CN103243054B - Strain capable of producing carboxyethylhydantoinase and application thereof - Google Patents
Strain capable of producing carboxyethylhydantoinase and application thereof Download PDFInfo
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Abstract
The invention relates to a strain capable of producing carboxyethylhydantoinase. The strain capable of producing carboxyethylhydantoinase is characterized in that classification name of the strain is alcaligenes faecalis subsp.faecalis, and the strain is preserved in China General Microbiological Culture Center on May 13th, 2013 with the preservation number of CGMCC No.7585. The invention also discloses an application of the strain capable of producing carboxyethylhydantoinase. Activity of carboxyethylhydantoinase produced by the strain alcaligenes faecalis subsp.faecalis is 0.064U/ml, the activity of purified carboxyethylhydantoinase can be 0.61U/ml, and enzyme activity of a constructed genetically engineered bacterium is 3.06U/ml, and a fermentation method of recombinant bacteria is simple. Conversion rate of substrate is high, the conversion rate of the substrate after reaction is carried out for 2 hours is 95%, the conversion rate of the substrate after reaction is carried out for 4 hours is 99.98%, and the strain has a better application prospect in catalyzing synthesis of carboxyethylhydantoinase.
Description
Technical field
The invention belongs to biotechnology and technical field of enzyme engineering, be specifically related to propyloic hydantoin enzyme and produced bacterial strain and application thereof.
Background technology
The catabolic pathway of L-Histidine mainly contains following 5: 1, decarboxylation becomes histamine, is further oxidized into imidazoles acetaldehyde and imidazoleacetic acid; 2, deaminizating becomes urocanic acid, is further degraded to L-glutamic acid, enters glutamic acid metabolism approach; 3, transamination forms imidazolylpyruvic acid, and then is reduced to imidazole lactic acid or imidazoleacetic acid; 4, methylation, forms 1-or 3-Methyl histidine; 5, form carnosine or anserine with Beta-alanine condensation reaction.Wherein the 2nd article of fundamental approach that approach is L-Histidine metabolism, all more conservative from prokaryotic organism to Eukaryotic each species, obtain Pidolidone by L-Histidine through stages such as deamination, urocanic acid hydrolysis, carbon-based group transfers, and be converted into α-ketoglutaric acid by Pidolidone and enter tricarboxylic acid cycle.For having had a lot of research by L-Histidine to the assignment of genes gene mapping, the relevant enzyme structure elucidation of the relevant enzyme of encoding in the various enzymes that relate in the degradation pathway of Pidolidone, intermediate product, some species in bacterium and Mammals.
But in all L-Histidine pathways metabolism figure, all exist one by L-Histidine the branch road degradation pathway to Pidolidone.It is indicated from 4-imidazolone-5-propionic acid, can produce L-glycolylurea-5-propionic acid through peroxidation, and then becomes Pidolidone by the effect hydrolysis of propyloic hydantoin enzyme and carbamyl hydrolysis enzyme.Than L-Histidine → urocanic acid → 4-imidazolone 5-propionic acid → N-formiminoglutamic acid → Pidolidone approach, except early stage a few studies person has carried out some preliminary researchs, at present, this approach becomes one " corner passing into silence " substantially.Wherein be oxidized into the step research of L-glycolylurea-5-propionic acid for 4-imidazolone-5-propionic acid more deep in the 50-60 age in 20th century, and be all unknown numbers for classification, character, structure and the gene regulating of two one-step hydrolysis enzymes in this approach.
Because glycolylurea-5-propionic acid has detected the existence of L-carbamylglutamic acid in microbial transformation process, therefore, existing report all thinks that glycolylurea-5-propionic acid passes through propyloic hydantoin enzyme (Carboxyethylhadantionase in microorganism cells, CEHase, E.C. unfiled) hydrolysis L-, D-or DL-glycolylurea-5-propionic acid generation N-carboxamide-Pidolidone, and further under the effect of L-carbamyl hydrolysis enzyme (L-Carbamoylase), be hydrolyzed and produce Pidolidone.
