CN105385621A - Acinetobacter junii zjutfet-1 and application thereof - Google Patents

Abstract

The invention discloses a novel strain, namely acinetobacter junii zjutfet-1 and an application of acinetobacter junii zjutfet-1 in detachment of methyl-2-chlorobutyrate. The acinetobacter junii zjutfet-1 is preserved in China Center for Type Culture Collection in China Wuhan University with a preservation number of CCTCC NO.M2015511 on September 6th, 2015. After methyl-2-chlorobutyrate is catalyzed for 2 hours by the acinetobacter junii zjutfet-1, (R)-chlorobutyric acid eep reaches 95%.

Description

Acinetobacter junii zjutfet-1 and application thereof

(1) technical field

The present invention relates to the preparation method of one (R)-chloro-butyric acid, particularly a strain Acinetobacter junii (Acinetobacterjunii) and the application in preparation (R)-chloro-butyric acid thereof.

(2) background technology

Make 2-chloro-butyric acid traditional technology in prior art many, the muriate of usual phosphorus and phosphorus is as catalyzer, and as taken butanic acid as raw material, phosphorus pentachloride is catalyzer, and logical chlorine carries out chlorination reaction.Normal pressure, temperature control 150-200 DEG C, reaction times 20-50h.After completion of the reaction, coarse fodder rectifying.Although this reaction transformation efficiency better (97%-99%), by product 3-chloro-butyric acid more (5%-10%), the reaction times is longer, and temperature is higher, and catalyzer cannot recycling, becomes solid waste.

So far, the method for biological resolution acquisition 2-chloro-butyric acid is not also employed.

(3) summary of the invention

The object of the invention is to provide one to be had better stereoselective bacterial strain-Acinetobacter junii (Acinetobacterjunii) zjutfet-1 and is splitting the application in 2-chlorobutanoate.

The technical solution used in the present invention is:

The invention provides a strain new strains--Acinetobacter junii (Acinetobacterjunii) zjutfet-1, be preserved in China typical culture collection center, preservation date is on September 6th, 2015, deposit number: CCTCCNO.M2015511, preservation address is Wuhan, China Wuhan University, postcode 430072.

The present invention also provides a kind of described Acinetobacter junii zjutfet-1 splitting the application in 2-chlorobutanoate preparation (R)-2-chloro-butyric acid, concrete described application is that the wet thallus that obtains through fermentation culture with Acinetobacter junii zjutfet-1 is for catalyzer, with 2-chlorobutanoate for substrate, with pH=7 phosphoric acid buffer for reaction medium, at 25-37 DEG C, catalyzed reaction is carried out under 100-180rpm condition, after reacting completely, obtain the mixed solution containing (S)-2-chloro-butyric acid and (R)-2-chloro-butyric acid, by mixed solution separation and purification, obtain (R)-2-chloro-butyric acid.

Further, described catalyst levels counts 10 ~ 80g/L damping fluid (preferably 20 ~ 80g/L, most preferably 20g/L) with wet thallus weight, and described substrate volume final concentration accounts for the 0.1-4.0% (preferably 1.0%) of damping fluid volume.

Further, the preparation method of wet thallus of the present invention is:

(1) Acinetobacter junii zjutfet-1 is seeded to slant medium, cultivates 1d at 30 DEG C, obtain inclined-plane bacterium colony; Slant medium final concentration used consists of: peptone 10g/L, yeast extract 5g/L, NaCl10g/L, agar 17g/L, and solvent is distilled water, and pH value is 7.0;

(2) by inclined-plane colony inoculation in seed culture medium, 30 DEG C cultivate 16h, obtain seed liquor; Described seed culture medium final concentration consists of: peptone 10g/L, yeast extract 5g/L, NaCl10g/L, and solvent is distilled water, and pH value is 7.0;

(3) seed liquor is inoculated in fermention medium with volumetric concentration 0.2% inoculum size, 30 DEG C, cultivate 48h under the condition of 180rpm, fermented liquid is centrifugal, abandon supernatant liquor, obtain wet thallus; Fermention medium final concentration forms: peptone 10g/L, sucrose 5g/L, K ₂hPO ₄10g/L, MgSO ₄0.5g/L, solvent is distilled water, and pH value is 7.0.

