CN1243096C - Recombined D-hydantoin enzyme and application thereof - Google Patents

Recombined D-hydantoin enzyme and application thereof Download PDF

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CN1243096C
CN1243096C CNB2004100008423A CN200410000842A CN1243096C CN 1243096 C CN1243096 C CN 1243096C CN B2004100008423 A CNB2004100008423 A CN B2004100008423A CN 200410000842 A CN200410000842 A CN 200410000842A CN 1243096 C CN1243096 C CN 1243096C
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enzyme
glycolylurea
hydantoinase
engineering bacteria
bacterium
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CN1560244A (en
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袁静明
石亚伟
赵莉霞
禇西宁
封霞
钮利喜
陈开明
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Shanxi University
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Abstract

The present invention relates to a recombined D-hydantoinase engineering bacterium and construction and applications thereof. The present invention has the technical scheme that a genomic DNA of a newly discovered high-quality D-hydantoinase producing strain is used as a mould plate, genes of D-hydantoinase are amplified by a PCR method, and the genes are cloned, recombined, transformed and expressed. The high-activity D-hydantoinase engineering bacterial strain is obtained; pure recombined D-hydantoinase is obtained through engineering bacterium fermentation and product purification; the activity of the D-hydantoinase engineering bacterium is 15 times higher than that of natural bacterial strains under respectively optimum conditions (when the fermentation liquor is 10OD, the engineering bacterium is 10.65 U/ml, and the natural bacterium is 0.70 U/ml), and the transformation rate of the recombined D-hydantoinase to substrate hydantoin and derivates thereof can reach 98%. The engineering bacterium can be used for biotransformation of non-natural D-amino acid and derivates thereof.

Description

A kind of reorganization D-glycolylurea enzyme and application thereof
One, technical field
The invention belongs to zymoprotein molecular engineering field, relate to artificial constructed engineering bacteria, specifically is a kind of reorganization D-glycolylurea enzyme engineering bacteria and structure and purposes.Engineering bacteria through fermentation can obtain highly active reorganization D-glycolylurea enzyme (claim Phenylhydantoinase again, or Hydantoinase, D-hydantoinase, EC3.5.2.2); Tunning can obtain pure reorganization D-glycolylurea enzyme through separation and purification; With artificial bacterial strain bio-transformation non-natural D-amino acid and derivative such as D-pHPG (D-HPG); For the synthetic amino acid whose peptide class of the D-product in fields such as medicine, food, agricultural chemicals provides intermediate.
Two, technical background
D-amino acid is widely used in the starting material that industries such as medicine, light industry, food, agricultural chemicals and foodstuff additive are used for more synthetic peptide series products, as pharmaceutical industries: polypeptide hormone, semi-synthetic antibiotic, antiviral agent, carcinostatic agent etc.; Food service industry: food-flavoring comps, nutritional additive, sweeting agent etc.; Cosmetic industry: hair care agent, permanent agent for permanent hair waving etc.; Also be used for agricultural herbicide such as pyrethroid etc. in addition, especially of many uses with pharmaceutical industries, the demand height, for example, D-pHPG (D-HPG) is the important side chain of synthetic beta-lactam semisynthetic antibiotics, new antibiotic medicines such as commercially available amoxycilline Trihydrate bp, Cefaclor, Cefalexin, Cephradine are exactly synthetic through an enzymatic or a chemical enzymatic by antibiotic parent nucleus 6APA or 7ACA and D-HPG.This class medicine can be treated general inflammation clinically, as illnesss such as stomach, duodenal ulcer disease and toxoplasma parasitic infections, all obtains better curative effect.The output of present domestic D-pHPG (D-HPG) far can not be met the need of market, dependence on import still, its basic reason is with natural bacterium bio-transformation efficient still lower, many technologic limiting factors are arranged again, await carrying out bio-transformation, so the present invention has great realistic significance with the engineering bacteria of artificial control.
D-amino acid derivatives such as D-pHPG (D-HPG) synthetic mainly contains two kinds of methods.First method is chemosynthesis, but this technology exists yield low, and the chemosynthesis process causes serious drawbacks such as environmental pollution, now need not.Second method is exactly to utilize D-glycolylurea enzyme to produce that bacterium splits hydantoin derivative and the thalline conversion method for preparing optically pure D-HPG derivative.The thalline conversion method is to utilize D-glycolylurea enzyme (D-hydantoinase, E.C.3.5.2.2) stereospecificity hydrolysis D-generates N-carboxamide-D-D-pHPG to hydroxyl-benzene glycolylurea, and the latter can finally obtain the D-HPG (see figure 1) by the diazotizing chemical method of nitrous acid or the enzyme process of N-carboxamide-D-amino acid amide lytic enzyme deamination formyl.All tend to double-enzyme method at present,, and can avoid environmental pollution, improve working conditions because the enzyme bio-transformation not only can obtain high yield.
The bacterial strain report of relevant D-amino acid bio conversion both at home and abroad is many in recent years, relate generally to the bacterial strain that produces the glycolylurea enzyme Pseudomonas fluorescens DSM84 (Lapointe et al.1994) is arranged, Bacillus stearothermophilrs NS1122 (Mukohara etal.1993), Pseudomonas putida CCRC12857 (Chien et al.1998), Agrobacteriumradiobacteria B11291 (Chao et al.1999), Burkholderia pickettii (Xu Zhen etc. 2002), wherein the D-Hydantoinase gene of most of bacterial strains is cloned and is expressed.Much contain D-Hydantoinase gene recon at expression in escherichia coli although reported, but most enzymic activity is not high or the formation inclusion body, therefore engineering bacteria is used for industrial bio-transformation production D-amino acid and still has certain distance, rite-directed mutagenesis or enzyme molecular orientation evolvement have been set about at present, attempt to improve enzymic activity, adapt to the application of industrial level.But under the lower situation of engineering bacteria basal enzyme activity level, obtain the artificial bacterium of ideal, still have certain degree of difficulty.Therefore the engineering bacteria of finding and make up high yield D-glycolylurea enzymic activity is one of the most effective means.
