CN103374556B - Method for producing low-temperature hydrogen-amino oxidase from heterotrophic nitrification acinetobacter L7 of Harbin Institute of Technology, and separation and purification method thereof - Google Patents
Method for producing low-temperature hydrogen-amino oxidase from heterotrophic nitrification acinetobacter L7 of Harbin Institute of Technology, and separation and purification method thereof Download PDFInfo
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Abstract
The invention discloses a method for producing low-temperature hydrogen-amino oxidase from heterotrophic nitrification acinetobacter L7 of Harbin Institute of Technology, and a separation and purification method of the low-temperature hydrogen-amino oxidase, and relates to a method for enzyme production and a separation and purification method of enzymes. The method comprises the following steps of: producing enzymes, namely, inoculating L7 strains into an enzyme production fluid nutrient medium to be cultured, separating and purifying, namely, firstly, inoculating the L7 strains into the enzyme production fluid nutrient medium to be cultured, and collecting thallus, secondly, crushing the thallus and preparing an extracellular matrix without lysozyme, and thirdly, separating the extracellular matrix, balancing, eluting, carrying out chromatography, carrying out electrophoresis, and collecting monomer proteins. The L7 strains inoculated into the enzyme production fluid nutrient medium are high in enzyme yield and stable in activity, and the enzyme activity is the highest and the enzyme yield is the highest at 15 DEG C. According to the separation and purification method disclosed by the invention, the produced low-temperature hydrogen-amino oxidase is a monomer protein with a molecular weight of 60.4KDa, the enzyme activity is stable at 2-25 DEG C when the pH value is 7.5 to 8.5, and ammonia nitrogen in low-temperature water is effectively eliminated.
Description
Technical field
The present invention relates to the method for producing enzyme.
Background technology
Biological denitrificaion be generally acknowledge at present both at home and abroad the most effectively, the technology of ammonia and nitrogen pollution in the removal water of most application prospect, be also research emphasis and the focus of current field of water pollution control.No matter be Autotrophic nitrification bacterium or nitrification bacteria, in ammonia nitrogen degradation process, all can be subject to temperature impact.Envrionment temperature reduces will cause ammonia nitrogen degradation deleterious, and especially when temperature is lower than 15 DEG C, microorganism stops growing and ammonia nitrogen cannot be degraded.The northern area of China water temperature in winter is usually less than 10 DEG C, and for Heilongjiang Province, water temperature in winter is minimum below 2 DEG C, and low temperature makes ammonia nitrogen removal become a difficult problem.
The electron transport chain that ammonia nitrogen degradation process in water is made up of a series of oxydo-reductase completes, wherein azanol (the NH that formed by ammonium oxidation of hydroxylamine oxidase (Hydroxylamine oxidoreductase, HAO)
2oH) nitrite nitrogen (NO is oxidized to
2 --N), play a crucial role in ammonia nitrogen degradation process.HAO in Autotrophic nitrification bacterium (Nitrosomonas europaea) is present in periplasmic, it is the reduced hematin of about 180kDa, comprise the α subunit of 3 63kDa and β subunit (Hooper, the A.in Autotrophic Bacteria Biochemistry of the Nitrifying Lithoautotrophic Bacteria of 3 11kDa; Schlegel and Bowien, Eds; Scinence Tech Publishers:Madison, WI 1989; Chapter13, pp239-265).In nitrification bacteria, also isolated HAO, but the HAO of different strains has specificity.In nitrification bacteria Thiosphaera pantotropha and Alcaligenes faecalis, isolated HAO is the small-sized monomeric protein of 20kDa, be present in (Wehrfritz in periplasmic, J.M., Reilly A., Spiro S.et al.Purification of hydroxylamine oxidase from Thiosphaera pantotropha identification of electron acceptors that couple heterotrophic nitrification to aerobic denitrification.FEBS, 1993,335:246-250; Otte, S., Schalk, J., Kuenen, J.G.et al.Hydroxylamine oxidation and subsequent nitrous oxide production by the heterotrophic ammonia oxidizer Alcaligenes faecalis.Appl Microbiol Biotechnol, 1999,51:255-261).The dimer of isolated HAO to be one-component be 132kDa in nitrification bacteria Pseudomonas PB16, α subunit (Jetten M.S.M. containing two 68kDa, de Bruijn P., Kuenen J.G.Hydroxylamine metabolism in Pseudomonas PB16:involvement of a novel hydroxylamine oxidoreductase.Antonie van Leeuwenhoek, 1997,71:69-74).Isolated HAO enzyme optimal reactive temperature is 25-35 DEG C above, and under cryogenic, enzymatic reaction speed declines rapidly, becomes the one of the main reasons of ammonia nitrogen degradation weak effect under low temperature.Therefore, oxyammonia oxidase activity under raising low temperature, high reactivity oxyammonia oxydase under separation low temperature, to releasing low temperature restraining effect, improves ammonia nitrogen degradation effect, significant.
