CN103074239A - Candida sorboxylosa and application thereof in preparation of (S)-4-chloro-3-hydroxybutanoate - Google Patents
Candida sorboxylosa and application thereof in preparation of (S)-4-chloro-3-hydroxybutanoate Download PDFInfo
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Abstract
The invention discloses a Candida sorboxylosa and the application of the Candida sorboxylosa in preparation of (S)-4-chloro-3-hydroxybutanoate (CHBE). The Candida sorboxylosa ZJB-12164 is preserved in China Center for Type Culture Collection, the preservation address is Wuhan University in Wuhan, China, the postcode is 430072, the preservation number is CTCC No: M2012420, and the preservation date is Oct. 22, 2012. The invention provides a new strain of Candida sorboxylosa ZJB-12164 and the application of the new strain in bioconversion of (S)-CHBE, the synthetic reaction condition is in mild condition, is environmental-friendly, has a simple process, and is high in conversion rate and product optical purity (e.e.>99%).
Description
(1) technical field
The present invention relates to a kind of new bacterial strain---sorb wood sugar candiyeast ZJB-12164(Candida sorboxylosa ZJB-12164), and the application in synthetic (the S)-4-chloro-3-hydroxyl ethyl butyrate of biocatalysis 4-chloroacetyl acetacetic ester.
(2) background technology
(S)-(Ethyl (S)-4-chloro-3-hydroxybutanoate (S)-CHBE) is a kind of colourless transparent oil liquid to 4-chloro-3-hydroxyl ethyl butyrate, and molecular formula is C
6H
11ClO
3, molecular weight 166.6, relative density 1.19g/mL, boiling point 93-95 ℃/5mmHg, (S)-the CHBE structure is shown in formula I.(S)-CHBE is as important chirality pharmaceutical intermediate compound, can be used for synthetic statins-hydroxymethyl glutaryl CoA (HMG-CoA) reductase inhibitor, its preparation Lipitor (atorvastatincalcuim) is that the U.S.'s first year sales volume surpasses 10,000,000,000 dollars medicine, also is at present the medicine of high sales volume of the whole world.In addition, (S)-CHBE can also transform generation Isosorbide-5-Nitrae-dihydropyridines beta-Blocking agent.Therefore, preparation optical activity (S)-CHBE enjoys investigator's concern.
(S)-preparation method of CHBE can be divided into racemic modification Split Method and asymmetry catalysis synthesis method.Because the method preparation efficiency that racemic modification splits is not high, theoretical yield only is 50%, and the separation and purification of product difficulty, therefore, has little significance for chiral separation preparation (the S)-CHBE of racemic modification.Compare with Split Method, adopt asymmetric synthesis (S)-CHBE to receive more concern.With chiral substrates 4-chloroacetyl acetacetic ester (the Ethyl 4-chloroacetoacetate that dives, COBE) be raw material, it is excessive to make its reaction generate (S)-CHBE under certain condition, even is single enantiomorph all, thereby avoids and reduce split process.Because substrate COBE low price is easy to synthesize, so dissymmetric synthesis is most economical effectively synthetic (S)-CHBE method.
Preparation (S)-CHBE can be divided into chemical catalysis and biological catalysis two classes take COBE as the raw material asymmetric synthesis.Chemical catalysis is the ruthenium compound that adopts chirality (RuX for example
2[(S)-BINAP]) as the reduction of catalyzer asymmetric hydrogenation, the e.e. value of product can reach more than 97%, but this method needs high-pressure hydrogenation, and reactor is had relatively high expectations, and catalyst system therefor is expensive, so production cost is higher; And the biological catalyst that adopts both can be the enzyme of purifying, and also can be microorganism cells.Because biological reducing needs expensive coenzyme to participate in, therefore, usually adopt to possess the microbe whole-cell of regenerating coenzyme system as catalyzer.Microbial method carbonyl asymmetric reduction preparation (S)-CHBE has the reaction conditions gentleness, and product is single, and the stereoselectivity advantages of higher is representing the developing direction of Green Chemistry.
(3) summary of the invention
The object of the invention provides a kind of new bacterial strain-sorb wood sugar candiyeast ZJB-12164(Candida sorboxylosa ZJB-12164), and the application in carbonyl asymmetric reduction preparation (S)-CHBE, this strain stability is good, and stereoselectivity is strict, and the product optical purity is high.
The technical solution used in the present invention is:
The present invention relates to a kind of sorb wood sugar candiyeast ZJB-12164(Candida sorboxylosa ZJB-12164), be preserved in Chinese Typical Representative culture collection center, preservation address: China, Wuhan, Wuhan University, postcode 430072, deposit number CCTCC No:M 2012420, preservation date on October 22nd, 2012.
Sorb wood sugar candiyeast ZJB-12164 of the present invention obtains by following program screening:
(1) will come from more than the 200 parts of pedotheques in ground such as Zhejiang, Sichuan, Yunnan and Jiangsu joins in the physiological saline, make soil supension and be inoculated in the enrichment medium, at 30 ℃, cultivate on the shaking table of 150r/min until after the nutrient solution change muddiness, by 2%(v/v) inoculum size is transferred in the new enrichment medium and continues to cultivate, and culture condition is the same.The enrichment culture based formulas is: glucose 50g/L, and soybean sprout 100g/L, COBE 12g/L, solvent are water, natural pH.
