CN101654677A - Gene for efficiently expressing nuclease P1 and application thereof - Google Patents
Gene for efficiently expressing nuclease P1 and application thereof Download PDFInfo
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Abstract
The invention discloses a gene for efficiently expressing nuclease P1, an expression vector containing the gene, a host cell containing the gene and application of the gene, and also discloses a recombinant pichia pastoris CGMCC No.3188 for expressing the nuclease P1 produced by penicillium citrinum and a construction method thereof. The recombinant engineering strain Pichia pastoris CGMCC No.3188 can efficiently express the penicillium citrinum nucP gene, secretes and expresses nuclease P1, has higher phosphodiesterase activity, is used for hydrolyzing RNA, has high product yield and less generated impurities, and is beneficial to reducing the working procedures of downstream separation and reducing the production cost. The recombinant engineering strain Pichia pastoris CGMCC No.3188 is used for high-density fermentation, and the enzyme activity is 1.5 times of that of a wild strain. The obtained recombinant enzyme is applied to enzymolysis of RNA solution, the hydrolysis rate reaches more than 40%, and the obtained hydrolysate by-products are greatly reduced.
Description
Technical field
The invention belongs to bioengineering field, gene and the application thereof of particularly a kind of express nucleic acid enzyme P1.
Background technology
5 '-Nucleotide [Wang Yuqin that plays an important role at aspects such as cellularstructure, metabolism, energy and regulatory functions, Cheng Wufeng, Li Xuanhai. Nucleotide and nutrition. foreign medical science hygiology fascicle, 1999,26 (5): 257-260], be indispensable active substance in the organism.In food service industry, by initial food fragrance adding agent, expand to and have the functional food additives that improves the organism immunologic function, can be added in the food such as bread, biscuit, especially the result of use in infant or baby food is very obvious, can effectively strengthen the ability that the infant resists bacillary dysentery, reduce the generation of diarrhoea; At field of medicaments, 5 '-Nucleotide not only can be as drug use, but also is the raw materials for production of many antiviral antitumor drugs; In addition, nucleotide preparation can also have tangible volume increase, weightening finish effect [production technology of the prosperous Nucleotide of Wang Ding and application prospect oil and foodstuffs science and technology, 2008,16 (3): 65-66] as growth regulating agent for animal and plant.The main generation method of 5 '-Nucleotide is to adopt phosphodiester enzymic hydrolysis RNA to obtain, and the nuclease P 1 in Penicillium citrinum source is to produce the most widely used phosphodiesterase of Nucleotide at present.
Nuclease P 1 (E.C.3.1.30.1) is the phosphodiesterase that is produced by Penicillium citrinum (Penicillium citrinum), can produce four kinds of mononucleotides (5 ' AMP, 5 ' CMP, 5 ' GMP, 5 ' UMP) by hydrolysis RNA.Penicillium citrinum (Penicilliumcitrinum) belongs to asymmetric mould group, velvet-like mould subgroup, and Penicillium citrinum system is present in soil, and the grain of rotten fruit, vegetables, meat and storage is first-class.By the nuclease P 1 that the Penicillium citrinum of fermentation method preparation is originated, the fermentation costs height, and the assorted enzyme that produces is more, influences hydrolysis efficiency, causes later stage four mononucleotide separation difficulty, has increased to add production cost.
The effective means that addresses the above problem is that the gene nucP that will produce nuclease P 1 in the Penicillium citrinum is integrated in the pichia spp, makes up engineering strain.Pichia spp is the energy high-density growth under aerobic conditions, helps scale operation and obtains the high density cell, thereby efficiently express; Pichia spp self is secreted in the substratum albumen seldom, makes the foreign protein of secretion property separate from the matrix of substratum easily; , be difficult for losing to the genomic foreign gene Stability Analysis of Structures of pichia spp by plasmid integration; Foreign gene can be incorporated in the pichia spp genome with high copy number, can screen high expression level bacterial strain [Li Zhi dragon Zhang Fuchun pichia pastoris phaff expression system progress, biotechnology circular 2006 (6); 9-13].Because above-mentioned advantage adopts the pichia spp system to express target enzyme, can reduce assorted enzyme and produce, obtain more enzymatic activity high, improve enzymolysis efficiency, reduce the later stage and separate the mononucleotide cost.
