CN1995336A - D-carboxamide hydrolase mutant and its uses - Google Patents
D-carboxamide hydrolase mutant and its uses Download PDFInfo
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Abstract
The invention discloses four mutants of D-carbamoyl hydrolase and application to manufacture D-p-hydroxybenzene glycine in the biological engineering domain, which is characterized by the following: mutating the amino acid at 18th position of mutation enzyme 1 from alanine into threonine and tyrosine at 30th position of mutation enzyme 2 to asparagine; switching the lysine at 30th of mutation enzyme 2 into glutacid; overlapping three mutation positions; obtaining the mutation enzyme 4.
Description
Technical field
The invention belongs to bioengineering field, particular content relates to the mutant and the application in producing the D-D-pHPG thereof of D-carbamyl hydrolysis enzyme.
Background technology
β-Nei Xiananleikangshengsu has antibiotic energetic, and advantages such as wide spectrum low toxicity are one of choice drugs of clinic control infectation of bacteria.D-D-pHPG (DHPG) is a kind of important intermediate of synthetic such medicine, is used to prepare the side chain of amoxycillin and cefaparole.The preparation of D-D-pHPG has chemical synthesis and two kinds of methods of enzyme process industrial, and wherein chemical synthesis cost height pollutes big; And enzyme process has lower, the free of contamination characteristics of cost, therefore comes into one's own day by day.The know-why of enzyme process is: with D-Hydantoinase (being also referred to as D-glycolylurea enzyme) DL-para hydroxybenzene glycolylurea asymmetric hydrolysis is become N-carbamyl-D-D-pHPG; (be also referred to as the D-carbamyl hydrolysis enzyme at N-carboxamide-D-amino acid lytic enzyme then; abbreviation DCase) under the effect, N-carbamyl-irreversible hydrolysis of D-D-pHPG is transformed into DHPG.
Find in the industrial production: produce in the process of D-D-pHPG at two step enzyme methods; because the DCase catalytic efficiency is low, the shortcoming of poor stability; cause intermediate N-carbamyl-D-D-pHPG to accumulate in a large number, end product D-D-pHPG productive rate is low.Therefore DCase becomes a key enzyme, the rate-limiting enzyme in the two step enzyme method reactive systems.
Along with the development of genetic engineering technique, the maturation day by day of escherichia expression system the DCase gene is carried out overexpression in escherichia expression system, thereby the engineering strain that obtains high expression level, high vigor becomes possibility.For example, Xu Zhen etc. can be converted into DL-para hydroxybenzene glycolylurea the bacterial strain of D-D-pHPG from a strain, successfully clone the DCase gene, and the Genebank number of landing is: AF320814 (Xu Zhen etc., biotechnology journal, 2002,18 (2): 149-54).We are going into the pET expression system with DCase is gene constructed, behind the Transformed E .coli overexpression, activity and SDS-PAGE to target protein analyze, the result shows: E.coli can great expression go out recombinant protein, but the recombinant protein about 70-80% is the form with inclusion body to be existed, and has seriously influenced the function performance of engineering enzyme.After being expressed, more target protein can correctly be folded into the direction that great-hearted albumen becomes research and makes great efforts.
Though the natural enzyme molecule was evolved millions upon millions of years under field conditions (factors), because the environment that the natural enzyme molecule exists is different with the environment of practical application, the enzyme molecule is still being contained huge evolution potentiality.Utilization molecular orientation evolvement technology is carried out molecule modification and artificial evolution such as fallibility round pcr (error-prone PCR), DNA reorganization (DNA shuffling) technology and site-directed mutagenesis technique etc. to microbe-derived enzyme and has been obtained the progress that attracts people's attention (referring to Wang Zhengxiang etc. in recent years, the biotechnology journal, 16 (3), 2000), the artificial orientation who has the become enzyme conventional means of evolving.Artificial orientation's evolution technology on this molecular level is to transform target gene by vitro recombination, and directed screening has the mutant of expection proterties, thereby creates the enzyme of the modification with new function, has quickened proteinic evolution process greatly.
Summary of the invention
Purpose of the present invention just is by the orthogenesis technology goal gene to be carried out random mutation, and uses high-throughout screening method, obtains the D-carbamyl hydrolysis enzyme mutant that solubility expression improves.
