CN104531577B - 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 PDFInfo
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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
(1) technical field
The present invention relates to a kind of synthetic method of N-BOC-L- homoserine lactones, more particularly to using thermophilic nicotine arthrobacterium
WYG001 (CCTCC M 2014054) is catalyzed racemic BOC-DL- homoserine lactones, and stereo selective hydrolysis are synthesized light
Pure N-BOC-L- homoserine lactones are learned, then the method that N-BOC-L- homoserine lactones are synthesized L- selenomethionines.
(2) background technology
L- selenomethionines, English is entitled：L- (+)-Selenomethionine, is a kind of important animal foodstuff addition
Agent amino acid, is also to prepare other a kind of important intermediates containing selenium medicine.Its major function has：The essence of animal can be strengthened
Sub- vigor；Increase antioxidant ability of organism, enhance immunity；Effect with cancer-resisting；Body can also be helped to discharge internal
Toxin (such as heavy metal).
The homoserine lactone of optical voidness form is important chiral intermediate, is widely used in medicine and food additives etc.
Field.The present invention is split using microbial enzyme method catalysis and a kind of chiral intermediate N-BOC-L- homoserine lactones is obtained, and with
It is that substrate successfully synthesizes L- selenomethionines.
Because the amino of homoserine lactone is more active, its existence form in different pH reaction solutions is different, to product
The extraction of thing is made troubles, it is therefore desirable to select amido protecting group to protect it.The DL- protected by catalytic amino is high
Serine lactone selective hydrolysis, a configuration is able to catalyzing hydrolysis open loop into acid in realizing raceme, and another configuration is constant,
And then according to the difference of physical property, two kinds of configurations of homoserine lactone are separated, finally obtain optically pure L- Kosé ammonia
Acid lactone.
At present, the synthesis N-BOC-L- homoserine lactones of document report mainly pass through L- homoserine and its lactone hydrogen bromine
Hydrochlorate or hydrochloride are substrate, and BOC amido protectings are obtained.The synthetic method of wherein L- homoserine and its lactone salt is main by phase
Answer L-type aspartic acid (such as Han Chong, Jiang Lijian, Zhai Lihai amino acid and the living resources .2008,30 (2) of configuration:33-35)
With L-type methionine (Troels Koch, Ole Buchardt, 1993,1065-1067；Min-Can Wang,et
al.Organic Chemistry.2008,73(1):168-176；Rise Chinese soldier's fine-chemical intermediates .2008,38 (3):20-
21) it is Material synthesis.Other chemical synthesis also have asymmetric with α-ethyl azidoacetate with the furans imines of N-Boc protections
The pure homoserine lactone of synthesizing optical.(Kumaraswamy G,et al.Tetrahedron Letters.2010,51
(50):6500-6502).Chemical synthesis process approach has that low yield, high cost, step are various, equipment requirement is high, product
The problems such as optical purity is relatively low, environmental pollution is serious.
, (M Venkataiah, G Reddipalli, the L Swarnalatha such as Mallam Venkataiah in 2011
Jasti et al.Chemo-enzymatic synthesis of the azasugars1,4-
dideoxyallonojirimycin and 1,4-dideoxymannojirimycin[J].Tetrahedron
Asymmetry.2011,22:Alpha-amido-the gamma-butyrolacton of racemic phenylacetyl first 1855-1860) is used into 10% liquefied ammonia
PH is into alkalescence for alkalization regulation, so that lactone open loop；Then hydrolyzed with Immobilized Pen Gacylase and obtain open loop
(R) derivative of the phenylacetyl of the homoserine of type homoserine and (S)-type；It is last to be acidified by HCl in methanol solution
(S) configuration, the e.e. ＞ 99% of N- phenylacetyl-L- homoserine lactones are obtained, yield is 47% (without conversion ratio).
, (the Kazuya M, Kentaro such as Kazuya Mochizuki, Kentaro Miyazaki in 2007
M.Microbial resolution of DL-homoserine for the production of D-homoserine
using a novel isolate,Arthrobacter nicotinovorans strain 2-3[J].Enzyme and
Microbial Technology.2007,41:318-321) first by racemic homoserine lactone lactone hydrobromate open loop
Homoserine is obtained, then with racemic homoserine as sole carbon source, being filtered out from soil being capable of selective hydrolysis
Fall L-configuration, and obtain the bacterial strain of the homoserine of D configurations.The microbial strains Arthrobacter for screening
Nicotinovorans Strain obtain optics in the racemic homoserine of catalysis of phosphate solution neutral body selectivity
The e.e. of pure enantiomerS>99.9%, D configuration yield are 25%, and the homoserine yield of L- configurations is about 4%, and conversion
Time is 48h, and the reaction time is long, is not easy to industrialized production.
By contrast, the bioanalysis reported of the present invention have that stereoselectivity is high, reaction condition is gentle, high income and product
The advantage of the easily separated purifying of thing.
(3) content of the invention
It is an object of the present invention to provide a kind of synthetic method of N-BOC-L- homoserine lactones, the method utilizes thermophilic nicotine section
Bacillus WYG001 (CCTCC NO:M 2014054) catalysis racemic N-BOC-DL- homoserine lactones, stereo selective hydrolysis
Optical voidness N-BOC-L- homoserine lactones are synthesized, then N-BOC-L- homoserine lactones are synthesized into L- selenomethionines,
The inventive method has the advantages that stereoselectivity is high, reaction condition gentle, high income and the easily separated purifying of product.
The technical solution adopted by the present invention is：
The present invention provides one plant of new strains -- thermophilic nicotine arthrobacterium (Arthrobacter nicotinovorans)
WYG001, is preserved in China typical culture collection center, deposit number CCTCC NO：M2014054, preservation date 2014 2
The moon 28, address is China, Wuhan, Wuhan University, postcode 430072.
The present invention also provides a kind of applications of thermophilic nicotine arthrobacterium WYG001 in N-BOC-L- homoserine lactones are prepared,
Described application is：It is dry after the wet thallus obtained with the fermented cultures of thermophilic nicotine arthrobacterium WYG001 or wet thallus freeze-drying
Thalline is catalyst, with N-BOC-DL- homoserine lactones as substrate, with 0.1M, pH7.0 phosphate buffer as reaction medium,
Conversion reaction (preferably reacting 1-30h) is carried out under the conditions of 20~50 DEG C, 200rpm, after reaction terminates, reaction solution is separated pure
Change, obtain N-BOC-L- homoserine lactones.
The preparation method of catalyst of the present invention is：(1) inclined-plane culture：Thermophilic nicotine arthrobacterium WYG001 is seeded to tiltedly
Face culture medium, 30 DEG C of culture 24h, obtains thalline inclined-plane；Every liter of slant medium (beef-protein medium) composition：Beef
Cream 5g, peptone 10g, NaCl 5g, agar 20g, pH 7.0-7.2, add water to 1000mL；121 DEG C of sterilizing 20min.
(2) seed culture：Seed culture medium is seeded to from the oese thalline of inclined-plane thalline picking one, in 30 DEG C, 200r/
Min cultivates 24h, obtains seed liquor；Seed culture medium is constituted：Peptone 10g/L, yeast extract 5g/L, NaCl 10g/L, solvent is
Water, pH 7.0；
(3) fermented and cultured：Aseptically, seed liquor is inoculated in into producing enzyme with the inoculum concentration of volumetric concentration 6-8% to train
Support in base, 30 DEG C, 200r/min culture 24h obtain zymotic fluid, and zymotic fluid 12000rpm is centrifuged 10min, abandoned by culture after terminating
Supernatant, obtains thermophilic nicotine arthrobacterium WYG001 wet thallus；Culture medium is constituted：Lactose 5-15g/L, yeast extract 10-
14g/L, NaCl 10g/L, MgSO40.22g/L、KH2PO40.136g/L, solvent is water, pH 7.0；
(4) in taking step (3) wet thallus with the phosphate buffer of the amount addition pH 7.0,0.1mol/L of 1.2g/ml, stir
Mix uniform, freeze-drying under the conditions of -80 DEG C obtains dry mycelium.
Further, the consumption of the wet thallus is calculated as 3~150g/L, preferably 10~100g/L with buffer solution volume, optimal
At the beginning of selecting 10~50g/L, the consumption of the dry mycelium to be calculated as 1~50g/L, preferably 5~30g/L, the substrate with buffer solution volume
Beginning concentration is 1~100g/L, preferably 5-50g/L.
The method that reaction solution of the present invention is isolated and purified is：After reaction terminates, reaction solution is centrifuged, takes supernatant, plus
Enter isometric ethyl acetate extraction, take ethyl acetate layer vacuum distillation to dry desolvation, obtain N-BOC-L- homoserine
Lactone.
Further, 1~30h is reacted in the reaction at 20~50 DEG C, and more preferably reaction temperature is 30~35 DEG C.
Culture medium composition of the present invention is preferably：Lactose 10g/L, yeast extract 12g/L, NaCl 10g/L,
MgSO40.22g/L、KH2PO40.136g/L, solvent is water, pH 7.0.
The present invention prepares the method for L- selenomethionines using N-BOC-L- homoserine lactones：
(1) N-Boc-L- homoserine lactones are mixed with the acetic acid solution of the hydrobromic acid of mass concentration 33%, 100 DEG C of heating
Backflow 10h, continues stirring reaction 10h, is slowly dropped to room temperature, stands overnight, and filters, and has solid to separate out, and is filtrated to get product L-
2- amino -4- bromobutanoic acid hydrobromides, and with ethyl acetate 30mL washed products, dry and obtain white solid, yield is 85%；
The volumetric usage of the acetic acid solution of the hydrobromic acid is calculated as 2.5ml/g with N-Boc-L- homoserine lactone quality；
(2) dimethyl diselenide ether is mixed with absolute methanol, sodium borohydride (hydroboration is dividedly in some parts under nitrogen protection
Sodium：Dimethyl diselenide ether=mass ratio 2:1), it is stirred at room temperature 0.5h-2.5h, solution is become colorless transparent by golden yellow, is obtained
CH3SeNa alcoholic solutions；To CH3The bromobutanoic acid hydrobromide of 2- amino -4 is added in SeNa alcoholic solutions, oil bath heating to 45 DEG C -65 DEG C,
Continue to stir 2-10h, filtering can obtain L- selenomethionine crude products after reaction terminates；The bromo-butyric acid hydrogen bromine of the 2- amino -4
Hydrochlorate is 1.4 with dimethyl diselenide ether mass ratio:1, the volumetric usage of the absolute methanol is calculated as with dimethyl diselenide ether quality
8ml/g。
N-BOC-L- homoserine lactones (I), obtain in the S-2- amino bromo- butyric acid of -4- (III) through hydrobromic acid open loop bromination,
With the L- selenomethionines (V) of dimethyl diselenide ether reaction system in the presence of sodium borohydride.
The beneficial effect major embodiment of the preparation method of medicine intermediate N-BOC-L- homoserine lactones of the present invention
：The present invention provides one plant of new strains -- thermophilic nicotine arthrobacterium (Arthrobacter nicotinovorans) WYG001, utilizes
Thermophilic nicotine arthrobacterium WYG001 prepares N-BOC-L- homoserine lactones, and the regioselectivity of enzyme is strong, and reaction conversion ratio is high, downstream
Separate simply, energy consumption is low, and environmental pollution is small, be adapted to industrialized production.
(4) illustrate
Fig. 1 racemic modification N-BOC-L- homoserine lactone reference substance gas chromatograms, 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 lactones, retention time 17.797 minutes, peak area 485.9, peak height 50.1, peak width 0.1501, it is symmetrical because
Son 0.905；
The gas chromatogram of Fig. 2 N-BOC-L- homoserine lactone standard items, peak b is N-Boc-L- homoserine lactones,
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 lactones, peak b is N-Boc-L- Kosé ammonia
Acid 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 light micrographs of the bacterial strain WYG001 under 100 times.
Fig. 7 is the 16S rDNA electrophoretograms of bacterial strain WYG001, and A is the electrophoretogram of bacterial strain WYG001, and left side swimming lane is bacterial strain
WYG001, right lanes are standard molecular weight, and B is the enlarged drawing of A.
(5) specific embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Embodiment 1
1st, the screening of microorganism
The present invention relates to microorganism screen isolated by following procedure：Weigh 1~2g of wet soil sample (from the whole nation not
Obtained with soil under environmental condition) it is suspended in the SPSSs of 10mL 0.5%, vibration is mixed；Connect 1~2mL suspensions
In 30mL liquid enriched mediums, after cultivating 2~3d under 30 DEG C, 200r/min, sterile working switching 1mL muddy bacterium solution
To fresh liquid enriched medium, continuous enrichment 3 times.It is unique with N-BOC-DL- homoserine lactones in enriched medium
Carbon source, is formulated as follows：NaNO34.0g/L, KH2PO42.0g/L, MgSO4·7H2O 0.5g/L, KCl 1.0g/L,
ZnSO40.05g/L, solvent is water, pH 7.0.The concentration 1g/L of the N-BOC-DL- homoserine lactones of first round enrichment, second
The concentration of wheel is 3g/L, and the concentration of third round is 5g/L, pH 7.0.Bacterium solution is through gradient dilution, coating point after third round is enriched with
From plating medium, separating for several times obtains single bacterium colony.Separating plating medium composition is：Enriched medium adds agar dense eventually
Degree 20g/L, substrate N-BOC-DL- homoserine lactones final concentration of 5g/L, pH 7.0.
The isolated single bacterium colony of picking is inoculated in slant medium, and 30 DEG C are cultivated 1-2 days, obtain inclined-plane thalline.From oblique
The oese thalline of face thalline picking one is seeded to the seed culture medium of 50mL, in 30 DEG C, 200r/min culture 24h, obtains seed
Liquid.Aseptically, take 3mL seed liquors to be inoculated in the culture medium of 50mL, 30 DEG C, 200r/min culture 24h are obtained
Zymotic fluid.Every liter of slant medium constitutes beef extract 5g, peptone 10g, NaCl 5g, agar 20g, pH 7.0-7.2, adds water to
1000mL；Seed culture based formulas are：Peptone 10g/L, yeast extract 5g/L, NaCl 10g/L, solvent is water, pH 7.0；Produce
Enzyme culture medium is constituted：Lactose 10g/L, yeast extract 12g/L, NaCl 10g/L, MgSO40.22g/L、KH2PO40.136g/L, it is molten
Agent is water, pH 7.0.15mL zymotic fluids 12000rpm centrifugations 10min in 50mL centrifuge tubes is taken, supernatant is abandoned, 5mL is added
The phosphate buffer of 0.1mol/L pH 7.0, vibration is mixed, and adds final concentration 8g/L substrate N-Boc-DL- homoserine
Lactone, under 30 DEG C, 200r/min, conversion reaction 24h.Centrifuge tube is taken out, the reaction solution of 1mL is taken, 2mL ethyl acetate is added,
Fully vibration, 6000rpm centrifugations 5min.Upper organic phase 1mL is taken, is removed water with a small amount of anhydrous sodium sulfate drying, with efficient liquid phase
Chromatogram detects the enantiomeric excess value of N-Boc-L- homoserine lactones
(e.e.), reaction conversion ratio C, screening enantiomeric excess value (e.e.) is more than 90%, and reaction conversion ratio C is (45%-
60%) microbial strains, are designated as bacterial strain WYG001.
2nd, the identification of microbial strains
Physiological and biochemical property：Bacterial strain WYG001 is Gram-negative bacteria, obligate aerobic, and growth changes more sensitive to pH, most
Suitable pH is 7.0, shown in colonial morphology as Fig. 5 and Fig. 6.Poor using inorganic nitrogen-sourced ability, growth and producing enzyme are subject to Cu2+、Fe2 +、Al3+、Zn2+Deng suppression, by Mg2+、K+、Na+Deng promotion.
Through sequencing identification, the 16S rDNA sequences of bacterial strain WYG001 are following (SEQ ID NO.1), and electrophoretogram is as shown in Figure 7：
TGCTTACACATGCAAGTCGAACGATGATCCCAGCTTGCTGGGGGATTAGTGGCGAACGGGTGAGTAACA
CGTGAGTAACCTGCCCTTGACTCTGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGACTCCTCATCGCATG
GTGGGGGGTGGAAAGCTTTTGTGGTTTTGGATGGACTCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAA
GGCGACGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGG
CAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTG
TAAACCTCTTTCAGTAGGGAAGAAGCGTAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGC
CGCGGTAATACGTAGGGCGCAAGCGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGC
TGTGAAAGACCGGGGCTCAACTCCGGTTCTGCAGTGGGTACGGGCAGACTAGAGTGCAGTAGGGGAGACTGGAATTC
CTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTCTCTGGGCTGTAACTGACGC
TGAGGAGCGAAAGCATGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGTTGGGCACTAGGTGT
GGGGGACATTCCACGTTTTCCGCGCCGTAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAA
ACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACC
AAGGCTTGACATGAACCGGAAAGACCTGGAAACAGGTGCCCCGCTTGCGGTCGGTTTACAGGTGGTGCATGGTTGTC
GTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCTATGTTGCCAGCGGTTCGGC
CGGGGACTCATAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGTC
TTGGGCTTCACGCATGCTACAATGGCCGGTACAAAGGGTTGCGATACTGTGAGGTGGAGCTAATCCCAAAAAGCCGG
TCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCG
GTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCACGAAAGTTGGTAACACCCGAAGCCGGTGGCCTAA
CCCTTGTGGGGGGAGCCGTCGAAGGTGGGACCGGCGATGGGACTAAGTC
According to physio-biochemical characteristics and molecular biology identification, the bacterial strain is accredited as thermophilic nicotine arthrobacterium bacterium
(Arthrobacter nicotinovorans), is named as thermophilic nicotine arthrobacterium bacterium (Arthrobacter
Nicotinovorans) WYG001, be preserved in China typical culture collection center (address China, Wuhan, Wuhan University,
430072), deposit number CCTCC NO：M 2014054, preservation date 2014 year 2 month 28 days.
Embodiment 2：
Inclined-plane culture：Thermophilic nicotine arthrobacterium WYG001 is seeded to slant medium, 30 DEG C of culture 24h obtain thalline oblique
Face；Slant medium is constituted with embodiment 1.
Seed culture：100mL seed culture mediums are seeded to from the oese thalline of inclined-plane thalline picking one, in 30 DEG C, 200r/
Min cultivates 24h, obtains seed liquor；Seed culture medium is constituted with embodiment 1.
Fermented and cultured：Aseptically, 10mL seed liquors are taken to be inoculated in the culture medium of 150mL, 30 DEG C,
200r/min cultivates 24h, obtains zymotic fluid, and zymotic fluid 12000rpm is centrifuged 10min, abandons supernatant, obtained by culture after terminating
Thermophilic nicotine arthrobacterium WYG001 wet thallus.Take the phosphate-buffered that wet thallus add pH 7.0,0.1mol/L with the amount of 1.2g/ml
In liquid, stir, freeze-drying obtains dry mycelium under the conditions of -80 DEG C.
Embodiment 3：
The wet thallus 0.34g that the method for Example 2 is obtained, addition N-BOC-DL- homoserine lactones 50mg and 0.1M,
PH7.0 phosphate buffer 5mL, under the conditions of 30 DEG C, 200rpm, stirring reaction 7.5h.Reacting liquid filtering is removed into enzyme, filtrate is taken
The content of the N-Boc-DL- homoserine lactones after conversion is determined with gas chromatographic analysis, enzyme activity, the zymotic fluid specific enzyme activity is calculated
Power is 36.85U/L.
Enzyme activity is defined as：U is 1 enzyme activity unit, and 1 described enzyme activity unit is every point under conditions of regulation
Clock the N-Boc- homoserine lactones of 1 μm of ol are hydrolyzed into N-Boc- homoserine needed for enzyme amount.
Analytical conditions for gas chromatography：Using the type gas chromatographs of gas chromatograph Agilent 6890；Chiral gas chromatography
Post BGB-175 (0.25mm × 30m)；Carrier gas N2(14.54kPa), split ratio 20:1,250 DEG C of injector temperature, detector temperature
220℃；Column temperature rise program：180 DEG C of initial temperature, keeps 20min；The μ L of sample size 1.0, hydrogen flowing quantity：30mL/min air
Flow：300mL/min；Make-up gas flow：N225mL/min；Hydrogen ion flame detector.N-BOC-D- homoserine lactones and
N-BOC-L- homoserine lactones are respectively in 16.831min and 17.797min appearances.
For the N-BOC-DL- homoserine lactone calibration curve equations of enzyme activity determination：
The racemic N-Boc-DL- that compound concentration is respectively 0.1%, 0.4%, 0.5%, 0.6%, 0.8%, 1.0% is high
Serine lactone (w/v), is detected using gas-chromatography, draws the linear relationship between the concentration of lactone and peak area, with
The mass concentration of N-Boc-DL- homoserine lactones is ordinate, and gas chromatographic detection gained peak area is marked for abscissa
Directrix curve equation：Y=1352x -26.79, in 0.1%-2% (w/v) concentration range, coefficient R2=0.999, reach
Requirement needed for external standard method, the analysis that the curve can be used for N-Boc-DL- homoserine lactones is determined.
Enzyme activity computing formula：
A=[(a1-a)/a1]×[C×V/(M×t)]×106Formula (1)
A --- the vigor (U) of enzyme in sample；
A --- conversion reaction terminates the peak area of N-BOC-DL- homoserine lactones in rear conversion fluid；
a1--- calculated according to above-mentioned standard curvilinear equation with initial substrate concentration identical N-BOC-DL- Kosé ammonia
The peak area of acid lactone standard specimen；
C --- in conversion fluid, the mass concentration of substrate, g/L；
V --- conversion fluid volume, L；
M --- the molal weight of substrate N-BOC-DL- homoserine lactones, g/mol；
T --- transformation time, Min.
Embodiment 4：
The dry mycelium 0.2g that the method for Example 2 is obtained, addition N-BOC-DL- homoserine lactones 0.1g, 0.1M,
PH7.0 phosphate buffer 10mL, under the conditions of 30 DEG C, 200rpm, stirring reaction 7.5h.Enzyme activity is determined using the method for embodiment 3,
The dry mycelium specific enzyme activity power is 5.53U/g.
Embodiment 5：
The method of Example 2 gained wet thallus 3.4g, addition N-BOC-DL- homoserine lactones 0.25g and 0.1M,
PH7.0 phosphate buffer 50mL, under the conditions of 30 DEG C, 200rpm, stirring reaction 4h after reaction terminates, reaction solution is centrifuged, and is taken
Supernatant, adds isometric ethyl acetate extraction, after taking ethyl acetate layer vacuum distillation to dry desolventizing, obtains N-BOC-L-
Homoserine lactone 0.12g.N-BOC-L- homoserine lactones are calculated using GC (testing conditions are with embodiment 3) detections
Enantiomeric excess value e.e.s99.9%, conversion ratio C are 51.8%, and yield is 96.4%.
Enantiomeric excess value (enantiomeric excesses) is the important indicator for weighing enzyme selectivity, N-BOC-L-
The enantiomeric excess value abbreviation e.e. of homoserine lactones, its computing formula is：
Formula (2)
Wherein S represents substrate, SRAnd SSRespectively (D)-and (L)-configuration substrate peak area.
The computing formula of reaction conversion ratio is：
Formula (3)
The computing formula of the yield (Yield) of N-BOC-L- homoserine lactones is
Wherein, SR0And SS0Respectively (D)-and (L)-configuration substrate initial peak area.
Embodiment 6：
Thermophilic nicotine arthrobacterium WYG001 dry mycelium 1g obtained in the method for Example 2, add in N-BOC-DL- homoserine
Ester 0.5g and 0.1M, pH7.0 phosphate buffer 100mL, under the conditions of 30 DEG C, 200rpm, stirring reaction 7.5h, reaction terminates
Afterwards, reaction solution is centrifuged, takes supernatant, add isometric ethyl acetate extraction, taken ethyl acetate layer vacuum distillation and taken off to dry
After solvent, N-BOC-L- homoserine lactones 0.24g is obtained.N- is calculated using GC (testing conditions are with embodiment 3) detections
The enantiomeric excess value e.e. of BOC-L- homoserine lactonessIt is 99.9%, conversion ratio C is 52.0%, and yield is 96.0%.
Embodiment 7
Thermophilic nicotine arthrobacterium WYG001 dry mycelium 4g obtained in the method for Example 2, add in N-BOC-DL- homoserine
Ester 2g and 0.1M, pH7.0 phosphate buffer 100mL, under the conditions of 30 DEG C, 200rpm, stirring reaction 8h, after reaction terminates, will
Reaction solution is centrifuged, and takes supernatant, adds isometric ethyl acetate extraction, takes ethyl acetate layer vacuum distillation to dry desolventizing
Afterwards, N-BOC-L- homoserine lactones 0.97g is obtained.The mapping of N-BOC-L- homoserine lactones is detected using the method for embodiment 3
Body excessive value e.e.99.9%, conversion ratio C are 51.0%, and yield is 97.0%.
Embodiment 8
Thermophilic nicotine arthrobacterium WYG001 dry mycelium 1.0g obtained in the method for Example 2, add N-BOC-DL- homoserine
Lactone 0.5g and 0.1M, pH7.0 phosphate buffer 20mL, under the conditions of 35 DEG C, 200rpm, stirring reaction 14h, reaction terminates
Afterwards, reaction solution is centrifuged, takes supernatant, add isometric ethyl acetate extraction, taken ethyl acetate layer vacuum distillation and taken off to dry
After solvent, N-BOC-L- homoserine lactones 0.233g is obtained.N-BOC-L- Kosé ammonia is calculated using the detection of the method for embodiment 3
The enantiomeric excess value e.e. of acid lactonesIt is 99.9%, conversion ratio C is 52.5%, and yield is 93.2%.
Embodiment 9
Thermophilic nicotine arthrobacterium WYG001 dry mycelium 1.0g obtained in the method for Example 2, add N-BOC-DL- homoserine
Lactone 1g and 0.1M, pH7.0 phosphate buffer 20mL, under the conditions of 35 DEG C, 200rpm, after stirring reaction 30h, reaction terminates
Afterwards, reaction solution is centrifuged, takes supernatant, add isometric ethyl acetate extraction, taken ethyl acetate layer vacuum distillation and taken off to dry
After solvent, N-BOC-L- homoserine lactones 0.453g is obtained.N-BOC-L- homoserine lactones are calculated using GC detections
Enantiomeric excess value e.e.sIt is 95.6%, conversion ratio C is 52.0%, and yield is 90.6%.
Embodiment 10：
Thermophilic nicotine arthrobacterium WYG001 dry mycelium 0.2g obtained in the method for Example 2, add N-BOC-DL- homoserine
Lactone 0.15g and 0.1M, pH7.0 phosphate buffer 10mL, under the conditions of 30 DEG C, 200rpm, stirring reaction 13h, reaction terminates
Afterwards, reaction solution is centrifuged, takes supernatant, add isometric ethyl acetate extraction, taken ethyl acetate layer vacuum distillation and taken off to dry
After solvent, N-BOC-L- homoserine lactones 0.144g is obtained.Using the method for embodiment 3 detection N-BOC-L- homoserine lactones
Enantiomeric excess value e.e.99.9%, conversion ratio C are 50.8%, and yield is 96.0%.
Embodiment 11：
Thermophilic nicotine arthrobacterium WYG001 dry mycelium 1.5g obtained in the method for Example 1, add N-BOC-DL- homoserine
Lactone 1.5g and 0.1M, pH7.0 phosphate buffer 100mL, under the conditions of 30 DEG C, 200rpm, stirring reaction 13h, reaction terminates
Afterwards, reaction solution is centrifuged, takes supernatant, add isometric ethyl acetate extraction, taken ethyl acetate layer vacuum distillation and taken off to dry
After solvent, N-BOC-L- homoserine lactones 0.725g is obtained.N-BOC-L- homoserine lactones are calculated using GC detections
Enantiomeric excess value e.e.sIt is 99.9%, conversion ratio C is 50.7%, and yield is 96.6%.
Embodiment 12：
Thermophilic nicotine arthrobacterium WYG001 dry mycelium 5g obtained in the method for Example 1, add in N-BOC-DL- homoserine
Ester 10g and 0.1M, pH7.0 phosphate buffer 100mL, under the conditions of 40 DEG C, 200rpm, stirring reaction 15h, after reaction terminates,
Reaction solution is centrifuged, supernatant is taken, isometric ethyl acetate extraction is added, ethyl acetate layer vacuum distillation to dry desolventizing is taken
Afterwards, N-BOC-L- homoserine lactones 4.70g is obtained.The enantiomer of N-BOC-L- homoserine lactones is calculated using GC detections
Excessive value e.e.sIt is 99.0%, conversion ratio C is 51.2%, and yield is 94.0%.
Embodiment 13：
Thermophilic nicotine arthrobacterium WYG001 dry myceliums in embodiment 11 are substituted into (dry bacterium with the dry mycelium 0.2g in table 1 respectively
The preparation of body is with embodiment 2), N-BOC-DL- homoserine lactones 0.1g and 0.1M, pH7.0 phosphate buffer 10mL is added,
30 DEG C, under the conditions of 200rpm, stirring reaction 24h after reaction terminates, reaction solution is centrifuged, and takes supernatant, adds isometric second
Acetoacetic ester is extracted, and after taking ethyl acetate layer vacuum distillation to dry desolventizing, obtains N-BOC-L- homoserine lactones.Detected using GC
It is calculated the enantiomeric excess value e.e. of N-BOC-L- homoserine lactonessWith conversion ratio C, 1 is shown in Table.
Table 1
The result of table 1 shows that the bacterial strain such as aspergillus oryzae wzz007 has certain catalytic effect and stereoselectivity to substrate, but
It is to be not so good as thermophilic nicotine arthrobacterium WYG001 of the invention in effect.
Embodiment 14：
(1) thermophilic nicotine arthrobacterium WYG001 dry mycelium 10g obtained in the method for Example 2, add N-BOC-DL- Kosé ammonia
Acid lactone 5g and 0.1M, pH7.0 phosphate buffer 200mL, under the conditions of 30 DEG C, 200rpm, stirring reaction 11h, terminating reaction,
Reaction solution is centrifuged off thalline, and supernatant is extracted with ethyl acetate (50mL × 5), takes ethyl acetate layer vacuum distillation to dry precipitation
After agent, optically pure N-Boc-L- homoserine lactones 2.40g (detection method is with embodiment 3) is obtained, yield is 96.0%；N-
The enantiomeric excess value e.e. of BOC-L- homoserine lactonessIt is 99.9%, its data structure characterizes as follows：
1H NMR(600MHz,CDCl3) δ 7.34 (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,CDCl3))δ174.5,
155.6,78.5,64.7,49.1,29.2,27.3.Mp 123-125℃.[α]D=+8.6 ° of (c=1, CHCl3)
(2) synthesis of 2- amino -4- bromobutanoic acid hydrobromides
In the round mouth flask of 50mL, N-Boc-L- homoserine lactone 2.40g are added, be subsequently adding 6mL mass concentrations
The acetic acid solution of 33% hydrobromic acid, oil bath heating continues stirring reaction 10h to 100 DEG C, and reaction solution fades to clarification by muddiness,
Room temperature is slowly dropped to, is stood overnight, there is solid to separate out, filtered, obtain the crude product of the bromobutanoic acid hydrobromide of 2- amino -4, be used in combination
Ethyl acetate washed product, is finally dried to obtain white solid 2.65g, and yield is 85%.
1H NMR(500MHz,D2O) δ 2.22-2.41 (m, 2H), 3.59-3.71 (m, 2H), 4.00 (d, J=5.6Hz,
1H).13C NMR(126MHz,D2O)δ174.6,51.7,32.9,28.3.
