CN103131649A - Pseudomonas fluorescens and application in preparation of transform-4-aminomethyl-naphthenic acid thereof - Google Patents
Pseudomonas fluorescens and application in preparation of transform-4-aminomethyl-naphthenic acid thereof Download PDFInfo
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Abstract
The invention discloses pseudomonas fluorescens (ZJB09-108) which is preserved in China representative culture conservation centre. The address is Wuhan University, in Wuhan, China. The postcode is 430072. The preserved number is CCTCC No: 2012539. The preserved date is December 20th, 2012. The Pseudomonas fluorescens and application in preparation of transform-4-aminomethyl-naphthenic acid thereof are provided. The pseudomonas fluorescens is temperate in synthetic reaction condition, environmentally friendly and simple in operational process and has good application prospects.
Description
(1) technical field
The present invention relates to a kind of method of the trans-4-aminomethyl-naphthenic acid that utilizes bioconversion method to prepare, particularly a kind of Pseudomonas fluorescens (Pseudomonas fluorescens) ZJB09-108 and the application in the trans-4-aminomethyl-naphthenic acid of preparation thereof.
(2) background technology
Trans-4-aminomethyl-naphthenic acid structural formula (referred to as tranexamic acid, tranamic acid) as shown in formula I, be a kind of hemostatic drug that acts on fibrinolytic enzyme system, be mainly used in clinically various hemorrhage due to acute or chronic, limitation or whole body hyperfibrinolysis.Also be combined with tyrosine oxidase because of contestable ground inhibition tyrosine, reduce melanic generation, be used for the treatment of chloasma.Also be widely used in makeup simultaneously, can block the process of the melanochrome deterioration that the uviolizing of preventing causes.Can also add toothpaste, prevent gingival hemorrhage.
Chemical method is all adopted in the production of trans-4-aminomethyl-naphthenic acid, mainly comprises following two kinds of methods:
1. the operational path of the cyclohexene method synthesis of trans-4-aminomethyl-naphthenic acid (formula (1))
Formula (1)
2. the operational path of paraaminomethyl benzoic acid method synthesis of trans-4-aminomethyl-naphthenic acid (formula (2))
Formula (2)
Must first synthesize cis-trans-4-aminomethyl-naphthenic acid (cis: trans=65:35) in these two kinds of operational paths, change into again trans-4-aminomethyl-naphthenic acid through High Temperature High Pressure under strong alkaline condition, conversion condition is harsh, transform not thorough, aftertreatment is complicated, cost is high, pollutes large.
Since the eighties in 20th century, along with the development of modern biotechnology, the biocatalysis technology take organism or enzyme as the basis receives much concern because of chemistry, regioselectivity and the stereoselectivity of height.But the report of the trans-4-aminomethyl-naphthenic acid that at present also do not utilize biocatalysis technology to prepare.The asymmetric conversion cis-trans of biological catalysis-4-aminomethyl-naphthenic acid prepares trans-4-aminomethyl-naphthenic acid and has the reaction conditions gentleness, transforms the advantages such as thorough, easy to operate.
(3) summary of the invention
The object of the invention is to provide new bacterial strain-Pseudomonas fluorescens (Pseudomonas fluorescens) ZJB09-108 of a kind of asymmetric conversion cis-trans-4-aminomethyl-naphthenic acid, and the application in the trans-4-aminomethyl-naphthenic acid of preparation.
The technical solution used in the present invention is:
Pseudomonas fluorescens (Pseudomonas fluorescens) ZJB09-108 is preserved in Chinese Typical Representative culture collection center, address: China, Wuhan, Wuhan University, postcode 430072, deposit number CCTCC No:M2012539, preservation date on December 20th, 2012.
Pseudomonas fluorescens ZJB09-108 of the present invention screens by the following method and obtains:
(1) take more than 150 parts of pedotheque 1g that are collected in zhejiang and other places, join in physiological saline, make soil supension and be inoculated in isolation medium, at 30 ℃, cultivate on the shaking table of 150r/min after 24 hours, get 1 mL and be transferred to and continue in new isolation medium to cultivate 24 hours.The separation and Culture based formulas is: extractum carnis 5 g/L, and peptone 10g/L, NaCl 5 g/L, cis-trans-4-aminomethyl-naphthenic acid 10 g/L, solvent is water, pH 7.2.
