CN102533599B - Pseudomonas putida for producing dimethylbenzene monooxygenase and applications of pseudomonas putida - Google Patents
Pseudomonas putida for producing dimethylbenzene monooxygenase and applications of pseudomonas putida Download PDFInfo
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Abstract
The invention provides pseudomonas putida ZJB-LLJ for producing a dimethylbenzene monooxygenase bacterial strain and applications of the pseudomonas putida to preparing 5-methylpyrazine-2-carboxylic acid through microbial fermentation. The bacterial strain is collected in China Center for Type Culture Collection (CCTCC for short), address: Wuhan University 430072, Wuhan, China, collection number: CCTCC No: M 2011395, and collection data: December, 13th ,2011. The invention has the main beneficial effects that a novel bacterial strain for producing dimethylbenzene monooxygenase is provided, and the 5-methylpyrazine-2-carboxylic acid which is a drug intermediate can be prepared through the bacterial strain. When the 5-methylpyrazine-2-carboxylic acid is produced by using the bacterial strain for producing the dimethylbenzene monooxygenase, the reaction condition is mild, the energy is saved, and the environment is protected. Even more important, the process is simple, and the product extraction is easy. The bacterial strain for producing the dimethylbenzene monooxygenase has a good industrial application prospect.
Description
(1) technical field
The present invention relates to a strain and produce dimethylbenzene monooxygenase bacterial strain---pseudomonas putida (Pseudomonas putida) ZJB-LLJ, and prepare application in 5-Methylpyrazine-2-carboxylic acid at microbial fermentation.
(2) background technology
5-Methylpyrazine-2-carboxylic acid (5-Methylpyrazine-2-carboxylic acid) is a kind of rice white solid crystal, and molecular formula is C
6H
6N
2O
2, molecular weight 138.12,166～169 ℃ of fusing points, outward appearance light yellow crystal, have irritating smell, being exposed in air can slow oxidation, becomes the thick solid of brownish black by brown oil, needs vacuum-sealing to preserve.5-Methylpyrazine-2-carboxylic acid has purposes widely in the medicine industry field, be mainly used in synthetic s-generation sulphur rise abruptly class hypoglycemic drug Glipizide, novel antihypertensive medicament Olbetam and antitubercular agent 5-Methylpyrazine-2-carboxylic acid first vinegar etc., therefore the research of 5-Methylpyrazine-2-carboxylic acid had important actual application value.
The chemosynthesis of 5-Methylpyrazine-2-carboxylic acid mainly contain following technique (old bright and etc., chemical reagent, 2008,30 (11): 869-870; Han Guang etc., 2000,17 (3): 208-210):
(1) oxidation, chlorination, be hydrolyzed, reoxidize: with hydrogen peroxide and diacetyl oxide 2, the 5-dimethylpyrazine is oxidized to single oxynitride and generates 5-methyl-2-methylol pyrazine through monomethyl side chain chlorination hydrolysis again, get 5-Methylpyrazine-2-carboxylic acid with potassium permanganate solution oxidation 5-methyl one 2-methylol pyrazine again, total recovery is 18%.
(2) chlorination, phthalein, hydrolysis, oxidation: under the protection of nitrogen, after causing N-chloro fourth two phthalimides (NCS) with initiator benzoyl peroxide first phthalein (BPO), again to 2, 5 one dimethylpyrazines carry out chlorination reaction, can obtain 5-methyl-2-chloromethyl pyrazine again with anhydrous sodium acetate in dehydrated alcohol, reflux under nitrogen protection, can obtain second phthalein oxygen methyl-derivatives, then process with solid sodium hydroxide, reaction generates 5-methyl-2-methylol pyrazine, use again potassium permanganate oxidation, use at low temperatures the concentrated hydrochloric acid adjust pH to the iso-electric point of 5-methylpyrazine-2-shuttle acid, use again the frozen water washing precipitation, get 5-Methylpyrazine-2-carboxylic acid with Vanadium Pentoxide in FLAKES vacuum-drying.Total recovery 47%.
(3) cyclization, oxidation, acidifying and decarboxylation: take pyruvic acid and adjacent benzene two ammoniums as basic raw material, cyclization under the existence of catalyzer Sodium Pyrosulfite, again through inorganic oxidizer oxidation, with sulfuric acid acidation, decarboxylation, in pH1.5-4.0 butanone extracting and separating, crystallization, drying, oven dry, pulverizing can obtain 5-Methylpyrazine-2-carboxylic acid.
