CN101058818B - A kind of hydantoin enzyme gene and its coded amino acid and application - Google Patents
A kind of hydantoin enzyme gene and its coded amino acid and application Download PDFInfo
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Abstract
The invention provides a hydantoin enzyme gene, an amino acid of the gene code, a recombinant plasmid with the gene and the gene application. The code amino acid sequence of hydantoin enzyme gene is different from the present known source gene, the expression product of the hydantoin enzyme gene with hydroxybenzene hydantoin as the substrate shows high enzymatic activity, the specific activity ishigher than the hydantoin enzyme from Agrobacterium sp. KNK712 in the present industrial production.
Description
Technical field
The invention belongs to the genetically engineered field, specifically, relate to a kind of Hydantoinase gene and amino acids coding thereof and application.
Background technology
D-amino acid and derivative thereof are the important raw and processed materials of medicine and field of food, are widely used in the intermediate of semisynthetic antibiotics, polypeptide hormone, pyrethroid, sterilant and sweeting agent etc.Wherein, D-D-pHPG (D-pHPG) is the important source material that there is SKF-60771 etc. synthetic semi-synthetic Broad spectrum antibiotics amoxycilline Trihydrate bp, S 578, cefoperazone, cephalo Luo Qi.Shortcomings such as the traditional processing technology of D-D-pHPG need be by chemosynthesis and fractionation (building-up process of D-D-pHPG as shown in Figure 1), and it is low that this technology exists yield, and environmental pollution is serious.
Since the last century the eighties, enzyme process prepares the research of D-D-pHPG and uses developed, its process is as follows: at first utilize chemical means to synthesize D, L-para hydroxybenzene glycolylurea (D, L-HPH), utilize biological enzyme to be translated into the D-D-pHPG then as the substrate of biological enzyme effect.Generally speaking, earlier obtain D by oxoethanoic acid, phenol, urea condensation, acyl benzene glycolylurea (pHPH) in the L-para hydroxybenzene second, pHPH can be by D-glycolylurea enzyme (D-hydantoinase, E.C.3.5.2.2) the stereospecificity hydrolysis generates D-N-carboxamide D-pHPG (D-CpHPG), at alkaline condition or exist under the condition of racemase, further transformed thereby do not turned to the D-type by the L-type enantiomorph racemization of open loop.By this enzymatic asymmetric hydrolysis reaction, can make the pHPH that mixes racemization be converted into D-HPG fully.Now, the report that carries out this reaction at industrial existing employing immobilized D-glycolylurea enzyme or immobilized cell.
Production by Enzymes D-D-pHPG has important use to be worth industrial, therefore it is produced the research of used relevant enzyme and transforms significant.And but D-glycolylurea enzyme catalysis 5 ' list substitutes the open loop of glycolylurea or dihydrouracil, form the intermediate of carbamyl class, this intermediate product can be degraded to the sweet amino acid of D-para hydroxybenzene by carbamyl hydrolysis enzyme, becomes the important intermediate of semi-synthetic β-Nei Xiananleikangshengsu.Thereby, the research of D-glycolylurea enzyme is become one of hot research problem of relevant industries gradually.
In recent years, people are from Pseudomonas fluorescens DSM84, Bacillus stearothermophilisNS1122A, Pseudomonas putida CCRC12857, Agrobacterium sp.KNK712, Agrobacteriumradiobacter NRRL B11291 clones the gene of coding glycolylurea enzyme, and it has been carried out the molecular biology transformation, to improve involved enzyme character.Yet, do not see the report that has the clone to derive from the glycolylurea enzyme of Jannaschia sp.CCS1 up to now as yet.
Summary of the invention
The objective of the invention is to, the Hydantoinase gene of a kind of Jannaschia of deriving from sp.CCS1 is provided.
Another object of the present invention is, a kind of described Hydantoinase gene amino acids coding is provided.
