CN103898081A - Keratinase mutant and application thereof - Google Patents
Keratinase mutant and application thereof Download PDFInfo
- Publication number
- CN103898081A CN103898081A CN201410164525.9A CN201410164525A CN103898081A CN 103898081 A CN103898081 A CN 103898081A CN 201410164525 A CN201410164525 A CN 201410164525A CN 103898081 A CN103898081 A CN 103898081A
- Authority
- CN
- China
- Prior art keywords
- mutant
- zyme
- keratinase
- enzyme
- keratinase mutant
- 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.)
- Granted
Links
Images
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
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Animal Husbandry (AREA)
- Food Science & Technology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Fodder In General (AREA)
Abstract
The invention provides a keratinase mutant. The keratinase mutant is obtained by transforming a 13th amino acid Ser of keratinase into Asn, a 27th amino acid Ser of the keratinase into Asn and a 46th amino acid Gln of the keratinase into Lys, wherein the amino acid sequence of the keratinase is SEQ ID No.1. The amino acid sequence of the keratinase mutant is SEQ ID No.3; and the nucleic acid sequence of a coding gene of the keratinase mutant is SEQ ID No.4. The optimal operative pH value of the keratinase mutant provided by the invention is 7.0, the relative enzyme activity level of the keratinase mutant under an alkaline condition is higher than that of a wild type keratinase mutant, and the keratinase mutant has stronger alkali resistance; and the optimal operative temperature of the keratinase mutant is 50 DEG C, more than 60% of relative enzyme activity can be maintained in the temperature of 45-60 DEG C, after the keratinase mutant is treated for 5 minutes at the temperature of 65 DEG C, more than 80% of enzyme activity can be maintained, after the keratinase mutant is treated for 5 minutes at the temperature of 70 DEG C, more than 55% of enzyme activity still can be maintained, and compared with the heat resistance of the wild type keratinase mutant, the heat resistance of the keratinase mutant is greatly improved.
Description
Technical field
The invention belongs to enzyme gene function renovation technique field, be specifically related to a kind of M-Zyme mutant and application thereof.
Technical background
Keratin sulfate (keratinase) is a kind of rigid albumen that is extensively present in occurring in nature, is mainly present in the structures such as the hair, scale, feather, hoof, angle of animal.Keratic peptide chain is with alpha-helix and β-laminated structure composition, form highly stable highly cross-linked three-dimensional structure by disulfide linkage, hydrogen bond and other cross-link bond effects, water insoluble, can not be by general proteolytic enzyme as hydrolysis such as trypsinase, stomach en-, papoids.But Keratin sulfate is nutritious, for example, in feather, more than 80%, contains various total amino acid contents more than 70% containing crude protein, and containing the needed indispensable amino acid of many animals, if dealt carefully with, be a kind of good feedstuff protein source.And along with modern poultry farming large-scale production; produce a large amount of cyokeratin refuses, as processed not in time, will cause the severe contamination of local environment; and occurring in nature certain micro-organisms can utilize the M-Zyme of self secreting to be decomposed utilization, as the required Carbon and nitrogen sources of growth.
M-Zyme is a species specificity keratic enzyme of degrading, and can be produced by multiple-microorganism, and it can be hydrolyzed into Keratin sulfate free amino acid or polypeptide.Generally believe at present the degradation process of M-Zyme in three steps, i.e. Denaturation, hydrolytic action and transamination.First disulfide bond reduction enzyme acts on keratin disulfide, and Gelucystine (S-S-) is reduced to halfcystine (SH), makes Keratin sulfate higher structure ladder and forms denatured keratin; Denatured keratin is hydrolyzed into gradually polypeptide, oligopeptides and total free aminoacids under the effect of polypeptide lytic enzyme; Finally produce ammonia and sulfide and Keratin sulfate is thoroughly hydrolyzed by transamination.This specific character of M-Zyme, can be used for processing discarded animal feather, hair etc., and the material of these original contaminate environment is turned waste into wealth, and it is being had broad application prospects aspect fodder industry, medicine industry, foodstuffs industry and environmental improvement.Therefore, become the study hotspot in this area by the M-Zyme of the method excellent that screens or transform.