Three, summary of the invention
The objective of the invention is to pass through gene engineering method, make up engineering strain and do not needing to add under the condition of any inductor, with regard to the D-glycolylurea enzyme that can obtain to efficiently express, can be used for the amino acid whose bio-transformation of D-type, it is low to overcome natural strain enzyme-producing amount, and need to add the defective that inductor could produce D-glycolylurea enzyme, and in producing, reduce cost, reduce the operational path that pollutes and lay the foundation.
A kind of isolating D-glycolylurea enzyme that the present invention provides from Pseudomonas putida YZ-II6 bacterium, and encoding gene, the transformation of gene and in various heterologous gene expression systems, efficiently express the genetic material that provides good for this reason.With Pseudomonas putida YZ-II6 strain gene group DNA is template, with PCR method separating clone this D-Hydantoinase gene, dna sequence analysis shows total length 1440bp, initiator codon is ATG, terminator codon is TGA, does not contain intron, 479 amino-acid residues of encoding altogether.
Described a kind of isolating D-glycolylurea enzyme, its aminoacid sequence is:
1 MSLLIRGATV VTHEESYPAD VLCADGLIRA IGQNLEPPTD CEILDGSGQY LMPGGIDPHT
61 HMQLPFMGTV ASEDFFSGTA AGLAGGTTSI IDFVIPNPQQ SLLEAFHTWR GWAQKSASDY
121 GFHVAITWWS EQVAEEMGEL VAKHGVNSFK HFMAYKNAIM AADNTLVASF ERCLQLGAVP
181 TVHAENGELV YHLQKKLLAQ GITGPEAHPL SRPSQVEGEA ASRAIRIAET LGTPLYLVHI
241 SSREALDEIA YARGKGQPVY GEVLPGHLLL DDSVYRDPDW ATAAGYVMSP PFRPREHQEA
301 LWRGLQSGNL HTTATDHCCF CAEQKAMGRD DFSRIPNGTA GIEDRMAVLW DAGVNSGRLS
361 MHEFVALTST NTAKIFNLFP RKGAIRVGAD ADLVLWDPQG TRTISAQTHH QQVDFNIFEG
421 RTVRGIPSHT ISQGKVLWAD GDLRAEPGAG RYVERPAYPS VYEVLGRRAE HQRPMPVQR
The polynucleotide of a kind of encoding D of the present invention-glycolylurea enzyme, its nucleotides sequence is classified as:
1 ATGTCCCTGT TGATCCGTGG CGCCACCGTG GTTACCCACG AAGAGAGTTA CCCCGCCGAT
61 GTCCTGTGTG CCGATGGCCT GATCCGTGCC ATCGGGCAAA ACCTCGAACC ACCCACCGAC
121 TGTGAGATCC TCGACGGCAG CGGCCAGTAC CTGATGCCCG GCGGCATCGA CCCGCACACC
181 CACATGCAGT TGCCGTTCAT GGGCACTGTA GCCAGCGAGG ACTTCTTCAG CGGCACCGCT
241 GCGGGCCTGG CAGGCGGCAC CACTTCGATC ATCGACTTCG TCATACCCAA CCCGCAGCAG
301 TCGTTGCTGG AGGCCTTCCA CACCTGGCGT GGCTGGGCGC AGAAAAGCGC CAGCGACTAT
361 GGCTTCCACG TCGCCATCAC CTGGTGGAGC GAGCAGGTGG CCGAAGAGAT GGGCGAGCTG
421 GTGGCCAAGC ACGGGGTGAA CAGCTTCAAG CATTTCATGG CCTACAAGAA TGCGATCATG
481 GCCGCCGACA ATACCTTGGT GGCCAGCTTC GAGCGTTGCC TGCAACTGGG CGCGGTGCCC
541 ACCGTGCATG CCGAGAACGG CGAGCTGGTG TACCACCTGC AGAAAAAACT GCTCGCCCAG
601 GGCATAACCG GGCCGGAAGC CCACCCGCTG TCACGCCCCT CGCAGGTTGA AGGCGAAGCA
661 GCCAGCCGCG CCATCCGCAT TGCCGAAACC CTCGGCACGC CGTTGTACCT GGTGCACATT
721 TCCAGCCGCG AGGCACTGGA TGAAATCGCT TATGCCCGAG GCAAGGGCCA GCCGGTGTAT
781 GGCGAGGTGT TGCCCGGCCA CCTGCTGCTG GATGACAGCG TCTACCGTGA CCCGGACTGG
841 GCCACCGCAG CCGGTTATGT AATGAGCCCG CCGTTCCGCC CACGCGAGCA CCAGGAAGCG
901 CTGTGGCGCG GCTTGCAGTC GGGCAACCTG CACACCACCG CCACCGACCA CTGCTGCTTC
961 TGCGCCGAGC AGAAAGCCAT GGGCCGCGAC GACTTCAGCC GCATCCCCAA CGGCACCGCC
1021 GGCATCGAAG ACCGCATGGC GGTACTGTGG GATGCCGGGG TCAACAGCGG GCGCCTGTCG
1081 ATGCACGAAT TCGTCGCGCT GACCTCTACC AATACGGCGA AAATCTTCAA CCTGTTCCCG
1141 CGCAAGGGCG CTATCCGCGT GGGTGCCGAT GCCGACCTGG TGCTGTGGGA CCCGCAGGGC
1201 ACCCGCACCA TCTCCGCCCA AACCCACCAC CAGCAGGTGG ACTTCAACAT TTTCGAAGGC
1261 CGCACCGTGC GCGGCATCCC AAGCCACACC ATCAGCCAGG GCAAGGTGCT CTGGGCCGAT
1321 GGCGACTTGC GTGCCGAACC GGGCGlGGGG CGCTATGTGG AACGGCCGGC GTACCCGTCA
1381 GTGTATGAGG TGCTGGGGCG CCGGGCCGAG CATCAGCGGC CGATGCCAGT TCAGCGCTGA
With above-mentioned aminoacid sequence DNAstar software analysis, the result show with the Pseudomonas putidaCCRC12857 homology reported be 91.4%, Pseudomonas aeruginosa PA01 homology is 89%, and has only 63% with Pseudomonasputida DSM84 homology.All be lower than 50% with the glycolylurea enzyme homology of other non-false monospore Bacillaceae.Although higher with Pseudomonas putida CCRC12857 homology, their zymologic properties, expression have the tall and erect difference of showing, D-glycolylurea enzyme in this patent has more good feature, do not need any inductor such as expressing, representation is solvable, and every liter of thalline vigor is the former more than ten times.