Summary of the invention
Technical problem to be solved by this invention provides a kind of gene of highly expressing nuclease P 1.
The technical problem that the present invention also will solve provides the expression vector that comprises said gene.
The technical problem that the present invention also will solve provides the host cell that comprises above-mentioned expression vector.
Another technical problem that the present invention also will solve provides the construction process of above-mentioned host cell.
The technical problem that the present invention will solve at last provides the application of gene in producing nuclease P 1 of above-mentioned highly expressing nuclease P 1.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A kind of gene of highly expressing nuclease P 1, its nucleotide sequence is shown in SEQ ID NO:1.This nuclease P 1 nucP mrna length is 810bp, 270 amino-acid residues of having encoded.
A kind of expression vector contains the described gene of claim 1.
Above-mentioned expression vector preferably contains the pichia spp secreted expression carrier pPIC9K-nucP just like gene shown in the SEQ ID NO:1.
A kind of host cell contains above-mentioned expression vector.
Above-mentioned host cell preferably contains the pichia pastoris of pPIC9K-nucP expression vector.
Above-mentioned host cell more has the pichia pastoris engineered strain that selects express nucleic acid enzyme P1, its called after pichia pastoris (Pichia pastoris) of classifying, deposit number CGMCC No.3188.This bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101 on July 10th, 2009.
Make up the method for host cell CGMCC No.3188, adopt the RT-PCR method from Penicillium citrinum (Penicilliumcitrinum) bacterial strain, to extract the nucP gene, make up recombinant expression plasmid carrier pPCI9K-nucP, utilize electric method for transformation that recombinant plasmid is incorporated on pichia pastoris (Pichia pastoris) the GS115 bacterial strain, on MD and MM substratum, screen, identify positive transformant through PCR, G418 screening multiple copied transformant, carry out abduction delivering then, screening obtains the highest bacterial strain CGMCC No.3188 of a strain yield of enzyme.
The application of the gene of above-mentioned highly expressing nuclease P 1 in producing nuclease P 1.
Above-mentioned application is embodied in and utilizes recombinant bacterial strain CGMCC No.3188 to produce nuclease P 1.Method is as follows: adopt high density fermentation technology enlarged culturing to produce recombinant bacterial strain CGMCC No.3188 in fermentor tank, substratum is every liter of phosphoric acid 15～20mL, calcium phosphate 0.1～0.5g, vitriolate of tartar 10～20g, sal epsom 12～20g, potassium hydroxide 2.0～4.0g, glycerine 20～40g, 28～30 ℃ of leavening temperatures, pH6.0, dissolved oxygen is controlled at 5LPM, fermentation is divided into two stages, and the fs is the thalli growth stage, is seeded in the above-mentioned substratum by 10% (v/v) and grows, fs incubation time 15～18 hours, after glycerine exhausted, dissolved oxygen rose rapidly, changed the multiplicative stage this moment over to, stream adds inductor methyl alcohol, the adding volume of inductor is 3% of a culture volume, and it is 6～10h that stream adds the time, makes dissolved oxygen maintain more than the 5LPM, total incubation time of multiplicative stage is 45～50h, and the elimination yeast cell obtains containing the supernatant liquor of nuclease P 1.
Beneficial effect: the present invention has found the gene nucP of highly expressing nuclease P 1 in the Penicillium citrinum (Penicillium citrinum) first, and has realized nuclease P 1 gene efficiently expressing in the eukaryotic expression system pichia spp.Recombinant strain pichia spp CGMCC No.3188 can efficiently express Penicillium citrinum nucP gene, the secreting, expressing nuclease P 1 has higher phosphodiesterase activity, is used for hydrolysis RNA, the efficiency of pcr product height, the impurity that produces is less, helps reducing the operation of downstream separation and reducing production costs.Utilize recombinant strain pichia spp CGMCCNo.3188 of the present invention to carry out high density fermentation, final thalline OD
600Be 210, the work of nuclease P 1 enzyme reaches 629U/mL, and 410U/mL compares with starting strain, and enzyme work is 1.5 times of wild strain.The recombinase that obtains is applied to enzymolysis RNA solution, and percent hydrolysis reaches more than 40%, and the hydrolysate by product that obtains significantly reduces.
