CN1088108C - 247 position single-mutant enzyme and 138, 247 position double-mutant enzyme of glucose isomerase and configuration method thereof - Google Patents
247 position single-mutant enzyme and 138, 247 position double-mutant enzyme of glucose isomerase and configuration method thereof Download PDFInfo
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- CN1088108C CN1088108C CN97119714A CN97119714A CN1088108C CN 1088108 C CN1088108 C CN 1088108C CN 97119714 A CN97119714 A CN 97119714A CN 97119714 A CN97119714 A CN 97119714A CN 1088108 C CN1088108 C CN 1088108C
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Abstract
The present invention provides a fixed-point mutagenesis scheme of protein engineering, a mutant enzyme of SM33GI and a double-mutant enzyme thereof, which is characterized in that the 247th site of SM33GI or Gly equivalent to the 247th site in homology comparison is replaced by Asp to obtain the mutant enzyme SM33GIG247D; compared with the enzyme activity of a wild type thick enzyme GI, the enzyme activity of the mutant enzyme SM33GIG247D is obviously enhanced; then, an SM33GI gene fragment with Asp247 is replaced by a corresponding fragment of an SM33GIGI38P gene to obtain a double-mutant enzyme SM33GI (GI38P, G247D), and the enzyme activity and the thermal stability of the double-mutant enzyme SM33GI(GI38P, G247D) are simultaneously enhanced.
Description
The present invention relates to utilize the technical field of the rite-directed mutagenesis method improvement glucose isomerase of protein engineering.
The mutant enzyme GI G138P of Chinese patent application 95112782.9-SM33 GI and a kind of sudden change scheme of the GI of raising thermostability " source, characteristic and the protein engineering that have provided relevant No. 7 streptomyces diastaticus M1033 glucose isomerases (SM33 GI) generalized case that this enzyme is transformed.But prior art fails to improve the vigor of this enzyme.
The objective of the invention is to propose the mutant enzyme SM33 GI G247D that a kind of SM33 of making GI enzyme activity improves, the double-mutant enzyme SM33 GI that thermostability and enzyme activity are improved simultaneously (G138P, G247D), and construction process.
The mutant enzyme SM33 GI G247D of SM33 GI of the present invention is characterized in that among the Gly247 of described SM33 GI or some GI that the glycine (Gly) that is equivalent to the Gly247 position of SM33 GI in homology on has relatively become aspartic acid (Asp).
The preparation method of SM33 GI G247D of the present invention, it is characterized in that gene order according to SM33 GI, design and synthesize the rite-directed mutagenesis primer of introducing the Asp247 codon, SM33 GI gene is carried out rite-directed mutagenesis, measure dna sequence dna, identify the Gly247 codon and become the mutant of Asp247 codon, and express.
The double-mutant enzyme SM33 GI (G138P of SM33 GI of the present invention, G247D), it is characterized in that to have the SM33 GI gene fragment of Asp247 codon, or be equivalent to the G247D of 5M33 GI among some GI on relatively in homology, substituted corresponding D NA fragment or corresponding site on the SM33 GI G138P.
SM33 GI double-mutant enzyme SM3 GI (G138P of the present invention, G247D) preparation method, obtain the mutant enzyme SM33 GI G138P of SM33 GI earlier by Chinese patent application number 95112782.9 technology, it is characterized in that: according to the gene order of SM33 GI, design and synthesize the rite-directed mutagenesis primer of introducing the Asp247 codon, SM33 GI gene is carried out rite-directed mutagenesis, measure dna sequence dna, identify the Gly247 codon and become the mutant of Asp247 codon, and express, obtain the mutant enzyme SM33 GI G247D of SM33 GI; Again SM33 GI G138P and SM33 GI G247D are used the XhoI enzymolysis respectively, the SM33GI gene 5 ' end dna fragmentation that will have a G138P and SM33 GI gene 3 ' the end dna fragmentation that has G247D are formed by connecting to having the SM33 GI gene in the two mutational sites of G138P, G247D through the T4 ligase enzyme, and express.