Summary of the invention
The object of this invention is to provide a kind of M-Zyme mutant and application thereof, the present invention is by carrying out protein engineering transformation to the M-Zyme that derives from subtilis (Bacillus subtilis), obtain mutant protein, its thermotolerance is significantly improved, pH sphere of action expands, and is conducive to its widespread use.
Applicant suddenlys change to the N-terminal of subtilis M-Zyme, through a large amount of screenings, finds S13N, and these three mutational sites of S27N and Q46K can cause M-Zyme alkali resistance and stable on heating change, thereby facilitate the present invention.
One aspect of the present invention provides a kind of M-Zyme mutant, is that aminoacid sequence is that the 13rd amino acids of the M-Zyme of SEQ ID NO:1 becomes Asn from Ser, and the 27th amino acids becomes Asn from Ser, and the 46th amino acids becomes Lys from Gln.
The aminoacid sequence of above-mentioned M-Zyme mutant is SEQ ID NO:3, and the nucleotide sequence of its a kind of encoding gene is SEQ ID NO:4.
One aspect of the present invention provides the recombinant plasmid that carries above-mentioned M-Zyme mutant code gene.
The present invention provides the subtilis recombinant bacterial strain that carries above-mentioned recombinant plasmid on the other hand.
The present invention also provides the application of above-mentioned M-Zyme mutant in feed.
M-Zyme mutant Optimun pH provided by the invention is 7.0, and the relative enzyme running water under alkaline condition is on average higher than wild-type, and alkali resistance is stronger; Its optimum temperature is 50 ℃, within the scope of 45 ℃-60 ℃, can keep more than 60% relative enzyme to live, process 5min under 65 ℃ of conditions after, can keep more than 85% enzyme to live, process 5min under 70 ℃ of conditions after, still can keep more than 55% enzyme to live, thermotolerance is highly improved compared with wild-type.In addition, M-Zyme mutant of the present invention is significantly higher than wild-type to the hydrolysis result of feed protein raw material, is therefore more conducive to improve the utilization ratio of cultivated animals to protein raw materials in feed, and then can reduce the addition of protein raw materials, reduce feed cost, increase economic benefit.
Accompanying drawing explanation
Fig. 1: M-Zyme mutant Ker7 of the present invention and wild-type KerI action pH-relative enzyme graphic representation alive;
Fig. 2: M-Zyme mutant Ker7 of the present invention and wild-type KerI pH stability comparison diagram;
Fig. 3: M-Zyme mutant Ker7 of the present invention and wild-type KerI operative temperature-relative enzyme graphic representation alive.
Embodiment
The present invention has used routine techniques and the method for genetic engineering and biology field use, for example MOLECULAR CLONING:A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and the method for recording in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003).These general reference provide definition well known by persons skilled in the art and method.But, on the basis of the technical scheme that those skilled in the art can record in the present invention, adopt other conventional method of this area, experimental program and reagent, and be not limited to the restriction of the specific embodiment of the invention.
Describe the present invention below in conjunction with embodiment.
The structure in embodiment 1 M-Zyme transgenation storehouse
Derive from the M-Zyme KerI of subtilis (aminoacid sequence is SEQ ID NO:1 in order to improve, coding nucleotide sequence is SEQ ID NO:2) thermotolerance, by orthogenesis technology, this enzyme has been carried out the screening of mass mutation, design PCR primer Ker-F, Ker-R are as follows:
Ker-F:GC
gTCGACtTCAACAACATGTCTGCGCAGGCTG (underscore is restriction enzyme Sal I recognition site)
Ker-R:CAT
gCATGCtTATTGTGCAGCTGCTTGTACGTTGATTAAC (underscore is restriction enzyme Sph I recognition site)
Take KerI gene as template, carry out pcr amplification with above-mentioned primer with GeneMorph II random mutation PCR test kit (Stratagene), glue reclaims PCR product, Sal I, Sph I carry out enzyme and cut the pWB980 carrier connection of processing afterwards and after same enzyme is cut, be converted in subtilis WB600, coat LB+Kan flat board, be inverted overnight incubation, on flat board, grow transformant for 37 ℃.