Another object of the present invention is, a kind of the have Hydantoinase gene of the aminoacid sequence shown in the coding SEQ ID NO:2 and a kind of recombinant plasmid of carrier DNA sequence are provided.
Last purpose of the present invention is, a kind of application of described Hydantoinase gene is provided.
For achieving the above object, the present invention obtains Hydantoinase gene by PCR method from Jannaschia sp.CCS1 genome amplification.
The dna fragmentation of the glycolylurea enzyme that will obtain after will increasing again is connected with carrier pET28a, thereby obtains the recombinant plasmid pETJ2 of glycolylurea enzyme.
The recombinant plasmid of described glycolylurea enzyme is transferred among the E.coli BL21 (DE3), and with described engineering bacteria BL21 (DE3)/pETJ2 fermentation culture, thereby described Hydantoinase gene amino acids coding obtained.
With the expression product of the Hydantoinase gene that obtained, with the para hydroxybenzene glycolylurea as substrate, preparation D-D-pHPG, and compare with the glycolylurea enzyme that derives from Agrobacterium sp.KNK712.
Hydantoinase gene amino acid sequence coded provided by the invention is different from the glycolylurea enzyme in present known source, and it is lived showing higher enzyme during as substrate with the para hydroxybenzene glycolylurea, apparently higher than the glycolylurea enzyme of Agrobacterium sp.KNK712.
Though, in the present invention, during construction of expression vector, that use is pET28a, still for the staff of this area, uses other carrier to make up corresponding expression vector, as the pET system, the pSU system, pTrc system, pMW system, the pKK system, the expression vector that RSF1010 etc. are commonly used also is conspicuous, so also should belong to scope of the present invention.Same, staff for this area, the corresponding expression vectors that builds is transformed into other microorganism, in Rhodopseudomonas (Pseudomonas), Flavobacterium (Flavobacterium), bacillus (Bacillus), Serratia (Serratia), Agrobacterium (Agrobacterium), corynebacterium (Corynebacterium) or brevibacterium sp (Brevibacterium) etc., obtain corresponding transformant microorganism, also be conspicuous, so also should belong to scope of the present invention.
Description of drawings
Fig. 1 is the synthetic synoptic diagram of D-D-pHPG.
Fig. 2 is the structure synoptic diagram of new glycolylurea expression of enzymes carrier pETJ2.
Fig. 3 crosses the electrophorogram of expressing the glycolylurea enzyme in intestinal bacteria.
Wherein each swimming lane is respectively: 1 is BL21 (the DE3)/pETJ2 before inducing; 2 are BL21 (the DE3)/pETJ2 after inducing; 3 is albumen Marker.
Fig. 4 utilizes HPLC to detect the graphic representation that the glycolylurea enzyme reaction generates product.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.Should be understood that following examples only are used to the present invention is described but not are used to limit scope of the present invention.
The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, the condition described in " molecular cloning experiment guide (third edition) " (work such as J. Sa nurse Brooker, 2003) is carried out.
Embodiment 1, contain the expression vector of goal gene and the structure of engineering bacteria
1.1, design of primers
The genome sequence of the Jannaschia sp.CCS1 that announces according to Genebank, design primer A and B, its sequence is as follows:
Primer A:5 '-AGGGGGATCCATGAGCAAGGTGATCAAGGG-3 ';
Primer B:5 '-CTAGAAGCTTTCAAACCCCCGCCGGAATG-3 '.
1.2, pcr amplification
With A and B is primer, is template with the genomic dna that derives from Jannaschia sp.CCS1, the pcr amplification goal gene.
Reaction system: 1 * PCR Buffer for KOD plus, 2mM MgSO
4, each 0.4 μ M of primer A and primer B, d.Jannaschia sp.CCS1 genomic dna 100ng, 0.2mM dNTP, 1U KOD plus/50 μ l.
Reaction conditions: 94 ℃ of 5min, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1.5min, 30 circulations; 72 ℃ of 10min.