(2) for the second time enrichment culture liquid is applied on the plate culture medium after dilution, 30 ℃ of incubator constant temperature culture 48 hours, and picking list bacterium colony is forwarded to preservation behind the slant culture, and the slant culture condition is 30 ℃ of incubator constant temperature culture 48 hours.Plate culture medium and slant culture based formulas are: glucose 50g/L, and soybean sprout 100g/L, agar 20g/L, solvent are water, natural pH.
(3) be forwarded to the shaking flask yeast culture base from the inclined-plane, cultivated 24 hours for 30 ℃, collect thalline, with 0.85% physiological saline washing 2 times, centrifugal collection thalline, gained thalline and substrate COBE 100 μ L are scattered in (0.1mol/L, pH 7.0) in the 10mL potassium phosphate buffer, add the glucose of 0.2g as cosubstrate, at 30 ℃, reaction is 24 hours under the 150r/min water-bath, after reaction finishes, conversion fluid is removed somatic cells through centrifugation, and supernatant liquor is by ethyl acetate extraction, anhydrous Na
2SO
4Dehydration, filtration, the employing gas chromatographic detection is analyzed, substrate COBE can be transformed the bacterial strain that generates (S)-CHBE is the purpose bacterial strain, finally therefrom selecting the highest bacterial strain of a strain stereoselectivity (be numbered ZJB-12164, namely CCTCC No:M 2012420) does follow-up strain identification and conversion.Described yeast culture based formulas is: glucose 50g/L, and soybean sprout 100g/L, solvent are water, pH 7.0.
The evaluation of sorb wood sugar candiyeast ZJB-12164 bacterial strain of the present invention:
(1) strain morphology feature and physiological and biochemical property:
Morphological specificity: cultivated 24 hours at bean sprout juice nutrient agar flat board in 30 ℃, bacterium colony is oval, and is smooth moistening, and neat in edge has wine flavour, and oyster white, size are about 2 ~ 5 * 4 ~ 10 μ m.
Physio-biochemical characteristics: utilize Biolog(GEN III) the automatic microbe identification systems are measured bacterial strain ZJB-12164 to the metabolism situation of 65 kinds of carbon sources, analyze Metabolic Fingerprinting through the Biolog readout instrument, bacterial strain ZJB-12164 can utilize more by force 4 kinds of carbon sources, maybe can not utilize a little less than other 61 kinds of utilization of carbon source abilities, as shown in Table 1 below.
(2) bacterial strain 18S rDNA sequencing and analysis:
Take the total DNA of the strain cell that extracts as template, utilize 18S rDNA and the order-checking of the primer amplification bacterial strain of design, this fragment physical length is 1470bp, carrying out similarity analysis with the GenBank related data finds, this bacterium and Candida sorboxylosa(AB054672.1) the highest (homology of homology, 99%/1470bps, based on 18S rDNA), therefore identify in conjunction with Physiology and biochemistry according to molecular biology identification, can determine that this bacterial strain is Candida sorboxylosa, called after sorb wood sugar candiyeast ZJB-12164(Candida sorboxylosa ZJB-12164).Sequence is shown in SEQ ID NO.1:
1 cctgcatgtc caagttagaa actgcgaatg gctcattaga tcagttatca tctccttgac
61 tcattacatg gataaccgtg gaaaatccag agctaataca cgccgtgcac atattaggtt
121 ccgactctga gtatttagcg aaccgtacgc tgggtcattc gagcatctgc cctatcaact
181 agacggtagg atctttgcct acggtggtgg tgacgggtaa cggggaataa gggttcggtt
241 ccggagaggg agcctgagag acggctacca catccaagga aggcagcagg cgcgcaaatt
301 gcacactggg aacgccccga tgcactagca ggcgtaccga tgcgtttacg caatcggata
361 aacacgcgta caggcccgaa tgtagcagtg gagggcaagt ctggtgccag cagccgcggt
421 aactccagct ccactagcgt atgttaaagt tgtagcagtt aaaacgctcg tagcgggggg
481 agcgcttata gctattactt tgagaaaatt agagtgttca aagcaggcct tgtgctcgga
541 tacgttagca tggaataatg gaacacgacg gcgtccatgg acgttgtaat gaccaagagg
601 ggcggaagag gcggtgcgta ttgggcggct aggggtgaaa tccgacgacc cgcccacgac
661 gagcgagtgc gaaggcacgc tgcagagacg tgcccgttcg tcaagaacga aagctaggga
721 atcgaaaatg atcagatacc attgtagtct tagccgtaaa cgatgtggag acacggggtg
781 cgattgtgct tcgtgggggg aaacttagtt cgttctgggg ggagtatgct cacaagggtg
841 aaacttaaag aaattggcgg aagactacct taagcaatgg gactgcggct taatttgact
901 caacacgggg aaactcacca ggtccagacg taataaggat tgaccagctg gagacttttc
961 ttgatcttac gggtggtggt gcatggccgt ttttcagtcc ttggagtgat ttgtctgctt
1021 aattgcgata acggacgaga ccttggcctc taactaggag agcgtatttg tacgcggact