Although propyloic hydantoin enzyme catalyzed reaction in form and hydantoin enzyme belong to a class together, due to many reasons, the research of propyloic hydantoin enzyme is almost to blank both at home and abroad at present.
Summary of the invention
Goal of the invention: first object of the present invention is to provide propyloic hydantoin enzyme and produces bacterial strain.Second object of the present invention is to provide propyloic hydantoin enzyme.The 3rd object of the present invention is to provide the recombinant vectors for expressing propyloic hydantoin enzyme.The 4th object of the present invention is to provide expresses the host cell of above-mentioned recombinant vectors and the preparation method of this host cell thereof.The 5th object of the present invention is to provide the method for utilizing above-mentioned host cell to produce N-carboxamide-Pidolidone.
Technical scheme: in order to solve the problems of the technologies described above, the technical solution adopted in the present invention is: a kind of propyloic glycolylurea enzyme-producing bacteria, its Classification And Nomenclature is Alcaligenes faecalis excrement subspecies (Alcaligenes faecalis subsp.faecalis), this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on May 13rd, 2013, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101, deposit number is CGMCC No.7585.Wherein, propyloic hydantoin enzyme is produced bacterial strain Alcaligenes faecalis excrement subspecies (Alcaligenes faecalis subsp.faecalis) and is abbreviated as CW221.
A kind of propyloic hydantoin enzyme, described propyloic hydantoin enzyme derives from above-mentioned propyloic glycolylurea enzyme-producing bacteria.
Described propyloic hydantoin enzyme, its nucleotide sequence is as shown in SEQ ID NO:1.
Described propyloic hydantoin enzyme, its aminoacid sequence is as shown in SEQ ID NO:2.
For expressing a recombinant vectors for propyloic hydantoin enzyme, it contains above-mentioned nucleotide sequence.
Described recombinant vectors is pET22b.
A kind of host cell, it contains above-mentioned recombinant vectors.
Described host cell is intestinal bacteria.
Prepare the method for above-mentioned host cell, build the recombinant vectors pET22b of above-mentioned nucleotide sequence, and express in intestinal bacteria E.coli BL21, the recombinant bacterium that has obtained product propyloic hydantoin enzyme is host cell.
Produce a method for N-carboxamide-Pidolidone, obtain fermented liquid with the host cell described in LB culture medium culturing, through cytoclasis, centrifuging and taking supernatant liquor adds propyloic glycolylurea solution to carry out conversion reaction.
Beneficial effect: compared with prior art, advantage of the present invention is: the activity that propyloic hydantoin enzyme is produced the propyloic hydantoin enzyme of bacterial strain Alcaligenes faecalis excrement subspecies (Alcaligenes faecalis subsp.faecalis) is 0.064U/ml, enzyme work after purifying can reach 0.61U/ml, the enzyme of the genetic engineering bacterium building is lived as 3.06U/ml, and recombinant bacterium fermentation process is simple.High to substrate conversion efficiency, it is 99.98% that reaction carries out after 2 hours the transformation efficiency of substrate to reach the transformation efficiency of 95%, 4 hour, in catalysis propyloic glycolylurea is synthetic, has good application prospect.
Brief description of the drawings
Fig. 1 is the SDS-PAGE electrophorogram of the propyloic hydantoin enzyme of bacterial strain Alcaligenes faecalis excrement subspecies (Alcaligenes faecalis subsp.faecalis), wherein swimming lane 1:Marker; Swimming lane 2:60%～80% sulphur ammonium precipitation; Enzyme liquid after swimming lane 3:DEAE-Sepharose purifying; Swimming lane 4, enzyme liquid after 5:15Q purifying.
Fig. 2 is the PCR electrophorogram of propyloic hydantoin enzyme, wherein swimming lane 1 and 2: propyloic Hydantoinase gene PCR fragment; Swimming lane 3:MarkerDL2000.
Fig. 3 is the design of graphics of propyloic Hydantoinase gene engineering bacteria.