(4) intermolecular cyclization: diamino maleic two eyeballs and pyruvic aldehyde generate the dicyano pyrazines derivatives through ring-closure reaction, pass through again acid hydrolytic reaction, generate 5-methylpyrazine-2,3-dicarboxyl pyrazine, decarboxylation obtain the mixture of 5-Methylpyrazine-2-carboxylic acid and 5-methylpyrazine-two kinds of isomer of 3-carboxylic acid.
(5) the inferior one-step oxidation process of N-bromo succinyl: add 2 in the reaction flask of drying, 5-dimethylpyrazine, ethyl acetate, N-bromo-succinimide, put reactant in infrared lamp intermittent irradiation 10h, detection reaction finishes, then steam ethyl acetate, add 10% sodium hydroxide in residuum to pH value of solution 8, use the 50mL chloroform extraction, tell water, the water decompression is steamed to substantially dried, transfer pH to 2 with concentrated hydrochloric acid, with butanone extraction 3 times, merge organic phase, steaming desolventizes, resistates adds 50mL water recrystallization and gets the yellow powder 5-Methylpyrazine-2-carboxylic acid, yield 84%.
(6) cobalt naphthenate one-step oxidation process: the cobalt naphthenate air oxidation process is adopted in this reaction, namely under normal pressure, passes into air in the mixed system of 2,5-dimethylpyrazine and cobalt naphthenate, reflux.Sherwood oil is added in the four-hole boiling flask that agitator, inlet pipe, reflux condensing tube and thermometer are housed, then add 2, the 5-dimethylpyrazine, add again cobalt naphthenate, for purplish red solution, pass into atmospheric oxidation after stirring, add TEBAC0, add again 30% sodium hydroxide solution, the strong stirring heating, solution becomes ash to take off look.After reactant begins to reflux, add the azobisisobutyronitrile initiation reaction, reflux temperature is controlled at 76 ℃ of left and right, and the reaction times is 5h.After stopped heating, standing demix, the upper strata is transparent brown oil reservoir, lower floor is that taupe brown is opaque and contain the solution of particulate solid.Pour out rear vibration separatory, upper strata brown jelly, lower floor is the pink colour clear solution.add 11% sodium hydroxide solution in the upper strata, after vibration shakes up, filtering and impurity removing matter, filtrate is yellowish brown, with lower floor's solution merging, logical nitrogen concentrating under reduced pressure, then be chilled to 0～5 ℃ with frozen water, placed 2 hours, adding the concentrated hydrochloric acid adjust pH is 2.0, place 2h at 0 ℃, filter, wash with frozen water, filter cake is dry with Vanadium Pentoxide in FLAKES, dissolve with butanone again, be brown solution, filter with activated carbon decolorizing, be yellow solution, logical nitrogen removes most of solvent under reduced pressure, the residuum nitrogen-filled seal, 0 ℃ of static spending the night in left and right, there is light yellow crystal to separate out, filter to get light yellow crystal, dry with Vanadium Pentoxide in FLAKES vacuum thousand, get 5-Methylpyrazine-2-carboxylic acid, yield 36.53%.
(7) use KMnO
4One-step oxidation process: the method is raw material with 2,5-dimethylpyrazine, under the existence of inhibitor, adds KMnO
4 Solution reaction 1～10 hour, heat filtering is removed MnO
2Filtrate through concentrating under reduced pressure, use the mineral acid acid out, filter and to obtain the portioned product 5-Methylpyrazine-2-carboxylic acid, filtrate again with the extraction agent extraction through underpressure distillation, then obtain the portioned product 5-Methylpyrazine-2-carboxylic acid.
Above method has the shortcomings such as production cost is high, and process is complicated, and productive rate is low, and product is difficult to be separated, and pollution is high.
Microbial method prepares 5-Methylpyrazine-2-carboxylic acid and has the reaction conditions gentleness, and product is single, and the row advantages of higher is selected in the zone, is representing the developing direction of Green Chemistry.
(3) summary of the invention
The bacterial strain that the purpose of this invention is to provide the product dimethylbenzene monooxygenase of the single Oxygenation of catalysis that a strain can high regioselectivity, and prepare application in 5-Methylpyrazine-2-carboxylic acid at microbial fermentation, to overcome the deficiency of chemical synthesis process.
The technical solution used in the present invention is:
Dimethylbenzene monooxygenase bacterial strain---pseudomonas putida (Pseudomonas putida) ZJB-LLJ is produced in one strain, be preserved in Chinese Typical Representative culture collection center, address: China, Wuhan, Wuhan University 430072, deposit number CCTCC No:M 2011395, preservation date on November 13rd, 2011.