(2) separation and Culture liquid is applied on plate culture medium after 10 times of serial dilutions for the second time, 30 ℃ of incubator constant temperature culture 24 hours, and picking list bacterium colony is forwarded to preservation after slant culture, and the slant culture condition is 30 ℃ of incubator constant temperature culture 24 hours.Plate culture medium and slant culture based formulas are: extractum carnis 5 g/L, and peptone 10g/L, NaCl 5 g/L, agar 18 g/L, solvent are water, pH 7.2.
(3) be forwarded to shaking flask yeast culture base from the inclined-plane, cultivated 24 hours for 30 ℃, collect thalline, with 0.85% physiological saline washing 2 times, centrifugal collection thalline, gained thalline and substrate cis-trans-4-aminomethyl-naphthenic acid 1 g is scattered in 1000 mL distilled water or damping fluid (phosphate buffered saline buffer, Tris-HCl damping fluid etc.) in, at 30 ℃, under the 150r/min water-bath, reaction is after 0.5-12 hour, conversion fluid is removed somatic cells through centrifugation, supernatant liquor is with 0.45 μ m micro-filtrate membrane filtration, filtrate is detected analysis by high performance liquid chromatography, the bacterial strain of cis-4-aminomethyl-naphthenic acid of degrading is the purpose bacterial strain, the final strain cis-4-bacterial strain (being numbered ZJB09-108) that aminomethyl-the naphthenic acid degrading activity is the highest of therefrom selecting.Described yeast culture based formulas is: extractum carnis 5 g/L, and peptone 10g/L, NaCl 5 g/L, agar 18 g/L, solvent are water, pH 7.2.
Bacterial strain ZJB09-108 physiological and biochemical property:
The straight-bar bacterium, 0.7-0.8 μ m * 2.3-2.8 μ m, polar flagella, motion, oxidase positive, catalase is positive.Liquefy gelatin, growth, do not produce gemma in 4-37 ℃, without folder film, Gram-negative.Motion.Aerobic.The bacterium colony projection, transparent, the smooth of the edge.Can utilize N-Acetyl-D-glucosamine, ribose, inositol, maltose, malonate, acetate, DL-LACTIC ACID salt, ALANINE, 5-ketone group-gluconate, N.F,USP MANNITOL, D-Glucose, propionic salt, caprate, valerate, Citrate trianion, Histidine, 2-ketone group-gluconate, 3-hydroxy-butyric acid salt, 4-hydroxy-benzoic acid salt, Serine and L-PROLINE.
Bacterial strain ZJB09-108 of the present invention is accredited as Pseudomonas fluorescens (Pseudomonas fluorescens), called after Pseudomonas fluorescens ZJB09-108 through morphology, Physiology and biochemistry and molecular biological characteristics.
the invention still further relates to the false unit cell ZJB09-108 of described fluorescence and prepare application in trans-4-aminomethyl-naphthenic acid in microbial transformation, the wet thallus that described application obtains after fermentation culture take Pseudomonas fluorescens ZJB09-108 is as catalyzer, take cis-trans-4-aminomethyl-naphthenic acid as substrate, carry out conversion reaction in the transformation system that the water of pH 5.0 ~ 9.0 or damping fluid consist of, after reacting completely, conversion reaction liquid is centrifugal, get the reaction solution that supernatant liquor namely obtains to contain trans-4-aminomethyl-naphthenic acid, with the reaction solution separation and purification, obtain described trans-4-aminomethyl-naphthenic acid.
Further, in described transformation system, the consumption of catalyzer is counted 0.5 ~ 50 g/L with the wet thallus quality, and the water content of described wet thallus is 80 ~ 90%, and in described transformation system, the starting point concentration of substrate is 0.5 ~ 50 g/L.
Further, described conversion reaction is at 20 ~ 45 ℃ of reaction 0.5 ~ 24h, obtains to contain the reaction solution of trans-4-aminomethyl-naphthenic acid.
Further, the product separation purifying is that the reaction solution of-4-aminomethyl-naphthenic acid trans with containing is centrifugal, add the gac boiling decoloring, filter, the concentrated gained concentrated solution of filtrate adds dehydrated alcohol to ethanol volume final concentration 95% and carries out crystallization, and is cooling, filters, crystallization is with absolute ethanol washing, filtration, drying, obtain described trans-4-aminomethyl-naphthenic acid.