The screening of the heat-resisting M-Zyme mutant of embodiment 2
With toothpick, the transformant growing on flat board is chosen one by one to 96 orifice plates, added 500 μ L LB+Kan substratum in each hole, 37 ℃ of 220rpm cultivate 24h left and right, and centrifugal collection supernatant, obtains the fermented liquid that contains M-Zyme.
Take out respectively suitably supernatant liquor to a two 96 new orifice plate of dilution of 40 μ L, wherein process after 10min in 70 ℃ for one, two 96 orifice plates all add 40 μ L substrates, after 40 ℃ of reaction 10min, measure enzyme live by the method for Folin reagent colour developing.Residual activity difference after different muton pyroprocessing.The thermotolerance of some M-Zyme muton does not change, and the thermotolerance of some muton has reduced, and can significantly improve the stable on heating sudden change combination S of M-Zyme 13N, S27N and Q46K to still keeping highly active muton to carry out DNA sequencing, finally having obtained.
Containing S13N, the M-Zyme mutant of S27N and Q46K tri-point mutation, its aminoacid sequence is SEQ ID NO:3, and coding nucleotide sequence is SEQ ID NO:4, and unnamed gene is Ker7.
Structure and the fermentation of embodiment 3 subtilis expression strains
Primer Ker-F, Ker-R for mutant gene Ker7 are carried out to pcr amplification, and Sal I, Sph I site are introduced in primer two ends.PCR reaction conditions is: 94 ℃ of sex change 5min; Then 94 ℃ of sex change 30s, 56 ℃ of renaturation 30s, 72 ℃ are extended 1.5min, after 30 circulations, 72 ℃ of insulation 10min.
Obtain the gene fragment of wild-type M-Zyme KerI by above-mentioned same PCR method amplification.
By after Sal I, Sph I double digestion for the KerI obtaining and Ker7 gene, carry out the expression vector pWB980 that enzyme cuts and connect with same, transform subtilis WB600 competent cell, coating LB+Kan flat board, 37 ℃ of incubated overnight.Next day, screening obtains recombinant bacterial strain.
The recombinant bacterial strain of KerI and Ker7 is inoculated in the liquid nutrient medium of 50mL LB+Kan, cultivates about 24h for 37 ℃, culture is carried out to 5000g, 5min is centrifugal, collects supernatant liquor, obtains M-Zyme crude enzyme liquid.
The zymologic property of embodiment 4 M-Zymes is measured
1, the suitableeest action pH and pH stability analysis
Employing pH value is respectively Sodium phosphate dibasic-citrate buffer solution of 5.5,6.0,6.5,7.0,7.5,8.0, pH value is borax-borate buffer of 8.5,9.0, the M-Zyme crude enzyme liquid that fermentation is obtained carries out dilution metering, M-Zyme substrate is also prepared with the damping fluid of corresponding pH value respectively, at 40 ℃, carry out enzyme activity determination, calculate enzyme and live, take the highest enzyme work as 100%, calculate relative enzyme and live, do the relative enzyme of pH-curve alive.As shown in Figure 1, the Optimun pH of wild-type M-Zyme KerI and M-Zyme mutant Ker7 is 7.0 to result, but the relative enzyme running water of mutant under alkaline condition is on average higher than wild-type, illustrates that the M-Zyme alkali resistance after sudden change is stronger.
Employing pH value is respectively 5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0 damping fluid, the crude enzyme liquid dilution that fermentation is obtained, process after 2h for 40 ℃, carry out dilution metering, calculating enzyme is lived, take the enzyme work of untreated samples as 100%, calculate the enzyme residual rate of living, do the pH-enzyme residual rate curve of living.Result as shown in Figure 2, within the scope of pH5.5-9.0, the enzyme of M-Zyme mutant Ker7 is lived residual rate all higher than wild-type, especially in the alkaline range of pH7.5-9.0, the enzyme residual rate alive of mutant Ker7 is significantly higher than wild-type, thereby the M-Zyme after explanation sudden change has stronger alkali resistance than wild-type.