This bacterial strain is the present application people from near Hangzhou, more than 100 parts, the ground soil samples such as Taizhou, Dongyang, Jinhua, the efficient oxidation 2 of energy that obtains through primary dcreening operation, multiple sieve and separation and purification, and the 5-methylpyrazine prepares the new bacterial strain of 5-Methylpyrazine-2-carboxylic acid.According to its physiological and biochemical property, be accredited as Pseudomonas putida.
The principal character of this bacterial strain is as follows:
Colonial morphology: cultivate 24h in 30 ℃ at the LB culture medium flat plate, bacterium colony is rounded, diameter, and corrugationless, the smooth of the edge, glossy, milk yellow is translucent.
Cellular form: present elliposoidal, be about 0.8 μ m, diameter is about 0.4 μ m.
Physio-biochemical characteristics: the carbon source positive is utilized project: alpha-D-glucose, D-MANNOSE, D-Fructose, glycerine, D-Fructose-6-PO4, ALANINE, L-arginine, L-Aspartic acid, Pidolidone, L-Histidine, L-Glutimic acid, Serine, D-galacturonic acid, D-glyconic acid, D-glucuronic acid, glucuronamide, mucic acid, quinic acid, D-saccharic acid, Pfansteihl, citric acid, α-ketoglutaric acid, L MALIC ACID, γ-aminobutyric acid, beta-hydroxy-D, L butyric acid, propionic acid, acetic acid.the carbon source feminine gender is utilized project: dextrin, D-Maltose, the D-trehalose, the D-cellobiose, gentiobiose, sucrose, the D-turanose, stachyose, the D-raffinose, α-D-lactose, the D-melibiose, Beta-methyl-D glucosides, the D-saligenin, N-acetyl-D glycosamine, N-acetyl-β-D mannosamine, N-acetyl-D GalN, N-acetyl-neuraminate, the D-semi-lactosi, the 3-methyl glucoside, the L-trehalose, the L-rhamnosyl, inosine, the D-Sorbitol Powder, PEARLITOL 25C, D-R alcohol, inositol, D-Glucose-6-PO4, D-Asp, D-Ser, gel, glycyl-L-PROLINE, pectin, the L-GaA lactone, p-hydroxyl-phenylacetic acid, the D-ALPHA-Hydroxypropionic acid methyl esters, D-malic acid, the bromine succsinic acid, polysorbate40, alpha-hydroxybutyric acid, α-ketone group-butyric acid, etheric acid.Positive sensitive items to chemical substance: pH 6,1%NaCl, 1% Sodium.alpha.-hydroxypropionate, potassium tellurite, Sodium propanecarboxylate.Negative sensitive items to chemical substance: pH 5,4%NaCl, 8%NaCl, fusidinic acid, D-Ser, troleomycin, Rifamycin Sodium, MINOCYCLINE HCL, lincomycin, Guanidinium hydrochloride, sulfuric acid sodium in four last of the ten Heavenly stems, vancomycin, tetrazolium violet, ditetrazolium chloride, Nalidixic Acid, lithium chloride, aztreonam, sodium bromate.
The physical length of this bacterial strain 16SrDNA amplified production is 1529bp, and sequence is as follows:
AGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGATGACGGGAGCTTGCTCCTTGATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGACAACGTTTCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGGGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCAAGCTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGAATCCTTGAGATTTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATGCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCTCACAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGGGGACGGTTACCACGGTGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGGGAACCTGCGGCTGGATCACCTCCTT
The invention still further relates to described pseudomonas putida ZJB-LLJ and prepare application in 5-Methylpyrazine-2-carboxylic acid at microbial fermentation.
Concrete, described being applied as: contain the enzyme somatic cells as catalyzer take what the cultivation of pseudomonas putida ZJB-LLJ multiparity enzyme obtained, with 2, the 5-dimethylpyrazine is substrate, time carried out conversion reaction 24～48 hours at 28～32 ℃, pH7.0～8.0, obtain described 5-Methylpyrazine-2-carboxylic acid in reaction solution.Reaction finishes the centrifugal supernatant that goes of afterreaction liquid can detect the generation 5-Methylpyrazine-2-carboxylic acid of telling with the high performance liquid chromatography detection.
Because be acid-producing, for improving reaction yield, the fermentation reaction process need be regulated pH between 7.0～8.0 with NaOH solution.Can make reaction complete as early as possible and productive rate the highest.