Fig. 4 is the SDS-PAGE electrophorogram of recombinant bacterium expressing protein, wherein swimming lane 1: albumen Marker swimming lane 2:E.coliBL21 band, the albumen that swimming lane 3-6:E.coliBL21 (DE3)/amd-his-pET-22b expresses
Embodiment
Below by specific embodiment, the present invention is further described; it should be pointed out that for the person of ordinary skill of the art, under the premise without departing from the principles of the invention; can also make some modification and improvement, these also should be considered as belonging to protection scope of the present invention.
Embodiment 1: this description of test produces the screening process of the natural bacterial strain of propyloic hydantoin enzyme.
Substratum:
A. broth culture (g/L): extractum carnis 3, peptone 10, NaCl5, agar 20, pH=7.2
B. enrichment medium (g/L): propyloic glycolylurea 5, glucose 3, peptone 3, potassium primary phosphate 5, magnesium sulfate 0.5, ferrous sulfate 0.055, manganous sulfate 0.0045, pH7.0
C. screening culture medium (g/L): add 20gL in enrichment medium
-1agar powder
D. seed culture medium (g/L): glucose 15, peptone 15, sodium-chlor 3, yeast extract paste 5, potassium primary phosphate 2,
Magnesium sulfate 0.25, pH=7.0-7.2
E. fermention medium (g/L): glucose 10, peptone 10, yeast extract paste 10, sodium-chlor 3, potassium primary phosphate 2, magnesium sulfate 0.25, cobalt chloride 0.05, propyloic glycolylurea 2, pH=7.0
All substratum are all with deionized water preparation, 0.1MPa, and 121 DEG C of sterilizings 15 minutes, cooling rear for subsequent use.
Prescreening method is as follows: get the soil sample 5g collecting, put into enrichment medium 50mL, cultivate 3d on shaking table; Leave standstill after 5min, draw 5mL upper liquid to new enrichment medium 50mL, and the corresponding propyloic glycolylurea add-on that doubles, continue to cultivate 3d.So repeat above-mentioned steps 2～3 times.Nutrient solution after enrichment leaves standstill after 10min, draws 1mL upper liquid, carries out gradient dilution, chooses 10
-3-10
-5part 1mL, to screening culture medium, is coated with and opens with spreading rod, cultivates 3d in incubator.By cultured dull and stereotyped taking-up, zoning overleaf, chooses single bacterium colony part with toothpick, proceeds to new broth culture numbering; Meanwhile, choose single bacterium colony part access seed culture medium (shaking pipe) with toothpick, after cultivation 1d, separate at the flat lining out of broth culture.The method obtains has bacterial strain 30 strains of propyloic hydantoin enzyme enzyme activity, and the bacterial strain that wherein 24h broth culture colony diameter exceedes 1mm only has 5 strains.
In order further to check the ability of bacterial strain secretion propyloic hydantoin enzyme, the bacterial strain by 5 strains of choosing in primary dcreening operation containing propyloic hydantoin enzyme vigor, access fermention medium, 3 bottles of each bacterial classifications (250mL bottle), 35 DEG C, 180rpm, cultivates 24h; After cultivation finishes, get 1mL fermented liquid, add in the substrate solution preparing, transform 2h, finally screening obtains the higher bacterial strain CW221 of 1 strain propyloic hydantoin enzyme vigor.
Propyloic hydantoin enzyme vigor testing methods: get 25mL fermented liquid, centrifugal (8000rpm, 15min), with physiological saline washing 1 time, recentrifuge (8000rpm, 15min) is got thalline; Thalline adds the propyloic glycolylurea solution of the 20mL2g/L preparing, and 37 DEG C, transform 1h, after conversion finishes, add 120g/L trichloroacetic acid solution stopped reaction; Centrifugal (8000rpm, 15min), draws 1mL supernatant liquor, adds 1mL paradimethy laminobenzaldehyde nitrite ion, after concussion mixes, measures absorbance value in 438nm place.One Ge Meihuo unit (U) is defined as under above-mentioned reaction conditions, generates the required enzyme amount of 1 μ molN-carboxamide-Pidolidone in 1min; The ratio vigor (U/mL) of enzyme is defined as every milliliter of enzyme units alive that fermented liquid is contained.