Further, the method for described mixed solution separation and purification: after reaction terminates, regulates mixed solution pH value to 1-2 with HCl, add isopyknic ethyl acetate to extract, the centrifugal 3min of 8000rpm, layering, obtain organic phase, get upper strata and namely obtain (R)-chloro-butyric acid.

Compared with prior art, beneficial effect of the present invention is mainly reflected in:

The invention provides a strain new strains--Acinetobacter junii zjutfet-1, this bacterial strain is to 2-chlorobutanoate catalysis 2h, ee _preach 95%, substrate conversion efficiency reaches 21.5%.

(4) accompanying drawing explanation

Fig. 1 be sweet oil as sole carbon source bacterial strain screening, rhodamine B is as the fluorescence photo of developer.

Fig. 2 be sweet oil as sole carbon source bacterial strain screening, purpurum bromocresolis is as the fluorescence photo of developer.

Fig. 3 is the colonial morphology of fine jade formula acinetobacter calcoaceticus zjutfet-1 after gramstaining.

Fig. 4 is fine jade formula acinetobacter calcoaceticus zjutfet-1 phylogenetic tree.

(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:

Experimental technique described in following embodiment, if no special instructions, is ordinary method; Described reagent and biomaterial, if no special instructions, all can obtain from commercial channels.

Embodiment 1: bacterial strain screening

1, Acinetobacter junii ( acinetobacterjunii) primary dcreening operation of zjutfet-1 and multiple sieve

(1) primary dcreening operation:

A), sweet oil as inductor, rhodamine B is as developer.Get 1g soil sample (being collected in colleges and universities of Zhejiang Province water drain) to join in 9mL physiological saline, fully shake up, carry out doubling dilution, gradient is 10 ^-9, 10 ^-11, 10 ^-13coating containing final concentration is that (polyvinyl alcohol of 3% mixes by 3:1 with sweet oil for 10g/L rhodamine B and final concentration 120mL/L sweet oil emulsion, refiner stir) primary dcreening operation culture medium flat plate in screen, 30 DEG C be cultured to colony growth after, flat board is placed in 365nm viewed under ultraviolet radiation periphery of bacterial colonies fluorescent ring and has nil case, the results are shown in Figure shown in 1.

Primary dcreening operation substratum final concentration composition (g/L): peptone 10, yeast extract 5, NaCl10, agar 17, solvent is distilled water, and pH value is 7.0.

B), pesticide intermediate as sole carbon source, purpurum bromocresolis is as developer

Get 1g soil sample (being collected in colleges and universities of Zhejiang Province water drain) to join in 9mL physiological saline, fully shake up, carry out doubling dilution, gradient is 10 ^-9, 10 ^-11, 10 ^-13coating containing final concentration is the bacterial classification primary dcreening operation solid medium of 20g/L2-chlorobutanoate and purpurum bromocresolis (10mg/ml) 50ml/L, cultivate after 2 days for 30 DEG C, substratum is placed in 365nm viewed under ultraviolet radiation periphery of bacterial colonies fluorescence variable color circle and size cases, the results are shown in Figure 2.

(2) multiple sieve: picking has the colony inoculation of fluorescent ring on LB solid medium under 365nm ultraviolet, cultivates 1-2d, separation and purification to flat board has single bacterium colony produce for 30 DEG C, picking list bacterium colony is in seed culture medium, after 30 DEG C of cultivation 16h, get 1mL bacterium liquid and dilute, be diluted to 10 ^-7, 10 ^-8, 10 ^-9after, coat in multiple sieve substratum, carry out cultivation 2d in 30 DEG C, obtain bacterial strain zjutfet-1.

LB solid medium final concentration composition (g/L): peptone 10, yeast extract 5, NaCl10, agar 17, solvent is distilled water, and pH value is 7.0.

Seed culture medium final concentration composition (g/L): peptone 10, yeast extract 5, NaCl10, solvent is distilled water, and pH value is 7.0.

Sieve substratum final concentration (g/L) again: peptone 10, yeast extract 5, sucrose 10, K ₂hPO ₄1, MgSO ₄7H ₂o0.5, solvent is distilled water, and pH value is 7.0.

