting production method of glutamate dehydrogenase by microorganisms - Google Patents
Fermenting production method of glutamate dehydrogenase by microorganisms Download PDFInfo
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- CN102653736A CN102653736A CN2012101067279A CN201210106727A CN102653736A CN 102653736 A CN102653736 A CN 102653736A CN 2012101067279 A CN2012101067279 A CN 2012101067279A CN 201210106727 A CN201210106727 A CN 201210106727A CN 102653736 A CN102653736 A CN 102653736A
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Abstract
The invention provides a fermenting production method of glutamate dehydrogenase (GDH) by microorganisms. The method comprises the following steps: activating microorganisms producing the GDH for 6 to 10 times; directionally domesticating the product for 4 to 6 times to lead the microorganisms to grow excellently at a temperature between 24 and 30 DEG C; gradually propagating the GDH producing strain at a temperature between 24 and 30 DEG C after product directional domestication according to a conventional method to prepare liquid primary seeds and liquid secondary seeds; inoculating into a liquid ferment culture medium in an inoculum size of 3 to 9 percent of the volume of a fermentation broth, culturing for 60 to 114 hours at a temperature between 24 and 30 DEG C to complete fermenting production of the GDH by the microorganisms; centrifuging the fermentation broth in 6000 to 8000rpm to collect strain, cleaning for multiple times, collecting precipitate, suspending in a buffer solution, grinding with quartz sands, and centrifugally collecting supernatant in 10000 to 14000rpm to obtain crude enzyme; and further concentrating, separating and purifying the crude enzyme according to different requirements and different use objects to prepare the enzyme preparations with different activities, purities and preparation forms.
Description
Technical field
The present invention relates to fields such as microbiology, enzyme engineering, fermentation engineering, biological chemistry, be specifically related to a kind of glutamate dehydrogenase microbial fermentation production method.The glutamate dehydrogenase that this invention is produced is mainly used in clinical hepatitis, stomach trouble, pancreatitic diagnosis and other glutamate dehydrogenase medical diagnosis industry.In view of glutamate dehydrogenase be a kind of protein of microorganisms, and nontoxic, have no side effect, can be widely used in the production of medical diagnosis box.
Technical background
Glutamate dehydrogenase (Glutamate dehydrogenase is abbreviated as GDH) is a kind of anaerobic dehydrogenase that relies on coenzyme, in amino acid metabolism, occupies critical role.It is divided into three types of NADH dependent form (EC.1.4.1.2), NAD (P) H dependent form (EC.1.4.1.3) and NADPH dependent forms (EC.1.4.1.4); Extensively be present in animal, plant and the mikrobe, and be widely used in (Hu Hanning etc., Wuhan University's journal in the medical diagnosis on disease industries such as clinical hepatitis, stomach trouble, pancreatitis; 2001); The zymin that detects hepatopathy at present mainly is gpt (ALT), but its specificity is relatively poor, can not reflect liver cell damage intensity (Guo Shaoli etc. fully; China's combination of Chinese tradiational and Western medicine digestion magazine, 2001).Along with of the application of GDH test kit in the medical diagnosis industry; With and the GDH specificity high; Advantage such as have no side effect makes it be more suitable for conventional liver functional testing and other disease detection projects, particularly the disease surveillance project of the judgement of chronic hepatitis patients degree of liver, observation of curative effect and state of illness monitoring (Cao Yan etc.; Shanghai Medicine check magazine, 2003).Early stage method of producing GDH is from animal viscera, to extract, and gained enzyme system is complicated, is difficult to obtain highly purified enzyme (Wang Yan etc., biotechnology journal, 2003), and makes easily through the microbial fermentation rule.Research mainly concentrates on strain selection, fermentation condition optimization, zymologic property about mikrobe GDH at present; Conceptual phase such as enzyme purification and gene clone (Wang Yan etc.; The biotechnology journal, 2003), and industrialization, the mass-producing of microbial fermentation production GDH also do not appear in the newspapers.The GDH purity that microbial fermentation is produced is high; Fermentation manufacturing technique is simple; Extraction step is simple and easy, and the purifying process of enzyme is stable, produces not influenced by raw material sources; More be applicable to batch process, and the GDH of microbial fermentation production to be higher than the GDH that additive method is produced on medical diagnosis, detection precision.Therefore, the GDH of microbial fermentation production has boundless potentiality to be exploited and application prospect.