Another object of the present invention is to provide the application of described D-carbamyl hydrolysis enzyme mutant in the D-D-pHPG is produced.
For achieving the above object, the method that the present invention uses fallibility round pcr and DNA shuffling technology to combine is carried out random mutation to the DCase gene, by a kind of protein solubility Monitoring systems gain mutant is detected again.
The present invention adopts a kind of protein solubility Monitoring systems (W.Christian Wigley etc.2001 NatureBiotechnology 19,131-136) solubility of detection recombinant protein.This Monitoring systems is to be based upon on the α complementary architecture basics of beta-galactosidase enzymes, and the ω fragment realizes that complementation comes the proteic solubility of testing goal in α fragment by the beta-galactosidase enzymes that merges on the carrier and the intestinal bacteria.
By aforesaid method, inventor's screening has obtained 3 mutant that the recombinant protein solubility expression increases, and is respectively A18T, Y30N and K34E.
The Genebank number of landing of D-carbamyl hydrolysis enzyme is: AF320814, to form by 304 amino acid, and its DNA sequences encoding comprises 915bp, its DNA sequences encoding and aminoacid sequence are shown in SEQ ID NO.1 and 2.The difference of the present invention obtained 3 mutant and DCase is respectively:
1, the 18th of A18T the amino acid is Threonine by alanine mutation, and accordingly, the GCG of 52-54bp becomes ACG in its DNA sequences encoding;
2, the 30th of Y30N the amino acid sports l-asparagine by tyrosine, and accordingly, the TAC of 88-90bp becomes AAC in its DNA sequences encoding;
3, the 34th of K34E the amino acid is L-glutamic acid by lysine mutation, and accordingly, the AAA of 100-102bp becomes GAA in its DNA sequences encoding.
After obtaining above-mentioned 3 kinds of mutant, contriver's sudden change that further above-mentioned 3 mutant superposeed, obtain a new mutant A18T/Y30N/K34E, the 18th amino acid of this stack mutant enzyme is Threonine by alanine mutation, the 30th amino acid sports l-asparagine by tyrosine, and the 34th amino acid is L-glutamic acid by lysine mutation.Accordingly, the GCG of 52-54bp becomes ACG in its DNA sequences encoding, and the TAC of 88-90bp becomes AAC, and the AAA of 100-102bp becomes GAA.
Than wild-type Dcase, the enzyme of every milliliter of fermentation thalline of 4 mutant A18T, Y30N, K34E and A18T/Y30N/K34E of the present invention is lived and is improved 1 times, 1.5 times, 4.7 times and 8.4 times respectively; Solubility expression is respectively 1.3,1.4,1.9,3.2 times of wild enzyme, is the best with the stack mutant especially wherein, demonstrates the potential using value in the D-D-pHPG is produced.
Embodiment
The present invention is further illustrated below in conjunction with embodiment, it is pointed out that present embodiment only is used to explain the present invention, but not limitation of the scope of the invention.
Embodiment 1, the DCase gene the clone
1.1, pcr amplification
Design a pair of primer:
5 '-CGCCATATGACACGTCAGATGATA-3 '; With
5’-CCCAAGCTTTCAGAGTTCCGCGAT-3’
(2002,18 (2): 149-54) DNA is a template, carries out pcr amplification for Xu Zhen etc., biotechnology journal with plasmid pXZ-total.The PCR condition is: behind 94 ℃ of sex change 10min, and 94 ℃ of sex change 30sec, 58 ℃ of annealing 30sec, 72 ℃ are extended 50sec, carry out 30 circulations altogether, and last 72 ℃ are fully extended 10min.
1.2, contain the plasmid construction of DCase gene
After pcr amplification finishes; using earlier 1% sepharose to carry out voltage is 100 volts Nucleotide electrophoresis; after the rubber tapping; reclaim test kit (Shanghai China Shun biotechnology company limited) with glue and reclaim the dna fragmentation about 900bp; then according to precious biotech firm (network address: the www.takara.com.cn) method described of 2005-2006 products catalogue specification sheets, dna fragmentation and the carrier pET-28b (Novagen company) that is obtained carried out double digestion with restriction enzyme NdeI and HindIII.
