CN106282148A - A kind of enzyme agedoite enzyme mutant improved alive - Google Patents
A kind of enzyme agedoite enzyme mutant improved alive Download PDFInfo
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- CN106282148A CN106282148A CN201610920514.8A CN201610920514A CN106282148A CN 106282148 A CN106282148 A CN 106282148A CN 201610920514 A CN201610920514 A CN 201610920514A CN 106282148 A CN106282148 A CN 106282148A
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- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
- C12N9/82—Asparaginase (3.5.1.1)
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- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01001—Asparaginase (3.5.1.1)
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Abstract
The invention discloses a kind of enzyme agedoite enzyme mutant improved alive, belong to technical field of enzyme engineering.The present invention, by asparaginase shown in SEQ ID NO.1 carries out fixed point saturation mutation, changes amino acid residue near protein molecule avtive spot, improves asparaginase catalytic efficiency, improves agedoite production of enzyme further.Relatively starting strain alive for asparaginase enzyme can be improved 2.37 times by the recombined bacillus subtilis that the asparaginase secretion capacity that the present invention builds strengthens.Improved genetic engineering bacterium enzymatic productivity significantly improves, and shake flask fermentation production asparaginase enzyme is lived and reached 320U/mL, for the highest yield of the shaking flask of report at present, is more suitable for commercial Application, it is possible to decrease production cost, improves production efficiency.
Description
Technical field
The present invention relates to a kind of enzyme agedoite enzyme mutant improved alive, belong to technical field of enzyme engineering.
Background technology
L-ASP (EC 3.5.1.1) is a kind of protease having active anticancer, can single-minded catalysis altheine
It is hydrolyzed into aspartic acid and NH3.The physiological action of L-ASP mainly shows as the inhibitory action to some tumor, especially
Effective to acute leukemia and malignant lymphoma.L-ASP has become the treatment very effective medicine of leukemia, right
Medullary cell does not has inhibitory action.
L-ASP can reduce the generation of acrylamide in food.Acrylamide is mainly by raw-food material
Reducing sugar and agedoite are generated by Maillard reaction in high-temperature heating process, add asparaginase permissible in food
Hydrolysis agedoite, reduces the generation of acrylamide from source.
Some microorganisms, mammal and plant are proved containing L-ASP.Because L-Radix Asparagi in animal serum
Amidase content is low, and extraction process is complicated, and microorganism has easily cultivation, low cost and other advantages, becomes the emphasis of scholar's research,
The at present product L-ASP microorganism of research mainly include Escherichia coli, Erwinia carotovora,
Erwinia chrysanthemi etc., but wild strain L-ASP yields poorly, and utilizes technique for gene engineering to incite somebody to action in recent years
L-ASP gene is cloned in escherichia coli, it is thus achieved that the high efficient expression of L-ASP, and utilizing works bacterium produces L-days
Winter amidase has become an important sources.
L-ASP has two types, L-ASP I and L-ASP II, Escherichia
Coli, Erwinia chrysanthemi, B.subtilis etc. all comprise both L-ASPs, and research confirms only L-days
Winter, amidase II had antitumaous effect, from Escherichia coli and the altheine of Erwinia chrysanthemi
Enzyme II has been developed that the active drug into treating acute lymphoblastic leukemia, and the major part of research is to have antitumor effect at present
The L-ASP II of fruit.
Current research shows, genetic engineering means is to improve the main method of agedoite production of enzyme, separate sources
Asparaginase escherichia coli, bacillus subtilis, the different hosts such as yeast obtains and expresses, but its yield is the highest.
The maximum output of report is 228U/mL (Wang Y, Qian S, Meng G, Zhang S (2001) Cloning and at present
expression of L-Asparaginase gene inEscherichiacoli.Appl BiochemBiotechnol 95
(2):93-101).It addition, the method for the expression of recombinant proteins level of the raising of routine mainly changes strong promoter, hypersecretion
Signal peptide that ability is high etc..
