CN103289928A - Pseudomonas fluorescens and application thereof in biosynthesizing methionine - Google Patents

Pseudomonas fluorescens and application thereof in biosynthesizing methionine Download PDF

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CN103289928A
CN103289928A CN2013102141122A CN201310214112A CN103289928A CN 103289928 A CN103289928 A CN 103289928A CN 2013102141122 A CN2013102141122 A CN 2013102141122A CN 201310214112 A CN201310214112 A CN 201310214112A CN 103289928 A CN103289928 A CN 103289928A
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pseudomonas fluorescens
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CN103289928B (en
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郑裕国
金利群
李晓庆
柳志强
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Zhejiang University of Technology ZJUT
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Abstract

The invention provides a rhodococcus rhodochrous strain, namely, pseudomonas fluorescens ZJB-12001, and an application thereof in 2-amino-4-methylmercapto-butyronitrile synthetic methionine. The strain is preserved in the Chinese Typical Culture Collection Center which is located in Wuhan College, Wuhan, China, the postcode is 430072, the preservation number is CCTCC NO: M 2012449, and the preservation date is November 8th, 2012. The pseudomonas fluorescens ZJB-12001 is applied to experiments for producing methionine in a biological catalysis mode, the result shows that the pseudomonas fluorescens ZJB-12001 can be used for effectively producing methionine in the biological catalysis mode, and the conversion rate of 20mM3-cyanopyridine within 60 minutes can be 60-75%, so that the pseudomonas fluorescens ZJB-12001 has a wide application prospect in the field of biological catalysis methionine production.

Description

Pseudomonas fluorescens and the application in the biosynthesizing methionine(Met) thereof
(1) technical field
The present invention relates to a strain and produce nitrilase bacterial strain---Pseudomonas fluorescens (Pseudomonas fluorescens) ZJB-12001, and the application in bio-transformation 2-amino-4-methylthio group butyronitrile synthetic methionine.
(2) background technology
Methionine(Met) (Methionine) has another name called methionine(Met), and chemical name is 2-amino-4-methylmercapto butyric acid, and molecular formula is C 5H 11NO 2S, outward appearance is white plates crystallization or powder, and special odor is arranged, it is little sweet to distinguish the flavor of, 281 ℃ of fusing points (decomposition), relative density 1.34, water-soluble, diluted acid and diluted alkaline, be slightly soluble in alcohol, be insoluble to ether, 1% methionine(Met) pH value of aqueous solution is 5.6～6.1, to heat and air-stable, to the strong acid instability, can cause demethylation.Methionine(Met) contains an amino and carboxyl, is the amino acid of unique sulfur-bearing, has opticity, be divided into D type and L type, in animal body, the L type easily by intestinal absorption, the D type to be the L type through enzymatic conversion, just can be utilized.
Methionine(Met) is a kind of very important restricted indispensable amino acid in the Animal nutrition.The reinforcer of a kind of important protein matter of Chang Zuowei feed and remedy the nutritional additive of amino acid balance, particularly in the bird feed, methionine(Met) is first limiting amino acid at mixed feed.It can promote that the fowl dirty swine is long, increases cutability, shorten breeding cycle, and the animal-feed that is added with methionine(Met) can help animal to grow up fast at short notice, saves about 40% feed.In addition, methionine(Met) can suppress the accumulation of fat in liver and the artery, the blood circulation of the metabolism of promotion lipid and brain, heart, kidney, promoting digestion, detoxifcation, drainage toxicant, heavy metal etc.; When every day, usage quantity was 800～1600mg, but antidepressant; The effect of anti-inflammatory is arranged in bone, joint disease, and promote the rehabilitation in joint; Anti-alopecia etc.Therefore, methionine(Met) is widely used in fields such as feed, medicine, foods and cosmetics, wherein the consumption maximum of fodder additives.