Enzyme is cut product, and to carry out voltage once more be 100 volts Nucleotide electrophoresis, reclaim corresponding size and be respectively fragment and carrier about 900bp, 5300bp, at last with carrier and fragment by after 1: 4 the mixed, add the T4 ligase enzyme of 1 unit, 16 ℃ connect more than 10 hours.
Connect product transformed into escherichia coli DH5 α competent cell, screen containing on the antibiotic LB flat board of kantlex, recon is through Nde I and HindIII double digestion and dna sequencing checking, obtain the correct clone of gene order, its sequence is shown in SEQ ID NO.1, with the Genebank number of landing is that the DCase gene of AF320814 is identical, is called wild-type, with its called after WT.
1.3, express the construction method of gene engineering strain of DCase
With ordinary method (Hao Fuying etc. write, molecular biology experiment technology, BJ University Press, 1998, the 12-15 pages or leaves) WT is transformed into E.coli BL21 (Novagen company), promptly obtains the escherichia coli expression bacterial strain of DCase gene.
Embodiment 2, the DCase gene random mutation
2.1, fallibility PCR
100 μ l fallibility PCR reaction systems are: the dna profiling pXZ-total of 20ng, a pair of primer 5 '-CGCCATATGACACGTCAGATGATA-3 ' and 5 '-CCCAAGCTTGAGTTCCGCGAT-3 ' of each 30pmol, 7mMMgCl
2, 50mM KCl, 10mM Tris-HCl (pH8.3), 0.01%gelatin, 0.2mM dGTP, 0.2mM dATP, 1mM dCTP, 1mM dTTP, 0.15mM MnCl
2And the Taq enzyme of 5 units (Shanghai sangon company).
The PCR reaction conditions is: behind 94 ℃ of sex change 10min, and 94 ℃ of sex change 30sec, 58 ℃ of annealing 30sec, 72 ℃ are extended 50sec, carry out 30 circulations altogether, and last 72 ℃ are fully extended 10min.
2.2, DNA reorganization
Use glue to reclaim test kit (Shanghai China Shun biotechnology company limited) purified pcr product.According to document (StemmerWPC.Nature, 1994,370 (6488): 389.) described method, the dna fragmentation that obtains is carried out DNA reorganization, comprising: degradation of dna, reclaim 50-200bp small segment, do not have primer PCR, primer PCR arranged.
DNA recombinant fragment through DNA reorganization back acquisition, method according to the precious 2005-2006 of biotech firm products catalogue specification sheets description, after Nde I and the digestion of HindIII double digestion, carry out ligation with pMAL-c2x (NEB company) carrier of cutting through same enzyme, reaction conditions is: carrier and fragment are pressed 1: 4 mixed, add the T4 ligase enzyme of 1 unit, 16 ℃ of connections are spent the night.Obtained surpassing 10
4Individual clone's mutant library.
Embodiment 3, mutant library screening
3.1, mutant transforms
Method by electric shock is transformed in the DH5 α bacterial strain making up the mutant that obtains in the step 2.2.Transformant is coated and is contained Amp
rOn the LB flat board of microbiotic, IPTG inductor and chromogenic substrate X-gal (peptone 1%, yeast extract 0.5%, sodium-chlor 1%, agar 2%), 37 ℃ of cultivations.
3.2, screening mutant and evaluation
Behind the strain growth 16h, the clone of every dull and stereotyped blueing color depth of picking with a small amount of plasmid extraction test kit (Shanghai China Shun biotechnology company limited) extracting plasmid, uses Nde I and HindIII to carry out double digestion evaluation and order-checking mensuration.
3.3, mutant gene amplification
Design a pair of primer:
5 '-CGCCATATGACACGTCAGATGATA-3 '; With
5’-CCCAAGCTTTCAGAGTTCCGCGAT-3’
With the mutant plasmid that is obtained in the step 3.2 is template, carries out pcr amplification.
The PCR condition is: behind 94 ℃ of sex change 10min, and 94 ℃ of sex change 30sec, 58 ℃ of annealing 30sec, 72 ℃ are extended 50sec, carry out 30 circulations altogether, and last 72 ℃ are fully extended 10min.
