CN105238710B - Bacillus subtilis for producing oxygenase and application thereof - Google Patents
Bacillus subtilis for producing oxygenase and application thereof Download PDFInfo
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- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 56
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 55
- 102000004020 Oxygenases Human genes 0.000 title claims abstract description 28
- 108090000417 Oxygenases Proteins 0.000 title claims abstract description 28
- 238000006243 chemical reaction Methods 0.000 claims abstract description 20
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims abstract description 17
- 238000004321 preservation Methods 0.000 claims abstract description 9
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 7
- SBOJXQVPLKSXOG-UHFFFAOYSA-N o-amino-hydroxylamine Chemical compound NON SBOJXQVPLKSXOG-UHFFFAOYSA-N 0.000 claims abstract 2
- 238000007254 oxidation reaction Methods 0.000 claims description 17
- 125000003277 amino group Chemical group 0.000 claims description 13
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 11
- 102000004190 Enzymes Human genes 0.000 claims description 9
- 108090000790 Enzymes Proteins 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 9
- 238000000855 fermentation Methods 0.000 claims description 8
- 230000004151 fermentation Effects 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 7
- 230000001590 oxidative effect Effects 0.000 claims description 7
- FYFDQJRXFWGIBS-UHFFFAOYSA-N 1,4-dinitrobenzene Chemical compound [O-][N+](=O)C1=CC=C([N+]([O-])=O)C=C1 FYFDQJRXFWGIBS-UHFFFAOYSA-N 0.000 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 230000003647 oxidation Effects 0.000 claims description 6
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 5
- 108010080698 Peptones Proteins 0.000 claims description 5
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 5
- 235000019319 peptone Nutrition 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 230000012010 growth Effects 0.000 claims description 3
- 230000009603 aerobic growth Effects 0.000 claims description 2
- 229920002261 Corn starch Polymers 0.000 claims 1
- 239000008120 corn starch Substances 0.000 claims 1
- 239000011734 sodium Substances 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 13
- 230000008569 process Effects 0.000 abstract description 9
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- 239000000126 substance Substances 0.000 abstract description 4
- 238000012258 culturing Methods 0.000 abstract description 3
- 238000005265 energy consumption Methods 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 238000012216 screening Methods 0.000 description 5
- 240000008042 Zea mays Species 0.000 description 4
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 4
- 235000005822 corn Nutrition 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000011218 seed culture Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
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- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 238000010564 aerobic fermentation Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
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- 239000003054 catalyst Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000003317 industrial substance Substances 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- -1 nitryl compound Chemical class 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 241001112741 Bacillaceae Species 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 238000011534 incubation Methods 0.000 description 1
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- 230000005764 inhibitory process Effects 0.000 description 1
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- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 150000002828 nitro derivatives Chemical class 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a bacillus subtilis for producing oxygenase and application thereof, wherein the bacterial strain is bacillus subtilis (B) for producing oxygenaseBacillus subtilis）AL001The culture is preserved in China center for type culture Collection, the preservation address is Wuhan university in Wuhan city, the preservation date is 2015, 9 months and 11 days, and the preservation number is CCTCC NO: m2015524. Bacillus subtilisAL001The generation of the nitro group from the amino oxide is realized by culturing and producing the oxygenase, and compared with the existing chemical method, the enzymolysis reaction condition is mild, the energy consumption is low, the pollution is small, and the process is easy to control.
Description
Technical field
The invention belongs to microbial technology field, and in particular to a kind of bacillus subtilis for producing oxygenase and its application.
Background technology
Paranitroanilinum is considered as a kind of typical hypertoxic pollutant, can all be caused during its synthesis and use tight
The environmental pollution of weight.Especially there is very big destruction to water body and soil, so as to influence biocoene therein.Therefore, mesh
Preceding most research is intended to the degraded and the wherein reduction of nitro of nitro compound.However, paradinitrobenzene is also a kind of
Important industrial chemical, in nature there are relatively fewer, is mainly incorporated into environment by mankind's activity.This is heavy
The industrial chemical wanted is widely used in the synthesis of many products, has important industrial application value.Commercial Application at present
On mostly use chemical method synthesizing nitryl compound, building-up process is difficult control, and cost is higher, seriously polluted, and danger coefficient is high.
Compared with chemical method, the amino of biological enzyme catalyst oxidation paranitroanilinum is changed into nitro, without adding strong acid as catalyst
And initiator, the whole reaction process of catalysis oxidation, simplifies production technology, reduces production cost so that whole building-up process
Efficient, safe green is simple controllable.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of bacillus subtilis for producing oxygenase and its application, this is withered
Oxygenase is produced in careless bacillus incubation, amino effectively can be converted into nitro, improve environment, expend resource
It is few.