Wherein propyloic glycolylurea is this laboratory self-control, its preparation method is D, Pidolidone (0.2mol, 29.4g) (be purchased from Yuan Ju bio tech ltd, Shanghai), Zassol (0.24mol, 15.6g) and sodium hydroxide (0.2mol, 8g) be dissolved in 100mL water, 70 DEG C of stirring and dissolving, reaction 2h, generate intermediate product N-carboxamide-D, Pidolidone, slowly drip 50mL concentrated hydrochloric acid, be acidified to pH=1, be warmed up to 100 DEG C, heating reflux reaction 2h, reaction finishes, filtered while hot, filtrate proceeds in beaker and leaves standstill, cooling, there are a large amount of solids to separate out, filter, crude product water recrystallization, obtain propyloic glycolylurea.
Embodiment 2: the biological property qualification of this description of test propyloic glycolylurea enzyme-producing bacteria CW221.
The biological property of propyloic glycolylurea enzyme-producing bacteria CW221: high power lens is observed, the main ovalize of cell, rod-short, long-width ratio, about 2-3 left and right, also has the cell of almost spherical, and cell arrangement is random, is Individual existence, without bunchiness or cell existence in groups.Gram-negative bacteria; On solid medium flat board, 24h growth bacterium colony is rounded, diameter 1mm, and smooth surface, oyster white, opaque, edge unfairness, centre is projection slightly; Streak culture on inclined-plane, bacterium colony prolongs line growth, and edge unfairness has spinule; In shaking flask, cultivate, nutrient solution is creamy white, opaque.Physio-biochemical characteristics show, this bacterium is that obligate is aerobic, can not utilize glucose all negative as carbon source, VP experiment and MR experimental result, and not hydrolyzed starch can not liquefy gelatin, and oxidable gluconate can not reduce nitrate.
The Biolog qualification result of CW221 bacterial strain is: Alcaligenes faecalis excrement subspecies (Alcaligenes faecalis subsp.faecalis), Proability(possibility): 100%, Sim(similarity value): 0.574.The PCR product that is 16s rDNA sequence is 1426bp, and this 16s rDNA sequence accession number in GenBank is GQ222232.Sequencing result compares by NCBI BLAST database and known array (homology >99%), and result shows that CW221 bacterial strain and Alcaligenes (Alkaligenes) homology can reach 99%.Comprehensive two qualification results, this bacterium can be identified to belonging to, and it belongs to Alcaligenes (Alcaligenes), called after Alcaligenes faecalis excrement subspecies (Alcaligenes faecalis subsp.faecalis).
Embodiment 3: the purification step of this description of test propyloic hydantoin enzyme.
Buffer A: 0.02mol/L Tris-HCl, pH9.0,
Take the fermentation process that fermentation thalline 15g(fermentation process refers to screening process in embodiment 1) be dissolved in the buffer A of 60ml, be mixed with bacteria suspension.After ultrasonication (power 400W, protects 25 DEG C of temperature for ultrasonic time 3s, intermittent time 5s, and omnidistance 10min, repeats 3 times), the centrifugal 30min of 10000r/min at 4 DEG C, collects supernatant liquor.Through 20%, 40%, 60%, 80% saturation ratio ammonium sulfate precipitation.Centrifugal collecting precipitation, is dissolved in buffer A, and is proceeded to dialysis tubing, 4 DEG C of dialysis of same buffer, more concentrated through PEG-400.Get DEAESepharose F.F anion-exchange column on concentrating sample, do linear gradient elution with buffer A and 1mol/L NaCl, flow velocity 2ml/min collects concentrated Peak Activity.After detection protein concentration and enzyme are lived, choose the rear upper Source15Q reinforcing yin essence ion exchange column of activated enzyme liquid dialysis, the concentrated Peak Activity of collection.Adopt SDS-PAGE to measure the molecular weight of the zymoprotein after purifying.By SDS-PAGE electrophoresis (Fig. 1), find that propyloic hydantoin enzyme has reached electrophoresis pure, the molecular weight of single subunit is 50kDa, purification is 11.929 and the rate of recovery 19.63%.Gather in table 1.
Table 1 propyloic hydantoin enzyme purifying is summed up
Note: protein concn adopts Xylene Brilliant Cyanine G method to measure.
Table 1
Embodiment 4: the separating clone program of this description of test propyloic Hydantoinase gene (amd).
The propyloic hydantoin enzyme of purifying (entrusting Research Centre for Proteome Analysis(Shanghai) to carry out) is carried out to MALDI-TOF/TOF and N end mensuration, hydroamidase (gi|393162052amidohydrolase[Alcaligenes faecalis subsp.faecalis the NCIB8687]) sequence that shows to derive from this enzyme and ncbi database the gi|393162052 that Alcaligenes faecalis NCIB8687 produces is close, find the gene order of this enzyme, find its conserved sequence, design thus primer, amplification obtains the gene order of propyloic hydantoin enzyme, the PCR fragment electrophoresis that contains this enzyme gene order is reclaimed, be cloned into pET-22b carrier, carry out sequential analysis.It is that primer 1amd-his-up and downstream primer are primer 2 amd-his-down that the primer of design comprises upstream primer, and wherein primer 1 and primer 2 are the primer that includes his-tag label, and primer is (underscore part is restriction enzyme site):
Primer 1:amd-his-up5 '-3 ' ggA ATT C
cA TAT gagCgA TTT TgA TCT ggTAgT
Primer 2: amd-his-down5 '-3 ' CCC
cTA gAgtgA CAA TTC GccTgC TTT g
CW221 bacterial classification is inoculated in the liquid nutrient medium of LB+Amp of 4ml, 37 DEG C are cultured to logarithmic phase, with adopt like pursue progress Bioisystech Co., Ltd produce AxyPrep
tMmultisource Genomic DNAMiniprep Kit50-prep Nucleic Acid Purification Kit test kit extracts its genome and obtains CW221 genome.
PCR reaction system (50 μ l system)
PCR reaction parameter is: 98 DEG C of sex change 10s, and 58 DEG C of annealing 15s, 72 DEG C are extended 90s, by above-mentioned parameter circulation 30 times.Last 72 DEG C are extended 10min.According to this reaction conditions, the PCR fragment (Fig. 2) of the amd gene of 1400bp has been arrived in amplification.This fragment is connected to pET-22b carrier and obtains product amd-his-pET-22b, carry out sequencing.Result shows, this fragment total length 1365bp, 455 aminoacid sequences of can encoding.Hydroamidase (amidohydrolase of Alcaligenesfaecalis subsp.FaecalisNCIB8687) the gene similarity of propyloic hydantoin enzyme sequence and Alcaligenes faecalis NCIB8687 is 99%.But on NCBI, this fragment gene sequence is not characterized, also, not about the research of this fragment gene sequence, therefore this blank has been filled up in research work of the present invention.
Embodiment 5: the structure of this description of test propyloic hydantoin enzyme recombinant bacterium.
The propyloic Hydantoinase gene (amd) amplifying is used restriction enzyme Xho I after glue reclaims purifying, Nde I is carried out double digestion, carrier pET-22b is carried out to double digestion with identical restriction enzyme simultaneously, fragment after enzyme is cut is carried out purifying through row rubber tapping recovery and ethanol precipitation respectively, then they is spent the night and obtains connecting product amd-his-pET-22b (Fig. 3) with 16 DEG C of connections of T4 ligase enzyme.Get the each 20 μ l of 2 pipe competent cell E.coliDH10B, wherein add 1 μ l connection product amd-his-pET-22b to be transformed.Mixed solution is proceeded in electric conversion pool, and 2.5V shocks by electricity.Every pipe adds 400 μ l SOC liquid nutrient mediums, and 37 DEG C of shaking tables are cultivated 1h(and shaken slowly).The competent cell of conversion is coated on (concentration of penbritin is 100 μ g/ml) on the LB flat board that contains ammonia benzyl resistance.Be inverted plate, 37 DEG C of constant temperature culture 12～16h, to occurring single bacterium colony.Picking positive colony, in the LB liquid nutrient medium of 100 μ g/ml, cultivates 10h for 37 DEG C, extracts plasmid, the order-checking of Hou Song Si Pujin Bioisystech Co., Ltd.To be converted into expressive host E.coliBL21 (DE3) bacterial strain through the recombinant plasmid amd-his-pET-22b that is accredited as positive colony, proceed to identical host as blank using pET-22b (+) plasmid simultaneously.The single bacterium colony growing on picking resistance screening flat board, access is containing in 100 μ g/ml penbritin LB liquid nutrient mediums, and 37 DEG C of shaking culture are spent the night.Then be inoculated in fresh culture (containing 100 μ g/ml penbritins) by 2% inoculum size, 37 DEG C are cultured to OD
600be about at 0.6 o'clock, add IPTG to final concentration 0.4mmolL
-1, 37 DEG C of abduction delivering 8h.
The preparation of competent escherichia coli cell
(1) inoculating one from intestinal bacteria flat board, to encircle in liquid amount be in the 250ml triangular flask of 25ml LB liquid nutrient medium, and 37 DEG C, 200rpm shaking culture is spent the night.
(2) be forwarded in the 250ml shaking flask of another 25ml LB liquid nutrient medium shaking culture 1.5～2h(37 DEG C, 200rpm with 1% inoculum size), make its OD
600be 0.4～0.6.
(3) get 1ml bacterium liquid and be placed in 1.5ml EP pipe, place 10min on ice.Operation afterwards remains on 0 DEG C as far as possible, and all containers are add must precooling before cell.
(4) 4 DEG C, the centrifugal 3min of 8000rpm, reclaims cell.
(5) pour out upper strata nutrient solution, blot nutrient solution residual in centrifuge tube with filter paper (sterilizing).
(6) with the ice-cold 0.1M CaCl of 0.5ml
2suspension cell (quick oscillation on vortex mixer), is incubated 10min on ice.4 DEG C, the centrifugal 3min of 8000rpm, abandons supernatant.
(7) with the ice-cold 0.1M CaCl of 0.5ml
2eddy diffusion cell, repeating step 6 once.
(8) with the ice-cold 0.1M CaCl of 100 μ l
2suspension cell, cell concn is 1～3 × 10
10cell/ml is advisable, and this cell is competent cell.
Electricity step of converting is as follows:
(1) get the each 20 μ l of 2 pipe competent cell E.coliDH10B, wherein add 1 μ l connection product amd-his-pet22b to be transformed.
(2) mixed solution is proceeded in electric conversion pool, 2.5V shocks by electricity.
(3) every pipe adds 400 μ l SOC liquid nutrient mediums, and 37 DEG C of shaking tables are cultivated 1h(and shaken slowly).
(6) competent cell of conversion is coated on to (concentration of penbritin is 100 μ g/ml) on the LB flat board that contains ammonia benzyl resistance.
(7) be inverted plate, 37 DEG C of constant temperature culture 12～16h, to occurring single bacterium colony.
SOC liquid culture based formulas (g/L): Tryptones 20, yeast soaks powder 5, sodium-chlor 0.5, Repone K 0.186, magnesium chloride 0.95, glucose 3.6.