1081 gcttagaggg acgacagacg ctaagtttgt ggaggcgcga ggcaacaaca ggtctgtgat
1141 gcccttagac gttctgggcc gcacgcgcgc tacactgacg gggggagcga gggccgggct
1201 gagaagccag ggcaatcagg aaaccgcgtc gtgctgggaa tagcggattg gaattgtttc
1261 ccttgaacgt ggaattgctt gtaagcgcag gtcatcagcc tgcgttgaat acgacccttg
1321 tctttgtaca caccgcccgt tgctactacc gattgaatgg cttagtgaga ggtcgggagg
1381 ccggcggagg gaactccgcg gggcgaactt gttctaactt ggctatttag agctcgtaaa
1441 agtcgtaaca aggtttccgt aggtgaacct
The invention still further relates to the application of described sorb wood sugar candiyeast ZJB-12164 in biocatalysis preparation (S)-4-chloro-3-hydroxyl ethyl butyrate, described being applied as: contain the enzyme wet thallus as catalyzer take what sorb wood sugar candiyeast ZJB-12164 fermentation culture obtained, take the 4-chloroacetyl acetacetic ester as substrate, in the transformation system a of pH4.0 ~ 9.0 water or buffered soln formation, under 20 ~ 45 ℃, carry out conversion reaction, after reacting completely, with the conversion fluid aftertreatment, obtain described (S)-4-chloro-3-hydroxyl ethyl butyrate.
The chemical equation of the application of described sorb wood sugar candiyeast ZJB-12164 in biocatalysis preparation (S)-4-chloro-3-hydroxyl ethyl butyrate is:
Further, initial substrate concentration is 10 ~ 2000mmol/L among the described transformation system a, preferred 50 ~ 1500 mmol/L.
Further, contain enzyme wet thallus addition among the transformation system a and count 10 ~ 200g/L with the wet thallus quality, preferred 50 ~ 100 g/L describedly contain the enzyme wet thallus to contain quality are 70 ~ 90%.
Further, realize the reprocessing cycle of coenzyme, also comprise cosubstrate among the described transformation system a, water or damping fluid by cosubstrate, substrate, catalyzer and pH4.0 ~ 9.0 consist of transformation system b, described cosubstrate is one of following: methyl alcohol, ethanol, n-propyl alcohol, Virahol, glucose, fructose, sucrose, maltose, lactose or glycerine, preferred glucose or fructose.
Further, described sorb wood sugar candiyeast ZJB-12164 being applied as in biocatalysis preparation (S)-4-chloro-3-hydroxyl ethyl butyrate: contain the enzyme wet thallus as catalyzer take what sorb wood sugar candiyeast ZJB-12164 fermentation culture obtained, take the 4-chloroacetyl acetacetic ester as substrate, add cosubstrate, in the transformation system b of pH4.0 ~ 9.0 water or buffered soln formation, under 20 ~ 45 ℃, carry out conversion reaction, after reacting completely, with the conversion fluid aftertreatment, obtain described (S)-4-chloro-3-hydroxyl ethyl butyrate; Initial substrate concentration is preferred 50 ~ 1500 mmol/L of 10 ~ 2000mmol/L(among the described transformation system b), contain enzyme wet thallus addition and count preferred 50 ~ 100 g/L of 10 ~ 200g/L(with the wet thallus quality), it is described that to contain the wet bacterium water content of enzyme be 70 ~ 90%, the cosubstrate starting point concentration is 10 ~ 2000mmol/L, described cosubstrate is one of following: methyl alcohol, ethanol, n-propyl alcohol, Virahol, glucose, fructose, sucrose, maltose, lactose or glycerine are preferably glucose or fructose.
Further, described conversion reaction is at 20 ~ 45 ℃ of lower reaction 0.1 ~ 36h, preferred 30 ~ 35 ℃ of reaction 1 ~ 12h.
Further, the method for described conversion fluid aftertreatment is: after reaction finishes that conversion fluid is centrifugal, remove somatic cells, and get supernatant liquor equal-volume ethyl acetate extraction, get the organic layer anhydrous Na
2SO
4Dehydration, filtration, filtrate being contains the crude product of (S)-4-chloro-3-hydroxyl ethyl butyrate,, namely gets (S)-4-chloro-3-hydroxyl ethyl butyrate with the crude product separation and purification.Described crude product separation and purification adopts technology well known in the art to carry out, and is generally rotary evaporation and underpressure distillation etc.
Further, described transformation system a or transformation system b all are the pH4.0 that formed by distilled water, tap water, phosphate buffered saline buffer or citrate buffer ~ 9.0 single_phase systems, perhaps the pH4.0 that forms of water and organic medium ~ 9.0 diphasic systems; Described organic medium is normal hexane, octane, octane-iso, normal heptane, dimethylbenzene, hexanaphthene, ethyl acetate or butylacetate, is preferably butylacetate.
