CN110982757B - Enterobacter cloacae ZJPH1903 and application - Google Patents
Enterobacter cloacae ZJPH1903 and application Download PDFInfo
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Abstract
The invention discloses enterobacter cloacae ZJPH1903 and application thereof in preparing (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol, the novel strain of the invention is used for preparing optically pure (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol by catalysis, and has the advantages of good stereoselectivity, high optical purity of products and the like, when the concentration of a substrate is 4g/L, the e.e. value of a target product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol is more than 99.9%, and the yield is 92.9%. When the surfactant is added into the reaction system, the catalytic efficiency can be effectively improved, the concentration of the catalytic substrate can be increased to 10g/L, the yield of the target product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol reaches 94.3 percent, and the e.e. value is more than 99.9 percent.
Description
(I) technical field
The invention relates to a new strain Enterobacter cloacae ZJPH1903 and application thereof in catalyzing asymmetric reduction of 2,2',4' -trichloroacetophenone to prepare (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol.
(II) background of the invention
The chemical structural formula of the (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol is as follows:
(R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol is a key chiral intermediate for synthesizing antifungal drugs, and the current clinically used antifungal drugs mainly comprise: miconazole, econazole, tioconazole, fenticonazole, isavuconazole and the like, and the optical purity of the chiral hydroxyl building block of the antifungal medicine has important influence on the medicine effect of the medicine. Imidazole drugs (miconazole, ketoconazole, etc.) have high antifungal activity, but are limited to external use due to their high toxicity. Fenticonazole nitrate was an imidazole-based broad-spectrum antifungal agent developed by the original factory of the RECORDATI large pharmaceutical factory, italy, and was approved for marketing in italy in 1986 for the treatment of superficial fungal and vaginal candida infections. Isavuconazole is a new generation of triazole system Drug, and is approved by the U.S. Food and Drug Administration (FDA) in 2015 for the treatment of adult invasive aspergillosis and mucormycosis.
At present, the preparation of antifungal drug key chiral intermediate (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol by catalytic asymmetric hydrogenation by using different catalysts including chiral oxazolecarboxane catalyst, chiral metal catalyst, biocatalyst and the like has been reported. The substrate spectrum of the chiral oxazoleborane catalyst is wide, and a compound taking rhodium, ruthenium and iridium as transition metals is the most commonly used catalyst in ketone hydrogenation reaction. However, transition metals require high selectivity of the additional chelating functional group enantiomer, the operation is difficult to control, and the high cost of transition metals limits their industrial application. The advantages of the biocatalyst compared to the chemical catalyst are its excellent reaction selectivity and environmental friendliness. With the increase of the market of novel antifungal drugs, the demand of (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol is rapidly increased, so that a high-efficiency biocatalyst is developed, and a novel process for preparing (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol by biocatalysis has good application prospects.
Xu et al (RSC. adv.2015,5: 22703-. Qin et al (ACS Catal.2016,6(9): 6135-. Yuhui bud et al (CN 106701698A) disclose that carbonyl reductase SsCR expressed by xylose fermenting yeast CBS6054 and a mutant of the carbonyl reductase SsCR catalyze asymmetric reduction of a latent chiral carbonyl compound to prepare (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol, the conversion rate is more than 99%, and the optical purity of the product is 99.9%. Thiago et al (org. chem.2018, (18): 2110-2116.) catalyse the biological reduction of 2,2',4' -trichloroacetophenone to (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol using the lipase Novozym 435 in a yield of 46.5% with an e.e. value > 99%.
Disclosure of the invention
The invention aims to provide a new strain, Enterobacter cloacae ZJPH1903, and application thereof in catalyzing asymmetric reduction of 2,2',4' -trichloroacetophenone to prepare (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol.
The technical scheme adopted by the invention is as follows:
the invention provides a new strain, Enterobacter cloacae ZJPH1903, which is preserved in China center for type culture collection with the preservation date of 2019, 10 months and 14 days and the preservation number: CCTCC NO: m2019821, deposit address: china, wuhan university, zip code 430072.
The invention also provides an application of the enterobacter cloacae ZJPH1903 in catalyzing asymmetric reduction of 2,2',4' -trichloroacetophenone to prepare (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol, wherein the application comprises the following steps: the method comprises the steps of taking 2,2',4' -trichloroacetophenone as a substrate, wet thalli obtained by fermentation culture of enterobacter cloacae ZJPH1903 as an enzyme source, taking distilled water or buffer solution (preferably phosphate buffer solution with the pH value of 6.5) with the pH value of 6.0-8.0 as a reaction medium to form a conversion system, carrying out reaction at the temperature of 30 ℃ and at the speed of 200rpm, centrifuging reaction liquid after the reaction is finished, and taking supernatant to obtain the conversion liquid containing the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol. The initial adding amount of the substrate is 2-6 g/L (preferably 4g/L) calculated by the volume of the reaction medium, and the using amount of wet thalli is 50-300 g/L (preferably 200g/L) calculated by the wet weight of thalli.