3.4, contain the plasmid construction of Dcase mutator gene
After pcr amplification finishes; using earlier 1% sepharose to carry out voltage is 100 volts Nucleotide electrophoresis; after the rubber tapping; reclaim test kit (Shanghai China Shun biotechnology company limited) with glue and reclaim the dna fragmentation about 900bp; then according to precious biotech firm (network address: the www.takara.com.cn) method described of 2005-2006 products catalogue specification sheets, dna fragmentation and the carrier pET-28b (Novagen company) that is obtained carried out double digestion with restriction enzyme Nde I and HindIII.
Enzyme is cut product, and to carry out voltage once more be 100 volts Nucleotide electrophoresis, reclaim corresponding size and be respectively fragment and carrier about 900bp, 5300bp, at last with carrier and fragment by after 1: 4 the mixed, the T4 ligase enzyme that adds 1 unit, 16 ℃ connect more than 10 hours, connect product transformed into escherichia coli DH5 α competent cell, screen containing on the antibiotic LB flat board of kantlex, recon obtains the correct plasmid that contains the Dcase mutator gene through Nde I and HindIII double digestion and dna sequencing checking.
3.5, the strain construction and the expression of DCase mutant
(Hao Fuying etc. write with ordinary method, the molecular biology experiment technology, BJ University Press, 1998, the 12-15 page or leaf) will contain the plasmid Transformed E .coli BL21 (Novagen company) that the Dcase mutator gene makes up, obtain the escherichia coli expression bacterial strain of mutant.
Escherichia coli expression bacterial strain elder generation (peptone 1%, yeast extract 0.5%, the sodium-chlor 1%) overnight incubation in the LB test tube that obtains will be made up, being connected to the 250ml that 30ml LB liquid nutrient medium is housed by the inoculum size of 1% volume ratio shakes in the bottle, grow to OD600 ≈ 0.6, IPTG (isopropylthio-) abduction delivering that adds 0.3mM then, be put in 200 rev/mins shaking table, overnight incubation.
4000 rev/mins, centrifugal 10 minutes, the collection thalline also is suspended in the PBS damping fluid, handles through ultrasonic disruption, and was centrifugal, gets supernatant, and throw out carries out SDS-PAGE and analyzes.
3.6, The selection result
By above-mentioned screening step, from mutant library, screening has obtained 3 mutant that the recombinant protein solubility expression increases.They are respectively A18T, Y30N and K34E, and its solubility expression amount is as shown in table 1, are respectively 1.3,1.4 and 1.9 times of wild enzyme.
The recombinant protein solubility expression of table 1, wild-type DCase and 4 mutant relatively
| Numbering | WT | A18T | Y30N | K34E | A18T/Y30N/K34E |
| Soluble proteins ratio (%) | 26.3±2.1 | 36.0±3.5 | 36.7±0.9 | 49.7±0.5 | 81.4±6.0 |
3.7, mutant order-checking
Through the aminoacid sequence and the gene order of three mutant are measured, find to compare with wild-type D-carbamyl hydrolysis enzyme, be respectively that variation has taken place the amino acid of 3 positions, change has also taken place in respective coding DNA, and is specifically as shown in table 2:
The aminoacid sequence of table 2, three DCase mutant and gene order result of variations
| Numbering | Amino acid position | Amino acid changes | Nucleotide position | Nucleotide changes |
| A18T | The 18th | L-Ala → Threonine | 52-54bp | GCG→ACG |
| Y30N | The 30th | Tyrosine → l-asparagine | 88-90bp | TAC→AAC |
| K34E | The 34th | Methionin → L-glutamic acid | 100-102bp | AAA→GAA |
Embodiment 4, three mutational sites stack
4.1, pcr amplification
With the DCase gene that contains K34E sudden change is template, uses primer right respectively:
5 '-CGCCATATGACACGTCAGATGATA-3 '; With
5’-GCGTGTCTCCGTGCGCGCGAT-3’；
Another primer is right:
5 '-CCCAAGCTTTCAGAGTTCCGCGAT-3 '; With
5’-ATCGCGCGCACGGAGACACGC-3’；
Carry out pcr amplification.
The PCR condition is: behind 94 ℃ of sex change 10min, and 94 ℃ of sex change 30sec, 63 ℃ of annealing 30sec, 72 ℃ are extended 30sec, carry out 30 circulations altogether, and last 72 ℃ are fully extended 10min.