Relatively express the character of the thick enzyme of mutant of acquisition by the said mutation body, can find: single-mutant enzyme SM33 GIG247D compares with the thick enzyme GI of wild-type, and enzyme has more significantly raising than living, but thermostability descends; And double-mutant enzyme SM33 GI (G138P G247D) all is better than wild-type GI, single-mutant enzyme GI G138P and GI G247D at thermostability and enzyme on living; Further relatively (G138P, G247D) and the pure enzyme of single-mutant enzyme GI G138P, the former is than living and thermostability all is better than the latter for double-mutant enzyme GI.
Hence one can see that, and the single-mutant enzyme SM33 GI G247D of SM33 GI of the present invention has the advantage that the raising enzyme is lived; And the fragment that will have this Asp247 position replaced the SM33 GI double-mutant enzyme GI that SM33 GI G138P respective segments is constituted (G138P, G247D), than any has higher enzyme and lives and thermostability in these two kinds of single-mutant enzymes.
According to the space structure of molecule, behind 247 introducing Asp, trickle adjustment takes place in local conformation, but can not produce big spatial obstacle.Because 247 bonding surfaces near the asymmetry unit disome, and this bonding surface mainly is made of the main chain of G260, A261, G262, and Gly residue main chain conformation allows bigger variation, allows the introducing of 247D.However, the introducing of 247D position forms the disome in the molecule and still has certain influence, can influence the thermostability of molecule.
247 effects of introducing the Asp generation are to introduce electric charge in molecule, thereby change the distribution of electrostatic of molecule.According to the corresponding result of study of subtilisin, this electric charge away from the active centre is introduced the catalysis activity that can influence enzyme.
Number of patent application 95112782.9 is analysis revealed, introduces the thermostability that Pro can increase enzyme for 138, and reason may be to introduce the flexibility of Pro meeting restriction enzyme, makes it become very inresponsive to temperature.
Space structure shows, 138 and 247 are far apart, and conformational change can not influence each other each other, so above-mentioned 138 and 247 introduced Pro and Asp respectively, its effect that influences to enzyme exists simultaneously.
Therefore, adopt construction process of the present invention, can make alive raising of enzyme of SM33 GI, and enzyme is lived and thermostability obtains to improve simultaneously; Especially double-mutant can make up engineering strain, is with a wide range of applications.
The preparation of embodiment 1.SM33 GI G247D:
1), synthetic oligonucleotide point mutation primer
According to the gene order of SM33 GI, design the oligonucleotide mutant primer that the codon of Gly247 (GGC) is made into the codon of Asp247 (GAC), for example: 5 '-ACC TCA ACG ACC AGT CCG-3 '; Adopt the DNA synthesis method to synthesize.
2), make up reorganization M13DNA
With recombinant expression plasmid pTKD-GI and M13mp19RF EcoRI+SphI double enzymolysis, through agarose gel electrophoresis, extracting 0.9kb and 7.2kb fragment from glue respectively, after connecting with the T4DNA ligase enzyme, transfection Escherichia coli XL1-blue.
3), site-directed mutagenesis
Adopt double-primer method, step is summarized as follows:
The ginseng U single-stranded template of a, preparation reorganization M13DNA is finished in intestinal bacteria CJ236;
B, phosphorylation oligonucleotide point mutation primer;
C, with phosphorylation point mutation primer, universal primer and the annealing of ginseng U single-stranded template, carry out external extension and be connected;
D, transfection jump reaction liquid are in the XL1-blue competent cell.
Detailed process can be referring to 27 (5) 470 pages of Chinese " Acta Biochimica et Biophysica Sinica " nineteen ninety-fives.
Picking plaque then, the extracting strand is measured dna sequence dna on a small scale, and the codon that identifies Gly247 becomes the mutant of the codon of Asp247.