Further, the described enzyme wet thallus that contains prepares as follows:
(1) slant culture: sorb wood sugar candiyeast ZJB-12164 inoculation in slant medium, in 20 ~ 37 ℃ of lower cultivations 12 ~ 48 hours, is obtained the slant activation bacterial classification; Described slant medium is the bean sprout juice nutrient agar, and collocation method is: the fresh soybean sprout of 50 ~ 200g is put into beaker, adds suitable quantity of water, boils half hour, and filtered through gauze gets supernatant, adds 10 ~ 50g glucose again, and 15 ~ 20g agar is mended and added water to 1L;
(2) seed culture: the slant activation bacterial classification inoculation in seed culture medium, is cultivated under 25 ~ 35 ℃, shaking table revolution 100 ~ 200r/min condition and obtained seed liquor in 12 ~ 36 hours; Described seed culture based formulas is: glucose 5 ~ 50g/L, yeast extract paste 5 ~ 40g/L, KH
2PO
40.5 ~ 5g/L, NaCl 0.5 ~ 5g/L, solvent are water, and initial pH is 5.0 ~ 8.0;
(3) fermentation culture: seed liquor according to volume ratio 1 ~ 5% access fermention medium, was cultivated 12 ~ 48 hours under 25 ~ 35 ℃, shaking table revolution 100 ~ 200r/min condition, with medium centrifugal, washing, collect and contain the enzyme wet thallus, refrigerate for subsequent use; Described fermentative medium formula is: glucose 5 ~ 50g/L, yeast extract powder 5 ~ 100g/L, KH
2PO
40.5 ~ 5g/L, NaCl 0.5 ~ 5g/L, CuCl
20.01 ~ 0.1g/L, solvent are water, initial pH is 5.0 ~ 8.0.
Product among the present invention (S)-CHBE molar yield and optical purity (enantiomeric excess value (e.e.%)) adopt gas Chromatographic Determination, and method is as follows:
After reaction finished, conversion fluid was removed somatic cells through centrifugation, supernatant liquor equal-volume ethyl acetate extraction, organic layer anhydrous Na
2SO
4Analyze with Shimadzu GC-14 gas chromatograph after dehydration, the filtration.Achirality GC analysis condition: HP-5 low-pole chromatographic column (30m * 0.32mm * 0.25 μ m), 120 ℃ of column temperatures keep 2.5min, with 50 ℃/min temperature programming to 165 ℃, keep 1.2min.Carrier gas is nitrogen, flow 1.0mL/min, and sample size 1 μ L, splitting ratio 50:1, detector temperature are 250 ℃, injector temperature is 230 ℃; The achirality chromatographic column can detection substrate COBE and the content of product C HBE, further calculates the molar yield of reaction.Chirality GC analysis condition: BGB-174 chiral capillary gas chromatography post (30m * 0.25mm * 0.25 μ m), 110 ℃ of column temperatures, with 0.5 ℃/min temperature programming to 125 ℃, carrier gas is helium, flow is 1.0mL/min, sample size 1 μ L, splitting ratio 40:1 detects and injector temperature is 220 ℃; Chiral chromatographic column can detect the content of (S)-CHBE and (R)-CHBE, further calculate product optical purity (enantiomeric excess value, e.e.%).
Transformation system a of the present invention and transformation system b are transformation system, name for ease of distinguishing transformation system Constitution Elements difference in the different step, and letter itself does not have implication.
Beneficial effect of the present invention is mainly reflected in: the invention provides the application in synthetic (the S)-CHBE of bio-transformation of a kind of new bacterial strain of Candida sorboxylosa ZJB-12164 and this Candida sorboxylosa ZJB-12164 bacterial strain, this synthetic reaction condition is gentle, environmental friendliness, flow process is simple, transformation efficiency is high, product optical purity high (e.e.＞99%).
(4) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1: the screening of microorganism, evaluation
(1) screening of microorganism
Bacterial classification enrichment culture based formulas: glucose 50g/L, soybean sprout 100g/L, COBE 12g/L, solvent are water, natural pH.
Plate culture medium and slant culture based formulas are: glucose 50g/L, and soybean sprout 100g/L, agar 20g/L, solvent are water, natural pH.
The enzymatic production culture medium prescription: glucose 50g/L, soybean sprout 100g/L, solvent are water, pH 7.0.
From gathering soil sample from the vegetable garden on the ground such as Zhejiang, Sichuan, Yunnan and Jiangsu, orchard vegetation bed, more than totally 200 parts in order to bacterial screening.Detailed process is as follows:
Getting a little soil sample joins in the physiological saline, make soil supension, 150r/min in the bacterial classification enrichment medium, on 30 ℃ the shaking table through twice enrichment culture, through dilution spread, choose single bacterium colony and be forwarded to the test tube slant and be numbered in 30 ℃ of biochemical cultivation cases and cultivated 48 hours.Be forwarded to the shake flask fermentation culture medium from the inclined-plane again, cultivated 24 hours for 30 ℃, collect thalline, with 0.85% physiological saline washing 2 times, centrifugal collection thalline, gained thalline and substrate COBE 100 μ L are scattered in (0.1mol/L, pH 7.0) in the 10mL potassium phosphate buffer, add the glucose of 0.2g as cosubstrate, at 30 ℃, reaction is 24 hours under the 150r/min water-bath, after reaction finishes, conversion fluid is removed somatic cells through centrifugation, and supernatant liquor is by ethyl acetate extraction, anhydrous Na
2SO
4Dehydration, filtration, the employing gas chromatographic detection is analyzed, substrate COBE can be transformed the bacterial strain that generates (S)-CHBE is the purpose bacterial strain, finally therefrom selecting the highest bacterial strain of a strain stereoselectivity (be numbered ZJB-12164, namely CCTCC No:M 2012420) does follow-up strain identification and conversion.