42, reclaim the PCR product
After pcr amplification finished, using 1.5% sepharose to carry out voltage earlier was 100 volts Nucleotide electrophoresis, after the rubber tapping, reclaimed dna fragmentation about test kit (Shanghai China Shun biotechnology company limited) recovery 50bp, 850bp with glue.
4.3, pcr amplification once more
With two fragments being reclaimed in the step 4.2 is template, uses primer right:
5 ,-CGCCATATGACACGTCAGATGATA-3 '; With
5’-CCCAAGCTTTCAGAGTTCCGCGAT-3’；
Carry out pcr amplification.
The PCR condition is: behind 94 ℃ of sex change 6min, and 94 ℃ of sex change 30sec, 58 ℃ of annealing 30sec, 72 ℃ are extended 50sec, carry out 30 circulations altogether, and last 72 ℃ are fully extended 10min.
4.4, PCR product order-checking
After pcr amplification finished, using 1% sepharose to carry out voltage earlier was 100 volts Nucleotide electrophoresis, after the rubber tapping, reclaims test kit (Shanghai China Shun biotechnology company limited) with glue and reclaims the dna fragmentation about 900bp, checks order.
Show that through order-checking the mutant of gained D-carbamyl hydrolysis enzyme is that the catastrophe point of A18T and K34E merges mutant, promptly the GCG of 52-54bp becomes ACG in the dna sequence dna of wild-type DCase, and the AAA of 100-102bp becomes GAA.
4.5, pcr amplification once more
DNA with the A18T/K34E mutant of gained in the step 4.4 is a template, uses primer right respectively:
5 '-CGCCATATGACACGTCAGATGATA-3 '; With
5’-CGTCAGCATGTTGAGAAGACGAA-3’；
Another is to primer:
5 '-CCCAAGCTTTCAGAGTTCCGCGAT-3 '; With
5’-TTCGTCTTCTCAACATGCTGACG-3’
Carry out pcr amplification.
The PCR condition is: behind 94 ℃ of sex change 10min, and 94 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ are extended 30sec, carry out 30 circulations altogether, and last 72 ℃ are fully extended 10min.
4.6, reclaim the PCR product
After pcr amplification finished, using 1.5% sepharose to carry out voltage earlier was 100 volts Nucleotide electrophoresis, after the rubber tapping, reclaimed dna fragmentation about test kit (Shanghai China Shun biotechnology company limited) recovery 90bp, 800bp with glue.
4.7, pcr amplification once more
With above-mentioned two fragments that step 4.6 was reclaimed is template, uses primer right:
5 '-CGCCATATGACACGTCAGATGATA-3 '; With
5’-CCCAAGCTTTCAGAGTTCCGCGAT-3’
Carry out pcr amplification, the PCR condition is: behind 94 ℃ of sex change 6min, and 94 ℃ of sex change 30sec, 58 ℃ of annealing 30sec, 72 ℃ are extended 50sec, carry out 30 circulations altogether, and last 72 ℃ are fully extended 10min.
4.8, PCR product order-checking
After pcr amplification finished, using 1% sepharose to carry out voltage earlier was 100 volts Nucleotide electrophoresis, after the rubber tapping, reclaims test kit (Shanghai China Shun biotechnology company limited) with glue and reclaims the dna fragmentation about 900bp, checks order.
Show through order-checking, the mutant of gained D-carbamyl hydrolysis enzyme is that the catastrophe point of A18T, Y30N and K34E merges mutant, be that the GCG of 52-54bp becomes ACG in the dna sequence dna of wild-type DCase, the TAC of 88-90bp becomes AAC, and the AAA of 100-102bp becomes GAA.
4.9, the structure of A18T/Y30N/K34E mutant
Method according to the precious 2005-2006 of biotech firm products catalogue specification sheets description; with the dna fragmentation that obtained in the step 4.8 and carrier pET-28b (Novagen company) through restriction enzyme Nde I and HindIII double digestion; carry out voltage once more and be 100 volts Nucleotide electrophoresis, reclaim test kit (Shanghai China Shun biotechnology company limited) with glue and reclaim fragment and carrier about 900bp, 5300bp.