Above-mentioned D-Hydantoinase gene sequence by GeneBank login (AY387829), is confirmed as a new gene.
The present invention utilizes molecule clone technology, encoding D-glycolylurea enzyme polynucleotide sequence directly is cloned into expression vector---plasmid pET-3a (Novagen company), this plasmid contains the T7 promotor, has ammonia benzyl resistance, constructed pET-HDT recon, utilize Calcium Chloride Method to transform the prokaryotic cell prokaryocyte system--in-the intestinal bacteria (E.coli BL21), obtain engineering bacteria.This project bacterium obtains solubility expression under the situation that does not need any inductor, expression amount accounts for 20% of total tropina, and enzyme work can reach 10.65U/mL (during fermented liquid 100D), far above the enzymic activity of having reported in the present document.
The engineering bacteria of above-mentioned structure, intestinal bacteria EHD-YZ26 (BL21/pET-HDT), this project bacterium is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC, China on November 24th, 2003, Beijing), preserving number is CGMCC No.1041.
The preparation method of D-glycolylurea enzyme provided by the invention, comprise the steps: that (a) is being fit to express under the condition of D-glycolylurea enzyme, cultivating above-mentioned preserving number is CGMCC No.1041 engineering bacteria, promptly cultivate 10～12h at 35--37 ℃, carry out second incubation with the switching of 4% inoculum size again, temperature is that 10h is cultivated in 30 ℃ of concussions, and at this moment enzyme is lived the highest.(b) with CGMCC No.1041 engineering bacteria through fermentation gained thalline, through centrifugal collection, in ice bath, use the ultrasonic disruption thalline, collect the ammonium sulfate precipitation part of 35%-60% saturation ratio then, obtain pure D-glycolylurea zymin through steps such as Phenyl-Sepharose hydrophobic chromatography post and Sephacryl S-200 gel chromatographies again, it can reach 62.0U/mg than work.
D-glycolylurea enzyme of the present invention and D-glycolylurea enzyme engineering bacteria EHD-YZ26 (pET-HDT/E.coilBL21) can be used for synthetic D-amino acid.Carrying out bio-transformation with described engineering bacteria cell, when being substrate as if the para hydroxybenzene glycolylurea, is 98% through HPLC analysis project bacterium transformation efficiency, high about 15 times of the natural bacterial strain of the efficiency ratio of engineering bacteria.
Four, description of drawings
The reaction mechanism synoptic diagram of Fig. 1 .D-glycolylurea enzyme catalysis 5 ' alternate glycolylurea
Fig. 2 .Pseudomonas putida YZ-II6 flagellar morphology (amplifying 2700 times)
Fig. 3 .Pseudomonas putida YZ-II6 bacterium 16SrDNA sequence
Fig. 4. the glycolylurea enzyme amino acid sequence comparison result of different sources
Pseudomonas putida YZ-II6 (the GenBank accession number is AY387829); Pseudomonas putidaCCRC12857 (the GenBank accession number is U84197); Pseudomonas putida DSM84 (the GenBank accession number is L24157); Agrobacterium radiobacter NRRLB11291 (the GenBank accession number is X91070); Ralstonia Pickettii (the GenBank accession number is AF320814)
Fig. 5. the building process of recombinant expression plasmid pET-HDT
Fig. 6. recombination bacillus coli EHD-YZ26 expresses D-glycolylurea enzyme SDS-PAGE and analyzes
The Mr standard molecular weight; Swimming lane 1:pET3a/E.coli.BL 21; Swimming lane 2:pET-HDT/E.coli.BL 21; Swimming lane 3: the supernatant of ultrasonication
Fig. 7. the purifying SDS-PAGE of reorganization D-glycolylurea enzyme analyzes
Mr: standard molecular weight; Swimming lane 1:pET3a/E.coli.BL 21; Swimming lane 2:pET-HDT/E.coli.BL 21; Swimming lane 3; The supernatant of ultrasonication; Swimming lane 4; 35%～60% (NH 4) 2SO 4Fractionation precipitation; Swimming lane 5:phenyl sepharose (16 * 10); Swimming lane 6; Sephacryl S-200 (16 * 100)
Fig. 8: the HPLC collection of illustrative plates of standard substance DL-para hydroxybenzene glycolylurea (DL-HPH) and N-carboxamide-D-D-pHPG (CpHPG)
Fig. 9: recombination bacillus coli EHD-YZ26 transforms the HPLC collection of illustrative plates 1.1ml thalline+1.5ml Tris-HCl (50mmol/L of DL-para hydroxybenzene glycolylurea (DL-HPH), pH8.0) contain saturated DL-para hydroxybenzene glycolylurea (DL-HPH), 1 hour product of 37 ℃ of reactions generates and base consumption; 2.1ml (50mmol/L pH8.0) contains saturated 4 hours product of 37 ℃ of conversion reactions of DL-para hydroxybenzene glycolylurea (DL-HPH) and generates and base consumption thalline+1.5mlTris-HCl;
Five, embodiment
The contrast accompanying drawing in conjunction with specific embodiments below, further set forth content of the present invention, the experimental technique of undeclared actual conditions among the embodiment, usually people's such as experiment condition such as Lu Shengdong " modern molecular biology experimental technique " (Higher Education Publishing House published in 1993) condition is carried out routinely, gives unnecessary details no longer in the present invention.