Described catalyzer prepares as follows:
(1) slant culture: Pseudomonas fluorescens ZJB09-108 is inoculated in slant medium, cultivated under 20 ~ 37 ℃ 20 ~ 48 hours, obtain the slant activation bacterial classification; Described slant medium final concentration consists of: extractum carnis 1 ~ 5 g/L, and peptone 1 ~ 15 g/L, NaCl 3 ~ 10g/L, agar 15 ~ 20g/L, solvent are water, initial pH is 6.5 ~ 8.0;
(2) seed culture: picking one transfering loop slant activation bacterial classification is inoculated in seed culture medium, cultivates under 25 ~ 37 ℃, shaking table revolution 100 ~ 200r/min condition and obtains seed liquor in 18 ~ 20 hours; Described seed culture medium final concentration consists of: extractum carnis 1 ~ 5 g/L, starch 1 ~ 20 g/L, glucose 1 ~ 20 g/L, glycerine 1 ~ 20 g/L, peptone 1 ~ 15 g/L, NaCl 3 ~ 10g/L, K
2HPO
43H
2O 0.5 ~ 2 g/L, solvent are water, and initial pH is 6.5 ~ 8.0;
(3) fermentation culture: seed liquor with volume ratio 1 ~ 10% access fermention medium, was cultivated 18 ~ 48 hours under 25 ~ 37 ℃, shaking table revolution 100 ~ 200r/min condition, with medium centrifugal, washing, collect wet thallus, refrigerate standby; Described fermention medium final concentration consists of: extractum carnis 1 ~ 5 g/L, starch 1 ~ 20 g/L, glucose 1 ~ 20 g/L, glycerine 1 ~ 20 g/L, peptone 1 ~ 15 g/L, NaCl 3 ~ 10g/L, K
2HPO
43H
2O 0.5 ~ 2 g/L, solvent are water, and initial pH is 6.5-8.0.
More specifically, the application of recommending Pseudomonas fluorescens ZJB09-108 of the present invention to prepare in microbial transformation in trans-4-aminomethyl-naphthenic acid is carried out as follows:
(1) seed culture
Pseudomonas fluorescens ZJB09-108 is inoculated in slant medium, and the slant culture based formulas is: extractum carnis 3 g/L, peptone 10 g/L, NaCl 10g/L, agar 18 g/L, solvent are water, regulating initial pH with 1M hydrochloric acid soln or 1M sodium hydroxide solution is 7.2, cultivates 24 hours under 30 ℃.
(2) seed culture: the thalline of slant culture is inoculated in seed culture medium cultivates.The formula of seed culture medium is: extractum carnis 3 g/L, starch 10 g/L, glucose 5 g/L, glycerine 5 g/L, peptone 5 g/L, NaCl 5 g/L, K
2HPO
43H
2O 1 g/L, solvent are water, and regulating initial pH with 1M hydrochloric acid soln or 1M sodium hydroxide solution is 7.5.Seed culture medium was cultivated 18 hours under the 150r/min condition at 30 ℃.
(3) fermentation culture: seed liquor is inoculated in fermention medium according to 5% volume ratio, cultivated 24 hours under 30 ℃, shaking speed 150r/min condition, with medium centrifugal, collect wet thallus, after the physiological saline washed twice, centrifugal collection thalline refrigerates standby; The formula of fermention medium is: extractum carnis 3 g/L, starch 10 g/L, glucose 5 g/L, glycerine 5 g/L, peptone 5 g/L, NaCl 5 g/L, K
2HPO
43H
2O 1 g/L, solvent are water, and regulating initial pH with 1M hydrochloric acid soln or 1M sodium hydroxide solution is 7.5.
(4) bio-transformation
Take cis-trans-4-aminomethyl-naphthenic acid as substrate, wet thallus after the Pseudomonas fluorescens ZJB09-108 fermentation culture is as catalyzer, be in the transformation system that consists of of 7.0 distilled water in the pH value, 37 ℃ of lower conversion reactions 2 hours, conversion reaction liquid is centrifugal, get the reaction solution that supernatant liquor namely obtains to contain trans-4-aminomethyl-naphthenic acid, in transformation system, initial substrate concentration is 2 g/L, the wet thallus addition is 40 g/L, and the moisture content of wet thallus is 85%.
(5) trans-4-aminomethyl-naphthenic acid separation and purification
The reaction solution that will contain trans-4-aminomethyl-naphthenic acid is centrifugal, add the gac boiling decoloring, filter, the concentrated gained concentrated solution of filtrate adds dehydrated alcohol to ethanol volume final concentration 95% and carries out crystallization, cooling, filter, crystallization is with absolute ethanol washing, filtration, drying, obtain described trans-4-aminomethyl-naphthenic acid.
