CN106011093A - Glucose oxidase mutant GOD-M01 with improved enzymatic activity and expression vector and application of glucose oxidase mutant GOD-M01 - Google Patents
Glucose oxidase mutant GOD-M01 with improved enzymatic activity and expression vector and application of glucose oxidase mutant GOD-M01 Download PDFInfo
- Publication number
- CN106011093A CN106011093A CN201610525903.0A CN201610525903A CN106011093A CN 106011093 A CN106011093 A CN 106011093A CN 201610525903 A CN201610525903 A CN 201610525903A CN 106011093 A CN106011093 A CN 106011093A
- Authority
- CN
- China
- Prior art keywords
- glucoseoxidase
- god
- gly
- ala
- leu
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03004—Glucose oxidase (1.1.3.4)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/102—Plasmid DNA for yeast
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Mycology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a glucose oxidase mutant GOD-M01 with improved enzymatic activity and an expression vector and application of the glucose oxidase mutant GOD-M01. Based on glucose oxidase optimized according to codon preference of Pichia pastoris, mutation is carried out on the gene of the glucose oxidase with an error-prone PCR method, then direct mutation is picked out with a high-flux screening method, and the glucose oxidase mutant GOD-M01 with improved enzymatic activity is obtained. Compared with the enzymatic activity generated through original genes, the enzymatic activity of the glucose oxidase mutant GOD-M01 is improved by 47%, and thus the purpose of reducing production cost in the industrial production of the glucose oxidase mutant GOD-M01 can be easily achieved; the glucose oxidase mutant GOD-M01 has good market application prospects and high industrial value.
Description
Technical field
The invention belongs to genetic engineering and enzyme engineering field, particular content relates to the glucose oxidation that a kind of enzymatic activity improves
Enzyme mutant GOD-M01 and expression vector thereof and application.
Background technology
Can be in specific manner under glucoseoxidase (glucose oxidase, E.C.1.1.3.4, GOD) aerobic conditions
Catalysis β-D-Glucose generates gluconic acid and hydrogen peroxide, has important using value at industrial circle, the most exists
It is used widely in the fields such as food industry, livestock-raising and medical treatment detection.Glucoseoxidase is distributed widely in animals and plants
With in microbial body, microorganism is its main source, and the main bacterial strain that produces is aspergillus niger and penicillium sp.Originate from Penicllium notatum and black fermented preparation
Mould glucoseoxidase has been put into the big class enzyme preparation of Ministry of Agriculture's " feed additive kind catalogue (2013) " the 4th.Along with Portugal
Glucoseoxidase is more and more applied to every field, and its existing performance has been had more to come by industrial especially feed industry
The highest requirement, wherein the catalytic efficiency of glucoseoxidase determines its application cost, only has the Portugal that higher enzyme is lived
Glucoseoxidase could effectively control cost, thus adapts to the requirement of large-scale industrial production, it is achieved product marketization and business
Industry.The natural grape carbohydrate oxidase screening high enzyme alive is an important channel, and uses protein engineering means to natural Portugal
The artificial reconstructed of glucoseoxidase is employed the most more and more widely.
Fallibility PCR (error-prone PCR) technology is when utilizing Taq enzyme to carry out PCR amplifying target genes, by adjusting
Whole reaction condition (as improved enzyme ion concentration, the concentration etc. of 4 kinds of dNTP in change system), changes the mutation frequency of Taq enzyme, from
And introduce sudden change with certain frequency in genes of interest, build mutation library, then select or screen the mutant of needs.In order to obtain
Obtain more preferable mutation effect, it is possible to use continuous error-prone PCR (sequential error prone PCR) strategy, Ji Jiangyi
The template that the direct mutation gene that secondary PCR amplification obtains expands as PCR next time, carries out random mutagenesis the most repeatedly, makes every
Once expand the forward mutation accumulation obtained and produce important beneficial mutation.
Summary of the invention
Glucoseoxidase mutant GOD-M01 and expression thereof that a kind of enzymatic activity improves are it is an object of the invention to provide
Carrier and application.The present invention by derive from aspergillus niger (Aspergillus niger) glucoseoxidase carry out albumen
Matter is engineered, uses fallibility PCR method to suddenly change glucoseoxidase carbohydrase GOD-1 gene, then is sieved by high flux
Direct mutation is detected by choosing method, it is thus achieved that mutant protein, compared with wild type, the enzymatic activity of mutant GOD-M01 is shown
Write and improve.
For achieving the above object, the present invention is achieved by the following technical solutions:
The invention provides the glucoseoxidase mutant GOD-M01 that a kind of enzymatic activity improves, described glucoseoxidase
The aminoacid sequence of mutant GOD-M01 is as shown in SEQ ID NO:3, and described mutant GOD-M01 by aminoacid sequence is
173rd amino acids of the glucoseoxidase of SEQ ID NO:1 is become valine from threonine and obtains.
Present invention also offers the glucoseoxidase encoding gene of described glucoseoxidase mutant GOD-M01.
Further, described glucoseoxidase encoding gene has the nucleotide sequence as shown in SEQ ID NO:4.
Present invention also offers the recombinant expression carrier containing described glucoseoxidase encoding gene.
Further, described recombinant expression carrier is restructuring expression plasmid of yeast pPIC9K-GOD-M01.
Present invention also offers the recombinant bacterial strain containing described recombinant expression carrier.
Further, described recombinant bacterial strain is recombinant yeast pichia pastoris.
Present invention also offers described glucoseoxidase mutant GOD-M01 for preparing animal feed additive
In application.
It is further: described glucoseoxidase encoding gene is connected with expression vector structure recombinant expression carrier,
Recombinant expression carrier is transformed in host cell, it is thus achieved that recombinant host bacterial strain, cultivates recombinant host bacterial strain and induce restructuring Portugal
The expression of glucoseoxidase, it is thus achieved that glucoseoxidase.
Further: described host cell is Pichia sp..
Advantages of the present invention and have the technical effect that first the present invention has synthesized glucoseoxidase GOD-1 full genome sequence
Row, are optimized the gene of acquisition optimization simultaneously, and adopt according to pichia pastoris phaff codon preference to its DNA sequence
By fallibility PCR method, it is carried out random mutation, with pET 28a (+) the efficient mutation library of vector construction, then will containing sudden change base
The plasmid of cause proceeds in expressive host, selects monoclonal expressing protein, the most each orifice plate inoculation wild type gene expression strain
As comparison.Centrifugal resuspended after, take part bacterium solution and measure enzymatic activity.Activity is transferred to new culture plate higher than the bacterial strain of comparison
In, carry out repeating screening.Enzymatic activity screening obtained, higher than the bacterial strain of comparison, send gene sequencing company to check order, it is thus achieved that sudden change
The DNA sequence of body GOD-M01.Obtain enzyme live improve mutant GOD-M01, with original gene produce enzyme live compared with, its
Enzyme is lived and is improve 47%, thus is advantageously implemented its purpose reducing production cost in industrialized production, provided by the present invention
Glucoseoxidase mutant GOD-M01 has good market application foreground and industrial value.
Accompanying drawing explanation
Fig. 1 is glucoseoxidase mutant GOD-M01 and wild-type amino acid sequence comparative result；
Fig. 2 is glucoseoxidase mutant GOD-M01 and the enzyme of wild type comparative measurements result alive.
Detailed description of the invention
With specific embodiment, technical scheme is further described in detail below in conjunction with the accompanying drawings.Below in conjunction with
The present invention will be further described for embodiment, it should be pointed out that the present embodiment is only used for explaining the present invention, rather than to this
The restriction of bright scope.
Embodiment 1: fallibility PCR (error prone PCR) method builds glucoseoxidase GOD-1 mutated library
The glucoseoxidase GOD-1 deriving from aspergillus niger is formed (see SEQ ID NO:1) by 589 aminoacid.Obtain for rapid
It is beneficial to the transformation to GOD-1 enzyme molecule to GOD-1 complete genome sequence, according to the GOD-1 gene sequence information reported,
Full genome synthetic method is used to synthesize glucoseoxidase GOD-1 complete genome sequence (such as Genbank ID KJ774107.1
Shown in), the gene two ends of synthesis are also with EcoR I and Not I restriction enzyme site.Simultaneously according to pichia pastoris phaff codon
Preference is optimized the glucose oxidase gene GOD-1 (see SEQ ID NO:2) obtained after optimizing to its DNA sequence,
Use with the GOD-1 after optimization for template amplification glucoseoxidase carbohydrase GOD-1 gene, use GeneMorph II random
Sudden change PCR kit (Stratagene) is randomly incorporated into sudden change.
The primer is:
5′-GCGCGAATTCTTGCCACATTACATTAGATCCAACGGTAT-3 ' (SEQ ID No:5),
5′-TAAAGCGGCCGCTTATTGCATAGAAGCGTAATCTTCCAAAATAGC-3 ' (SEQ ID No:6)；
EcoR I and Not I restriction enzyme site it is respectively at underscore.
Reaction condition is: 94 ° of C denaturations 10min, 94 ° of C degeneration 60s, and 58 ° of C annealing 60s and 72 ° of C extend
2min, totally 30 circulations, 1% sepharose electrophoresis, test kit reclaims genes of interest fragment.
After double digested for purpose fragment EcoR I and Not I, with through identical enzyme action pET 21a (+) carry
Body (ampicillin resistance) Ligase is attached reaction.The fragment connected is converted to e. coli bl21-DE3, is coated with
Cloth contains the LB flat board of ampicillin, is inverted for 37 DEG C and cultivates, and after there is transformant on flat board, picking monoclonal is to 96 holes
Plate, containing 150uL LB culture medium (containing 1mM IPTG, 50ng/mL ampicillin) in every hole, the most each orifice plate is inoculated
Wild type gene expression strain is as comparison, and 12h is cultivated in 30 DEG C of 220rpm concussions, and orifice plate is placed in-20 DEG C, and multigelation breaks
Wall, it is thus achieved that containing the crude enzyme liquid of glucoseoxidase.Take out 5uL crude enzyme liquid and contain dianisidine to 96 new orifice plates, addition
Methanol buffer, dextrose buffer liquid, the nitrite ion 145uL altogether of horseradish peroxidase solution, add after 37 DEG C of reaction 3min
100uL2M sulphuric acid terminates reaction, measures enzyme according to chromogenic reaction and lives.
Take the enzyme activity bacterial strain higher than wild type GOD-1 in 96 new well culture plates, carry out repeating screening.Screen
One of them mutant is GOD-M01, and enzyme activity is 1-2 times of wild type control, and picking monoclonal send gene sequencing company to survey
Sequence.
Sequencing result shows, as it is shown in figure 1, epicycle fallibility PCR obtains a mutant containing T173V simple point mutation
Its aminoacid sequence of GOD-M01(is SEQ ID NO:3), coding produces the glucose oxidase gene sequence of mutant GOD-M01
Row are as shown in SEQ ID No:4.
Compared with GOD-M01:Mutation:173T → V(SEQ ID No:4 and SEQ ID No:2, in its DNA sequence
173 are become GTT for the nucleotide sequence that aminoacid is corresponding from ACT, and remaining base sequence is identical).
Embodiment 2: the structure of pichia pastoris engineered strain
Use primer described in embodiment 1, carry out PCR amplification using the mutant that embodiment 1 obtains as template, obtained 3
Two ends are with EcoR I and the glucoseoxidase mutant gene of Not I restriction enzyme site.PCR reaction condition is: 94 DEG C of degeneration
5min；94 DEG C of degeneration 30s, 56 DEG C of renaturation 30s, 72 DEG C extend 2min, 30 circulations, and 72 DEG C extend 10min.
The glucoseoxidase mutant gene fragment that above-mentioned clone is obtained and the base of raw glucose oxidase GOD-1
Because of fragment, it is connected with Expression vector pPIC9K by EcoR I and Not I site, construction of expression vector pPIC9K-GOD-
M01 and pPIC9K-GOD-1.
Above expression vector SalI is carried out linearisation, linearized fragment fragment purification test kit (TaKaRa
MiniBEST DNA Fragment Purifibation Kit) after purified pool, convert Pichia sp. by electricity method for transformation
GS115, is coated with MD flat board.The bacterium colony grown on MD flat board is applied to concentration and gradually rises (1mg/mL, 2mg/ successively
ML, 4mg/mL, 8mg/mL) Geneticin YPD flat board on screen the positive transformant of multicopy, obtain Pichia sp. restructuring
Bacterial strain.
The transformant named Pichia sp. GOD-M01(obtainedPichia pastoris And Pichia sp. GOD-M01)
GOD-1(Pichia pastorisGOD-1), the transformant of two genes of picking is transferred in BMGY culture medium respectively, 30 DEG C,
After 220rpm shaken cultivation 18h, centrifugal acquisition thalline, proceed to appropriate thalline, in BMMY culture medium, make cell concentration reach
OD600=1,30 DEG C, 220rpm continues shaken cultivation, and every 24h adds the methanol of volume of culture 1%.After abduction delivering 4d, will cultivate
Liquid is centrifugal obtains supernatant, and supernatant is carried out glucoseoxidase vitality test.
Glucoseoxidase enzyme activity detection method:
(0.1mL1% dianisidine methanol storing solution joins 12mL 0.1M pH6.0 to take dianisidine buffer 2.5mL
Phosphate buffer is made into), 18% glucose solution 0.3mL, 0.03% Peroxidase Solution 0.1mL, join in color comparison tube 37
DEG C insulation 5 min, add 0.1 mL glucoseoxidase enzyme liquid (blank tube add 0.1mL distilled water), after reaction 3min, add
Enter 2M sulphuric acid 2mL, mix to terminate reaction.With standard blank sample as blank, at 540 nm wavelength, measure blank (A0)
With sample solution (A1) light absorption value.Draw Δ A=A1- A0
Sample enzyme activity calculates:
X=(Δ A × n × 3)/(11.3 × t × 0.1)
T minute, min
0.1 sample volume, mL
11.3 extinction coefficient
N extension rate
3 reactant liquor volumes, mL
Enzyme is lived unit and definition: at pH5.5, under conditions of 37 DEG C, per minute β-the D-Glucose of 1.0 μm ol can be oxidized to Portugal
Grape saccharic acid and H2O2Enzyme amount be a unit.
Enzyme activity determination result: obtain Pichia sp. GOD-1 fermented supernatant fluid as in figure 2 it is shown, measure according to the method described above
Enzyme work is 53 U/mL, and the work of the enzyme of Pichia sp. GOD-M01 fermented supernatant fluid is 78 U/mL, and enzyme running water is flat improves 47%.
Embodiment 3: the cultivation application experiment of glucoseoxidase
3.1 experimental design
Experimentation selects body condition anosis Ross 308 broiler chicks 160000 similar, healthy, is divided into matched group and experimental group,
Often group 80000, often group arranges 4 repetitions, 20000 chickens of each repetition, and 1,3,5,7 hen houses are experimental group, 2,4,6,8
Hen house is matched group, and wherein experimental group adds the glucoseoxidase GOD-M01 that the present invention provides, feeding period 40 in daily ration
My god, concrete packet is shown in Table 1.
Table 1: experiment packet design
| Process group | Daily ration |
| 1 house | Basal diet+0.2% glucoseoxidase GOD-M01 |
| 2 houses | Basal diet |
| 3 houses | Basal diet+0.2% glucoseoxidase GOD-M01 |
| 4 houses | Basal diet |
| 5 houses | Basal diet+0.2% glucoseoxidase GOD-M01 |
| 6 houses | Basal diet |
| 7 houses | Basal diet+0.2% glucoseoxidase GOD-M01 |
| 8 houses | Basal diet |
The mensuration of 3.2 production performances
Experiment start the 1st day and the 21st, 40 day early morning each group of chicken is weighed on an empty stomach, record just starting weight and end are heavy.In feeding process,
Observed and recorded respectively organizes the health status (search for food, drink water, the mental status etc.) of chicken, and itemized record every cage feed intake weekly, statistics
Average daily gain in experiment periods, average daily ingestion amount and feed-weight ratio.Average daily ingestion amount (g/d)=experiment periods often organize feed intake/
Experiment natural law often organizes chicken number；Average daily gain (g/d)=experiment periods often organizes weightening finish/(experiment natural law often organizes chicken number)；Feed-weight ratio
=feed consumption/weightening finish.
3.3 data statisticss and analysis
Experimental data uses SPSS17.0 statistical software to carry out ANOVA analysis, and carries out Duncan multiplicity and compare,P< 0.05 is
Significant difference.Data represent with mean+SD (Mean ± SE).
3.4 experimental result and analysis
3.4.1 average daily gain
Broiler average daily gain of each stage is shown in Table 2, the average daily gain of broiler 1-21 age in days and 22-40 age in days experimental group than
Matched group has improvement in various degree, has been respectively increased 0.90% and 6.29%.At the whole breeding cycle of 1-40 age in days, experimental group
Average daily gain 3.81% can be improved.
Table 2: broiler average daily gain of each stage (gram)
| Process | 1-21 age in days | 22-40 age in days | 1-40 age in days |
| Matched group | 45.45±1.23 | 86.63±1.24 | 65.32±1.09 |
| Experimental group | 45.86±0.97 | 92.08±2.75 | 67.81±1.32 |
3.4.2 average daily ingestion amount
Broiler each stage, average daily ingestion amount was shown in Table 3, and at whole breeding process, experimental group is with matched group broiler daily ingestion amount difference not
Significantly (P> 0.05), and matched group and experimental group daily ingestion amount basically identical.
Table 3: broiler average daily ingestion amount of each stage (gram)
| Process | 1-21 age in days | 22-40 age in days | 1-40 age in days |
| Matched group | 66.96±1.32 | 149.24±1.22 | 106.16±0.54 |
| Experimental group | 66.82±0.43 | 151.09±2.83 | 106.63±1.34 |
3.4.3 feed-weight ratio
Broiler each stage feed-weight ratio is shown in Table 4, has in various degree than matched group at 1-21 age in days and 22-40 age in days experimental group
Improve, reduce 7.90% and 5.03% respectively.From the point of view of the full phase (1-40 age in days), experimental group FCR relatively matched group reduces 6.06%
(P> 0.05).
Table 4: broiler each stage feedstuff-meat ratio (FCR)
| Process | 1-21 age in days | 22-40 age in days | 1-40 age in days |
| Matched group | 1.52±0.029 | 1.79±0.032 | 1.65±0.016 |
| Experimental group | 1.40±0.016 | 1.70±0.017 | 1.55±0.020 |
From above experimental result it can be seen that with the addition of the experimental group of glucoseoxidase GOD-M01 compared with matched group, adopting
In the case of appetite is consistent, average daily gain increased, and feed-weight ratio has declined, and adds glucoseoxidase in daily ration
In the case of, feed conversion rate can be effectively improved, increase culture benefit.
The present embodiment 3 is for the ease of embodying the application of glucoseoxidase of the present invention, however it is not limited to answering of broiler
With, because described glucoseoxidase can add in basal diet, may be used for feeding of other fowl poultry kinds.Generally may be used
To add in its mixed feed in the breeding process such as pig, rabbit and milch cow.
Above example is only in order to illustrate technical scheme, rather than is limited；Although with reference to aforementioned reality
Execute example the present invention has been described in detail, for the person of ordinary skill of the art, still can be to aforementioned enforcement
Technical scheme described in example is modified, or wherein portion of techniques feature is carried out equivalent；And these are revised or replace
Change, do not make the essence of appropriate technical solution depart from the spirit and scope of claimed technical solution of the invention.
SEQUENCE LISTING
<110>Qingdao red cherry Bioisystech Co., Ltd
<120>a kind of enzymatic activity improves glucoseoxidase carbohydrase mutant GOD-M01 and expression vector and application
<130>
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 589
<212> PRT
<213>aspergillus niger
<400> 1
Leu Pro His Tyr Ile Arg Ser Asn Gly Ile Glu Ala Ser Leu Leu Thr
1 5 10 15
Asp Pro Lys Asp Val Ser Gly Arg Thr Val Asp Tyr Ile Ile Ala Gly
20 25 30
Gly Gly Leu Thr Gly Leu Thr Thr Ala Ala Arg Leu Thr Glu Asn Pro
35 40 45
Asn Ile Ser Val Leu Val Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg
50 55 60
Gly Pro Ile Ile Glu Asp Leu Asn Ala Tyr Gly Asp Ile Phe Gly Ser
65 70 75 80
Ser Val Asp His Ala Tyr Glu Thr Val Glu Leu Ala Thr Asn Asn Gln
85 90 95
Thr Ala Leu Ile Arg Ser Gly Asn Gly Leu Gly Gly Ser Thr Leu Val
100 105 110
Asn Gly Gly Thr Trp Thr Arg Pro His Lys Ala Gln Val Asp Ser Trp
115 120 125
Glu Thr Val Phe Gly Asn Glu Gly Trp Asn Trp Asp Asn Val Ala Ala
130 135 140
Tyr Ser Leu Gln Ala Glu Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile
145 150 155 160
Ala Ala Gly His Tyr Phe Asn Ala Ser Cys His Gly Thr Asn Gly Thr
165 170 175
Val His Ala Gly Pro Arg Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val
180 185 190
Lys Ala Leu Met Ser Ala Val Glu Asp Arg Gly Val Pro Thr Lys Lys
195 200 205
Asp Phe Gly Cys Gly Asp Pro His Gly Val Ser Met Phe Pro Asn Thr
210 215 220
Leu His Glu Asp Gln Val Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu
225 230 235 240
Pro Asn Tyr Gln Arg Pro Asn Leu Gln Val Leu Thr Gly Gln Tyr Val
245 250 255
Gly Lys Val Leu Leu Ser Gln Asn Gly Thr Thr Pro Arg Ala Val Gly
260 265 270
Val Glu Phe Gly Thr His Lys Gly Asn Thr His Asn Val Tyr Ala Glu
275 280 285
His Glu Val Leu Leu Ala Ala Gly Ser Ala Val Ser Pro Thr Ile Leu
290 295 300
Glu Tyr Ser Gly Ile Gly Met Lys Ser Ile Leu Glu Pro Leu Gly Ile
305 310 315 320
Asp Thr Val Val Asp Leu Pro Val Gly Leu Asn Leu Gln Asp Gln Thr
325 330 335
Thr Ala Thr Val Arg Ser Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly
340 345 350
Gln Ala Ala Trp Phe Ala Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ser
355 360 365
Glu Lys Ala His Glu Leu Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu
370 375 380
Glu Ala Val Ala Arg Gly Gly Phe His Asn Thr Thr Ala Leu Leu Ile
385 390 395 400
Gln Tyr Glu Asn Tyr Arg Asp Trp Ile Val Asn His Asn Val Ala Tyr
405 410 415
Ser Glu Leu Phe Leu Asp Thr Ala Gly Val Ala Ser Phe Asp Val Trp
420 425 430
Asp Leu Leu Pro Phe Thr Arg Gly Tyr Val His Ile Leu Asp Lys Asp
435 440 445
Pro Tyr Leu His His Phe Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu
450 455 460
Leu Asp Leu Leu Gly Gln Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile
465 470 475 480
Ser Asn Ser Gly Ala Met Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro
485 490 495
Gly Asp Asn Leu Ala Tyr Asp Ala Asp Leu Ser Ala Trp Thr Glu Tyr
500 505 510
Ile Pro Tyr His Phe Arg Pro Asn Tyr His Gly Val Gly Thr Cys Ser
515 520 525
Met Met Pro Lys Glu Met Gly Gly Val Val Asp Asn Ala Ala Arg Val
530 535 540
Tyr Gly Val Gln Gly Leu Arg Val Ile Asp Gly Ser Ile Pro Pro Thr
545 550 555 560
Gln Met Ser Ser His Val Met Thr Val Phe Tyr Ala Met Ala Leu Lys
565 570 575
Ile Ser Asp Ala Ile Leu Glu Asp Tyr Ala Ser Met Gln
580 585
<210> 2
<211> 1770
<212> DNA
<213>artificial sequence
<400> 2
ttgccacatt acattagatc caacggtatt gaagcatcct tgttgactga tcctaaggat 60
gtttcaggta gaactgttga ttacattatt gctggaggtg gattgactgg tttgactact 120
gctgctagat tgactgaaaa cccaaacatt tcagttttgg ttattgaatc aggttcttac 180
gaatccgata gaggtcctat tattgaagat ttgaacgctt acggggacat ttttggttct 240
tccgttgatc atgcttacga aactgttgaa ttggctacta acaaccaaac tgctttgatt 300
agatccggta acggattggg tggttccact ttggttaacg gaggtacttg gactagacct 360
cataaggctc aagttgattc ctgggaaact gtttttggta acgaaggatg gaactgggat 420
aacgttgctg cttactcctt gcaagctgaa cgagcgcggg cccctaacgc taagcaaatt 480
gctgctggtc attactttaa cgcttcctgt catggaacta acggaactgt tcatgctggt 540
ccaagagata ctggagacga ttactcccca attgttaagg ctttgatgtc cgctgttgaa 600
gataggggtg ttccaactaa gaaggatttt ggatgtggcg acccacatgg agtttctatg 660
tttccaaaca ctttgcatga agatcaagtt agatccgatg ctgctagaga atggttgttg 720
cctaactacc aaagacctaa cttgcaagtt ttgactggtc aatacgttgg taaagttttg 780
ttgtcacaaa acggaactac tcctagagct gttggagttg aatttggtac tcataagggt 840
aacactcata acgtttacgc tgaacatgaa gttttgttgg ctgctggttc cgctgtttca 900
ccaactattt tggaatactc cggtattggt atgaagtcta ttttggaacc attgggtatt 960
gatactgttg ttgatttgcc agttggattg aacttgcaag atcaaactac tgctactgtt 1020
agatccagaa ttacttccgc tggagctgga caaggacaag ctgcttggtt tgctactttt 1080
aacgaaactt ttggagatta ctccgaaaag gctcatgaat tgttgaacac taagttggaa 1140
caatgggctg aagaagctgt tgctagaggt ggttttcata acactactgc tttgttgatt 1200
caatacgaaa actacagaga ttggattgtt aaccataacg ttgcttactc agaattgttt 1260
ttggatactg ctggagttgc ttcctttgat gtttgggatt tgttgccctt cactagagga 1320
tacgttcata ttttggataa agacccatac ttgcatcatt ttgcttacga tccacaatac 1380
tttttgaacg aattggattt gttgggacaa gctgctgcta ctcaattggc tagaaacatt 1440
tctaactcgg gtgctatgca aacttacttt gctggagaaa ctattccagg tgacaacttg 1500
gcttacgatg ctgatttgtc cgcttggact gaatacattc cataccattt tagacctaac 1560
taccacggag ttggtacttg ttctatgatg ccaaaggaaa tgggaggtgt tgttgataac 1620
gctgctagag tttacggagt tcaaggattg agagttattg atggttcaat tccaccaact 1680
caaatgtctt cacatgttat gactgtgttt tacgctatgg ctttgaagat ttcagatgct 1740
attttggaag attacgcttc tatgcaataa 1770
<210> 3
<211> 589
<212> PRT
<213>artificial sequence
<400> 3
Leu Pro His Tyr Ile Arg Ser Asn Gly Ile Glu Ala Ser Leu Leu Thr
1 5 10 15
Asp Pro Lys Asp Val Ser Gly Arg Thr Val Asp Tyr Ile Ile Ala Gly
20 25 30
Gly Gly Leu Thr Gly Leu Thr Thr Ala Ala Arg Leu Thr Glu Asn Pro
35 40 45
Asn Ile Ser Val Leu Val Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg
50 55 60
Gly Pro Ile Ile Glu Asp Leu Asn Ala Tyr Gly Asp Ile Phe Gly Ser
65 70 75 80
Ser Val Asp His Ala Tyr Glu Thr Val Glu Leu Ala Thr Asn Asn Gln
85 90 95
Thr Ala Leu Ile Arg Ser Gly Asn Gly Leu Gly Gly Ser Thr Leu Val
100 105 110
Asn Gly Gly Thr Trp Thr Arg Pro His Lys Ala Gln Val Asp Ser Trp
115 120 125
Glu Thr Val Phe Gly Asn Glu Gly Trp Asn Trp Asp Asn Val Ala Ala
130 135 140
Tyr Ser Leu Gln Ala Glu Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile
145 150 155 160
Ala Ala Gly His Tyr Phe Asn Ala Ser Cys His Gly Val Asn Gly Thr
165 170 175
Val His Ala Gly Pro Arg Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val
180 185 190
Lys Ala Leu Met Ser Ala Val Glu Asp Arg Gly Val Pro Thr Lys Lys
195 200 205
Asp Phe Gly Cys Gly Asp Pro His Gly Val Ser Met Phe Pro Asn Thr
210 215 220
Leu His Glu Asp Gln Val Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu
225 230 235 240
Pro Asn Tyr Gln Arg Pro Asn Leu Gln Val Leu Thr Gly Gln Tyr Val
245 250 255
Gly Lys Val Leu Leu Ser Gln Asn Gly Thr Thr Pro Arg Ala Val Gly
260 265 270
Val Glu Phe Gly Thr His Lys Gly Asn Thr His Asn Val Tyr Ala Glu
275 280 285
His Glu Val Leu Leu Ala Ala Gly Ser Ala Val Ser Pro Thr Ile Leu
290 295 300
Glu Tyr Ser Gly Ile Gly Met Lys Ser Ile Leu Glu Pro Leu Gly Ile
305 310 315 320
Asp Thr Val Val Asp Leu Pro Val Gly Leu Asn Leu Gln Asp Gln Thr
325 330 335
Thr Ala Thr Val Arg Ser Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly
340 345 350
Gln Ala Ala Trp Phe Ala Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ser
355 360 365
Glu Lys Ala His Glu Leu Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu
370 375 380
Glu Ala Val Ala Arg Gly Gly Phe His Asn Thr Thr Ala Leu Leu Ile
385 390 395 400
Gln Tyr Glu Asn Tyr Arg Asp Trp Ile Val Asn His Asn Val Ala Tyr
405 410 415
Ser Glu Leu Phe Leu Asp Thr Ala Gly Val Ala Ser Phe Asp Val Trp
420 425 430
Asp Leu Leu Pro Phe Thr Arg Gly Tyr Val His Ile Leu Asp Lys Asp
435 440 445
Pro Tyr Leu His His Phe Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu
450 455 460
Leu Asp Leu Leu Gly Gln Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile
465 470 475 480
Ser Asn Ser Gly Ala Met Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro
485 490 495
Gly Asp Asn Leu Ala Tyr Asp Ala Asp Leu Ser Ala Trp Thr Glu Tyr
500 505 510
Ile Pro Tyr His Phe Arg Pro Asn Tyr His Gly Val Gly Thr Cys Ser
515 520 525
Met Met Pro Lys Glu Met Gly Gly Val Val Asp Asn Ala Ala Arg Val
530 535 540
Tyr Gly Val Gln Gly Leu Arg Val Ile Asp Gly Ser Ile Pro Pro Thr
545 550 555 560
Gln Met Ser Ser His Val Met Thr Val Phe Tyr Ala Met Ala Leu Lys
565 570 575
Ile Ser Asp Ala Ile Leu Glu Asp Tyr Ala Ser Met Gln
580 585
<210> 4
<211> 1770
<212> DNA
<213>artificial sequence
<400> 4
ttgccacatt acattagatc caacggtatt gaagcatcct tgttgactga tcctaaggat 60
gtttcaggta gaactgttga ttacattatt gctggaggtg gattgactgg tttgactact 120
gctgctagat tgactgaaaa cccaaacatt tcagttttgg ttattgaatc aggttcttac 180
gaatccgata gaggtcctat tattgaagat ttgaacgctt acggggacat ttttggttct 240