Described product enzyme is cultivated and is carried out in take p-Xylol as the culture medium of unique carbon source and the energy, time cultivates 12～24 hours in 28～32 ℃, pH7.0～8.0.For preventing p-Xylol to the toxic effect of bacterial strain, p-Xylol provides carbon source as good take the form of steam as strain growth, and specific practice is that welding one glass pipe in the middle of the glass shaking flask is in the small tubes in the middle of p-Xylol is joined.
Common, before producing the enzyme cultivation, bacterial strain need carry out enlarged culturing with the seed culture fluid of the applicable pseudomonas putida of routine and obtain seed liquor, and seed liquor is inoculated in culture medium with the inoculum size of 5% volume ratio again.Described seed culture medium concentration forms can be as follows: peptone 10g/L, and yeast extract paste 5g/L, NaCl5g/L, solvent are water.
Described culture medium also can add other common trace elements take p-Xylol as unique carbon source and the energy, and in the present invention, culture medium quality composition can be as follows: (NH
4)
2SO
41.0～3.0g/L; Na
2HPO
42H
2O 1.0～5.0g/L; KH
2PO4 0.5～2.0g/L; NaCl 1.0～5.0g/L; MgCl
26H
2O 0.1～0.5g/L; CaCl
22H
2O 1.0～5.0g/L; FeCl
36H
2O 0.1～0.5g/L; ZnSO
47H
2O 0.05～0.2g/L; MnCl
24H
2O 0.05～0.1g/L; H
3BO
30.1～0.5g/L; CaCl
26H
2O 0.1～0.3g/L; CuCl
22H
2O 0.05～0.02g/L; NiCl
26H
2O 0.01～0.03g/L; Na
2MoO
42H
2O 0.01～0.05g/L; EDTANa
22H
2O 3.0～8.0g/L; FeSO
47H
2O 1.0～3.0g/L, p-Xylol 0.5～2mL/L, solvent are water, pH 6.8～7.2.For preventing p-Xylol to the toxic effect of bacterial strain, p-Xylol provides carbon source take the form of steam as strain growth, and specific practice is welding one glass pipe in the middle of the glass shaking flask, and p-Xylol is in small tubes in the middle of joining.
Concrete, described culture medium quality is composed as follows: (NH
4)
2SO
42.0g/L; Na
2HPO
42H
2O 2.5g/L; KH
2PO4 1.0g/L; NaCl 3.0g/L; MgCl
26H
2O 0.4g/L; CaCl
22H
2O 2.5g/L; FeCl
36H
2O 0.25g/L; ZnSO
47H
2O 0.1g/L; MnCl
24H
2O 0.09g/L; H
3BO
30.3g/L; CaCl
26H
2O 0.2g/L; CuCl
22H
2O 0.01g/L; NiCl
26H
2O 0.02g/L; Na
2MoO
42H
2O 0.03g/L; EDTANa
22H
2O 5.0g/L; FeSO
47H
2O 2.0g/L, p-Xylol 1mL/L, solvent are water, pH 7.0.For preventing p-Xylol to the toxic effect of bacterial strain, p-Xylol provides carbon source take the form of steam as strain growth, and specific practice is welding one glass pipe in the middle of the glass shaking flask, in the small tubes in the middle of p-Xylol joins.
Described conversion reaction is directly to carry out in aforementioned culture medium, namely adds substrate after producing the enzyme cultivation and carries out conversion reaction, and the starting point concentration of substrate is 2～6g/L.
Beneficial effect of the present invention is mainly reflected in: a kind of new bacterial strain that produces the dimethylbenzene monooxygenase is provided, can have prepared the pharmaceutical intermediate 5-Methylpyrazine-2-carboxylic acid by this bacterial strain, the accumulation of shake flask fermentation cultured products is 1.4g/L, and productive rate is 40%.Use the present invention to produce 5-Methylpyrazine-2-carboxylic acid, reaction conditions is gentle, and is energy-conservation, environmental protection, and the process of the more important thing is is simple, and product extracts easily, has good prospects for commercial application.