Further, the buffer solution is selected from citrate buffer solution (sodium citrate buffer solution), phosphate buffer solution (potassium phosphate buffer solution, sodium phosphate buffer solution), and most preferably potassium phosphate buffer solution.
Furthermore, in order to promote coenzyme regeneration and improve reaction efficiency, an auxiliary substrate is added into the conversion system, and the auxiliary substrate is one of the following substances: glucose, maltose, sucrose, lactose, glycerol, methanol, ethanol, isopropanol, cysteine, alanine, methionine, preferably glucose; the adding amount of the auxiliary substrate is 50-300 g/L (preferably 150g/L) based on the volume of the reaction medium.
Further, the reaction is preferably a conversion reaction at 30 ℃ and 200rpm for 2-36 h, preferably 24-36 h.
Further, it is preferable that the use of said Enterobacter cloacae ZJPH1903 for the production of (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol is carried out by the following steps: taking 2,2',4' -trichloroacetophenone as a substrate, glucose as an auxiliary substrate, taking wet thalli obtained by fermentation culture of enterobacter cloacae ZJPH1903 as an enzyme source, taking a phosphate buffer solution with the pH of 6.5 as a reaction medium to form a conversion system, reacting for 24 hours at the temperature of 30 ℃ and the speed of 200rpm, and obtaining a conversion solution containing a target product after the reaction is finished; the initial adding amount of the substrate is 4g/L buffer solution according to the volume of the buffer solution, the adding amount of the auxiliary substrate is 150g/L buffer solution according to the volume of the buffer solution, the using amount of wet thalli is 200g/L buffer solution according to the wet weight of the thalli, the e.e. value of the target product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol is more than 99.9 percent, and the yield is 92.9 percent.
In addition, the conversion system consists of an enzyme source, a substrate, an auxiliary substrate and a surfactant, wherein the surfactant is tween-80, tween-20 and sodium dodecyl sulfate, and preferably tween-80; the addition amount of the surfactant is 2-20 g/L, most preferably 6g/L, calculated by the volume of the reaction medium.
The most preferred transformation system of the invention consists of an enzyme source, a substrate, a co-substrate and a surfactant, and the application of the Enterobacter cloacae ZJPH1903 in the preparation of (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol is carried out according to the following steps: taking 2,2',4' -trichloroacetophenone as a substrate, glucose as an auxiliary substrate, tween-80 as a surfactant, wet thalli obtained by fermentation culture of enterobacter cloacae ZJPH1903 as an enzyme source, and a phosphate buffer solution with pH of 6.5 as a reaction medium to form a conversion system, reacting for 24 hours at 30 ℃ and 200rpm, and obtaining a conversion solution containing a target product after the reaction is finished; the adding amount of the substrate is 5-15 g/L (preferably 10 g/L) calculated by the volume of the reaction medium, and the adding amount of the enzyme source is 200g/L calculated by the volume of the reaction medium; the auxiliary substrate is glucose, and the addition amount of the glucose is 150g/L in terms of the volume of the reaction medium; the surfactant is tween-80, and the addition amount of the surfactant is 6g/L in terms of the volume of the reaction medium; the yield of the conversion system reaches 94.3 percent.
The preparation method of the enzyme source comprises the following steps:
(1) slant culture: inoculating Enterobacter cloacae ZJPH1903 to a slant culture medium, and culturing at 25-30 ℃ for 36h to obtain a slant strain; the final concentration composition of the slant culture medium is as follows: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH)4)2SO42g/L，KH2PO4 2g/L，NaCl 1g/L，MgSO4·7H20.5g/L of O, 15-20 g/L of agar powder, water as a solvent and 6.5 of pH value;
(2) seed culture: inoculating slant strains into a seed culture medium, culturing at 25-30 ℃ and 150-250 rpm for 10-24 h (preferably at 30 ℃ and 200rpm for 12h) to obtain a seed solution; the final concentration composition of the seed culture medium is as follows: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH)4)2SO4 2g/L，KH2PO4 2g/L，NaCl 1g/L，MgSO4·7H2O is 0.5g/L, the solvent is water, and the pH value is 6.5;
(3) fermentation culture: mixing the seed liquid with the volumeInoculating the inoculum size with the concentration of 4-10% (preferably 8%) into a fermentation culture medium, filling the inoculum size of 80mL/250mL shake flask, culturing at 25-30 ℃ and 150-250 rpm for 12-32 h (preferably culturing at 30 ℃ and 200rpm for 16h), centrifuging the fermentation liquor after the fermentation is finished, washing the obtained precipitate with 0.1M, pH 6.5.5 phosphate buffer solution, and collecting wet bacteria, namely the enzyme source; the final concentration of the fermentation medium is as follows: 30g/L of sucrose, 41.43g/L of yeast extract and KH2PO4 2.53g/L，MgSO4·7H2O0.5 g/L, solvent is water, pH 7.5.