Summary of the invention
The present invention seeks to the problem cannot effectively removed to solve existing low temperature ammonia nitrogen, and provide Harbin Institute of Technology's heterotrophic nitrification motionless bacterium L7 to produce the oxidasic method of low temperature oxyammonia.
Harbin Institute of Technology's heterotrophic nitrification motionless bacterium L7 produces the oxidasic method of low temperature oxyammonia, carries out according to the following steps:
By motionless for Harbin Institute of Technology's heterotrophic nitrification bacterium L7 (Acinetobacter Heteronitrifihit L7) inoculation in culture medium, inoculum size is 2%, at 160rpm/min, 15 DEG C of cultivation 14 ~ 24h, namely complete the motionless bacterium L7 of Harbin Institute of Technology's heterotrophic nitrification and produce low temperature oxyammonia oxydase; Wherein the component of culture medium is as follows: NH
4cl0.1g/L, CH
3cH
2oNa1.0g/L, NH
2oH0.7g/L, MgSO
47H
2o0.05g/L, K
2hPO
40.2g/L, NaCl0.12g/L, CuSO
45H
2o0.01g/L, NaMoO
42H
2o0.05g/L, BaCl
20.01g/L, MnSO
44H
2o0.01g/L, FeSO
40.01g/L, K [Fe (CN)
6] distilled water of 0.01g/L, CtyC0.02g/L and surplus, pH value 7.0 ~ 7.4.
Harbin Institute of Technology's heterotrophic nitrification motionless bacterium L7 produces the oxidasic separation purification method of low temperature oxyammonia, carries out according to the following steps:
One, by motionless for Harbin Institute of Technology's heterotrophic nitrification bacterium L7 (Acinetobacter Heteronitrifihit L7) inoculation in culture medium, inoculum size is 2%, at 160rpm/min, 15 DEG C of cultivation 14 ~ 24h, then after the centrifugal 5min of 6000rpm, collect thalline;
Two, ultrasonication 15min in ice-water bath is placed on by resuspended for the Tris-HCI damping fluid of thalline 10mmol/L, pH8.5, then N,O-Diacetylmuramidase is adopted to carry out fragmentation to cell broken liquid, the thalline of collection is divided into tenuigenin, periplasmic and cytolemma three part, again by osmosis, prepare the extracellular matrix of not lysozyme;
Three, apply DEAE-CL6B ion exchange column to be separated the extracellular matrix collected, and balance with the Tris-HCl of 5mM, pH8.0, in identical damping fluid, wash-out is carried out with 0-500mM gradient NaCl, oxyammonia oxydase (HAO) elutes under the NaCl condition of 80mM from ion exchange column, then removes cytochrome C by cross-linked polyacrylamide dextran S-100 gel chromatography
551, then denaturing polyacrylamide gel electrophoresis (SDS-PAGE), collect the monomeric protein of molecular weight 60.4KDa, namely complete Harbin Institute of Technology's heterotrophic nitrification motionless bacterium L7 and produce the oxidasic separation and purification of low temperature oxyammonia;
Wherein in step one, the component of culture medium is as follows: NH
4cl0.1g/L, CH
3cH
2oNa1.0g/L, NH
2oH0.7g/L, MgSO
47H
2o0.05g/L, K
2hPO
40.2g/L, NaCl0.12g/L, CuSO
45H
2o0.01g/L, NaMoO
42H
2o0.05g/L, BaCl
20.01g/L, MnSO
44H
2o0.01g/L, FeSO
40.01g/L, K [Fe (CN)
6] distilled water of 0.01g/L, CtyC0.02g/L and surplus, pH value 7.0 ~ 7.4.
