CN1258745A - New-type subtilisin and its preparation and application - Google Patents
New-type subtilisin and its preparation and application Download PDFInfo
- Publication number
- CN1258745A CN1258745A CN 98126745 CN98126745A CN1258745A CN 1258745 A CN1258745 A CN 1258745A CN 98126745 CN98126745 CN 98126745 CN 98126745 A CN98126745 A CN 98126745A CN 1258745 A CN1258745 A CN 1258745A
- Authority
- CN
- China
- Prior art keywords
- subtilisin
- mutain
- expression
- host cell
- present
- 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
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention provides mutase derived from subtilysin E and containing Ser226CYs mutatant. It contains also mutant selected from Ala15Asp/Gly20His and Ser24His/Lys27Asp. It has high thermal stability and stability in non-water medium, and may be used widely in protein hydrolyzing reaction. The present invention also provides corresponding coding sequence, expression carrier, converted host cell and their preparation process.
Description
The present invention relates to genetically engineered and enzyme engineering field, more specifically, relate to the subtilisin that new thermostability improves.
Bacillus alkaline protease E is a kind of serine hydrolase, and it is widely used in industry such as washing composition, process hides, is a kind of important industrial enzymes.But shortcomings such as poor stability are also arranged, the existing report that improves its stability both at home and abroad.
Active and the stability (Bryan:Proteins:Struct. of Asn218Ser sudden change both can raising subtilisin BPN ', Funct., Genet.1986,1,326-34), also can improve the activity of Sumizyme MP E and stability (Chen Keqin:Proc.Natl.Acad.Sci.USA 1993,90,5618-22).
Asn118Ser sudden change can improve the activity of Sumizyme MP E and thermostability equally, and (Zhu Liuqin: Chinese patent CN 1113953A), this is that a domestic unique example is suddenlyd change on unknown site and the report that makes the proteolytic enzyme performance obtain improveing.
Yet the thermostability of existing subtilisin E and variant protein thereof still can not be satisfactory, therefore presses for the subtilisin E that thermostability further improves.
Purpose of the present invention is exactly the above-mentioned shortcoming that overcomes in the prior art, provides a kind of at the high subtilisin E of thermostability, thereby solves the problem of poor heat stability, has enlarged the range of application in fields such as washing, process hides.
In one aspect of the invention, provide a kind of subtilisin E mutain, it is characterized in that, it contains the Ser236Cys sudden change.In addition, it can also contain the sudden change that is selected from down group: Ala15Asp/Gly20His and Ser24His/Lys27Asp.
In another aspect of this invention, provide a kind of separated DNA sequence, the subtilisin E mutain that contains the Ser236Cys sudden change that its coding is above-mentioned.
On the other hand, the present invention also provides a kind of expression vector, and it contains the dna sequence dna of above-mentioned code book invention New-type subtilisin E.In addition, also provide a kind of host cell, this host cell is transformed by above-mentioned expression vector.
The present invention also provides a kind of production New-type subtilisin E of the present invention the method for mutain, and the method comprising the steps of:
(a) dna sequence dna with code book invention New-type subtilisin E mutain operationally is connected in expression regulation sequence, forms subtilisin E mutain expression vector;
(b) expression vector in the step (a) is transformed into host cell;
(c) under the condition that is fit to this subtilisin of expression E mutain, cultivate, thereby give expression to this subtilisin E mutain;
(d) separation and purification goes out this subtilisin E mutain.
In addition, also provide the purposes of New-type subtilisin E mutain of the present invention, it is used to the proteolysis reaction.
In appended accompanying drawing,
Fig. 1 is New-type subtilisin E mutain BP-1 of the present invention and BW-1, and the thermostability graphic representation of wild-type protease E.
Fig. 2 is New-type subtilisin E mutain of the present invention and wild-type protease E beta stability line figure in dimethyl formamide (DMF).
Fig. 3 is under the DMF of different concns, the activity curve figure of New-type subtilisin E mutain of the present invention and wild-type protease E.
Fig. 4 is the dna sequence dna of natural subtilisin E.
The present invention is based on and finishes on the following unexpected basis of finding: the 236th serine (Ser) among withered grass (bacillus) the protease E is sported cysteine (Cys) afterwards, can significantly improve the heat endurance of albumen. And the stability of novel mutain of the present invention in non-aqueous media also has significantly to be provided.