Carrier and fragment by after 1: 4 the mixed, are added the T4 ligase enzyme of 1 unit, and 16 ℃ connect 10 hours.
Connect product transformed into escherichia coli DH5 α competent cell, screen containing on the antibiotic LB flat board of kantlex, recon finally obtains the clone of 3 point mutation, called after A18T/Y30N/K34E through Nde I and HindIII double digestion and dna sequencing checking.
Embodiment 5, mutant solubility expression contrast
According to the method described in the step 3.5, measure wild-type and D-carbamyl hydrolysis enzyme mutant (A18T, Y30N, K34E and A18T/Y30N/K34E) solubility situation, the results are shown in Table 1.Test shows that the synergetic D-carbamyl hydrolysis enzyme of a plurality of sudden changes mutant is compared single mutant, and the recombinant protein solubility expression further increases, and all is better than wild-type.Such as, the solubility expression albumen ratio of A18T/Y30N/K34E is 3.2 times of wild enzyme up to 81.4%, promptly the ratio of inclusion body only is 18.6%.Therefore these 4 mutant all have the potential application advantage in the production of D-D-pHPG.
Embodiment 6, wild-type enzyme and mutant enzyme the unit bacterial enzyme live relatively
6.1, thalline (thick enzyme) preparation
Get each 1ml of thalline (A18T, Y30N, K34E and A18T/Y30N/K34E) of fermentation culture among the embodiment 5 respectively, add in the centrifuge tube of 1.5ml, centrifugal, abandon supernatant.Frozen more than 2 hours, standby at-20 ℃ of refrigerators.
6.2, enzymically hydrolyse reaction
Each thalline is suspended in respectively in the phosphoric acid buffer of 0.1M, pH7.5 of 1ml, gets the suspension of 400ul, adds N-carboxamide-D-D-pHPG substrate of the 100mM of 400 μ l, and reaction is hydrolyzed.At 40 ℃ shaking bath, with 150 rev/mins velocity fluctuation.
React after 30 minutes, add the 10% hydrochloric acid stopped reaction of 800 μ l.
6.3, enzyme activity determination
After getting an amount of reaction solution and diluting, 13000 rev/mins centrifugal 5 minutes, get supernatant and carry out HPLC and measure.
The method that HPLC measures the D-carbamyl hydrolysis enzyme is: with phosphoric acid 2.25mM, potassium primary phosphate 20mM, the methyl alcohol of pH5.0 and water are moving phase, its ratio is 20: 80, flow velocity is 1.0ml/min, detect the different peak heights and the retention value of different content material and same substance at the 210nm place, draw out typical curve.
The work of 1U enzyme is defined as per minute and produces the required enzyme amount of 1 micromole D-D-pHPG.
6.4, bacterial enzyme lives relatively
Enzyme activity determination the results are shown in Table 3:
The unit bacterial enzyme of table 3, wild-type DCase and 4 mutant is lived relatively
| Numbering | WT | A18T | Y30N | K34E | A18T/Y30N/K34E |
| Unit bacterial enzyme (U/ml) alive | 0.124± 0.014 | 0.249± 0.032 | 0.308± 0.005 | 0.707± 0.046 | 1.165±0.029 |
Than wild-type D-carbamyl hydrolysis enzyme, the enzyme of the unit thalline of mutant enzyme A18T, Y30N, K34E and A18T/Y30N/K34E is lived and is improved 1 times, 1.5 times, 4.7 times and 8.4 times respectively, and the advantage of the mutant enzyme that wherein superposes is especially obvious.
The foregoing description shows, 4 mutant enzymes all are better than wild-type at solubility expression and unit bacterial enzyme aspect living, and also further show that the sudden change stack has very big potentiality simultaneously, and adopting orthogenesis technological transformation industrial enzymes is highly effective means.
Be understood that in addition, read of the present invention tell about content after, under the prerequisite of the spirit and scope that do not depart from the present invention, those skilled in the art do any modification or revise the present invention all is should be included within the application's appended claims institute restricted portion.