(3) synthesis of L- selenomethionines hydrobromate
In two mouthfuls of flasks of 50mL, 1.88g dimethyl diselenide ethers are added, be subsequently adding the absolute methanol of 15mL, make it
Mixing, one end is connected with nitrogen protection device.Sodium borohydride 3.76g is dividedly in some parts under nitrogen protection, to prevent reaction excessively acute
It is strong.(25 DEG C) of room temperature is stirred 10 minutes, and solution is become colorless transparent by golden yellow, obtains CH3SeNa alcoholic solutions.By what is prepared
CH3The bromobutanoic acid hydrobromide of 2.65g 2- amino -4 is added in SeNa alcoholic solutions, oil bath heating continues to stir 5h to 60 DEG C.Instead
There is solid to separate out after should terminating, filtering can obtain the crude product of L- selenomethionines and then use ethanol washed product again, dry
Obtain white solid.Yield is 61.2%.
1H NMR(500MHz,D2O)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,D2O)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. thermophilic nicotine arthrobacterium (Arthrobacter nicotinovorans) WYG001, it is preserved in Chinese Typical Representative culture guarantor
Tibetan center, deposit number CCTCC NO：M 2014054, preservation date 2014 year 2 month 28 days, preservation address is Chinese, Wuhan,
Wuhan University, postcode 430072.
2. prepared by thermophilic nicotine arthrobacterium WYG001 described in a kind of claim 1NApplication in-BOC-L- homoserine lactones.
3. application as claimed in claim 2, it is characterised in that described application is：It is fermented with thermophilic nicotine arthrobacterium WYG001
The dry mycelium cultivated after the wet thallus or wet thallus freeze-drying for obtaining is catalyst, is with N-BOC-DL- homoserine lactones
Substrate, with 0.1M, pH7.0 phosphate buffer as reaction medium, carries out converting instead under the conditions of 30 ~ 35 DEG C or 40 DEG C, 200rpm
Should, after reaction terminates, reaction solution is isolated and purified, obtainN- BOC-L- homoserine lactones.
4. application as claimed in claim 3, it is characterised in that the preparation method of the catalyst is：
（1）Inclined-plane culture：Thermophilic nicotine arthrobacterium WYG001 is seeded to slant medium, 30 DEG C of 24 h of culture obtain thalline oblique
Face；Every liter of slant medium composition：The g of beef extract 5, peptone 10 g, NaCl 5 g, agar 20 g, pH 7.0-7.2, add water
To 1000 mL；121 C sterilizings, 20 min；
（2）Seed culture：Seed culture medium is seeded to from the oese thalline of inclined-plane thalline picking one, in 30 DEG C, 200 r/min
24 h are cultivated, seed liquor is obtained；Seed culture medium is constituted：The g/L of peptone 10, yeast extract 5 g/L, NaCl 10 g/L, solvent
It is water, pH 7.0；
（3）Fermented and cultured：Aseptically, seed liquor is inoculated in by culture medium with the inoculum concentration of volumetric concentration 6-8%
In, 30 DEG C, 200 r/min culture 24h obtain zymotic fluid, and the rpm of zymotic fluid 12000 is centrifuged 10 min, abandoned by culture after terminating
Supernatant, obtains thermophilic nicotine arthrobacterium WYG001 wet thallus；Culture medium is constituted：Lactose 5-15 g/L, yeast extract 10-14
G/L, NaCl 10 g/L, MgSO4 0.22g/L、KH2PO40.136g/L, solvent is water, pH 7.0；
（4）Take step（3）Wet thallus are added in pH 7.0, the phosphate buffer of 0.1 mol/L with the amount of 1.2g/ml, stirring
Uniformly, freeze-drying under the conditions of -80 DEG C, obtains dry mycelium.
5. application as claimed in claim 3, it is characterised in that the consumption of the wet thallus is calculated as 3 ~ 150 g with buffer solution volume
/L。
6. application as claimed in claim 3, it is characterised in that the consumption of the dry mycelium is calculated as 1 ~ 50 g with buffer solution volume
/L。
7. application as claimed in claim 3, it is characterised in that the initial substrate concentration is 1 ~ 100g/L.
8. application as claimed in claim 3, it is characterised in that the method that the reaction solution is isolated and purified is：After reaction terminates,
Reaction solution is centrifuged, supernatant is taken, isometric ethyl acetate extraction is added, ethyl acetate layer vacuum distillation is taken molten to dry removing
Agent, obtainsN- BOC-L- homoserine lactones.
9. application as claimed in claim 3, it is characterised in that the reaction reacts 1 ~ 30h at 30 ~ 35 DEG C or 40 DEG C.
10. application as claimed in claim 4, it is characterised in that culture medium constitutes and is：The g/L of lactose 10, yeast extract 12
G/L, NaCl 10 g/L, MgSO4 0.22g/L、KH2PO40.136g/L, solvent is water, pH 7.0.
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