In the present invention, " subtilisin E " refers to the protease E from hay bacillus, and it can be the protease E of natural wild type, also can be the protease E that biology is derived or recombinated. The amino acid sequence of the subtilisin E of natural wild type is shown among the SEQ ID No.2, and its nucleotide sequence (the part non-coding region that comprises both sides) is shown in Fig. 4.
As used herein, term " non-aqueous media " refers to contain the medium of organic solvent, and this medium can be only to contain a kind of organic solvent, also can be the mixed organic solvents that two or more organic solvents form. Can be the mixed solvent of the formation of organic solvent and water in addition. Representativeness is that organic solvent has: acetonitrile, methyl-sulfoxide, dimethyl formamide (DMF) etc. Preferably, contain DMF in this non-aqueous media.
Novel protease of the present invention (variant enzyme) can be prepared like this: the sequence according to published subtilisin E is come synthetic primer, amplifies the coded sequence of subtilisin E from hay bacillus by the PCR method. Then, dna sequence dna with subtilisin E, carry out genetic modification by the sudden change form of pointing out among the present invention, the technology of carrying out genetic modification is as known in the art, for example can be referring to " Mutagenesis:a Practical Approach ", M.J.McPherson, Ed., (IRL Press, Oxford, UK. (1991) wherein for example comprise direct mutagenesis, cassette mutagenesis and polymerase chain reaction (PCR) mutagenesis.
After the dna sequence dna that has obtained the code book invention neomorph enzyme behind the rite-directed mutagenesis, it is connected into suitable expression vector, changing suitable host cell over to. At last, cultivate the host cell after transforming, obtain new protease mutant of the present invention by separation and purification.
In the present invention, can select various carrier known in the art, such as commercially available carrier. Such as, select commercially available carrier, the nucleotide sequence with code book invention neomorph enzyme operationally is connected in expression regulation sequence then, can form protein expression vector.
As used herein, " operationally being connected in " refers to so a kind of situation, and namely some part of linear DNA sequence can affect the activity of same other parts of linear DNA sequence. For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion targeting sequencing) DNA operationally is connected in polypeptid DNA so; If transcribing of promoter control sequence, it is operationally to be connected in coded sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in coded sequence so. Generally, " operationally being connected in " means adjoining, then means in reading frame adjacent for the secretion targeting sequencing.
In the present invention, term " host cell " comprises prokaryotic and eukaryotic. The example of protokaryon host cell commonly used comprises Escherichia coli, hay bacillus etc. Eukaryotic host cell commonly used comprises yeast cells, insect cell and mammalian cell. Preferably, this host cell is prokaryotic, more preferably is hay bacillus.
The inventor is through for many years research, with 118Ser, 222Ala, 60Asn, 218Ser, the various sudden change forms such as 103Arg and combination thereof are introduced in the subtilisin E, and in conjunction with computer research crystal structure (the Chu:Protein Eng.1995 of alkaline protease E-PMSF compound, 8,211-5). Research finds, Ser221 is in the surface that Ser236 on the same alpha-helix also is positioned at the enzyme molecule, and is distant with the distance in activated centre, therefore, introduces the Ser236Cys sudden change and should not produce big impact to the activated centre. Yet behind Ser236Cys sudden change introducing subtilisin E, unexpected discovery can significantly improve the heat endurance of mutain and/or the stability in non-aqueous media, thereby has finished the present invention on this discovery basis.
In addition, by the further mutagenesis on other sites, also be created in the higher and/or active higher subtilopeptidase A of stability in the non-aqueous media.
In one embodiment of the invention, 236 are suddenlyd change, thereby formed the Ser236Cys mutant, this mutant enzyme (abbreviating variant enzyme as) is named as BP-1.
In another embodiment of the present invention, also 15 and 20 are suddenlyd change, thereby formed the Ala15Asp/Gly20His/Ser236Cys mutant, this mutant enzyme (abbreviating variant enzyme as) is named as BU-1.
In another embodiment, also 24 and 27 are suddenlyd change, thereby formed the Ser24His/Lys27Asp/Ser236Cys mutant, this mutant enzyme (abbreviating variant enzyme as) is named as BW-1.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The preparation of variant enzyme BP-1
EcoR I/Sal I site with Sumizyme MP E gene (comprise signal peptide and the gene of leading peptide moiety, be total to 1.9Kb, SEQ ID No.3) insertion pAlter-I (available from Promega company).When rite-directed mutagenesis, suddenly change with reference to Promega company sudden change specification sheets, wherein, used mutagenic primer is: 5 '-GCG TTA ATTCTT T
GCAAG CAC CCG (introducing the Ser236Cys sudden change)
In above-mentioned primer sequence, tiltedly the mutational site represented in boldface type, and " Ser236Cys " expression becomes Cys with the 236th Ser in the original acid sequence, and all the other mutational sites are represented with similar fashion.