tccgttgatc atgcttacga aactgttgaa ttggctacta acaaccaaac tgctttgatt 300
agatccggta acggattggg tggttccact ttggttaacg gaggtacttg gactagacct 360
cataaggctc aagttgattc ctgggaaact gtttttggta acgaaggatg gaactgggat 420
aacgttgctg cttactcctt gcaagctgaa cgagcgcggg cccctaacgc taagcaaatt 480
gctgctggtc attactttaa cgcttcctgt catggagtta acggaactgt tcatgctggt 540
ccaagagata ctggagacga ttactcccca attgttaagg ctttgatgtc cgctgttgaa 600
gataggggtg ttccaactaa gaaggatttt ggatgtggcg acccacatgg agtttctatg 660
tttccaaaca ctttgcatga agatcaagtt agatccgatg ctgctagaga atggttgttg 720
cctaactacc aaagacctaa cttgcaagtt ttgactggtc aatacgttgg taaagttttg 780
ttgtcacaaa acggaactac tcctagagct gttggagttg aatttggtac tcataagggt 840
aacactcata acgtttacgc tgaacatgaa gttttgttgg ctgctggttc cgctgtttca 900
ccaactattt tggaatactc cggtattggt atgaagtcta ttttggaacc attgggtatt 960
gatactgttg ttgatttgcc agttggattg aacttgcaag atcaaactac tgctactgtt 1020
agatccagaa ttacttccgc tggagctgga caaggacaag ctgcttggtt tgctactttt 1080
aacgaaactt ttggagatta ctccgaaaag gctcatgaat tgttgaacac taagttggaa 1140
caatgggctg aagaagctgt tgctagaggt ggttttcata acactactgc tttgttgatt 1200
caatacgaaa actacagaga ttggattgtt aaccataacg ttgcttactc agaattgttt 1260
ttggatactg ctggagttgc ttcctttgat gtttgggatt tgttgccctt cactagagga 1320
tacgttcata ttttggataa agacccatac ttgcatcatt ttgcttacga tccacaatac 1380
tttttgaacg aattggattt gttgggacaa gctgctgcta ctcaattggc tagaaacatt 1440
tctaactcgg gtgctatgca aacttacttt gctggagaaa ctattccagg tgacaacttg 1500
gcttacgatg ctgatttgtc cgcttggact gaatacattc cataccattt tagacctaac 1560
taccacggag ttggtacttg ttctatgatg ccaaaggaaa tgggaggtgt tgttgataac 1620
gctgctagag tttacggagt tcaaggattg agagttattg atggttcaat tccaccaact 1680
caaatgtctt cacatgttat gactgtgttt tacgctatgg ctttgaagat ttcagatgct 1740
attttggaag attacgcttc tatgcaataa 1770
<210> 5
<211> 39
<212> DNA
<213>artificial sequence
<400> 5
gcgcgaattc ttgccacatt acattagatc caacggtat 39
<210> 6
<211> 45
<212> DNA
<213>artificial sequence
<400> 6
taaagcggcc gcttattgca tagaagcgta atcttccaaa atagc 45
Claims (10)
1. the glucoseoxidase mutant GOD-M01 that an enzymatic activity improves, it is characterised in that described glucoseoxidase
The aminoacid sequence of mutant GOD-M01 is as shown in SEQ ID NO:3, and described mutant GOD-M01 by aminoacid sequence is
173rd amino acids of the glucoseoxidase of SEQ ID NO:1 is become valine from threonine and obtains.
2. the glucoseoxidase encoding gene of the glucoseoxidase mutant GOD-M01 described in claim 1.
Glucoseoxidase encoding gene the most according to claim 2, it is characterised in that it has such as SEQ ID NO:4 institute
The nucleotide sequence shown.
4. contain the recombinant expression carrier of glucoseoxidase encoding gene described in claim 2.
Recombinant expression carrier the most according to claim 4, it is characterised in that described recombinant expression carrier is recombination yeast table
Reach plasmid pPIC9K-GOD-M01.
6. contain the recombinant bacterial strain of recombinant expression carrier described in claim 4.
Recombinant bacterial strain the most according to claim 6, it is characterised in that described recombinant bacterial strain is recombinant yeast pichia pastoris.
8. the glucoseoxidase mutant GOD-M01 described in claim 1 is for preparing answering in animal feed additive
With.
Glucoseoxidase mutant GOD-M01 the most according to claim 8 is prepared in animal feed additive being used for
Application, it is characterised in that: described glucoseoxidase encoding gene is connected with expression vector structure recombinant expression carrier,
Recombinant expression carrier is transformed in host cell, it is thus achieved that recombinant host bacterial strain, cultivates recombinant host bacterial strain and induce restructuring Portugal
The expression of glucoseoxidase, it is thus achieved that glucoseoxidase.
Glucoseoxidase mutant GOD-M01 the most according to claim 9 is prepared in animal feed additive being used for
Application, it is characterised in that: described host cell is Pichia sp..