(4) description of drawings
Fig. 1 is enrichment and fermentation culture schematic diagram;
Fig. 2 is the form (B) under the single colonial morphology (A) of bacterial strain ZJB-LLJ and scanning electron microscope;
Fig. 3 is bacterial strain 16S rDNA sequence pcr amplification argrose electrophorogram;
Fig. 4 is the phylogenetic analysis result of the bacterial strain on ZJB-LLJ bacterial strain and NCBI;
Fig. 5 is substrate in the fermentation culture process, the change in concentration of product and the variation of biomass; ● the change in concentration of expression substrate, ■ represents the change in concentration of product, the variation of ▲ expression biomass,
The variation of expression pH;
Fig. 6 is the variation of production concentration in different inductor culturing process; ● the expression p-Xylol, ▲ expression m-xylene, ■ represents dimethylbenzene;
Fig. 7 is that the substrate joining day is on the impact of reaction;
Fig. 8 is that different pH substratum are on the impact of reaction;
Fig. 9 is that in constant different pH fermenting processs, product changes; ■ pH6, ● pH7, ▲ pH8,
◆ pH10.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1: the screening of microorganism, evaluation
1. the screening of microorganism
Culture presevation: yeast extract paste 5g/L, peptone 10g/L, NaCl 5g/L, agar 20g/L, solvent are water, pH 7.0;
The seed liquor substratum: yeast extract paste 5g/L, peptone 10g/L, NaCl 5g/L, solvent are water, pH 7.0;
Enzymatic production substratum, bacterial classification enrichment medium:
(NH
4)
2SO
42.0g/L; Na
2HPO
42H
2O 2.5g/L; KH
2PO4 1.0g/L; NaCl 3.0g/L; MgCl
26H
2O 0.4g/L; CaCl
22H
2O 2.5g/L; FeCl
36H
2O 0.25g/L; ZnSO
47H
2O 0.1g/L; MnCl
24H
2O 0.09g/L; H
3BO
30.3g/L; CaCl
26H
2O 0.2g/L; CuCl
22H
2O 0.01g/L; NiCl
26H
2O 0.02g/L; Na
2MoO
42H
2O 0.03g/L; EDTANa
22H
2O 5.0g/L; FeSO
47H
2O 2.0g/L, p-Xylol 1mL/L, solvent are water, pH 7.0.P-Xylol provides carbon source take the form of steam as strain growth, and specific practice is welding one glass pipe in the middle of the glass shaking flask, and the 1ml p-Xylol is in small tubes in the middle of joining.
Near the sewage draining exit in chemical plant, orchard, vegetable garden vegetation bed gathers soil sample, and the difference of the regional distribution in soil sample source also can be brought more opportunities of success to screening operation simultaneously.Thereby near Hangzhou, the ground such as Taizhou, Dongyang, Jinhua collected more than totally 100 parts of soil samples in order to bacterial screening.Detailed process is as follows:
Get a little soil sample 150r/min temperature in the enzymatic production substratum and be on the shaking table of 30 ℃ through twice enrichment culture, be forwarded to the test tube slant and be numbered in biochemical cultivation case and cultivated 1 day through single bacterium colony of choosing of dilution spread.Be forwarded to the shake flask fermentation culture medium from the inclined-plane again, to add 100 μ L 2, the 5-dimethylpyrazine after p-Xylol steam inducing culture 12h.Cultivate after 2 days, take a sample centrifugal, cross and detect (pillar: C with high performance liquid chromatography after film is processed
18Post, acetonitrile: water=3: 7, flow velocity 1mL/min detects wavelength 275nm, 40 ℃ of column temperatures).Selection has the bacterial strain of activity of conversion, finally selects the highest bacterial strain of a strain activity of conversion (be numbered ZJB-LLJ, namely CCTCCNO:M 2011395) and does follow-up strain identification and conditions of flask fermentation optimization experiment.
2. microorganism strains is numbered the Physiology and biochemistry evaluation of " ZJB-LLJ " bacterial strain
Then use method of dilution butteron on plate through enrichment culture from soil, obtaining three strains through high performance liquid chromatography detection screening can be with 2, the 5-dimethylpyrazine is oxidized to the bacterial strain of 5-Methylpyrazine-2-carboxylic acid, therefrom selecting a strongest label of strain oxidation activity is ZJB-LLJ such as Fig. 2 (A), and colonial morphology is less, circle, the smooth of the edge, glossy, milk yellow is translucent.Obtain cellular form by electron microscope scanning and present cell and present elliposoidal, be about 0.8 μ m, diameter is about 0.4 μ m Fig. 2 (B).