2, optimum temperature and thermal stability analysis
At 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, under pH5.5 condition, the crude enzyme liquid that fermentation is obtained carries out M-Zyme enzyme activity determination respectively, take the highest enzyme work as 100%, calculates relative enzyme and lives, and does temperature-enzyme curve alive relatively.As shown in Figure 3, the optimum temperature of wild-type M-Zyme KerI and mutant Ker7 is 50 ℃ to result, but mutant Ker7 is within the scope of 45 ℃-60 ℃, and enzyme is lived all higher than 60% relatively, stronger than wild-type thermotolerance.
After the dilution of the acetic acid of pH5.5 for the crude enzyme liquid that fermentation is obtained-sodium acetate buffer, under 65 ℃, 70 ℃ conditions, process respectively after 2min and 5min, measure enzyme and live, take the enzyme work of untreated samples as 100%, calculate the enzyme residual rate of living.Result demonstration, wild-type M-Zyme KerI can keep more than 60% enzyme to live process 5min under 65 ℃ of conditions after, only remain 12% but residual enzyme is alive process 5min under 70 ℃ of conditions after; And M-Zyme mutant Ker7 can keep more than 85% enzyme to live process 5min under 65 ℃ of conditions after, process 5min under 70 ℃ of conditions after, still can keep more than 55% enzyme to live, illustrate that the M-Zyme thermotolerance compared with wild-type after sudden change is highly improved.
Above-mentioned result shows that M-Zyme mutant Ker7 gene has stronger alkali resistance and thermotolerance than wild-type.
Embodiment 5 impacts of M-Zyme mutant on feed protein raw material digestibility
Dregs of beans, peanut meal, corn, wheat, fish meal, DDGS are being the protein raw materials being most widely used in feed, and this test detects the impact of M-Zyme mutant of the present invention on protein raw materials digestibility by the digestive process of in-vitro simulated monogastric animal stomach and small intestine.
test sample:
(1) blank: additionally do not add zymin;
(2) wild-type M-Zyme KerI (enzyme 5000U/g alive)
(3) M-Zyme mutant Ker7 (enzyme 5000U/g alive)
substrate:
Dregs of beans: crude protein content 48%
Peanut meal: crude protein content 44.34%;
Corn: crude protein content 9.25%;
Wheat: crude protein content 13.37%;
Fish meal: crude protein content 69.87%;
DDGS: crude protein content 30%
digestive ferment:
Stomach en-(sigma p-70001:10000); Trypsin sigma p-7545).
External enzymolysis profile test:
crude protein content is measured:
With reference to " measuring method of crude protein in GBT6432-1994 feed ".
the external enzymolysis test method of two-step approach:
1) take 3g substrate and be placed in 100mL tool plug triangular flask, add 10mL distilled water, fully dissolve.
2) in 0min, add 10mL0.1M pH4.6 citric acid-Sodium phosphate dibasic damping fluid, pH is adjusted to 4.6, add the above-mentioned test sample of 0.1g (blank group does not add).
3) in 120min, add 0.1M hydrochloric acid, regulate pH to 2.5-3.0, add 5ml0.1M pH2.8 damping fluid (containing stomach en-).
4) in 140min, add 0.2M NaOH to regulate pH to 6.2, add 5ml0.2M pH6.2 Sodium phosphate dibasic-citrate buffer solution (containing trypsinase).
5), in 320min, add 5mL20% sulphosalicylic acid, 4 ℃ of precipitation 30min (precipitating not digestible protein).
6) in 350min, suction filtration method is filtered and is obtained not slaking residue, is specially: chyme is transferred in strainer with 1% sulphosalicylic acid, with ethanol and the acetone rinsing residue of 2 × 10ml.
7) 80 ℃ of dried overnight, obtain dry residue.