Summary of the invention
The present invention provides the method that a kind of microbial fermentation is produced GDH; This invention mainly is after mikrobe is tamed through the product orientation; Produce the method for GDH at 24～30 ℃ of liquid fermentings; The GDH liquid activity that this working method obtains can reach 145U/mL, through separating and purifying, can obtain the zymin of different concns and purity as again.The microbial fermentation GDH that this method is produced is simple, convenient, quick, cost is low, the loaded down with trivial details technology and expensive equipment that can fundamentally avoid animal tissues to extract.
The method that a kind of microbial fermentation of the present invention is produced glutamate dehydrogenase specifically may further comprise the steps:
The mikrobe that (1) will produce glutamate dehydrogenase carries out 6～10 activation, through the directed domestication of product 4～6 times, makes its well-grown under 24～30 ℃ of envrionment conditionss again;
(2) produce bacterium 24～30 ℃ of enlarged culturing step by step by the glutamate dehydrogenase of ordinary method after, be prepared into liquid first order seed and secondary seed the directed domestication of product;
(3) with liquid first order seed or secondary seed, 3～9% inoculum sizes of pressing fermentating liquid volume insert in the liquid fermentation medium, and when cultivating 60～114h for 24～30 ℃, promptly microbial fermentation is produced the glutamate dehydrogenase end;
(4) with the fermented liquid of (3) 6,000～8, the centrifugal collection liquid of 000rpm, with distilled water wash for several times after centrifugal collecting precipitation;
(5) deposition is suspended in the damping fluid, adds silica sand and grind fragmentation, 10,000～14,000rpm is centrifugal, and the supernatant of collecting is the glutamate dehydrogenase crude enzyme liquid;
(6) different with the use object according to different needs, the crude enzyme liquid that (5) are obtained further concentrates, separation and purification, is prepared into the zymin of different activities, purity and formulation.
The bacterial classification that uses among the present invention derives from China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), and initial stage activation and growth conditions are undertaken by the explanation that culture presevation unit provides.Glutamate dehydrogenase produces bacterium (CGMCC bacterium numbering: 1.495 or 1.581); After the activation of bacterial strain elder generation, the directed domestication; By condition of enzyme production fermentative prodn glutamate dehydrogenase of the present invention; Bacterial strain after the directed domestication can be preserved 2 months in 4 ℃ of environment, bacteria suspension, the preservation for a long time under subzero 80 ℃ of conditions of processing with 10～25% glycerine.
Embodiment
Embodiment one:
(1) medium preparation
1. strain activation and culture base: Carnis Bovis seu Bubali cream 10.0g, peptone 10.0g, NaCl 5.0g, glucose 10.0g, zero(ppm) water 1.0L, 7.0～7.2,121 ℃ of high pressure steam sterilization 30min of pH value.
2. the directed acclimation shaking culture base of bacterial classification: glucose 8.0～12.0g, peptone 18.0～22.0g, Carnis Bovis seu Bubali cream 16.0～24.0g, NaCl 4.0～6.0g, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
3. liquid seed culture medium: glucose 22.0～26.0g, steeping water 24.0～28.0g, urea 1.5～3.5g, K
2HPO
41.0～2.5g, MgSO
40.4～0.8g, FeSO
40.002～0.006g, MnSO
40.004～0.008g, tap water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
4. enzymatic production substratum: glucose 25.0～45.0g, steeping water 4.0～8.0g, urea 4.0～6.0g, K
2HPO
40.5～2.5g, FeSO
40.001～0.004g, MgSO
45.0～7.0g, MnSO
40.002～0.005g, tap water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
The mikrobe that (2) will produce glutamate dehydrogenase through the directed domestication of product 4～6 times, makes its well-grown under 24～30 ℃ of envrionment conditionss again by 6～10 activation of bacterial strain explanation carrying out that Chinese common micro-organisms culture presevation administrative center (CGMCC) provides;
(3) produce bacterium by the glutamate dehydrogenase of ordinary method after and cultivate 36～60h at 24～30 ℃ and then carry out enlarged culturing step by step, be prepared into liquid first order seed and secondary seed by 5% inoculum size with the directed domestication of product;
(4) 3%～5% inoculum size that the secondary seed that (3) is prepared is pressed fermentating liquid volume inserts in the enzymatic production substratum of 10L, and when cultivating 96～114h for 24～26 ℃, promptly microbial fermentation is produced the glutamate dehydrogenase end;
(5) with the fermented liquid of (4) 6,000～8, the centrifugal collection liquid of 000rpm, with distilled water wash for several times after centrifugal collecting precipitation;
(6) deposition is suspended in the damping fluid, adds silica sand and grind fragmentation, 10,000～14,000rpm is centrifugal, and the supernatant of collecting is the glutamate dehydrogenase crude enzyme liquid;
(7) different with the use object according to different needs, the crude enzyme liquid that (6) are obtained further concentrates, separation and purification, is prepared into the zymin of different activities, purity and formulation.