Reaction is pressed operational manual after finishing, and the PCR product is reclaimed the product of the about 1.5kb of test kit recovery purifying with the glue of Hua Shun company.
Check order to reclaiming product, sequencing result shows that the gene that amplification obtains has the dna sequence dna shown in the SEQ ID NO:3, and its corresponding amino acid sequence is shown in SEQ ID NO:2.
As Query, in GenBank, carry out homology search with the aminoacid sequence (SEQ ID NO:1) of the D-glycolylurea enzyme that derives from Agrobacterium sp.KNK712.The result shows, this aminoacid sequence (SEQ ID NO:2) is 40% with the homology of the aminoacid sequence (Q409E0) with SEQ ID NO:1 record, according to the BLAST interpretation of result of NCBI, find the conserved domain that there is the glycolylurea enzyme in it, infer that it may have the function of glycolylurea enzyme.
1.3, carrier construction and engineering bacteria
Building process as shown in Figure 2, concrete operations are as follows:
With reference to operational manual, respectively pcr amplification product and pET28a carrier (available from Novagen company) are carried out double digestion with BamHI (available from MBI company) and HindIII (available from MBI company), adopt the damping fluid of the double digestion damping fluid R of MBI company as endonuclease reaction.
After enzyme is cut, product detects with 1% agarose gel electrophoresis, and the purpose fragment is reclaimed in rubber tapping, and (PCR product enzyme is cut the back clip size and is about 1.5kb, the pET28a enzyme is cut the back size and is about 5.4kb), then with reference to operational manual, cut the back fragment with the enzyme that the glue of Hua Shun company reclaims that test kit reclaims respectively and the enzyme of purified pcr product is cut back fragment and carrier pET28a.
Get PCR product enzyme and cut back fragment and each 4 μ l of pET28a carrier segments, 1 μ l T4 ligase enzyme damping fluid and 1 μ l T4 ligase enzyme, in 16 ℃, connection is spent the night, and results connect product, are expression vector pETJ2.
Get 5 μ l and connect product transformed into escherichia coli DH5 α competent cell, 37 ℃ of overnight incubation.
Picking list bacterium colony inserts in the liquid nutrient medium, and in 37 ℃, 250rpm cultivated after 6-8 hour, extracting plasmid, and checking clone.Choose enzyme and cut the correct cloning and sequencing of checking, whether suddenly change with the checking sequence.
Sequencing result shows that the sequence of goal gene is not undergone mutation.
The expression vector pETJ2 that builds is transformed host bacterium E.coli BL21 (DE3) (available from Novagen company), engineering bacteria called after BL21 (the DE3)/pETJ2 that obtains.
Though, in the present embodiment, during construction of expression vector, that use is pET28a, still for the staff of this area, uses other carrier to make up corresponding expression vector, as the pET system, the pSU system, pTrc system, pMW system, the pKK system, the expression vector that RSF1010 etc. are commonly used also is conspicuous, so also should belong to scope of the present invention.Same, staff for this area, the corresponding expression vectors that builds is transformed into other microorganism, in Rhodopseudomonas (Pseudomonas), Flavobacterium (Flavobacterium), bacillus (Bacillus), Serratia (Serratia), Agrobacterium (Agrobacterium), corynebacterium (Corynebacterium) or brevibacterium sp (Brevibacterium) etc., obtain corresponding transformant microorganism, also be conspicuous, so also should belong to scope of the present invention.
Embodiment 2, goal gene expression
Single bacterium colony of picking E.coli BL21 (DE3)/pETJ2 inserts and contains in the LB liquid nutrient medium of 50 μ g/ml kantlex, and 37 ℃, 200rpm, overnight incubation.
BL21 (the DE3)/pETJ2 of incubated overnight inserted by 1% inoculum size contain in the LB liquid nutrient medium of 50 μ g/ml kantlex, 37 ℃, 200rpm, when being cultured to OD600 ≈ 0.6, adding final concentration is the IPTG of 0.25mM, in 20 ℃, inducing culture 5 hours.