Summary of the invention
First purpose of the present invention is to provide a kind of agedoite enzyme mutant, is by Radix Asparagi shown in SEQ ID NO.1
The 29th glutamic acid rite-directed mutagenesis of amidase amino acid sequence is other aminoacid；Other aminoacid described includes: glutamine,
Arginine, threonine, lysine, agedoite.
In one embodiment of the invention, other aminoacid described also include alanine, cysteine, histidine,
Isoleucine, glycine, leucine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, valine.
In one embodiment of the invention, described asparaginase mutant sequence such as SEQ ID NO.2, or
SEQ ID NO.3, or SEQ ID NO.4, or SEQ ID NO.5, or shown in SEQ ID NO.6.
Second object of the present invention is to provide the gene encoding described agedoite enzyme mutant.
Third object of the present invention is to provide the carrier containing described gene or cell line.
Fourth object of the present invention is to provide a kind of genetic engineering bacterium expressing described mutant.
In one embodiment of the invention, described genetic engineering bacterium is with pP43NMK as carrier, with bacillus subtilis
For host.
In one embodiment of the invention, described bacillus subtilis is bacillus subtilis WB600.
5th purpose of the present invention is to provide a kind of method improving asparaginase, is to asparaginase activity position
Near point, aminoacid carries out pinpointing saturation mutation；Described active site amino is the 29th glutamic acid.
In one embodiment of the invention, described agedoite enzyme sequence is as shown in SEQ ID NO.1.
6th purpose of the present invention is to provide the application in terms of preparing medicine of the described mutant.
The present invention also provides for the application in preparation contains the product of agedoite of the described genetic engineering bacterium.
Beneficial effects of the present invention: the present invention changes amino near protein molecule avtive spot by fixed point saturation mutation
Acid residue, improves asparaginase catalytic efficiency, improves agedoite production of enzyme further.The asparaginase that the present invention builds
Relatively starting strain alive for asparaginase enzyme can be improved 2.37 times by the recombined bacillus subtilis that secretion capacity strengthens.Improved
Genetic engineering bacterium enzymatic productivity significantly improves, and shake flask fermentation production asparaginase enzyme is lived and reached 320U/mL, for report at present
The highest yield of shaking flask, is more suitable for commercial Application, it is possible to decrease production cost, improves production efficiency.
Accompanying drawing explanation
ASN yield after Fig. 1 rite-directed mutagenesis E29；
ASN protein expression level after Fig. 2 rite-directed mutagenesis E29；Swimming lane 1～6 is respectively original strain WT, mutant E29Q,
Mutant E29R, mutant E29T, mutant E29K, mutant E29N；
Fig. 3 SDS-PAGE analyzes the level of purification of mutant；Swimming lane 1～6 is respectively original strain WT, mutant E29Q,
Mutant E29R, mutant E29T, mutant E29K, mutant E29N.
Detailed description of the invention
Culture medium:
LB culture medium: tryptone 10g/L, yeast powder 5g/L, NaCl 10g/L, pH7.0；
Fermentation medium: soy peptone 10g/L, Semen Maydis pulp 5g/L, carbamide 1g/L, sucrose 35g/L, dipotassium hydrogen phosphate
2.3g/L, potassium dihydrogen phosphate 1.7g/L, magnesium sulfate 0.75g/L, sodium chloride 5g/L；Regulation pH6.8-7.0.
The mensuration of asparaginase enzyme activity:
Spectrophotometry asparaginase enzyme is used to live.1 unit asparaginase enzyme is lived and is defined as: anti-at 37 DEG C
Under the conditions of Ying, the interior altheine that can be catalyzed per minute discharges 1 μm ol NH3Required enzyme amount is enzyme unit (U/ alive
ml).Enzyme activity determination condition: under the conditions of 37 DEG C, 1ml 10mM K2HPO4-KH2PO4(pH7.5), 0.1ml 189mM agedoite,
0.1ml fermented supernatant fluid, is incubated 30 minutes, and 0.5ml 1.5M TCA terminates reaction.Utilize ShimadzuUV-1240 at 436nm
Place measures light absorption value, draws standard curve by ammonium sulfate, calculates enzyme according to standard curve and lives.
