CN104531577A - Arthrobacter nicotinovorans WYG001 and application thereof in preparation of N-BOC-L-homoserine lactone - Google Patents

Arthrobacter nicotinovorans WYG001 and application thereof in preparation of N-BOC-L-homoserine lactone Download PDF

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CN104531577A
CN104531577A CN201410800564.3A CN201410800564A CN104531577A CN 104531577 A CN104531577 A CN 104531577A CN 201410800564 A CN201410800564 A CN 201410800564A CN 104531577 A CN104531577 A CN 104531577A
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homoserine lactone
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王宇光
朱冰春
吴冬梅
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Zhejiang University of Technology ZJUT
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Abstract

The invention discloses arthrobacter nicotinovorans WYG001 and application thereof in preparation of N-BOC-L-homoserine lactone. The N-BOC-L-homoserine lactone is prepared by taking a wet bacterium body obtained from the fermentation cultivation of the arthrobacter nicotinovorans WYG001 or a dry bacterium body obtained by freeze-drying the wet bacterium body as a catalyst, N-BOC-DL-homoserine lactone as a substrate and 0.1M of a phosphate buffer solution at pH7.0 as a reaction medium through carrying out a conversion reaction at 20-50 DEG C, and separating and purifying a reaction liquid after the reaction, so as to obtain the N-BOC-L-homoserine lactone. The method disclosed by the invention is strong in enzyme regioselectivity, high in reaction conversion rate, simple in downstream separation, low in energy consumption, slight in environmental pollution and suitable for industrial production.

Description

Addicted to nicotine Arthrobacter WYG001 and the application in preparation N-BOC-L-homoserine lactone thereof
(1) technical field
The present invention relates to a kind of synthetic method of N-BOC-L-homoserine lactone, in particular to utilization addicted to nicotine Arthrobacter WYG001 (CCTCC M 2014054) catalysis racemize BOC-DL-homoserine lactone, stereo selective hydrolysis Reactive Synthesis optical purity N-BOC-L-homoserine lactone, then by the method for N-BOC-L-homoserine lactone synthesis L-selenomethionine.
(2) background technology
L-selenomethionine, English by name: L-(+)-Selenomethionine is a kind of important animal food additive amino acid, be also other a kind of important intermediate containing selenium medicine of preparation.Its major function has: the motility of sperm that can strengthen animal; Increase antioxidant ability of organism, improve immunizing power; There is the effect of cancer-resisting; Body can also be helped to discharge vivotoxin (as heavy metal) etc.
The homoserine lactone of optical purity form is important chiral intermediate, is widely used in the fields such as medicine and foodstuff additive.The present invention adopts microbial enzyme method catalysis to split obtained a kind of chiral intermediate N-BOC-L-homoserine lactone, and with it for substrate successfully synthesizes L-selenomethionine.
Because the amino of homoserine lactone is relatively more active, its existence form in different pH reaction solutions is different, makes troubles to the extraction of product, therefore needs to select amido protecting group to protect it.The DL-homoserine lactone selective hydrolysis protected by catalytic amino; realize a configuration in raceme and be able to the acid of catalytic hydrolysis open loop one-tenth; another configuration is constant; and then according to the difference of physical properties; by two of homoserine lactone kinds of configurations separately, optically pure L-homoserine lactone is finally obtained.
At present, the synthesis N-BOC-L-homoserine lactone of bibliographical information is substrate mainly through L-homoserine and lactone hydrobromate thereof or hydrochloride, and BOC amido protecting obtains.Wherein the synthetic method of L-homoserine and lactone salt thereof is primarily of the L-type aspartic acid (Han Chong of respective configuration, Jiang Lijian, Zhai Lihai etc. amino acid and Biological resources .2008,30 (2): 33-35) and L-type methionine(Met) (TroelsKoch, Ole Buchardt, 1993,1065-1067; Min-Can Wang, et al.OrganicChemistry.2008,73 (1): 168-176; Rise Chinese soldier. fine-chemical intermediate .2008,38 (3): 20-21) be Material synthesis.Other chemical synthesis also has with the furans imines of N-Boc protection and α-optically pure homoserine lactone of ethyl azidoacetate asymmetric synthesis.(Kumaraswamy G,etal.Tetrahedron Letters.2010,51(50):6500-6502)。Chemical synthesis process approach also exists the problems such as yield is low, cost is high, step is various, equipment requirements is high, product optical purity is lower, environmental pollution is serious.
2011, (the M Venkataiah such as Mallam Venkataiah, G Reddipalli, LSwarnalatha Jasti et al.Chemo-enzymatic synthesis of the azasugars1,4-dideoxyallonojirimycin and 1,4-dideoxymannojirimycin [J] .TetrahedronAsymmetry.2011,22:1855-1860) first by the alpha-amino group-gamma-butyrolactone of racemic phenylacetyl, regulate pH alkalize with 10% liquefied ammonia alkalization, thus make lactone open loop; Then the derivative of the phenylacetyl of (R) type homoserine of open loop and the homoserine of (S)-type is obtained with ImmobilizedPen Gacylase hydrolysis; Last in methanol solution, obtain (S) configuration by HCl acidifying, the e.e. > 99% of N-phenylacetyl-L-homoserine lactone, yield is 47% (not having transformation efficiency).