2, the qualification of bacterial strain zjutfet-1

Bacterial strain zjutfet-1 morphologic observation: the bacterial strain zjutfet-1 screened is lined in LB solid medium, be inverted for 30 DEG C and cultivate 2d, observe the features such as single colony shape, size, color and projection, and gramstaining (Fig. 3) is carried out to it, Gram-negative bacteria, shaft-like, without gemma; Colony characteristics, rounded on substratum, protuberance, bacterium colony is yellowish, opaque, and the smooth of the edge is smooth, moistening.

16SrDNA molecular biology identification: bacterial strain zjutfet-1 delivers to raw work biotechnology (Shanghai), and limited-liability company identifies, the sequence length 1446bp (shown in SEQIDNO.1) of 16SrDNA.Obtained by sequence alignment:

From comparison result display, bacterial strain belongs to the bacterium that Acinetobacter belongs to.At LPSN (http; //www.bactrrio.net/) search for the pattern bacterium that Acinetobacter belongs to, the simultaneously 16SrDNA of search pattern bacterial strain on NCBI, obtained 16SrDNA and bacterial strain zjutfet-1 is carried out the analysis of similarity, evolutionary tree is as shown in Figure 4.By 16SrDNA molecular biology identification in conjunction with bacterial strain zjutfet-1 physiological and biochemical property, by bacterial strain zjutfet-1 called after fine jade formula acinetobacter calcoaceticus (Acinetobacterjunii) zjutfet-1, be preserved in China typical culture collection center, preservation date is on September 6th, 2015, deposit number: CCTCCNO.M2015511, preservation address is Wuhan, China Wuhan University, postcode 430072.

Embodiment 2

Acinetobacter junii zjutfet-1 is seeded to slant medium, cultivates 1d for 30 DEG C, obtain inclined-plane thalline.Slant medium final concentration consists of (g/L): peptone 10, yeast extract 5, NaCl10, agar 17, and solvent is distilled water, and pH value is 7.0.

By inclined-plane colony inoculation in seed culture medium, cultivate 16h at 30 DEG C, obtain seed liquor; Described seed culture medium final concentration consists of: peptone 10g/L, yeast extract 5g/L, NaCl10g/L, and solvent is distilled water, and pH value is 7.0;

Seed liquor is seeded to (volume liquid amount is 50mL/250mL Erlenmeyer flask) in fermention medium with the inoculum size of volumetric concentration 0.2%, 30 DEG C, cultivate 48h in 180rpm temperature control shaking table, at 4 DEG C after fermentation, under 8000rpm condition, centrifugal 10min, obtains wet thallus.Fermention medium final concentration composition (g/L): peptone 10, sucrose 5, anhydrous K ₂hPO ₄10, anhydrous MgSO ₄0.5, sweet oil 10, pH value is 7.0, and solvent is distilled water.

Embodiment 3

The total system 5mL of catalyzed reaction: wet thallus 0.1g prepared by embodiment 2,2-chlorobutanoate 50 μ l, phosphoric acid buffer (pH=7.0) 5ml, at 37 DEG C, catalyzed reaction 2h under 180rpm condition, after reaction terminates, regulate mixed solution pH value to 1-2 with concentrated hydrochloric acid (mass concentration 36-38%), add isopyknic ethyl acetate and extract, layering, obtain organic phase, the centrifugal 3min of 8000rpm, gets upper strata and namely obtains (R)-chloro-butyric acid, ee _preach 95%, substrate conversion efficiency reaches 21.5%.

The chemical formula of 2-chlorobutanoate:

GC temperature programming: initial temperature is 80 DEG C, is warming up to 120 DEG C with the speed of 4 DEG C/min.

Enantiomeric excess value (ee _p) by formulae discovery:

Substrate ${\mathrm{ee}}_s\left(\%\right)=\left|\frac{{\[S\]}_S-{\[S\]}_R}{{\[S\]}_S+{\[S\]}_R}\right|\×100\%$

Product ${\mathrm{ee}}_p\left(\%\right)=\left|\frac{{\[P\]}_S-{\[P\]}_R}{{\[P\]}_S+{\[P\]}_R}\right|\×100\%$

Substrate conversion efficiency $C\left(\%\right)=\frac{{\mathrm{ee}}_s}{{\mathrm{ee}}_s+{\mathrm{ee}}_p}\×100\%$

In formula, [S] s and [S] _rbe respectively the content of S and R type substrate in sample, [P] _s[P] _rfor recording the content of S and R type substrate enantiomer in sample.Ee _sand ee _pbe respectively substrate and product enantiomeric excess value.