Bacterium liquid after 12000rpm is centrifugal 2 minutes, is abandoned supernatant.
To precipitate with the Tris-HCl damping fluid of 50mM pH8.0 resuspended, ultrasonication.With the cell after the fragmentation, by the proteic expression amount of polyacrylamide gel electrophoresis analysis purposes, the result as shown in Figure 3.
According to the result of Fig. 3 as seen, the expression amount of target protein accounts for 30% of total protein, can be beneficial to the immobilization of follow-up zymoprotein and the suitability for industrialized production that transforms continuously by at intestinal bacteria reorganization overexpression J2.
Embodiment 3, the target protein enzyme mensuration of living
BL21 (the DE3)/pETJ2 of incubated overnight among the embodiment 2 inserted by 1% inoculum size contain in the TB liquid nutrient medium of 50 μ g/ml kantlex, 37 ℃, 200rpm, when being cultured to OD600 ≈ 0.6, adding final concentration is the IPTG of 0.25mM, in 20 ℃, and inducing culture 5 hours.
Collect bacterium liquid, centrifugal, supernatant discarded, and in-20 ℃ of freeze thawing once, use the Tris-HCl damping fluid of 50mM pH8.0 that thalline is resuspended then.
Thalline after resuspended is joined in the Tris-HCl damping fluid that contains 1% para hydroxybenzene glycolylurea, pH8.0, and 40 ℃ were reacted 10 minutes, added 3.7% HCl termination enzyme reaction and mixing.
With above-mentioned reaction solution centrifugal 5 minutes in 12000rpm, get the amount (result is as shown in Figure 4) of utilizing HPLC analytical reaction product to generate after 4 times of the supernatant dilutions, calculate enzyme and live, the result is as shown in table 1.
Table 1, target protein enzyme activity determination result
| O.D.600 | μmol/min/ml/OD | |
| BL21/pETJ2 | 2.2±0.1 | 0.016±0.002 |
Embodiment 4, target protein is than the mensuration of living
BL21 (the DE3)/pETJ2 of incubated overnight among the embodiment 2 inserted by 1% inoculum size contain in the TB liquid nutrient medium of 50 μ g/ml kantlex, 37 ℃, 200rpm, when being cultured to OD600 ≈ 0.6, adding final concentration is the IPTG of 0.25mM, in 20 ℃, and inducing culture 5 hours.
Collect bacterium liquid, centrifugal, supernatant discarded uses the Tris-HCl damping fluid of 50mM pH8.0 that thalline is resuspended then.Resuspended bacterium hydraulic pressure is pressed broken wall, adopt the GE Ni Sepharose High Performance of company purifying target protein, and measure target protein concentration with the Bradford method.
Is final concentration 0.06mg/mL with target protein with the Tris-HCl of 50mM pH8.0 dilution, joins in the Tris-HCl damping fluid that contains 1% para hydroxybenzene glycolylurea pH8.0, and 40 ℃ of reactions 30 minutes add 3.7% HCl and end enzyme reaction and mixing.
With above-mentioned reaction solution centrifugal 5 minutes in 12000rpm, get the amount (result is as shown in Figure 4) of utilizing HPLC analytical reaction product to generate after 10 times of the supernatant dilutions, calculate enzyme than living, the result is as shown in table 2.
Use the same method can from contain the engineering bacteria (CGMCCNo.0520.2) that derives from Agrobacterium sp.KNK712 glycolylurea enzyme, express, glycolylurea enzyme that purifying obtains deriving from Agrobacterium sp.KNK712, and under above-mentioned same condition, measure its activity and ratio and live.