2007, Kazuya Mochizuki, (the Kazuya M such as Kentaro Miyazaki, KentaroM.Microbial resolution of DL-homoserine for the production ofD-homoserine using a novel isolate, Arthrobacter nicotinovorans strain 2-3 [J] .Enzyme and Microbial Technology.2007, 41:318-321) first the open loop of racemic homoserine lactone lactone hydrobromate is obtained homoserine, then with racemic homoserine for sole carbon source, filter out from soil and can fall L configuration by selective hydrolysis, and obtain the bacterial strain of the homoserine of D configuration.The microorganism strains Arthrobacter nicotinovorans Strain screened, at the phosphate solution neutral body optionally racemic homoserine of catalysis, obtains the e.e. of optically pure enantiomorph s>99.9%, D configuration productive rate is 25%, and the homoserine yield of L-configuration is about 4%, and transformation time is 48h, and the reaction times is long, is not easy to suitability for industrialized production.
By contrast, the biological process that the present invention reports has that stereoselectivity is high, reaction conditions is gentle, yield is high and the advantage of the easily separated purifying of product.
(3) summary of the invention
The object of the invention is to provide a kind of synthetic method of N-BOC-L-homoserine lactone, the method utilizes addicted to nicotine Arthrobacter WYG001 (CCTCC NO:M 2014054) catalysis racemize N-BOC-DL-homoserine lactone, stereo selective hydrolysis Reactive Synthesis optical purity N-BOC-L-homoserine lactone, again by N-BOC-L-homoserine lactone synthesis L-selenomethionine, the inventive method has that stereoselectivity is high, reaction conditions is gentle, yield is high and the advantage of the easily separated purifying of product.
The technical solution used in the present invention is:
The invention provides a strain new strains--addicted to nicotine Arthrobacter (Arthrobacter nicotinovorans) WYG001, be preserved in China typical culture collection center, deposit number CCTCC NO:M2014054, preservation date on February 28th, 2014, address is China, Wuhan, Wuhan University, postcode 430072.
The present invention also provides a kind of addicted to the application of nicotine Arthrobacter WYG001 in preparation N-BOC-L-homoserine lactone, described is applied as: with the dry mycelium after the wet thallus obtained through fermentation culture addicted to nicotine Arthrobacter WYG001 or wet thallus lyophilize for catalyzer, with N-BOC-DL-homoserine lactone for substrate, with 0.1M, pH7.0 phosphoric acid buffer for reaction medium, 20 ~ 50 DEG C, carry out conversion reaction (preferred reaction 1-30h) under 200rpm condition, after reaction terminates, by reaction solution separation and purification, obtain N-BOC-L-homoserine lactone.
The preparation method of catalyzer of the present invention is: (1) slant culture: will be seeded to slant medium addicted to nicotine Arthrobacter WYG001, cultivates 24h for 30 DEG C, obtains thalline inclined-plane; Often liter of slant medium (beef-protein medium) composition: extractum carnis 5g, peptone 10g, NaCl 5g, agar 20g, pH 7.0-7.2, adds water to 1000mL; 121 DEG C of sterilizing 20min.
(2) seed culture: be seeded to seed culture medium from inclined-plane thalline picking one transfering loop thalline, 30 DEG C, 200r/min cultivates 24h, obtains seed liquor; Seed culture medium forms: peptone 10g/L, yeast extract paste 5g/L, NaCl 10g/L, and solvent is water, pH 7.0;
(3) fermentation culture: aseptically, with the inoculum size of volumetric concentration 6-8%, seed liquor is inoculated in culture medium, 30 DEG C, 200r/min cultivates 24h, obtain fermented liquid, by centrifugal for fermented liquid 12000rpm 10min after cultivation terminates, abandon supernatant liquor, obtain addicted to nicotine Arthrobacter WYG001 wet thallus; Culture medium consists of: lactose 5-15g/L, yeast extract paste 10-14g/L, NaCl 10g/L, MgSO 40.22g/L, KH 2pO 40.136g/L, solvent is water, pH 7.0;
(4) get step (3) wet thallus to add in the phosphate buffered saline buffer of pH 7.0,0.1mol/L with the amount of 1.2g/ml, stir, lyophilize under-80 DEG C of conditions, obtains dry mycelium.
Further, the consumption of described wet thallus counts 3 ~ 150g/L with damping fluid volume, preferably 10 ~ 100g/L, most preferably 10 ~ 50g/L, the consumption of described dry mycelium counts 1 ~ 50g/L with damping fluid volume, preferably 5 ~ 30g/L, described initial substrate concentration is 1 ~ 100g/L, preferred 5-50g/L.