(2) microorganism strains is numbered the Physiology and biochemistry evaluation of " ZJB-12164 " bacterial strain
The strain morphology feature: cultivated 24 hours at bean sprout juice nutrient agar flat board in 30 ℃, bacterium colony is oval, and is smooth moistening, and neat in edge has wine flavour, and oyster white, size are about 2 ~ 5 * 4 ~ 10 μ m.
Physio-biochemical characteristics: utilize Biolog automatic microbe identification systems to investigate bacterial strain ZJB-12164 to the metabolism situation of 65 kinds of different carbon sources: with inoculation in plate culture medium, 28 ℃ of constant temperature culture 2 days, with aseptic cotton carrier the thalline on the flat board is washed, mix with inoculation liquid (sterilized water), make bacteria suspension, be adjusted to 47%T with turbidometer.Bacteria suspension is added in respectively in each hole of Biolog YT micropore identification plate every hole 100 μ L with the electronic liquid fillers in 8 holes.The micropore identification plate is placed in 28 ℃ of incubators, after cultivating 24h, 48h and 72h, is placed on reading result on the Biolog readout instrument respectively.Analyze Metabolic Fingerprinting through the Biolog readout instrument, bacterial strain can utilize more by force 4 kinds of carbon sources, can not utilize or utilize ability to other 61 kinds of carbon sources a little less than, as shown in Table 1 below.
Table 1 bacterial strain ZJB-12164 is to the ability of utilizing of 65 kinds of carbon sources on the Biolog GEN III plate
Annotate :+, the positive; , feminine gender; B, the boundary line
(3) 18S rDNA complete sequence determination and analysis
Take the total DNA of the strain cell that extracts as template, utilize 18S rDNA and the order-checking of this bacterial strain of primer amplification of design, physical length is 1470bp, carrying out similarity analysis with the GenBank related data finds, this bacterium and Candida sorboxylosa(AB054672.1) homology the highest (homology, 99%/1470bps, based on 18S rDNA), therefore identify in conjunction with Physiology and biochemistry according to molecular biology identification, can determine that this bacterial strain is Candida sorboxylosa.
Through above-mentioned evaluation, with the present invention " ZJB-12164 " bacterial strain called after sorb wood sugar candiyeast ZJB-12164(Candida sorboxylosa ZJB-12164).
The fermentation culture of embodiment 2 Candida sorboxylosa ZJB-12164
(1) slant culture: Candida sorboxylosa ZJB-12164 is inoculated in slant medium, in 30 ℃ of lower cultivations 24 hours, obtains the inclined-plane thalline.Slant medium is bean sprout juice nutrient agar (fresh soybean sprout 100g/L, glucose 20g/L, agar 20g/L), natural pH.
(2) seed culture: be inoculated in seed culture medium with transfering loop from inclined-plane thalline picking one ring thalline, 30 ℃, cultivated 24 hours under the 150r/min condition, obtain seed liquor.The prescription of seed culture medium is: glucose 20g/L, yeast extract paste 20g/L, KH
2PO
42.5g/L NaCl 1g/L, solvent are water, initial pH is 6.0.
(3) fermentation culture: cultured seed liquor is inoculated in the fermention medium according to 2% volume ratio, under 28 ℃, shaking table revolution 150r/min condition, cultivated 24 hours, obtain the mycetocyte fermented liquid.The mycetocyte fermented liquid that obtains is centrifugal, abandoning supernatant, after precipitation was used the physiological saline washed twice, centrifugal collecting precipitation obtained wet thallus, and the output of wet thallus is 58g/L, water content 80%.The prescription of fermention medium is: glucose 10g/L, yeast extract powder 20g/L, K
2HPO
41.0g/L, NaCl 1g/L, CuCl
20.05g/L solvent is water, initial pH is 6.0.
The fermentation culture of embodiment 3 Candida sorboxylosa ZJB-12164
(1) slant culture: with embodiment 2.
(2) seed culture: with embodiment 2.
(3) fermentation culture: cultured seed liquor is inoculated in the yeast culture base according to 2% volume ratio, under 30 ℃, shaking table revolution 150r/min condition, cultivated 24 hours, obtain the mycetocyte fermented liquid.The mycetocyte fermented liquid that obtains is centrifugal, abandoning supernatant, after precipitation was used the physiological saline washed twice, centrifugal collecting precipitation obtained wet thallus, and the output of wet thallus is 75g/L, water content 80%.The prescription of yeast culture base is: glucose 20g/L, yeast extract powder 50g/L, K
2HPO
42.5g/L, NaCl 1g/L, CuCl
20.02g/L solvent is water, initial pH is 5.0.
Embodiment 4 Candida sorboxylosa ZJB-12164 biocatalysis preparation (S)-CHBE
The preparation of wet thallus is with embodiment 2.Add 100mL sodium phosphate buffer (pH7.0,0.1M) in transforming bottle, wherein contain wet thallus 5g, substrate COBE 5mmol was 35 ℃ of water bath with thermostatic control stirring reactions 4 hours.After reaction finished, conversion fluid was removed somatic cells through centrifugation, supernatant liquor equal-volume ethyl acetate extraction, organic layer anhydrous Na
2SO
4Gas chromatographic analysis after dehydration, the filtration.Product (S)-CHBE optical purity＞99%, transformation efficiency are 65%.
Embodiment 5 Candida sorboxylosa ZJB-12164 biocatalysis preparation (S)-CHBE
The preparation of wet thallus is with embodiment 2.Add 100mL sodium citrate buffer solution (pH6.0,0.1M) in transforming bottle, wherein contain wet thallus 5g, substrate COBE 5mmol, cosubstrate glucose were 5mmol, 35 ℃ of water bath with thermostatic control stirring reactions 1 hour.After reaction finished, conversion fluid was removed somatic cells through centrifugation, supernatant liquor equal-volume ethyl acetate extraction, organic layer anhydrous Na
2SO
4Gas chromatographic analysis after dehydration, the filtration.Product (S)-CHBE optical purity＞99%, transformation efficiency are 93%.
Embodiment 6 Candida sorboxylosa ZJB-12164 biocatalysis preparation (S)-CHBE
The preparation of wet thallus is with embodiment 3.Add 100mL sodium phosphate buffer (pH7.0,0.1M) in transforming bottle, wherein contain wet thallus 10g, substrate COBE 10mmol, cosubstrate glucose were 10mmol, 35 ℃ of water bath with thermostatic control stirring reactions 2 hours.After reaction finished, conversion fluid was removed somatic cells through centrifugation, supernatant liquor equal-volume ethyl acetate extraction, organic layer anhydrous Na
2SO
4Gas chromatographic analysis after dehydration, the filtration.Product (S)-CHBE optical purity＞99%, transformation efficiency are 94%.
Embodiment 7 Candida sorboxylosa ZJB-12164 biocatalysis preparation (S)-CHBE
The preparation of wet thallus is with embodiment 3.Add 100mL sodium phosphate buffer (pH7.5,0.1M) in transforming bottle, wherein contain wet thallus 10g, substrate COBE 100mmol, cosubstrate glucose were 100mmol, 30 ℃ of water bath with thermostatic control stirring reactions 12 hours.After reaction finished, conversion fluid was removed somatic cells through centrifugation, supernatant liquor equal-volume ethyl acetate extraction, organic layer anhydrous Na
2SO
4Gas chromatographic analysis after dehydration, the filtration.Product (S)-CHBE optical purity＞99%, transformation efficiency are 89%.
Embodiment 8 Candida sorboxylosa ZJB-12164 biocatalysis preparation (S)-CHBE
The preparation of wet thallus is with embodiment 3.Add 100mL sodium phosphate buffer (pH7.5,0.1M) in transforming bottle, wherein contain wet thallus 10g, substrate COBE 100mmol, cosubstrate fructose were 200mmol, 35 ℃ of water bath with thermostatic control stirring reactions 12 hours.After reaction finished, conversion fluid was removed somatic cells through centrifugation, supernatant liquor equal-volume ethyl acetate extraction, organic layer anhydrous Na
2SO
4Gas chromatographic analysis after dehydration, the filtration.Product (S)-CHBE optical purity＞99%, transformation efficiency are 93%.
Embodiment 9 Candida sorboxylosa ZJB-12164 biocatalysis preparation (S)-CHBE
The preparation of wet thallus is with embodiment 3.Add 50mL sodium phosphate buffer (pH7.5,0.1M) and 50mL butylacetate in transforming bottle, wherein contain wet thallus 20g, substrate COBE 200mmol, cosubstrate glucose were 300mmol, 35 ℃ of water bath with thermostatic control stirring reactions 8 hours.After reaction finishes, conversion fluid centrifugal 5 minutes at 8000rpm, organic layer anhydrous Na
2SO
4Gas chromatographic analysis after dehydration, the filtration.Product (S)-CHBE optical purity＞99%, transformation efficiency are 85%.
Embodiment 10 Candida sorboxylosa ZJB-12164 biocatalysis preparation (S)-CHBE
The preparation of wet thallus is with embodiment 3.In transforming bottle, add 60mL sodium phosphate buffer (pH8.0,0.1M) and 40mL butylacetate, wherein contain wet thallus 20g, substrate COBE 300mmol, cosubstrate glucose are 300mmol, at 35 ℃ of water bath with thermostatic control stirring reactions after 4 hours, add wet thallus 20g, continue reaction 4 hours.After reaction finishes, conversion fluid centrifugal 5 minutes at 8000rpm, organic layer anhydrous Na
2SO
4Gas chromatographic analysis after dehydration, the filtration.Product (S)-CHBE optical purity 99%, transformation efficiency are 92%.