Table 2, target protein are than measurement result alive
| Than μ mol/ alive (min.mg albumen) | |
| Agrobacterium?sp.KNK712 | 2.0±0.02 |
| J2 | 9.1±0.4 |
According to experimental result in the foregoing description, can infer the albumen of aminoacid sequence with SEQ ID NO:2, have the function of glycolylurea enzyme, be a kind of glycolylurea enzyme therefore; And glycolylurea enzyme of the present invention is different from the glycolylurea enzyme in present known source, it is with the para hydroxybenzene glycolylurea during as substrate, showing higher ratio lives, it is about 4 times of the glycolylurea enzyme that derives from Agrobacterium sp.KNK712 than living, therefore, in the industrial production of utilization double-enzyme method production D-pHPG, more wide utilization prospect can be arranged.
Sequence table
＜110〉Shanghai Inst. of Life Science, CAS
＜120〉a kind of glycolylurea enzyme
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Met?Asp?Ile?Ile?Ile?Lys?Asn?Gly?Thr?Ile?Val?Thr?Ala?Asp?Gly?Ile
1 5 10 15
Ser?Arg?Ala?Asp?Leu?Gly?Ile?Lys?Asp?Gly?Lys?Ile?Thr?Gln?Ile?Gly
20 25 30
Gly?Ala?Leu?Gly?Pro?Ala?Glu?Arg?Thr?Ile?Asp?Ala?Ala?Gly?Arg?Tyr
35 40 45
Val?Phe?Pro?Gly?Gly?Ile?Asp?Val?His?Thr?His?Val?Glu?Thr?Val?Ser
50 55 60
Phe?Asn?Thr?Gln?Ser?Ala?Asp?Thr?Phe?Ala?Thr?Ala?Thr?Val?Ala?Ala
65 70 75 80
Ala?Cys?Gly?Gly?Thr?Thr?Thr?Ile?Val?Asp?Phe?Cys?Gln?Gln?Asp?Arg
85 90 95
Gly?His?Ser?Leu?Ala?Glu?Ala?Val?Ala?Lys?Trp?Asp?Gly?Met?Ala?Gly
100 105 110
Gly?Lys?Ser?Ala?Ile?Asp?Tyr?Gly?Tyr?His?Ile?Ile?Val?Leu?Asp?Pro
115 120 125
Thr?Asp?Ser?Val?Ile?Glu?Glu?Leu?Glu?Val?Leu?Pro?Asp?Leu?Gly?Ile
130 135 140
Thr?Ser?Phe?Lys?Val?Phe?Met?Ala?Tyr?Arg?Gly?Met?Asn?Met?Ile?Asp
145 150 155 160
Asp?Val?Thr?Leu?Leu?Lys?Thr?Leu?Asp?Lys?Ala?Val?Lys?Thr?Gly?Ser
165 170 175
Leu?Val?Met?Val?His?Ala?Glu?Asn?Gly?Asp?Ala?Ala?Asp?Tyr?Leu?Arg
180 185 190
Asp?Lys?Phe?Val?Ala?Glu?Gly?Lys?Thr?Ala?Pro?Ile?Tyr?His?Ala?Leu
195 200 205
Ser?Arg?Pro?Pro?Arg?Val?Glu?Ala?Glu?Ala?Thr?Ala?Arg?Ala?Leu?Ala
210 215 220