Compared with the prior art, the invention has the following beneficial effects: the invention provides a new strain, Enterobacter cloacae (Enterobacter cloacae) ZJPH1903, which can be used for catalyzing asymmetric reduction of 2,2',4' -trichloroacetophenone to prepare (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol, thereby providing beneficial reference in the aspect of preparing antifungal drug key chiral intermediate (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol by microbial transformation; the new strain is adopted to catalyze and prepare the optically pure (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol, and has the advantages of good stereoselectivity, high optical purity of the product and the like; according to the invention, wet thalli obtained by fermentation of Enterobacter cloacae (Enterobacter cloacae) ZJPH1903 is used as a chiral biocatalysis of a catalyst, and in a phosphate buffer solution system with pH 6.5, when the concentration of a substrate is 4g/L, the e.e. value of a target product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol is more than 99.9%, and the yield is 92.9%. When the surfactant is added into a reaction system, the catalytic efficiency can be effectively improved, the concentration of a catalytic substrate can be increased to 10g/L, the reaction lasts for 24 hours, the yield of the target product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol reaches 94.3 percent, and the e.e. value is more than 99.9 percent.
(IV) description of the drawings
FIG. 1 is a liquid phase detection map of (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol product standard, an (S) -2-chloro-1- (2, 4-dichlorophenyl) ethanol product standard, and a 2,2',4' -trichloroacetophenone substrate standard.
FIG. 2 is a liquid chromatogram of a transformation solution of Enterobacter cloacae ZJPH 1903.
FIG. 3 shows the colony morphology of Enterobacter cloacae ZJPH 1903.
FIG. 4 shows the cell morphology of Enterobacter cloacae ZJPH1903 under a light microscope (10X 100, oil mirror).
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
in the embodiment of the invention, the adding amounts of the wet thallus, the substrate, the auxiliary substrate and the surfactant are calculated by the volume of the reaction medium (distilled water or buffer solution).
Example 1 Strain screening, bioconversion and analytical testing of the products of transformation, Strain identification
1) Strain screening:
the strain source is as follows: enterobacter cloacae ZJPH1903 is obtained by separating and screening soil samples collected near Binjiang pharmaceutical factories in Hangzhou city, Zhejiang by the following specific screening method: weighing 1g of soil sample, adding into a 250ml conical flask filled with 100ml of sterile water, and oscillating at 30 ℃ and 200rpm for 0.5h to obtain a soil sample diluent. Adding 1ml of the diluent into a 250ml conical flask filled with 100ml of enrichment medium, culturing for 5 days at 30 ℃ and 200rpm, adding 1ml of the culture solution into a fresh enrichment medium after the culture solution becomes turbid, culturing for several days under the same condition, and repeating enrichment culture for 3-4 times. 2,2',4' -trichloroacetophenone is used as the only carbon source in the enrichment medium. The final concentration of the enrichment medium is as follows: 2,2',4' -trichloroacetophenone 2g/L, (NH)4)2SO4 2g/L，K2HPO4 2g/L，KH2PO4 1g/L，NaCl 1g/L，MgSO4·7H2O0.5 g/L, water as solvent, and pH 6.5.
Diluting the enriched culture solution to 10% with sterile water-5、10-6、10-70.2mL of the suspension was applied to a plate containing a screening medium, and cultured at 30 ℃ for 2 days, and colonies of different morphologies were selected from the colonies grown on the plate and separated by streaking the plate to obtain single colonies. The plate culture medium is composed of enrichment medium added with 20g/L agar.
Selecting single colony, inoculating to seed culture medium, culturing at 30 deg.C and 200rpm for 24 hr, inoculating to seed solution with volume concentration of 10%Culturing at 30 deg.C and 200rpm for 24 hr in fermentation medium, and centrifuging the fermentation liquid to obtain wet thallus. The seed culture medium and the fermentation culture medium have the same formula, and the formula is as follows: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH)4)2SO4 2g/L，KH2PO4 2g/L，NaCl 1g/L，MgSO4·7H2O0.5 g/L, water as solvent, and pH 6.5.
2) And (3) biotransformation process: 2,2',4' -trichloroacetophenone was used as a substrate, and microbial cells obtained by screening (i.e., wet cells after fermentation culture) were used as a catalyst and were subjected to bioconversion in a phosphate buffer solution of pH 6.5 at 30 ℃ for 24 hours. After the reaction is finished, extracting the conversion solution by using ethyl acetate with the same volume, centrifuging to obtain a supernatant, filtering an organic phase by using a 0.45 mu m microporous filter membrane, and determining the enantiomer excess value (e.e. value) of a target product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol in the conversion solution by using a high performance liquid chromatography so as to screen and obtain the microbial strain ZJPH1903 with high enantiomer excess value (e.e. value is more than 99.9%) and high yield.
