CN104531729A - Method for improving heat stability of ferulic acid esterase A by adding disulfide bond - Google Patents
Method for improving heat stability of ferulic acid esterase A by adding disulfide bond Download PDFInfo
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Abstract
The invention aims to provide a method for reforming heat stability of ferulic acid esterase A, constructing a mutant ferulic acid esterase A engineering bacterium and realizing high-efficiency heterologous expression of mutant ferulic acid esterase A. According to the space structure design mutant A126C and N152C derived from A.usamii E001 ferulic acid esterase A (AuFaeA), mutant ferulic acid esterase A: AuFaeA A126C-N152C is obtained, the mature peptide gene sequence thereof is SEQ ID NO:1, and C126 and C152 can form one disulfide bond. Experimental results indicate that the heat stability of the enzyme is obviously improved after mutation, thus providing a new technological path for reforming of other enzymes. As a heat-resistant enzyme preparation, the ferulic acid esterase has great industrial production potential and economic value.
Description
Technical field
The present invention relates to the thermostability transformation of Aspergillus usamii (Aspergillus usamii) E001 bacterial strain feruloyl esterase A (AuFaeA) gene, the structure of sudden change feruloyl esterase A engineering bacteria and the high expression of recombination mutation feruloyl esterase A, belong to technical field of bioengineering.
Background technology
Feruloyl esterase (EC 3.1.1.73) is also known as Ferulic acid esterase, it is a subclass of carboxylic acid lytic enzyme, belong to extracellular enzyme, can interrupt between hemicellulose, crosslinked between hemicellulose and xylogen ester bond, be one of moiety of hemicellulose degrading enzymes system.Since Faulds in 1991 etc. isolate feruloyl esterase first, existing to be cloned more than 30 kinds of feruloyl esterases, Expression and purification, its primary biological function is the ester bond that in hydrolyzing plant cell walls, polysaccharide and forulic acid link, and discharges free monomer ferulic acid or ferulic acid dimers.But result of study shows, the thermostability of natural ferulaic acid esterase is generally poor, becomes the bottleneck that it applies in the numerous areas such as food-processing, feed (granulation), paper industry (association with pulp bleaching).
Because high-temperature biological engineering-environment limits the application of feruloyl esterase, so people strengthen the research to enzyme heat stability.Wish by improving enzyme operative temperature and increasing enzyme heat stability, thus enzyme functioning efficiency to be improved, to reach the object reducing industrial production cost.At present, the research of exploitation heat resistant type feruloyl esterase mainly comprises the super heat-resisting ferulic acid ester enzyme-producing bacteria of screening occurring in nature and utilizes protein engineering etc. to carry out directional transformation to enzyme molecule, relative to the blindness of sieve bacterium technology, the latter has stronger specific aim, is subject to the favor of Chinese scholars.Along with the fast development of genetic engineering technique, information biology etc., for molecular biological research opens new road.By using extensive high performance computer and related software thereof, in conjunction with genetic engineering means, utilize simulation test to carry out directional transformation to improve thermostability to enzyme molecule, thus expand the application surface of feruloyl esterase.
Summary of the invention
The object of this invention is to provide the transformation of a kind of feruloyl esterase A thermostability, sudden change feruloyl esterase A engineering bacteria builds and the method for the efficient heterogenous expression of sudden change feruloyl esterase A.
Technical scheme of the present invention: according to space structure design sudden change A126C and N152C deriving from A.usamii E001 feruloyl esterase A (AuFaeA), obtain sudden change feruloyl esterase A:AuFaeA
a126C-N152C, its mature peptide Gene A ufaeA
a126C-N152Csequence is SEQ ID NO:1, C126 and C152 can form disulfide linkage.
The described AuFaeA obtained by mature peptide genes encoding
a126C-N152Caminoacid sequence is SEQ ID NO:2.
The activity determination method of described restructuring feruloyl esterase A:
With Ferulic acid methylester (MFA) for substrate, high performance liquid phase (HPLC) analyzes the burst size of forulic acid.Concrete operations are: pipette 900 μ L MFA (1mM, pH 6.0) in 2mL EP pipe, 45 DEG C of insulation 10min, add the enzyme liquid that 100 μ L suitably dilute, add 400 μ L glacial acetic acid termination reactions after reaction 10min, high-efficient liquid phase analysis is carried out in mixing immediately.First to add 400 μ L glacial acetic acids in enzyme solution, then add substrate solution for contrast.Chromatographic condition: with methyl alcohol, 1% acetic acid one step gradient elution, in 10min, methanol concentration rises to 80% by 50%, flow velocity 1mL/min, column temperature 30 DEG C, determined wavelength 320nm, applied sample amount 20 μ L.According to peak area, find corresponding forulic acid amount from typical curve, thus it is alive to calculate enzyme.Enzyme unit definition alive: (45 DEG C, pH 6.0) per minute enzyme amount produced needed for 1 μm of ol forulic acid is a Ge Meihuo unit (U) under condition determination.
The design of described sudden change feruloyl esterase A gene, Cloning and Expression:
(1) amino-acid residue mutational site is selected: with the three-dimensional structure of aspergillus niger feruloyl esterase A (1UWC) for template, go out the three-dimensional structure of AuFaeA with Modeller 9.9 software simulation.The three-dimensional structure of Discovery Studio 3.0Client, PyMOL and Disulfide By Design software to simulation is utilized to analyze, and with gromacs-4.5 routine package, dynamics simulation is carried out to the three-dimensional structure before and after transformation, finally determine amino-acid residue mutational site.
(2) mutator gene AufaeA
a126C-N152Cand the structure of expression plasmid: according to applying for patent of invention (application number: 201210181372.X) nucleotide sequence and mutational site feature design mutant primer 126-F, 152-R:
126-F：5’-ATCCGGACTATTGCCTTACCGTGACA-3’，
152-R：5’-GTACAGACGGACGCAGTCATATGTCGC-3’，
The pUCm-T-AufaeA preserved with this laboratory is template, is that primer carries out first round PCR with 126-F, 152-R, obtains gene fragment AufaeA
126-152; Take pUCm-T-AufaeA as template, with first round PCR primer AufaeA
126-152for primer with to have applied in patent of invention (application number: 201210181372.X) that Auele Specific Primer FAE-F carries out second and takes turns large primer PCR, obtain gene fragment AufaeA
f-152; Take pUCm-T-AufaeA as template, take turns PCR primer AufaeA with second
f-152for primer with to have applied for that in patent of invention (application number: 201210181372.X), Auele Specific Primer FAE-R carries out third round large primer PCR.By third round PCR primer AufaeA
a126C-N152Cwith 1% agarose gel electrophoresis analysis, rubber tapping is reclaimed object band and is connected (pUCm-T-AufaeA with pUCm-T
a126C-N152C).Transform JM109, cut after qualification correctly through enzyme and serve Hai Shenggong order-checking.
(3) structure of recombinant expression plasmid: by pUCm-T-AufaeA correct for sequencing result
a126C-N152Call carry out double digestion with EcoR I and Not I with pPIC9K plasmid, the digestion products that rubber tapping is reclaimed connects under the effect of T4DNA ligase enzyme, obtains recombinant expression plasmid pPIC9K-AufaeA
a126C-N152C, and sequencing is carried out to recombinant expression plasmid (Fig. 1).
(4) GS115/AufaeA
a126C-N152Cthe mensuration of the structure of recon, expression and enzymatic property: with Sal I couple of pPIC9K-AufaeA
a126C-N152Ccarry out linearizing, carry out electricity according to Pichia anomala expression handbook and turn, screen, obtain the pichia spp recon GS115/AufaeA of high copy
a126C-N152C; Abduction delivering is carried out according to the normal process on handbook; Use SDS-PAGE testing goal molecular weight of albumen after fermentation liquor preliminary purification, and measure the temperature profile etc. of this enzyme.
Beneficial effect of the present invention: the invention provides a kind of novel easy method improving feruloyl esterase A thermostability by adding disulfide linkage, the sudden change feruloyl esterase A called after AuFaeA obtained after transformation
a126C-N152C, its corresponding gene is AufaeA
a126C-N152C, its thermostability is significantly improved, and has larger suitability for industrialized production and application potential and economic worth, also for the research of other feruloyl esterase is laid a good foundation.
Accompanying drawing explanation
Fig. 1: recombinant plasmid pPIC9K-AufaeA
a126C-N152Cstructure schematic diagram
Embodiment
Below in conjunction with specific embodiment, set forth working method of the present invention further.But these embodiments are only for describing the present invention in detail, and are not used in and limit the scope of the invention.