Leu?Ala?Glu?Ile?Val?Asn?Ala?Pro?Ile?Tyr?Ile?Val?His?Val?Thr?Cys
225 230 235 240
Glu?Glu?Ser?Leu?Glu?Glu?Val?Met?Arg?Ala?Lys?Ser?Arg?Gly?Val?Arg
245 250 255
Ala?Leu?Ala?Glu?Thr?Cys?Thr?His?Tyr?Leu?Tyr?Leu?Thr?Lys?Glu?Asp
260 265 270
Leu?Glu?Arg?Pro?Asp?Phe?Glu?Gly?Ala?Lys?Tyr?Val?Phe?Thr?Pro?Pro
275 280 285
Ala?Arg?Ala?Lys?Lys?Asp?His?Asp?Val?Leu?Trp?Asn?Ala?Leu?Arg?Asn
290 295 300
Gly?Val?Phe?Glu?Thr?Val?Ser?Ser?Asp?His?Cys?Ser?Trp?Leu?Phe?Lys
305 310 315 320
Gly?His?Lys?Asp?Arg?Gly?Arg?Asn?Asp?Phe?Arg?Ala?Ile?Pro?Asn?Gly
325 330 335
Ala?Pro?Gly?Val?Glu?Glu?Arg?Leu?Met?Met?Val?Tyr?Gln?Gly?Val?Asn
340 345 350
Glu?Gly?Arg?Ile?Ser?Leu?Thr?Gln?Phe?Val?Glu?Leu?Val?Ala?Thr?Arg
355 360 365
Pro?Ala?Lys?Val?Phe?Gly?Met?Phe?Pro?Gln?Lys?Gly?Thr?Ile?Ala?Val
370 375 380
Gly?Ser?Asp?Ala?Asp?Ile?Val?Leu?Trp?Asp?Pro?Glu?Ala?Glu?Met?Val
385 390 395 400
Ile?Glu?Gln?Thr?Ala?Met?His?Asn?Ala?Met?Asp?Tyr?Ser?Ser?Tyr?Glu
405 410 415
Gly?His?Lys?Val?Lys?Gly?Val?Pro?Lys?Thr?Val?Leu?Leu?Arg?Gly?Lys
420 425 430
Val?Ile?Val?Asp?Glu?Gly?Ser?Tyr?Val?Gly?Glu?Pro?Thr?Asp?Gly?Lys
435 440 445
Phe?Leu?Lys?Arg?Arg?Lys?Tyr?Lys?Gln
450 455
<210>2
<211>487
<212>PRT
<213>Jannaschia?sp.CCS1
<400>2
Met?Ser?Lys?Val?Ile?Lys?Gly?Gly?Thr?Ile?Val?Thr?Ala?Asp?Arg?Gln
1 5 10 15
Trp?Gln?Ala?Asp?Val?Leu?Ile?Glu?Gly?Glu?Lys?Ile?Ala?Glu?Ile?Gly
20 25 30
Glu?Asn?Leu?Arg?Gly?Asp?Glu?Val?Ile?Asp?Ala?Glu?Gly?Ala?Tyr?Val
35 40 45
Ile?Pro?Gly?Gly?Ile?Asp?Pro?His?Thr?His?Leu?Glu?Met?Pro?Phe?Met
50 55 60
Gly?Thr?Thr?Ala?Ala?Glu?Thr?Phe?Glu?Thr?Gly?Thr?Phe?Ala?Ala?Ala
65 70 75 80
Ala?Gly?Gly?Thr?Thr?Met?Leu?Val?Asp?Phe?Cys?Leu?Pro?Gly?Glu?Asp
85 90 95
Gly?Ser?Leu?Leu?Ser?Ala?Ile?Asp?Ala?Trp?Asp?Ala?Lys?Ser?Lys?Asp
100 105 110
Gln?Ile?Cys?Val?Asp?Ile?Ser?Tyr?His?Met?Ala?Ile?Thr?Gly?Trp?Ser
115 120 125
Glu?Ser?Ile?Phe?Asn?Glu?Met?Ala?Asp?Val?Val?Asn?Val?Arg?Gly?Ile
130 135 140
Asn?Thr?Phe?Lys?His?Phe?Met?Ala?Tyr?Lys?Gly?Ala?Leu?Met?Ile?Glu
145 150 155 160
Asp?Asp?Glu?Met?Phe?Ser?Ser?Phe?Lys?Arg?Cys?Ala?Glu?Leu?Gly?Ala
165 170 175
Leu?Pro?Leu?Val?His?Ala?Glu?Asn?Gly?Asp?Ile?Val?Gln?Glu?Leu?Gln