In the present invention, Harbin Institute of Technology's heterotrophic nitrification motionless bacterium L7 produces the oxidasic method of low temperature oxyammonia, culture medium used is independent development, be prepared from for bacterial strain L7 feature and oxyammonia oxydase characteristic, the yield of enzyme of inoculation Harbin Institute of Technology's heterotrophic nitrification motionless bacterium L7 is high, and when 15 DEG C, enzymic activity is the highest (is worth for 0.31mmolO
2min
-1mg
-1), activity stabilized, yield of enzyme is the highest, and (value is 0.3mmolmin
-1mg
-1), active higher than having been reported middle ammona monooxygenase.
The comparison of carbon source in culture medium used in the present invention, sodium acetate can promote to produce enzyme, and propionic salt, butyrates take second place.
In the present invention, Harbin Institute of Technology's heterotrophic nitrification motionless bacterium L7 produces the oxidasic separation purification method of low temperature oxyammonia, and gained low temperature oxyammonia oxydase is the monomeric protein of molecular weight 60.4KDa, and at 2 ~ 25 DEG C, pH value 7.5 ~ 8.5 enzymic activity is stablized.Fe
3+there is activation to isolated oxyammonia oxydase, need cytochrome C to make electron acceptor(EA).High density Mn
2+, Ca
2+, Cu
2+have restraining effect to enzyme, chemical reagent SDS, urea, DTT have high inhibition effect to enzyme, can effectively remove ammonia nitrogen in low temperature water difficulty.
Accompanying drawing explanation
Fig. 1 is the low temperature oxyammonia oxydase obtained after separation and purification in embodiment three, and wherein M represents Marker, and 1 represents the low temperature oxyammonia oxydase that separation and purification goes out.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: present embodiment Harbin Institute of Technology heterotrophic nitrification motionless bacterium L7 produces the oxidasic method of low temperature oxyammonia, carries out according to the following steps:
By motionless for Harbin Institute of Technology's heterotrophic nitrification bacterium L7 (Acinetobacter Heteronitrifihit L7) inoculation in culture medium, inoculum size is 2%, at 160rpm/min, 15 DEG C of cultivation 14 ~ 24h, namely complete the motionless bacterium L7 of Harbin Institute of Technology's heterotrophic nitrification and produce low temperature oxyammonia oxydase; Wherein the component of culture medium is as follows: NH
4cl0.1g/L, CH
3cH
2oNa1.0g/L, NH
2oH0.7g/L, MgSO
47H
2o0.05g/L, K
2hPO
40.2g/L, NaCl0.12g/L, CuSO
45H
2o0.01g/L, NaMoO
42H
2o0.05g/L, BaCl
20.01g/L, MnSO
44H
2o0.01g/L, FeSO
40.01g/L, K [Fe (CN)
6] distilled water of 0.01g/L, CtyC0.02g/L and surplus, pH value 7.0 ~ 7.4.
The motionless bacterium L7 of the heterotrophic nitrification of Harbin Institute of Technology described in present embodiment (Acinetobacter Heteronitrifihit L7), be derived from patent " a strain low temperature nitrification bacteria (application number: 201310192043X, the applying date: 2013.5.22) "; It is in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, and deposit number is CGMCC No.7486, and preservation address is No. 3, No. 1, Beichen Lu, Chaoyang District, Beijing City institute.
Cultivate after 14 ~ 24h and measure oxyammonia oxidase activity for 15 DEG C in present embodiment, result to show that 15 DEG C time the highest (value is 0.31mmolmin to enzymic activity
-1mg
-1), low temperature is less on enzymic activity impact, when 2 DEG C, and when enzymic activity is 15 DEG C 77.4%.
In present embodiment, during 15 DEG C of cultivation 14 ~ 24h, yield of enzyme is the highest (is worth for 0.3mmolmin
-1mg
-1).