In the present invention, substrate and product adopt high-performance liquid chromatogram determination, and method is as follows:
After reaction finishes, conversion reaction liquid is removed somatic cells through centrifugation, supernatant liquor is with 0.45 μ m micro-filtrate membrane filtration, filtrate is detected with the Shimadzu LC-20AT high performance liquid chromatograph that the SPD-20A ultraviolet-visible(light)detector is housed, and moving phase is 0.1 M pH 2.6 phosphate buffered saline buffers: methyl alcohol=60:40(v/v); And employing ultrasonic degas.The HPLC analysis condition: Diamonsil C18 ODS post (250 mm * 4.6 mm * 5 μ m), 39 ℃ of column temperatures, flow velocity 1.0 mL/min detect wavelength 220 nm, sample size 20 μ L.
Beneficial effect of the present invention is mainly reflected in: the invention provides a kind of Pseudomonas fluorescens ZJB09-108 and Pseudomonas fluorescens ZJB09-108 and prepare application in trans-4-aminomethyl-naphthenic acid in bio-transformation, this synthetic reaction condition is gentle, environmental friendliness, operating process are simple, have a good application prospect.
(4) 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) screening of microorganism
Isolation and Culture base final concentration consists of: extractum carnis 5 g/L, and peptone 10g/L, NaCl 5 g/L, cis-trans-4-aminomethyl-naphthenic acid 10 g/L, solvent is water, pH 7.2.
Plate culture medium and slant medium final concentration composition are: extractum carnis 5 g/L, and peptone 10g/L, NaCl 5 g/L, agar 18 g/L, solvent are water, pH 7.2.
Seed liquor substratum final concentration consists of: extractum carnis 3 g/L, starch 10 g/L, glucose 5 g/L, glycerine 5 g/L, peptone 5 g/L, NaCl 5 g/L, K
2HPO
43H
2O 1 g/L, solvent are water, and pH is 7.5.
The fermention medium final concentration consists of: extractum carnis 3 g/L, starch 10 g/L, glucose 5 g/L, glycerine 5 g/L, peptone 5 g/L, NaCl 5 g/L, K
2HPO
43H
2O 1 g/L, solvent are water, and pH is 7.5.
Gather soil sample from rice field, vegetable garden, orchard from Zhejiang Hangzhou, Quzhou, Jinhua, Ningbo, Wenzhou, Lishui, Taizhou and other places, more than totally 150 parts are used for bacterial screening.Detailed process is as follows:
Getting 1 g soil sample joins in the physiological saline that 50 mL fill granulated glass sphere, fully vibration, make soil supension, 150 r/min in the Isolation and Culture base, on the shaking table of 30 ℃ through twice cultivation, pass through dilution spread, picking list bacterium colony is forwarded to test tube slant and numbering again, is placed in 30 ℃ of biochemical cultivation cases and cultivates 24 hours.be forwarded to Medium of shaking flask fermentation from the inclined-plane again, cultivated 24 hours for 30 ℃, collect thalline, with 0.85% physiological saline washing 2 times, centrifugal collection thalline, gained thalline and substrate cis-trans-4-aminomethyl-naphthenic acid 1.0 g are scattered in distilled water, at 30 ℃, under the 150r/min water-bath, reaction is after 1-8 hour, remove somatic cells through centrifugation, supernatant liquor is with 0.45 μ m micro-filtrate membrane filtration, filtrate is detected analysis by high performance liquid chromatography, the bacterial strain of cis-4-aminomethyl-naphthenic acid of degrading is the purpose bacterial strain, finally therefrom selecting a highest bacterial strain of strain degrading activity (is numbered ZJB09-108, be CCTCC M 2012539) do follow-up research.
(2) microorganism strains is numbered the Physiology and biochemistry evaluation of " ZJB09-108 " bacterial strain
I) strain morphology feature:
Colonial morphology: the dull and stereotyped cultivation of beef-protein medium 20 hours, bacterium colony was rounded in 30 ℃, and projection is translucent, corrugationless, the smooth of the edge.
Cellular form: straight-bar bacterium, 0.7-0.8 μ m * 2.3-2.8 μ m, polar flagella, motion.