180 185 190
Gln?Lys?Tyr?Met?Ala?Met?Gly?Val?Thr?Gly?Pro?Glu?Gly?His?Ala?Tyr
195 200 205
Ser?Arg?Pro?Pro?Glu?Val?Glu?Gly?Glu?Ala?Ala?Asn?Arg?Ala?Ile?Met
210 215 220
Ile?Ala?Asp?Ala?Ala?Gly?Thr?Pro?Leu?Tyr?Ile?Val?His?Val?Ser?Cys
225 230 235 240
Glu?Gln?Ala?His?Glu?Ala?Ile?Arg?Arg?Ala?Arg?Gln?Lys?Gly?Met?Arg
245 250 255
Val?Phe?Gly?Glu?Pro?Leu?Ile?Gln?His?Leu?Thr?Leu?Asp?Glu?Ser?Glu
260 265 270
Tyr?Phe?Asn?Lys?Asp?Trp?Gln?Tyr?Ala?Ala?Arg?Arg?Val?Met?Ser?Pro
275 280 285
Pro?Phe?Arg?Asn?Lys?Glu?His?Gln?Asp?Gly?Leu?Trp?Ala?Gly?Leu?Ala
290 295 300
Ala?Gly?Ser?Leu?Gln?Val?Val?Ala?Thr?Asp?His?Ala?Ala?Phe?Thr?Asp
305 310 315 320
Glu?Gln?Lys?Arg?Met?Gly?Val?Asp?Asn?Phe?Gly?Met?Ile?Pro?Asn?Gly
325 330 335
Thr?Gly?Gly?Leu?Glu?Glu?Arg?Met?Ala?Met?Leu?Trp?Thr?Arg?Gly?Val
340 345 350
Glu?Thr?Gly?Arg?Leu?Thr?Pro?Glu?Glu?Phe?Val?Ala?Val?Thr?Ser?Ser
355 360 365
Asn?Ile?Ala?Lys?Ile?Leu?Asn?Ile?Tyr?Pro?Met?Lys?Gly?Gly?Ile?Asn
370 375 380
Val?Gly?Gly?Asp?Ala?Asp?Ile?Val?Val?Trp?Asp?Pro?Lys?Leu?Gly?Arg
385 390 395 400
Thr?Ile?Thr?Thr?Ala?Thr?Ala?Lys?Ser?Ile?Leu?Asp?Tyr?Asn?Val?Phe
405 410 415
Glu?Gly?Met?Glu?Val?Ser?Ala?Ser?Pro?Arg?Tyr?Thr?Leu?Ser?Arg?Gly
420 425 430
Asp?Val?Val?Trp?Ala?Ala?Gly?Gln?Asn?Ser?Gln?Pro?Gln?Pro?Gly?Arg
435 440 445
Gly?Lys?Phe?Val?Lys?Arg?Pro?Pro?Ala?Ala?Ser?Ala?Ser?Gln?Ala?Leu
450 455 460
Ser?Lys?Trp?Lys?Ala?Leu?Asn?Thr?Pro?Arg?Lys?Ile?Glu?Arg?Asp?Pro
465 470 475 480
Met?Asn?Ile?Pro?Ala?Gly?Val
485
<210>3
<211>1464
<212>DNA
<213>Jannaschia?sp.CCS1
<400>3
atgagcaagg?tgatcaaggg?cggcacgatt?gtgaccgcag?accgtcaatg?gcaggcggac 60
gtgttgatcg?agggcgaaaa?gattgccgag?atcggggaga?acctgcgcgg?ggatgaggtg 120
atcgacgcgg?aaggcgccta?tgtgatcccg?ggcggcatag?acccccacac?gcatcttgag 180
atgcccttca?tgggcaccac?ggcggcggag?acgttcgaga?cgggcacctt?tgcggcggca 240
gcgggcggca?ccacgatgct?ggtcgatttc?tgccttccgg?gcgaggatgg?cagccttttg 300
tccgccatcg?atgcctggga?cgccaaatcg?aaggatcaga?tctgcgttga?tatctcctac 360
cacatggcga?tcaccggctg?gtcggagagc?attttcaatg?agatggcgga?cgttgttaat 420
gtgcgcggca?tcaacacatt?taagcatttc?atggcctata?aaggcgcgct?gatgatcgag 480