Embodiment:
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, reaction conditions and result thereof only are used to illustrate the present invention, and should also can not limit the present invention described in detail in claims.
Embodiment 1: the structure of recombination microzyme.
1, the clone of Penicillium citrinum (Penicillium citrinum) nuclease P 1 gene nucP.
(1) extraction of the total RNA of Penicillium citrinum.
Get 50ml penicillium citrinum liquid, 4 ℃, 8000 ℃ rmin
-1Centrifugal 15min collects thalline, and with mortar liquid nitrogen precooling, the limit adds the liquid nitrogen limit and is ground into powder, and adds 3～5 times of Trizol reagent; Room temperature is put 15min, adds the 1/5V chloroform, votex mixing 10s, room temperature 10min, 4 ℃, centrifugal 1200rpm, 15min; Remove supernatant liquor packing 500～600 μ L, add the equal-volume Virahol, mixing is 5 times up and down, leaves standstill 10min, 12000g, centrifugal 4 ℃, 15min; Fall supernatant liquor, thieving paper blots, and adds 1mL 75% alcohol flushing precipitation, leaves standstill 1min, and 4 ℃, the centrifugal 10min of 12000g; Abandon supernatant, be inverted in cystose, super clean bench dries up, and adds DEPC water 10 μ L, and is centrifugal slightly behind the votex, and 55 ℃ of water-bath 10min obtain the total RNA of Penicillium citrinum, and 1% agarose carries out electrophoresis detection, the concentration of spectrophotometric determination RNA.
(2) cDNA reverse transcription.
The MLVRT reversed transcriptive enzyme has been adopted in experiment, adds the RNA sample of about 5 μ L in removing the 0.5mL centrifuge tube of RNase, and 70 ℃ of incubation 5min behind 2 μ L Oligo dT and the 0.5 μ L Ribonuclease inhibitor mixing take out ice bath 5min.Add reaction soln subsequently, behind 42 ℃ of insulation 90min, can make mRNA reverse transcription generation cDNA among total RNA.Reaction solution is as follows: 5 * damping fluid, 5 μ L, dNTP 5 μ L, RNase inhibitor (40U μ L
-1) 0.5 μ L, MLV RTase (10U μ L
-1) 6 μ L, remove RNase water to 25 μ L.
(3) amplification of nuclease P 1 gene.
Upstream primer 5 '-TGGGGCAACCTGGGGCATG-3 ', downstream primer 5 '-CTTAGCAATTTCAGAACCGTGAATTTCGTT-3 '.Getting 1 μ L reverse transcription product is template, adopts the Taq archaeal dna polymerase to carry out the pcr amplification of gene.Reaction soln is as follows: sterilized water 40.5 μ L, 10 * damping fluid, 5 μ L, dNTP 1 μ L, forward and reverse primer (50 μ molL
-1) each 1 μ L, template 1 μ L, archaeal dna polymerase 0.5 μ L.The PCR condition is 94 ℃ of 1min of sex change; 94 ℃ of 1min; 54 ℃ of 1min; 72 ℃ of 1.5min; Totally 30 circulations; Last 72 ℃ are extended 10min.Getting 3 μ L amplified productions detects with 1.0% agarose gel electrophoresis.
(4) clone of nuclease P 1 gene.