Embodiment 6: the fermentation of this experiment propyloic hydantoin enzyme recombinant bacterium
Select E.coliBL21 (the DE3)/amd-his-pET-22b that is accredited as positive colony, be inoculated in containing in 100 μ g/ml penbritin LB liquid nutrient mediums, 37 DEG C are cultured to OD
600be about at 0.6 o'clock, add IPTG to final concentration 0.4mmolL
-1, 37 DEG C of abduction delivering 8h, 8000rpm,, centrifugal 10 minutes, abandons supernatant and obtains bacterium mud by 4 DEG C.
Embodiment 7: the protein expression of this description of test recombinant bacterium
Get the bacterium mud of embodiment 6, wash bacterium twice with the 0.02MTris-HCl buffered soln (pH9.0) of precooling, thoroughly reject supernatant liquor.In 300 μ l damping fluids for cell after washing (0.02MTris-HCl buffered soln pH9.0), be placed on ice ultrasonication cell.With 10mm probe, in 300w ultrasonication on ice, super 3s, stops 7s, altogether ultrasonic 2min.Enzyme liquid after supersound process is in 12000rpm, and 4 DEG C of centrifugal 10min collect supernatant.The separation gel that is 12% with massfraction and 5% concentrated glue carry out sds polyacrylamide gel electrophoresis investigates the expression (Fig. 4) of albumen.Electrophoresis result shows that recombinant bacterium has an obvious protein band in 50kDa left and right, in the same size with the protein band that purifying obtains from CW221 bacterial strain fermentation liquor.
Embodiment 8: the conversion capability of this description of test recombinant bacterium to substrate
Get the enzyme liquid of the above-mentioned processing of 10 μ l, then add the 2g/L propyloic glycolylurea solution 150 μ l with 0.05mol/L Tris-HCl damping fluid (pH9.0) preparation, the sampling in 1 hour of 37 DEG C of water-baths is surveyed enzyme and is lived.Find the enzyme 3.06U/mL of being alive of recombinant bacterium.Separately get the enzyme liquid of 100 μ l, add the substrate of 1.5ml, every half an hour sampling, detect the conversion capability to substrate, find 1 hour be that 85%, 2 hour transformation efficiency is the transformation efficiency of substrate to be reached to 99.98% in 95%, 4 hour to substrate conversion efficiency.
Above are only the preferred embodiment of the invention, be not restricted to the present invention.To those of ordinary skill in the art, can also make on the basis of the above description other multi-form variation or variations.Here without also illustrating all embodiment.And the apparent variation that scheme is extended out thus or variation are still within protection scope of the present invention.
Claims (8)
1. a propyloic glycolylurea enzyme-producing bacteria, it is characterized in that, its Classification And Nomenclature is Alcaligenes faecalis excrement subspecies (Alcaligenes faecalis subsp.faecalis), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 13rd, 2013, and deposit number is CGMCC No.7585.
2. a propyloic hydantoin enzyme, it is characterized in that, described propyloic hydantoin enzyme derives from propyloic glycolylurea enzyme-producing bacteria claimed in claim 1, described propyloic hydantoin enzyme, its nucleotide sequence is as shown in SEQ ID NO:1, and its aminoacid sequence is as shown in SEQ ID NO:2.
3. for expressing a recombinant vectors for propyloic hydantoin enzyme, it contains nucleotide sequence claimed in claim 2.
4. recombinant vectors according to claim 3, is characterized in that, described recombinant vectors is pET22b.
5. a host cell, it contains the recombinant vectors described in claim 3 or 4 any one.
6. host cell according to claim 5, is characterized in that, described host cell is intestinal bacteria.
7. prepare the method for host cell described in claim 5 or 6 any one, it is characterized in that, the recombinant vectors pET22b that structure comprises nucleotide sequence claimed in claim 2, and express in intestinal bacteria E.coli BL21, the recombinant bacterium that has obtained product propyloic hydantoin enzyme is host cell.
8. a method of producing N-carboxamide-Pidolidone, is characterized in that, obtains fermented liquid with the host cell described in LB culture medium culturing claim 5 or 6 any one, and through cytoclasis, centrifuging and taking supernatant liquor adds in propyloic glycolylurea solution and carries out conversion reaction.
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