Sequence table
＜110〉Shanghai Inst. of Life Science, CAS
＜120〉mutant of D-carbamyl hydrolysis enzyme and application thereof
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Thr Lys Ala Ala Ser Arg Gly Ala Asn Phe Ile Val Phe Pro Glu Leu
35 40 45
Ala Leu Thr Thr Phe Phe Pro Arg Trp His Phe Thr Asp Glu Ala Glu
50 55 60
Leu Asp Ser Phe Tyr Glu Thr Glu Met Pro Gly Pro Val Val Arg Pro
65 70 75 80
Leu Phe Glu Lys Ala Ala Glu Leu Gly Ile Gly Phe Asn Leu Gly Tyr
85 90 95
Ala Glu Leu Val Val Glu Gly Gly Val Lys Arg Arg Phe Asn Thr Ser
100 105 110
Ile Leu Val Asp Lys Ser Gly Lys Ile Val Gly Lys Tyr Arg Lys Ile
115 120 125
His Leu Pro Gly His Lys Glu Tyr Glu Ala Tyr Arg Pro Phe Gln His
130 135 140
Leu Glu Lys Arg Tyr Phe Glu Pro Gly Asp Leu Gly Phe Pro Val Tyr
145 150 155 160
Asp Val Asp Ala Ala Lys Met Gly Met Phe Ile Cys Asn Asp Arg Arg
165 170 175
Trp Pro Glu Ala Trp Arg Val Met Gly Leu Arg Gly Ala Glu Ile Ile
180 185 190
Cys Gly Gly Tyr Asn Thr Pro Thr His Asn Pro Pro Val Pro Gln His
195 200 205
Asp His Leu Thr Ser Phe His His Leu Leu Ser Met Gln Ala Gly Ser
210 215 220
Tyr Gln Asn Gly Ala Trp Ser Ala Ala Ala Gly Lys Val Gly Met Glu
225 230 235 240
Glu Asn Cys Met Leu Leu Gly His Ser Cys Ile Val Ala Pro Thr Gly
245 250 255
Glu Ile Val Ala Leu Thr Thr Thr Leu Glu Asp Glu Val Ile Thr Ala
260 265 270
Ala Val Asp Leu Asp Arg Cys Arg Glu Leu Arg Glu His Ile Phe Asn
275 280 285
Phe Lys Gln His Arg Gln Pro Gln His Tyr Gly Leu Ile Ala Glu Leu
290 295 300
Claims (10)
1, a kind of mutant of D-carbamyl hydrolysis enzyme is characterized in that, has following each described aminoacid sequence:
The 18th alanine mutation is a Threonine in the aminoacid sequence of the D-carbamyl hydrolysis enzyme shown in A, the SEQ ID NO:2;
The 30th tyrosine sports l-asparagine in the aminoacid sequence of the D-carbamyl hydrolysis enzyme shown in B, the SEQ ID NO:2;
The 34th lysine mutation is a glutamine in the aminoacid sequence of the D-carbamyl hydrolysis enzyme shown in C, the SEQ ID NO:2;
The 18th alanine mutation is a Threonine in the aminoacid sequence of the D-carbamyl hydrolysis enzyme shown in D, the SEQ IDNO:2, and the 30th tyrosine sports l-asparagine, and the 34th lysine mutation is a glutamine.
2, the mutant of D-carbamyl hydrolysis enzyme as claimed in claim 1, it is characterized in that, the aminoacid sequence of this mutant is that the 18th alanine mutation is a Threonine in the aminoacid sequence of the D-carbamyl hydrolysis enzyme shown in the SEQ ID NO:2, the 30th tyrosine sports l-asparagine, and the 34th lysine mutation is a glutamine.
3, the dna sequence dna of the described D-carbamyl hydrolysis enzyme of coding claim 1 mutant.
4, dna sequence dna as claimed in claim 3 is characterized in that, has following each described dna sequence dna:
The GCG of 52-54bp sports ACG in the gene order of the D-carbamyl hydrolysis enzyme shown in A, the SEQ ID NO:1;
The TAC of 88-90bp sports AAC in the gene order of the D-carbamyl hydrolysis enzyme shown in B, the SEQ ID NO:1;
The AAA of 100-102bp sports GAA in the gene order of the D-carbamyl hydrolysis enzyme shown in C, the SEQ ID NO:1;
The GCG of 52-54bp sports ACG in the gene order of the D-carbamyl hydrolysis enzyme shown in D, the SEQ ID NO:1, and the TAC of 88-90bp sports AAC, and the AAA of 100-102bp sports GAA.