Embodiment 1 mutator gene AufaeA
a126C-N152Cand the structure of expression plasmid
Adopt large primer PCR technical construction fusion gene, be mainly divided into four steps: be that primer carries out first round PCR (94 DEG C of 5min with 126-F, 152-R; 2 circulations, 94 DEG C of 30s, 45 DEG C of 30s, 72 DEG C of 15s; 28 circulations, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 15s; 72 DEG C of 10min; 10 DEG C of preservations), obtain gene fragment AufaeA
126-152; Take pUCm-T-AufaeA as template, with first round PCR primer AufaeA
126-152for primer with to have applied in patent of invention (application number: 201210181372.X) that Auele Specific Primer FAE-F carries out second and takes turns large primer PCR (94 DEG C of 5min; 2 circulations, 94 DEG C of 30s, 45 DEG C of 30s, 72 DEG C of 30s; 28 circulations, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C, 30s; 72 DEG C of 10min; 10 DEG C of preservations), obtain gene fragment AufaeA
f-152; Take pUCm-T-AufaeA as template, take turns PCR primer AufaeA with second
f-152for primer with to have applied for that in patent of invention (application number: 201210181372.X), Auele Specific Primer FAE-R carries out third round large primer PCR (94 DEG C of 5min; 2 circulations, 94 DEG C of 30s, 45 DEG C of 30s, 72 DEG C of 60s; 28 circulations, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 60s; 72 DEG C of 10min; 10 DEG C of preservations).By third round PCR primer AufaeA
a126C-N152Cwith 1% agarose gel electrophoresis analysis, rubber tapping is reclaimed object band and is connected (pUCm-T-AufaeA with pUCm-T
a126C-N152C), transform JM109, cut after qualification correctly through enzyme and serve Hai Shenggong order-checking.
By pUCm-T-AufaeA correct for sequencing result
a126C-N152Call carry out double digestion with EcoR I and Not I with pPIC9K plasmid, the digestion products of recovery connects under the effect of T4DNA ligase enzyme, obtains recombinant expression plasmid pPIC9K-AufaeA
a126C-N152C, and sequencing is carried out to recombinant expression plasmid.
Embodiment 2GS115/AufaeA
a126C-N152Cthe mensuration of the structure of recon, expression and character
With Sal I couple of pPIC9K-AufaeA
a126C-N152Ccarry out linearizing, carry out electricity according to Pichia anomala expression handbook and transform, screen, obtain the pichia spp recon GS115/AufaeA of high copy
a126C-N152C.This project bacterium 1.0% methanol induction 72h.Centrifuged supernatant is restructuring feruloyl esterase A crude enzyme liquid, and be detected as single band through SDS-PAGE, recombinate after transformation AuFaeA
a126C-N152Coptimal reactive temperature be 50 DEG C, comparatively protoenzyme improves 5 DEG C, and the transformation period at 60 DEG C reaches 40min, and thermostability comparatively protoenzyme there has also been and significantly improves.
Claims (2)
1. the improved feruloyl esterase A Gene A ufaeA being derived from Aspergillus usamii (Aspergillus usamii) E001
a126C-N152C, the Nucleotide of its correspondence and protein sequence are respectively SEQ ID NO:1 and SEQ ID NO:2.