Embodiment two: present embodiment and embodiment one are unlike CH in the component of described culture medium
3cH
2oNa can be substituted by propionic salt or butyrates.Other is identical with embodiment one.
Embodiment three: present embodiment Harbin Institute of Technology heterotrophic nitrification motionless bacterium L7 produces the oxidasic further separation purification method of low temperature oxyammonia, carries out according to the following steps:
One, by motionless for Harbin Institute of Technology's heterotrophic nitrification bacterium L7 (Acinetobacter Heteronitrifihit L7) inoculation in culture medium, inoculum size is 2%, at 160rpm/min, 15 DEG C of cultivation 14 ~ 24h, then after the centrifugal 5min of 6000rpm, collect thalline;
Two, ultrasonication 15min in ice-water bath is placed on by resuspended for the Tris-HCI damping fluid of thalline 10mmol/L, pH8.5, then N,O-Diacetylmuramidase is adopted to carry out fragmentation to cell broken liquid, the thalline of collection is divided into tenuigenin, periplasmic and cytolemma three part, again by osmosis, prepare the extracellular matrix of not lysozyme;
Three, apply DEAE-CL6B ion exchange column to be separated the extracellular matrix collected, and balance with the Tris-HCl of 5mM, pH8.0, in identical damping fluid, wash-out is carried out with 0-500mM gradient NaCl, oxyammonia oxydase (HAO) elutes under the NaCl condition of 80mM from ion exchange column, then removes cytochrome C by cross-linked polyacrylamide dextran S-100 gel chromatography
551, then denaturing polyacrylamide gel electrophoresis (SDS-PAGE), collect the monomeric protein of molecular weight 60.4KDa, namely complete Harbin Institute of Technology's heterotrophic nitrification motionless bacterium L7 and produce the oxidasic separation and purification of low temperature oxyammonia;
Wherein in step one, the component of culture medium is as follows: NH
4cl0.1g/L, CH
3cH
2oNa1.0g/L, NH
2oH0.7g/L, MgSO
47H
2o0.05g/L, K
2hPO
40.2g/L, NaCl0.12g/L, CuSO
45H
2o0.01g/L, NaMoO
42H
2o0.05g/L, BaCl
20.01g/L, MnSO
44H
2o0.01g/L, FeSO
40.01g/L, K [Fe (CN)
6] distilled water of 0.01g/L, CtyC0.02g/L and surplus, pH value 7.0 ~ 7.4.
Carry out wash-out with 0-500mM gradient NaCl in step 3 in present embodiment, measure absorbance at 410nm, collecting cell pigment part, cytopigment, as the electron acceptor(EA) of HAO, do not need to be further purified.
In present embodiment, in step 3, oxyammonia oxydase (HAO) elutes under the NaCl condition of 80mM from ion exchange column, and oxyammonia oxydase is at this moment easily by cytochrome C
551pollute, so need to remove cytochrome C by cross-linked polyacrylamide dextran S-100 gel chromatography
551.
The monomeric protein (see Fig. 1) of gained molecular weight 60.4KDa in present embodiment, i.e. low temperature oxyammonia oxydase, stablizes 2 ~ 25 DEG C of enzymic activitys, when temperature is more than 40 DEG C, enzyme deactivation.Stablize in pH value 7.5 ~ 8.5 enzymic activity, when pH value is 8.0, enzymic activity is the highest.Fe
3+there is activation to isolated oxyammonia oxydase, need cytochrome C to make electron acceptor(EA).High density Mn
2+, Ca
2+, Cu
2+have restraining effect to enzyme, chemical reagent SDS, urea, DTT have high inhibition effect to enzyme.
Embodiment four: present embodiment and embodiment three are unlike CH in the component of culture medium described in step one
3cH
2oNa can be substituted by propionic salt or butyrates.Other step and parameter identical with embodiment three.
