CN101407436A - Novel biological nitrogen fixation enzyme preparation (HSE preparation) - Google Patents
Novel biological nitrogen fixation enzyme preparation (HSE preparation) Download PDFInfo
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- CN101407436A CN101407436A CNA2008100337486A CN200810033748A CN101407436A CN 101407436 A CN101407436 A CN 101407436A CN A2008100337486 A CNA2008100337486 A CN A2008100337486A CN 200810033748 A CN200810033748 A CN 200810033748A CN 101407436 A CN101407436 A CN 101407436A
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Abstract
The invention relates to a biological reagent, which is a novel biological azotase preparation (HSE preparation) generated by the fermentation of a microorganism (Haitobacter sufflavus Tih-w37) which has proprietary intellectual property rights and is separated from soil, and the preparation can fix the nitrogen in the air or fix the nitrogen to ammonia. The method comprises the following steps: under a certain temperature and pH value, the HSE preparation is directly cast into the water with air or nitrogen to turn the nitrogen which is hard to be fixed into ammonia, wherein, the equilibrium concentration of the ammonia can be higher than 6mM; under the optimum condition, the thalli with 0.1g of green weight can produce 0.23mM of ammonia in 120 hours. The biological reagent is characterized in that the HSE preparation prepared by the fermentation of the microorganism in a culture medium can stabilize oxygen, nitrogen, and the like and has the function of catalyzing the nitrogen to react with water to generate ammonia.
Description
New bio nitrogenase preparation (HSE preparation) is a kind of biological enzyme formulation of making by the microbial fermentation that independent intellectual property right is arranged that is located away from soil.Reaction generates ammoniacal liquor to the HSE preparation to the nitrogen G﹠W in the stabilization energy catalytic airs such as oxygen, ammonia.The HSE preparation is a kind of biological reagent that has the challenge meaning, still is that theoretical investigation all has unusual value in practical application.
One, affiliated field
The invention belongs under a kind of normal temperature and pressure the biological nitrogen fixation zymin (HSE preparation) that airborne nitrogen is fixed up.
Two, technical background
Biological nitrogen fixation is one of great fundamental research problem in the life science, and Biological Nitrogen Fixation Researches enters a new phase both at home and abroad, is characterized in subject crossing, and fundamental research and application prospect are combined.For these key subjects being had breakthrough and simulating biological nitrogen fixation and use, current Biological Nitrogen Fixation Researches is just launched on molecule and atomic level
[1]People have verified traditional nitrogenase to the inactivation very easily of instability such as oxygen, ammonia etc. especially, and biological nitrogen fixation mechanism is further illustrated and used is the thing that the mankind dream of.
Germanization scholar Haber Fritz had invented with Fe in 1909
3O
4Be main catalyzer, under high-temperature and high-pressure conditions, make nitrogen and hydrogen reaction, the method for synthetic ammonia.
In the last hundred years, this is called as the invention of 20th century maximum, has made great contribution for promoting the well-being of mankind, but it is not only a high investment, hangs down the process of output, and has brought suitable negative impact to the environment that the mankind depend on for existence.However, the mankind still must not and for it.Therefore, under the Wen He condition, the repercussion study that the very inactive nitrogen molecule of chemical property is changed ammonification is the important process that is related to the environment and the energy more.
The nitrogen element is a nucleic acid, amino acid, the important component part of protein etc., it is the element of activity necessity of earning a bare living, be to build the necessary plastics of modern civilization life simultaneously, fiber, pharmaceuticals, one of indispensable important element such as industrial chemicals, yet, nitrogen abundant in the atmosphere contains very big, " N ≡ N " triple bond demonstrates its extremely difficult reaction properties in conjunction with energy (946KJ/MEL), thereby, under normal temperature and pressure conditions, making it change nitrogen compound into is not an easy thing.On the other hand, the nitrogenase that vinelandii produced at normal temperatures and pressures, just airborne nitrogen can be fixed up, from the total amount of the annual fixed nitrogen of gauge ball mould entirely: the fixed nitrogen of commercial run only account for total amount less than 20%, remaining 80% major part is that Institute of Micro-biology is
1)As reality less and less such as human important energy coal, oil, paid close attention to by the world, and the quick growth of world population, also more and more to the demand of fertilizer.To the low-down nitrogen of reactivity, if can by or touch bionical thing nitrogenase, it is reacted, therefore, the chemical simulation biological nitrogen fixation become the thing that the mankind dream of will be the significant problem of a Entries.
People have carried out extensive studies in order to utilize the catalytic mechanism of nitrogen-fixing microorganism and announcement (understanding) nitrogenase to the nitrogen-fixing microorganism source.From the anaerobion to the aerobic microbiological, from symbiotic microorganism to free microorganism, found 30 surplus class
2)(table 1-1)
Table 1-1 nitrogen-fixing microorganism kind table
2)
Since the sixties in last century, institute of U.S. Du Pont de Nemours central authorities, from vinelandii, successfully extracted out since the nitrogenase first, the investigator has also carried out the research of different levels to the nitrogenase of various types of nitrogen-fixing microorganisms and generation thereof.
Many microorganisms (nif) wherein and the nitrogen fixing capacity of nitrogenase, structure etc. are studied widely
3) 5) 6), the general character of its nitrogenase is: 1) be made of two kinds of protein; 2) susceptibility to oxygen is big, easily inactivation; 3) all containing metal iron and molybdenum; 4) magnesium ion is necessary to their activity; 5) ATP is converted into ADP when activity, and ADP encumbers its activity; 6) its major part has the ability of reduction triplen molecule, as nitrogen, and second hydrocarbon etc., in the performance active process, hydrogen ion is reduced into hydrogen and emits
3) 8) 9)7) ammonia of Sheng Chenging is to its encumbering property of activity
2)Its fatal part is that oxygen, ammonia etc. have infringement to it, and making to the research of nitrogenase and directly using becomes difficulty.
However, now people Dui Bi the Azotobacter of the easy research of More vinelandii; The nitrogenase that nitrogen-fixing microorganisms such as Clostridium are produced has carried out the analysis of X-crystal structure, and the active centre of its nitrogenase has been configured with higher level understanding
3) 7) 8) 9)Azotobacter vinelandii is to produce to contain one of bacterial classification of molybdenum-iron nitrogenase, by two kinds independently the responsive albumen of oxygen form: Mo-Fe protein (MoFe albumen, component I) and ferritin (Fe albumen, component I I).Nineteen sixty-five is a material with Clostridium baratii (Cp) first, and separation and purification has obtained two protein ingredients of nitrogenase
2), i.e. ferritin (Fe albumen) and Mo-Fe protein (MoFe albumen), the about 60KD of ferritin molecular weight by the dimer that two identical subunits are formed, contains one 4Fe-4S bunch, and bridging is between two subunits.The about 220KD of Mo-Fe protein molecular weight is by α
2β
2Form the tetramer, contain 2 about 32 Fe of Mo and equal amounts of S
2-Atom.These metals form two special 4F-4S bunch (claim P-bunch to) and two iron-molybdenum cofactors (FeMo2co)
20) 8-14)Do not present nitrogenase activity during above-mentioned two protein ingredient Individual existences, when only combining, the fixed nitrogen function is arranged just under certain condition.
Nitrogenase two component proteins are all extremely responsive to oxygen, and responsive more with the raising of purity.The transformation period of ferritin is 30～45 seconds in air, and the transformation period of Mo-Fe protein is 10 minutes
18)Need under the strictly anaerobic condition, carry out for keeping active during the vitro study nitrogenase.Slightly variant between the different diazotrophs to the susceptibility of oxygen, and all have different anti-oxygen protected modes
18)Nitrogenase is present in the bacteroid in root nodule.And the special construction of root nodule and physiological environment not only protected nitrogenase but also suitable fixed nitrogen condition be provided, and wherein leghemoglobin plays important oxygen therapy effect.Nitrogenase is to cold sensitivity, generally easy inactivation about 0 ℃.Wherein ferritin is to cold the most responsive, but there are differences between the different diazotroph.Yet all nitrogenases all are stable under-200 ℃, so the nitrogenase of purifying all can be kept in the liquid nitrogen
19)
Under field conditions (factors), N
2With proton be the substrate of nitrogenase.The every reduction a part of nitrogenase N
2Will discharge a part H
2When without any other substrate, whole electronics of nitrogenase all are used for hydrionic reduction and discharge hydrogen.The hydrogen of putting of nitrogenase also needs adenosine triphosphate (ATP), and this then is different from the hydrogen enzyme
22)Except that above-mentioned natural substrate, nitrogenase is that separate in the biology unique can reduce the enzyme of three key compounds.Its substrate majority is to have triple bond or potential triple-linked molecule, as H-S-C ≡ N, CH
2-N
+≡ C-S, N ≡ C-C=N, CH
2=CH-N-C, and just obtained the carbon oxysulfide (COS) and the CO that prove recently
2Also be the substrate of nitrogenase, however its analogue CS
2It then is inhibitor
23)What cause people's widespread use in substrate is acetylene, because of the acetylene reduction is used as the easy sensitive method of measuring nitrogenase activity by people.
The adenosine triphosphate of nitrogenase and reductive agent
24)Diazotroph can be because of there being the biological catalyst nitrogenase in its body with airborne nitrogen reduction ammonification.Biological nitrogen fixation also is the reaction of a power consumption, in order to reduce above-mentioned substrate, then needs ATP and reductive agent (electronics).General ATP and Mg in complete fixed nitrogen organism
2+Be the composite form existence with 1: 1, a large amount of experiments show that nitrogenase whenever provides an electronics to N
2The ATP (generating product A DP and inorganic phosphorus) that needs 2 molecules of hydrolysis, i.e. ATP/e
-Than being 2.Therefore, nitrogenase catalyzing N
2The reductive reaction formula can be write:
N
2+8H
++8e
-+16MgATP→2NH
3+H
2+16MgAD+16Pi。
In the equation:
ATP: adenosine triphosphate is provided by breathing, anaerobic respiration, fermentation or photosynthesis;
ADP: adenosine diphosphate (ADP);
Pi: inorganic phosphate.
The nitrogenase catalyticing mechanism.The nitrogenase mechanism of action comprises the transmission of electronics, complexing and reduction two portions of substrate.Stream of electrons by the nitrogenase system enters by ferritin, and emitted by Mo-Fe protein with the substrate form that has been reduced.From the results presumption of nitrogenase two component protein structural analyses, the order of electron transport is in the nitrogenase system:
The proteic P of Fe albumen → MoFe
2Bunch right → FeMo2co → substrate
The hydrolysis of MgATP is the first step coupling with electron transport, and when substrate and FeMo2co complexing, electronics and proton transfer are given substrate
25)
Dynamics research shows that also ferritin once transmits an electronics and gives Mo-Fe protein, and a this electron transfer process is comprising coupling reconciliation link coupled working cycle between ferritin and the Mo-Fe protein again, and uncoupling also is the conditioning step of decision enzyme ' s reaction speeding
26)
At present, the research of being engaged in the chemical simulation biological nitrogen fixation in the world roughlly speaking has three aspects, and the one, the structure of simulation nitrogenase is by the synthetic similar catalyzer of chemical process
1)The 2nd, seek the microorganism that new nitrogen-fixing microorganism or mutagenesis make new advances with special nitrogen fixing capacity; The 3rd, do genetic modification or clone unconventional nitrogen-fixing microorganism or plant.Up to the present, really can be applied to also not emerging in the industry on a large scale.No matter the research of that aspect, the deep layer understanding that nitrogenase is constructed is very important.Our country also just had the investigator to separate as far back as 1978 and the molybdoiron protein matter crystallization of having purified out, had made very big contribution for the mankind further are familiar with nitrogenase.