2. to suddenly change the construction process of feruloyl esterase A engineering bacteria and expression method:
(1) Mutated residues site is selected: with the three-dimensional structure of aspergillus niger feruloyl esterase A (1UWC) for template, the three-dimensional structure of AuFaeA is gone out with Modeller 9.9 software simulation, Discovery Studio 3.0 Client, PyMOL and Disulfide By Design software is utilized to analyze the three-dimensional structure simulated, and with gromacs-4.5 routine package, dynamics simulation is carried out to the three-dimensional structure before and after transformation, finally determine amino-acid residue mutational site;
(2) mutator gene AufaeA
a126C-N152Cclone and the structure of expression plasmid: according to applying for patent of invention (application number: 201210181372.X) nucleotide sequence and mutational site feature design mutant primer 126-F, 152-R:
126-F：5’-ATCCGGACTATTGCCTTACCGTGACA-3’，
152-R：5’-GTACAGACGGACGCAGTCATATGTCGC-3’，
The pUCm-T-AufaeA preserved with this laboratory is template, is that primer carries out first round PCR with 126-F, 152-R, obtains gene fragment AufaeA
126-152; Take pUCm-T-AufaeA as template, with first round PCR primer AufaeA
126-152for primer with to have applied in patent of invention (application number: 201210181372.X) that Auele Specific Primer FAE-F carries out second and takes turns large primer PCR, obtain gene fragment AufaeA
f-152; Take pUCm-T-AufaeA as template, take turns PCR primer AufaeA with second
f-152for primer with to have applied for that in patent of invention (application number: 201210181372.X), Auele Specific Primer FAE-R carries out third round large primer PCR, by third round PCR primer AufaeA
a126C-N152Cwith 1% agarose gel electrophoresis analysis, rubber tapping is reclaimed object band and is connected (pUCm-T-AufaeA with pUCm-T
a126C-N152C), transform JM109, cut after qualification correctly through enzyme and serve Hai Shenggong order-checking;
(3) structure of recombinant expression plasmid: by pUCm-T-AufaeA correct for sequencing result
a126C-N152Call carry out double digestion with EcoR I and Not I with pPIC9K plasmid, the digestion products that rubber tapping is reclaimed connects under the effect of T4DNA ligase enzyme, obtains recombinant expression plasmid pPIC9K-AufaeA
a126C-N152C, and sequencing is carried out to recombinant expression plasmid (Fig. 1);
(4) GS115/AufaeA
a126C-N152Cthe mensuration of the structure of recon, expression and enzymatic property: with Sal I couple of pPIC9K-AufaeA
a126C-N152Ccarry out linearizing, carry out electricity according to Pichia anomala expression handbook and turn, screen, obtain the pichia spp recon GS115/AufaeA of high copy
a126C-N152C; Abduction delivering is carried out, with 1.0% methanol induction 72h according to the normal process on handbook; Centrifuged supernatant is recombination mutation feruloyl esterase A crude enzyme liquid, is detected as single band through SDS-PAGE, and this restructuring feruloyl esterase optimal reactive temperature is 50 DEG C, and comparatively protoenzyme improves 5 DEG C, and the transformation period at 60 DEG C reaches 40min.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105950581A (en) * | 2016-06-21 | 2016-09-21 | 浙江科技学院 | Disulfide-bond-introduced omega-aminotransferase mutant and application thereof |
| CN106479988A (en) * | 2016-11-08 | 2017-03-08 | 江南大学 | A kind of enzyme activity and stability-enhanced formic dehydrogenase mutant and its construction method |
| CN108841801A (en) * | 2018-05-29 | 2018-11-20 | 江南大学 | A kind of method of amino acid residue relevant to enzyme activity in screening enzyme |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220299A (en) * | 2011-04-22 | 2011-10-19 | 中国科学院成都生物研究所 | Feruloyl esterase A mutant and purpose thereof |
| CN102703403A (en) * | 2012-06-05 | 2012-10-03 | 江南大学 | Cloning of feruloyl esterase gene (Fae-A) and preparation of recombinant enzyme |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220299A (en) * | 2011-04-22 | 2011-10-19 | 中国科学院成都生物研究所 | Feruloyl esterase A mutant and purpose thereof |
| CN102703403A (en) * | 2012-06-05 | 2012-10-03 | 江南大学 | Cloning of feruloyl esterase gene (Fae-A) and preparation of recombinant enzyme |
Non-Patent Citations (1)
| Title |
|---|
| SHUAI-BING ZHANG ET AL.: "Multiple amino acid substitutions significantly improve the thermostability of feruloyl esterase A from Aspergillus niger", 《BIORESOURCE TECHNOLOGY》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105950581A (en) * | 2016-06-21 | 2016-09-21 | 浙江科技学院 | Disulfide-bond-introduced omega-aminotransferase mutant and application thereof |
| CN106479988A (en) * | 2016-11-08 | 2017-03-08 | 江南大学 | A kind of enzyme activity and stability-enhanced formic dehydrogenase mutant and its construction method |
| CN106479988B (en) * | 2016-11-08 | 2019-08-06 | 江南大学 | A kind of enzyme activity and stability-enhanced formic dehydrogenase mutant and its construction method |
| CN108841801A (en) * | 2018-05-29 | 2018-11-20 | 江南大学 | A kind of method of amino acid residue relevant to enzyme activity in screening enzyme |
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