Utilize Biolog (GEN III) automatic microbe identification systems to carry out 94 kinds of phenotype tests (the Biolog microorganism identifies that automatically special agent and substratum etc. are available from Biolog company) to bacterial strain ZJB-LLJ, comprise that 71 kinds of utilization of carbon source situations detect and 23 kinds of chemosensitivities detect: with inoculation in the BUG plate culture medium, 33 ℃ of constant temperature culture 2 days, with aseptic cotton carrier, the thalline on flat board is washed, (IF-A) mixes with inoculation liquid, make bacteria suspension, be adjusted to 95%T with turbidometer.With the 8 electronic liquid fillerss in hole, bacteria suspension is added in respectively in each hole of Biolog GEN III micropore identification plate every hole 100 μ L.The micropore identification plate is placed in 33 ℃ of incubators, is placed on reading result on the Biolog readout instrument respectively after cultivating 12h and 24h.Analyze Metabolic Fingerprinting through the Biolog readout instrument, bacterial strain ZJB-LLJ can utilize more by force 27 kinds of carbon sources, a little less than can not utilizing or utilize ability to other 44 kinds of carbon sources; Bacterial strain ZJB-LLJ is to 16 kinds of chemical substance sensitivities.The Biolog system provides the 24h qualification result, as shown in Table 1 and Table 2.
Table 1: the utilize ability of bacterial strain ZJB-LLJ to 71 kinds of carbon sources on Biolog GEN III plate
Table 2: the chemosensitivity of bacterial strain ZJB-LLJ to 23 kinds of chemical substances on Biolog GEN III plate
3.16S rDNA complete sequence determination and analysis
3.1.16S rDNA sequence amplification
Take the total DNA of the cell that extracts as template, utilize the 16S rDNA sequence of the primer amplification bacterial strain ZJB-LLJ of design, the PCR product is carried out 0.9% agarose gel electrophoresis, successfully obtained a fragment that is about as 1.5kb through pcr amplification as can be seen from Figure 2, meet expected results.
3.2.16S rDNA analyzes
To be cloned into through the fragment of pcr amplification the recombinant plasmid that contains the 16S rDNA fragment that this experiment obtains after T carrier, extracting plasmid, confirm that through order-checking this fragment physical length is 1529bp, as shown in Figure 3.
3.3. bacterial classification belongs to determining of planting
The data of preserving in the sequence that obtains in 6.2.2 and GenBank are carried out similarity analysis to be found, the Identifying micro-organisms ZJB-LLJ of institute of the present invention and the highest (homology of Pseudomonas putida (EU439422.1) homology, 99%/1529bps, based on 16S rDNA), according to microorganism molecular genetics identity principle, higher than 95%, identify that bacterium belongs to the contrast bacterium substantially based on the homology of 16S rDNA sequence.Therefore, the microorganism of this experimental identification belongs to the putida kind that Pseudomonas belongs to, and Chinese is pseudomonas putida.
With using blast program to compare and further analyze in the sequence information input National Center Biontechnology Information that records, then use related software bacterial strain to be carried out the structure of sequence alignment and phylogenetic tree.The results are shown in Figure 4.
4. fermentation reaction process
Record the growth curve of seed liquor through experiment, the result demonstration begins to enter logarithmic phase from 5h, and 12h finishes.Enter stationary phase afterwards.So the enzymatic production of back is cultivated the seed liquor of selecting 12h.
As shown in Figure 5, in the enzymatic production culturing process, biomass constantly increases, and cultivating 60h OD maximum value is 0.48.Induce to produce to add substrate after enzyme is cultivated 12h, concentration of substrate is diminished gradually by 1.71g/L, minimum value 0.69g/L occurs to 44h.After adding substrate, production concentration becomes large gradually by zero, reaches maximum value 0.65g/L after 44h.PH reduces gradually by 6.8, and reaching minimum by 52 hours is that after 5.2, pH gos up again to some extent.
4.2 the impact of inductor on reaction
Select dimethylbenzene, p-Xylol, m-xylene, o-Xylol (concentration is 1mL/L) as inductor research inductor on the impact of reaction (for to prevent p-Xylol to the toxic effect of bacterial strain, dimethylbenzene, p-Xylol, m-xylene, o-Xylol provides carbon source take the form of steam as strain growth respectively, and specific practice is welding one glass pipe in the middle of the glass shaking flask, and inductor is in small tubes in the middle of joining).
Experimental result reacts slower as shown in Figure 6 when dimethylbenzene and m-xylene begin, production concentration is less.The 12h afterreaction is accelerated, and peak value appears in 36h, raises again after concentration diminishes a little.The p-Xylol initial reaction is very fast, and production concentration is larger, and the 12h afterreaction is slack-off, and peak value appears in 36h, raises after concentration diminishes again.That final product concentration is the highest is dimethylbenzene 0.39g/L, is secondly p-Xylol 0.37g/L, is then m-xylene 0.36g/L, and o-Xylol does not have activity substantially.Consider the generation of inducing monooxygenase that p-Xylol can be best.