The method of reaction solution separation and purification of the present invention is: after reaction terminates, reaction solution is centrifugal, gets supernatant liquor, adds isopyknic extraction into ethyl acetate, gets ethyl acetate layer underpressure distillation to dry desolvation, obtains N-BOC-L-homoserine lactone.
Further, described reaction is at 20 ~ 50 DEG C of reaction 1 ~ 30h, and more preferably temperature of reaction is 30 ~ 35 DEG C.
Culture medium composition of the present invention is preferably: lactose 10g/L, yeast extract paste 12g/L, NaCl 10g/L, MgSO 40.22g/L, KH 2pO 40.136g/L, solvent is water, pH 7.0.
The method that the present invention utilizes N-BOC-L-homoserine lactone to prepare L-selenomethionine is:
(1) N-Boc-L-homoserine lactone is mixed with the hydrobromic acetic acid solution of mass concentration 33%, 100 DEG C of reflux 10h, continue stirring reaction 10h, be slowly down to room temperature, hold over night, filter, there is solid to separate out, filter and obtain product L-2-amino-4-bromobutanoic acid hydrobromide, and by ethyl acetate 30mL washed product, oven dry obtains white solid, and yield is 85%; The volumetric usage of described hydrobromic acetic acid solution counts 2.5ml/g with N-Boc-L-homoserine lactone quality;
(2) dimethyl diselenide ether is mixed with anhydrous methanol; add sodium borohydride (sodium borohydride: dimethyl diselenide ether=mass ratio 2:1) under nitrogen protection in batches; stirring at room temperature 0.5h-2.5h, solution is become colorless transparent by golden yellow, obtain CH 3seNa alcoholic solution; To CH 3add 2-amino-4 bromobutanoic acid hydrobromide in SeNa alcoholic solution, oil bath is heated to 45 DEG C-65 DEG C, continues to stir 2-10h, and reaction terminates rear filtration and can obtain the thick product of L-selenomethionine; Amino-4 bromobutanoic acid hydrobromide of described 2-and dimethyl diselenide ether mass ratio are 1.4:1, and the volumetric usage of described anhydrous methanol counts 8ml/g with dimethyl diselenide ether quality.
N-BOC-L-homoserine lactone (I), obtains in the bromo-butyric acid of S-2-amino-4-(III) through Hydrogen bromide open loop bromination, under sodium borohydride exists, react the L-selenomethionine (V) made with dimethyl diselenide ether.
The beneficial effect of the preparation method of medicine intermediate N-BOC-L-homoserine lactone of the present invention is mainly reflected in: the invention provides a strain new strains--addicted to nicotine Arthrobacter (Arthrobacternicotinovorans) WYG001, utilize and prepare N-BOC-L-homoserine lactone addicted to nicotine Arthrobacter WYG001, the regioselectivity of enzyme is strong, reaction conversion ratio is high, downstream separation is simple, energy consumption is low, and environmental pollution is little, is applicable to suitability for industrialized production.
(4) accompanying drawing explanation
Fig. 1 racemic modification N-BOC-L-homoserine lactone reference substance gas chromatogram, peak a is N-Boc-D-homoserine lactone, retention time 16.831 minutes, peak area 489.4, peak height 51.2, peak width 0.1465, symmetrical factor 1.16, peak b is N-Boc-L-homoserine lactone, retention time 17.797 minutes, peak area 485.9, peak height 50.1, peak width 0.1501, symmetrical factor 0.905;
The gas chromatogram of Fig. 2 N-BOC-L-homoserine lactone standard substance, peak b is N-Boc-L-homoserine lactone, retention time 17.791 minutes, peak area 677.1, peak height 73.1, peak width 0.1111, symmetrical factor 0.788;
The gas chromatogram of Fig. 3 microorganism catalysis product N-BOC-L-homoserine lactone, peak b is N-Boc-L-homoserine lactone, retention time 17.792 minutes, peak area 628.2, peak height 67.4, peak width 0.1327, symmetrical factor 0.815.
Fig. 4 is reacting flow chart of the present invention.
Fig. 5 is the colonial morphology figure of bacterial strain WYG001.
Fig. 6 is the light micrograph of bacterial strain WYG001 under 100 times.