The structure of reorganization D-glycolylurea enzyme engineering bacteria
Embodiment 1: the screening and the morphologic observation of pseudomonas putida YZ-II6 (Pseudomonas putida YZ-II6) bacterial strain
The natural strain growth that this study group is screened voluntarily is on the M9 solid medium of improvement.Every liter of substratum contains Na 2HPO 46g, KH 2PO 43g, NaCl 0.5g, glycolylurea 1g adds water to 800ml, stirs, and solute is fully dissolved, and regulates pH value to 7.4, adds 15g agar then, 1.034 * 10 5Pa high pressure steam sterilization 20 minutes when treating that temperature is lower than 50 ℃, adds the following solution of degerming respectively: 2ml 1mol/L MgSO 4, 10ml 20% glucose, 0.1ml 1mol/L CaCL 2, 500 μ l 100mg/mL Amp, adding sterilized water again, to make volume be 1L, falls plate culture medium, cultivated 10 days at 30 ℃.The bacterium colony that to grow on improvement M9 flat board is used culture medium A (yeast powder 0.5%, peptone 0.5%, glycerine 0.5%, K respectively 2HPO 40.2%, glycolylurea 0.1% pH7.0) is cultivated again, is the enzyme activity determination that substrate carries out thalline then with the glycolylurea.Select the highest bacterial strain of enzymic activity as further research object.
Strain morphology and physico-chemical property are all undertaken by the big spade-shaped farm tool used in ancient China chief editor of king " systematic bacteriology " (1997):
1. gram staining liquid:
Viola crystallina liquid: first liquid: Viola crystallina 2g, 95% ethanol 20ml; Second liquid: ammonium oxalate 0.8g, distilled water 80ml.With first, second two liquid-phase mixing, leave standstill after 48 hours and use.Staining fluid is positioned in the brown bottle and preserves.
Iodine liquid: crystalline flake of iodine 1g, potassiumiodide 2g, distilled water 300ml.Earlier, drop into the crystalline flake of iodine again, treat that iodine all after the dissolving, adds entry and is diluted to 300ml with a small amount of (3-5ml) dissolved in distilled water iodate clock.Be positioned in the brown bottle and preserve.
Discoloring agent: 95% ethanol
Redye liquid: 0.5% the luxuriant red aqueous solution luxuriant red-ethanol liquid (2.5%) 20ml, distilled water 80ml.
2. flagella staining liquid:
A liquid: Weibull 5g, FeCL 31.5g, distilled water 100mL, formalin (15%) 2.0mL, NaOH (1%) 1.0mL.After preparing, used the same day, and next day, weak effect then should not use on the 3rd.
B liquid: AgNO 32g, distilled water 100mL.
Treat AgNO 3After the dissolving, it is standby to take out 10mL, to remaining 90mLAgNO 3In splash into dense NH 4OH makes it to become dense thick suspension, continues to be added dropwise to NH again 4OH is till the precipitation of new formation has just been dissolved again.The 10mLAgNO that will use again 3Mist then appears in very long splashing into, and after shaking gently, thin thing shape precipitation disappears again, splashes into AgNO again 3After shaking, still present till the slight and stable thin thing shape precipitation.As being mist heavy, this dye liquor can use a week, mist is heavy, then silver salt is settled out, and should not use.
Examine under a microscope thalline YZ-II6, its form is shaft-like, microbend, the blunt circle in two ends, one end is point slightly, and size is 0.5-0.6 μ m * 2.0-3.0 μ m, and the culture on the LB begins by faint yellow, old culture is a brown, and the bacterium colony circle is smooth, cymbidium Albert'stain Albert feminine gender.Single polar flagella (see figure 2).
In addition, the oxydase of this bacterial strain and superoxide enzyme test are positive reaction, and the glucose oxidase fermentation is determined as acid-producing bacteria; This bacterial strain can also resist certain density penbritin.With the increase of Amp concentration, biomass and glycolylurea enzymic activity are linear and descend, and thalline stops growing substantially when concentration reaches 400 μ g/mL, and the contrast bacterium promptly stops growing in 50-100 μ g/mL Amp concentration, so this strains has certain tolerance.
Embodiment 2: bacterial strain 16S rDNA amplification and sequential analysis
Bacterial strain YZ-II6 is seeded on the LB flat board (1% tryptone, 0.5% yeast extract, 1%NaCl, 1.5% agar) transfers pH to 7.2-7.4,1.034 * 10 with NaOH 5Pa high pressure steam sterilization 20 minutes, 28 ℃ of overnight incubation.Picking list bacterium colony is suspended in the 50 μ L sterile distilled waters, and in 100 ℃ of water-baths 5 minutes, centrifugal back supernatant was as pcr amplification 16S rDNA template, and the primer is pressed the universal primer of bibliographical information, and sequence is as follows:
P0：5′-GAGAGTTTGATCCTGGCTCAG-3′
P6：5′-CTACGGCTACCTTGTTACGA-3′
The pcr amplification reaction system
10*PCR buffer 25mM MgCl 2 4mM dNTP P0(150ng/uL) P6(150ng/uL) Template Taq DNA polymerase ddH2O 2.5uL 2.0uL 2.5uL 1.0uL 1.0uL 2.0uL 0.5U 13.5uL
Total volume 25uL
Earlier at 95 ℃ of sex change 5min, 94 ℃ of 1min of follow procedure then, 50 ℃ of 1min, 72 ℃ of 2min carry out 30 circulations altogether, and last 72 ℃ are extended 6min.Amplified production detects with 1% agarose gel electrophoresis, the extraordinary purpose band of specificity appears at the 1.5kb place, after the target DNA fragment recovery, be connected to carrier pGEM-T easy by molecule clone technology, after restriction analysis or PCR detection, the recon that contains goal gene entrusts the precious biotech firm in Dalian to measure 16S rDNA sequence.