Embodiment 4

The consumption of the wet thallus in embodiment 3 changes 0.05g, 0.1g, 0.2g, 0.3g, 0.4g into, other implementation and operation with example 3, catalyzed reaction time 2h, sampling analysis.Result shows ee _pfirst increase then to reduce to some extent.0.1g wet thallus, reaction 2h, makes ee _preaching maximum value is 95%, and substrate conversion efficiency reaches 21.5%.During wet thallus consumption 0.4g, transformation efficiency is increased to 48.3% (maximum) but ee _pdrop to 60.1%, primary product is (R)-chloro-butyric acid.

Embodiment 5

2-chlorobutanoate volumetric usage in embodiment 3 changes into and accounts for 0.1%, 0.5%, 1%, 2%, 3%, 4% of damping fluid volume, other implementation and operations with example 3, catalyzed reaction 2h sampling analysis.Result shows the increase ee along with amount of substrate _pfirst increase and then promptly reduce.When 2-chlorobutanoate consumption is 1% of reaction system, ee _preaching maximum value is 95%.

Claims (6)

1. Acinetobacter junii (Acinetobacterjunii) zjutfet-1, be preserved in China typical culture collection center, preservation date is on September 6th, 2015, deposit number: CCTCCNO.M2015511, preservation address is Wuhan, China Wuhan University, postcode 430072.

2. Acinetobacter junii zjutfet-1 described in a claim 1 is splitting the application in 2-chlorobutanoate preparation (R)-2-chloro-butyric acid.

3. apply as claimed in claim 2, it is characterized in that described being applied as: the wet thallus obtained through fermentation culture with Acinetobacter junii zjutfet-1 is for catalyzer, with 2-chlorobutanoate for substrate, with the phosphoric acid buffer of pH=7.0 for reaction medium, 25-37 DEG C, carry out catalyzed reaction under 100-180rpm condition, after reacting completely, obtain the mixed solution containing (R)-2-chloro-butyric acid, by mixed solution separation and purification, obtain (R)-2-chloro-butyric acid.

4. apply as claimed in claim 3, it is characterized in that described catalyst levels counts 10 ~ 80g/L damping fluid with wet thallus weight, described substrate volume final concentration counts 0.1-4.0% with damping fluid volume.

5. apply as claimed in claim 3, it is characterized in that the preparation method of described wet thallus is:

(1) Acinetobacter junii zjutfet-1 is seeded to slant medium, cultivates 1d at 30 DEG C, obtain inclined-plane bacterium colony; Slant medium final concentration used consists of: peptone 10g/L, yeast extract 5g/L, NaCl10g/L, agar 17g/L, and solvent is distilled water, and pH value is 7.0;

(2) by inclined-plane colony inoculation in seed culture medium, 30 DEG C cultivate 16h, obtain seed liquor; Described seed culture medium final concentration consists of: peptone 10g/L, yeast extract 5g/L, NaCl10g/L, and solvent is distilled water, and pH value is 7.0;

(3) seed liquor is inoculated in fermention medium with volumetric concentration 0.2% inoculum size, 30 DEG C, cultivate 48h under the condition of 180rpm, fermented liquid is centrifugal, abandon supernatant liquor, obtain wet thallus; Fermention medium final concentration forms: peptone 10g/L, sucrose 5g/L, K ₂hPO ₄10g/L, MgSO ₄0.5g/L, sweet oil 10g/L, solvent is distilled water, and pH value is 7.0.

6. apply as claimed in claim 3, it is characterized in that the method for described mixed solution separation and purification: after reaction terminates, regulate mixed solution pH value to 1-2, add isopyknic ethyl acetate to extract, layering, obtain organic phase, the centrifugal 3min of 8000rpm, get upper strata and namely obtain (R)-chloro-butyric acid.