4.3 the impact of substrate joining day on reaction
Add substrate in the different time after the seed liquor of switching 12h, cultivate respectively after 48h sampling centrifugal, detect with HPLC.Experimental result such as Fig. 7: the 0h substrate joining day adds substrate after namely transferring immediately, and production concentration is 0.24g/L.Before 12h, along with the increase of induction time, final product concentration increases thereupon.When maximum value 0.63g/L appears at 12h.After this elongated along with the substrate joining day, production concentration begins to diminish.So the substrate joining day is defined as 12h.
4.4 the impact of the initial different pH of enzymatic production substratum (reaction process is no longer regulated pH) on reaction.As shown in Figure 8, pH3 and 4 o'clock production concentrations are zero, and illustrating that pH is too small has restraining effect to thalli growth.During pH5, production concentration is 0.10g/L, and along with the increase of pH, production concentration also increases gradually, the pH10h production concentration reaches maximum 0.91g/L, pH11 and 12 o'clock production concentrations are zero, and reaction stops substantially, illustrates alkalinely excessive thalli growth also to be had restraining effect.
Experimental result such as Fig. 9 under ensuing constant pH show, the product accumulation volume of whole fermenting process is all maximum when pH is 8, final stage pH7,8,9 to fermentation reaches unanimously substantially, and conversion fully of substrate, and the product amount reaches 1.41g/L.And the output of less pH6 is 1.18g/L, and the output of pH10 is less, only has 0.55g/L.The yeasting of the neutral meta-alkali of this explanation is more suitable for the accumulation of product.
SEQUENCE LISTING
＜110〉Zhejiang Polytechnical University
＜120〉pseudomonas putida and the application thereof of dimethylbenzene monooxygenase produced in a strain
<130>
<160> 1
<170> PatentIn version 3.4
<210> 1
<211> 1529
<212> DNA
<213> Pseudomonas putida
<400> 1
agagtttgat cctggctcag attgaacgct ggcggcaggc ctaacacatg caagtcgagc 60
ggatgacggg agcttgctcc ttgattcagc ggcggacggg tgagtaatgc ctaggaatct 120
gcctggtagt gggggacaac gtttcgaaag gaacgctaat accgcatacg tcctacggga 180
gaaagcaggg gaccttcggg ccttgcgcta tcagatgagc ctaggtcgga ttagctagtt 240
ggtggggtaa tggctcacca aggcgacgat ccgtaactgg tctgagagga tgatcagtca 300
cactggaact gagacacggt ccagactcct acgggaggca gcagtgggga atattggaca 360
atgggcgaaa gcctgatcca gccatgccgc gtgtgtgaag aaggtcttcg gattgtaaag 420
cactttaagt tgggaggaag ggcagtaagt taataccttg ctgttttgac gttaccgaca 480
gaataagcac cggctaactc tgtgccagca gccgcggtaa tacagagggt gcaagcgtta 540
atcggaatta ctgggcgtaa agcgcgcgta ggtggtttgt taagttggat gtgaaagccc 600
cgggctcaac ctgggaactg catccaaaac tggcaagcta gagtacggta gagggtggtg 660
gaatttcctg tgtagcggtg aaatgcgtag atataggaag gaacaccagt ggcgaaggcg 720
accacctgga ctgatactga cactgaggtg cgaaagcgtg gggagcaaac aggattagat 780
accctggtag tccacgccgt aaacgatgtc aactagccgt tggaatcctt gagattttag 840
tggcgcagct aacgcattaa gttgaccgcc tggggagtac ggccgcaagg ttaaaactca 900
aatgaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg 960
aagaacctta ccaggccttg acatgcagag aactttccag agatggattg gtgccttcgg 1020
gaactctgac acaggtgctg catggctgtc gtcagctcgt gtcgtgagat gttgggttaa 1080
gtcccgtaac gagcgcaacc cttgtcctta gttaccagca cgttatggtg ggcactctaa 1140
ggagactgcc ggtgacaaac cggaggaagg tggggatgac gtcaagtcat catggccctt 1200
acggcctggg ctacacacgt gctacaatgg tcggtacaga gggttgccaa gccgcgaggt 1260
ggagctaatc tcacaaaacc gatcgtagtc cggatcgcag tctgcaactc gactgcgtga 1320
agtcggaatc gctagtaatc gcgaatcaga atgtcgcggt gaatacgttc ccgggccttg 1380
tacacaccgc ccgtcacacc atgggagtgg gttgcaccag aagtagctag tctaaccttc 1440
ggggggacgg ttaccacggt gtgattcatg actggggtga agtcgtaaca aggtagccgt 1500
aggggaacct gcggctggat cacctcctt 1529
Claims (6)
1. pseudomonas putida (Pseudomonas putida) ZJB-LLJ of dimethylbenzene monooxygenase is produced in a strain, and its deposit number is CCTCC No:M 2011395, and preservation date is on November 13rd, 2011.