8) measure the weight that is dried residue.
9) calculate the protein digestibility that is dried residue.
Result is as follows:
As can be seen from the above table, compared with blank group, wild-type M-Zyme KerI and M-Zyme mutant Ker7 all can effectively improve the digestibility of conventional protein raw materials in feed, and the hydrolysis result of mutant Ker7 will be significantly higher than wild-type, thereby illustrate that the zymologic property of M-Zyme mutant provided by the invention is more conducive to its application in feed.The interpolation of described M-Zyme mutant in feed can effectively improve the utilization ratio of cultivated animals to protein raw materials, and then can reduce the addition of protein raw materials, reduces feed cost, increases economic benefit.
Claims (8)
1. a M-Zyme mutant, it is characterized in that, described M-Zyme mutant is that aminoacid sequence is that the 13rd amino acids of the M-Zyme of SEQ ID NO:1 becomes Asn from Ser, and the 27th amino acids becomes Asn from Ser, and the 46th amino acids becomes Lys from Gln.
2. M-Zyme mutant as claimed in claim 1, is characterized in that the aminoacid sequence of described M-Zyme mutant is SEQ ID NO:3.
3. a gene, is characterized in that, described genes encoding M-Zyme mutant claimed in claim 1.
4. gene as claimed in claim 3, is characterized in that the nucleotide sequence of described gene is SEQ IDNO:4.
5. a recombinant plasmid, is characterized in that described recombinant plasmid carries gene claimed in claim 3.
6. recombinant plasmid as claimed in claim 5, is characterized in that described recombinant plasmid carries gene claimed in claim 4.
7. a recombinant bacterial strain, is characterized in that, described recombinant bacterial strain is to transform or the have the right subtilis of the recombinant plasmid described in requirement 5 of transfection.
8. the application of M-Zyme mutant claimed in claim 1 in feed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410164525.9A CN103898081B (en) | 2014-04-22 | 2014-04-22 | A kind of M-Zyme mutant and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410164525.9A CN103898081B (en) | 2014-04-22 | 2014-04-22 | A kind of M-Zyme mutant and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103898081A true CN103898081A (en) | 2014-07-02 |
| CN103898081B CN103898081B (en) | 2016-04-13 |
Family
ID=50989653
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410164525.9A Active CN103898081B (en) | 2014-04-22 | 2014-04-22 | A kind of M-Zyme mutant and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103898081B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636042A (en) * | 2016-10-18 | 2017-05-10 | 江南大学 | Keratinase mutants with thermostability and catalytic activity improved |
| CN107475229A (en) * | 2015-03-18 | 2017-12-15 | 江南大学 | Keratinase mutant that a kind of substrate specificity improves and preparation method thereof |
| CN110343689A (en) * | 2019-08-23 | 2019-10-18 | 四川大学 | A kind of novel streptomycete trypsin GM2938 and its heterogenous expression |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1405305A (en) * | 2001-09-19 | 2003-03-26 | 姚斌 | Efficient, broad-spectrum keratinase and its gene |
| CN1800358A (en) * | 2005-12-27 | 2006-07-12 | 云南师范大学 | Keratinase-proudicng bacterium and its preparation method |
| CN102936588A (en) * | 2012-12-10 | 2013-02-20 | 江南大学 | Protease with improved thermal stability as well as construction method and application thereof |
-
2014
- 2014-04-22 CN CN201410164525.9A patent/CN103898081B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1405305A (en) * | 2001-09-19 | 2003-03-26 | 姚斌 | Efficient, broad-spectrum keratinase and its gene |
| CN1800358A (en) * | 2005-12-27 | 2006-07-12 | 云南师范大学 | Keratinase-proudicng bacterium and its preparation method |