3) Analyzing and detecting:
HPLC chromatographic conditions: adopting an Agilent1200 high performance liquid chromatograph; zheda N2000 chromatography workstation; a chromatographic column: polysaccharide derivative chiral chromatographic column CHIRALCEL OB-H (250 mm. times.4.6 mm); detection wavelength: 220 nm; mobile phase: n-hexane/isopropanol 97:3 (v/v); flow rate: 1 mL/min; column temperature: room temperature; sample introduction amount: 5 μ L. Retention time: (R) -configuration product 8.17min, substrate 13.84 min.
The calculated yield is:
Y(yield)＝CProduct of/CSubstrate×100％
In the formula, CProduct ofTo convert the mass concentration of the resulting product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol, CSubstrateThe mass concentration of the substrate 2,2',4' -trichloroacetophenone at the beginning of the reaction was used.
The optical purity of the product is expressed as enantiomeric excess (enantiomeric excess, e.e.):
e.e.＝(CR-CS)/(CR+CS)×100％
in the formula，CRIs the concentration of (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol, CSThe concentration of (S) -2-chloro-1- (2, 4-dichlorophenyl) ethanol.
4) Physiological and biochemical characteristics
Colony morphology: after culturing at 30 ℃ for 36h, large and wet slime-like colonies are formed, the surfaces are wet, smooth and the middle part is raised. The growth is white or milky white, and is opaque and sticky.
Physiological and biochemical characteristics: is gram-negative crude brevibacterium, has a whole body flagellum, has no spore, and is facultative anaerobic. Carbon sources that can be utilized are: d-cellobiose, β -galactosidase, β -N-acetylglucosaminidase, D-glucose, γ -glutamyltransferase, ferment/glucose, β -glucosidase, D-maltose, D-mannitol, D-mannose, β -xylosidase, gulose, tyrosine arylamine, D-sorbitol, sucrose, D-trehalose, citrate (sodium), malonate, L-lactate salinization, succinate basification, β -N-acetylgalactosaminidase, α -galactosidase, phosphatase, glycine arylamine, ornithine decarboxylase, O/129 drug resistance (comp. Carbon sources that cannot be utilized are: alanine-phenylalanine proline arylamine, adonitol, L-pyrrolidone arylamine, L-arabitol, H2S generation, glutamyl arylamine pNA, beta-alanine arylamine pNA, L-proline arylamine, lipase, urease, D-tagatose, 5-keto-D-gluconate, alpha-glucosidase, lysine decarboxylase, L-histidine assimilation, coumaric acid, beta-glucuronidase, glutamic acid-glycine-arginine arylamine, L-malate assimilation, ELLMAN and L-lactate assimilation.
5)16S rDNA sequence characteristics
Using cell total DNA extracted by an Ezup column type bacterial genome DNA extraction kit as a template, amplifying a 16S rDNA gene of a strain by using universal primers 27F (5 '-AGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'), wherein a PCR reaction system is 25 mu l, the PCR reaction system comprises 20-25 ng of genome DNA, 0.5 mu l of 10 xBuffer, 2.5 mu l of DNA polymerase, 0.2 mu l of forward and reverse primers, 0.5 mu l of forward and reverse primers, 1 mu l of dNTP (2.5 mM of each), and double distilled water is added to 25 mu l; and (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 4min, 30 cycles at 94 ℃ for 45s, 55 ℃ for 45s, and 72 ℃ for 1min, final extension at 72 ℃ for 10min, and termination at 4 ℃. After extracting genome DNA and amplifying 16S rDNA fragment by PCR, DNA fragment with 1468bp size is obtained. The 16S rDNA gene sequence of the strain obtained by purifying the PCR product and sequencing the PCR product by biological engineering (Shanghai) corporation is shown as SEQ ID NO. 1.
The 16S rDNA sequence of strain ZJPH1903 was subjected to homology alignment (BLAST) on the NCBI website (http:// www.ncbi.nlm.nih.gov), and the results showed that: the strain ZJPH1903 has higher sequence homology with partial strains of Enterobacter cloacae (Enterobacter cloacae), and has 99 percent of sequence homology with Enterobacter cloacae NIBSM-OsR 12 strain (GenBank accession No.: KY930712.1).
According to the physiological and biochemical characteristics and combined with molecular biological identification, the strain ZJPH1903 is identified as Enterobacter cloacae (Enterobacter cloacae) named as Enterobacter cloacae ZJPH1903, is preserved in China center for type culture Collection with the preservation date of 2019, 10 months and 14 days, and has the preservation number: CCTCC NO: m2019821, deposit address: china, wuhan university, zip code 430072.
Example 2: obtaining of Wet cells
1) Slant culture: selecting Enterobacter cloacae ZJPH1903 single colony, inoculating to slant culture medium, culturing at 30 deg.C for 36 hr, and storing in refrigerator at 4 deg.C; the final concentration composition of the slant culture medium is as follows: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH)4)2SO4 2g/L，KH2PO4 2g/L，NaCl 1g/L，MgSO4·7H20.5g/L of O, 20g/L of agar, water as a solvent and 6.5 of pH value.
2) Seed culture: selecting a ring thallus from a mature culture inclined plane, inoculating the ring thallus into a seed culture medium, and culturing at 30 ℃ and 200rpm for 12h to obtain a seed solution; the seed culture medium formula comprises: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH)4)2SO42g/L，KH2PO4 2g/L，NaCl 1g/L，MgSO4·7H2O0.5 g/L, water as solvent, and pH 6.5.
3) Fermentation culture: transferring the seed solution into a fermentation culture medium by an inoculation amount with the volume concentration of 8%, culturing at 30 ℃ and 200rpm for 16h to obtain a fermentation liquid, centrifuging the fermentation liquid, washing the obtained precipitate with 0.1M, pH 6.5.5 phosphoric acid buffer solution, and collecting wet bacteria, namely an enzyme source; the formula of the culture medium of the fermentation liquid is as follows: 30g/L of sucrose, 41.43g/L of yeast extract and KH2PO4 2.53g/L，MgSO4·7H2O 0.5g/L，pH 7.5。
Example 3:
the wet cells obtained in example 2 were suspended in 10mL of different reaction media (pH 7.0) at a concentration of 100g/L wet weight of the reaction media; 2g/L of 2,2',4' -trichloroacetophenone substrate is added, 100g/L of glucose is added as an auxiliary substrate, and the mixture is placed in a shaking table at 30 ℃ and 200rpm for reaction for 24 hours. After the reaction, the reaction solution was centrifuged, and the supernatant was collected, and the yield and optical purity of the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol were measured by the detection method of example 1, and as can be seen from table 1, when the buffer was phosphate buffer, the yield of (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol was the highest, 55.3%, and the e.e. value was greater than 99.9%.
TABLE 1 Effect of different reaction media on yield and e.e. value
Example 4:
the wet cells obtained in example 2 were suspended in 10mL of phosphate buffers having different pH values, and the concentration of the wet cells was 100g/L in terms of wet weight; 2g/L of 2,2',4' -trichloroacetophenone substrate is added, 100g/L of glucose is added as an auxiliary substrate, and the mixture is placed in a shaking table at 30 ℃ and 200rpm for reaction for 24 hours. After the reaction, the reaction solution was centrifuged, and the supernatant was collected, and the yield and optical purity of the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol were measured by the detection method of example 1, and as can be seen from table 2, when the pH of the buffer was 6.5, the yield of (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol was the highest, 67.5%, and the e.e. value was more than 99.9%.
Table 2 effect of different pH values of the buffer on yield and e.e. value
Example 5:
the wet cells obtained in example 2 were suspended in 10mL of a phosphate buffer (pH 6.5) at a concentration of 100g/L on a wet weight basis; 2g/L of 2,2',4' -trichloroacetophenone substrate was added, various cosubstrates were added (control with no addition of cosubstrates) and the mixture was reacted for 24 hours in a shaker at 30 ℃ and 200 rpm. After the reaction, the reaction solution was centrifuged, and the supernatant was collected, and the yield and optical purity of the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol were measured by the detection method of example 1, and as can be seen from table 3, when the co-substrate was glucose, the yield of (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol was the highest, 66.9%, and the e.e. value was more than 99.9%.
TABLE 3 Effect of different classes of cosubstrates on yield and e.e. value
Example 6:
the wet cells obtained in example 2 were suspended in 10mL of a phosphate buffer (pH 6.5) at a concentration of 100g/L on a wet weight basis; 2g/L of 2,2',4' -trichloroacetophenone substrate is added, glucose with different concentrations is added as an auxiliary substrate, and the mixture is placed in a shaking table with the temperature of 30 ℃ and the rpm of 200 for reaction for 24 hours. After the reaction, the reaction solution was centrifuged, and the supernatant was collected, and the yield and optical purity of the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol were measured by the detection method of example 1, and as can be seen from table 4, when the glucose concentration was 150g/L, the yield of (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol was the highest, the yield was 72.1%, and the e.e. value was more than 99.9%.