In order to solve the above technical problems, the technical solution adopted by the present invention is as follows：
A kind of bacillus subtilis for producing oxygenase, realizes the bacillus subtilis that amino group is nitro（Bacillus subtilis）AL001, it is preserved in China typical culture collection center, preservation address is Wuhan University of Wuhan City, preservation date
For September in 2015 11 days, deposit number was CCTCC NO：M 2015524.
The bacillus subtilis bacterium colony rough surface of above-mentioned production oxygenase is opaque, and dirty white, edge is not whole, aerobe
Long, 28 ~ 39 DEG C of cultivation temperature, optimum temperature is 30 DEG C, is grown in the range of pH 5.0 ~ 10.0, not chromogenic element, strain morphology length
It is rod-shaped, Gram-positive.
The bacillus subtilis of above-mentioned production oxygenase is in the application for realizing that amino group is nitro.
The bacillus subtilis of above-mentioned production oxygenase is realizing application of the amino group for nitro, comprises the following steps：
Step 1, by bacillus subtilis（Bacillus subtilis）AL001It is seeded in culture medium, in temperature
20 ~ 37 DEG C, ferment under 200 rpm of rotating speed 60 ~ 96h, and centrifuging and taking supernatant obtains thick enzyme；
Step 2, toward thick enzyme is added in paranitroanilinum, oxidation reaction obtains paradinitrobenzene.
It is that the culture medium component described in step 1 is 10 ~ 40g/L of glucose as improved, peptone 10 ~
20g/L, 10 ~ 20g/L of corn pulp, dipotassium hydrogen phosphate 0.7g/L, potassium dihydrogen phosphate 0.3g/L, magnesium sulfate 0.5g/L, NaCl
5g/L, initial pH 6 ~ 8；30 DEG C of fermentation temperature, throughput are 0.5 ~ 1vvm.
It is 20 ~ 50 DEG C of oxidizing reaction temperature in step 2 as improved.
It is that oxidizing reaction temperature is 40 DEG C in step 2 as further improved.
It is that oxidation reaction pH is 6.0 ~ 8.0 in step 2 as improved.
It is that oxidation reaction pH is 7.0 ~ 7.5 in step 2 as further improved.
It is that oxidation reaction is that Na is added into oxidation system in step 2 as improved+、Fe2+Or Mg2+。
It is that oxidation reaction is that final concentration of 10mmol/L is added into oxidation system in step 2 as further improved
Fe2+。
Beneficial aspects
Compared with prior art, the present invention there is following advantage：
Compared with chemical method, reaction is gentle, and process easily manipulates, the high reaction rate under room temperature, excellent chemo-selective,
Regioselectivity and stereoselectivity, the features such as, there is the advantages that energy consumption is low, material consumption is few, environmentally friendly.
Brief description of the drawings
Fig. 1 is bacillus subtilis（Bacillus subtilis）AL001For bacterium Gram's staining form, wherein thalline
In purple.
Fig. 2 is bacillus subtilis（Bacillus subtilis）AL001Colonial morphology figure.
Fig. 3 is bacillus subtilis（Bacillus subtilis）AL001 16s comparison diagrams.
Fig. 4 is bacillus subtilis（Bacillus subtilis）AL001Aoxidize paranitroanilinum generation paradinitrobenzene
Gas chromatographic detection（Agilent GC-7890B）Collection of illustrative plates, wherein, 5.157 peak is the response peak of paranitroanilinum.
Fig. 5 is bacillus subtilis（Bacillus subtilis）AL001Influences of the pH to conversion ratio in oxidation reaction.
Fig. 6 is bacillus subtilis（Bacillus subtilis）AL001Shadow of the temperature to conversion ratio in oxidation reaction
Ring.
Embodiment
According to following embodiments, the present invention may be better understood.It is however, as it will be easily appreciated by one skilled in the art that real
Apply that example is described to be merely to illustrate the present invention, without this hair described in detail in claims should will not be limited
It is bright.
1 bacillus subtilis of embodiment（Bacillus subtilis）AL001Screening and identification
（One）Sample collection
Totally 30 parts of seawater sample is obtained from Taizhou, 4 DEG C is stored in time and saves backup.
（Two）Enrichment culture
50ml brain heart infusion broths culture medium is taken to be loaded in 500ml triangular flasks, access 1ml samples to be sieved after sterilizing, 37 DEG C,
Shaking culture 24h under the conditions of 200rpm, obtains enrichment culture liquid.
