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 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, 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 IDNO.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.
4.2, 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.
<110〉Shanghai Inst. of Life Science, CAS