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610525903.0A CN106011093A (en) | 2016-07-06 | 2016-07-06 | Glucose oxidase mutant GOD-M01 with improved enzymatic activity and expression vector and application of glucose oxidase mutant GOD-M01 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610525903.0A CN106011093A (en) | 2016-07-06 | 2016-07-06 | Glucose oxidase mutant GOD-M01 with improved enzymatic activity and expression vector and application of glucose oxidase mutant GOD-M01 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106011093A true CN106011093A (en) | 2016-10-12 |
Family
ID=57106666
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610525903.0A Pending CN106011093A (en) | 2016-07-06 | 2016-07-06 | Glucose oxidase mutant GOD-M01 with improved enzymatic activity and expression vector and application of glucose oxidase mutant GOD-M01 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106011093A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107012130A (en) * | 2017-06-02 | 2017-08-04 | 中国农业科学院饲料研究所 | A kind of glucose oxidase mutant and its encoding gene and application |
| CN107189991A (en) * | 2017-05-08 | 2017-09-22 | 中国农业科学院饲料研究所 | A kind of glucose oxidase mutant and its encoding gene and application |
| CN113528476A (en) * | 2021-08-20 | 2021-10-22 | 福建福大百特生物科技有限公司 | Glucose oxidase mutant and coding gene and efficient recombinant expression thereof |
| CN114410662A (en) * | 2022-02-18 | 2022-04-29 | 中国农业大学 | Method for improving expression efficiency of glucose oxidase gene in pichia pastoris |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525778A (en) * | 2013-10-18 | 2014-01-22 | 江南大学 | Glucose oxidase mutant with high catalytic activity |
| CN103981159A (en) * | 2014-06-05 | 2014-08-13 | 青岛蔚蓝生物集团有限公司 | Glucose oxidase mutant and application thereof |
-
2016
- 2016-07-06 CN CN201610525903.0A patent/CN106011093A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525778A (en) * | 2013-10-18 | 2014-01-22 | 江南大学 | Glucose oxidase mutant with high catalytic activity |
| CN103981159A (en) * | 2014-06-05 | 2014-08-13 | 青岛蔚蓝生物集团有限公司 | Glucose oxidase mutant and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| JULIA MARÍN-NAVARRO ET AL.: "Identification and Structural Analysis of Amino Acid Substitutions that Increase the Stability and Activity of Aspergillus niger Glucose Oxidase", 《PLOS ONE》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107189991A (en) * | 2017-05-08 | 2017-09-22 | 中国农业科学院饲料研究所 | A kind of glucose oxidase mutant and its encoding gene and application |
| CN107012130A (en) * | 2017-06-02 | 2017-08-04 | 中国农业科学院饲料研究所 | A kind of glucose oxidase mutant and its encoding gene and application |
| CN107012130B (en) * | 2017-06-02 | 2020-05-22 | 中国农业科学院饲料研究所 | Glucose oxidase mutant and coding gene and application thereof |
| CN113528476A (en) * | 2021-08-20 | 2021-10-22 | 福建福大百特生物科技有限公司 | Glucose oxidase mutant and coding gene and efficient recombinant expression thereof |
| CN113528476B (en) * | 2021-08-20 | 2023-01-31 | 福建福大百特生物科技有限公司 | Glucose oxidase mutant and coding gene and efficient recombinant expression thereof |
| CN114410662A (en) * | 2022-02-18 | 2022-04-29 | 中国农业大学 | Method for improving expression efficiency of glucose oxidase gene in pichia pastoris |
| CN114410662B (en) * | 2022-02-18 | 2024-02-06 | 中国农业大学 | Method for improving expression efficiency of glucose oxidase gene in pichia pastoris |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6341936B2 (en) | High production strain of 5-aminolevulinic acid and its production method and use | |
| CN105950577A (en) | Glucose oxidase mutant with improved thermal stability as well as encoding genes and application thereof | |
| CN105936910B (en) | The glucose oxidase gene GOD and its expression vector of a kind of optimization and application | |
| CN105950578A (en) | Heat-resisting glucose oxidase mutant as well as encoding gene and application thereof | |
| CN106011093A (en) | Glucose oxidase mutant GOD-M01 with improved enzymatic activity and expression vector and application of glucose oxidase mutant GOD-M01 | |
| CN107937361B (en) | A kind of alanine dehydrogenase mutant and its application | |
| CN101918539A (en) | Light-driven CO2 reduction to organic compounds to serve as fuels or as industrial half products by an autotroph containing a fermentative gene cassette | |
| CN102787130B (en) | Acid and high temperature resistant alpha-amylase, and its gene, engineering bacterium and preparation method | |
| CN110577939B (en) | Glucose oxidase mutant with improved heat resistance as well as coding gene and application thereof | |
| CN102071171A (en) | Bifunctional glutathione synthetase and method for producing glutathione by using same | |
| CN106119219A (en) | Glucoseoxidase mutant that enzymatic activity improves and expression vector and application | |
| CN107083374B (en) | The beta-mannase enzyme mutant and its encoding gene and application that enzymatic activity improves | |
| CN110713996B (en) | Trehalase, and carrier and application thereof | |
| CN109321586A (en) | Recombinant Aspergillus niger Glucose Oxidase optimization gene and its expression vector and application | |
| CN107406821A (en) | For producing the mutation host cell of 3 hydracrylic acids | |
| CN107164344A (en) | One class heat-resistance phytase mutant and its encoding gene and application | |
| CN106434393B (en) | Recombinant aspergillus niger expresses bacterial strain | |
| CN107109347A (en) | Recombinant host cell for producing 3 hydracrylic acids | |
| CN110452861A (en) | A kind of genetic recombination engineering bacteria and its catalyzing and synthesizing the application in D- pantoyl internal ester | |
| CN103773746B (en) | Lipase and mutant thereof | |
| CN100491533C (en) | Improved xylanase with high specific activity, its gene expression vector and recombinant yeast cell, and expression method | |
| CN1224757A (en) | Method for producing phytase | |
| CN111424048A (en) | Gene for expressing acidic β -mannase, vector and application thereof | |
| CN112877306B (en) | Super-heat-resistant glucose oxidase AtGOD and gene and application thereof | |
| CN108517329A (en) | A kind of recombination Yarrowia lipolytica lipase and its expression bacterial strain and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20161012 |