Cut out 1.9Kb fragment after the sudden change respectively with EcoR I/Sal I, insert (Guo Xinghua: biotechnology journal 1991,7 in the pBE-2 shuttle plasmid, 224-9), structure obtains expression plasmid pBP-1, changes Bacillus subtilus DB104 over to, and the Bacillus subtilus after the conversion is expressed variant enzyme BP-1.
Express the Bacillus subtilus BP-1 (Ap of variant enzyme BP-1
rE) be preserved in Chinese typical culture collection center C CTCC (China, Wuhan) on December 11st, 1998, preserving number is CCTCC M98021.
In addition, the aminoacid sequence after the variant enzyme BP-1 maturation is shown in respectively among the SEQ ID No.1.
The preparation of variant enzyme BU-1 and BW-1
Basically press the identical step of embodiment 1, difference is: the Ser236Cys mutant gene with acquisition among the embodiment 1 is a template, with primer 5 '-G CCG G
AT CTT CAC TCT CAA
CACTAC ACA GG introduces Ala15Asp/Gly20His sudden change (producing BU-1);
Be template with the Ser236Cys mutant gene equally, with primer 5 '-C ACA GGC
CAT AAC GTA
GA
CGTA GCT GT introduces Ser24His/Lys27Asp sudden change (producing BW-1)
Wherein, in above-mentioned primer sequence, tiltedly the mutational site represented in boldface type, and " Ser236Cys " expression becomes Cys with the 236th Ser in the original acid sequence, and all the other mutational sites are represented with similar fashion.
The result makes the host cell after expression plasmid pBU-1 and pBW-1 and corresponding the conversion, and makes variant enzyme BU-1 and BW-1.
Embodiment 3
Performance measurement
(1) thermostability
Variant enzyme BP-1 of the present invention and BW-1 and wild-type subtilisin E are placed water, in 50 ℃ of insulations, sampling and measuring residual enzyme vigor at regular intervals, thus draw relative thermal stability data, the results are shown in Fig. 1.
As seen from Figure 1, the half life of BP-1, surpass 1 hour, and the half life of BW-1 is about 0.5 hour, and the half life of Sumizyme MP E is no more than 20 minutes, and the thermotolerance of BP-1 is higher about 3 times than natural enzyme.Therefore, compare with natural enzyme, the proteolytic enzyme BP-1 of 236 generation Ser236Cys sudden change and the thermotolerance of BW-1 all have significantly raising, and especially the thermostability of BP-1 is higher.
(2) stability in non-aqueous media
Each variant enzyme and wild-type protease E are placed the DMF of different concns, sampling and measure residue esterlysis activity behind the certain hour at interval.The result shows, the stability of variant enzyme of the present invention (especially BP-1) in non-aqueous media also be significantly increased (Fig. 2).
(3) enzymic activity in non-aqueous media
Press Abrahmsen:Biochemistry (1991), 30, the method described in the 4151-9 is a substrate with s-Ala-ala-Pro-Phe-pNA, and the amido bond hydrolytic activity is measured.Measure each variant enzyme and the wild-type protease E esterlysis activity in DMF, wherein DMF concentration is as shown in table 1 in the system.
Determination data is listed in table 1 and Fig. 3.As shown in Figure 3, as seen, the activity of BP-1, BW-1, BU-1 and WT all reduces with the rising of DMF solubility, and wherein the stability of BP-1 is the highest, and BW-1 is similar to WT, and BU-1 is minimum.This shows that the thermotolerance and the activity of Ser236Cys mutant all are improved.
In sum, variant enzyme with Ser236Cys sudden change is thermotolerance and active all high than Sumizyme MP E proteolytic enzyme, stability in DMF is also than natural enzyme height, the variant enzyme BP-1 that produced through the Ser236Cys sudden change of Sumizyme MP E especially, and performance is particularly splendid.