2. pseudomonas putida ZJB-LLJ as claimed in claim 1 prepares application in 5-Methylpyrazine-2-carboxylic acid at microbial fermentation.
3. application as claimed in claim 2, it is characterized in that described being applied as: contain the enzyme somatic cells as catalyzer take what the cultivation of pseudomonas putida ZJB-LLJ multiparity enzyme obtained, with 2, the 5-dimethylpyrazine is substrate, time carried out conversion reaction 24 ~ 48 hours at 28 ~ 32 ℃, pH7.0 ~ 8.0, obtain described 5-Methylpyrazine-2-carboxylic acid in reaction solution.
4. application as claimed in claim 3, it is characterized in that described product enzyme is cultivated carries out in take p-Xylol as the culture medium of unique carbon source and the energy, time cultivates 12 ~ 24 hours in 28 ~ 32 ℃, pH7.0 ~ 8.0.
5. application as claimed in claim 4 is characterized in that described culture medium quality is composed as follows: (NH
4)
2SO
41.0 ~ 3.0 g/L; Na
2HPO
42H
2O 1.0 ~ 5.0 g/L; KH
2PO4 0.5 ~ 2.0 g/L; NaCl 1.0 ~ 5.0 g/L; MgCl
26H
2O 0.1 ~ 0.5 g/L; CaCl
22H
2O 1.0 ~ 5.0 g/L; FeCl
36H
2O 0.1 ~ 0.5 g/L; ZnSO
47H
2O 0.05 ~ 0.2 g/L; MnCl
24H
2O 0.05 ~ 0.1 g/L; H
3BO
30.1 ~ 0.5 g/L; CaCl
26H
2O 0.1 ~ 0.3 g/L; CuCl
22H
2O 0.05 ~ 0.02 g/L; NiCl
26H
2O 0.01 ~ 0.03 g/L; Na
2MoO
42H
2O 0.01 ~ 0.05 g/L; EDTANa
22H
2O 3.0 ~ 8.0 g/L; FeSO
47H
2O 1.0 ~ 3.0 g/L, p-Xylol 0.5 ~ 2 mL/L, solvent are water, pH 6.8 ~ 7.2.
6. application as claimed in claim 3, the starting point concentration that it is characterized in that described substrate is 2～6g/L.
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| CN108753862A (en) * | 2018-06-27 | 2018-11-06 | 泰州市惠利生物科技有限公司 | A method of continuously fermented using recombination bacillus coli and prepares 5-Methylpyrazine-2-carboxylic acid |
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| CN106434434B (en) * | 2016-09-12 | 2017-12-22 | 迪沙药业集团有限公司 | One plant of strains A rthrobacter woluwensis for producing dimethylbenzene monooxygenase and its application |
| CN107541532A (en) * | 2017-09-29 | 2018-01-05 | 迪沙药业集团有限公司 | A kind of preparation method of the carboxylic acid of 5 methylpyrazine 2 |
| CN107974428A (en) * | 2017-12-13 | 2018-05-01 | 迪沙药业集团有限公司 | A kind of recombination bacillus coli and the method for converting production 5-Methylpyrazine-2-carboxylic acid |
| CN110527656B (en) * | 2019-09-04 | 2021-07-20 | 江南大学 | Engineering bacterium for efficiently synthesizing 5-methylpyrazine-2-carboxylic acid and construction method and application thereof |
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| CN102181375A (en) * | 2010-12-09 | 2011-09-14 | 江苏商达水务有限公司 | Aerobic phosphorus-removing bacterium-pseudomonas putida WSH 1002 and application thereof |
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| CN108753862A (en) * | 2018-06-27 | 2018-11-06 | 泰州市惠利生物科技有限公司 | A method of continuously fermented using recombination bacillus coli and prepares 5-Methylpyrazine-2-carboxylic acid |
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