Adopt TaKaRa Agarose Gel DNA purification Kit to reclaim pcr amplification product, under the effect of T4DNA ligase enzyme with Pgem
R-T Vector carries out ligation.In 100 μ LDH5a competent cells, add 5 μ L and connect liquid mixing in aseptic centrifuge tube, place 20min on ice.Beginning transformation experiment, ice bath 2min behind 42 ℃ of water-bath 50s, every pipe add 37 ℃ of shaking tables of 1mL LB substratum and cultivate 1-1.5h.Last 5000rmin
-1The centrifugal 5min of room temperature removes supernatant, adds 200 μ L LB substratum vibration suspension cell.Draw 100 μ L conversion fluids to the LB solid medium that contains Amp, conversion fluid evenly is applied to agar plate surface with spreading rod.Flat board is absorbed until conversion fluid fully as for room temperature, is inverted dull and stereotyped 37 ℃ of cultivation 12-16h and single bacterium colony occurs.
(5) extraction of plasmid DNA: subtraction extracts plasmid DNA.
(6) order-checking of reorganization T vector plasmid: reorganization T vector plasmid send the Nanjing peculiar limit of gold think of company to finish order-checking on ABI3730 type sequenator.Nuclease P 1 nucP mrna length is 810bp, 270 amino-acid residues of having encoded.
2, the structure of gene nucP expression vector.
Primer is expressed in nucleotide sequence design according to the nucP gene that obtains, 5 ' end and 3 ' end difference primer SnaBI and NotI restriction enzyme site at primer, pcr amplification, adopt SnaBI and NotI double digestion PCR product and plasmid pPIC9K, two enzymes are cut product to be connected with the T4 ligase enzyme, obtain recombinant plasmid pPIC9K-nucP, and adopt the method for chemical conversion to enter bacillus coli DH 5 alpha, obtain containing the recombinant expression plasmid of nuclease P 1 gene through the screening of bacterium colony PCR method, go forward side by side performing PCR and enzyme cut evaluation, and order-checking confirms that the reading frame of recombinant plasmid is correct.
3, express the structure and the screening of the pichia pastoris engineered strain of Penicillium citrinum nuclease P 1 nucP gene.
(1) linearizing of recombinant expression plasmid: with recombinant expression plasmid pPIC9K-nucP restriction enzyme Sal I linearizing.
(2) transform: electric shock transformed yeast Pichia pastoris GS115 yeast competent cell, method for transformation is referring to Invitrogen company pichia spp operational manual.
(3) screening: on MM and MD flat board, cultivate 2-3d with the corresponding dibbling of sterilization toothpick picking transformant for 30 ℃, picking is the positive transformant of all well-grown transformant on the MD/MM flat board.Picking positive transformant dibbling respectively screens the multiple copied transformant on the flat board of 250 μ g/mL, 300 μ g/mL, 400 μ g/mL, 500 μ g/mL and 600 μ g/mL G418.
Embodiment 2: the abduction delivering of recombinant pichia yeast strain in shaking bottle.
Select a single bacterium colony, place the 250mL that 25mL BMGY substratum is housed to shake bottle, in 30 ℃, 250-300rmin
-1Be cultured to OD
600To 2-6.The centrifugal 5min of 1500-3000g under the room temperature collects thalline, with the resuspended thalline of BMMY, makes OD
600Reach about 1.0 (about 100-200mL).The bacterium liquid of gained is placed the bottle that shakes of 1L, seal, be positioned over 28-30 ℃, 250-300rmin with double gauze or cheese cloth
-1Shaking table on continued growth.Every 24h adds 100% methyl alcohol 1～5% (v/v) in substratum.After testing, enzyme work reaches 625U/mL, and the wild strain enzyme is lived high by 52.43% than setting out.
Embodiment 3: the high density fermentation of recombinant pichia yeast strain in the 5L fermentor tank.
Fermention medium is that substratum is every liter of phosphoric acid 16mL, calcium phosphate 0.3g, and vitriolate of tartar 12g, sal epsom 15g, potassium hydroxide 3.0g, glycerine 27g, 28 ℃ of leavening temperatures, pH6.0, dissolved oxygen is controlled at 5LPM.Fermentation is divided into two stages, and the fs is the thalli growth stage, is seeded in the above-mentioned substratum by 10% (v/v) and grows, fs incubation time 16 hours, after glycerine exhausted, dissolved oxygen rose rapidly, changed the multiplicative stage this moment over to, stream adds inductor methyl alcohol, the adding volume of inductor is 3% of a culture volume, and it is 16h that stream adds the time, makes dissolved oxygen maintain more than the 5LPM, total incubation time of multiplicative stage is 48h, and the elimination yeast cell obtains supernatant liquor.Final thalline OD
600Be 210, the work of nuclease P 1 enzyme reaches 621U/mL.