Table 1 BP-1, BW-1, BU-1 and the WT kinetic constant in different DMF systems
| Enzyme | DMF | K cat(s -1) | K m(mM) | ?K cat/K m(M -1s -1) |
| | 0％ | 27.83 | 0.61 | ?4.57×10 4 |
| 5％ | 26.76 | 1.16 | ?2.31×10 4 | |
| 10％ | 24.85 | 2.56 | ?9.71×10 3 | |
| 20％ | 22.36 | 9.32 | ?2.4×10 3 | |
| 40％ | 3.73 | 17.76 | ?2.10×10 2 | |
| BP-1 | 0％ | 31.02 | 0.40 | ?7.72×10 4 |
| 5％ | 53.81 | 0.93 | ?5.80×10 4 | |
| 10％ | 29.11 | 0.96 | ?3.02×10 4 | |
| 20％ | 16.98 | 2.61 | ?6.51×10 3 | |
| 40％ | 0.74 | 5.46 | ?1.35×10 2 | |
| BW-1 | 0％ | 5.74 | 0.15 | ?3.73×10 4 |
| 5％ | 8.35 | 0.44 | ?1.89×10 4 | |
| 10％ | 11.34 | 1.30 | ?8.73×10 3 | |
| 20％ | 21.87 | 12.27 | ?1.78×10 3 | |
| 40％ | 0.1245 | 0.20 | ?6.20×10 2 | |
| BU-1 | 0％ | 12.76 | 0.44 | ?2.92×10 4 |
| 5％ | 23.93 | 4.28 | ?5.58×10 3 | |
| 10％ | 3.64 | 1.31 | ?2.78×10 3 | |
| 20％ | 0.66 | 0.96 | ?6.87×10 2 |
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
(1) general information:
(i) applicant: Shanghai Research Center of Biotechnology
(ii) denomination of invention: New-type subtilisin and method for making and purposes
(iii) sequence number: 2
(2) information of SEQ ID NO.1:
(i) sequence signature:
(A) length: 275 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ii) molecule type: polypeptide
(xi) sequence description: SEQ ID NO.1:AQSVPYGISQ IKAPALHSQG YTGSNVKVAV IDSGIDSSHP DLNVRGGASF 50VPSETNPYQD GSSHGTHVAG TIAALNNSIG VLGVSPSASL YAVKVLDSTG 100SGQYSWIING IEWAISNNMD VINMSLGGPS GSTALKTVVD KAVSSGIVVA 150AAAGNEGSSG SSSTVGYPAK YPSTIAVGAV NSSNQRASFS SAGSELDVMA 200PGVSIQSTLP GGTYGAYNGT SMATPHVAGA AALILCKHPT WTNAQVRDRL 250ESTATYLGNS FYYGKGLINV QAAAQ 275
(2) information of SEQ ID NO.2:
(i) sequence signature:
(A) length: 275 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ii) molecule type: polypeptide
(xi) sequence description: SEQ ID NO.2:AQSVPYGISQ IKAPALHSQG YTGSNVKVAV IDSGIDSSHP DLNVRGGASF 50VPSETNPYQD GSSHGTHVAG TIAALNNSIG VLGVSPSASL YAVKVLDSTG 100SGQYSWIING IEWAISNNMD VINMSLGGPS GSTALKTVVD KAVSSGIVVA 150AAAGNEGSSG SSSTVGYPAK YPSTIAVGAV NSSNQRASFS SAGSELDVMA 200PGVSIQSTLP GGTYGAYNGT SMATPHVAGA AALILSKHPT WTNAQVRDRL 250ESTATYLGNS FYYGKGLINV QAAAQ 275
Claims (10)
1. a subtilisin E mutain is characterized in that, it contains the Ser236Cys sudden change.
2. subtilisin E mutain as claimed in claim 1 is characterized in that, it also contains the sudden change that is selected from down group:
Ala15Asp/Gly20His and Ser24His/Lys27Asp.
3. subtilisin E mutain as claimed in claim 1 is characterized in that it is variant enzyme BP-1, and it has the aminoacid sequence shown in the SEQ ID NO.1.
4. subtilisin E mutain as claimed in claim 2 is characterized in that it is variant enzyme BU-1 or BW-1.
5. a separated DNA sequence is characterized in that, the described subtilisin E of its coding claim 1 mutain.
6. an expression vector is characterized in that, it contains the described dna sequence dna of claim 5.
7. a host cell is characterized in that, it is transformed by the described expression vector of claim 6.
8. host cell as claimed in claim 7 is characterized in that, it is Bacillus subtilus BP-1 (Ap
rE) CCTCC No.M98021.