Ii) physiological and biochemical property:
Utilize bioMerieux ATB Expression automatic microbe identification systems and ID32 GN lath to identify, reagent and 0.85%NaCl solution are available from bioMerieux company.With bacterial strain ZJB09-108 at the beef extract-peptone plate culture medium streak culture 20 hours, get the consistent bacterium colony of one or several form to the ampere bottle that 0.85%NaCl is housed with aseptic cotton carrier, the preparation turbidity is equivalent to the bacteria suspension of 0.5 Maxwell unit: use than turbid instrument and measure.Open an ampoule (without the dropper cap) that the AUX substratum is housed, get 200 μ L(approximately 4-8 drip) bacteria suspension adds in ampoule, with inoculator automatic mixing AUX substratum and bacteria suspension.Divide to be added in each hole of identifying strip and add 135 μ L., cover the lid of identifying strip.30 ℃ of moisturizings were cultivated 24 hours.Strip is placed in ATB Expression Reader sentence read result.
Table 1 bacterial strain ZJB-physiological and biochemical property
| NO. | Carbon source | ZJB09-108 | NO. | Carbon source | ZJB09-108 |
| 1 | Rhamnosyl | - | 17 | D-Glucose | + |
| 2 | N-Acetyl-D-glucosamine | + | 18 | Salicin | - |
| 3 | Ribose | ＋ | 19 | The D-melibiose | - |
| 4 | Inositol | ＋ | 20 | L-fucose | - |
| 5 | Sucrose | - | 21 | D-glucitol | - |
| 6 | Maltose | + | 22 | L-arabinose | - |
| 7 | Methylene-succinic acid | - | 23 | Propionic salt | + |
| 8 | The zinc diacid salt | 24 | Caprate | + | |
| 9 | Malonate | + | 25 | Valerate | + |
| 10 | Acetate | + | 26 | Citrate trianion | + |
| 11 | DL-LACTIC ACID salt | + | 27 | Histidine | + |
| 12 | ALANINE | + | 28 | 2-ketone group-gluconate | + |
| 13 | 5-ketone group-gluconate | + | 29 | 3-hydroxy-butyric acid salt | + |
| 14 | Glycogen | - | 30 | 4-hydroxy-benzoic acid salt | + |
| 15 | 3-hydroxy-benzoic acid salt | - | 31 | Serine | + |
| 16 | N.F,USP MANNITOL | + | 32 | L-PROLINE | + |
[0060]Iii) 16S rDNA sequencing and analysis:
Take the total DNA of the strain cell that extracts as template, utilize the 16S rDNA of the primer amplification bacterial strain ZJB09-108 of design, by Sangon Biotech (Shanghai) Co., Ltd. check order (nucleotide sequence is seen shown in SEQ ID NO.1), this fragment physical length is 1427 bp, carrying out similarity analysis with the GenBank related data finds, the highest (the homology of false unit cell (Pseudomonas fluorescens) homology of this bacterium and fluorescence, 100%/1469 bps, based on 16S rDNA).
Therefore identify the binding molecule biological assay according to Physiology and biochemistry, can determine that this bacterial strain is the false unit cell (Pseudomonas fluorescens) of fluorescence, called after Pseudomonas fluorescens (Pseudomonas fluorescens) ZJB09-108.
The preparation of embodiment 2 Pseudomonas fluorescens ZJB09-108 wet thallus
(1) slant culture: Pseudomonas fluorescens ZJB09-108 is inoculated in slant medium, cultivated 24 hours under 30 ℃, obtain the inclined-plane thalline.The slant culture based formulas is: extractum carnis 5 g/L, and peptone 10g/L, NaCl 5 g/L, agar 18 g/L, solvent are water, pH 7.2.
(2) seed culture: be inoculated in seed culture medium with transfering loop from inclined-plane thalline picking one ring thalline, 30 ℃, cultivated 18 hours under the 150r/min condition, obtain seed liquor.The formula of seed culture medium is: extractum carnis 3 g/L, starch 10 g/L, glucose 5 g/L, glycerine 5 g/L, peptone 5 g/L, NaCl 5 g/L, K
2HPO
43H
2O 1 g/L, solvent are water, and pH is 7.5.
(3) fermentation culture: cultured seed liquor is inoculated into fermention medium according to 5% volume ratio, cultivated 24 hours under 30 ℃, 200 r/min conditions, obtain the mycetocyte fermented liquid, fermented liquid is centrifugal, and abandoning supernatant is after precipitation is used the physiological saline washed twice, centrifugal collecting precipitation, obtain wet thallus, the output of wet thallus is 43 g/L, and water content is 90%.The formula of fermention medium is: extractum carnis 3 g/L, starch 10 g/L, glucose 5 g/L, glycerine 5 g/L, peptone 5 g/L, NaCl 5 g/L, K
2HPO
43H
2O 1 g/L, solvent are water, and pH is 7.5.
