CN104263713A - Tistrella mobilis, halohydrin dehalogenase, gene, vector, recombinant strain and application of halohydrin dehalogenase - Google Patents
Tistrella mobilis, halohydrin dehalogenase, gene, vector, recombinant strain and application of halohydrin dehalogenase Download PDFInfo
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Abstract
The invention discloses a tistrella mobilis, a halohydrin dehalogenase, a gene, a vector, a recombinant strain and application of the halohydrin dehalogenase. The amino acid sequence of the halohydrin dehalogenase is as shown in SEQ ID No: 2; the halohydrin dehalogenase is capable of asymmetrically catalyzing 1,3-dichloropropanol and 1,3-dibromopropanol to generate (S)-epoxy chloropropane and (S)-epoxy bromopropane, and meanwhile, the activity of the halohydrin dehalogenase to (S)-4-chloro-3-hydroxy butyric acid ethyl ester is obviously far higher than that of other halohydrin dehalogenases; as a result, the halohydrin dehalogenase is a natural halohydrin dehalogenase discovered at present having the highest activity to the substrate, and can be applied to preparing epoxides or other chiral epoxides as well as atorvastatin intermediates as a catalyst; besides, the halohydrin dehalogenase is high in catalytic efficiency, excellent in enantioselectivity and high in substrate adaptability, and has brilliant industrial application prospect.
Description
(1) technical field
The present invention relates to a kind of come source movement for the halide alcohol dehalogenase gene of this Cui Na bacterium, recombinant expression vector containing this gene and recombinant bacterium, recombinant bacterium is utilized to prepare the method for recombinase, and this restructuring halide alcohol dehalogenase prepares epoxide, chiral epoxy compound, beta substitution alcohol, and preparing the application in atorvastatin crucial chiral intermediate (R)-4-cyano-3-hydroxy ethyl butyrate.
(2) background technology
Halide alcohol dehalogenase, is also halogenohydrin-hydrogen halide lyase, and being converted into epoxide and hydrogen halide by internal nucleophilic substitution mechanism catalyze aromatic or the adjacent halogenohydrin of aliphatics, is one of key enzyme of microbiological deterioration organic halogen compound.Hhe A, Hhe B and Hhe C3 class is divided into according to its sequence homology.Organic halogen compound has become one of current important environmental pollutant, mainly because waste discharge and the halid widespread use of synthetic cause.At occurring in nature, most of heteroplasia matter halogenide is very poor from degradation capability, and chemical compound lot is suspected to be carcinogenic or high mutagen simultaneously.Therefore, using microbe and dehalogenase degraded Organohalogen compounds have caused people to pay close attention to widely, and in environmental pollution improvement, particularly chiral epoxy compound synthesis aspect etc. have a very important role.
Halide alcohol dehalogenase is mainly through forming hydrogen bond between Serine conservative in protein structure and substrate hydroxyl oxygen atom, stablize and Binding Capacity, the pKa value of tyrosine is reduced by arginine, tyrosine from the Sauerstoffatom substrate as nucleophilic reagent, the carbon atom of attack ortho position halogen substiuted, and then release halogen ion, form epoxide.Halide alcohol dehalogenase not only the fracture of catalysis carbon-halogen bond can carry out the dehalogenation reaction, highly selective catalysis can accept except halogen ion a series of non-natural nucleophilic reagents, as N
3 -, NO
2 -, CN
-deng mediated epoxide ring-opening reaction, in order to generate a series of optically pure beta substitution alcohol.Therefore can be used for synthesizing some non-natural chipal compounds.
Halide alcohol dehalogenase can be widely used in epoxide and beta substitution alcohol.Wherein azide alcohol, cyanogen substituted alcohols and nitroalcohol are the precursors of synthesizing amino alcohol; Chiral amino alcohol is the very important compound of a class in field of biological pharmacy, can be used to synthesize various bioactivators.Isothiocyanic acid substituted alcohols is with oxazolidone extensive application in agrochemicals reagent, medicine and chemical field.Halide alcohol dehalogenase has been successfully applied to the synthesis of the crucial chiral intermediate of statins.Can know from current existing bibliographical information, the halide alcohol dehalogenase being most suitable for catalysis (S)-4-chloro-3-hydroxyl ethyl butyrate synthesis (R)-4-cyano-3-hydroxy ethyl butyrate is the Hhe C deriving from Agrobacterium radiobacter AD1.But original Hhe C vigor is still very low.Space-time yield only has 6 × 10
-3g/L/h.The most successfully scientist from Codexis company to Hhe C transformation, the Study on Directed Evolution of Proteins strategy that they utilize protein sequence-activity relationships (ProSAR) to drive is transformed Hhe C, through the sudden change that 18 take turns, obtain the mutant strain that a strain vigor is very high, the vigor of its catalysis (S)-CHBE is about 4000 times of wild-type enzyme.Concentration of substrate is 140g/L, and enzyme amount is 1.2g/L, and reaction 5h, productive rate is that 92%, ee value is greater than 99.5%.Current can apply to suitability for industrialized production only have this mutant enzyme, therefore excavate the novel halide alcohol dehalogenase with independent intellectual property right this substrate to higher catalytic activity, and applied to suitability for industrialized production, there is market potential application prospect.
Utilize the document of the direct asymmetric dehalogenation synthesizing chiral epoxy chloropropane of halide alcohol dehalogenase less.Tetsuji etc. utilize Hhe B catalysis 5mM 1, the 3-dichlorohydrine derived from Corynebacterium sp.N-1074 to generate (R)-epoxy chloropropane.The ee value of reaction starting stage (R)-epoxy chloropropane is maximum reaches 90%, but along with the carrying out reacted, ee value slowly declines until be finally zero.
Lutje Spelberg etc. utilize halide alcohol dehalogenase Hhe C catalysis 1, the 3-dichlorohydrine or 2, the 3-bis-trimethylewne chlorohydrin 3-synthesizing epoxy chloropropanes that derive from Agrobacterium radiobacter AD1, and its ee value is less than 5%.
In recent years, from multiple-microorganism, found halide alcohol dehalogenase, the gene of part halide alcohol dehalogenase has been cloned and at expression in escherichia coli, has obtained and produce the higher genetic engineering bacterium of enzyme activity, and is applied to catalysis formation epoxy compounds and beta substitution alcohol.Halide alcohol dehalogenase known at present, only there are 7 kinds, come from agrobacterium radiobacter (Agrobacterium radiobacter AD1) respectively, Arthrobacter (Agrobacter sp.AD2), coryneform bacteria (Corynebacterium sp.N-1074), Agrobacterium tumefaciens (Agrobacterium tumefaciens), tubercule bacillus (Mycobacterium sp.), earth mould (Agromyces mediolanus ZJB120203).So while studying existing halide alcohol dehalogenase, excavate new microbe-derived halide alcohol dehalogenase gene, and the application prospect excavating novel halide alcohol dehalogenase is the focus studied from now on.At present the research of halide alcohol dehalogenase is concentrated on and find that the catalyzed reaction time is short, products collection efficiency is high, the high and halide alcohol dehalogenase that side reaction is few of enantio-selectivity.
(3) summary of the invention
The object of the invention is to provide a kind of base sequence of restructuring halide alcohol dehalogenase of encoding, and the restructuring halide alcohol dehalogenase of coding, provides the recombinant vectors containing above-mentioned base sequence simultaneously, and the transformant transforming host by this recombinant vectors and obtain.Present invention also offers restructuring halide alcohol dehalogenase to 1,3-dichlorohydrine, 1,3-dibromo-propanol and (S)-4-chloro-3-hydroxyl ethyl butyrate demonstrate high reactivity, utilize this enzyme by 1,3-dichlorohydrine, 1,3-dibromo-propanol and (S)-4-chloro-3-hydroxyl ethyl butyrate prepare the method for (S)-epoxy chloropropane, (S)-epoxy bromopropane and (R)-4-cyano-3-hydroxy ethyl butyrate.And the application of this restructuring halide alcohol dehalogenase in other halohydrin Biodehalogenation of catalysis or asymmetric fractionation epoxide synthesizing chiral epoxy compound and beta substitution alcohol.
The technical solution used in the present invention is:
The invention provides one and derive from the restructuring halide alcohol dehalogenase of motion for this Cui Na bacterium (Tistrella mobilis) ZJB1405, the aminoacid sequence of described restructuring halide alcohol dehalogenase is as shown in SEQ ID No:2.
The present invention also provides a kind of encoding gene of described restructuring halide alcohol dehalogenase, and the nucleotides sequence of described encoding gene is classified as shown in SEQ ID No:1.
Halide alcohol dehalogenase gene source of the present invention is in Tistrella mobilis ZJB1405.Concrete preparation method can be: according to halohydrin epoxidase A gene order design PCR primer in the Tistrella mobilis KA081020-065 including prediction in Genbank.Preferably, primer 1CGC
cATATGaTGCCTGTCACCGACACCGC (underscore is Nde I restriction enzyme site), primer 2 ATTT
gCGGCCGCtTACGGCCAGCCGCCGGTG (underscore is Not I restriction enzyme site).Then with the genomic dna of Tistrella mobilis ZJB1405 for template, polymerase chain reaction (PCR) is utilized to carry out gene amplification, obtain a complete halide alcohol dehalogenase full-length gene order, the gene of base sequence as shown in SEQ ID No.1 in sequence table, called after HHDH
tm, total length 747bp.Wherein, from the 1st base to the 744th base only, initiator codon is ATG to its encoding sequence, and terminator codon is TAA.The aminoacid sequence of its coding is as shown in SEQ ID No.2 in sequence table.
The present invention relates to the recombinant vectors that a kind of described encoding gene builds.Restructuring halide alcohol dehalogenase gene of the present invention is connected to structure on various expression vector by this area ordinary method and forms by it.Described carrier can be the various carriers of this area routine, and as commercially available plasmid, clay, phage or virus vector etc., preferred plasmid is pET28a (b).Preferably, obtain recombinant expression vector of the present invention by following method: the halide alcohol dehalogenase gene product by pcr amplification gained be connected with carrier pGEM-T, obtain cloning vector pGEM-T-HHDH
tm, afterwards by cloning vector and expression vector pET28b restriction enzyme Nde I and Not I double digestion, form complementary sticky end, then connect through T4DNA ligase enzyme, form the recombinant expression vector pET28b-HHDH containing halide alcohol dehalogenase gene of the present invention
tm.
The present invention relates to a kind of recombination engineering bacteria of described construction of recombinant vector.It is obtained by being converted into by recombinant expression vector of the present invention in host microorganism.Described host microorganism can be the various host microorganisms of this area routine, as long as can meet copying voluntarily of recombinant expression vector Absorbable organic halogens, and entrained restructuring halide alcohol dehalogenase gene of the present invention can by effective expression.The preferred intestinal bacteria of the present invention, more preferably escherichia coli (E.coli) BL21 (DE3).By aforementioned recombinant expression plasmid pET28b-HHDH
tmbe converted in (E.coli) BL21 (DE3), the preferred engineering strain of the present invention, i.e. E.coli BL21 (DE3)/pET28b-HHDH
tm.
The present invention relates to the application of a kind of described encoding gene in preparation restructuring halide alcohol dehalogenase, described is applied as: build the recombinant vectors containing described restructuring halide alcohol dehalogenase gene, described recombinant vectors is converted in intestinal bacteria, the recombination engineering bacteria obtained carries out inducing culture, and nutrient solution is separated the somatic cells obtained containing restructuring halide alcohol dehalogenase.It comprises the steps: to cultivate recombinant bacterial strain of the present invention, obtains recombinant expressed halide alcohol dehalogenase.Wherein, the same foregoing description of described recombinant bacterial strain, obtains by recombinant expression vector of the present invention is converted into host microorganism.Wherein, described cultivation recombination bacillus coli substratum used can be that this area is any to be made recombined engineering bacteria growing and produce the substratum of halide alcohol dehalogenase of the present invention, preferred LB substratum: peptone 10g/L, yeast extract paste 5g/L, NaCl10g/L, solvent is deionized water, pH7.0.Cultural method and culture condition do not have special restriction, appropriate selection can be carried out by this area general knowledge, as long as enable recombinant bacterial strain grow and produce halide alcohol dehalogenase of the present invention according to the difference of the factor such as host type and cultural method.Other are cultivated transformant concrete operations and all can be undertaken by this area routine operation, preferred following method: recombinant expressed for the recombination bacillus coli that the present invention relates to transformant is seeded in the LB substratum containing final concentration 50mg/L kantlex and cultivates, as the optical density(OD) OD of nutrient solution
600when reaching 0.8, under the induction of isopropyl-beta D-thio galactopyranoside (IPTG) that final concentration is 0.2mM, high expression restructuring halide alcohol dehalogenase of the present invention.
The invention still further relates to a kind of described restructuring halide alcohol dehalogenase and prepare application in chiral epoxy compound at catalysis halohydrin, described is applied as: the wet thallus obtained through fermentation culture with the engineering bacteria containing restructuring halide alcohol dehalogenase gene is for catalyzer, take halohydrin as substrate, be in the damping fluid of 8 ~ 10 in pH value, in 35 DEG C, react under 150r/min condition, after reaction terminates, by reaction solution separation and purification, obtain chiral epoxy compound; Described halohydrin is 1,3-dichlorohydrine, 1, the chloro-1-phenylethyl alcohol of 3-dibromo-propanol, 2-, 1, the bromo-2-propyl alcohol of 3-bis-or 2, the bromo-2-propyl alcohol of 3-bis-, the consumption of described wet thallus is 10 ~ 50g/L damping fluid (preferred 20-40g/L), and the starting point concentration of described substrate is 5 ~ 50mmol/L damping fluid (preferred 20-40mmol/L).
Further, catalyzer of the present invention (i.e. wet thallus) is prepared as follows: be seeded in the LB substratum containing final concentration 50mg/L sulphuric acid kanamycin by the engineering bacteria containing restructuring halide alcohol dehalogenase gene, 37 DEG C of shaking culture are spent the night, by volume the inoculum size of concentration 1% accesses in LB liquid nutrient medium, put 37 DEG C, 180rpm shaking table shaking culture, when the OD600 of nutrient solution reaches 0.6, adding final concentration is that the IPTG of 0.5mM is as inductor, 28 DEG C of induction 10h, by medium centrifugal, collect wet thallus.
The invention still further relates to the application of a kind of described restructuring halide alcohol dehalogenase in catalysis halogenated hydroxyl ethyl butyrate dehalogenation, described is applied as: the wet thallus obtained through fermentation culture with the engineering bacteria containing restructuring halide alcohol dehalogenase gene is for catalyzer, with halogenated hydroxyl ethyl butyrate for substrate, be in the damping fluid of 7 ~ 10 in pH value, in 35 DEG C, react under 150r/min condition, after reaction terminates, by reaction solution separation and purification, obtain (R)-4-cyano-3-hydroxy ethyl butyrate; Described halogenated hydroxyl ethyl butyrate is (S)-4-chloro-3-hydroxyl ethyl butyrate, the consumption of described wet thallus is 10 ~ 60g/L damping fluid (preferred 20-40g/L), and the starting point concentration of described substrate is 10 ~ 150g/L damping fluid (preferred 50-100g/L).
The invention still further relates to a kind of described restructuring halide alcohol dehalogenase to split epoxide in catalysis and prepare application in chiral epoxy compound, described is applied as: the wet thallus obtained through fermentation culture with the engineering bacteria containing restructuring halide alcohol dehalogenase gene is for catalyzer, take epoxide as substrate, add nucleophilic reagent, be in the damping fluid of 4 ~ 7 in pH value, in 35 DEG C, react under 150r/min condition, after reaction terminates, by reaction solution separation and purification, obtain chiral epoxy compound; Described epoxide is phenyl ethylene oxide or epoxy chloropropane, and the consumption of described wet thallus is 10 ~ 50g/L damping fluid, and the starting point concentration of described substrate is 10 ~ 100mmol/L damping fluid (preferred 20-40mmol/L), and described nucleophilic reagent is NaN
3, NaNO
2or NaCN, the add-on of described nucleophilic reagent is 20 ~ 200mmol/L damping fluid (preferred 50-100mmol/L).
In above-mentioned application, each condition of described reaction can be selected by the normal condition of this type of reaction of this area.
Agents useful for same of the present invention and raw material are all commercially.
In addition, the present invention from soil microorganisms through primary dcreening operation, multiple sieve and separation and purification, obtain the motion of a kind of new strains that can produce halide alcohol dehalogenase-----for this Cui Na bacterium (Tistrella mobilis) ZJB1405, be preserved in China typical culture collection center, preservation date is on May 7th, 2014, deposit number is CCTCC NO:M 2014187, and preservation address is Wuhan, China Wuhan University, postcode 430072.
Motion of the present invention can grow for this Cui Na bacterium within the scope of temperature 25-35 DEG C, survives in the environment of pH5-9.
Described motion forms for the substratum of this Cui Na bacterium ZJB1405: glycerine 1-30g/L, yeast extract paste 1-30g/L, peptone 1-20g/L, (NH
4)
2sO
40.1-3g/L, K
2hPO
40.1-3g/L, Na
2hPO
42H
2o0.1-3g/L, NaH
2pO
412H
2o0.1-3g/L, MgSO
47H
2o0.1-3g/L, NaCl0.1-3g/L, solvent is deionized water, pH5-8,121 DEG C of sterilizing 20min.
Described motion preferably comprises shake-flask culture for the cultural method of this Cui Na bacterium (Tistrella mobilis) ZJB1405: wherein, described shaking flask liquid amount is 10%-30% (v/v), inoculum size is 1-8%, culture temperature is 20-40 DEG C, shaking speed is 100-200rpm, incubation time is 24-72h, and medium centrifugal, washing obtain resting cell.
Beneficial effect of the present invention is mainly reflected in: for current report product halide alcohol dehalogenase bacterial strain and the halide alcohol dehalogenase studied of clonal expression is less, and only have the mutant of Hhe C to be applied to the problems such as suitability for industrialized production.Screen a strain and produce halide alcohol dehalogenase bacterial strain: motion is for this Cui Na bacterium (Tistrella mobilis ZJB1405).Halide alcohol dehalogenase of the present invention obtains from motion for clonal expression this Cui Na bacterium first, this halide alcohol dehalogenase and other halide alcohol dehalogenase similarity confirmed very low (being less than 40%), this halide alcohol dehalogenase can asymmetry catalysis 1, 3-dichlorohydrine (Rate activity: 2696.7U/g) and 1, 3-dibromo-propanol (Rate activity: 6188.7U/g) generates (S)-epoxy chloropropane and (S)-epoxy bromopropane, and the ee value of Rate activity and (S)-epoxy chloropropane and (S)-epoxy bromopropane is all at present the highest (>60%).This original halide alcohol dehalogenase is compared with other wild-type halide alcohol dehalogenase simultaneously, the activity of (S)-4-chloro-3-hydroxyl ethyl butyrate is wanted obviously far above other halide alcohol dehalogenase, be the one natural halide alcohol dehalogenase the highest for this substrate active found at present, suitability for industrialized production can be directly used in without transformation.Restructuring halide alcohol dehalogenase of the present invention can be used as catalyst application in preparing epoxide or chiral epoxy compound and atorvastatin intermediate, and catalytic efficiency is high, and enantioselectivity is strong, and substrate wide adaptability, has good prospects for commercial application.
(4) accompanying drawing explanation
Fig. 1 is cloning vector pGEM-T-HHDH
tmphysical map;
Fig. 2 is pET28b-HHDH
tmrecombinant plasmid physical map;
Fig. 3 is halide alcohol dehalogenase gene PCR amplification argrose electrophorogram; Wherein, 2 ~ 5 to increase the halide alcohol dehalogenase gene fragment obtained for utilizing primer 1 and primer 2; 1 is DL2000DNA Marker;
The positive recombinant plasmid pET28b-HHDH of Fig. 4
tmenzyme cut structure iron; Wherein, 1 is λ DNA/Hind III DNA Marker; 2 is pET28b-HHDH
tm/ Nde I sample; 3 is pET28b-HHDH
tm/ Xho I sample; 4 is pET28b-HHDH
tm/ Nde I and Not I sample; 5 is DL2000DNA Marker fragment.
Fig. 5 is halide alcohol dehalogenase SDS-PAGE figure; The E.coli BL21/pET28b-HHDH that 1:IPTG does not induce
tm; 2: the E.coli BL21/pET28b-HHDH of induction
tm; 3: cytoclasis supernatant liquor, 4: cytoclasis precipitates, 5: protein molecular weight Marker;
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: motion is for the acquisition of this Cui Na bacterium (Tistrella mobilis) ZJB1405
The present invention from Zhejiang, Jiangsu, coastal areas of Fujian province get the samples such as ooze more than 100 part, utilize 1,3-dichlorohydrine or (S)-4-chloro-3-hydroxyl ethyl butyrate are sole carbon source, take Bromothymol blue as pH indicator, carry out the cultivation of two-wheeled enrichment isolation, screening halide alcohol dehalogenase producing strains, by repeated screening, is separated the halide alcohol dehalogenase producing strains of the plant height vigor that obtains.The concrete grammar of screening: ooze sample sterilized water is made Soil Slurry, gets 1.0mL supernatant liquor, join in screening culture medium, in 30 DEG C, shaking culture 4 days on 150rpm shaking table, gets this nutrient solution stroke-physiological saline solution afterwards and carry out gradient dilution, get 10
-6, 10
-7, 10
-8gradient dilution liquid be applied on LB solid medium, put 30 DEG C of constant temperature culture 3 days.Single colony inoculation that picking grows is cultivated in liquid fermentation medium.30 DEG C of shaking tables are cultivated 2 days, collected by centrifugation thalline, and with phosphate buffered saline buffer (pH8.0) washing, collect wet thallus.Wet thallus is used for transforming, conversion condition is as follows: phosphate buffered saline buffer (200mM, pH8.0) 30g/L wet thallus is added in, 1,3-dichlorohydrine 20mmol/L damping fluid, is placed in 35 DEG C of shaking baths and transforms 2h, get 1mL conversion fluid, after extraction into ethyl acetate, get organic layer and carry out vapor detection analysis.Adopt the concentration of vapor detection epoxy chloropropane, adopt GC-14A system, chromatographic column type: BGB-175 capillary column; Chromatographic condition: column temperature 90 DEG C, Sample Room temperature 220 DEG C, fid detector 220 DEG C, helium gas flow is 1.6mL/min; Splitting ratio is 40:1.Mei Huo unit (U) is defined as: 35 DEG C, under pH10.0 condition, the enzyme amount that catalysis 1,3-dichlorohydrine generates required for 1 μm of ol epoxy chloropropane in 1min is defined as 1U.Know recombinant bacterium enzyme by inference according to the growing amount of epoxy chloropropane in system to live.According to viability examination, obtain an enzyme the highest strain bacterial strain alive, numbering ZJB1405, and identify.
The present invention screens the bacterial strain ZJB1405 obtained, by the Physiology and biochemistry qualification of " common bacteria system identification handbook " and " uncle Jie Shi Bacteria Identification handbook (the 8th edition) ", Gram-negative, shaft-like.Colony characteristics on solid plate substratum: colony colour is white, opaque, smooth surface, moistening, regular edges, medium and small without halo, colonial morphology, circle, center projections.
Described screening culture medium final concentration composition: 1,3-DCP2.7g/L or (S)-4-chloro-3-hydroxyl ethyl butyrate 2.4g/L, (NH
4)
2sO
41g/L, K
2hPO
41g/L, Na
2hPO
42H
2o1g/L, NaH
2pO
412H
2o2g/L, MgSO
47H
2o0.5g/L, FeSO
47H
2o0.01g/L, CuSO
45H
2o0.01g/L, MnSO
45H
2o0.01g/L, pH6.8, solvent is deionized water.
Described solid LB media final concentration composition: peptone 10g/L, yeast extract paste 5g/L, NaCl10g/L, pH7.0, solvent is deionized water, pH value nature.
Described fermention medium final concentration composition: glycerine 10g/L, yeast extract paste 10g/L, peptone 5g/L, (NH
4)
2sO
41g/L, K
2hPO
41g/L, Na
2hPO
42H
2o1g/L, NaH
2pO
412H
2o2g/L, MgSO
47H
2o0.5g/L, NaCl3g/L, pH7.0, solvent is deionized water.
Screen the bacterial strain ZJB1405 obtained, carry out Physiology and biochemistry qualification, and press fine works molecular biology experiment Directory Method extraction chromosomal DNA, with the cell STb gene extracted for template, utilize the 16SrDNA gene of primer 16S-8:AGAGTTTGATCCTGGCTCAG and 16S-1541:AAGGAGGTGATCCAGCCGCA amplification bacterial strain, after gene product is connected with carrier T, rear trust Shanghai Sani increases and order-checking to this bacterium 16S rDNA, after obtaining the 16S rDNA sequence of this bacterial strain, NCBI website is retrieved with BLAST the 16S rRNA gene order of related strain in GenBank, and carry out sequence analysis.Based on the qualification of form, physiological and biochemical property and the aspect such as 16S rDNA sequence and phylogenetic analysis, determine that this bacterium is for motion is for this Cui Na bacterium, called after motion is for this Cui Na bacterium (Tistrella mobilis) ZJB1405, this bacterial strain is deposited in China typical culture collection center on May 7th, 2014, and deposit number is CCTCC No:M 2014187.
Bacterial strain ZJB140516S rDNA sequence
1 GGGCCTTCGA CTTCCTTGTT ACGACTTCAC CCCAGTCGCT GACCTTACCG TGGACGGCTG CCTCCCTTAC
71 GGGTCAGCTC ACCGGCTTCG GGTAAAACCA ACTCCCATGG TGTGACGGGC GGTGTGTACA AGGCCCGGGA
141 ACGTATTCAC CGCGGCATGC TGTTCCGCGA TTACTAGCGA TTCCGACTTC ATGCACTCGA GTTGCAGAGT
211 ACAATCCGAA CTGAGACGAC TTTTTGAGAT TAGCTTCACC TCGCGATGTC GCTGCCCACT GTAGTCGCCA
281 TTGTAGCACG TGTGTAGCCC AGCCCATAAG GGCCATGAGG ACTTGACGTC ATCCCCACCT TCCTCCGGCT
351 TATCACCGGC AGTTTCCCTA GAGTGCCCAG CCGAACTGAT GGCAACTAAG GATGAGGGTT GCGCTCGTTG
421 CGGGACTTAA CCCAACATCT CACGACACGA GCTGACGACA GCCATGCAGC ACCTGTGTGA CGTCCGGCCG
491 AACCGAAAAC CCCGTCTCCA GGGTCGCGAC GTCCATGTCA AGGGCTGGTA AGGTTCTGCG CGTTGCTTCG
561 AATTAAACCA CATGCTCCAC CGCTTGTGCG GGCCCCCGTC AATTCCTTTG AGTTTTAACC TTGCGGCCGT
631 ACTCCCCAGG CGGAGTGCTT AACGCGTTAG CTACGACACT GCGATACTAA GTATCCCAAC GTCCAGCACT
701 CATCGTTTAC GGCGTGGACT ACCAGGGTAT CTAATCCTGT TTGCTCCCCA CGCTTTCGTG CCTCAGCGTC
771 AGTTTCGGGC CAGGCAGCCG CCTTCGCCAC CGGTGTTCTT CCCAATATCT ACGAATTTCA CCTCTACACT
841 GGGAATTCCA CTACCCTCTC CCGACTCCAG CCTACCAGTC TCAAAGCAGT TCCGGAGTTG AGCCCGGGCT
911 TTCACTTCTG ACTTGATAAG CCGCCTACGC ACGCTTTACG CCCAGTAAAT CCGAACACGC TAGCCCCCTT
981 CGTATTACGC GGCTGCTGGC ACGAAGTTAG CGGGGCTTCT TCTACGGGTA CGTCATTATC TTCCCGTCGA
1051 AAGAGCTTAC AATCGAAGAC CTTCATCACT CACGCGCATG CTGGAATCAA GCTTCGGCAA TGTCATAATC
1121 CCACTGCTGC TCCGTAGGGA GTCTGACAG
Embodiment 2: the clone of halide alcohol dehalogenase goal gene
According to include in Genbank to be predicted as Tistrella mobilis enzyme gene order (Genbank accession number: CP003236.1) be foundation, design PCR primer is as follows:
Upstream primer: CGC
cATATGaTGCCTGTCACCGACACCGC
Downstream primer: ATTT
gCGGCCGCtTACGGCCAGCCGCCGGTG
Wherein, in upstream primer, be with underscore part to be Nde I restriction enzyme site, in downstream primer, be with underscore part to be Not I restriction enzyme site.
The total genomic dna replacing this Cui Na bacterium (Tistrella mobilis) ZJB1405 with motion is that template carries out pcr amplification.The each component add-on of PCR reaction system (cumulative volume 100 μ L): 10 × Pfu DNA Polymerase Buffer10 μ L (Mg
2+), 10mM dNTP mixture (each 2.5mM of dATP, dCTP, dGTP and dTTP) 0.5 μ L, concentration is cloning primer 1, each 0.5 μ L of primer 2, the genomic dna 1 μ L of 50 μMs, Pfu DNA Polymerase1 μ L, seedless sour water 86.5 μ L.
Adopt the PCR instrument of Biorad, PCR reaction conditions is: denaturation 94 DEG C of 3min, then enters temperature cycle 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, totally 30 circulations, and last 72 DEG C extend 10min, and final temperature is 4 DEG C.
PCR primer, through agarose gel electrophoresis purifying, utilizes sepharose DNA to reclaim test kit recovery about 750bp and occurs obvious band (Fig. 3).Then add A with Taq enzyme PCR, then be connected with carrier T, obtain cloning recombinant plasmids pGEM-T-HHDH
tm(see Fig. 1).This recombinant plasmid electricity is converted in e. coli jm109, bacterium colony PCR is utilized to check order, the positive bacterium colony of picking cultivates sample presentation order-checking, utilize software analysis sequencing result, result shows: the nucleotide sequence length increased through primer 1 and primer 2 is 747bp (its nucleotide sequence is as shown in SEQ ID NO:1), the open reading frame that this sequence encoding one is complete.
1 ATGCCTGTCA CCGACACCGC GCCCCGCGTG GCGCTGGTTA CCAATGCGAC CAAATATGCC
61 GGCGCCCCCA CGGTGGCGGC GCTTGCCAGC CAGGGCTGGC AGATCATCGC CCATGACGCA
121 TCCTTCACCG ACGTGGCGGC CCGTGCCGCC TGGGAGGCGG ACAACCAGGG CATGACCGCC
181 GCCGAGGCTC AGGATCCGGC CGGGTTGATC GCCGAAGTGC GCGACCGGAT GGGCGGGCTG
241 CACGGTATCG TTTCCAACGA CGCCTATCCC GCGATCCGTC GCCGTATCGA AGAGACCGAG
301 GCGGAGGCGC TGCGCGAGAT GCTGGAGGCG CTGACCGTCT TCCCCTTCGC GCTCGCCTCG
361 GCAGTGACCC CACACCTGAA GGCGCAGGGC GCCGGCGCCA TCGTGATGGT CACCTCCGCA
421 TCGCCGCGCC GTCCCTACCC GGGGTTCGCG ATGTACGCAA CCGCCCGCTC GGCCTCGACC
481 GGGCTTGCCA AGGCACTGGC GAACGAGCTG GCGCCCCATG GCATCCGGGT GAATGCCGTG
541 GCGCCGAACT TCCTCTACAG CGAGACCTAT TACCCGCGCG CCAAATGGAT CGACGATCCC
601 GCCGGTGCCG CCCGGGTGCG CGAGATGGTA CCGCTCGGCC GTCTGGGCCG GCCCGAGGAG
661 ATCGGTGAAC TGATTGCTTT CCTGCTGTCC GACAAGGCCG GCTTTGTGGT CGGCGAGACC
721 GTGGGCTTCA CCGGCGGCTG GCCGTAA
Utilize software to analyze this gene order, know the aminoacid sequence shown in described halide alcohol dehalogenase genes encoding SEQ ID NO:2 by inference:
1 MPVTDTAPRV ALVTNATKYA GAPTVAALAS QGWQIIAHDA SFTDVAARAA WEADNQGMTA
61 AEAQDPAGLI AEVRDRMGGL HGIVSNDAYP AIRRRIEETE AEALREMLEA LTVFPFALAS
121 AVTPHLKAQG AGAIVMVTSA SPRRPYPGFA MYATARSAST GLAKALANEL APHGIRVNAV
181 APNFLYSETY YPRAKWIDDP AGAARVREMV PLGRLGRPEE IGELIAFLLS DKAGFVVGET
241 VGFTGGWP*
The preparation of embodiment 3 recombinant expression plasmid and recombinant expressed transformant
Halide alcohol dehalogenase gene clone plasmid is contained by what obtain in embodiment 2,37 DEG C with restriction enzyme Nde I and Not I double digestion 5h, through agarose gel electrophoresis purifying, utilize sepharose DNA to reclaim test kit (love pursue progress biotechnology (Hangzhou) company limited) and reclaim target fragment.Under the effect of T4DNA ligase enzyme, by target fragment and same vector plasmid pET28b after Nde I and Not I enzyme cut, at 16 DEG C, connection is spent the night and is obtained recombinant expression plasmid pET28b-HHDH
tm.
Above-mentioned recombinant expression plasmid is transformed in intestinal bacteria (E.coli) BL21 (DE3) competent cell, LB flat board containing final concentration 50mg/L sulphuric acid kanamycin screens positive recombinants, picking mono-clonal, bacterium colony PCR verifies positive colony.Obtain positive recombinant conversion body intestinal bacteria (E.coli) BL21 (DE3)/pET28b-HHDH
tm.Random picked clones extracting plasmid carries out enzyme and cuts qualification, qualification result as shown in Figure 4, positive recombinant plasmid pET28b-HHDH as can be seen from Figure 4
tmthere is single band in line at 3 swimming lanes and 4 swimming lanes in single endonuclease digestion, after double digestion, two bands appear in 5 swimming lanes, and a band is consistent with goal gene clip size.This result illustration purpose gene has been cloned into Nde I and the Not I site of pET28b.
Embodiment 4 is recombinated the expression of halide alcohol dehalogenase
By the recombination bacillus coli of embodiment 3 gained, be seeded in the LB substratum containing final concentration 50mg/L sulphuric acid kanamycin, 37 DEG C of shaking culture are spent the night, be equipped with in the 500mL triangular flask of 100mL LB substratum by the inoculum size access of 1% (v/v), put 37 DEG C, 180rpm shaking table shaking culture, when the OD 600 of nutrient solution reaches 0.6, adding final concentration is that the IPTG of 0.5mM is as inductor, 28 DEG C of induction 10h, by medium centrifugal, collecting cell (i.e. wet thallus), and wash twice with phosphate buffered saline buffer, obtain resting cell.Gained resting cell is suspended in the damping fluid of 20mL pH8.0, ultrasonication in ice bath, collected by centrifugation supernatant, is the crude enzyme liquid of restructuring halide alcohol dehalogenase.Crude enzyme liquid is through Polyacrylamide Gel Electrophoresis (Fig. 5), and recombinant protein exists with partly soluble form in cell, has Partial Protein to be present in cell debris in addition.
Embodiment 5: halide alcohol dehalogenase catalysis 1,3-dichlorohydrine synthesis (S)-epoxy chloropropane
With recombination bacillus coli (E.coli) BL21 (the DE3)/pET28b-HHDH containing intracellular expression recombinant plasmid obtained in embodiment 4
tmwet thallus as conversion enzyme, and with intestinal bacteria (E.coli) BL21 (DE3), intestinal bacteria (E.coli) BL21 (DE3)/pET28b, recombination bacillus coli (E.coli) BL21 (DE3)/pET28b-HHDH
tmnon-induction fermentation thalline bacterium in contrast.With 1,3-dichlorohydrine for substrate, carry out conversion reaction and prepare epoxy chloropropane.Transformation system composition and conversion operation as follows: add 1 of 0.2g wet thallus and 40mmol/L damping fluid in 10mL glycine-NaOH buffer (pH10.0), 3-dichlorohydrine is not (to add the reaction system of thalline in contrast, the epoxy chloropropane of 0.5mmol/L racemization is had to generate), 1min is reacted under 35 DEG C of shaking table 150r/min conditions, extract by equal-volume ethyl acetate, extracting twice, combining extraction liquid, and add 1-chlorine normal hexane, transformation efficiency and the ee value of substrate is measured with gas chromatographic analysis.The transformation efficiency of 1,3-dichlorohydrine reaches more than 90%, and the yield of (S)-epoxy chloropropane reaches 82.4%, ee value and reaches 60.1%.
Adopt the concentration of vapor detection epoxy chloropropane, adopt GC-14A system, chromatographic column type: BGB-175 capillary column; Chromatographic condition: column temperature 90 DEG C, Sample Room temperature 220 DEG C, fid detector 220 DEG C, helium gas flow is 1.6mL/min; Splitting ratio is 40:1.
Mei Huo unit (U) is defined as: 35 DEG C, under pH10.0 condition, the enzyme amount that catalysis 1,3-dichlorohydrine generates required for 1 μm of ol epoxy chloropropane in 1min is defined as 1U.Know recombinant bacterium enzyme by inference according to the growing amount of epoxy chloropropane in system to live.Measurement result is in table 1.
Table 1: with recombination bacillus coli BL21/pET28b-HHDH
tmfor the halide alcohol dehalogenase vigor that enzyme source measures
| Bacterial strain/plasmid | Enzyme lives (U/g (wet cells)) |
| (E.coli)BL21(DE3) | 0 |
| (E.coli)BL21(DE3)/pET28b | 0 |
| (E.coli)BL21(DE3)/pET28b-HHDH Tm(induction) | 40.5 |
| (E.coli)BL21(DE3)/pET28b-HHDH Tm(induction) | 2696.7 |
Embodiment 6: halide alcohol dehalogenase catalysis 2,3-dichlorohydrine dehalogenation synthesizing epoxy chloropropane
With recombination bacillus coli (E.coli) BL21 (the DE3)/pET28b-HHDH containing intracellular expression recombinant plasmid obtained in embodiment 4
tmwet thallus as conversion enzyme, and with intestinal bacteria (E.coli) BL21 (DE3), intestinal bacteria (E.coli) BL21 (DE3)/pET28b, recombination bacillus coli (E.coli) BL21 (DE3)/pET28b-HHDH
tmnon-induction fermentation thalline bacterium in contrast.With 2,3-dichlorohydrine for substrate, carry out conversion reaction, investigate its dehalogenation to 2,3-dichlorohydrine active.Transformation system composition and conversion operation as follows: add 2 of 0.4g wet thallus and 20mmol/L damping fluid in 10mL phosphate buffered saline buffer (pH8.0), 3-dichlorohydrine is not (to add the reaction system of thalline in contrast, generate without product), 10 are reacted under 35 DEG C of shaking table 150r/min conditions, 20, 30min samples, after reaction terminates, reaction solution is centrifugal, get supernatant liquor and carry out gas phase analysis, or reaction solution equal-volume ethyl acetate is extracted, extracting twice, combining extraction liquid, and add internal standard substance, the transformation efficiency of substrate and enzyme is measured to 2 with gas chromatographic analysis, the enzyme of 3-dichlorohydrine is lived.After reaction 30min, the transformation efficiency of 2,3-dichlorohydrines reaches 10.4%, and the yield of (S)-epoxy chloropropane reaches 9.6%, ee value and reaches 71.5%.
Adopt the concentration of vapor detection epoxy chloropropane, adopt GC-14A system, chromatographic column type: BGB-175 capillary column; Chromatographic condition: column temperature 90 DEG C, Sample Room temperature 220 DEG C, fid detector 220 DEG C, helium gas flow is 1.6mL/min; Splitting ratio is 40:1.Mei Huo unit (U) is defined as: 35 DEG C, under pH8.0 condition, the enzyme amount that catalysis 2,3-dichlorohydrine generates required for 1 μm of ol epoxy chloropropane in 1min is defined as 1U.Know recombinant bacterium enzyme by inference according to the growing amount of epoxy chloropropane in system to live.Measurement result is in table 2.
Table 2: with recombination bacillus coli BL21/pET28b-HHDH
tmfor the halide alcohol dehalogenase vigor that enzyme source measures
| Bacterial strain/plasmid | Enzyme lives (U/g (wet cells)) |
| (E.coli)BL21(DE3) | 0 |
| (E.coli)BL21(DE3)/pET28b | 0 |
| (E.coli)BL21(DE3)/pET28b-HHDH Tm(induction) | 0 |
| (E.coli)BL21(DE3)/pET28b-HHDH Tm(induction) | 6.60 |
Embodiment 7: halide alcohol dehalogenase catalysis (the S)-4-chloro-3-hydroxyl ethyl butyrate dehalogenation reaction
With recombination bacillus coli (E.coli) BL21 (the DE3)/pET28b-HHDH containing intracellular expression recombinant plasmid obtained in embodiment 4
tmwet thallus as conversion enzyme, and with intestinal bacteria (E.coli) BL21 (DE3), intestinal bacteria (E.coli) BL21 (DE3)/pET28b, recombination bacillus coli (E.coli) BL21 (DE3)/pET28b-HHDH
tmnon-induction fermentation thalline bacterium in contrast.With (S)-4-chloro-3-hydroxyl ethyl butyrate for substrate, carry out conversion reaction, investigate its dehalogenation to (S)-4-chloro-3-hydroxyl ethyl butyrate active.Transformation system composition and conversion operation as follows: add (S)-4-chloro-3-hydroxyl ethyl butyrate of 0.2g wet thallus and 20mmol/L damping fluid in 10mL phosphate buffered saline buffer (pH8.0) (not add the reaction system of thalline in contrast, contrast and generate without product), 10 are reacted under 35 DEG C of shaking table 150r/min conditions, 20, 30min samples, after reaction terminates, reaction solution is centrifugal, get supernatant liquor and carry out gas phase analysis, or reaction solution equal-volume ethyl acetate is extracted, extracting twice, combining extraction liquid, and add internal standard substance, the transformation efficiency of substrate is measured with gas chromatographic analysis, after 30min, the transformation efficiency of substrate is 90%, ee value reaches 99%.
In reaction solution, the content of (S)-4-chloro-3-hydroxyl ethyl butyrate is by gas chromatography determination.Gas-chromatography model is Agilent 7890A, capillary column model used is HP-5 (column length 30m, internal diameter 0.25mm, thickness of liquid film 0.25 μm, filler is 5%Phenyl/95%dimethylpolysiloxane), chromatographic condition: column temperature 120 DEG C keeps 4min, 20 DEG C/min temperature programming to 180 DEG C, keeps 1min.Injector temperature 220 DEG C, fid detector 220 DEG C, helium gas flow is 1.6mL/min; Splitting ratio is 40:1.Substrate (S)-4-chloro-3-hydroxyl ethyl butyrate retention time is 4.9min.Mei Huo unit (U) is defined as: 35 DEG C, under pH8.0 condition, the enzyme amount in 1min required for catalysis 1 μm of ol (S)-4-chloro-3-hydroxyl ethyl butyrate dehalogenation is defined as 1U.Know recombinant bacterium enzyme by inference according to the reduction of (S)-4-chloro-3-hydroxyl ethyl butyrate in system to live.Measurement result is in table 3.
Table 3: with recombination bacillus coli BL21/pET28b-HHDH
tmfor the halide alcohol dehalogenase vigor that enzyme source measures
| Bacterial strain/plasmid | Enzyme lives (U/g (wet cells)) |
| (E.coli)BL21(DE3) | 0 |
| (E.coli)BL21(DE3)/pET28b | 0 |
| (E.coli)BL21(DE3)/pET28b-HHDH Tm(induction) | 0.39 |
| (E.coli)BL21(DE3)/pET28b-HHDH Tm(induction) | 226.42 |
Embodiment 8: halide alcohol dehalogenase catalysis 2-chloro-1-phenylethyl alcohol dehalogenation synthesis of phenyl oxyethane
With recombination bacillus coli (E.coli) BL21 (the DE3)/pET28b-HHDH containing intracellular expression recombinant plasmid obtained in embodiment 4
tmwet thallus as conversion enzyme, and with intestinal bacteria (E.coli) BL21 (DE3), intestinal bacteria (E.coli) BL21 (DE3)/pET28b, recombination bacillus coli (E.coli) BL21 (DE3)/pET28b-HHDH
tmnon-induction fermentation thalline bacterium in contrast.With the chloro-1-phenylethyl alcohol of 2-for substrate, carry out conversion reaction, investigate its dehalogenation to the chloro-1-phenylethyl alcohol of 2-active.Transformation system composition and conversion operation as follows: add the chloro-1-phenylethyl alcohol of 2-of 0.2g wet thallus and 20mmol/L damping fluid in 10mL phosphate buffered saline buffer (pH8.0) (not add the reaction system of thalline in contrast, generate without product), 10 are reacted under 35 DEG C of shaking table 150r/min conditions, 20, 30min samples, after reaction terminates, reaction solution centrifuging and taking supernatant liquor is carried out gas phase analysis, or reaction solution equal-volume ethyl acetate is extracted, extracting twice, combining extraction liquid, and add internal standard substance, the transformation efficiency of substrate is measured and enzyme is lived to the enzyme of the chloro-1-phenylethyl alcohol of 2-with gas chromatographic analysis.After the transformation efficiency of the chloro-1-phenylethyl alcohol of 2-reaches 15%, 30min, the yield of (R)-phenyl ethylene oxide reaches 34.4%, ee value and reaches 20.6%.
Adopt the concentration of vapor detection phenyl ethylene oxide, adopt GC-14A system, chromatographic column type: BGB-175 capillary column; Chromatographic condition: column temperature 100 DEG C, Sample Room temperature 220 DEG C, fid detector 220 DEG C, helium gas flow is 1.6mL/min; Splitting ratio is 40:1.Mei Huo unit (U) is defined as: 35 DEG C, under pH8.0 condition, the enzyme amount that the chloro-1-phenylethyl alcohol of catalysis 2-generates required for 1 μm of ol phenyl ethylene oxide in 1min is defined as 1U.Know recombinant bacterium enzyme by inference according to the growing amount of phenyl ethylene oxide in system to live.Measurement result is in table 4.
Table 4: with recombination bacillus coli BL21/pET28b-HHDH
tmfor the halide alcohol dehalogenase vigor that enzyme source measures
| Bacterial strain/plasmid | Enzyme lives (U/g (wet cells)) |
| (E.coli)BL21(DE3) | 0 |
| (E.coli)BL21(DE3)/pET28b | 0 |
| (E.coli)BL21(DE3)/pET28b-HHDH Tm(induction) | 1.19 |
| (E.coli)BL21(DE3)/pET28b-HHDH Tm(induction) | 80.61 |
Embodiment 9: the halide alcohol dehalogenase catalysis 4-chloro-3-hydroxyl butyronitrile dehalogenation reaction
With recombination bacillus coli (E.coli) BL21 (the DE3)/pET28b-HHDH containing intracellular expression recombinant plasmid obtained in embodiment 4
tmwet thallus as conversion enzyme, and with intestinal bacteria (E.coli) BL21 (DE3), intestinal bacteria (E.coli) BL21 (DE3)/pET28b, recombination bacillus coli (E.coli) BL21 (DE3)/pET28b-HHDH
tmnon-induction fermentation thalline bacterium in contrast.With 4-chloro-3-hydroxyl butyronitrile for substrate, carry out conversion reaction, investigate its dehalogenation to 4-chloro-3-hydroxyl butyronitrile active.Transformation system composition and conversion operation as follows: add the 4-chloro-3-hydroxyl butyronitrile of 0.2g wet thallus and 20mmol/L damping fluid in 10mL phosphate buffered saline buffer (pH8.0) (not add the reaction system of thalline in contrast, generate without product), 10 are reacted under 35 DEG C of shaking table 150r/min conditions, 20, 30min samples, after reaction terminates, reaction solution is centrifugal, get supernatant liquor and carry out gas phase analysis, or reaction solution equal-volume ethyl acetate is extracted, extracting twice, combining extraction liquid, and add internal standard substance, the transformation efficiency of substrate is measured and enzyme is lived to the enzyme of 4-chloro-3-hydroxyl butyronitrile with gas chromatographic analysis.After 30min, the transformation efficiency of 4-chloro-3-hydroxyl butyronitrile reaches 67%.
Gas-chromatography Agilent6890N is adopted to measure, chromatographic column type: capillary column model used is HP-5 (filler is 5%Phenyl/95%dimethylpolysiloxane for column length 30m, internal diameter 0.25mm, thickness of liquid film 0.25 μm); Chromatographic condition: column temperature 60 DEG C keeps 4min, 20 DEG C/min temperature programming to 160 DEG C, keeps 2min.Injector temperature 220 DEG C, fid detector 220 DEG C, helium gas flow is 1.6mL/min; Splitting ratio is 40:1.4-chloro-3-hydroxyl butyronitrile retention time is 8.5min.Mei Huo unit (U) is defined as: 35 DEG C, under pH8.0 condition, the enzyme amount that catalysis consumes required for 1 μm of ol4-chloro-3-hydroxyl butyronitrile in 1min is defined as 1U.Know recombinant bacterium enzyme by inference according to the consumption of 4-chloro-3-hydroxyl butyronitrile in system to live.Measurement result is in table 5.
Table 5: with recombination bacillus coli BL21/pET28b-HHDH
tmfor the halide alcohol dehalogenase vigor that enzyme source measures
| Bacterial strain/plasmid | Enzyme lives (U/g (wet cells)) |
| (E.coli)BL21(DE3) | 0 |
| (E.coli)BL21(DE3)/pET28b | 0 |
| (E.coli)BL21(DE3)/pET28b-HHDH Tm(induction) | 0.25 |
| (E.coli)BL21(DE3)/pET28b-HHDH Tm(induction) | 116.2 |
Embodiment 10: the bromo-2-propyl alcohol dehalogenation synthesis of halide alcohol dehalogenase catalysis 1,3-bis-(S)-epoxy bromopropane
With recombination bacillus coli (E.coli) BL21 (the DE3)/pET28b-HHDH containing intracellular expression recombinant plasmid obtained in embodiment 4
tmwet thallus as conversion enzyme, and with intestinal bacteria (E.coli) BL21 (DE3), intestinal bacteria (E.coli) BL21 (DE3)/pET28b, recombination bacillus coli (E.coli) BL21 (DE3)/pET28b-HHDH
tmnon-induction fermentation thalline bacterium in contrast.With the bromo-2-propyl alcohol of 1,3-bis-for substrate, carry out conversion reaction, investigate its dehalogenation to the bromo-2-propyl alcohol of 1,3-bis-active.Transformation system composition and conversion operation as follows: add 1 of 0.05g wet thallus and 20mmol/L damping fluid in 10mL phosphate buffered saline buffer (pH10.0), the bromo-2-propyl alcohol of 3-bis-is not (to add the reaction system of thalline in contrast, micro-racemic epoxide N-PROPYLE BROMIDE is had to generate), certain hour sampling is reacted under 35 DEG C of shaking table 150r/min conditions, control reaction conversion ratio within 10%, after reaction terminates, reaction solution is centrifugal, supernatant liquor is gone to carry out gas phase analysis, or reaction solution equal-volume ethyl acetate is extracted, extracting twice, combining extraction liquid, and add internal standard substance, the transformation efficiency of substrate and enzyme is measured to 1 with gas chromatographic analysis, the enzyme of the bromo-2-propyl alcohol of 3-bis-is lived.After reaction 1.5min, the transformation efficiency of the bromo-2-propyl alcohol of 1,3-bis-reaches more than 95%, and the yield of (S)-epoxy bromopropane reaches 90.1%, ee value and reaches 50%.
Adopt the concentration of vapor detection epoxy bromopropane, adopt GC-14A system, chromatographic column type: BGB-175 capillary column; Chromatographic condition: column temperature 90 DEG C, Sample Room temperature 220 DEG C, fid detector 220 DEG C, helium gas flow is 1.6mL/min; Splitting ratio is 40:1.Mei Huo unit (U) is defined as: 35 DEG C, under pH8.0 condition, the enzyme amount that catalysis 1,3-dibromo-propanol generates required for 1 μm of ol epoxy bromopropane in 1min is defined as 1U.Know recombinant bacterium enzyme by inference according to the growing amount of epoxy bromopropane in system to live.Measurement result is in table 6.
Table 6: with recombination bacillus coli BL21/pET28b-HHDH
tmfor the halide alcohol dehalogenase vigor that enzyme source measures
| Bacterial strain/plasmid | Enzyme lives (U/g (wet cells)) |
| (E.coli)BL21(DE3) | 0 |
| (E.coli)BL21(DE3)/pET28b | 0 |
| (E.coli)BL21(DE3)/pET28b-HHDH Tm(induction) | 80.32 |
| (E.coli)BL21(DE3)/pET28b-HHDH Tm(induction) | 6188.7 |
Embodiment 11: the bromo-2-propyl alcohol dehalogenation synthesis of halide alcohol dehalogenase catalysis 2,3-bis-(S)-epoxy bromopropane
With recombination bacillus coli (E.coli) BL211 (the DE3)/pET28b-HHDH containing intracellular expression recombinant plasmid obtained in embodiment 4
tmwet thallus as conversion enzyme, and with intestinal bacteria (E.coli) BL21 (DE3), intestinal bacteria (E.coli) BL211 (DE3)/pET28b, recombination bacillus coli (E.coli) BL211 (DE3)/pET28b-HHDH
tmnon-induction fermentation thalline bacterium in contrast.With the bromo-2-propyl alcohol of 2,3-bis-for substrate, carry out conversion reaction, investigate its dehalogenation to the bromo-2-propyl alcohol of 2,3-bis-active.Transformation system composition and conversion operation as follows: add 2 of 0.2g wet thallus and 20mmol/L damping fluid in 10mL phosphate buffered saline buffer (pH10.0), the bromo-2-propyl alcohol of 3-bis-is not (to add the reaction system of thalline in contrast, micro-racemic epoxide N-PROPYLE BROMIDE is had to generate), 10min sampling is reacted under 35 DEG C of shaking table 150r/min conditions, control reaction conversion ratio within 10%, after reaction terminates, reaction solution is centrifugal, get supernatant liquor and carry out gas phase analysis, or reaction solution equal-volume ethyl acetate is extracted, extracting twice, combining extraction liquid, and add internal standard substance, product (S)-epoxy bromopropane ee is measured with gas chromatographic analysis, with enzyme to 2, the enzyme of the bromo-2-propyl alcohol of 3-bis-is lived.After reaction 1min, the transformation efficiency of substrate is 8%, and the yield of epoxy bromopropane is that 7.4%, ee reaches 90%.
Adopt the concentration of vapor detection epoxy bromopropane, adopt GC-14A system, chromatographic column type: BGB-175 capillary column; Chromatographic condition: column temperature 90 DEG C, Sample Room temperature 220 DEG C, fid detector 220 DEG C, helium gas flow is 1.6mL/min; Splitting ratio is 40:1.Mei Huo unit (U) is defined as: 35 DEG C, under pH8.0 condition, the enzyme amount that catalysis 2,3-dibromo-propanol generates required for 1 μm of ol epoxy bromopropane in 1min is defined as 1U.Know recombinant bacterium enzyme by inference according to the growing amount of epoxy bromopropane in system to live.Measurement result is in table 7.
Table 7: with recombination bacillus coli BL21/pET28b-HHDH
tmfor the halide alcohol dehalogenase vigor that enzyme source measures
| Bacterial strain/plasmid | Enzyme lives (U/g (wet cells)) |
| (E.coli)BL21(DE3) | 0 |
| (E.coli)BL21(DE3)/pET28b | 0 |
| (E.coli)BL21(DE3)/pET28b-HHDH Tm(induction) | 2.86 |
| (E.coli)BL21(DE3)/pET28b-HHDH Tm(induction) | 193.1 |
Embodiment 12: the ring-opening reaction of halide alcohol dehalogenase Epichlorohydrin
With recombination bacillus coli (E.coli) BL21 (the DE3)/pET28b-HHDH containing intracellular expression recombinant plasmid obtained in embodiment 4
tmwet thallus as conversion enzyme, and with intestinal bacteria (E.coli) BL21 (DE3), intestinal bacteria (E.coli) BL21 (DE3)/pET28b, recombination bacillus coli (E.coli) BL21 (DE3)/pET28b-HHDH
tmnon-induction fermentation thalline bacterium in contrast.Take epoxy chloropropane as substrate, carry out the ring-opening reaction of epoxide.Reaction system and reaction conditions as follows: the epoxy chloropropane adding 0.2g wet thallus and 10mmol/L damping fluid in 10mL citrate-phosphate salt buffer (pH5.0) is substrate, the nucleophilic reagent (NaNO of 20mmol/L damping fluid
2) (not add the reaction system of thalline in contrast, there is not epoxy chloropropane generation ring-opening reaction), after reacting 60min under 35 DEG C of shaking table 150r/min conditions, reaction solution equal-volume ethyl acetate is extracted, extracting twice, combining extraction liquid, and add internal standard substance, the transformation efficiency of detection ring oxide compound and selectivity.Obtain (R)-epoxy chloropropane and 1-nitro-3-chlorine 2-propyl alcohol.The transformation efficiency of epoxy chloropropane reaches more than 91%, and the yield of (R)-epoxy bromopropane reaches 8.2%, ee value and reaches 90%.
Adopt concentration and the selectivity of vapor detection epoxy chloropropane, adopt GC-14A system, chromatographic column type: BGB-175 capillary column; Chromatographic condition: column temperature 90 DEG C, Sample Room temperature 220 DEG C, fid detector 220 DEG C, helium gas flow is 1.6mL/min; Splitting ratio is 40:1.Mei Huo unit (U) is defined as: 35 DEG C, under pH5.0 condition, the enzyme amount that Epichlorohydrin open loop generates required for 1 μm of ol1-nitro-3-chlorine 2-propyl alcohol in 1min is defined as 1U.
Know recombinant bacterium enzyme by inference according to the consumption of epoxy chloropropane in system to live.Measurement result is in table 8.
Table 8: with recombination bacillus coli BL21/pET28b-HHDH
tmfor the halide alcohol dehalogenase vigor that enzyme source measures
| Bacterial strain/plasmid | Enzyme lives (U/g (wet cells)) |
| (E.coli)BL21(DE3) | 0 |
| (E.coli)BL21(DE3)/pET28b | 0 |
| (E.coli)BL21(DE3)/pET28b-HHDH Tm(induction) | 0.28 |
| (E.coli)BL21(DE3)/pET28b-HHDH Tm(induction) | 19.1 |
Embodiment 13: the ring-opening reaction of halide alcohol dehalogenase catalysis phenyl ethylene oxide
With recombination bacillus coli (E.coli) BL21 (the DE3)/pET28b-HHDH containing intracellular expression recombinant plasmid obtained in embodiment 4
tmwet thallus as conversion enzyme, and with intestinal bacteria (E.coli) BL21 (DE3), intestinal bacteria (E.coli) BL21 (DE3)/pET28b, recombination bacillus coli (E.coli) BL21 (DE3)/pET28b-HHDH
tmnon-induction fermentation thalline bacterium in contrast.Take phenyl ethylene oxide as substrate, carry out the ring-opening reaction of epoxide.Reaction system and reaction conditions as follows: the phenyl ethylene oxide adding 0.2g wet thallus and 10mmol/L damping fluid in 10mL citrate-phosphate salt buffer (pH5.0) is substrate, the nucleophilic reagent (NaNO of 20mmol/L damping fluid
2) (not add the reaction system of thalline in contrast, substrate does not reduce), after reacting 30min under 35 DEG C of shaking table 150r/min conditions, reaction solution equal-volume ethyl acetate extracts, extracting twice, combining extraction liquid, and add internal standard substance, the transformation efficiency of detection ring oxide compound and selectivity.Obtain (S)-phenyl ethylene oxide and 1-Nitrophenethyl alcohol.The transformation efficiency of phenyl ethylene oxide is 30.5%, and the ee value of (S)-phenyl ethylene oxide is 27.6%.
Adopt concentration and the selectivity of vapor detection epoxy chloropropane, adopt GC-14A system, chromatographic column type: BGB-175 capillary column; Chromatographic condition: column temperature 100 DEG C, Sample Room temperature 220 DEG C, fid detector 220 DEG C, helium gas flow is 1.6mL/min; Splitting ratio is 40:1.Mei Huo unit (U) is defined as: 35 DEG C, under pH5.0 condition, the enzyme amount that the open loop of catalysis phenyl ethylene oxide generates required for 1 μm of ol1-Nitrophenethyl alcohol in 1min is defined as 1U.
Know recombinant bacterium enzyme by inference according to the consumption of phenyl ethylene oxide in system to live.Measurement result is in table 9.
Table 9: with recombination bacillus coli BL21/pET28b-HHDH
tmfor the halide alcohol dehalogenase vigor that enzyme source measures
| Bacterial strain/plasmid | Enzyme lives (U/g (wet cells)) |
| (E.coli)BL21(DE3) | 0 |
| (E.coli)BL21(DE3)/pET28b | 0 |
| (E.coli)BL21(DE3)/pET28b-HHDH Tm(induction) | 0.11 |
| (E.coli)BL21(DE3)/pET28b-HHDH Tm(induction) | 7.5 |
Embodiment 14:(R) the catalyzing and synthesizing of-4-cyano-3-hydroxy ethyl butyrate
With recombination bacillus coli (E.coli) BL21 (the DE3)/pET28b-HHDH containing intracellular expression recombinant plasmid obtained in embodiment 4
tmwet thallus as conversion enzyme, and with intestinal bacteria (E.coli) BL21 (DE3), intestinal bacteria (E.coli) BL21 (DE3)/pET28b, recombination bacillus coli (E.coli) BL21 (DE3)/pET28b-HHDH
tmnon-induction fermentation thalline bacterium in contrast.With (S)-4-chloro-3-hydroxyl ethyl butyrate for substrate, carry out conversion reaction preparation (R)-4-cyano-3-hydroxy ethyl butyrate.Transformation system composition and conversion operation as follows: in 50mL flask, add 30mL50mM NaH
2pO
4the aqueous solution is as damping fluid, (S)-4-chloro-3-hydroxyl ethyl butyrate of 100g/L damping fluid, water-bath is preheated to 40 DEG C, utilize constant pH/potentiometric titrator stream to add NaCN and regulate pH to 7.5, the wet thallus of 1.5g is added (not add the reaction system of thalline in contrast in system, generate without product), with NaCN control pH 7.8, reaction 6h.Sampling, uses isopyknic extraction into ethyl acetate, operates the same, vapor detection (R)-4-cyano-3-hydroxy ethyl butyrate content.The productive rate of product reaches 90%, and optical purity (ee) value reaches 99%.
In reaction solution, the content of (S)-4-chloro-3-hydroxyl ethyl butyrate and (R)-4-cyano-3-hydroxy ethyl butyrate is by gas chromatography determination.Gas-chromatography model is Agilent7890A, capillary column model used is HP-5 (column length 30m, internal diameter 0.25mm, thickness of liquid film 0.25 μm, filler is 5%Phenyl/95%dimethylpolysiloxane), chromatographic condition: column temperature 120 DEG C keeps 4min, 20 DEG C/min temperature programming to 180 DEG C, keeps 1min.Injector temperature 220 DEG C, fid detector 220 DEG C, helium gas flow is 1.6mL/min; Splitting ratio is 40:1.Substrate (S)-4-chloro-3-hydroxyl ethyl butyrate retention time is 4.9min, and the retention time of product (R)-4-cyano-3-hydroxy ethyl butyrate is 6.2min.(S) retention time of-4-chloro-3-hydroxyl ethyl butyrate and (R)-4-cyano-3-hydroxy ethyl butyrate is respectively 4.9min and 6.2min.Enzyme is lived and is defined: at 40 DEG C, under pH7.8 condition, the enzyme amount that per minute catalyzed reaction generates required for 1 μm of ol HN is defined as 1U.
Know recombinant bacterium enzyme by inference according to the growing amount of HN in system to live.Measurement result is in table 10.
Table 10: with recombination bacillus coli BL21/pET28b-HHDH
tmfor the halide alcohol dehalogenase vigor that enzyme source measures
| Bacterial strain/plasmid | Enzyme lives (U/g (wet cells)) |
| (E.coli)BL21(DE3) | 0 |
| (E.coli)BL21(DE3)/pET28b | 0 |
| (E.coli)BL21(DE3)/pET28b-HHDH Tm(induction) | 6.8 |
| (E.coli)BL21(DE3)/pET28b-HHDH Tm(induction) | 459.1 |
In sum, although the present invention preferably implements to disclose as above with one, so itself and be not used to limit the present invention.The technical staff in the technical field of the invention, without departing from the spirit and scope of the present invention, can make various changes or modifications.Therefore, protection scope of the present invention is when being as the criterion of limiting depending on appended application claims.
Claims (10)
1. derive from the restructuring halide alcohol dehalogenase of motion for this Cui Na bacterium (Tistrella mobilis) ZJB1405, it is characterized in that: the aminoacid sequence of described restructuring halide alcohol dehalogenase is as shown in SEQ ID No:2.
2. an encoding gene for halide alcohol dehalogenase of recombinating described in claim 1, is characterized in that the nucleotides sequence of described encoding gene is classified as shown in SEQ ID No:1.
3. the recombinant vectors of encoding gene structure described in a claim 2.
4. the recombination engineering bacteria of construction of recombinant vector described in a claim 3.
5. the application of encoding gene described in a claim 2 in preparation restructuring halide alcohol dehalogenase, it is characterized in that described being applied as: build the recombinant vectors containing described restructuring halide alcohol dehalogenase gene, described recombinant vectors is converted in intestinal bacteria, the recombination engineering bacteria obtained carries out inducing culture, and nutrient solution is separated the somatic cells obtained containing restructuring halide alcohol dehalogenase.
6. halide alcohol dehalogenase of recombinating described in a claim 1 prepares the application in chiral epoxy compound at catalysis halohydrin, it is characterized in that described being applied as: the wet thallus obtained through fermentation culture with the engineering bacteria containing restructuring halide alcohol dehalogenase gene is for catalyzer, take halohydrin as substrate, be in the damping fluid of 8 ~ 10 in pH value, in 35 DEG C, react under 150r/min condition, after reaction terminates, obtain the mixed solution containing chiral epoxy compound; Described halohydrin is 1,3-dichlorohydrine, 1,3-dibromo-propanol, the chloro-1-phenylethyl alcohol of 2-, 1, the bromo-2-propyl alcohol of 3-bis-or 2, the bromo-2-propyl alcohol of 3-bis-, the consumption of described wet thallus is 10 ~ 50g/L damping fluid, and the starting point concentration of described substrate is 5 ~ 50mmol/L damping fluid.
7. apply as claimed in claim 6, it is characterized in that described catalyzer is prepared as follows: be seeded in the LB substratum containing final concentration 50mg/L sulphuric acid kanamycin by the engineering bacteria containing restructuring halide alcohol dehalogenase gene, 37 DEG C of shaking culture are spent the night, by volume the inoculum size of concentration 1% accesses in LB liquid nutrient medium, put 37 DEG C, 180rpm shaking table shaking culture, when the OD600 of nutrient solution reaches 0.6, adding final concentration is that the IPTG of 0.5mM is as inductor, 28 DEG C of induction 10h, by medium centrifugal, collect wet thallus.
8. the application of halide alcohol dehalogenase of recombinating described in a claim 1 in catalysis halogenated hydroxyl ethyl butyrate dehalogenation, it is characterized in that described being applied as: the wet thallus obtained through fermentation culture with the engineering bacteria containing restructuring halide alcohol dehalogenase gene is for catalyzer, with halogenated hydroxyl ethyl butyrate for substrate, be in the damping fluid of 7 ~ 10 in pH value, in 35 DEG C, react under 150r/min condition, after reaction terminates, obtain the mixed solution containing (R)-4-cyano-3-hydroxy ethyl butyrate; Described halogenated hydroxyl ethyl butyrate is (S)-4-chloro-3-hydroxyl ethyl butyrate or (S)-4-cyano-3-hydroxy ethyl butyrate, the consumption of described wet thallus is 10 ~ 60g/L damping fluid, and the starting point concentration of described substrate is 10 ~ 150g/L damping fluid.
9. halide alcohol dehalogenase of recombinating described in a claim 1 prepares the application in chiral epoxy compound at fractionation epoxide, it is characterized in that described being applied as: the wet thallus obtained through fermentation culture with the engineering bacteria containing restructuring halide alcohol dehalogenase gene is for catalyzer, take epoxide as substrate, add nucleophilic reagent, be in the damping fluid of 4.0 ~ 7.0 in pH value, in 35 DEG C, react under 150r/min condition, after reaction terminates, obtain the mixed solution containing chiral epoxy compound; Described epoxide is phenyl ethylene oxide or epoxy chloropropane, and the consumption of described wet thallus is 10 ~ 50g/L damping fluid, and the starting point concentration of described substrate is 10 ~ 100moml/L damping fluid, and described nucleophilic reagent is NaN
3, NaNO
2or NaCN, the add-on of described nucleophilic reagent is 20 ~ 200mmol/L damping fluid.
10. motion is for this Cui Na bacterium (Tistrella mobilis) ZJB1405, be preserved in China typical culture collection center, preservation date is on May 7th, 2014, and deposit number is CCTCC NO:M 2014187, preservation address is Wuhan, China Wuhan University, postcode 430072.
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