The result shows that whole 16S rDNA total length is the 1439bp (see figure 3), on Internet, carry out data analysis with Blast software, it is the highest with the 16S rDNA sequence homology of false unit cell strain bacterium to disclose bacterial strain YZ-II6 16S rDNA sequence, wherein Score is the highest, the bacterial classification that the E value is minimum is Pseudomonas sp WBC-3, Pseudomonas PutidiaATCC17453, Pseudomonas Putidia ATCC 17514.Combining form is observed and biochemical reactions, can regard as the subspecies of Pseudomonas Putidia, names the YZ-II6 into Pseudomonas Putidia.
Embodiment 3: obtain the D-Hydantoinase gene from bacterial strain YZ-II6
The preparation of genomic dna will be at 5ml culture medium A (yeast powder 0.5%, peptone 0.5%, glycerine 0.5%, K 2HPO 40.2%, glycolylurea 0.1%, pH7.0) the middle bacterial strain YZ-II6 that cultivates gets the 1.5ml culture, the centrifugal 3min of 5000rpm.Throw out added the TE damping fluid of 567 μ L, blows and beats repeatedly with suction pipe and makes it resuspended, adds the Proteinase K mixing of 30 μ L 10%SDS and 3 μ L 20mg/mL, in 37 ℃ of incubations 1 hour.Add 100 μ L 5mol/L NaCl then, fully mixing adds 80 μ L CTAB/NaCl solution (the 0.7mol/L NaCl of 10%CTAB) again, and mixing is in 65 ℃ of incubation 10min.Add equal-volume chloroform/primary isoamyl alcohol, the centrifugal 5min of mixing.Supernatant liquor is changed in the new pipe, add isopyknic phenol/chloroform/primary isoamyl alcohol again, mixing, centrifugal 5min, supernatant liquor is changed in the new pipe of another, add 0.6 times of volume Virahol, mix up to DNA precipitating gently, with the thin glass tube of an end closure, transfer DNA washs to the ethanol of 1mL 70%.And then centrifugal 5min, abandon supernatant, dry a little on Freeze Drying Equipment, heavily be dissolved in the TE damping fluid of 100 μ L.-20 ℃ of preservations.Through the 1%Agarose detected through gel electrophoresis, a band is being arranged greater than 23kb, consistent with bacillus coli gene group DNA size.
Result according to strain identification, in GeneBank, look for the gene order of the possible D-glycolylurea enzyme in false pseudomonas bacillus source, with login gene order U84197, L24157, and a series of PCR primers of two tip designs of relevant bibliographical information test, wherein forward primer and RV1 combination primer amplification the best as a result.
Forward primer: 5 '-CCC GAA TTCCAT ATG TCC CTG TTG ATC CGT 3 '
Reverse primer: RV1:5 '-TTA GGA TCCTCA (AG) CG CTG AAC (TG) GG-3 '
RV2：5’-TTA GGA TCC TTA ACG CTG CAC GGG CGT CGG 3′
RV3：5’-TTA GGA TCC TCA(AG)CG CTG (CA)AC GGG (TC)GT CGG3′
RV4：5’-TTA GGA TCC T(AC)T (TC)TG TGC CGG CG(AT) TGG 3′
RV5：5’-TTA GGA TCC TTA T(AC)T TTG GCT TGC (CT)GG 3′
RV6：5’-TTA GGA TCC TTA TAT TTG CGA GCT TGC CGG 3′
The pcr amplification reaction system:
10*PCR buffer 25mM MgCl 2 4mM dNTP FW(10pmol/L) RV(10pmol/L) Template Taq DNA polymerase ddH 2O 2.5uL 4.0uL 2.5uL 2.5uL 2.5uL 1.0uL 0.5U 9.5uL
Total volume 25uL
After being sequentially added into reactant, pre-sex change 5min in the time of 95 ℃, add 0.5U Taq polymerase then, add isopyknic mineral oil (Sigma company) behind the mixing, 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 2min, 30 circulations, 72 ℃ are extended 10min, detect specific fragment about 1.5kb at 1.0% sepharose.
With the PCR product; by the gel reagents box operation of Shanghai China Shun bio-engineering corporation; carry out recovery, the purifying of PCR product; use Nde I/BamH I double digestion then; enzyme is cut the back fragment be connected to expression vector pET-3a (Novagen company) (see figure 5) of cutting through same enzyme; ligation system 10 μ L, external source fragment 5 μ L and pET-3a carrier 1 μ L and T 4Dna ligase damping fluid 1 μ L, aseptic deionized water 2.5 μ L, T 4Dna ligase is 0.5 μ L, spends the night 16 ℃ of connections.Get 5 μ L connection product and add the E.coli BL21 competent cell of 100 μ L with the calcium chloride preparation, ice bath 30min, 42 ℃ of heat-shocked 2min, ice bath 5min then, the LB liquid medium that adds 37 ℃ of preheatings of 900 μ L, cultivate 1h down at 37 ℃, get 200 μ L nutrient solutions to be coated on the flat board of 100 μ g/mL penbritins 37 ℃ of overnight incubation.