The preparation of embodiment 3 Pseudomonas fluorescens ZJB09-108 wet thallus
(1) slant culture: with embodiment 2.
(2) seed culture: with embodiment 2.
(3) fermentation culture: cultured seed liquor is inoculated in fermention medium according to 2% volume ratio, cultivated 24 hours under 37 ℃, shaking table revolution 150 r/min conditions, obtain the mycetocyte fermented liquid.Fermented liquid is centrifugal, abandoning supernatant, after precipitation was used the physiological saline washed twice, centrifugal collecting precipitation obtained wet thallus, and the output of wet thallus is 35 g/L, and water content is 84%.The formula of fermention medium is: extractum carnis 5 g/L, starch 5 g/L, glucose 10 g/L, glycerine 10 g/L, peptone 10 g/L, NaCl 5 g/L, K
2HPO
43H
2O 2 g/L, solvent are water, and pH is 7.5.
Embodiment 4 microbial transformations
The preparation of wet thallus is with embodiment 2.With wet thallus 4 g, substrate cis-trans-4-aminomethyl-naphthenic acid 0.2 g dissolves with distilled water, adds in 500 mL triangular flasks, and regulating pH with 1 M sodium hydroxide is 7.0, complement to 100mL with distilled water again and consist of the conversion reaction system, 25 ℃ of water bath with thermostatic control stirring reactions 2 hours.After reaction finished, conversion fluid was removed somatic cells through centrifugation, and supernatant liquor is with 0.45 μ m micro-filtrate membrane filtration, filtrate adopts HPLC to analyze, cis-4-aminomethyl-naphthenic acid is degradable, trans-and 4-aminomethyl-naphthenic acid content is 0.06 g, and substrate conversion efficiency is 85.7%.Centrifuged supernatant 98 mL(are contained trans-4-aminomethyl-naphthenic acid 0.058 g), add gac 5.5 g, boiling decoloring.Filter evaporation concentration filtrate to 5 mL(contains trans-4-aminomethyl-naphthenic acid 0.058 g under room temperature), add dehydrated alcohol to final concentration 95%(v/v) crystallization.Be cooled to 0 ℃, filter, crystallization is with absolute ethanol washing, filtration, drying, obtains described trans-4-aminomethyl-naphthenic acid (0.045 g), always extract yield 77.6%.
Embodiment 5 microbial transformations
The preparation of wet thallus is with embodiment 3.Wet thallus 2 g, (pH 7.5 with buffer solution of sodium phosphate for substrate cis-trans-4-aminomethyl-naphthenic acid 1.0g, 0.1M) dissolving, and adjusting pH value is 7.5, (pH 7.5 with buffer solution of sodium phosphate, 0.1M) complement to 100 mL and consist of the conversion reaction systems, 37 ℃ of water bath with thermostatic control stirring reactions 1 hour.After reaction finished, conversion fluid was removed somatic cells through centrifugation, and supernatant liquor is with 0.45 μ m micro-filtrate membrane filtration, filtrate adopts Shimadzu HPLC to analyze, cis-4-aminomethyl-naphthenic acid is degradable, trans-and 4-aminomethyl-naphthenic acid content is 0.32 g, and substrate conversion efficiency is 91.4%.Centrifuged supernatant 95 mL(are contained trans-4-aminomethyl-naphthenic acid 0. 32 g), add gac 8.0 g, boiling decoloring.Filter evaporation concentration filtrate to 6 mL(contains trans-4-aminomethyl-naphthenic acid 0.3g under room temperature), add dehydrated alcohol to ethanol final concentration 95%(v/v) crystallization.Be cooled to 0 ℃, filter, crystallization is with absolute ethanol washing, filtration, drying, obtains described trans-4-aminomethyl-naphthenic acid (0.28 g), always extract yield 88%.
SEQUENCE LISTING
＜110〉Zhejiang Polytechnical University, Yangzhou Tianhe Pharmaceutical Co., Ltd.