Embodiment two:
(1) medium preparation
1. strain activation and culture base: Carnis Bovis seu Bubali cream 10.0g, peptone 10.0g, NaCl 5.0g, glucose 10.0g, zero(ppm) water 1.0L, 7.0～7.2,121 ℃ of high pressure steam sterilization 30min of pH value.
2. the directed acclimation shaking culture base of bacterial classification: glucose 8.0～12.0g, peptone 18.0～22.0g, Carnis Bovis seu Bubali cream 16.0～24.0g, NaCl 4.0～6.0g, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
3. liquid seed culture medium: glucose 22.0～26.0g, steeping water 24.0～28.0g, urea 1.5～3.5g, K
2HPO
41.0～2.5g, MgSO
40.4～0.8g, FeSO
40.002～0.006g, MnSO
40.004～0.008g, tap water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
4. enzymatic production substratum: glucose 25.0～45.0g, steeping water 4.0～8.0g, urea 4.0～6.0g, K
2HPO
40.5～2.5g, FeSO
40.001～0.004g, MgSO
45.0～7.0g, MnSO
40.002～0.005g, tap water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
The mikrobe that (2) will produce glutamate dehydrogenase through the directed domestication of product 4～6 times, makes its well-grown under 24～30 ℃ of envrionment conditionss again by 6～10 activation of bacterial strain explanation carrying out that Chinese common micro-organisms culture presevation administrative center (CGMCC) provides;
(3) produce bacterium by the glutamate dehydrogenase of ordinary method after and cultivate 36～60h at 24～30 ℃ and then carry out enlarged culturing step by step, be prepared into liquid first order seed and secondary seed by 5% inoculum size with the directed domestication of product;
(4) 5%～7% inoculum size that the secondary seed that (3) is prepared is pressed fermentating liquid volume inserts in the enzymatic production substratum of 50L, and when cultivating 78～96h for 26～28 ℃, promptly microbial fermentation is produced the glutamate dehydrogenase end;
(5) with the fermented liquid of (4) 6,000～8, the centrifugal collection liquid of 000rpm, with distilled water wash for several times after centrifugal collecting precipitation;
(6) deposition is suspended in the damping fluid, adds silica sand and grind fragmentation, 10,000～14,000rpm is centrifugal, and the supernatant of collecting is the glutamate dehydrogenase crude enzyme liquid;
(7) different with the use object according to different needs, the crude enzyme liquid that (6) are obtained further concentrates, separation and purification, is prepared into the zymin of different activities, purity and formulation.
Embodiment three:
(1) medium preparation
1. strain activation and culture base: Carnis Bovis seu Bubali cream 10.0g, peptone 10.0g, NaCl 5.0g, glucose 10.0g, zero(ppm) water 1.0L, 7.0～7.2,121 ℃ of high pressure steam sterilization 30min of pH value.
2. the directed acclimation shaking culture base of bacterial classification: glucose 8.0～12.0g, peptone 18.0～22.0g, Carnis Bovis seu Bubali cream 16.0～24.0g, NaCl 4.0～6.0g, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
3. liquid seed culture medium: glucose 22.0～26.0g, steeping water 24.0～28.0g, urea 1.5～3.5g, K
2HPO
41.0～2.5g, MgSO
40.4～0.8g, FeSO
40.002～0.006g, MnSO
40.004～0.008g, tap water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
4. enzymatic production substratum: glucose 25.0～45.0g, steeping water 4.0～8.0g, urea 4.0～6.0g, K
2HPO
40.5～2.5g, FeSO
40.001～0.004g, MgSO4 5.0～7.0g, MnSO
40.002～0.005g, tap water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
The mikrobe that (2) will produce glutamate dehydrogenase through the directed domestication of product 4～6 times, makes its well-grown under 24～30 ℃ of envrionment conditionss again by 6～10 activation of bacterial strain explanation carrying out that Chinese common micro-organisms culture presevation administrative center (CGMCC) provides;
(3) produce bacterium by the glutamate dehydrogenase of ordinary method after and cultivate 36～60h at 24～30 ℃ and then carry out enlarged culturing step by step, be prepared into liquid first order seed and secondary seed by 5% inoculum size with the directed domestication of product;
(4) 7%～9% inoculum size that the secondary seed that (3) is prepared is pressed fermentating liquid volume inserts in the enzymatic production substratum of 100L, and when cultivating 60～78h for 28～30 ℃, promptly microbial fermentation is produced the glutamate dehydrogenase end;
(5) with the fermented liquid of (4) 6,000～8, the centrifugal collection liquid of 000rpm, with distilled water wash for several times after centrifugal collecting precipitation;
(6) deposition is suspended in the damping fluid, adds silica sand and grind fragmentation, 10,000～14,000rpm is centrifugal, and the supernatant of collecting is the glutamate dehydrogenase crude enzyme liquid;
(7) different with the use object according to different needs, the crude enzyme liquid that (6) are obtained further concentrates, separation and purification, is prepared into the zymin of different activities, purity and formulation.