Enzyme biopsy survey method: the substrate solution of 1.9ml (is contained concentration and be about 1% RNA; 0.2M the acetate buffer solution of pH5.2 is with the ZnSO4 of 0.0005M) behind 70 ℃ of water bath with thermostatic control 10min, add the enzyme liquid of 0.1ml through suitably diluting, add 2.0ml nucleic acid precipitation agent (0.25% ammonium molybdate-2.5 is crossed chloric acid) behind 70 ℃ of insulation 15min, centrifugal behind the ice-water bath 20min, get supernatant distilled water diluting certain multiple, the light absorption value of measuring its 260nm place is A260.With the person that adds the precipitation agent earlier in contrast, other operation is the same.Under these conditions, the Nucleotide amount that per minute generated is to be defined as 1 enzyme activity unit at 1.0 o'clock in the difference of the light absorption value at 260nm place.Its calculation formula is as follows:
Wherein α is the extension rate of original enzyme liquid, and β is the extension rate of centrifugal supernatant.
Embodiment 4: recombinant pichia yeast strain produces nuclease P 1 enzymolysis RNA.
Enzymatic hydrolysis condition: prepare the RNA solution 100ml of 5% content, phosphoric acid buffer control pH7.0,65 ℃ of reactions add the recombinant nucleic acid enzyme P11～2ml that obtains, and hydrolysis 3～5 hours, sampling are carried out HPLC and are analyzed.Final hydrolysis result is 24.323g/L, and enzymolysis reaches 46.67%.
Detection method: adopt HPLC
The system LichrospherC18 of Huaiyin Han Bang Science and Technology Ltd. post (5mm, 250mm * 4.6mm); Moving phase is A:20mmolL
-1(NH
4)
2HPO
4Solution (preparing) with ultrapure water; B: methyl alcohol, gradient elution.Flow velocity: 1mLmin
-1Detect wavelength: 254nm; Sample size: 20 μ L.
Precision takes by weighing the mark product of four kinds of Nucleotide, is mixed with mass concentration with damping fluid and is about 50mgL
-1Standard solution; To be diluted to about 50mgL with damping fluid through pretreated sample
-1, and with after O.45 μ m microporous membrane filters, as need testing solution.
SEQUENCE?LISTING
＜110〉Nanjing University of Technology
＜120〉a kind of gene of highly expressing nuclease P 1 and application thereof
<130>njut090915
<160>2
<170>PatentIn?version?3.3
<210>1
<211>810
<212>DNA
＜213〉Penicillium citrinum (Penicillium citrinum)