＜120〉Pseudomonas fluorescens and the application in the trans-4-aminomethyl-naphthenic acid of preparation thereof
<130>
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1437
<212> DNA
<213> Psedomonas fluorescens
<400> 1
accctggcga gtctaccatg caagtcgagc ggtagagagg tgcttgcacc tcttgagagc 60
ggcggacggg tgagtaatgc ctaggaatct gcctggtagt gggggataac gctcggaaac 120
ggacgctaat accgcatacg tcctacggga gaaagcaggg gaccttcggg ccttgcgcta 180
tcagatgagc ctaggtcgga ttagctagtt ggtgaggtaa tggctcacca aggcgacgat 240
ccgtaactgg tctgagagga tgatcagtca cactggaact gagacacggt ccagactcct 300
acgggaggca gcagtgggga atattggaca atgggcgaaa gcctgatcca gccatgccgc 360
gtgtgtgaag aaggtcttcg gattgtaaag cactttaagt tgggaggaag ggcagttacc 420
taatacgtaa ttgttttgac gttaccgaca gaataagcac cggctaactc tgtgccagca 480
gccgcggtaa tacagagggt gcaagcgtta atcggaatta ctgggcgtaa agcgcgcgta 540
ggtggttcgt taagttggat gtgaaagccc cgggctcaac ctgggaactg cattcaaaac 600
tgtcgagcta gagtatggta gagggtggtg gaatttcctg tgtagcggtg aaatgcgtag 660
atataggaag gaacaccagt ggcgaaggcg accacctgga ctgatactga cactgaggtg 720
cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccgt aaacgatgtc 780
aactagccgt tgggagcctt gagctcttag tggcgcagct aacgcattaa gttgaccgcc 840
tggggagtac ggccgcaagg ttaaaactca aatgaattga cgggggcccg cacaagcggt 900
ggagcatgtg gtttaattcg aagcaacgcg aagaacctta ccaggccttg acatccaatg 960
aactttccag agatggattg gtgccttcgg gagcattgag acaggtgctg catggctgtc 1020
gtcagctcgt gtcgtgagat gttgggttaa gtcccgtaac gagcgcaacc cttgtcctta 1080
gttaccagca cgtaatggtg ggcactctaa ggagactgcc ggtgacaaac cggaggaagg 1140
tggggatgac gtcaagtcat catggccctt acggcctggg ctacacacgt gctacaatgg 1200
tcggtacaga gggttgccaa gccgcgaggt ggagctaatc ccacaaaacc gatcgtagtc 1260
cggatcgcag tctgcaactc gactgcgtga agtcggaatc gctagtaatc gcgaatcaga 1320
atgtcgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc atgggagtgg 1380
gttgcaccag aagtagctag tctaaccttc ggggggacgg ttaccacggt gtgattc 1437
Claims (5)
1. Pseudomonas fluorescens (Pseudomonas fluorescens) ZJB09-108 is preserved in Chinese Typical Representative culture collection center, address: China, Wuhan, Wuhan University, postcode 430072, deposit number CCTCC No:M2012539, preservation date on December 20th, 2012.
2. the described Pseudomonas fluorescens ZJB09-108 of claim 1 prepares the application in trans-4-aminomethyl-naphthenic acid in microbial transformation, it is characterized in that wet thallus that described application obtains take Pseudomonas fluorescens ZJB09-108 is as catalyzer after fermentation culture, take cis-trans-4-aminomethyl-naphthenic acid as substrate, carry out conversion reaction in the transformation system that the water of pH 5.0 ~ 9.0 or damping fluid consist of, after reacting completely, conversion reaction liquid is centrifugal, get the reaction solution that supernatant liquor namely obtains to contain trans-4-aminomethyl-naphthenic acid, with the reaction solution separation and purification, obtain trans-4-aminomethyl-naphthenic acid.
3. Pseudomonas fluorescens ZJB09-108 prepares application in trans-4-aminomethyl-naphthenic acid in microbial transformation as claimed in claim 2, the consumption that it is characterized in that catalyzer in described transformation system is counted 0.5 ~ 50 g/L with the wet thallus quality, the water content of described wet thallus is 80 ~ 90%, and in described transformation system, the starting point concentration of substrate is 0.5 ~ 50 g/L.
4. Pseudomonas fluorescens ZJB09-108 prepares application in trans-4-aminomethyl-naphthenic acid in microbial transformation as claimed in claim 2, it is characterized in that described conversion reaction is at 20 ~ 45 ℃ of reaction 0.5 ~ 24h, obtains to contain the reaction solution of trans-4-aminomethyl-naphthenic acid.