Fig. 7 is the 16S rDNA electrophorogram of bacterial strain WYG001, and A is the electrophorogram of bacterial strain WYG001, and left side swimming lane is bacterial strain WYG001, and right lanes is standard molecular weight, and B is the enlarged view of A.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1
1, the screening of microorganism
The microorganism that the present invention relates to is obtained by following program screening and separating: take wet soil sample 1 ~ 2g (obtaining from national varying environment Soil Under Conditions) and be suspended in 10mL 0.5% stroke-physiological saline solution, vibration mixing; Connect 1 ~ 2mL suspension in 30mL liquid enrichment medium, 30 DEG C, cultivate 2 ~ 3d under 200r/min after, the bacterium liquid of aseptic technique switching 1mL muddiness to fresh liquid enrichment medium, continuous enrichment 3 times.In enrichment medium, with N-BOC-DL-homoserine lactone for sole carbon source, fill a prescription as follows: NaNO 34.0g/L, KH 2pO 42.0g/L, MgSO 47H 2o 0.5g/L, KCl 1.0g/L, ZnSO 40.05g/L, solvent is water, pH 7.0.The concentration 1g/L of the N-BOC-DL-homoserine lactone of first round enrichment, the second concentration of taking turns is 3g/L, and the concentration of third round is 5g/L, pH 7.0.By bacterium liquid after third round enrichment through gradient dilution, coating separating plate substratum, separating for several times, obtains single bacterium colony.Separating plate substratum consists of: enrichment medium adds agar final concentration 20g/L, and substrate N-BOC-DL-homoserine lactone final concentration is 5g/L, pH 7.0.
Picking is separated single colony inoculation of obtaining in slant medium, cultivates 1-2 days for 30 DEG C, obtains inclined-plane thalline.Be seeded to the seed culture medium of 50mL from inclined-plane thalline picking one transfering loop thalline, 30 DEG C, 200r/min cultivates 24h, obtains seed liquor.Aseptically, get 3mL seed liquor and be inoculated in the culture medium of 50mL, 30 DEG C, 200r/min cultivates 24h, obtains fermented liquid.Often liter of slant medium composition extractum carnis 5g, peptone 10g, NaCl 5g, agar 20g, pH 7.0-7.2, adds water to 1000mL; Seed culture based formulas is: peptone 10g/L, yeast extract paste 5g/L, NaCl 10g/L, and solvent is water, pH 7.0; Culture medium consists of: lactose 10g/L, yeast extract paste 12g/L, NaCl 10g/L, MgSO 40.22g/L, KH 2pO 40.136g/L, solvent is water, pH 7.0.Get 15mL fermented liquid centrifugal 10min of 12000rpm in 50mL centrifuge tube, abandon supernatant liquor, add the phosphate buffered saline buffer of 5mL 0.1mol/L pH 7.0, vibration mixing, add final concentration 8g/L substrate N-Boc-DL-homoserine lactone again, 30 DEG C, under 200r/min, conversion reaction 24h.Take out centrifuge tube, get the reaction solution of 1mL, add 2mL ethyl acetate, fully vibrate, the centrifugal 5min of 6000rpm.Get upper organic phase 1mL, dewater with a small amount of anhydrous sodium sulfate drying, detect the enantiomeric excess value of N-Boc-L-homoserine lactone with high performance liquid chromatography
(e.e.), reaction conversion ratio C, screening enantiomeric excess value (e.e.) is greater than 90%, and reaction conversion ratio C is the microorganism strains of (45%-60%), is designated as bacterial strain WYG001.
2, the qualification of microorganism strains
Physiological and biochemical property: bacterial strain WYG001 is Gram-negative bacteria, obligate aerobic, grow pH change more responsive, optimal pH is 7.0, and colonial morphology is shown in shown in Fig. 5 and Fig. 6.Utilize inorganic nitrogen-sourced ability poor, growth and product enzyme are subject to Cu 2+, Fe 2+, Al 3+, Zn 2+deng suppression, be subject to Mg 2+, K +, Na +deng promotion.
Through order-checking qualification, the 16S rDNA sequence of bacterial strain WYG001 following (SEQ ID NO.1), electrophorogram as shown in Figure 7:
TGCTTACACATGCAAGTCGAACGATGATCCCAGCTTGCTGGGGGATTAGTGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCTTGACTCTGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGACTCCTCATCGCATGGTGGGGGGTGGAAAGCTTTTGTGGTTTTGGATGGACTCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGTAGGGAAGAAGCGTAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGCTGTGAAAGACCGGGGCTCAACTCCGGTTCTGCAGTGGGTACGGGCAGACTAGAGTGCAGTAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTCTCTGGGCTGTAACTGACGCTGAGGAGCGAAAGCATGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGTTGGGCACTAGGTGTGGGGGACATTCCACGTTTTCCGCGCCGTAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATGAACCGGAAAGACCTGGAAACAGGTGCCCCGCTTGCGGTCGGTTTACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCTATGTTGCCAGCGGTTCGGCCGGGGACTCATAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAAAGGGTTGCGATACTGTGAGGTGGAGCTAATCCCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCACGAAAGTTGGTAACACCCGAAGCCGGTGGCCTAACCCTTGTGGGGGGAGCCGTCGAAGGTGGGACCGGCGATGGGACTAAGTC
According to physio-biochemical characteristics and molecular biology identification, this bacterial strain is accredited as addicted to nicotine Arthrobacter bacterium (Arthrobacter nicotinovorans), called after is addicted to nicotine Arthrobacter bacterium (Arthrobacternicotinovorans) WYG001, be preserved in China typical culture collection center (address China, Wuhan, Wuhan University, 430072), deposit number CCTCC NO:M 2014054, preservation date on February 28th, 2014.