Table 1 primer sequence
The asparaginase crystal structure simulation of embodiment 1 bacillus subtilis source
With the Erwinia chrysanthemi asparaginase (PDB code:1hg0) reported as template
(Structural basis for the activity and substrate specificity ofErwinia
Chrysanthemi L-Asparaginase, calendar year 2001 is open) (both amino acid similarity degree are 58.1%), utilize online mould
Intend software SWISS-MODEL, the crystal structure of the asparaginase in simulation bacillus subtilis source.
Embodiment 2 rite-directed mutagenesis E29 asparaginase strain construction
Upstream and downstream primer P1, P2 (as shown in table 1) of design, carries out PCR with pP43H-D30 plasmid for masterplate, builds E29
Saturation mutation bacterial strain.PCR condition is: 98 DEG C of 3min, 98 DEG C of 30S, 55 DEG C of 90S, 72 DEG C of 8min, 34 circulations.PCR expands body
System: template 1 μ L, each 1 μ L of upstream and downstream primer, dNTP Mix4 μ L, 5 × primeSTARBuffer10 μ L, the distilled water of sterilizing
32.5 μ L, prime STARDNA polymerase 0.5 μ L.Use glue to reclaim test kit PCR primer is purified and reclaims, electrophoresis
The concentration of product is reclaimed in inspection.DpnI digestion process PCR reclaims product, is transformed into competence E.coil JM109, uses ammonia
Benzylpcnicillin LB flat board, picking positive bacterium colony.Extraction plasmid after 37 DEG C of incubator overnight cultivations, named pP43H-D30/E29X,
Proceed to bacillus subtilis WB600 again, to obtain the mutant strain WB43H-D30/E29X of E36 saturation mutation (after X represents sudden change
Aminoacid).
Embodiment 3 hypersecretion ability asparaginase produces the checking of bacterial strain
Select transformant in embodiment 2 to be inoculated into equipped with in LB fluid medium 96 orifice plate, 37 DEG C, cultivate 6h, be transferred to
In fermentation medium, inoculum concentration is 5% by volume, cultivates 48h.Collecting fermentation supernatant, detection fermentation supernatant enzyme is lived.Select product
The bacterial strain that amount improves carries out shake flask fermentation, is measured agedoite enzyme activity, and result is as shown in Figure 1.With starting strain ratio
Relatively, the enzyme of mutant is lived and is significantly improved, and wherein E29R enzyme is lived and reached 320U/mL.Protein electrophoresis shows, bacterial strain target egg after sudden change
White expression does not has significant change (Fig. 2).Do not affect ASN after showing E29 fixed point saturation mutation to exist simultaneously
Secretion in B.subtilis.
Fermented supernatant fluid is through Ni2+Affinity chromatograph column purification obtains electrophoretically pure ASN saturated mutant (Fig. 3), and detects enzyme
Learning character, as shown in table 2, compared with wild enzyme, Km reduces 1.3-1.8 times, and Kcat value improves 1.41-2.2 times, and catalytic efficiency
(Kcat/Km) 1.8-2.8 times of wild enzyme is risen to.Property in contrast, it has been found that the ratio enzyme of the mutant of output increased
Living and also significantly improve, wherein the wilder enzyme of E29Q specific enzyme activity improves 2.15 times, reaches 258.8U/mg.Existed by detection mutant
The half-life of 65 DEG C shows, in addition to E29K, and mutant half-life (t1/2) significantly improve, wherein E29Q reaches 150min, wilder
Raw enzyme improves 2.37 times.Analyzing free energy result to show, mutant free energy is respectively increased 1.5-2.2kJ mol-1.Above-mentioned knot
Fruit shows, fixed point saturation mutation E29 can significantly improve the substrate binding ability of ASN, catalytic efficiency and heat stability.
Table 2 mutant zymologic property
Although the present invention is open the most as above with preferred embodiment, but it is not limited to the present invention, any is familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention
Enclosing should be with being as the criterion that claims are defined.