<220>
<221>CDS
<222>(1)..(810)
<400>1
tgg?ggc?aac?ctg?ggg?cat?gca?aca?gtt?gct?tat?gtt?gct?caa?cat?tat 48
Trp?Gly?Asn?Leu?Gly?His?Ala?Thr?Val?Ala?Tyr?Val?Ala?Gln?His?Tyr
1 5 10 15
gta?tcg?ccc?gaa?gca?gct?tca?tgg?gcg?caa?ggg?atc?ctt?ggg?agc?tca 96
Val?Ser?Pro?Glu?Ala?Ala?Ser?Trp?Ala?Gln?Gly?Ile?Leu?Gly?Ser?Ser
20 25 30
tcc?agc?tcg?tat?ctg?gcc?agt?att?gcc?tca?tgg?gca?gac?gaa?tat?cgc 144
Ser?Ser?Ser?Tyr?Leu?Ala?Ser?Ile?Ala?Ser?Trp?Ala?Asp?Glu?Tyr?Arg
35 40 45
ttg?aca?agc?gcc?ggg?aaa?tgg?tca?gct?tca?ttg?cat?ttc?atc?gat?gca 192
Leu?Thr?Ser?Ala?Gly?Lys?Trp?Ser?Ala?Ser?Leu?His?Phe?Ile?Asp?Ala
50 55 60
gaa?gat?aat?ccc?ccc?acg?aac?tgc?aac?gtg?gat?tat?gag?cga?gac?tgc 240
Glu?Asp?Asn?Pro?Pro?Thr?Asn?Cys?Asn?Val?Asp?Tyr?Glu?Arg?Asp?Cys
65 70 75 80
ggg?agc?tca?ggc?tgc?tca?atc?tca?gca?att?gca?aat?tac?acc?cag?cgt 288
Gly?Ser?Ser?Gly?Cys?Ser?Ile?Ser?Ala?Ile?Ala?Asn?Tyr?Thr?Gln?Arg
85 90 95
gtg?agt?gat?agc?agt?ctt?tca?tca?gag?aac?cat?gca?gaa?gct?ctg?agg 336
Val?Ser?Asp?Ser?Ser?Leu?Ser?Ser?Glu?Asn?His?Ala?Glu?Ala?Leu?Arg
100 105 110
ttc?ctt?gtt?cac?ttc?att?gga?gac?atg?acc?cag?ccg?cta?cac?gac?gag 384
Phe?Leu?Val?His?Phe?Ile?Gly?Asp?Met?Thr?Gln?Pro?Leu?His?Asp?Glu
115 120 125
gcc?tac?gca?gtc?ggg?ggt?aat?aaa?atc?aat?gtc?acc?ttc?gac?gga?tac 432
Ala?Tyr?Ala?Val?Gly?Gly?Asn?Lys?Ile?Asn?Val?Thr?Phe?Asp?Gly?Tyr
130 135 140
cac?gac?aac?ctc?cat?tca?gat?tgg?gat?act?tat?atg?ccc?cag?aaa?ctg 480
His?Asp?Asn?Leu?His?Ser?Asp?Trp?Asp?Thr?Tyr?Met?Pro?Gln?Lys?Leu
145 150 155 160
atc?ggt?ggc?cat?gca?ctc?tca?gat?gca?gag?tca?tgg?gca?aag?acc?ttg 528
Ile?Gly?Gly?His?Ala?Leu?Ser?Asp?Ala?Glu?Ser?Trp?Ala?Lys?Thr?Leu
165 170 175
gtc?caa?aat?atc?gaa?tcg?ggg?aat?tat?acg?gct?caa?gct?atc?gga?tgg 576
Val?Gln?Asn?Ile?Glu?Ser?Gly?Asn?Tyr?Thr?Ala?Gln?Ala?Ile?Gly?Trp
180 185 190
atc?aag?ggt?gat?aat?atc?agc?gag?cca?att?act?acc?gca?act?cga?tgg 624
Ile?Lys?Gly?Asp?Asn?Ile?Ser?Glu?Pro?Ile?Thr?Thr?Ala?Thr?Arg?Trp
195 200 205
gct?tca?gat?gcg?aat?gct?tta?gtc?tgc?act?gtt?gtt?atg?cct?cat?gga 672
Ala?Ser?Asp?Ala?Asn?Ala?Leu?Val?Cys?Thr?Val?Val?Met?Pro?His?Gly
210 215 220
gct?gcc?gct?tta?cag?acc?ggt?gat?ctt?tat?ccc?acc?tat?tat?gat?tca 720
Ala?Ala?Ala?Leu?Gln?Thr?Gly?Asp?Leu?Tyr?Pro?Thr?Tyr?Tyr?Asp?Ser
225 230 235 240
gtc?atc?gac?acc?atc?gaa?ttg?cag?att?gct?aag?ggc?ggc?tat?cga?ttg 768
Val?Ile?Asp?Thr?Ile?Glu?Leu?Gln?Ile?Ala?Lys?Gly?Gly?Tyr?Arg?Leu
245 250 255
gca?aat?tgg?gtc?aac?gaa?att?cac?ggt?tct?gaa?att?gct?aag 810