Embodiment 2:
Slant culture: will be seeded to slant medium addicted to nicotine Arthrobacter WYG001, cultivates 24h for 30 DEG C, obtains thalline inclined-plane; Slant medium composition is with embodiment 1.
Seed culture: be seeded to 100mL seed culture medium from inclined-plane thalline picking one transfering loop thalline, 30 DEG C, 200r/min cultivates 24h, obtains seed liquor; Seed culture medium composition is with embodiment 1.
Fermentation culture: aseptically, gets 10mL seed liquor and is inoculated in the culture medium of 150mL, 30 DEG C, 200r/min cultivates 24h, obtain fermented liquid, by centrifugal for fermented liquid 12000rpm 10min after cultivation terminates, abandon supernatant liquor, obtain addicted to nicotine Arthrobacter WYG001 wet thallus.Get wet thallus to add in the phosphate buffered saline buffer of pH 7.0,0.1mol/L with the amount of 1.2g/ml, stir, under-80 DEG C of conditions, lyophilize obtains dry mycelium.
Embodiment 3:
The wet thallus 0.34g that Example 2 method obtains, adds N-BOC-DL-homoserine lactone 50mg and 0.1M, pH7.0 phosphoric acid buffer 5mL, 30 DEG C, under 200rpm condition, stirring reaction 7.5h.Reacting liquid filtering is removed enzyme, and get filtrate measures the N-Boc-DL-homoserine lactone after transforming content with gas chromatographic analysis, calculate enzyme and live, this fermented liquid is 36.85U/L than enzyme activity.
Enzyme is lived and is defined as: U is 1 enzyme activity unit, and 1 described enzyme activity unit is under defined terms, and the N-Boc-homoserine lactone of 1 μm of ol is hydrolyzed into the enzyme amount needed for N-Boc-homoserine by per minute.
Analytical conditions for gas chromatography: adopt gas chromatograph Agilent 6890 type gas chromatograph; Chiral gas chromatography post BGB-175 (0.25mm × 30m); Carrier gas N 2(14.54kPa), splitting ratio 20:1, injector temperature 250 DEG C, detector temperature 220 DEG C; Column temperature rise program: initial temperature 180 DEG C, keeps 20min; Sample size 1.0 μ L, hydrogen flowing quantity: 30mL/min air flow quantity: 300mL/min; Make-up gas flow: N 225mL/min; Hydrogen ion flame detector.N-BOC-D-homoserine lactone and N-BOC-L-homoserine lactone go out peak at 16.831min and 17.797min respectively.
N-BOC-DL-homoserine lactone typical curve equation for enzyme activity determination:
Compound concentration is respectively the racemize N-Boc-DL-homoserine lactone (w/v) of 0.1%, 0.4%, 0.5%, 0.6%, 0.8%, 1.0%, gas-chromatography is utilized to detect, draw the linear relationship between the concentration of lactone and peak area, with the mass concentration of N-Boc-DL-homoserine lactone for ordinate zou, gas chromatographic detection gained peak area is that X-coordinate obtains typical curve equation: y=1352x – 26.79, in 0.1%-2% (w/v) concentration range, coefficient R 2=0.999, reach the requirement needed for external standard method, the analysis that this curve may be used for N-Boc-DL-homoserine lactone measures.
Enzyme calculation formula alive:
A=[(a 1-a)/a 1c × V/] × [(M × t)] × 10 6formula (1)
A---the vigor (U) of enzyme in sample;
A---conversion reaction terminates the peak area of N-BOC-DL-homoserine lactone in rear conversion fluid;
A 1---according to the peak area of the N-BOC-DL-homoserine lactone standard specimen identical with initial substrate concentration that above-mentioned typical curve Equation for Calculating goes out;
C---in conversion fluid, the mass concentration of substrate, g/L;
V---conversion fluid volume, L;
M---the molar mass of substrate N-BOC-DL-homoserine lactone, g/mol;
T---transformation time, Min.
Embodiment 4:
The dry mycelium 0.2g that Example 2 method obtains, adds N-BOC-DL-homoserine lactone 0.1g, 0.1M, pH7.0 phosphoric acid buffer 10mL, 30 DEG C, under 200rpm condition, stirring reaction 7.5h.Adopt embodiment 3 method to measure enzyme to live, this dry mycelium is 5.53U/g than enzyme activity.