5, dna sequence dna as claimed in claim 4, it is characterized in that, this dna sequence dna is that the GCG of 52-54bp sports ACG in the gene order of the D-carbamyl hydrolysis enzyme shown in the SEQ ID NO:1, and the TAC of 88-90bp sports AAC, and the AAA of 100-102bp sports GAA.
6, the application of D-carbamyl hydrolysis enzyme mutant as claimed in claim 1 or 2 in the D-D-pHPG is produced.
7, the application of the encoding gene of D-carbamyl hydrolysis enzyme mutant as claimed in claim 3 in the D-D-pHPG is produced.
8, application as claimed in claim 7 is characterized in that, dna sequence dna as claimed in claim 3 is cloned into host cell, and the zymophyte body of this host cell is used for the production of D-D-pHPG.
9, application as claimed in claim 8 is characterized in that, described host cell is intestinal bacteria.
10, application as claimed in claim 9 is characterized in that, described host cell is intestinal bacteria E.coli BL21.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101544969B (en) * | 2008-03-25 | 2012-07-18 | 中国科学院上海生命科学研究院 | Mutant of D-carbamyl hydrolysis enzyme and application thereof |
| CN102146365B (en) * | 2010-02-08 | 2013-07-10 | 中国科学院上海生命科学研究院 | D-carbamoyl hydrolase mutant |
| CN106011117A (en) * | 2015-07-30 | 2016-10-12 | 重庆桑禾动物药业有限公司 | N-carbamoyl-D-p-hydroxyphenylglycine hydrolase mutants and construction of engineering bacteria thereof |
| CN111454933A (en) * | 2020-05-08 | 2020-07-28 | 江南大学 | D-carbamoyl hydrolase mutant and application thereof in synthesis of D-aromatic amino acid |
| CN114807103A (en) * | 2022-06-10 | 2022-07-29 | 铜陵利夫生物科技有限公司 | Carbamyl hydrolase mutant, gene and application |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE69531725T2 (en) * | 1994-06-24 | 2004-07-08 | Kanegafuchi Kagaku Kogyo K.K. | METHOD FOR PRODUCING D-AMINO ACID BY A COMPOSED IMMOBILIZED ENZYME COMPOUND |
| IT1277125B1 (en) * | 1995-12-21 | 1997-11-04 | Eniricerche Spa | THERMOSTABLE MUTANTS OF D-N-ALPHA-CARBAMYLASE |
| CN1062603C (en) * | 1996-01-31 | 2001-02-28 | 中国科学院大连化学物理研究所 | Film reaction technology for producing D-p-hydroxy-phenyl glycine by enzyme method |
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2006
- 2006-01-06 CN CNB2006100231478A patent/CN100447240C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101544969B (en) * | 2008-03-25 | 2012-07-18 | 中国科学院上海生命科学研究院 | Mutant of D-carbamyl hydrolysis enzyme and application thereof |
| CN102146365B (en) * | 2010-02-08 | 2013-07-10 | 中国科学院上海生命科学研究院 | D-carbamoyl hydrolase mutant |
| CN106011117A (en) * | 2015-07-30 | 2016-10-12 | 重庆桑禾动物药业有限公司 | N-carbamoyl-D-p-hydroxyphenylglycine hydrolase mutants and construction of engineering bacteria thereof |
| CN106011117B (en) * | 2015-07-30 | 2019-06-18 | 重庆桑禾动物药业有限公司 | The building of N- carbamyl-D-pHPG hydrolysis enzyme mutant and its engineering bacteria |
| CN111454933A (en) * | 2020-05-08 | 2020-07-28 | 江南大学 | D-carbamoyl hydrolase mutant and application thereof in synthesis of D-aromatic amino acid |
| CN114807103A (en) * | 2022-06-10 | 2022-07-29 | 铜陵利夫生物科技有限公司 | Carbamyl hydrolase mutant, gene and application |
| CN114807103B (en) * | 2022-06-10 | 2023-07-04 | 铜陵利夫生物科技有限公司 | Carbamoyl hydrolase mutant, gene and application |
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|---|---|
| CN100447240C (en) | 2008-12-31 |
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