Half single bacterium colony of picking inserts 96 orifice plates from the culture plate, and each hole contains 50uL culture medium A (0.5% tryptone, 0.5% yeast extract, 0.5% glycerine, 0.2%K 2HPO 4, 0.1% glycolylurea, pH7.0) and 37 ℃ cultivated 18 hours, survey then and live.Surveying live body is the dihydro uracil that adds the 50mM of 50uL 50mmol/L Tris-HCl (pH8.0) damping fluid preparation in above-mentioned system, 37 ℃ were reacted l hour, add again 50uL 10% to dimethylin phenyl aldehyde (DMBA) solution in each hole, according to the depth of displaing yellow, carry out scalping.With second half picking from the culture plate of the bright recon bacterium colony of colour developing, in the 5mL test tube, cultivate 8h, it is centrifugal to get 100 μ L bacterium liquid, abandons supernatant, and 100 μ L sterilized waters suspend.Get 2 μ L as template, carry out pcr amplification again, the l%agarose detected through gel electrophoresis, residue bacterium liquid continues 37 ℃ of cultivations.The reorganization bacterium that pcr amplification is positive carries out plasmid extraction.Method adopts conventional alkaline lysis.Acquisition has the segmental recon of insertion.
Utilize the T7 promotor on the pET-3a carrier to carry out dna sequence analysis to the fragment of insertion recon pET-HDT is arranged for primer.Show that through sequencing the frame of amplified production from ATG to TGA is the 1440bp nucleotide fragments, go up Blast software with Internet then and compare this sequence, the result shows, with the D-Hydantoinase gene of false pseudomonas bacillus higher homology being arranged, is 63% with Pseudomonas Putidia DSM 84 (L24157) homology of known array on amino acid levels respectively; Pseudomonas Putidia CCRCl2857 (U84197) homology is 91.4%; Agrobacterium radiobacterNRRCBll291 (X91070) homology is 35.2%.But be about 34.8% (see figure 4) with Burkholderia pickettii MMR003 (AF320814).
Embodiment 4: the expression vector of reorganization D-Hydantoinase gene and the structure of engineering bacteria
Referring to accompanying drawing 5, the present invention in fact finishes the structure of recombinant vectors and screening gas in embodiment 3 of recombinant chou.Here concise and to the point again narration once is convenient to implement.The D-Hydantoinase gene be with 5 '-CCC GAA TTCCAT ATG TCC CTGTTG ATC CGT 3 ' and 5 '-TTA GGA TCCTwo primers of TCA (AG) CG CTG AAC (TG) GG-3 ' are template with the genomic dna, pre-sex change 5min in the time of 95 ℃, add 0.5U Taq polymerase then, add isopyknic mineral oil (Sigma company) behind the mixing, 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 2min, 30 circulations, 72 ℃ are extended 10min, pcr amplified fragment, then through Nde I/BamH I double digestion, enzyme is cut the back fragment be connected to the expression vector pET-3a (Novagen company) that cuts through same enzyme, ligation system 10 μ L, external source fragment 5 μ L and pET-3a carrier 1 μ L and T 4Dna ligase damping fluid 1 μ L, aseptic deionized water 2.5 μ L, T 4Dna ligase is 0.5 μ L, spends the night 16 ℃ of connections.Get 5 μ L connection product and add the E.coli BL21 competent cell of 100 μ L with the calcium chloride preparation, ice bath 30min, 42 ℃ of heat-shocked 2min, ice bath 5min then, the LB liquid medium that adds 37 ℃ of preheatings of 900 μ L, cultivate 1h down at 37 ℃, get 200 μ L nutrient solutions to be coated on the flat board of 100 μ g/mL penbritins 37 ℃ of overnight incubation.
Half single bacterium colony of picking inserts 96 orifice plates from the culture plate, and each hole contains 50uL YCG substratum (0.5% tryptone, 0.5% yeast extract, 0.5% glycerine, 0.2%K 2HPO 4, 0.1% glycolylurea pH7.0) was cultivated 18 hours for 37 ℃, surveyed then and lived.Surveying live body is the dihydro uracil that adds the 50mM of 50uL 50mmol/L Tris-HCl (pH8.0) damping fluid preparation in above-mentioned system, 37 ℃ were reacted 1 hour, add again 50uL 10% to dimethylin phenyl aldehyde (DMBA) solution in each hole, according to the depth of displaing yellow, carry out scalping.With second half picking from the culture plate of the bright recon bacterium colony of colour developing, in the 5mL test tube, cultivate 8h, it is centrifugal to get 100 μ L bacterium liquid, abandons supernatant, and 100 μ L sterilized waters suspend.Get 2 μ L as template, carry out pcr amplification again, the 1%agarose detected through gel electrophoresis, residue bacterium liquid continues 37 ℃ of cultivations.The reorganization bacterium that pcr amplification is positive carries out plasmid extraction.Method adopts conventional alkaline lysis.Acquisition has the segmental recon (seeing accompanying drawing 5) of insertion.
To show the vigor height, and the BL21/pET-HDT engineering bacteria of stable good stability, called after EHD-YZ26, so this project bacterium on November 24th, 2003 was deposited in the common micro-organisms center (CGMCC of China Committee for Culture Collection of Microorganisms, China, Beijing), preserving number is CGMCC No.1041.
Embodiment 5: the expression of reorganization D-Hydantoinase gene
Picking list bacterium colony is connected to 5ml LB (the containing 100ug/mL Amp) liquid nutrient medium from flat board, cultivates 10～12h for 37 ℃.Get above-mentioned pre-cultivation bacterium and transfer in the fresh LB nutrient solution that contains microbiotic Amp (100ug/mL) with 4% inoculum size, 10h is cultivated in 30 ℃ of concussions.4 ℃ of centrifugal collection thalline of 5000rpm carry out enzyme activity determination.And with SDS-PAGE gel electrophoresis analysis expression product and expression amount, the amount of solubility expression product accounts for 20% (seeing accompanying drawing 6) of whole cell.