4), express mutant gene
Mutant gene is cloned back former reorganization expression plasmid pTKD-GI, transformed into escherichia coli K38/pGP1-2.Can adopt China's " hi-tech communication " 3 (7) 10 pages of described methods in 1993, culture expression goes out SM33 GI G247D.
Embodiment 2.SM33 GI (G138P, structure G247D) and the preparation of double-mutant enzyme:
1), pTKD-GI (G138P, structure G247D)
Accompanying drawing 1 is that (A-I is pTKD-GI G138P to pTKD-GI among the figure for G138P, design of graphics G247D), and its DNA size is 4.3Kb; A-II be A-I behind the XhoI enzymolysis, through the big fragment that electrophoretic separation obtains, its DNA size is 3.6Kb, it is made of big fragment of pTKD carrier and the GI gene 5 ' end dna fragmentation that has a G138P mutational site.B-I is pTKD-GI G247D among the figure, its DNA size is 4.3Kb, B-II be B-I behind the XhoI enzymolysis, the small segment that obtains through electrophoretic separation, its DNA size is 700bp, and it is to be made of pTKD carrier small segment and GI gene 3 ' the end dna fragmentation that has a G247D mutational site.
AB is meant pTKD-GI (G138P, G247D), be that A-II dna fragmentation and B-II dna fragmentation are connected and the pTKD expression plasmid of the complete S M33 GI gene that has the two mutational sites of G138P, G247D that constitutes through T4 ligase enzyme (T4Ligase), its DNA size is 4.3Kb.
2), express SM33 GI (G138P, G247D) double-mutant enzyme
(culture expression can obtain SM33 GI (G138P, G247D) double-mutant enzyme for G138P, G247D) transformed into escherichia coli K38/pGP1-2 with pTKD-GI to press embodiment 1 described method.
Embodiment 3.SM33 GI, SM33 GI G138P, SM33 GI G247D and SM33 GI (G138P, G247D) thick enzymatic property is relatively:
1), measures than living
Adopting halfcystine-carbazole method, is substrate with glucose.Specifically pressed " China Science ﹠ Technology University's journal " 1992 22 (2), the described method of 232-235 page or leaf is measured, and the results are shown in table 1:
Table 1
| GI (wild-type) | 13.27(u/mg) |
| GI(G138P) | 12.71(u/mg) |
| GI(G247D) | 17.65(u/mg) |
| GI(G138P，G247D) | 19.23(u/mg) |
2), the mensuration of thermostability
70 ℃, heated 2 hours, adopt halfcystine-carbazole method, be substrate with glucose, measure the relative vigor of its heating front and back, the results are shown in table 2:
Table 2
Can be drawn by above-mentioned measurement result to thick enzymatic property: (G138P's double-mutant enzyme SM33 GI, enzyme G247D) live and thermostability is best.
By Chinese patent application number 95112782.9 " the mutant enzyme GI G138P of SM33GI and a kind of mutation methods that improve the GI thermostability " that propose, the thermostability of mutant enzyme GI G138P is significantly improved than wild-type, and enzyme is lived in not having obviously and descended; And double-mutant enzyme SM33 GI (G138P G247D) in fact just introduces the sudden change of Asp again at 247 Gly of SM33 GI G138P mutant enzyme; So be necessary to be further purified and relatively double-mutant enzyme SM33 GI (G138P is G247D) with the change of properties of single-mutant enzyme SM33 GI G138P.
Embodiment 4. double-mutant enzyme SM33 GI (G138P, G247D) separation and purification and the zymologic property with single-mutant enzyme SM33 GI G138P compares:
1), purifying
I), heat denatured is removed foreigh protein removing
Zymoprotein and enzyme variation alive after 70 ℃ of heating 15 ':
Respectively with SM33 GI (G138P, G247D) and two kinds of crude enzyme liquids of SM33 GI G138P take out in 70 ℃ of heating 15 ' back, be cooled to 4 ℃ immediately, centrifugal 20 minutes of 10000rmp, go precipitation, by supernatant record single-mutant enzyme SM33 GI G138P and double-mutant enzyme SM33 GI (G138P, G247D) total protein and the total activity (to be 100% before heating) before and after the heating the results are shown in table 3:
Table 3
As seen adopt the method for heat denatured to remove most of foreign protein.
| GI G138P single-mutant enzyme | GI (G138P, G247D) double-mutant enzyme | |||
| Before the heating | After the heating | Before the heating | After the heating | |
| | 100％ | 26.9％ | 100％ | 23.6％ |
| | 100％ | 93％ | 100％ | ～100％ |
Ii), purifying
By " Acta Biochimica et Biophysica Sinica " nineteen ninety-five 27 (5) 470 pages of described methods, supernatant through DEAE-sepharose FF primary column chromatography, is promptly obtained the pure enzyme of SDS-PAGE electrophoresis.
2), (G138P, G247D) character with single-mutant enzyme SM33 GIG138P compares double-mutant enzyme SM33 GI
I), the mensuration of thermostability: select 10mM MgCl for use
2/ 50mM Tris-HCl (PH7.0) damping fluid is as the damping fluid of thermostability experiment.Enzyme liquid is incubated in 80 ℃ of water-baths, and per 20 minutes sampling 100ul are substrate with wood sugar and glucose respectively, and with after substrate mixes respectively, reaction is 15 minutes in 35 ℃ of water-baths with enzyme liquid, surveys alively with halfcystine-carbazole method, and data are listed in table 4 and table 5 respectively:
Table 4 is relative vigor of enzyme after the different soaking times when being substrate with the wood sugar;
Table 5 is relative vigor of enzyme after the different soaking times when being substrate with glucose.
Table 4
Table 5
Get X-coordinate and be the time (unit: minute), ordinate zou is relative vigor, with soaking time-logarithm mapping of vigor relatively.Fig. 2 be SM33 GI G138P and SM33 GI (G138P, G247D) at 80 ℃, the thermostability curve when being substrate with the wood sugar; Fig. 3 be SM33 GI G138P and SM33 GI (G138P, G247D) at 80 ℃, the thermostability curve when being substrate with glucose.Bullet is that (black box is the vigor data of SM33 GI G138P to SM33 GI for G138P, vigor data G247D) among the figure; Solid line represent SM33 GI (G138P, G247D) time-relative vigor curve, dotted line represent SM33 GI G138P time-vigor curve relatively.
With reference to the accompanying drawings 2 and 3, the pairing X-coordinate of lg50 from the ordinate zou, can try to achieve the transformation period of mutant glucose isomerase and list in table 6:
Table 6
| Transformation period (minute) | GIG138P | GI(G138P，G247D) |
| Wood sugar is a substrate | 67 | 272 |
| Glucose is substrate | 131 | 163 |
As can be seen, and double-mutant enzyme SM33 GI (G138P, thermostability G247D) is better than single-mutant enzyme SM33 GIG138P, and its heat inactivation transformation period is being brought up to 1.24 times with glucose during for substrate; Bringing up to 4.06 times with the wood sugar during for substrate.
Ii), measure than living
Through above-mentioned first heat denatured, the double-mutant enzyme that obtains through DEAE-sepharose FF column chromatography again, adopt halfcystine-carbazole method, with glucose is substrate, the ratio that records SM33 GI G138P is lived and to be 113.3U/mg, and SM33 GI (G138P G247D) is 179.2 U/mg, be that (G138P, ratio work G247D) is 1.58 times of SM33 GI G138P to SM33 GI.