At present, the production method of methionine(Met) is mainly chemical synthesis, as glycolylurea method, amino lactone process, malonic ester method, solid-liquid phase transfer catalysis process etc.:
(1) glycolylurea method: propenal and thiomethyl alcohol carry out addition reaction and generate methylthiopropionaldehyde under catalyst action; Methylthiopropionaldehyde and NaCN or HCN, NH 4HCO 3Condensation reaction takes place make methylmercaptoethyl glycolylurea (glycolylurea); Then the methylmercaptoethyl glycolylurea is at NaOH or K 2CO 3Effect hydrolysis down generates Sodium L-methioninate/sylvite; Further adopt H 2SO 4, HCl or CO acidifying namely get DL-methionine, concentrate at last, crystallization, separation obtain the methionine(Met) finished product.Glycolylurea method Technology maturation, reaction yield height, level of automation height; By product such as sodium sulfate, carbonic acid gas, ammonia etc. all can circulate in technological process, therefore, glycolylurea method technology becomes the production method that company of external most of methionine(Met) manufacturer U.S. promise Victory-idea, French Luo Na-Rhone-Poulenc and goldschmidt chemical corporation, Japanese Cao Da and Sumitomo company etc. generally adopt.
(2) amino lactone process: amino lactone process has another name called the gamma-butyrolactone method, is the raw material synthetic methionine with γ-butyl lactone mainly.γ-butyl lactone bromination obtains alpha, gamma-dibromo-butyric acid, and heating is carried out condensation reaction and generated α-bromo-γ-butyl lactone, obtains alpha-amino group-γ-butyl lactone with Liquid Ammonia Treatment, and last and sodium methyl mercaptide reacts and makes DL-methionine, and productive rate is 40%.
(3) malonic ester method: mainly one of them is the feedstock production methionine(Met) to the malonic ester method with 2-methylthio group Narcotile and phthalimide-based malonic ester sodium salt, ethyl acetamide base cyano-acetate, diethyl kharophen malonate, ethyl acetoacetic ester, productive rate is respectively 58%, 48%, 60.5%, 70%, wherein 2-methylthio group Narcotile and ethyl acetoacetic ester are reacted, obtain β-methylthio ethyl methyl aceto acetate, then with NH 3Reaction uses the NaOH hydrolysis to obtain methionine sulphoxide subsequently, uses calcium sulfate reduction methionine sulphoxide to be worth DL-methionine, and productive rate reaches 70%.
(4) solid-liquid phase transfer catalysis process: in the mixed solution of glycine ethyl ester hydrochloride, phenyl aldehyde, anhydrous magnesium sulfate, methylene dichloride, drip catalyst of triethylamine and prepare benzene methylene ethyl aminoacetate, benzene methylene ethyl aminoacetate and 2-chloroethyl methyl thioether be the amino first and second thioether group ethyl acetate of synthetic benzene methylene under the saponification of potassium hydroxide and Anhydrous potassium carbonate, use concentrated hydrochloric acid hydrolysis to obtain methionine(Met) at last, productive rate reaches 59%.
Although chemosynthesis methionine(Met) technology is comparatively ripe at present, the growing amount of by product reduces to some extent relatively, or recycle and reuse, but still inevitably used hazardous compounds such as having corrosive acid, alkali, various vitriol have been produced, this not only need increase extra-pay recycling salt, and has increased investment goods, does not meet the requirement of Green Chemistry and atom economy.Also have some scholars to pass through the fermentative Production methionine(Met) in research, although the methionine(Met) of fermentative Production all is the L type, there is serious feedback inhibition in this approach, and output is little, and yield is extremely low, and cost is higher, and industrialization is slow, does not still possess industrial value.Microbial enzyme method transforms has advantages such as raw materials cost is low, flow process is simple, facility investment is few, reaction conditions is gentle, by product is few, environmental pollution is little, meet the requirement of Green Chemistry and atom economy, therefore, under the contrast, microbial enzyme method transforms the production methionine(Met) and has great application prospect.
(3) summary of the invention
The object of the invention provides the new bacterial strain that nitrilase is produced in a strain, and prepares the application in the methionine(Met) acid at biocatalysis 2-amino-4-methylthio group butyronitrile.
The technical solution used in the present invention is:
Pseudomonas fluorescens (Pseudomonas fluorescens) ZJB-12001 is preserved in Chinese typical culture collection center, address: China, Wuhan, Wuhan University, postcode 430072, deposit number CCTCC No:M2012449, preservation date on November 08th, 2012.