The preparation of D-glycolylurea enzyme
Embodiment 6: the preparation of reorganization D-glycolylurea enzyme
PET-HDT/E.coli BL21 engineering bacteria is cultivated in a large number by above-mentioned expression condition, obtain fermented liquid, through 4 ℃ of centrifugal collection thalline, with 50mmol/L Tris-HCl (pH8.0) damping fluid, suspension thalline (1g thalline weight in wet base/10mL damping fluid), use the ultrasonic disruption cell in ice bath, 4 ℃ of centrifugal 30min of 15000rpm remove insoluble substances such as cell debris then.Collect the ammonium sulfate precipitation part of 35%-60% saturation ratio.To precipitate with above-mentioned damping fluid and dissolve again and 1mol/L (NH 4) 2SO 4Carry out dialysed overnight, then with dialyzate (～50-60mg) by using 1mol/L (NH in advance 4) 2SO 4Equilibrated Phenyl-Sepharose hydrophobic chromatography post (16cm * 10cm).(NH with 0.2mol 4) 2SO 4Above-mentioned buffer solution elution do not have to absorb until 280nm and get back to baseline, and then use the distilled water wash-out.Distilled water wash-out sample segment concentrates through Amico ultra-fine filter PM30, and then goes up sample Sephacryl S-200 gel (LKB chromatographic system) chromatography, obtains the pure glycolylurea enzyme of SDS-PAGE, and enzymic activity always is recovered as 32.37%, is 62U/mg (seeing accompanying drawing 7) than vigor.
The enzyme assay of embodiment 7:D-glycolylurea
According to the textural property of D-glycolylurea enzymic catalytic reaction and product, can use indirect method (to the dimethylin phenyl aldehyde, DMBA), and behind the reaction terminating, through color measurenent OD 430nmEnzymic activity is calculated in photoabsorption.Also available direct method (HPLC), when the phenyl ring that is had a conjugated double bond when the substrate glycolylurea substituted, enzyme reaction can use HPLC to carry out the quantitative assay (see figure 8) after stopping.
1. to dimethylin phenyl aldehyde (DMBA) method
Enzyme assay improves a little by literature method.There were significant differences because the enzyme of original bacterium and engineering bacteria is lived, the biomass difference of getting, usually get original bacterium 1mL or engineering bacteria 300 μ L, centrifugal 10min, the gained thalline is washed once with 50mmol/L Tris-HCl (pH8.0), uses the same buffer suspended bacteria of 1.5mL 2% glycolylurea again, react certain hour respectively in 37 ℃ of reactions, add 0.25mL 10% trichoroacetic acid(TCA) termination reaction, add again 0.25mL 10% to dimethylin phenyl aldehyde (DMBA) solution, read OD 430nmPhotoabsorption.N-carbamino alanine with concentration known is the standard product, and under the same system situation, the drawing standard curve is obtained the K value, according to formula, calculates enzymic activity.
2.HPLC method
Get an amount of thalline and substrate (phenylglycine or D-D-pHPG) in D-para hydroxybenzene glycolylurea (D-HPH)-after fixing time, 10000rpm is centrifugal, get supernatant liquor and directly go up sample, post is C18 (280mm * 4.6mm), with 50mmol/LNaAc-HAc (pH4.2): methyl alcohol (90: 10) wash-out, can carry out quantitative assay according to the appearance time and the peak area of standard substance.
Unit of enzyme activity (U) is defined as under these conditions, when cell concentration is 10 OD 600nmThe time, every mL thalline conversion of substrate in 1min generates the required enzyme amount of 1 μ mol carbamino alanine.
Enzymic activity (U/mL)=K * OD 430nm* extension rate/t
Enzyme is to have narrow spectrum biological catalyst, different substrates there is different catalytic activitys, because the alternative group difference of substrate molecule, they with the collision probability of enzyme molecule, interaction sterically hindered and hydrophilic and hydrophobic grouping all might be different, therefore show the difference of enzymatic activity under the same conditions.Engineering bacteria and reactivity such as the following table of natural bacterium D-glycolylurea enzyme to different substrates.Dihydrouracil is its suitableeest substrate as can be seen, so the name of this enzyme claims the dihydrouracil enzyme again.
Original bacterium and catalyzed reaction such as the following table of engineering bacteria to different substrates
Contain the application of D-glycolylurea enzyme engineering bacteria
Embodiment 8: contain D-glycolylurea enzyme engineering bacteria catalytic production N-carboxamide D-pHPG
Get by embodiment 5, picking list bacterium colony is connected to 5ml LB (the containing 100ug/mL Amp) liquid nutrient medium from flat board, cultivates 10～12h for 37 ℃.Get above-mentioned pre-cultivation bacterium and transfer in the fresh LB nutrient solution that contains microbiotic Amp (100ug/mL) with 4% inoculum size, 10h is cultivated in 30 ℃ of concussions.Get the 1ml thalline, 4 ℃ of centrifugal collection thalline of 5000rpm, wash thalline twice with 0.1mol/LTris-HCl (pH8.0), add 0.5% D-para hydroxybenzene glycolylurea (D-HPH) then with 0.1mol/L Tris-HCl (pH8.0) preparation, 37 ℃ of shaking tables reactions, the reaction times is 1 hour, after 4 hours, sampling is measured N-carboxamide D-pHPG (CpHPG) (see figure 9) of its generation through HPLC, and the reaction times is 5 hours, and its transformation efficiency can reach 98%.