Embodiment 5:
Example 2 method gained wet thallus 3.4g, add N-BOC-DL-homoserine lactone 0.25g and 0.1M, pH7.0 phosphoric acid buffer 50mL, 30 DEG C, under 200rpm condition, stirring reaction 4h is after reaction terminates, centrifugal by reaction solution, get supernatant liquor, add isopyknic extraction into ethyl acetate, after getting ethyl acetate layer underpressure distillation to dry desolventizing, obtain N-BOC-L-homoserine lactone 0.12g.GC (testing conditions is with embodiment 3) detection computations is adopted to obtain the enantiomeric excess value e.e. of N-BOC-L-homoserine lactone s99.9%, transformation efficiency C is 51.8%, and productive rate is 96.4%.
Enantiomeric excess value (enantiomeric excesses) is the important indicator weighing enzyme selectivity, and the enantiomeric excess value of N-BOC-L-homoserine lactone is called for short e.e. s, its calculation formula is:
e . t . S ( % ) = S S - S R S S + S R × 100 % Formula (2)
Wherein S represents substrate, S rand S sbe respectively (D)-and the peak area of substrate of (L)-configuration.
The calculation formula of reaction conversion ratio is:
C ( % ) = 1 - S R + S S S R 0 + S S 0 × 100 % Formula (3)
The calculation formula of the yield (Yield) of N-BOC-L-homoserine lactone is
Yield ( % ) = S S S S 0 × 100 %
Wherein, S r0and S s0be respectively (D)-and the initial peak area of substrate of (L)-configuration.
Embodiment 6:
Example 2 method obtained addicted to nicotine Arthrobacter WYG001 dry mycelium 1g, add N-BOC-DL-homoserine lactone 0.5g and 0.1M, pH7.0 phosphoric acid buffer 100mL, 30 DEG C, under 200rpm condition, stirring reaction 7.5h is after reaction terminates, centrifugal by reaction solution, get supernatant liquor, add isopyknic extraction into ethyl acetate, after getting ethyl acetate layer underpressure distillation to dry desolventizing, obtain N-BOC-L-homoserine lactone 0.24g.GC (testing conditions is with embodiment 3) detection computations is adopted to obtain the enantiomeric excess value e.e. of N-BOC-L-homoserine lactone sbe 99.9%, transformation efficiency C is 52.0%, and productive rate is 96.0%.
Embodiment 7
Example 2 method obtained addicted to nicotine Arthrobacter WYG001 dry mycelium 4g, add N-BOC-DL-homoserine lactone 2g and 0.1M, pH7.0 phosphoric acid buffer 100mL, 30 DEG C, under 200rpm condition, stirring reaction 8h is after reaction terminates, centrifugal by reaction solution, get supernatant liquor, add isopyknic extraction into ethyl acetate, after getting ethyl acetate layer underpressure distillation to dry desolventizing, obtain N-BOC-L-homoserine lactone 0.97g.Adopt embodiment 3 method to detect the enantiomeric excess value e.e.99.9% of N-BOC-L-homoserine lactone, transformation efficiency C is 51.0%, and productive rate is 97.0%.
Embodiment 8
Example 2 method obtained addicted to nicotine Arthrobacter WYG001 dry mycelium 1.0g, add N-BOC-DL-homoserine lactone 0.5g and 0.1M, pH7.0 phosphoric acid buffer 20mL, 35 DEG C, under 200rpm condition, stirring reaction 14h is after reaction terminates, centrifugal by reaction solution, get supernatant liquor, add isopyknic extraction into ethyl acetate, after getting ethyl acetate layer underpressure distillation to dry desolventizing, obtain N-BOC-L-homoserine lactone 0.233g.Embodiment 3 method detection computations is adopted to obtain the enantiomeric excess value e.e. of N-BOC-L-homoserine lactone sbe 99.9%, transformation efficiency C is 52.5%, and productive rate is 93.2%.
Embodiment 9
Example 2 method obtained addicted to nicotine Arthrobacter WYG001 dry mycelium 1.0g, add N-BOC-DL-homoserine lactone 1g and 0.1M, pH7.0 phosphoric acid buffer 20mL, 35 DEG C, under 200rpm condition, after stirring reaction 30h, after reaction terminates, reaction solution is centrifugal, get supernatant liquor, add isopyknic extraction into ethyl acetate, after getting ethyl acetate layer underpressure distillation to dry desolventizing, obtain N-BOC-L-homoserine lactone 0.453g.GC detection computations is adopted to obtain the enantiomeric excess value e.e. of N-BOC-L-homoserine lactone sbe 95.6%, transformation efficiency C is 52.0%, and productive rate is 90.6%.
Embodiment 10:
Example 2 method obtained addicted to nicotine Arthrobacter WYG001 dry mycelium 0.2g, add N-BOC-DL-homoserine lactone 0.15g and 0.1M, pH7.0 phosphoric acid buffer 10mL, 30 DEG C, under 200rpm condition, stirring reaction 13h is after reaction terminates, centrifugal by reaction solution, get supernatant liquor, add isopyknic extraction into ethyl acetate, after getting ethyl acetate layer underpressure distillation to dry desolventizing, obtain N-BOC-L-homoserine lactone 0.144g.Adopt embodiment 3 method to detect the enantiomeric excess value e.e.99.9% of N-BOC-L-homoserine lactone, transformation efficiency C is 50.8%, and productive rate is 96.0%.