Three, summary of the invention
Title: a kind of new bio nitrogenase preparation (HSE preparation).The HSE preparation can be at normal temperatures and pressures, with the airborne nitrogen generation ammoniacal liquor that is fixed up.New bio nitrogenase preparation (HSE preparation) is a kind of biological enzyme formulation of making by the microbial fermentation that is located away from soil.
Function: the HSE preparation fixedly is converted into ammoniacal liquor with airborne nitrogen at an easy rate to stabilization energies such as oxygen, ammonia, and the ammoniacal liquor equilibrium concentration can be up to more than the 6mM; Under the optimum condition, included enzyme thalline weight in wet base 0.1 and restrain the ammonia that to produce 0.23mM approximately in 120 hours.On biotechnology, basic scientific research, be widely used and the research prospect.
The scope of application: the function that ammonia is produced in airborne nitrogen or the reaction of purity nitrogen G﹠W.
The contents are as follows: (Dang Time studied abroad in Japan in 1999), I have isolated the novel nitrogen-fixing microorganism of a strain (Haitobacter sufflavus Tih-w37) from soil.The biotechnological formulation (HSE preparation) that mainly utilizes this microbial fermentation to produce has the function of the nitrogen G﹠W reaction generation ammoniacal liquor in the catalytic air.Concrete grammar is, by measuring the weight in wet base throw out 0.1g that obtains by the centrifugation of HSE preparation as the nitrogenase agent, add 10ml distilled water then, in air, carry out centrifugation behind the reaction certain hour, supernatant liquor is the ammoniacal liquor for producing then, and precipitation then be the zymin of usefulness next time---, like this, move in circles and carry out ammoniacal liquor production (accompanying drawing 1, accompanying drawing 2-4).Find that this biotechnological formulation has the specific competence that produces ammoniacal liquor, and the ammonia concn that obtains of each centrifugation production in time prolongation and increase
14) 15) 16)The ammonia concn test is finished (accompanying drawing 1) by liquid chromatography (HPLC).
By with the comparative studies of the nitrogenase of the stronger Azotobacter Vinelandii type strain of known nitrogen fixing capacity, find under the same conditions, this new bio nitrogenase preparation is at least 5 times of type strain nitrogenase nitrogen fixing capacity, and it is reusable, be difficult for inactivation, the concentration of its each synthetic ammonia does not almost have anything to change (accompanying drawing 2).The then very fast inactivation of type strain nitrogenase loses the ammoniacal liquor ability of producing.
By (the NH of 0.1g weight in wet base precipitation enzyme agent (HSE preparation) in various concentration
4)
2SO
4React in the solution, and the concentration of reacting back ammoniacal liquor is tested, learn that ammonia is not obvious to the nitrogen fixing capacity influence of this novel nitrogenase, stable to ammonia.The equilibrium concentration of finding HSE preparation ammonia in water can be up to (accompanying drawing 3) more than the 6mM.
On the contrary, the nitrogenase that type strain Azotobacter vinclandii is produced is inactivation in a short period of time, can not be repeated to utilize (accompanying drawing 2,4)
16)In one month, 0.1 gram weight in wet base preparation can constantly have been emitted ammoniacal liquor (accompanying drawing 4) by recycling, inactivation reparation.
In order further to confirm the nitrogenase activity of HSE preparation, also carried out
15N
2Isotropic substance produce the ammoniacal liquor test.Confirmed the fixed nitrogen performance of HSE preparation.
Four, HSE preparation constituent material
This ASE preparation is to be made of zymoprotein, microorganism, water, small amounts of inorganic salt etc.;
Main enzyme: the nitrogenase that Fe albumen and MoFe albumen etc. constitute.
Mechanics: microorganism and molybdate, phosphoric acid salt, molysite, magnesium salts, the biotechnological formulation that fermentations such as sucrose form.
Five, embodiment
The HSE preparation directly is sprinkled in the water that contains air or nitrogen, and the HSE preparation just can be fixed nitrogen, change into ammoniacal liquor as catalyzer.At 30 ℃ of temperature, pH7, have under the condition of enough substrate nitrogen G﹠W reactions, thalline weight in wet base 0.1 gram can be produced the ammonia of 0.23mM approximately in 120 hours.
Concrete diagram illustration, the accompanying drawing 1-4. of implementing
Description of drawings is as follows:
Accompanying drawing 1 is that 0.1 gram weight in wet base thalline is produced ammoniacal liquor and analytical procedure thereof respectively in 10ml water;
Accompanying drawing 2 is the novel nitrogen-fixing microorganism Haitobacter sufflavus Tih-w37 of 0.1 gram weight in wet base thalline and the comparative result of the nitrogen fixing capacity of nitrogenase under differing temps that type strain Azotobacter vinelandii produces;
Accompanying drawing 3 is different concns (NH
4)
2SO
4The nitrogenase nitrogen fixing capacity that solution is produced novel nitrogen-fixing microorganism Haitobacter sufflavus Tih-w37 influence the result;
Accompanying drawing 4 is the novel nitrogen-fixing microorganism Haitobacter sufflavus Tih-w37 of 0.1 gram weight in wet base thalline and type strain Azotobacter vinelandii continued to produce ammoniacal liquor in one month result.
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Claims (5)
1. a new bio nitrogenase preparation (HSE preparation) is a biological nitrogen fixation zymin of utilizing fermentation outputs such as the microorganism that independent intellectual property right is arranged (Haitobacter sufflavus Tih-w37) be located away from soil, phosphoric acid salt, molysite, molybdate.Its fixed nitrogen method is under certain temperature, pH value, and the HSE preparation directly is sprinkled in the water that is dissolved with air or nitrogen, just can change into ammoniacal liquor with being difficult to fixed nitrogen.It is characterized in that: the HSE preparation that utilizes microorganism to ferment in substratum and make has the function that the reaction of catalysis nitrogen G﹠W generates ammonia.
2. a kind of new bio nitrogenase preparation according to claim 1 (HSE preparation), it is characterized in that: utilize the HSE preparations that fermentation is made in substratum such as microorganism (Haitobacter sufflavus Tih-w37), at temperature 0-40 ℃, the pH value is under the 4.5-8.5 condition, and the reaction of catalysis nitrogen G﹠W generates the biological method of ammonia.
3. a kind of new bio nitrogenase preparation according to claim 2 (HSE preparation) is characterized in that: when the HSE preparation was brought into play nitrogenase activity under conditions suitable, the product that is generated was the resource of our company.
4. a kind of new bio nitrogenase preparation according to claim 1 (HSE preparation) is characterized in that: the HSE preparation is the biomaterial of being made up of microorganism, enzyme etc. of fermentation such as microorganism (Haitobacter sufflavus Tih-w37), phosphoric acid salt, molysite, molybdate outputs.
5. a kind of new bio nitrogenase preparation according to claim 1 (HSE preparation) is characterized in that: HSE preparation and to use as the agent of diazotroph enzyme be peer-to-peer, the development and use of other purposes of HSE preparation are our company's resources.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102160920A (en) * | 2011-02-18 | 2011-08-24 | 上海居知园生物技术有限公司 | Novel biological material for degrading and transforming organic matters and poisonous things thereof |
| CN101948182B (en) * | 2009-12-21 | 2016-12-14 | 上海居知园生物技术有限公司 | A kind of biological preparation is for the Degradation and Transformation method of nitrite |
| CN115500256A (en) * | 2022-11-05 | 2022-12-23 | 北京化工大学 | Photocatalysis nitrogen fixation plant water planting growth device |
-
2008
- 2008-02-21 CN CNA2008100337486A patent/CN101407436A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948182B (en) * | 2009-12-21 | 2016-12-14 | 上海居知园生物技术有限公司 | A kind of biological preparation is for the Degradation and Transformation method of nitrite |
| CN102160920A (en) * | 2011-02-18 | 2011-08-24 | 上海居知园生物技术有限公司 | Novel biological material for degrading and transforming organic matters and poisonous things thereof |
| CN115500256A (en) * | 2022-11-05 | 2022-12-23 | 北京化工大学 | Photocatalysis nitrogen fixation plant water planting growth device |
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