Ala?Asn?Trp?Val?Asn?Glu?Ile?His?Gly?Ser?Glu?Ile?Ala?Lys
260 265 270
<210>2
<211>270
<212>PRT
＜213〉Penicillium citrinum (Penicillium citrinum)
<400>2
Trp?Gly?Asn?Leu?Gly?His?Ala?Thr?Val?Ala?Tyr?Val?Ala?Gln?His?Tyr
1 5 10 15
Val?Ser?Pro?Glu?Ala?Ala?Ser?Trp?Ala?Gln?Gly?Ile?Leu?Gly?Ser?Ser
20 25 30
Ser?Ser?Ser?Tyr?Leu?Ala?Ser?Ile?Ala?Ser?Trp?Ala?Asp?Glu?Tyr?Arg
35 40 45
Leu?Thr?Ser?Ala?Gly?Lys?Trp?Ser?Ala?Ser?Leu?His?Phe?Ile?Asp?Ala
50 55 60
Glu?Asp?Asn?Pro?Pro?Thr?Asn?Cys?Asn?Val?Asp?Tyr?Glu?Arg?Asp?Cys
65 70 75 80
Gly?Ser?Ser?Gly?Cys?Ser?Ile?Ser?Ala?Ile?Ala?Asn?Tyr?Thr?Gln?Arg
85 90 95
Val?Ser?Asp?Ser?Ser?Leu?Ser?Ser?Glu?Asn?His?Ala?Glu?Ala?Leu?Arg
100 105 110
Phe?Leu?Val?His?Phe?Ile?Gly?Asp?Met?Thr?Gln?Pro?Leu?His?Asp?Glu
115 120 125
Ala?Tyr?Ala?Val?Gly?Gly?Asn?Lys?Ile?Asn?Val?Thr?Phe?Asp?Gly?Tyr
130 135 140
His?Asp?Asn?Leu?His?Ser?Asp?Trp?Asp?Thr?Tyr?Met?Pro?Gln?Lys?Leu
145 150 155 160
Ile?Gly?Gly?His?Ala?Leu?Ser?Asp?Ala?Glu?Ser?Trp?Ala?Lys?Thr?Leu
165 170 175
Val?Gln?Asn?Ile?Glu?Ser?Gly?Asn?Tyr?Thr?Ala?Gln?Ala?Ile?Gly?Trp
180 185 190
Ile?Lys?Gly?Asp?Asn?Ile?Ser?Glu?Pro?Ile?Thr?Thr?Ala?Thr?Arg?Trp
195 200 205
Ala?Ser?Asp?Ala?Asn?Ala?Leu?Val?Cys?Thr?Val?Val?Met?Pro?His?Gly
210 215 220
Ala?Ala?Ala?Leu?Gln?Thr?Gly?Asp?Leu?Tyr?Pro?Thr?Tyr?Tyr?Asp?Ser
225 230 235 240
Val?Ile?Asp?Thr?Ile?Glu?Leu?Gln?Ile?Ala?Lys?Gly?Gly?Tyr?Arg?Leu
245 250 255
Ala?Asn?Trp?Val?Asn?Glu?Ile?His?Gly?Ser?Glu?Ile?Ala?Lys
260 265 270
Claims (9)
1, a kind of gene of highly expressing nuclease P 1, its nucleotide sequence is shown in SEQ ID NO:1.
2, a kind of expression vector is characterized in that containing the described gene of claim 1.
3, expression vector according to claim 2 is characterized in that described expression vector is the pichia spp secreted expression carrier pPIC9K-nucP that contains the described gene of claim 1.
4, a kind of host cell is characterized in that containing the described expression vector of claim 2.
5, host cell according to claim 4 is characterized in that described host cell is the pichia pastoris that contains the pPIC9K-nucP expression vector.
6, host cell according to claim 5 is characterized in that described host cell is the pichia pastoris engineered strain of express nucleic acid enzyme P1, its called after pichia pastoris (Pichia pastoris) of classifying, deposit number CGMCCNo.3188.
