CN103642777A - Method of improving thermal stability of aspergillus oryzae xylanase - Google Patents
Method of improving thermal stability of aspergillus oryzae xylanase Download PDFInfo
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- CN103642777A CN103642777A CN201310666413.9A CN201310666413A CN103642777A CN 103642777 A CN103642777 A CN 103642777A CN 201310666413 A CN201310666413 A CN 201310666413A CN 103642777 A CN103642777 A CN 103642777A
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- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
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- C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
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Abstract
The invention discloses a novel method for carrying out directed modification on normal-temperature xylanase by utilizing a computer technology and a bioinformatics method, which constructs mutant xylanase engineering bacteria with efficient expression ability by applying a genetic engineering technology. A xylanase mutant is obtained by carrying out directed modification of thermal stability on the normal-temperature xylanase from an aspergillus oryzae CICC40186 bacterial strain, wherein thermal stability of the xylanase mutant is improved to a certain extent, and catalytic activity of the xylanase mutant is not changed obviously in comparison with proenzyme. As a heat-resisting enzyme preparation, the xylanase has great industrial production potential and economic value.
Description
Technical field
The present invention relates to be derived from the directional transformation of normal temperature zytase (AoXyn11A) thermostability of aspergillus oryzae (Aspergillus oryzae) CICC40186 bacterial strain, the engineering bacteria structure of the xylanase mutant of expression Heat stability is good and the method for the high efficient expression of recombined xylanase mutant, belong to technical field of bioengineering.
Background technology
Zytase (EC3.2.1.8) is the important industrial enzymes of a class.It is mainly to act on xylan backbone, cuts randomly the wood sugar glycosidic bond of xylan inside, and hydrolysate is mainly the wood oligose of different polymerization degree and a small amount of wood sugar, is the enzyme of most critical in xylanolytic enzyme system.In recent years, because zytase is in fodder industry, food-processing and the industry such as brewage and have potential industrial application and economic worth, especially in the preparation of xylo-oligosaccharide, and also increasing about its research report.In the enzyme process preparation of xylo-oligosaccharide, suitably improve the speed that temperature of reaction can add fast response, can also reduce the risk of microbiological contamination simultaneously.Therefore, the enzyme of Heat stability is good also has more advantage in the preparation of xylo-oligosaccharide.At present, the research of exploitation heat resistant type zytase mainly comprises from hot environment that screening obtains and resistant to elevated temperaturesly can produce the microorganism strains of zytase and utilize genetic engineering technique to carry out directional transformation to enzyme molecule, blindness with respect to sieve bacterium technology, the latter has stronger specific aim, is more subject to the favor of Chinese scholars.
Based on to the structure of zytase catalysis region and property analysis, can be classified as different families, wherein in the majority with the glycoside hydrolase 10 He of family 11 families.Due to the zytase molecular weight of G/11 family, and relatively simple for structure, be more suitable for the molecular model as theoretical investigation, can be for the research of zytase catalysis modular system and protein molecule folding mechanism etc. under extreme condition.The fast development of genetic engineering technique, information biology and computer science, for new prospect has been opened up in molecular biological research.We can use extensive high performance computer and related software thereof, in conjunction with genetic engineering means, utilize simulation test to carry out directional transformation to improve its thermostability to enzyme molecule, accelerate the application process of zytase in various fields.
Summary of the invention
The object of this invention is to provide a kind of novel utilize computer technology and bioinformatic analysis and the thermostability of normal temperature zytase is carried out to the method for rationality transformation, construct the xylanase mutant engineering bacteria that can express Heat stability is good the high efficient expression of realizing recombined xylanase mutant.The sudden change zytase called after AoXyn11A being obtained by directional transformation
n9H, its corresponding gene is Aoxyn11A
n9H, its thermostability improves, and the catalytic activity of enzyme does not obviously change compared with protoenzyme, has larger industrial application potentiality and economic worth.
Technical scheme of the present invention: the zytase of a kind of A.oryzae of deriving from CICC40186 (AoXyn11A) is carried out to design and rational under area of computer aided, obtain a kind of xylanase mutant: AoXyn11A
n9H, the DNA that it is complete and aminoacid sequence are respectively SEQ ID NO:1 and SEQ ID NO:2.
The zytase called after AoXyn11A of the described 11 family that is derived from A.oryzae CICC40186, its corresponding unnamed gene is Aoxyn11A (GenBank accession NO.JQ326257); The super heat resistant xylanase of the template Wei Tong family adopting, by its called after EvXyn11
tS, gene order is this laboratory synthetic gene Syxyn11 (GenBank accession NO.JX459567).