TABLE 4 yield and ee value of the co-substrate glucose at different concentrations
Example 7:
the wet cells obtained in example 2 were suspended in 10mL of a phosphate buffer (pH 6.5) at a concentration of 100g/L on a wet weight basis; 2g/L of 2,2',4' -trichloroacetophenone substrate is added, 150g/L of glucose is added as an auxiliary substrate, and the mixture is placed in a shaking table with 200rpm at different temperatures for reaction for 24 hours. After the reaction, the reaction solution was centrifuged, and the supernatant was collected, and the yield and optical purity of the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol were measured by the detection method of example 1, and as can be seen from table 5, when the reaction temperature was 30 ℃, the yield of (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol was the highest, the yield was 72.6%, and the e.e. value was more than 99.9%.
TABLE 5 yield and e.e. value at different temperatures
Example 8:
the wet cells obtained in example 2 were suspended in 10mL of phosphate buffer (pH 6.5), added with wet cells of different concentrations, 2g/L of 2,2',4' -trichloroacetophenone substrate and 150g/L of glucose as an auxiliary substrate, and reacted for 24 hours in a shaker at 30 ℃ and 200 rpm. After the reaction, the reaction solution was centrifuged, and the supernatant was collected, and the yield and optical purity of the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol were measured by the detection method of example 1, and as can be seen from Table 6, when the wet cell concentration was 200g/L, the yield of (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol was the highest, 93.7%, and the e.e. value was more than 99.9%.
TABLE 6 influence of wet cell concentration on yield and product e.e. value
Example 9:
the wet cells obtained in example 2 were suspended in 10mL of a phosphate buffer (pH 6.5) at a concentration of 200g/L on a wet weight basis; adding 2,2',4' -trichloroacetophenone substrates with different concentrations, adding 150g/L glucose as an auxiliary substrate, and reacting in a shaker at 30 ℃ and 200rpm for 24 h. After the reaction, the reaction solution was centrifuged, and the supernatant was collected, and the yield and optical purity of the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol were measured by the detection method of example 1, and as can be seen from table 7, when the substrate concentration was 4g/L, the yield of (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol was 92.9%, and the e.e. value was more than 99.9%.
TABLE 7 Effect of different substrate concentrations on yield and product e.e. value
Example 10:
the wet cells obtained in example 2 were suspended in 10mL of a phosphate buffer (pH 6.5) at a concentration of 200g/L on a wet weight basis; adding 5 g/L2, 2',4' -trichloroacetophenone substrate, adding 150g/L glucose as auxiliary substrate, adding 6g/L surfactant, and reacting in a shaker at 30 deg.C and 200rpm for 2 h. After the reaction, the reaction solution was centrifuged, and the supernatant was collected, and the yield and optical purity of the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol were measured by the detection method of example 1, and as can be seen from table 8, when the surfactant was tween-80, the yield of (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol was the highest, the yield was 75.5%, and the e.e. value was more than 99.9%.
Table 8 effect of different surfactants on yield and product e.e. value
Example 11:
the wet cells obtained in example 2 were suspended in 10mL of a phosphate buffer (pH 6.5) at a concentration of 200g/L on a wet weight basis; adding 5 g/L2, 2',4' -trichloroacetophenone substrate, adding 150g/L glucose as auxiliary substrate and 6g/L Tween-80, and reacting in a shaker at 30 deg.C and 200rpm for 24 h. After the reaction, the reaction solution was centrifuged, and the supernatant was collected, and the yield and optical purity of the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol were measured by the detection method of example 1, wherein the yield of (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol was 95.5% and the e.e. value was more than 99.9%.
Example 12:
the wet cells obtained in example 2 were suspended in 10mL of a phosphate buffer (pH 6.5) at a concentration of 200g/L on a wet weight basis; adding 10 g/L2, 2',4' -trichloroacetophenone substrate, adding 150g/L glucose as auxiliary substrate and 6g/L Tween-80, and reacting in a shaker at 30 deg.C and 200rpm for 24 h. After the reaction, the reaction solution was centrifuged, and the supernatant was collected, and the yield and optical purity of the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol were measured by the detection method of example 1, wherein the yield of (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol was 94.3%, and the e.e. value was more than 99.9%.
Example 13:
the wet cells obtained in example 2 were suspended in 10mL of a phosphate buffer (pH 6.5) at a concentration of 200g/L on a wet weight basis; adding 15 g/L2, 2',4' -trichloroacetophenone substrate, adding 150g/L glucose as auxiliary substrate and 6g/L Tween-80, and reacting in a shaker at 30 deg.C and 200rpm for 24 h. After the reaction, the reaction solution was centrifuged, and the supernatant was collected, and the yield and optical purity of the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol were measured by the detection method of example 1, wherein the yield of (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol was 68.3% and the e.e. value was more than 99.9%.