SEQUENCE LISTING
＜110〉Zhejiang Polytechnical University
＜120〉sorb wood sugar candiyeast and the application in preparation (S)-4-chloro-3-hydroxyl ethyl butyrate thereof
<130>
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1470
<212> DNA
<213> Candida sorboxylosa
<400> 1
cctgcatgtc caagttagaa actgcgaatg gctcattaga tcagttatca tctccttgac 60
tcattacatg gataaccgtg gaaaatccag agctaataca cgccgtgcac atattaggtt 120
ccgactctga gtatttagcg aaccgtacgc tgggtcattc gagcatctgc cctatcaact 180
agacggtagg atctttgcct acggtggtgg tgacgggtaa cggggaataa gggttcggtt 240
ccggagaggg agcctgagag acggctacca catccaagga aggcagcagg cgcgcaaatt 300
gcacactggg aacgccccga tgcactagca ggcgtaccga tgcgtttacg caatcggata 360
aacacgcgta caggcccgaa tgtagcagtg gagggcaagt ctggtgccag cagccgcggt 420
aactccagct ccactagcgt atgttaaagt tgtagcagtt aaaacgctcg tagcgggggg 480
agcgcttata gctattactt tgagaaaatt agagtgttca aagcaggcct tgtgctcgga 540
tacgttagca tggaataatg gaacacgacg gcgtccatgg acgttgtaat gaccaagagg 600
ggcggaagag gcggtgcgta ttgggcggct aggggtgaaa tccgacgacc cgcccacgac 660
gagcgagtgc gaaggcacgc tgcagagacg tgcccgttcg tcaagaacga aagctaggga 720
atcgaaaatg atcagatacc attgtagtct tagccgtaaa cgatgtggag acacggggtg 780
cgattgtgct tcgtgggggg aaacttagtt cgttctgggg ggagtatgct cacaagggtg 840
aaacttaaag aaattggcgg aagactacct taagcaatgg gactgcggct taatttgact 900
caacacgggg aaactcacca ggtccagacg taataaggat tgaccagctg gagacttttc 960
ttgatcttac gggtggtggt gcatggccgt ttttcagtcc ttggagtgat ttgtctgctt 1020
aattgcgata acggacgaga ccttggcctc taactaggag agcgtatttg tacgcggact 1080
gcttagaggg acgacagacg ctaagtttgt ggaggcgcga ggcaacaaca ggtctgtgat 1140
gcccttagac gttctgggcc gcacgcgcgc tacactgacg gggggagcga gggccgggct 1200
gagaagccag ggcaatcagg aaaccgcgtc gtgctgggaa tagcggattg gaattgtttc 1260
ccttgaacgt ggaattgctt gtaagcgcag gtcatcagcc tgcgttgaat acgacccttg 1320
tctttgtaca caccgcccgt tgctactacc gattgaatgg cttagtgaga ggtcgggagg 1380
ccggcggagg gaactccgcg gggcgaactt gttctaactt ggctatttag agctcgtaaa 1440
agtcgtaaca aggtttccgt aggtgaacct 1470
Claims (10)
1. sorb wood sugar candiyeast ZJB-12164(Candida sorboxylosa ZJB-12164), be preserved in Chinese Typical Representative culture collection center, preservation address: China, Wuhan, Wuhan University, postcode 430072, deposit number CCTCC No:M 2012420, preservation date on October 22nd, 2012.
2. the application of the described sorb wood sugar of claim 1 candiyeast ZJB-12164 in biocatalysis preparation (S)-4-chloro-3-hydroxyl ethyl butyrate, it is characterized in that described being applied as: contain the enzyme wet thallus as catalyzer take what sorb wood sugar candiyeast ZJB-12164 fermentation culture obtained, take the 4-chloroacetyl acetacetic ester as substrate, in the transformation system a that the water of pH4.0 ~ 9.0 or damping fluid consist of, under 20 ~ 45 ℃, carry out conversion reaction, after reacting completely, with the conversion fluid aftertreatment, obtain described (S)-4-chloro-3-hydroxyl ethyl butyrate.
3. the as claimed in claim 2 application of sorb wood sugar candiyeast ZJB-12164 in biocatalysis preparation (S)-4-chloro-3-hydroxyl ethyl butyrate is characterized in that initial substrate concentration is 10 ~ 2000mmol/L among the described transformation system a.
4. the as claimed in claim 2 application of sorb wood sugar candiyeast ZJB-12164 in biocatalysis preparation (S)-4-chloro-3-hydroxyl ethyl butyrate, it is characterized in that containing among the transformation system a enzyme wet thallus quality and count 10 ~ 200g/L, describedly contain the enzyme wet thallus to contain quality be 70 ~ 90%.