Embodiment 11:
Example 1 method obtained addicted to nicotine Arthrobacter WYG001 dry mycelium 1.5g, add N-BOC-DL-homoserine lactone 1.5g and 0.1M, pH7.0 phosphoric acid buffer 100mL, 30 DEG C, under 200rpm condition, stirring reaction 13h is after reaction terminates, centrifugal by reaction solution, get supernatant liquor, add isopyknic extraction into ethyl acetate, after getting ethyl acetate layer underpressure distillation to dry desolventizing, obtain N-BOC-L-homoserine lactone 0.725g.GC detection computations is adopted to obtain the enantiomeric excess value e.e. of N-BOC-L-homoserine lactone sbe 99.9%, transformation efficiency C is 50.7%, and productive rate is 96.6%.
Embodiment 12:
Example 1 method obtained addicted to nicotine Arthrobacter WYG001 dry mycelium 5g, add N-BOC-DL-homoserine lactone 10g and 0.1M, pH7.0 phosphoric acid buffer 100mL, 40 DEG C, under 200rpm condition, stirring reaction 15h is after reaction terminates, centrifugal by reaction solution, get supernatant liquor, add isopyknic extraction into ethyl acetate, after getting ethyl acetate layer underpressure distillation to dry desolventizing, obtain N-BOC-L-homoserine lactone 4.70g.GC detection computations is adopted to obtain the enantiomeric excess value e.e. of N-BOC-L-homoserine lactone sbe 99.0%, transformation efficiency C is 51.2%, and productive rate is 94.0%.
Embodiment 13:
Substitute in embodiment 11 (preparation of dry mycelium is with embodiment 2) with the dry mycelium 0.2g in table 1 respectively addicted to nicotine Arthrobacter WYG001 dry mycelium, add N-BOC-DL-homoserine lactone 0.1g and 0.1M, pH7.0 phosphoric acid buffer 10mL, 30 DEG C, under 200rpm condition, stirring reaction 24h, after reaction terminates, reaction solution is centrifugal, get supernatant liquor, add isopyknic extraction into ethyl acetate, after getting ethyl acetate layer underpressure distillation to dry desolventizing, obtain N-BOC-L-homoserine lactone.GC detection computations is adopted to obtain the enantiomeric excess value e.e. of N-BOC-L-homoserine lactone swith transformation efficiency C, in table 1.
Table 1
The bacterial strains such as table 1 result shows, aspergillus oryzae wzz007 have certain catalytic effect and stereoselectivity to substrate, but not as the present invention is addicted to nicotine Arthrobacter WYG001 in effect.
Embodiment 14:
(1) Example 2 method obtained addicted to nicotine Arthrobacter WYG001 dry mycelium 10g, add N-BOC-DL-homoserine lactone 5g and 0.1M, pH7.0 phosphoric acid buffer 200mL, 30 DEG C, under 200rpm condition, stirring reaction 11h, termination reaction, the centrifugal removing thalline of reaction solution, supernatant liquor is extracted with ethyl acetate (50mL × 5), after getting ethyl acetate layer underpressure distillation to dry desolventizing, obtain optically pure N-Boc-L-homoserine lactone 2.40g (detection method is with embodiment 3), productive rate is 96.0%; The enantiomeric excess value e.e. of N-BOC-L-homoserine lactone sbe 99.9%, its data structure characterizes as follows:
1H NMR(600MHz,CDCl 3)δ7.34(d,J＝8.4Hz,1H),4.37-4.28(m,2H),4.19-4.15(m,1H),2.39-2.34(m,1H),2.17-2.12(m,1H),1.39(s,9H). 13C NMR(150MHz,CDCl 3))δ174.5,155.6,78.5,64.7,49.1,29.2,27.3.Mp 123-125℃.[α] D＝+8.6°(c＝1,CHCl 3)
(2) synthesis of 2-amino-4-bromobutanoic acid hydrobromide
In the round mouth flask of 50mL, add N-Boc-L-homoserine lactone 2.40g, then add the hydrobromic acetic acid solution of 6mL mass concentration 33%, oil bath is heated to 100 DEG C, and continue stirring reaction 10h, reaction solution fades to clarification by muddiness, slowly be down to room temperature, hold over night, has solid to separate out, and filters, obtain the thick product of 2-amino-4 bromobutanoic acid hydrobromide, and use ethyl acetate washed product, final drying obtains white solid 2.65g, and yield is 85%.
1H NMR(500MHz,D 2O)δ2.22-2.41(m,2H),3.59-3.71(m,2H),4.00(d,J＝5.6Hz,1H). 13C NMR(126MHz,D 2O)δ174.6,51.7,32.9,28.3.