Claims (2)
1. Harbin Institute of Technology's heterotrophic nitrification motionless bacterium L7 produces the oxidasic method of low temperature oxyammonia, it is characterized in that it carries out according to the following steps:
By motionless for Harbin Institute of Technology's heterotrophic nitrification bacterium L7 (Acinetobacter Heteronitrifihit L7) inoculation in culture medium, inoculum size is 2%, at 160rpm, 15 DEG C of cultivation 14 ~ 24h, namely complete the motionless bacterium L7 of Harbin Institute of Technology's heterotrophic nitrification and produce low temperature oxyammonia oxydase; Wherein the component of culture medium is as follows: NH
4cl 0.1g/L, propionic salt or butyrates 1.0g/L, NH
2oH 0.7g/L, MgSO
47H
2o 0.05g/L, K
2hPO
40.2g/L, NaCl 0.12g/L, CuSO
45H
2o 0.01g/L, NaMoO
42H
2o 0.05g/L, BaCl
20.01g/L, MnSO
44H
2o 0.01g/L, FeSO
40.01g/L, K [Fe (CN)
6] distilled water of 0.01g/L, Cty C 0.02g/L and surplus, pH value 7.0 ~ 7.4.
2. Harbin Institute of Technology's heterotrophic nitrification motionless bacterium L7 produces the oxidasic method of low temperature oxyammonia according to claim 1, it is characterized in that Harbin Institute of Technology's heterotrophic nitrification motionless bacterium L7 produces the oxidasic separation purification method of low temperature oxyammonia and carries out according to the following steps:
One, by motionless for Harbin Institute of Technology's heterotrophic nitrification bacterium L7 (Acinetobacter Heteronitrifihit L7) inoculation in culture medium, inoculum size is 2%, at 160rpm, 15 DEG C of cultivation 14 ~ 24h, then after the centrifugal 5min of 6000rpm, collect thalline;
Two, ultrasonication 15min in ice-water bath is placed on by resuspended for the Tris-HCI damping fluid of thalline 10mmol/L, pH8.5, then N,O-Diacetylmuramidase is adopted to carry out fragmentation to cell broken liquid, the thalline of collection is divided into tenuigenin, periplasmic and cytolemma three part, again by osmosis, prepare the extracellular matrix of not lysozyme;
Three, apply DEAE-CL6B ion exchange column to be separated the extracellular matrix collected, and balance with the Tris-HCl of 5mM, pH8.0, in identical damping fluid, wash-out is carried out with 0-500mM gradient NaCl, oxyammonia oxydase elutes under the NaCl condition of 80mM from ion exchange column, then removes cytochrome C by cross-linked polyacrylamide dextran S-100 gel chromatography
551, then denaturing polyacrylamide gel electrophoresis, collect the monomeric protein of molecular weight 60.4KDa, namely complete Harbin Institute of Technology's heterotrophic nitrification motionless bacterium L7 and produce the oxidasic separation and purification of low temperature oxyammonia; Wherein in step one, the component of culture medium is as follows: NH
4cl 0.1g/L, propionic salt or butyrates 1.0g/L, NH
2oH 0.7g/L, MgSO
47H
2o 0.05g/L, K
2hPO
40.2g/L, NaCl 0.12g/L, CuSO
45H
2o 0.01g/L, NaMoO
42H
2o 0.05g/L, BaCl
20.01g/L, MnSO
44H
2o0.01g/L, FeSO
40.01g/L, K [Fe (CN)
6] distilled water of 0.01g/L, Cty C 0.02g/L and surplus, pH value 7.0 ~ 7.4.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1584014A (en) * | 2004-06-04 | 2005-02-23 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Acinetobacter for denitrification and decomposing ammonia in wastewater |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1584014A (en) * | 2004-06-04 | 2005-02-23 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Acinetobacter for denitrification and decomposing ammonia in wastewater |
Non-Patent Citations (3)
| Title |
|---|
| "低温好氧反硝化菌的分离鉴定及脱氮特性研究";魏巍 等;《供水技术》;20121231;第6卷(第6期);第12-16页 * |
| "好氧异养硝化菌Acinetobacter sp. YY-5的分离鉴定及脱氮机理";金敏 等;《应用与环境生物学报》;20091025;第15卷(第5期);第692页摘要 * |
| "异养硝化菌Alcaligenes faecalis strain NR的硝化性能及其酶活性";安强 等;《上海交通大学学报》;20120531;第46卷(第5期);第774-779页 * |
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