By The above results as can be known, SM33 GI (the G138P of the present invention's preparation, G247D) be the significant double-mutant enzyme that thermostability and enzyme activity than single-mutant enzyme SM33 GI G138P are improved simultaneously, its enzyme work is brought up to 1.58 times than GI G138P, its heat inactivation transformation period is being brought up to 1.24 times with glucose during for substrate, is bringing up to 4.06 times with the wood sugar during for substrate.
Claims (4)
1. the mutant enzyme SM33 GI G247D of No. 7 streptomyces diastaticus M1033 glucose isomerase SM33 GI is characterized in that the 247th the glycine Gly.G of SM33 GI has been mutated into aspartic acid Asp.D.
2. the preparation method of 247 mutant enzyme SM33 of No. 7 streptomyces diastaticus M1033 glucose isomerases GI G247D, it is characterized in that, gene order according to SM33 GI, design and synthesize and introduce Asp in the rite-directed mutagenesis primer of 247 codons, SM33 GI gene is carried out rite-directed mutagenesis, measure dna sequence dna, identify 247 glycine Gly and become the mutant of aspartic acid Asp, and express.
3. 138 of No. 7 streptomyces diastaticus M1033 glucose isomerase SM33 GI and 247 double-mutant enzyme SM33 GI G138P G247D is characterized in that the gene fragment that has 247 aspartic acid Asp of SM33 GI G247D has been substituted the respective segments of 138 mutant enzyme SM33 GI G138P of SM33 GI.
4. the preparation method of the double-mutant enzyme SM33 GI G138PG247D of No. 7 streptomyces diastaticus M1033 glucose isomerase SM33 GI, elder generation is according to the gene order of SM33 GI, design and synthesize the rite-directed mutagenesis primer of introducing the Pro138 codon, SM33 GI gene is carried out rite-directed mutagenesis, measure dna sequence dna, identify 138 glycine Gly and become the mutant of proline(Pro) Pro, express, obtain mutant enzyme SM33 GI G138P; It is characterized in that: again according to the gene order of SM33 GI, design and synthesize and introduce Asp in the rite-directed mutagenesis primer of 247 codons, SM33 GI gene is carried out rite-directed mutagenesis, measure dna sequence dna, identify 247 glycine Gly and become the mutant of aspartic acid Asp, and express, obtain mutant enzyme SM33 GI G247D; Respectively the gene of SM33 GI G138P and SM33 GI G247D is cut with the XhoI enzyme then, hold dna fragmentation and SM33 GI G247D gene to have 3 ' the end dna fragmentation of G247D 5 ' of the G138P that has of SM33 GI G138P gene, be connected to become the SM33 GI gene that has the two mutational sites of G138P, G247D with the T4 ligase enzyme, and express.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN97119714A CN1088108C (en) | 1997-09-30 | 1997-09-30 | 247 position single-mutant enzyme and 138, 247 position double-mutant enzyme of glucose isomerase and configuration method thereof |
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| CN97119714A CN1088108C (en) | 1997-09-30 | 1997-09-30 | 247 position single-mutant enzyme and 138, 247 position double-mutant enzyme of glucose isomerase and configuration method thereof |
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| CN1088108C true CN1088108C (en) | 2002-07-24 |
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| CN1743454A (en) * | 2004-09-02 | 2006-03-08 | 百瑞全球有限公司 | Glucose isomerase mutant and its use |
| CN1966702B (en) * | 2005-11-18 | 2011-12-14 | 百瑞全球有限公司 | Use of glucose isomerase mutant |
| CN1966679B (en) | 2005-11-18 | 2011-08-17 | 百瑞全球有限公司 | Glucose isomerase mutant, its application and DNA encoding same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1156182A (en) * | 1995-11-27 | 1997-08-06 | 中国科学技术大学 | Mutational enzyme GIG138P of SM33GI and mutational plan for increasing GI thermal stability |
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| CN1156182A (en) * | 1995-11-27 | 1997-08-06 | 中国科学技术大学 | Mutational enzyme GIG138P of SM33GI and mutational plan for increasing GI thermal stability |
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