(3) synthesis of L-selenomethionine hydrobromate
In two mouthfuls of flasks of 50mL, add 1.88g dimethyl diselenide ether, then add the anhydrous methanol of 15mL, make it mix, one end is connected with nitrogen protection device.Add sodium borohydride 3.76g under nitrogen protection in batches, reacted in acutely to prevent.Room temperature (25 DEG C) stirs 10 minutes, and solution is become colorless transparent by golden yellow, obtain CH 3seNa alcoholic solution.By the CH of preparation 3add amino-4 bromobutanoic acid hydrobromide of 2.65g 2-in SeNa alcoholic solution, oil bath is heated to 60 DEG C, continues to stir 5h.Have solid to separate out after reaction terminates, filtration can obtain the thick product of L-selenomethionine and then use washing with alcohol product, and drying obtains white solid.Yield is 61.2%.
1H NMR(500MHz,D 2O)δ3.73-3.71(m,1H),2.52-2.49(m 2H),2.15-2.04(m,2H),1.92-1.90(m,3H). 13C NMR(125MHz,D 2O)δ174.2,54.9,31.0,19.4,3.4.MS(ESI):m/z[M+H]+:198.Mp 267.2-269.3℃.[α] D＝+18.8°(c＝1,1N HCl)。

Claims (10)

1. addicted to nicotine Arthrobacter (Arthrobacter nicotinovorans) WYG001, be preserved in China typical culture collection center, deposit number CCTCC NO:M 2014054, preservation date on February 28th, 2014, preservation address is China, Wuhan, Wuhan University, postcode 430072.
2. preparing the application in N-BOC-L-homoserine lactone addicted to nicotine Arthrobacter WYG001 described in a claim 1.
3. apply as claimed in claim 2, it is characterized in that described being applied as: with the dry mycelium after the wet thallus obtained through fermentation culture addicted to nicotine Arthrobacter WYG001 or wet thallus lyophilize for catalyzer, with N-BOC-DL-homoserine lactone for substrate, with 0.1M, pH7.0 phosphoric acid buffer for reaction medium, 20 ~ 50 DEG C, carry out conversion reaction under 200rpm condition, after reaction terminates, by reaction solution separation and purification, obtain N-BOC-L-homoserine lactone.
4. apply as claimed in claim 3, it is characterized in that the preparation method of described catalyzer is:
(1) slant culture: will be seeded to slant medium addicted to nicotine Arthrobacter WYG001, cultivates 24h for 30 DEG C, obtains thalline inclined-plane; Often liter of slant medium composition: extractum carnis 5g, peptone 10g, NaCl 5g, agar 20g, pH 7.0-7.2, adds water to 1000mL; 121 DEG C of sterilizing 20min;
(2) seed culture: be seeded to seed culture medium from inclined-plane thalline picking one transfering loop thalline, 30 DEG C, 200r/min cultivates 24h, obtains seed liquor; Seed culture medium forms: peptone 10g/L, yeast extract paste 5g/L, NaCl 10g/L, and solvent is water, pH 7.0;
(3) fermentation culture: aseptically, with the inoculum size of volumetric concentration 6-8%, seed liquor is inoculated in culture medium, 30 DEG C, 200r/min cultivates 24h, obtain fermented liquid, by centrifugal for fermented liquid 12000rpm 10min after cultivation terminates, abandon supernatant liquor, obtain addicted to nicotine Arthrobacter WYG001 wet thallus; Culture medium consists of: lactose 5-15g/L, yeast extract paste 10-14g/L, NaCl 10g/L, MgSO 40.22g/L, KH 2pO 40.136g/L, solvent is water, pH 7.0;
(4) get step (3) wet thallus to add in the phosphate buffered saline buffer of pH 7.0,0.1mol/L with the amount of 1.2g/ml, stir, lyophilize under-80 DEG C of conditions, obtains dry mycelium.
5. apply as claimed in claim 3, it is characterized in that the consumption of described wet thallus counts 3 ~ 150g/L with damping fluid volume.
6. apply as claimed in claim 3, it is characterized in that the consumption of described dry mycelium counts 1 ~ 50g/L with damping fluid volume.
7. apply as claimed in claim 3, it is characterized in that described initial substrate concentration is 1 ~ 100g/L.
8. as claim 3 application, it is characterized in that the method for described reaction solution separation and purification is: reaction terminate after, reaction solution is centrifugal, get supernatant liquor, add isopyknic extraction into ethyl acetate, get ethyl acetate layer underpressure distillation to dry desolvation, obtain N-BOC-L-homoserine lactone.
9. as claim 3 application, it is characterized in that described reaction 20 ~ 50 DEG C reaction 1 ~ 30h.
10. as claim 4 application, it is characterized in that culture medium consists of: lactose 10g/L, yeast extract paste 12g/L, NaCl 10g/L, MgSO 40.22g/L, KH 2pO 40.136g/L, solvent is water, pH 7.0.
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