5. Pseudomonas fluorescens ZJB09-108 prepares application in trans-4-aminomethyl-naphthenic acid in microbial transformation as claimed in claim 2, it is characterized in that described catalyzer prepares as follows:
(1) slant culture: Pseudomonas fluorescens ZJB09-108 is inoculated in slant medium, cultivated under 20 ~ 37 ℃ 20 ~ 48 hours, obtain the slant activation bacterial classification; Described slant medium final concentration consists of: extractum carnis 1 ~ 5 g/L, and peptone 1 ~ 15 g/L, NaCl 3 ~ 10g/L, agar 15 ~ 20g/L, solvent are water, initial pH is 6.5 ~ 8.0;
(2) seed culture: picking one transfering loop slant activation bacterial classification is inoculated in seed culture medium, cultivates under 25 ~ 37 ℃, shaking table revolution 100 ~ 200r/min condition and obtains seed liquor in 18 ~ 20 hours; Described seed culture medium final concentration consists of: extractum carnis 1 ~ 5 g/L, starch 1 ~ 20 g/L, glucose 1 ~ 20 g/L, glycerine 1 ~ 20 g/L, peptone 1 ~ 15 g/L, NaCl 3 ~ 10g/L, K
2HPO
43H
2O 0.5 ~ 2 g/L, solvent are water, and initial pH is 6.5 ~ 8.0;
(3) fermentation culture: seed liquor with volume ratio 1 ~ 10% access fermention medium, was cultivated 18 ~ 48 hours under 25 ~ 37 ℃, shaking table revolution 100 ~ 200r/min condition, with medium centrifugal, washing, collect wet thallus, refrigerate standby; Described fermention medium final concentration consists of: extractum carnis 1 ~ 5 g/L, starch 1 ~ 20 g/L, glucose 1 ~ 20 g/L, glycerine 1 ~ 20 g/L, peptone 1 ~ 15 g/L, NaCl 3 ~ 10g/L, K
2HPO
43H
2O 0.5 ~ 2 g/L, solvent are water, and initial pH is 6.5 ~ 8.0.
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| CN108893455A (en) * | 2018-05-30 | 2018-11-27 | 浙江工业大学 | A kind of transaminase mutant and its application for producing L-glufosinate-ammonium |
| CN109207410A (en) * | 2018-10-29 | 2019-01-15 | 四川大宇中和农业科技发展有限公司 | One plant of potassium decomposing pseudomonad and its application |
| WO2024005155A1 (en) * | 2022-06-30 | 2024-01-04 | キリンホールディングス株式会社 | Method for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid |
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| CN101864376A (en) * | 2010-02-10 | 2010-10-20 | 浙江省农业科学院 | Pseudomonas fluorescens strain, microbial inoculum and use thereof as seedling culture medium for controlling tomato bacterial wilt |
| CN102517226A (en) * | 2011-10-27 | 2012-06-27 | 云南省烟草农业科学研究院 | Pseudomonas fluorescens preparation and application thereof |
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| CN101575583A (en) * | 2009-03-12 | 2009-11-11 | 南京林业大学 | Pseudomonas fluorescens and application thereof in promoting growth of poplar |
| CN101864376A (en) * | 2010-02-10 | 2010-10-20 | 浙江省农业科学院 | Pseudomonas fluorescens strain, microbial inoculum and use thereof as seedling culture medium for controlling tomato bacterial wilt |
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| CN108660122A (en) * | 2018-05-30 | 2018-10-16 | 浙江工业大学 | A kind of application of transaminase, mutant and its production L-glufosinate-ammonium |
| CN108893455A (en) * | 2018-05-30 | 2018-11-27 | 浙江工业大学 | A kind of transaminase mutant and its application for producing L-glufosinate-ammonium |
| CN108893455B (en) * | 2018-05-30 | 2020-07-28 | 浙江工业大学 | Transaminase mutant and application thereof in producing L-glufosinate-ammonium |
| CN109207410A (en) * | 2018-10-29 | 2019-01-15 | 四川大宇中和农业科技发展有限公司 | One plant of potassium decomposing pseudomonad and its application |
| CN109207410B (en) * | 2018-10-29 | 2021-05-07 | 四川大宇中和农业科技发展有限公司 | Potassium-solubilizing pseudomonas and application thereof |
| WO2024005155A1 (en) * | 2022-06-30 | 2024-01-04 | キリンホールディングス株式会社 | Method for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid |
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