Claims (11)

1, a kind of isolating D-glycolylurea enzyme is characterised in that its aminoacid sequence is:
1 MSLLIRGATV VTHEESYPAD VLCADGLIRA IGQNLEPPTD CEILDGSGQY LMPGGIDPHT
61 HMQLPFMGTV ASEDFFSGTA AGLAGGTTSI IDFVIPNPQQ SLLEAFHTWR GWAQKSASDY
121 GFHVAITWWS EQVAEEMGEL VAKHGVNSFK HFMAYKNAIM AADNTLVASF ERCLQLGAVP
181 TVHAENGELV YHLQKKLLAQ GITGPEAHPL SRPSQVEGEA ASRAIRIAET LGTPLYLVHI
241 SSREALDEIA YARGKGQPVY GEVLPGHLLL DDSVYRDPDW ATAAGYVMSP PFRPREHQEA
301 LWRGLQSGNL HTTATDHCCF CAEQKAMGRD DFSRIPNGTA GIEDRMAVLW DAGVNSGRLS
361 MHEFVALTST NTAKIFNLFP RKGAIRVGAD ADLVLWDPQG TRTISAQTHH QQVDFNIFEG
421 RTVRGIPSHT ISQGKVLWAD GDLRAEPGAG RYVERPAYPS VYEVLGRRAE HQRPMPVQR
2, the polynucleotide of the described D-glycolylurea of a kind of claim 1 of encoding enzyme, its nucleotides sequence is classified as:
1 ATGTCCCTGT TGATCCGTGG CGCCACCGTG GTTACCCACG AAGAGAGTTA CCCCGCCGAT
61 GTCCTGTGTG CCGATGGCCT GATCCGTGCC ATCGGGCAAA ACCTCGAACC ACCCACCGAC
121 TGTGAGATCC TCGACGGCAG CGGCCAGTAC CTGATGCCCG GCGGCATCGA CCCGCACACC
181 CACATGCAGT TGCCGTTCAT GGGCACTGTA GCCAGCGAGG ACTTCTTCAG CGGCACCGCT
241 GCGGGCCTGG CAGGCGGCAC CACTTCGATC ATCGACTTCG TCATACCCAA CCCGCAGCAG
301 TCGTTGCTGG AGGCCTTCCA CACCTGGCGT GGCTGGGCGC AGAAAAGCGC CAGCGACTAT
361 GGCTTCCACG TCGCCATCAC CTGGTGGAGC GAGCAGGTGG CCGAAGAGAT GGGCGAGCTG
421 GTGGCCAAGC ACGGGGTGAA CAGCTTCAAG CATTTCATGG CCTACAAGAA TGCGATCATG
481 GCCGCCGACA ATACCTTGGT GGCCAGCTTC GAGCGTTGCC TGCAACTGGG CGCGGTGCCC
541 ACCGTGCATG CCGAGAACGG CGAGCTGGTG TACCACCTGC AGAAAAAACT GCTCGCCCAG
601 GGCATAACCG GGCCGGAAGC CCACCCGCTG TCACGCCCCT CGCAGGTTGA AGGCGAAGCA
661 GCCAGCCGCG CCATCCGCAT TGCCGAAACC CTCGGCACGC CGTTGTACCT GGTGCACATT
721 TCCAGCCGCG AGGCACTGGA TGAAATCGCT TATGCCCGAG GCAAGGGCCA GCCGGTGTAT
781 GGCGAGGTGT TGCCCGGCCA CCTGCTGCTG GATGACAGCG TCTACCGTGA CCCGGACTGG
841 GCCACCGCAG CCGGTTATGT AATGAGCCCG CCGTTCCGCC CACGCGAGCA CCAGGAAGCG
901 CTGTGGCGCG GCTTGCAGTC GGGCAACCTG CACACCACCG CCACCGACCA CTGCTGCTTC
961 TGCGCCGAGC AGAAAGCCAT GGGCCGCGAC GACTTCAGCC GCATCCCCAA CGGCACCGCC
1021 GGCATCGAAG ACCGCATGGC GGTACTGTGG GATGCCGGGG TCAACAGCGG GCGCCTGTCG
1081 ATGCACGAAT TCGTCGCGCT GACCTCTACC AATACGGCGA AAATCTTCAA CCTGTTCCCG
1141 CGCAAGGGCG CTATCCGCGT GGGTGCCGAT GCCGACCTGG TGCTGTGGGA CCCGCAGGGC
1201 ACCCGCACCA TCTCCGCCCA AACCCACCAC CAGCAGGTGG ACTTCAACAT TTTCGAAGGC
1261 CGCACCGTGC GCGGCATCCC AAGCCACACC ATCAGCCAGG GCAAGGTGCT CTGGGCCGAT
1321 GGCGACTTGC GTGCCGAACC GGGCGlGGGG CGCTATGTGG AACGGCCGGC GTACCCGTCA
1381 GTGTATGAGG TGCTGGGGCG CCGGGCCGAG CATCAGCGGC CGATGCCAGT TCAGCGCTGA
3, a kind of carrier is characterized in that, it contains the described polynucleotide of claim 2.
4, the carrier of claim 3, it is a plasmid.
5, the carrier of claim 3, it contains the T7 promotor.
6, a kind of engineering bacteria is characterized in that it contains the described carrier of claim 3.
7, the described engineering bacteria of claim 6 is the prokaryotic cell prokaryocyte system.
8, the prokaryotic cell prokaryocyte system of claim 7 is intestinal bacteria EHD-YZ26 (BL21-pET-HDT), and preserving number is CGMCCNo.1041.
9, a kind of preparation method of D-glycolylurea enzyme is characterized in that comprising the steps:
(a) under the condition that is fit to expression D-glycolylurea enzyme, cultivating above-mentioned preserving number is CGMCC No.1041 engineering bacteria;
(b) separation and purification D-glycolylurea enzyme from culture.
10, claim 1 described D-glycolylurea enzyme and the described engineering bacteria of claim 6 purposes in synthetic D-amino acid.
11, the purposes of claim 10, wherein said D-amino acid is the D-D-pHPG.
CNB2004100008423A 2004-01-05 2004-01-05 Recombined D-hydantoin enzyme and application thereof Expired - Fee Related CN1243096C (en)

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