9. method of producing the described subtilisin E of claim 1 mutain is characterized in that the method comprising the steps of:
(a) will the encode dna sequence dna of the described subtilisin E of claim 1 mutain operationally is connected in expression regulation sequence, forms subtilisin E mutain expression vector;
(b) expression vector in the step (a) is transformed into host cell;
(c) under the condition that is fit to this subtilisin of expression E mutain, cultivate, thereby give expression to this subtilisin E mutain;
(d) separation and purification goes out this subtilisin E mutain.
10. the purposes of subtilisin E mutain as claimed in claim 1 is characterized in that, it is used to the proteolysis reaction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 98126745 CN1258745A (en) | 1998-12-31 | 1998-12-31 | New-type subtilisin and its preparation and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 98126745 CN1258745A (en) | 1998-12-31 | 1998-12-31 | New-type subtilisin and its preparation and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1258745A true CN1258745A (en) | 2000-07-05 |
Family
ID=5229762
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 98126745 Pending CN1258745A (en) | 1998-12-31 | 1998-12-31 | New-type subtilisin and its preparation and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1258745A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102648277A (en) * | 2009-09-25 | 2012-08-22 | 诺维信公司 | Use of protease variants |
| CN101218343B (en) * | 2005-07-08 | 2013-11-06 | 诺维信公司 | Subtilase variants |
| CN101243182B (en) * | 2005-08-16 | 2014-08-06 | 诺维信公司 | Subtilases |
| WO2017162428A1 (en) * | 2016-03-23 | 2017-09-28 | Henkel Ag & Co. Kgaa | Improved cleaning performance on protein sensitive soiling |
-
1998
- 1998-12-31 CN CN 98126745 patent/CN1258745A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101218343B (en) * | 2005-07-08 | 2013-11-06 | 诺维信公司 | Subtilase variants |
| CN101243182B (en) * | 2005-08-16 | 2014-08-06 | 诺维信公司 | Subtilases |
| CN102648277A (en) * | 2009-09-25 | 2012-08-22 | 诺维信公司 | Use of protease variants |
| CN102648277B (en) * | 2009-09-25 | 2015-05-20 | 诺维信公司 | Use of protease variants |
| WO2017162428A1 (en) * | 2016-03-23 | 2017-09-28 | Henkel Ag & Co. Kgaa | Improved cleaning performance on protein sensitive soiling |
| US11535817B2 (en) | 2016-03-23 | 2022-12-27 | Henkel Ag & Co. Kgaa | Proteases with improved enzyme stability in detergents |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4263248B2 (en) | Library creation method by DNA shuffling | |
| JP4189035B2 (en) | Recombinant human interferon-beta with improved solubility | |
| JP2005512545A5 (en) | ||
| JP2005512537A5 (en) | ||
| KR890700675A (en) | New proteolytic enzymes | |
| AU2001279674A1 (en) | New nucleotide sequences which code for the mdha gene | |
| EP2256206B1 (en) | Method and gene for imparting or enhancing nonspecific adherence and/or aggregability to microorganism | |
| CN1258745A (en) | New-type subtilisin and its preparation and application | |
| ATE465259T1 (en) | NUCLEOTIDE SEQUENCES ENCODING THE DAPC GENE AND METHOD FOR PRODUCING L-LYSINE | |
| CN101268186B (en) | Novel endoribonuclease | |
| CN1235978A (en) | Protein participating in activation of nitrile hydratase and gene encoding the same | |
| CN102796719B (en) | (+)Gamma-lactamase, its coding gene and application | |
| CN1995336A (en) | D-carboxamide hydrolase mutant and its uses | |
| CN105296513A (en) | Marine esterase as well as coding gene E22 and application thereof | |
| WO2023039377A1 (en) | Class ii, type v crispr systems | |
| CN101268187B (en) | Novel endoribonuclease | |
| ATE274061T1 (en) | NUCLEOTIDE SEQUENCES CODING FOR THE LYSR2 GENE | |
| CN1258744A (en) | Polypeptide ligase being stable in non-water medium and its preparation and application | |
| EP3550014B1 (en) | Directed evolution of cyp52a12 gene and its use in dicarboxylic acid production | |
| CN1366056A (en) | Lipase gene sequence and its application in yeast | |
| CN1818068A (en) | Construction of recombinant human leucocyte medium-2(125ser) expression carrier | |
| WO2002012291A3 (en) | Novel nucleotide sequences coding for the luxr gene and a process for the production of l-amino acids | |
| CN1900276A (en) | High temperature lipase gene and its using method | |
| CN1178835A (en) | Hear resisting basic phosphatase and its expression | |
| CN1361279A (en) | Lichenized bacillus L-25 keratinase and its encoding DNA |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |