CN107365726A - A kind of the nitrilase producing strains mutant and its selection of high nitrile substrate tolerance - Google Patents
A kind of the nitrilase producing strains mutant and its selection of high nitrile substrate tolerance Download PDFInfo
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Abstract
It is an object of the invention to provide a kind of combination ARTP mutagenesis and the method for the production nitrilase pseudomonas putida mutant of the micro enzyme reaction method high flux screening nitrile substrate tolerance liftings of OPC TCA, belong to industrial biotechnology field.Through one plant mutant body mut D3 of the micro enzyme reaction primary dcreening operations of OPC TCA, shaking flask secondary screening and the final acquisition of passage, in the case of higher Final substrate concentrations are 125mM, the substrate tolerance of the bacterial strain significantly improves, the nitrilase enzyme activity of simultaneous mutation body is 1.51 times of wild mushroom, and enzyme activity still keeps stable after 20 passages.The cyanopyridine of mutant strain energy Efficient Conversion 3 synthesizes nicotinic acid, and nicotinic acid cumulative production significantly improves compared to wild mushroom.The present invention complies fully with mutagenesis and the screening needs of industrialization nitrilase producing bacterial strain, and can be applied to the biosynthesis of nicotinic acid, has important application value.
Description
Technical field
The invention belongs to industrial biotechnology field, and in particular to ARTP mutagenesis and the high nitrile substrate of high-throughput screening method seed selection
The method of the pseudomonas putida mutant of tolerance.
Background technology
Nicotinic acid and niacinamide belong to pyridine compounds and their, are collectively referred to as vitamin B3.In human body, nicotinic acid is the coenzyme of dehydrogenase
(NAD+And NADH+) carbon skeleton is provided, and these coenzyme play weight in vivo as electron carrier in various redox reactions
Act on.But human body itself can only convert a small amount of tryptophan generation nicotinic acid, remaining required amount need to be provided by food.If intake
Amount deficiency, can cause pellagra, so nicotinic acid is widely used in food and feed additive, market demand is very big.In addition, cigarette
Acid also has the function that stronger expansion peripheral vessels, can be used as clinical drug treatment headache, antimigraine, tinnitus, interior Ménière
Disease etc.；Nicotinic acid can suppress lipid mobilization, decline VLDL synthesis in liver, so as to reduce plasma cholesterol, consolidate for treating high courage
Alcoholemia.Meanwhile nicotinic acid can also be used as medicine intermediate, the production for isoniazid, acid amides, peptamine etc..In dye industry, cigarette
Acid is also important organic synthesis intermediate as pyridine compounds and their.At present, chemical method synthesis nicotinic acid be primarily present yield it is low,
The features such as accessory substance is more, reaction condition is violent, it significantly limit the large-scale production of nicotinic acid.And the microorganism containing nitrilase
Energy one step catalysis nicotinonitrile generation nicotinic acid, because process is easy, selectivity is strong, reaction condition is gentle, has become in recent years
Carry out the study hotspot of nicotinic acid synthesis.But wild mushroom generally existing enzyme activity less stable, it is not high to the tolerance of substrate and product,
The low problem of enzyme protein expression amount in thalline.Such as can effectively improve these problems will be helpful to the development of greenization nicotinic acid industry with
Further lift economic benefit.
ARTP mutagenesis (plasma mutagenesis) is a kind of new and effective induced-mutation technique, can produce temperature under atmospheric pressure in 25-
Between 40 DEG C, have high activity particle (including helium atom in excitation state, oxygen atom, nitrogen-atoms, OH free radicals etc.) dense
The plasma jet of degree.The active-energy particle that ARTP is rich in causes to damage to the inhereditary material of bacterial strain/plant/cell etc.,
And induce biological cell and start SOS repair mechanisms, so a large amount of different mutant strains can be produced.In addition, ARTP equipment operations are simple
It is single, quick and safe, played a significant role in industrial microorganism mutation breeding field.
If the Screening And Fermenting Cultivation technology based on high-throughput screening method and ARTP is combined, then mutation can be significantly lifted
Storehouse screening efficiency.Ammonia and organic acid are the generations of equimolar ratio in nitrilase catalytic reaction, using the micro enzyme reactions of OPA-TCA,
Ammonia can generate leuco-compounds with O-phthalic aldehyde reaction, and the compound can generate the soluble chemical combination of blueness in acid condition
Thing, and there is maximum light absorption value at 600nm, the high flux screening of nitrilase producing strains can be efficiently applied to.
The content of the invention
It is an object of the invention to provide a kind of based on ARTP mutagenesis and the high nitrile substrate tolerance of High Throughput Screening Assay seed selection
The method of pseudomonas putida mutant, the mutant strain obtained using this method are significantly carried to the tolerance of substrate nicotinonitrile
Height, corresponding nicotinic acid yield are consequently increased.
The technical scheme is that：By pseudomonas putida P.putida wild mushrooms carry out ARTP mutagenesis, then after cultivate with
Obtain stable mutant strain, high flux screening carried out using the OPA-TCA micro-reactions method based on 96 orifice plates, then by enzyme activity compared with
High mutant strain inoculation shaking flask carries out secondary screening checking and passed on to investigate gene stability, finally obtains high nitrile substrate tolerance
Mutant strain.Comprise the following steps that：
It is prepared by bacteria suspension：Pseudomonas putida wild mushroom is used into physiology salt under 25-37 DEG C of shaken cultivation 20-48h, aseptic condition
Water washing and resuspension cell, the glycerine for adding final concentration 20% simultaneously dilute regulation cell number to 106-108Individual/mL, cell is made and hangs
Liquid.
ARTP mutagenesis and its fatal rate are drawn：Draw the upper surface that above-mentioned 10 μ L cell suspensions are spread evenly across metal slide glass.With
99.99% helium is as working gas, under power (100W), throughput (10slm), operating distance 2mm parameter at mutagenesis
Manage 0s, 10s, 15s, 30s, 45s, 60s, 90s, 120s, 150s, 180s.
After cultivate：Metal slide glass with bacterium solution is put under aseptic condition in the EP pipes equipped with fresh liquid culture medium,
25-37 DEG C of shaken cultivation 2-5h, dilute bacterium solution and be coated on solid medium.Culture medium nutritional requirement used by cultivating afterwards
Abundant, culture medium prescription (g/L) is：Peptone 5-20, dusty yeast 5-20, glycerine 10-30, urea 0.5-5, NaCl 0.1-2,
Potassium dihydrogen phosphate 1-5, dipotassium hydrogen phosphate 1-5.
The screening of high nitrile substrate tolerance mutant strain：By the mutant bacteria numbering in mutation library, simultaneously picking is fresh to 500 μ L are contained
In 96 deep-well plates of fluid nutrient medium, 25-37 DEG C of shaken cultivation 24-48h in orifice plate incubator is placed in.Supernatant is removed in centrifugation, per hole
Add sodium phosphate buffer (100mM, pH 7.2) to wash and be resuspended, 900 μ L bacteria suspensions are made.Add 100 μ L nicotinonitriles
Solution (final concentration 125mM), 30 DEG C of oscillating reactions 10min, centrifuge terminating reaction.10 μ L are drawn respectively reacts supernatant to containing
In the new 96 hole microwell plate of 100 μ L OPA reagents (20mM), 20 DEG C of oscillating reactions 20min, 100 μ L DMSO dissolvings are added, then add
Enter 50 μ L TCA reagent colour developments, absorbance is determined under 600nm.
Secondary screening：The higher mutant bacteria of enzyme activity in primary dcreening operation is seeded in 50mL fluid nutrient mediums, 25-37 DEG C of shaken cultivation 24-
48h.Take appropriate 200 μ L bacterium solutions measure enzyme activity.
Passage：Excellent direct mutation will be showed in primary dcreening operation and secondary screening 20 times are passed in solid medium to investigate gene stabilization
Property.
Batch converts：Mutant strain being seeded in 50mL fluid nutrient mediums, 25-37 DEG C of oscillation and fermentation 24-48h, supernatant is removed in centrifugation,
Resuspension is washed with sodium phosphate buffer (100mM, pH 7.2), 50mL bacteria suspensions are made.Substrate is added using batch transformation mode
Nicotinonitrile detects nicotinic acid and nicotinonitrile content, HPLC conditions are as follows, instrument in 30 DEG C of oscillating reactions by HPLC：
Wear 3000 serial HPLC of peace Ultimate, chromatographic column：Atlantis dC18 (5 μm, 4.6 × 150mm；Waters, USA), stream
Dynamic phase：Acetonitrile：Water=3:2 (containing 0.02% trifluoroacetic acid), flow velocity：0.5mL/min, Detection wavelength：268nm, column temperature：30℃、
Sample size：10μL.
Investigate influence of the different nicotinonitrile concentration to nicotinic acid yield：Respectively choose 50mM, 100mM, 150mM,
200mM, 250mM, 300mM are as nicotinonitrile feed concentrations.
Beneficial effect：With existing screening technique ratio, the invention provides one kind to combine ARTP mutagenesis and the micro enzyme reactions of OPC-TCA
The method of the production nitrilase pseudomonas putida mutant of method high flux screening nitrile substrate tolerance lifting, the present invention also obtain
The mutant strain mut-D3 that significantly improves of one plant of substrate tolerance and enzyme activity, the bacterial strain can convert accumulation and obtain 189g/L nicotinic acid,
Nicotinic acid yield improves 42.11% compared to wild mushroom.
Brief description of the drawings
Fig. 1 Pseudomonas putida ARTP mutagenesis fatal rate curves
Fig. 2 ammonia standard curves
Fig. 3 Pseudomonas putida wild mushrooms growth curve and enzyme activity curve in 96 deep-well plates
Fig. 4 Pseudomonas putida mutant strains mut-D3 primary dcreening operation
The secondary screening of Fig. 5 Pseudomonas putida mutant strains and mutant strain mut-D3 passage
Fig. 6 wild mushrooms and mut-D3 batch convert
Influence and batch conversion of Fig. 7 difference nicotinonitrile concentration to nicotinic acid yield
Specific implementation method
In order to be further described the present invention, it is described in detail below in conjunction with the accompanying drawings.
Embodiment 1：The ARTP mutation breedings of nitrilase producing strains
Make OD600- number of cells curves：Inoculation P.putida is inoculated in 50mL fluid nutrient mediums, and 30 DEG C of vibrations are cultivated extremely
14th, 16,18,20,22,24,26,28h, takes 200 μ L bacterium solution dilution meterings OD600 respectively.100 μ L bacterium solution gradient dilutions are taken, are taken
Dilution spread plate under 50 μ L acceptable diluent degree, it is i.e. countable to treat that bacterium colony is grown.
Fatal rate Drawing of Curve：P.putida is inoculated in fluid nutrient medium, in 30 DEG C of shaken cultivation 24h to logarithmic growth
Phase.Under aseptic condition with brine and be resuspended cell, add final concentration 20% glycerine and dilute regulation cell number to
106-108Individual/mL.Draw the upper surface that above-mentioned 10 μ L cell suspensions are spread evenly across metal slide glass.Using 99.99% helium as
Working gas, under power (100W), throughput (10slm), operating distance 2mm parameter mutagenic treatment 0s, 10s, 15s,
30s、45s、60s、90s、120s、150s、180s.Slide glass is transferred in the EP pipes containing 1mL culture mediums after processing, shaken
Vibration elution 1min on device is swung, forms new bacteria suspension, then EP pipes are placed in 30 DEG C of culture 3h of shaken cultivation case.To each processing
Mutagenesis bacteria suspension under time carries out gradient dilution, takes 50 μ L dilution spread plates to be respectively placed in 30 DEG C of incubators and cultivates.
Treat that bacterium colony is grown, take flat board of the flat-plate bacterial colony number between 30-300 under each processing time to be counted.Calculate fatal rate simultaneously
Draw destruction curve.
Fatal rate %=(without mutagenesis clump count-through mutagenesis clump count)/without mutagenesis clump count × 100%
ARTP mutagenesis：P.putida wild mushrooms are more sensitive to ARTP mutagens as shown in Figure 1, and 10s fatal rates have reached
It is all lethal when 95.13%, mutagenesis 35s.Oversize in view of mutation time, cellular damage is more serious to be difficult to survive, and selects 10
S is plasma mutation time.
Embodiment 2：The screening of high nitrile substrate tolerance positive mutating strain
The ammonia standard curve of OPA-TCA micro-reactions：A certain amount of pure chloride solid of analysis is taken to be dried in 105 DEG C of drying boxes
2-3h accurately weighs 0.3147g, is dissolved in and removes in right amount in ammoniacal liquor, be settled to 100mL, obtaining concentration is to solid constant weight
1.0mg/mL ammonia mother liquor, take 1mL to dilute 50 times, that is, obtain 20 μ L/mL ammonia titer.Respectively draw 0,10,20,30,
40th, 50,60,70,80,90,100 μ L ammonia titers are into 96 orifice plates, and 100 μ L are settled to ammoniacal liquor is removed, ammonia density is 0,2,
4、6、8、10、12、14、16、18、20μg/mL.The OPA reagents for adding 100 μ L 20mM are placed in the 20 DEG C of vibrations of orifice plate incubator
React 20min.Add 50 μ L DMSO dissolving, add 50 μ L TCA reagent colour developments, then with ultraviolet specrophotometer in
Absorbance is determined at 600nm.The wherein compound method of OPA reagents：Take 134.13 mg OPAs to be dissolved in 10mL methanol to obtain
100mM OPA solution, then with sodium tetraborate solution (15mM, pH 9.5) 1:5 dilutions obtain 20mM OPA reagents.TCA reagents
Compound method：Take 10gTCA to be dissolved in 50mL to remove in ammoniacal liquor, obtain 10% (w/v) TCA reagents.As shown in Figure 2, OPA-TCA
The sensitivity of micro-reaction preferably (R2=0.9991) it, may be used as high-throughput screening method.
Determine wild P.putida growth curve and enzyme activity curve in 96 deep-well plates：P.putida is inoculated in containing 500 μ L
In 96 deep-well plates of fluid nutrient medium, the shaken cultivation in 30 DEG C of orifice plate incubator.Taken respectively in 20,24,28,32h suitable
Measure bacterium solution measure growth curve and enzyme activity curve.From the figure 3, it may be seen that wild mushroom is limited to dissolved oxygen and nutrition bar in 96 deep-well plates
Part, reach maximum simultaneously in 24h biomass and enzyme activity.So selected 24h, which is mutagenic bacteria primary dcreening operation, determines the enzyme activity time.
The micro enzyme reaction high flux screenings of OPA-TCA：Mutant bacteria in the mutation library built is numbered and picking is to containing 500
In 96 deep-well plates of μ L fresh liquid culture mediums, in 30 DEG C of orifice plate incubator, 300rpm shaken cultivations 24h.Centrifuge and discard
Clearly, sodium phosphate buffer (100mM, pH 7.2) is added per hole to wash and be resuspended, 900 μ L bacteria suspensions are made.With multi-pore channel liquid relief
Rifle is drawn 100 μ L nicotinonitrile solution (final concentration 125mM) and put in bacteria suspension, reaction system 1mL.Cover orifice plate lid, 30 DEG C
Oscillating reactions 10min, centrifuge terminating reaction.10 μ L reactions supernatant, which is drawn, with multi-pore channel liquid-transfering gun extremely contains 100 μ L OPA reagents
In the new 96 hole microwell plate of (20mM), in 20 DEG C of oscillating reactions 20min of orifice plate incubator, 100 μ L DMSO dissolvings are added, then add
Enter 50 μ L TCA reagent colour developments, absorbance (Fig. 4) is determined under 600nm.
Shaking flask secondary screening and passage：The higher mutant bacteria of the enzyme activity of primary dcreening operation acquisition is seeded in 50mL fluid nutrient mediums, 30 DEG C are shaken
Swing culture 36h.Take appropriate bacterium solution measure enzyme activity.Passed on excellent direct mutation is showed in primary dcreening operation and secondary screening in solid medium
20 times to investigate gene stability.Mut-D3 enzyme activity is up to 1.51 times of wild mushroom as shown in Figure 5, and enzyme activity after passing on 20 times
Still stablize.The mutant strain is now preserved in China General Microbiological culture presevation administrative center, deposit number CGMCC
No.14276, preservation date are on June 26th, 2017.
Embodiment 3：Batch conversion is carried out using the mutant of high substrate tolerance
Mutant strain is seeded in 50mL fluid nutrient mediums, 30 DEG C of oscillation and fermentation 36h, supernatant is removed in centrifugation, uses sodium phosphate buffer
(100mM, pH 7.2) washing is resuspended, and bacteria suspension is made and carries out batch conversion.Final concentration of 100mM nicotinonitrile is added,
And fed intake in 30 DEG C of oscillating reactions, preceding 10 batch every 10-20min, fed intake after 10 batches every 35-60min.Examined with HPLC
It is instrument to survey nicotinic acid and nicotinonitrile content, wherein HPLC analysis conditions：Wear 3000 serial HPLC of peace Ultimate, color
Compose post：Atlantis dC18 (5 μm, 4.6 × 150mm；Waters, USA), mobile phase：Acetonitrile：Water=3:2 (contain 0.02% 3
Fluoroacetic acid), flow velocity：0.5mL/min, Detection wavelength：268nm, column temperature：30 DEG C, sample size： 10μL.It will be appreciated from fig. 6 that wild mushroom
The nicotinonitrile (final concentration 100mM) of 15 batches can be converted, is accumulated through 450min and obtains 133g/L nicotinic acid.And mut-D3
The nicotinonitrile of 20 batches can be converted, is accumulated through 610min and obtains 182 g/L nicotinic acid, nicotinic acid output increased 36.84%.
50mM, 100mM, 150mM, 200mM, 250mM, 300Mm are chosen respectively as nicotinonitrile feed concentrations, are investigated different
Influence of the nicotinonitrile concentration to nicotinic acid yield.As shown in Figure 7, it is in 50mM to 150mM, mut-D3 nicotinic acid conversion ratio
201mmol/L/h, significantly larger than wild mushroom 147mmol/L/h (50mM to 100mM), so can be by mut-D3 3- cyano group pyrroles
The feed concentrations of pyridine are improved to 150mM from 100mM.By Fig. 7, the 3- cyano group pyrroles of 15 batches can be converted through 450min wild mushrooms
Pyridine (final concentration 100mM), accumulation obtain 133g/L nicotinic acid.And the 3- cyanogen of 13 batches can be equally converted through 450min mut-D3
Yl pyridines (final concentration 150mM), accumulation obtain 189g/L nicotinic acid, nicotinic acid output increased 42.11%.
Claims (4)
1. the nitrilase producing strains mutant of a plant height nitrile substrate tolerance, is named as pseudomonas putida
Pseudomonas putida mut-D3, the China being now preserved in positioned at BeiChen West Road, Chaoyang District, BeiJing City No.1 institute 3 are common
Microbiological Culture Collection administrative center, deposit number are CGMCC No.14276, and preservation date is on June 26th, 2017.
2. the ARTP mutagenesis of nitrilase producing strains mutant and its side of high flux screening of a kind of high nitrile substrate tolerance
Method, it is characterised in that prepare CGMCC No.14276 bacteria suspensions and dilute regulation cell number to 106-108Individual/mL, by bacteria suspension
Using ARTP mutagenesis, processing time 10-180s, the bacterium solution after processing accesses fresh liquid culture medium, 25- under aseptic condition
37 DEG C of shaken cultivation 2-5h, 125mM-250mM nicotinonitrile concentration of substrate is selected, using the OPA-TCA based on 96 orifice plates
Micro-reaction method carries out high flux screening.
3. method as claimed in claim 2, it is characterised in that CGMCC No.14276 reaction substrate nicotinonitrile is most suitable
Reaction density is 125mM.
4. a kind of method for the biodegradable synthesis nicotinic acid of nitrile compounds, it is characterized in that, using CGMCC No.14276, phase
Compareed than wild mushroom, nicotinic acid yield is promoted to 189g/L by 133g/L.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486209A (en) * | 2018-03-22 | 2018-09-04 | 浙江工业大学 | A kind of method of high-flux fast screening high yield R-2- (4- hydroxyphenoxies) propionic acid bacterial strain |
| CN108823098A (en) * | 2018-06-30 | 2018-11-16 | 浙江工业大学 | A kind of high-throughput screening method of R-2- (4- hydroxyphenoxy) propionic acid synthesis bacterial strain |
| CN111733192A (en) * | 2020-07-03 | 2020-10-02 | 湖北大学 | Novel enzyme catalysis method for preparing cinnamic acid from cinnamaldehyde and application |
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- 2017-09-28 CN CN201710666935.7A patent/CN107365726A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486209A (en) * | 2018-03-22 | 2018-09-04 | 浙江工业大学 | A kind of method of high-flux fast screening high yield R-2- (4- hydroxyphenoxies) propionic acid bacterial strain |
| CN108486209B (en) * | 2018-03-22 | 2022-03-18 | 浙江工业大学 | Method for rapidly screening (R) -2- (4-hydroxyphenoxy) propionic acid producing strains in high flux |
| CN108823098A (en) * | 2018-06-30 | 2018-11-16 | 浙江工业大学 | A kind of high-throughput screening method of R-2- (4- hydroxyphenoxy) propionic acid synthesis bacterial strain |
| CN111733192A (en) * | 2020-07-03 | 2020-10-02 | 湖北大学 | Novel enzyme catalysis method for preparing cinnamic acid from cinnamaldehyde and application |
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