Example 14:
selecting Pseudomonas aeruginosa (Pseudomonas aeruginosa) ZJPH1504, storing in China center for type culture Collection, with the storage number: CCTCC NO: m2016188, deposit date: 2016, 4 months and 11 days, wherein the preservation address is Wuhan, Wuhan university, Zip code 430072. This strain has been disclosed in a prior patent application (publication No. CN 105925506A, published: 2016, 9/7). The culture method of the strain and the enzyme-derived cell preparation process were carried out in accordance with the prior patent application (publication No. CN 105925506A, published: 2016, 9/7/2016).
The wet cells thus obtained were suspended in 10mL of a phosphate buffer (pH 6.5) at a concentration of 200g/L on a wet basis; adding 4 g/L2, 2',4' -trichloroacetophenone substrate, adding 150g/L glucose as auxiliary substrate, and reacting in a shaker at 30 deg.C and 200rpm for 24 h. After the reaction is finished, the reaction solution is centrifuged, the supernatant is taken, the yield and the optical purity of the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol are determined by adopting the detection method of example 1, and the experimental result shows that the Pseudomonas aeruginosa (Pseudomonas aeruginosa) ZJPH1504 can not convert 2,2',4' -trichloroacetophenone to prepare (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol.
Example 15:
rhodococcus erythropolis XS1012 was selected and deposited in China center for type culture Collection with the following addresses: china, wuhan university, accession number: CCTCC NO: m2013650, preservation date of 2013, 12 months and 11 days. This species has been disclosed in a prior patent application (publication No. CN103773724A, published: 5/7/2014). The culture method of the strain and the enzyme-derived cell preparation process were in accordance with the previous patent application (publication No. CN103773724A, published: 5/7/2014).
The wet cells thus obtained were suspended in 10mL of a phosphate buffer (pH 6.5) at a concentration of 200g/L on a wet basis; adding 4 g/L2, 2',4' -trichloroacetophenone substrate, adding 150g/L glucose as auxiliary substrate, and reacting in a shaker at 30 deg.C and 200rpm for 24 h. After the reaction, the reaction solution was centrifuged, the supernatant was collected, and the yield and optical purity of the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol were determined by the detection method of example 1, and the experimental results showed that Rhodococcus erythropolis XS1012 could not convert 2,2',4' -trichloroacetophenone to prepare (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol.
Sequence listing
<110> Zhejiang industrial university
<120> Enterobacter cloacae ZJPH1903 and application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1468
<212> DNA
<213> Enterobacter cloacae
<400> 1
tcagattgaa cgctggcggc aggcctaaca catgcaagtc gaacggtagc acagagagct 60
tgctctcggg tgacgagtgg cggacgggtg agtaatgtct gggaaactgc ctgatggagg 120
gggataacta ctggaaacgg tagctaatac cgcataacgt cgcaagacca aagaggggga 180
ccttcgggcc tcttgccatc agatgtgccc agatgggatt agctagtagg tggggtaacg 240
gctcacctag gcgacgatcc ctagctggtc tgagaggatg accagccaca ctggaactga 300
gacacggtcc agactcctac gggaggcagc agtggggaat attgcacaat gggcgcaagc 360
ctgatgcagc catgccgcgt gtatgaagaa ggccttcggg ttgtaaagta ctttcagcgg 420
ggaggaaggt gttgtggtta ataaccgcag caattgacgt tacccgcaga agaagcaccg 480
gctaactccg tgccagcagc cgcggtaata cggagggtgc aagcgttaat cggaattact 540
gggcgtaaag cgcacgcagg cggtctgtca agtcggatgt gaaatccccg ggctcaacct 600
gggaactgca ttcgaaactg gcaggctaga gtcttgtaga ggggggtaga attccaggtg 660
tagcggtgaa atgcgtagag atctggagga ataccggtgg cgaaggcggc cccctggaca 720
aagactgacg ctcaggtgcg aaagcgtggg gagcaaacag gattagatac cctggtagtc 780
cacgccgtaa acgatgtcga tttggaggtt gtgcccttga ggcgtggctt ccggagctaa 840
cgcgttaaat cgaccgcctg gggagtacgg ccgcaaggtt aaaactcaaa tgaattgacg 900
ggggcccgca caagcggtgg agcatgtggt ttaattcgat gcaacgcgaa gaaccttacc 960
tggtcttgac atccacagaa ctttccagag atggattggt gccttcggga actgtgagac 1020
aggtgctgca tggctgtcgt cagctcgtgt tgtgaaatgt tgggttaagt cccgcaacga 1080
gcgcaaccct tatcctttgt tgccagcggt taggccggga actcaaagga gactgccagt 1140
gataaactgg aggaaggtgg ggatgacgtc aagtcatcat ggcccttacg accagggcta 1200
cacacgtgct acaatggcgc atacaaagag aagcgacctc gcgagagcaa gcggacctca 1260
taaagtgcgt cgtagtccgg attggagtct gcaactcgac tccatgaagt cggaatcgct 1320
agtaatcgta gatcagaatg ctacggtgaa tacgttcccg ggccttgtac acaccgcccg 1380
tcacaccatg ggagtgggtt gcaaaagaag taggtagctt aaccttcggg agggcgctta 1440
ccactttgtg attcatgact ggggtgaa 1468
Claims (10)
1. Enterobacter cloacae: (Enterobacter cloacae) ZJPH1903, preserved in China center for type culture Collection, with a preservation date of 2019, 10 months and 14 days, and a preservation number: CCTCC NO: m2019821, deposit address: china, wuhan university, zip code 430072.