| CN102936588A (en) * | 2012-12-10 | 2013-02-20 | 江南大学 | Protease with improved thermal stability as well as construction method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| LIU,B. ET AL.: "《Bacillus licheniformis strain BBE11-1 keratinase gene, complete cds》", 《NCBI GENBANK: JX504681.1，》, 3 December 2012 (2012-12-03) * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107475229A (en) * | 2015-03-18 | 2017-12-15 | 江南大学 | Keratinase mutant that a kind of substrate specificity improves and preparation method thereof |
| CN107475229B (en) * | 2015-03-18 | 2019-12-24 | 江南大学 | Keratinase mutant with improved substrate specificity and preparation method thereof |
| CN106636042A (en) * | 2016-10-18 | 2017-05-10 | 江南大学 | Keratinase mutants with thermostability and catalytic activity improved |
| CN110343689A (en) * | 2019-08-23 | 2019-10-18 | 四川大学 | A kind of novel streptomycete trypsin GM2938 and its heterogenous expression |
| CN110343689B (en) * | 2019-08-23 | 2021-11-05 | 四川大学 | Streptomyces trypsin GM2938 and heterologous expression thereof in bacillus subtilis |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103898081B (en) | 2016-04-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| TWI635178B (en) | Phytase | |
| CN103525790B (en) | Optimized high-temperature resistant mannanase MAN5gy, and preparation method and application thereof | |
| TWI654304B (en) | Lysozyme having improved enzymatic activity | |
| CN107384900B (en) | The acid protease 6749 and its gene of a kind of originated from fungus and application | |
| CN105950578A (en) | Heat-resisting glucose oxidase mutant as well as encoding gene and application thereof | |
| CN102757947A (en) | Xylanase xyn-CDBFV-m with modified thermal stability, gene thereof, and application thereof | |
| CN106520642B (en) | Bacillus amyloliquefaciens and application thereof | |
| CN103589705B (en) | A kind of M-Zyme and encoding gene thereof | |
| CN104371993B (en) | A kind of enzyme agedoite enzyme mutant improved alive | |
| DK3072962T3 (en) | PROCEDURE FOR THE PREPARATION OF PHYTASE VARIANT WITH IMPROVED THERMOSTABILITY AND A PHYTASE VARIANT AND APPLICATION THEREOF | |
| CN103898081B (en) | A kind of M-Zyme mutant and application thereof | |
| CN109371004B (en) | Acid protease Bs2688 mutant K203E with improved thermal stability and gene and application thereof | |
| CN107400666A (en) | A kind of aminopeptidase and its encoding gene and application | |
| CN109415749A (en) | The method of tunning is produced in trichoderma | |
| CN103695394B (en) | Acid resistance mannase MAN26gy of a kind of optimization and its preparation method and application | |
| CN103773746B (en) | Lipase and mutant thereof | |
| CN109022396A (en) | The alpha-amylase mutant and its application that a kind of enzyme activity improves | |
| CN103555739A (en) | Recombined microorganism glutamine transaminase gene and preparation method thereof | |
| CN114774396B (en) | Keratinase mutant, compound preparation of keratinase mutant and bile acid and application of compound preparation in additive | |
| CN103013950B (en) | Lipase | |
| CN110663813B (en) | Fish meal microbial fermentation and enzymolysis method | |
| CN102978182B (en) | Lipase mutant | |
| CN110656100A (en) | Heat-resistant acidic beta-mannase derived from bacillus amyloliquefaciens and coding gene thereof | |
| CN115029337A (en) | Additive compounded by keratinase mutant and eucommia ulmoides leaf extract and application thereof | |
| CN114736881A (en) | Glucose oxidase GoxM10 mutant A4D with improved acid stability and derivative mutant and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20190523 Address after: 266061 Shandong 12A07 Shandong High Speed Building, 29 Miaoling Road, Laoshan District, Qingdao City, Shandong Province Co-patentee after: Shaanxi Shiyang agricultural Polytron Technologies Inc Patentee after: Qingdao Weilan Biology Group Co., Ltd. Address before: 266061 Shandong 12A07 Shandong High Speed Building, 29 Miaoling Road, Laoshan District, Qingdao City, Shandong Province Patentee before: Qingdao Weilan Biology Group Co., Ltd. |
|
| TR01 | Transfer of patent right |