7, make up the method for the described host cell of claim 6, it is characterized in that adopting the RT-PCR method from the Penicillium citrinum bacterial strain, to extract the nucP gene, make up recombinant expression plasmid carrier pPCI9K-nucP, utilize electric method for transformation that recombinant plasmid is incorporated on pichia pastoris (Pichia pastoris) GS 115 bacterial strains, on MD and MM substratum, screen, identify positive transformant through PCR, G418 screening multiple copied transformant, carry out abduction delivering then, screening obtains the highest bacterial strain CGMCC No.3188 of a strain yield of enzyme.
8, the application of the gene of the described highly expressing nuclease P 1 of claim 1 in producing nuclease P 1.
9, the application of the gene of highly expressing nuclease P 1 according to claim 8 in producing nuclease P 1, it is characterized in that in fermentor tank, adopting high density fermentation technology enlarged culturing to produce recombinant bacterial strain CGMCCNo.3188, substratum is every liter of phosphoric acid 15～20mL, calcium phosphate 0.1～0.5g, vitriolate of tartar 10～20g, sal epsom 12～20g, potassium hydroxide 2.0～4.0g, glycerine 20～40g, 28～30 ℃ of leavening temperatures, pH6.0, dissolved oxygen is controlled at 5LPM, and fermentation is divided into two stages, fs is the thalli growth stage, be seeded in the above-mentioned substratum by 10% (v/v) and grow, fs incubation time 15～18 hours is after glycerine exhausts, dissolved oxygen rises rapidly, change the multiplicative stage this moment over to, and stream adds inductor methyl alcohol, and the adding volume of inductor is 3% of a culture volume, it is 6～10h that stream adds the time, make dissolved oxygen maintain more than the 5LPM, total incubation time of multiplicative stage is 45～50h, and the elimination yeast cell obtains containing the supernatant liquor of nuclease P 1.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102757977A (en) * | 2012-07-19 | 2012-10-31 | 中国科学技术大学 | Expression and preparation method of granulysin |
| WO2015143638A1 (en) * | 2014-03-26 | 2015-10-01 | 南京工业大学 | Nucleotide production process |
| CN106318926A (en) * | 2015-06-19 | 2017-01-11 | 安琪酵母股份有限公司 | Method for producing nuclease P1 through fermentation of penicillium citrinum |
| CN113025634A (en) * | 2021-03-05 | 2021-06-25 | 中国食品发酵工业研究院有限公司 | Gene sequence containing sugar alcohol and ATP binding domain and expression and function verification method of gene sequence in yarrowia lipolytica |
| WO2023103543A1 (en) * | 2021-12-07 | 2023-06-15 | 南京工业大学 | Method for preparing nuclease p1 |
| CN118109448A (en) * | 2024-03-18 | 2024-05-31 | 武汉新华扬生物股份有限公司 | Auxiliary agent for improving nuclease stability and preparation method and application thereof |
-
2009
- 2009-09-17 CN CN200910183311A patent/CN101654677A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102757977A (en) * | 2012-07-19 | 2012-10-31 | 中国科学技术大学 | Expression and preparation method of granulysin |
| WO2015143638A1 (en) * | 2014-03-26 | 2015-10-01 | 南京工业大学 | Nucleotide production process |
| CN106318926A (en) * | 2015-06-19 | 2017-01-11 | 安琪酵母股份有限公司 | Method for producing nuclease P1 through fermentation of penicillium citrinum |
| CN106318926B (en) * | 2015-06-19 | 2019-10-25 | 安琪酵母股份有限公司 | A kind of Penicillium citrinum fermentation produces the production method of nuclease P 1 |
| CN113025634A (en) * | 2021-03-05 | 2021-06-25 | 中国食品发酵工业研究院有限公司 | Gene sequence containing sugar alcohol and ATP binding domain and expression and function verification method of gene sequence in yarrowia lipolytica |
| WO2023103543A1 (en) * | 2021-12-07 | 2023-06-15 | 南京工业大学 | Method for preparing nuclease p1 |
| CN118109448A (en) * | 2024-03-18 | 2024-05-31 | 武汉新华扬生物股份有限公司 | Auxiliary agent for improving nuclease stability and preparation method and application thereof |
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