（Three）Plate screening
Enrichment culture liquid is filtered with sterile individual layer filter paper, gradient dilution 10-6 to 10-10, draw 0.2ml dilution spreads
In screening and culturing medium tablet, 30 DEG C of culture 5d, by all bacterium colony slant preservations grown.
（Four）Shaking flask is screened
By in the bacterium access liquid screening medium of preservation on tablet, 5d is cultivated, surveys the content of substrate and product, and is identified
It is nitro that amino group, which whether can be made,.
Screening and culturing medium：Paranitroanilinum, 5g/L glucose, 5g/L yeast extracts, 5g/L corn pulps, the phosphoric acid hydrogen two of 1g/L
Potassium 0.7g/L, potassium dihydrogen phosphate 0.3g/L, magnesium sulfate 0.5g/L.
The form of the bacterium, physiological and biochemical property are as follows：
Colonial morphology and color：Edge is not whole, canescence.Thalli morphology：Elongated rod shape.Gram's staining：It is positive.Aerobic side
Formula：Aerobic growth.Growth temperature：28-39℃.Grow pH：5.0-10.0, salt tolerant growth scope 0%-10% NaCl.
Bacterial strain is identified：Extract postgenome design primer pair its 16S rDNA to be expanded and be sequenced, by sequencing result
Compared with the sequence in GeneBank, it is found that the bacterium belongs to Bacillus Pseudomonas with the highest type strain of its homology.
The bacterium is judged for bacillus guiding principle, bacillus head, Bacillaceae, and bacillus, is named asBacillus subtilis AL001。
Embodiment 2
Bacillus subtilis（Bacillus subtilis）AL001In fermentation production oxygenase and convert paranitroanilinum
Using
Seed culture medium：Glucose 5g/L, peptone 10g/L, yeast extract 10 g/L, dipotassium hydrogen phosphate 0.7g/L, phosphoric acid
Potassium dihydrogen 0.3g/L, magnesium sulfate 0.5g/L, NaCl 5g/L, 37 DEG C of cultivation temperature, 200 rpm；
Strain enzyme-producing culture medium：Glucose 10g/L, peptone 10g/L, corn pulp 10g/L, dipotassium hydrogen phosphate 0.7g/L,
Potassium dihydrogen phosphate 0.3g/L, magnesium sulfate 0.5g/L, 20g/L NaCl, initial pH7.0.1.7L fermentation tank liquid amounts 1L, 121
DEG C sterilizing 15 minutes.
By glycerol stocks bacillus subtilis（Bacillus subtilis）AL001, access 50mL seed culture mediums, 37
DEG C, 200rpm culture 12h, then transfer into 1L fermentation mediums, blowing air 1v/vm, rotating speed according to 1% (v/v) inoculum concentration
200rpm, after aerobic fermentation 70h, collects fermented liquid supernatant, 4 DEG C save backup by 30 DEG C.
The bacterial strain, which produces oxygenase, can make paranitroanilinum be oxidized to paradinitrobenzene, and 24h product yields are up to 70%.
Embodiment 3
Bacillus subtilis（Bacillus subtilis）AL001In fermentation production oxygenase and aoxidize paranitroanilinum
Using
Seed culture medium：With embodiment 2
Strain enzyme-producing culture medium：Glucose 15g/L, peptone 15g/L, corn pulp 10g/L, dipotassium hydrogen phosphate 0.7g/L,
Potassium dihydrogen phosphate 0.3g/L, magnesium sulfate 0.5g/L, 40g/L NaCl, initial pH7.0.1.7L fermentation tank liquid amounts 1L, 121
DEG C sterilizing 15 minutes.
By glycerol stocks bacillus subtilis（Bacillus subtilis）AL001, access 50mL seed culture mediums, 37
DEG C, 200rpm culture 12h, then transfer into 1L fermentation mediums, blowing air 1v/vm, rotating speed according to 1% (v/v) inoculum concentration
200rpm, after aerobic fermentation 70h, collects fermented liquid supernatant, 4 DEG C save backup by 30 DEG C.
The bacterial strain, which produces oxygenase, can make paranitroanilinum be oxidized to paradinitrobenzene, and 24h product yields are up to 86%.
Embodiment 4
Bacillus subtilis（Bacillus subtilis）AL001Influences of the pH to conversion ratio in oxidizing process.
The fermented liquid supernatant collected in centrifugation embodiment 2, using saturated ammonium sulfate precipitation classification precipitation target protein simultaneously
Using the excessive salt ion of dialysis removal, thick enzyme is obtained through vacuum freeze drying after acquisition crude enzyme liquid.Respectively into transformation system
Add different pH value and obtain phosphate buffer, carry out enzymolysis experiment under the same conditions, final research shows that peak enzymolysis-ability pH is
7.2-7.5（See attached drawing 5）.