5. the as claimed in claim 2 application of sorb wood sugar candiyeast ZJB-12164 in biocatalysis preparation (S)-4-chloro-3-hydroxyl ethyl butyrate, it is characterized in that also comprising cosubstrate among the described transformation system a, consist of transformation system b by the water of cosubstrate, substrate, catalyzer and pH4.0 ~ 9.0 or damping fluid, described cosubstrate is one of following: methyl alcohol, ethanol, n-propyl alcohol, Virahol, glucose, fructose, sucrose, maltose, lactose or glycerine.
6. the as claimed in claim 5 application of sorb wood sugar candiyeast ZJB-12164 in biocatalysis preparation (S)-4-chloro-3-hydroxyl ethyl butyrate, it is characterized in that described being applied as: contain the enzyme wet thallus as catalyzer take what sorb wood sugar candiyeast ZJB-12164 fermentation culture obtained, take the 4-chloroacetyl acetacetic ester as substrate, add cosubstrate, in the transformation system b that the water of pH4.0 ~ 9.0 or damping fluid consist of, under 20 ~ 45 ℃, carry out conversion reaction, after reacting completely, with the conversion fluid aftertreatment, obtain described (S)-4-chloro-3-hydroxyl ethyl butyrate; Initial substrate concentration is 10 ~ 2000mmol/L among the described transformation system b, contain enzyme wet thallus addition and count 10 ~ 200g/L with the wet thallus quality, describedly contain the enzyme wet thallus to contain quality be 70 ~ 90%, the cosubstrate starting point concentration is 10 ~ 2000mmol/L, and described cosubstrate is one of following: methyl alcohol, ethanol, n-propyl alcohol, Virahol, glucose, fructose, sucrose, maltose, lactose or glycerine.
7. such as the application of sorb wood sugar candiyeast ZJB-12164 as described in claim 2 or 5 in biocatalysis preparation (S)-4-chloro-3-hydroxyl ethyl butyrate, it is characterized in that described conversion reaction is at 20 ~ 45 ℃ of lower reaction 0.1 ~ 36h.
8. such as the application of sorb wood sugar candiyeast ZJB-12164 as described in claim 2 or 5 in biocatalysis preparation (S)-4-chloro-3-hydroxyl ethyl butyrate, the method that it is characterized in that described conversion fluid aftertreatment is: after reaction finishes, conversion fluid is centrifugal, remove somatic cells, get supernatant liquor equal-volume ethyl acetate extraction, get the organic layer anhydrous Na
2SO
4Dehydration, filtration, filtrate being contains the crude product of (S)-4-chloro-3-hydroxyl ethyl butyrate,, namely gets (S)-4-chloro-3-hydroxyl ethyl butyrate with the crude product separation and purification.
9. such as the application of sorb wood sugar candiyeast ZJB-12164 as described in claim 2 or 5 in biocatalysis preparation (S)-4-chloro-3-hydroxyl ethyl butyrate, it is characterized in that described transformation system a or transformation system b all are the pH4.0 that formed by distilled water, tap water, phosphate buffered saline buffer or citrate buffer ~ 9.0 single_phase systems, perhaps the pH4.0 that forms of water and organic medium ~ 9.0 diphasic systems; Described organic medium is normal hexane, octane, octane-iso, normal heptane, dimethylbenzene, hexanaphthene, ethyl acetate or butylacetate.
10. such as the application of sorb wood sugar candiyeast ZJB-12164 as described in claim 2 or 5 in biocatalysis preparation (S)-4-chloro-3-hydroxyl ethyl butyrate, it is characterized in that the described enzyme wet thallus that contains prepares as follows:
(1) slant culture: sorb wood sugar candiyeast ZJB-12164 inoculation in slant medium, in 20 ~ 37 ℃ of lower cultivations 12 ~ 48 hours, is obtained the slant activation bacterial classification; Described slant medium is the bean sprout juice nutrient agar, and collocation method is: the fresh soybean sprout of 50 ~ 200g is put into beaker, adds entry, boils half hour, and filtered through gauze gets supernatant, adds 10 ~ 50g glucose again, and 15 ~ 20g agar is mended and added water to 1L;
(2) seed culture: the slant activation bacterial classification inoculation in seed culture medium, is cultivated under 25 ~ 35 ℃, shaking table revolution 100 ~ 200r/min condition and obtained seed liquor in 12 ~ 36 hours; Described seed culture based formulas is: glucose 5 ~ 50g/L, yeast extract paste 5 ~ 40g/L, KH
2PO
40.5 ~ 5g/L, NaCl 0.5 ~ 5g/L, solvent are water, and initial pH is 5.0 ~ 8.0;
(3) fermentation culture: seed liquor according to volume ratio 1 ~ 5% access fermention medium, was cultivated 12 ~ 48 hours under 25 ~ 35 ℃, shaking table revolution 100 ~ 200r/min condition, with medium centrifugal, washing, collect and contain the enzyme wet thallus, refrigerate for subsequent use; Described fermentative medium formula is: glucose 5 ~ 50g/L, yeast extract powder 5 ~ 100g/L, KH
2PO
40.5 ~ 5g/L, NaCl 0.5 ~ 5g/L, CuCl
20.01 ~ 0.1g/L, solvent are water, initial pH is 5.0 ~ 8.0.
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