Described Pseudomonas fluorescens ZJB-12001 bacterial strain is from from the Zhejiang Province surplus in the of 80 part soil sample and the sewage work's sewage, and the 2-of the catalysis efficiently amino-4-methylthio group butyronitrile that obtains through primary dcreening operation, multiple sieve and separation and purification prepares the new bacterial strain of methionine(Met).According to its physiological and biochemical property, be accredited as Pseudomonas fluorescens (Pseudomonas fluorescens).
The major physiological biochemical character of described Pseudomonas fluorescens ZJB-12001 bacterial strain is as follows:
Colonial morphology: cultivate 1-2d in 30 ℃ at the beef extract-peptone plate culture medium, about colony diameter 1-2cm, bacterium colony is creamy white, spheroidal, light, moistening, neat in edge, surperficial microprotrusion, easy picking.
Physiological and biochemical property: the carbon source positive is utilized project: glycerine, flesh nucleosides, D-fructose-6-phosphate, D-Serine, L-arginine, L-L-glutamic acid, L-Histidine, maltonic acid, glucuronamide, quinic acid, right-hydroxyl-toluylic acid, D-lactic acid, D-lactic acid, citric acid, α-Tong Wuersuan, D-oxysuccinic acid, L MALIC ACID, gamma-amino-butyric acid, Alpha-hydroxy-butyric acid, beta-hydroxy-D, L-butyric acid, acetic acid.
The carbon source feminine gender is utilized project: dextrin, D-maltose, N-acetyl-D-amino glucose, the D-saligenin, Beta-methyl-D-glucoside, the D-melibiose, α-D-lactose, the D-raffinose, stachyose, the D-turanose, sucrose, sucrose, the D-cellobiose, the D-trehalose, D-maltose, N-acetyl-β-D-mannosamine, gelatin, glycyl (base)-L-proline(Pro), the L-Pyrrolidonecarboxylic acid, the L-Serine, pectin, the D-galacturonic acid, the L-galactonolactone, the D-glucuronic acid, N-acetyl-β-D-mannosamine, the D-G-6-P, the D-aspartic acid, α-ketone group-butyric acid, etheric acid, formic acid, the D-Fucose, the L-Fucose, the L-Fucose, the D-sorbyl alcohol, D-N.F,USP MANNITOL, the D-arabitol, inositol, polysorbate40, the N-n acetylneuraminic acid n, glactaric acid, the D-saccharic acid, the D-methyl lactate, the D-seminose, the D-semi-lactosi, the 3-methyl glucoside.
The 16S rDNA amplified production sequence (1527bp) of this bacterial strain is as follows:
AGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGTAGAGAGAAGCTTGCTTCTCTTGAGAGAGGCGGACGGGTGAGTAAAGCCTAGGAATCTGCCTGGTAGTGGGGGATAACGTTCGGAAACGGACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTTAAGAAGGTCTTCGGGTTGTAAAGCACTTTAAGTTGGGAGGAAGGGCATTAACCTAATACGTTAGTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCAAAACTGACTGACTAGAGTATGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGCAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTAATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGAAGCCTTGAGCTCTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAGCCTTACCAGGCCTTGACATCCAATGAACTTTCTAGAGATAGATTGGTGCCTTCGGGAACATTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTAATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCTCAAAACCGATCGTAGTCCGGATCGTAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGAGGGCGGTTACCACGGTGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGGGAACCTGCGGCTGGATCACCTCCTT。
The invention still further relates to the application in described Pseudomonas fluorescens ZJB-12001 bio-transformation 2-amino-4-methylthio group butyronitrile synthetic methionine.
Concrete, described being applied as: be substrate with 2-amino-4-methylthio group butyronitrile, the enzyme wet thallus that contains that obtains with Pseudomonas fluorescens ZJB-12001 fermentation culture is catalyzer, in the buffered soln of pH7.0～10.0, under 25～55 ℃ of conditions, carry out conversion reaction, make described methionine(Met).After reacting completely, conversion reaction liquid can obtain described methionine(Met) through separation and purification.
Preferably, the starting point concentration of substrate is 1.0～5.0g/L in the described damping fluid, and the quality consumption that contains the enzyme wet thallus that described Pseudomonas fluorescens ZJB-12001 fermentation culture obtains is counted 2.0～7.0g/L with dry cell weight.
The described conversion reaction time is preferably 10～120min.
Described catalyzer can obtain as follows:
(1) slant culture: ZJB-12001 is seeded to slant medium with Pseudomonas fluorescens, cultivates 12～36h at 28～30 ℃, obtains the inclined-plane thalline; The final concentration of described slant medium consists of: peptone 7.5～15g/L, and extractum carnis 3.0～8.0g/L, sodium-chlor 3.0～8.0g/L, agar 15.0～20.0g/L, solvent are water, pH6.5～7.5;
(2) seed culture: be seeded to seed culture medium from inclined-plane thalline picking one transfering loop thalline, cultivate 16～36h, obtain seed liquor for 28～30 ℃; Described seed culture medium final concentration consists of: glucose 10.0～20.0g/L, Sodium Glutamate 5.0～10.0, yeast powder 3.0～6.0g/L, hexanolactam 0.5～2.0g/L, KH 2PO 40.5～1.0g/L, K 2HPO 43H 2O0.5～1.0g/L, MgSO 47H 2O0.1～0.5g/L, solvent are water, pH6.5～7.5;
(3) fermentation culture: the seed liquor that step (2) is obtained is seeded to fermention medium with 2～6% volume ratios, 36～72h is cultivated in concussion under 28～30 ℃, initial pH6.5～7.5 conditions, medium centrifugal separates, and supernatant discarded is got precipitation and namely obtained the described enzyme wet thallus that contains; Described fermention medium final concentration consists of: glycerine 10.0～20.0g/L, trisodium citrate 10.0～20.0g/L, Sodium Glutamate 2.5～7.5g/L, yeast powder 10.0～40.0g/L, hexanolactam 0.5～2.0g/L, KH 2PO 40.5～1.0g/L, K 2HPO 43H 2O0.5～1.0g/L, MgSO 47H 2O0.1～0.5g/L, solvent are water, pH6.5～7.5;
Further, the method for described conversion reaction liquid separation and purification is: after transforming end that conversion reaction liquid is centrifugal, abandon precipitation, and get supernatant liquor and obtain described methionine(Met).
Further, the application of described Pseudomonas fluorescens ZJB-12001 in the bio-transformation synthetic methionine carried out as follows: the enzyme thalline that contains that Pseudomonas fluorescens ZJB-12001 fermentation culture is obtained mixes with 2-amino-4-methylthio group butyronitrile, in the damping fluid of pH7.0～8.5, constitute the conversion reaction system, conversion reaction 20min under 40 ℃ of conditions, after reaction finishes, reaction solution is abandoned precipitation at the centrifugal 10min of 10000rpm, gets supernatant liquor and obtains described methionine(Met); The quality consumption that contains the enzyme somatic cells that described Pseudomonas fluorescens ZJB-12001 fermentation culture obtains is counted 6.67g/L to contain the enzyme dry mycelium, and the initial final concentration of described 2-amino-4-methylthio group butyronitrile is 2.6g/L.
The dry cell weight measuring method that contains the enzyme thalline that Pseudomonas fluorescens ZJB-12001 fermentation culture of the present invention obtains is: it is centrifugal to get a certain amount of fermented liquid, abandon supernatant, dry to constant weight for 80～95 ℃, somatic cells dry weight in unit of measure's volume fermented liquid, and then can obtain the dry cell weight in each stage of fermented liquid.
The high-efficient liquid phase chromatogram condition of detection product methionine(Met) of the present invention is: day island proper body fluid chromatography, stainless steel packed column, filler are C 18, long 0.25m, internal diameter 4.6mm, flow velocity 1ml/min, column temperature room temperature, moving phase are methyl alcohol: 20mM aqueous phosphatic (20V:80V), sample size are 20 μ l.Wherein, the methionine(Met) appearance time is 2.8min.Adopt external standard method to determine the content of methionine(Met) in the sample.