gatgacgaga?tgttttcgtc?gttcaagcgc?tgcgctgaat?tgggcgcgct?gccgctggtc 540
catgccgaaa?acggcgatat?cgtccaggag?ttgcaacaga?aatacatggc?gatgggcgtg 600
acggggccgg?agggtcacgc?atattcccgt?ccgcctgagg?tcgaagggga?agccgccaac 660
cgcgcgatca?tgatcgccga?cgccgctggc?acgccgttgt?atatcgtcca?tgtgtcgtgt 720
gagcaggccc?atgaggccat?ccgccgtgcc?cgtcagaagg?ggatgcgggt?cttcggggag 780
ccactgatcc?agcacctgac?gctggatgag?agcgagtatt?tcaacaagga?ttggcaatat 840
gcggcccgcc?gggtcatgtc?cccgccgttt?cgcaacaaag?agcatcagga?cggtctttgg 900
gcaggtcttg?ccgctgggtc?cttgcaggtt?gtggccacgg?accacgccgc?cttcaccgac 960
gagcaaaagc?gcatgggcgt?ggacaatttc?ggcatgatcc?ccaacggcac?cggcgggctt 1020
gaggagcgca?tggcaatgtt?gtggacgcgc?ggcgtggaaa?cgggccgcct?gacgccggaa 1080
gaattcgttg?cggtgacgtc?atcgaacatc?gccaagatcc?tcaacattta?cccaatgaag 1140
ggtggcatca?acgtcggcgg?cgacgcggat?atcgtggtct?gggacccgaa?actgggccgc 1200
acgatcacga?cggcaacggc?gaaatctatc?cttgattaca?atgtgttcga?gggaatggag 1260
gtgagcgcct?ccccccgcta?caccctgtcg?cgcggggatg?tggtgtgggc?ggcggggcaa 1320
aacagccagc?cgcaaccggg?ccgtgggaaa?ttcgtgaaac?ggcccccggc?ggcgagtgcg 1380
tcccaggcgc?tgagcaagtg?gaaggcgttg?aacacgccgc?gcaagatcga?gcgcgacccg 1440
atgaacattc?cggcgggggt?ttga 1464
Claims (8)
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| CN2007100392296A CN101058818B (en) | 2007-04-06 | 2007-04-06 | A kind of hydantoin enzyme gene and its coded amino acid and application |
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| CN103981228B (en) * | 2014-03-31 | 2016-05-18 | 浙江工业大学 | A kind of lipase-catalyzed benzene glycolylurea is prepared the method for N-carbamyl phenylglycine |
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Non-Patent Citations (5)
| Title |
|---|
| CP000264.EMBL-EBI.2006, |
| CP000264.EMBL-EBI.2006,;李隽,胡卓逸.诱导物对二氢嘧啶酶的作用.中国药科大学学报30 3.1999,30(3),220-223. * |
| 李志强，胡卓逸，刘景晶.假单胞菌海因酶基因在大肠杆菌中的高效表达.中国生物化学与分子生物学报18 2.2002,18(2),145-150. |
| 李志强,胡卓逸,刘景晶.假单胞菌海因酶基因在大肠杆菌中的高效表达.中国生物化学与分子生物学报18 2.2002,18(2),145-150. * |
| 李隽，胡卓逸.诱导物对二氢嘧啶酶的作用.中国药科大学学报30 3.1999,30(3),220-223. |
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