Claims (10)
1. an agedoite enzyme mutant, it is characterised in that by agedoite enzyme amino acid sequence shown in SEQ ID NO.1
29 glutamic acid rite-directed mutagenesises are other aminoacid；Other aminoacid described includes: glutamine, arginine, threonine, relies ammonia
Acid, agedoite.
Agedoite enzyme mutant the most according to claim 1, it is characterised in that sequence such as SEQ ID NO.2, or SEQ
ID NO.3, or SEQ ID NO.4, or SEQ ID NO.5, or shown in SEQ ID NO.6.
3. the gene of agedoite enzyme mutant described in coding claim 1 or 2.
4. contain carrier or the cell line of gene described in claim 3.
5. a genetic engineering bacterium, it is characterised in that express agedoite enzyme mutant described in claim 1 or 2.
Genetic engineering bacterium the most according to claim 5, it is characterised in that with pP43NMK as carrier, with bacillus subtilis
For host.
Genetic engineering bacterium the most according to claim 6, it is characterised in that described bacillus subtilis is bacillus subtilis
WB600。
8. the method improving asparaginase, it is characterised in that asparaginase activity location proximate aminoacid is carried out
Fixed point saturation mutation；Near described avtive spot, aminoacid is the 29th glutamic acid.
9. the application in terms of preparing medicine of the mutant described in claim 1 or 2.
10. claim 5-7 arbitrary described genetic engineering bacterium application in preparation contains the product of agedoite.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828768A (en) * | 2017-12-13 | 2018-03-23 | 江南大学 | A kind of L asparagines enzyme mutant and its construction method |
| CN109266635A (en) * | 2018-11-20 | 2019-01-25 | 江南大学 | A kind of altheine enzyme mutant and its construction method that enzyme activity improves |
| CN109593749A (en) * | 2018-12-24 | 2019-04-09 | 浙江工业大学 | Halide alcohol dehalogenase mutant and its application in synthesizing chiral epoxy chloropropane |
| CN110938555A (en) * | 2019-06-14 | 2020-03-31 | 南京农业大学 | Bacillus licheniformis Z-1 and L-asparaginase gene and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104371993A (en) * | 2014-10-22 | 2015-02-25 | 江南大学 | Asparaginase mutant with enhanced enzyme activity |
| CN105349515A (en) * | 2015-11-25 | 2016-02-24 | 江南大学 | Asparaginase mutant with increased secretion ability and application of asparaginase mutant |
-
2016
- 2016-10-21 CN CN201610920514.8A patent/CN106282148A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104371993A (en) * | 2014-10-22 | 2015-02-25 | 江南大学 | Asparaginase mutant with enhanced enzyme activity |
| CN105349515A (en) * | 2015-11-25 | 2016-02-24 | 江南大学 | Asparaginase mutant with increased secretion ability and application of asparaginase mutant |
Non-Patent Citations (1)
| Title |
|---|
| 龙水清 等: "点突变H125L和E145A对L-天冬酰胺酶酶活和热稳定性的改善", 《食品与发酵工业》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828768A (en) * | 2017-12-13 | 2018-03-23 | 江南大学 | A kind of L asparagines enzyme mutant and its construction method |
| CN107828768B (en) * | 2017-12-13 | 2020-10-09 | 江南大学 | L-asparaginase mutant and construction method thereof |
| CN109266635A (en) * | 2018-11-20 | 2019-01-25 | 江南大学 | A kind of altheine enzyme mutant and its construction method that enzyme activity improves |
| CN109266635B (en) * | 2018-11-20 | 2020-12-01 | 江南大学 | L-asparaginase mutant with improved enzyme activity and construction method thereof |
| CN109593749A (en) * | 2018-12-24 | 2019-04-09 | 浙江工业大学 | Halide alcohol dehalogenase mutant and its application in synthesizing chiral epoxy chloropropane |
| CN109593749B (en) * | 2018-12-24 | 2020-09-25 | 浙江工业大学 | Halogen alcohol dehalogenase mutant and application thereof in synthesis of chiral epichlorohydrin |
| CN110938555A (en) * | 2019-06-14 | 2020-03-31 | 南京农业大学 | Bacillus licheniformis Z-1 and L-asparaginase gene and application thereof |
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