Beneficial effect of the present invention is mainly reflected in: (1) but the present invention from micropopulation, screen obtain the new bacterial strain Pseudomonas fluorescens ZJB-12001(Pseudomonas fluorescens ZJB-12001 that biocatalysis is produced methionine(Met)) CCTCC NO:M2012449, it can effectively produce methionine(Met) under the condition of being fit to; (2) the present invention is used for the experiment of biocatalysis production methionine(Met) with Pseudomonas fluorescens ZJB-12001, the result shows, this bacterium effectively biocatalysis produces methionine(Met), 20mM3-cyanopyridine transformation efficiency can reach 60～75% in 60 minutes, so this bacterium is with a wide range of applications in the field of biocatalysis production methionine(Met).
(4) description of drawings
Fig. 1 is the single colonial morphology photo (10 * 100) of bacterial strain ZJB-12001;
Fig. 2 is the single bacterium colony sem photograph (1 * 12000) of bacterial strain ZJB-12001;
Fig. 3 is the evolutionary tree analytical results of the bacterial strain on bacterial strain ZJB-12001 and the NCBI.
(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 new bacterial strain ZJB-12001
From being inoculated into the enrichment medium by soil sample and the pharmaceutical factory sewage sample that gather in Zhejiang Province; At 30 ℃, on the shaking table of 150rpm behind the enrichment culture 72h, get the 2ml nutrient solution again and be inoculated into and carry out second incubation on the fresh enrichment medium, so repeat 3 circulations.Be applied on the beef extract-peptone plate culture medium after the last nutrient solution dilution, cultivate 24～72h for 30 ℃ and obtain single bacterium colony, picking list colony inoculation carries out preservation to slant medium.To fermention medium, 48h is cultivated in concussion under 30 ℃, initial pH7.0～7.5 conditions from slant medium picking one transfering loop bacterial classification inoculation, centrifugation, and supernatant discarded is got precipitation and is namely obtained containing the enzyme wet thallus; To contain in the phosphoric acid buffer water solution system that the enzyme wet thallus is suspended in pH7.0 respectively, carry out transformation experiment by adding 3-cyanopyridine 2-amino-4-methylthio group butyronitrile as substrate.Above-mentioned 0.15g is contained the enzyme wet thallus to add respectively in the reaction system of the phosphoric acid buffer aqueous solution of 3-cyanopyridine 2-amino-4-methylthio group butyronitrile that initial final concentration is 120mM, behind 30～90 minutes min of 35～40 ℃ of reactions, reaction solution is centrifugal, get supernatant liquor detects methionine(Met) acid respectively with high performance liquid chromatography content, selection has the bacterial strain of activity of conversion, (be numbered ZJB-0915012001, namely CCTCC NO:M2012449 does follow-up strain identification and conversion condition optimization experiment finally to select the highest bacterial strain of a strain activity of conversion.
Described enrichment culture based formulas is (final concentration): glucose 5g/L, K 2HPO 43H 2O0.5g/L, KH 2PO 40.5g/L, MgSO 40.5g/L, FeSO 47H 2O0.02g/L, hexanolactam 4g/L, pH7.0, solvent are water.121 ℃ of substratum, sterilization 20min.Adding the 3-cyanopyridine then, to make ultimate density be 5mM.
Described fermentative medium formula is (final concentration): glycerine 15.80g/L, trisodium citrate 14.70g/L, yeast powder 32g/L, Sodium Glutamate 5g/L, other composition hexanolactam 0.5g/L, MgSO 4.7H 2O0.15g/L, KH 2PO 40.75g/L, K 2HPO 4.3H 2O0.75g/L, solvent are water.
Embodiment 2: the evaluation of new bacterial strain ZJB-09150
From soil, use the dilution coating method then through enrichment culture among the embodiment 1, obtaining the strongest bacterial strain label of a strain activity through high performance liquid chromatography detection screening is ZJB-12001, the Photomicrograph of single bacterium colony is seen shown in Figure 1, about colony diameter 1.0-2.0cm, bacterium colony is creamy white, spheroidal, light, moistening, neat in edge, surperficial microprotrusion.Electron microscope photo scanning is seen shown in Figure 2, polar flagella, 3～6.
Utilizing the Biolog(GEN III) the automatic microbe identification systems have carried out the test of 94 kinds of phenotypes to bacterial strain ZJB-12001, comprise that 71 kinds of utilization of carbon source situations detect and 23 kinds of chemosensitivities detect (the Biolog microorganism identifies that automatically special agent and substratum etc. are available from Biolog company), analyze the metabolism fingerprint through the Biolog readout instrument, bacterial strain ZJB-12001 qualification result as shown in Table 1 and Table 2.