Claims (3)
1. a microbial fermentation is produced the method for glutamate dehydrogenase (being abbreviated as GDH), and it may further comprise the steps:
The mikrobe that (1) will produce GDH carries out 6～10 activation, through the directed domestication of product 4～6 times, makes its well-grown under 24～30 ℃ of envrionment conditionss again;
(2) produce bacterium 24～30 ℃ of enlarged culturing step by step by the GDH of ordinary method after, be prepared into liquid first order seed and secondary seed the directed domestication of product;
(3) with liquid first order seed or secondary seed, 3～9% inoculum sizes of pressing fermentating liquid volume insert in the liquid fermentation medium, and when cultivating 60～114h for 24～30 ℃, promptly microbial fermentation is produced the GHD end;
(4) with the fermented liquid of (3) 6,000～8, the centrifugal collection liquid of 000rpm, with distilled water wash for several times after centrifugal collecting precipitation;
(5) deposition is suspended in the damping fluid, adds silica sand and grind fragmentation, 10,000～14,000rpm is centrifugal, and the supernatant of collecting is the DGH crude enzyme liquid;
(6) different with the use object according to different needs, the crude enzyme liquid that (5) are obtained further concentrates, separation and purification, is prepared into the zymin of different activities, purity and formulation.
2. method according to claim 1 further comprises in step (6) afterwards: the crude enzyme liquid that step (6) is obtained further concentrates, separation and purification, is prepared into the zymin of different activities, purity and formulation.
3. method according to claim 1 and 2, wherein the directed acclimation shaking culture base of bacterial classification, liquid seed culture medium, enzymatic production substratum are respectively:
(1) the directed acclimation shaking culture base of bacterial classification: glucose 8.0～12.0g, peptone 18.0～22.0g, Carnis Bovis seu Bubali cream 16.0～24.0g, NaCl 4.0～6.0g, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
(2) liquid seed culture medium: glucose 22.0～26.0g, steeping water 24.0～28.0g, urea 1.5～3.5g, K
2HPO
41.0～2.5g, MgSO
40.4～0.8g, FeSO
40.002～0.006g, MnSO
40.004～0.008g, tap water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
(3) enzymatic production substratum: glucose 25.0～45.0g, steeping water 4.0～8.0g, urea 4.0～6.0g, K
2HPO
40.5～2.5g, FeSO
40.001～0.004g, MgSO4 5.0～7.0g, MnSO
40.002～0.005g, tap water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1266905A (en) * | 1999-02-20 | 2000-09-20 | 德古萨－于尔斯股份公司 | Method for producing L-amino acid by fermentation with corynebacteria |
| CN1807610A (en) * | 2006-01-23 | 2006-07-26 | 迟乃玉 | Method for producing low temperature cellulase using microbe fermentation |
| CN101580812A (en) * | 2008-05-13 | 2009-11-18 | 长春大成实业集团有限公司 | Bacteria and method for producing L-glutamic acid |
| CN102093987A (en) * | 2009-12-11 | 2011-06-15 | 大连大学 | Method for producing low-temperature phytase by fermenting microorganisms |
-
2012
- 2012-04-12 CN CN2012101067279A patent/CN102653736A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1266905A (en) * | 1999-02-20 | 2000-09-20 | 德古萨－于尔斯股份公司 | Method for producing L-amino acid by fermentation with corynebacteria |
| CN1807610A (en) * | 2006-01-23 | 2006-07-26 | 迟乃玉 | Method for producing low temperature cellulase using microbe fermentation |
| CN101580812A (en) * | 2008-05-13 | 2009-11-18 | 长春大成实业集团有限公司 | Bacteria and method for producing L-glutamic acid |
| CN102093987A (en) * | 2009-12-11 | 2011-06-15 | 大连大学 | Method for producing low-temperature phytase by fermenting microorganisms |
Non-Patent Citations (1)
| Title |
|---|
| 顾薇 等: "谷氨酸脱氢酶发酵工艺的正交试验设计", 《化工时刊》, vol. 24, no. 1, 31 January 2010 (2010-01-31), pages 16 - 19 * |
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Application publication date: 20120905 |