Embodiment 5
Bacillus subtilis（Bacillus subtilis）AL001Influence of the temperature to conversion ratio in oxidizing process
Under identical conversion condition（With embodiment 4）, variable is changed to temperature, by adjusting conversion temperature discovery with temperature
The rise of degree, the conversion ratio of amino persistently rise, and peak enzymolysis-ability temperature is 45 DEG C, then the rise with temperature, conversion ratio can be fast
Speed declines（See attached drawing 6）.
Embodiment 6
Bacillus subtilis（Bacillus subtilis）AL001Influence of the metal ion to conversion ratio in oxidizing process
Research to the oxygenase shows Na+, Fe2+, Mg2+To having facilitation in conversion process, add into transformation system
Enter the Fe of final concentration of 10mmol/L2+Conversion ratio can be made to improve 53%, Na+, Mg2+29% and 35%, Al is respectively increased3+, Cu2+With
Ca2+Strong inhibition conversion process, wherein Al3+Then completely inhibit.
The foregoing is merely the embodiment of the present invention, but protection scope of the present invention is not limited thereto, any
Those skilled in the art disclosed herein technical scope in, can without the change that creative work is expected or
Person replaces, and should be covered by the protection scope of the present invention.Therefore, protection scope of the present invention should be with claims institute
Subject to the protection domain of restriction.
Claims (11)
1. a kind of bacillus subtilis for producing oxygenase, it is characterised in that produce the bacillus subtilis (Bacillus of oxygenase
Subtilis) AL001, is preserved in China typical culture collection center, and preservation address is Wuhan University of Wuhan City, preservation date
For September in 2015 11 days, deposit number was CCTCC NO：M2015524.
2. the bacillus subtilis of oxygenase is produced according to claim 1, it is characterised in that：Bacterium colony rough surface is opaque,
Dirty white, edge is not whole, aerobic growth, 28~39 DEG C of cultivation temperature, and optimum temperature is 30 DEG C, in the range of pH5.0~10.0
Growth, not chromogenic element, strain morphology elongated rod shape, Gram-positive.
3. application of the bacillus subtilis of oxygenase in realizing that amino group is nitro is produced described in claim 1.
4. application of the bacillus subtilis of oxygenase in realizing that amino group is nitro is produced according to claim 3, its
It is characterized in that, comprises the following steps：
Step 1, bacillus subtilis (Bacillus subtilis) AL001 is seeded in culture medium, temperature 20~
37 DEG C, ferment under rotating speed 200rpm 60~96h, and centrifuging and taking supernatant obtains crude enzyme liquid；
Step 2, toward crude enzyme liquid is added in paranitroanilinum, amino-oxide group obtains paradinitrobenzene.
5. application of the bacillus subtilis of oxygenase in realizing that amino group is nitro is produced according to claim 4, its
It is characterized in that：The component of culture medium described in step 1 is 10~40g/L of glucose, 10~20g/L of peptone, corn
Starch 10~20g/L, dipotassium hydrogen phosphate 0.7g/L, potassium dihydrogen phosphate 0.3g/L, magnesium sulfate 0.5g/L, sodium chloride 5g/L, initial pH6
~8；30 DEG C of fermentation temperature, throughput are 0.5~1v/ (vmin).
6. application of the bacillus subtilis of oxygenase in realizing that amino group is nitro is produced according to claim 4, its
It is characterized in that：20~50 DEG C of oxidizing reaction temperature in step 2.
7. application of the bacillus subtilis of oxygenase in realizing that amino group is nitro is produced according to claim 6, it is special
Sign is：Oxidizing reaction temperature is 40 DEG C in step 2.
8. application of the bacillus subtilis of oxygenase in realizing that amino group is nitro is produced according to claim 4, its
It is characterized in that：Oxidation reaction pH is 6.0~8.0 in step 2.
9. application of the bacillus subtilis of oxygenase in realizing that amino group is nitro is produced according to claim 8, its
It is characterized in that：Oxidation reaction pH is 7.0~7.5 in step 2.
10. application of the bacillus subtilis of oxygenase in realizing that amino group is nitro is produced according to claim 4, its
It is characterized in that：Oxidation reaction is that Na is added into oxidation system in step 2+、Fe2+Or Mg2+。
11. application of the bacillus subtilis of oxygenase in realizing that amino group is nitro is produced according to claim 10,
It is characterized in that：Oxidation reaction is that final concentration of 10mmol/L Fe are added into oxidation system in step 22+。
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| WO2008044614A1 (en) * | 2006-09-28 | 2008-04-17 | Ajinomoto Co., Inc. | Method for producing 4-hydroxy-l-isoleucine |
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