2. Use of enterobacter cloacae ZJPH1903 of claim 1 for catalyzing the asymmetric reduction of 2,2',4' -trichloroacetophenone for the preparation of (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol.
3. The use according to claim 2, characterized in that said use is: taking 2,2',4' -trichloroacetophenone as a substrate, taking wet thalli obtained by fermentation culture of enterobacter cloacae ZJPH1903 as an enzyme source, taking distilled water or phosphate buffer solution with the pH of 6.0-8.0 as a reaction medium to form a conversion system, carrying out reaction at 30 ℃ and 200rpm, centrifuging the reaction solution after the reaction is finished, and taking supernatant to obtain the conversion solution containing the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol.
4. The use according to claim 3, wherein the substrate is initially added in an amount of 2 to 6g/L based on the volume of the reaction medium, and the wet biomass is used in an amount of 50 to 300g/L based on the wet weight of the biomass.
5. Use according to claim 3, wherein a co-substrate is added to the conversion system, wherein the co-substrate is one of the following: glucose, maltose, sucrose, lactose, glycerol, methanol, ethanol, isopropanol, alanine, methionine.
6. The use according to claim 5, wherein the cosubstrate is added in an amount of 50 to 300g/L based on the volume of the reaction medium.
7. Use according to claim 5, characterized in that the conversion system consists of an enzyme source, a substrate, a co-substrate and a surfactant, the surfactant being tween-80, tween-20 or sodium dodecyl sulphate.
8. The use according to claim 7, wherein the surfactant is added in an amount of 2 to 20g/L based on the volume of the reaction medium.
9. The use according to claim 7, characterized in that the use of Enterobacter cloacae ZJPH1903 for the preparation of (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol is carried out as follows: taking 2,2',4' -trichloroacetophenone as a substrate, glucose as an auxiliary substrate, tween-80 as a surfactant, wet thalli obtained by fermentation culture of enterobacter cloacae ZJPH1903 as an enzyme source, forming a conversion system in a phosphate buffer solution with the pH of 6.5, reacting for 24 hours at the temperature of 30 ℃ and the rpm of 200, and obtaining a conversion solution containing a target product after the reaction is finished; the initial adding amount of the substrate is 5-15 g/L buffer solution by volume of the buffer solution, the adding amount of the auxiliary substrate is 150g/L buffer solution by volume of the buffer solution, the using amount of wet thalli is 200g/L buffer solution by wet weight of the thalli, and the adding amount of the surfactant is 6g/L by volume of the buffer solution.
10. Use according to claim 3, characterized in that the enzyme source is prepared by a process comprising:
(1) slant culture: inoculating Enterobacter cloacae ZJPH1903 to a slant culture medium, and culturing at 25-30 ℃ for 36h to obtain a slant strain; the final concentration composition of the slant culture medium is as follows: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH)4)2SO4 2g/L，KH2PO4 2g/L，NaCl 1g/L，MgSO4·7H20.5g/L of O, 15-20 g/L of agar powder, water as a solvent and 6.5 of pH value;
(2) seed culture: inoculating slant strains into a seed culture medium, and culturing at 25-30 ℃ and 150-250 rpm for 10-24 hours to obtain a seed solution; the final concentration composition of the seed culture medium is as follows: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH)4)2SO4 2g/L，KH2PO4 2g/L，NaCl 1g/L，MgSO4·7H2O is 0.5g/L, the solvent is water, and the pH value is 6.5;
(3) fermentation culture: inoculating the seed solution into a fermentation culture medium in an inoculation amount with the volume concentration of 4-10%, shaking the flask with the liquid loading amount of 80mL/250mL, culturing at 25-30 ℃ and 150-250 rpm for 12-32 h, centrifuging the fermentation liquid after the fermentation is finished, washing the obtained precipitate with 0.1M, pH 6.5.5 phosphoric acid buffer solution, and collecting wet thalli, namely an enzyme source; the final concentration of the fermentation medium is as follows: 30g/L of sucrose, 41.43g/L of yeast extract and KH2PO4 2.53g/L，MgSO4·7H2O0.5 g/L, solvent is water, pH 7.5.
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