Table 1: 71 kinds of carbon sources utilizes ability on the Biolog GEN of the bacterial strain ZJB-12001 III plate
Figure BDA00003281507100101
Figure BDA00003281507100111
Table 2: the chemosensitivity of 23 kinds of chemical substances on the Biolog GEN of the bacterial strain ZJB-12001 III plate
Figure BDA00003281507100112
Figure BDA00003281507100121
On the basis of the above, further (F.M. Ao Sibai edits by " fine works molecular biology experiment guide method " with bacterial strain ZJB-12001, Science Press, 2008) extract chromosomal DNA, be template with the total DNA of the cell that extracts, utilize primer: the 16S rDNA gene of p1 5'-AGAGTTTGATCCTGGCTCAG-3' and p2 5'-AAGGAGGTGATCCAGCCGCA-3' amplification bacterial strain, after amplified production connected with the T carrier, entrust Shanghai Sani's bio tech ltd to this bacterium 16S rDNA amplification and order-checking, after obtaining the 16S rDNA sequence (shown in the SEQ ID NO.1) of this bacterial strain, on the NCBI website, retrieve the 16S rDNA gene order of relevant bacterial strain among the GenBank with BLAST, and carry out the homology comparison; Evaluation based on form, physiological and biochemical property and aspects such as 16S rDNA sequence and phylogenetic systematics analysis is accredited as Pseudomonas fluorescens with bacterial strain ZJB-12001, intends called after (Pseudomonas fluorescens ZJB-12001).Use blast program to compare among the sequence information input National Center Biontechnology Information that 16S rDNA is recorded and further analyze, use related software bacterial strain to be carried out the structure of sequence alignment and phylogenetic tree then, the results are shown in shown in Figure 3ly, can help to understand the evolutionary history of Pseudomonas fluorescens ZJB-12001.
Embodiment 3: biological catalyst Pseudomonas fluorescens ZJB-12001 contains the preparation of enzyme wet thallus
(1) slant culture:
The Pseudomonas fluorescens ZJB-12001(CCTCC No:M2012449 that embodiment 2 is obtained) is seeded to slant medium, cultivates 48h for 30 ℃, obtain the inclined-plane thalline; Described slant medium final concentration consists of: peptone 10.0g/L, extractum carnis 5.0g/L, sodium-chlor 5.0g/L, agar 20.0g/L, pH7.2.
(2) seed culture:
From step (1) inclined-plane thalline picking one ring thalline, be seeded to 50.0ml aseptic seed substratum, cultivate 24h under 30 ℃ of conditions, obtain seed liquor; The seed culture medium final concentration consists of: glucose 10.0g/L, Sodium Glutamate 10.0g/L, yeast powder 3.0g/L, KH 2PO 40.75g/L, K 2HPO 43H 2O0.75g/L, MgSO 47H 2O0.2g/L, hexanolactam 1.0g/L, pH7.0, solvent are water.121 ℃ of sterilizations of substratum 20 minutes.
(3) fermentation culture:
The seed liquor that step (2) is obtained is seeded to fermention medium with the inoculum size of volume ratio 4%, 30 ℃, concussion is cultivated under the 150rpm condition, at different time (26h, 30h, 34h, 38h, 42h, 48h, 54h, 64h and 72h) sampling and measuring biomass and enzyme are lived, and (above-mentioned time corresponding biomass and enzyme work are respectively 5.11,5.92,6.40,6.55,7.24,7.33,7.49,7.36,7.14gDCW/L and 55.37,71.19,80.15,93.48,99.69,129.15,101.56,93.57,88.74U/L), therefore selecting best fermented incubation time is 48h, collecting incubation time is the fermented liquid of 48h, behind the centrifugal 10min of 10000rpm, abandon supernatant and collect wet thallus, wet thallus washs 3 times with mass concentration 0.85% physiological saline, the bacterium mud of collecting is and contains the enzyme wet thallus, biological catalyst with 2-amino-4-methylthio group butyronitrile hydrolytic activity, 4 ℃ of refrigerator preservations, standby.
Described fermention medium final concentration consists of: glycerine 15.0g/L, trisodium citrate 13.0g/L, Sodium Glutamate 5.0g/L, yeast powder 20.0g/L, KH 2PO 40.75g/L, K 2HPO 43H 2O0.75g/L, MgSO 47H 2O0.2g/L, hexanolactam 1.0g/L, pH7.0, solvent are water.
Embodiment 4: the preparation of methionine(Met)
High-efficient liquid phase chromatogram condition is: day island proper body fluid chromatography, stainless steel packed column, filler are C 18, long 0.25m, internal diameter 4.6mm, flow velocity 1ml/min, column temperature room temperature, moving phase are methyl alcohol: 20mM aqueous phosphatic (20V:80V), sample size are 20 μ l.Wherein, the methionine(Met) appearance time is 2.8min.Adopt external standard method to determine the content of methionine(Met) in the sample.
With embodiment 3 preparation contain the enzyme wet thallus and substrate 2-amino-4-methylthio group butyronitrile is blended in the Tris-HCl damping fluid (50.0mM) of pH8.5, wherein containing enzyme wet thallus concentration is that 6.67g/L(is with dry weight basis), 2-amino-4-methylthio group butyronitrile concentration is 2.6g/L, at 40 ℃ of conversion reaction 60min, after reaction finishes, with conversion fluid 10, the centrifugal 10min of 000rpm abandons precipitation, collects supernatant liquor aperture 0.45 μ m micro-filtrate membrane filtration, filtrate is carried out efficient liquid phase chromatographic analysis, the experiment carry out 3 parallel, transformation efficiency is respectively 72.5%, 69.3% and 65.8%, product methionine(Met) concentration is respectively 14.50mM, 13.86mM and 13.16mM.
Contain methionine(Met) filtrate by vacuum concentration, add the dehydrated alcohol extraction, the subcooling crystallization can get methionine crystal.
Figure IDA00003281508100011
Figure IDA00003281508100021

Claims (5)

1. Pseudomonas fluorescens (Pseudomonas fluorescens) ZJB-12001 is preserved in Chinese typical culture collection center, address: China, Wuhan, Wuhan University, postcode 430072, deposit number CCTCC No:M2012449, preservation date on November 08th, 2012.
(2) as claimed in claim 1, wherein the Pseudomonas fluorescens ZJB-12001, characterized in that the amplification product of 16S rDNA of strain sequence is as follows:AGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGTAGAGAGAAGCTTGCTTCTCTTGAGAGAGGCGGACGGGTGAGTAAAGCCTAGGAATCTGCCTGGTAGTGGGGGATAACGTTCGGAAACGGACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTTAAGAAGGTCTTCGGGTTGTAAAGCACTTTAAGTTGGGAGGAAGGGCATTAACCTAATACGTTAGTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCAAAACTGACTGACTAGAGTATGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGCAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTAATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGAAGCCTTGAGCTCTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAGCCTTACCAGGCCTTGACATCCAATGAACTTTCTAGAGATAGATTGGTGCCTTCGGGAACATTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTAATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCTCAAAACCGATCGTAGTCCGGATCGTAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGAGGGCGGTTACCACGGTGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGGGAACCTGCGGCTGGATCACCTCCTT。
3. the application in the described Pseudomonas fluorescens ZJB-12001 of claim 1 bio-transformation 2-amino-4-methylthio group butyronitrile synthetic methionine.
4. application as claimed in claim 3, it is characterized in that described being applied as: be substrate with 2-amino-4-methylthio group butyronitrile, the enzyme wet thallus that contains that obtains with Pseudomonas fluorescens ZJB-12001 fermentation culture is catalyzer, in the buffered soln of pH7.0～10.0, under 25～55 ℃ of conditions, carry out conversion reaction, make described methionine(Met).
5. application as claimed in claim 4, the starting point concentration that it is characterized in that substrate in the described damping fluid is 1.0～5.0g/L, and the quality consumption that contains the enzyme wet thallus that described Pseudomonas fluorescens ZJB-12001 fermentation culture obtains is counted 2.0～7.0g/L with dry cell weight.
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