The activity determination method of described recombined xylanase:
In 25mL tool plug test tube A and B, respectively add with pH5.5, citric acid-Na
2hPO
4beech xylan (Sigma, USA) the solution 2.4mL that the mass concentration of damping fluid preparation is 0.5%, 50 ℃ of preheating 10min add the enzyme liquid of the suitable extension rate of 0.1mL, 50 ℃ of accurate response 15min in A pipe; Respectively add immediately 2.5mL3,5-dinitrosalicylic acid (DNS) reagent is added 0.1mL enzyme liquid in B pipe again, and A and B pipe all boil 7min; After cooling, respectively add deionized water 5mL, shake up; 540nm sentences B pipe for blank mensuration A pipe absorbance, and finds corresponding reducing sugar (in wood sugar) content and be converted to unit of enzyme activity from wood sugar typical curve.Unit of enzyme activity's definition: under this condition determination, produce the required enzyme amount of 1 μ mol reducing sugar with per minute and be defined as 1 xylanase activity unit (IU).
Design and the expression method of described heat resistant xylanase mutant:
(1) simulation of the space structure of AoXyn11A: login protein structure database Protein data bank (http://rcsb.org), through BLAST comparison, finds the sequence with AoXyn11A with high homology.This experimental selection has the EvXyn11 of very high thermostability
tS(Protein Data Bank accession number is 2VUL) is template, and both homologys are 66.49%, and using SWISS-MODEL server on-line Full is AoXyn11A virtual space structure.Use software Verify_3D to evaluate and optimize the structural models of homology modeling, obtain the higher space-filling model of accuracy.
(2) design and rational of the xylanase mutant that thermostability improves: by sequential analysis and prediction, N end the 9th amino acids of AoXyn11A is carried out to rite-directed mutagenesis, original l-asparagine is replaced with to Histidine, structurally introduce imidazole ring.Recycling molecular dynamics simulation software package carries out molecular dynamics simulation to zytase protoenzyme and mutant.
(3) containing mutator gene Aoxyn11A
n9Hthe structure of recombinant expression plasmid: design 2 primers according to the gene order (GenBank accession No.JQ326257) of above-mentioned analytical results and the upper Aoxyn11A of GenBank:
Xyn11-F
n9H: 5 '-
cTCGAGaAAAGATCCACACCCAGTAGCACGGGCTATCACAATGGCTA-3 ', contains Xho I restriction enzyme site;
Xyn11-R:5 '-
gCGGCCGCtCAATAAACAGTGATAGCAG-3 ', contains Not I restriction enzyme site:
The plasmid that contains Aoxyn11A of take is template, Xyn11-F
n9Hwith Xyn11-R be that primer carries out PCR, reaction conditions is: 94 ℃ of 5min; 94 ℃ of 30s, 51 ℃ of 30s, 72 ℃ of 58s, 30 circulations; 72 ℃ of 10min; This PCR product is carried out to nucleic acid electrophoresis and rubber tapping recovery, then connect the checking of pUCm-T carrier, order-checking, correct pUCm-T-Aoxyn11A checks order
n9Hwith carrier pPIC9K
m(patent applied for, the patent No. 201110410391.0) plasmid all carries out double digestion with Xho I and Not I, and the enzyme of recovery is cut product and connected under the effect of T4DNA ligase enzyme, obtains recombinant plasmid pPIC9K
m-Aoxyn11A
n9H, and recombinant expression plasmid is carried out to sequencing.
(4) GS115/Aoxyn11A
n9Hthe structure of recon, expression and enzyme assay: use Sac I to pPIC9K
m-Aoxyn11A
n9Hcarry out linearizing, according to Pichia anomala expression handbook, carry out electricity and transform, screen, obtain the pichia spp recon GS115/Aoxyn11A of high copy
n9H; 1.0% methanol induction 96h for this project bacterium, centrifuged supernatant is recombination mutation zytase crude enzyme liquid, and the zymologic property of this enzyme and protoenzyme is compared: in the situation that 0.5% beech xylan substrate, reaction times be 15min, reAoXyn11A
n9Hoptimum temperuture by 55 ℃ of protoenzyme, rise to 60 ℃; Under 60 ℃ of pre-treatment, reAoXyn11A
n9Htransformation period by the 1min of protoenzyme, extend to 18min.
Beneficial effect of the present invention: the present invention has carried out directional transformation to aspergillus oryzae normal temperature zytase, keeping, on the basis of former enzymatic activity, having improved the thermostability of this enzyme, makes it have better prospects for commercial application.
Accompanying drawing explanation
Fig. 1: recombinant plasmid pPIC9K
m-Aoxyn11A
n9Hstructure schematic diagram
Embodiment
Below in conjunction with specific embodiment, further set forth working method of the present invention.But these embodiment only, for describing the present invention in detail, limit the scope of the invention and be not used in.
The simulation of embodiment 1AoXyn11A and mutant space structure thereof
Login protein structure database Protein data bank (http://rcsb.org), through BLAST comparison, finds the sequence with AoXyn11A with high homology.The EvXyn11TS (Protein Data Bank accession number is 2VUL) that this experimental selection has very high thermostability is template, both homologys are 66.49%, use that SWISS-MODEL server is on-line Full simulates space structure for AoXyn11A and mutant thereof.Use software Verify_3D to evaluate and optimize the structural models of homology modeling, obtain the higher space-filling model of accuracy.
The molecular dynamics simulation of embodiment 2AoXyn11A and mutant thereof
Respectively to the AoXyn11A simulating and AoXyn11A
n9Hspace structure carry out molecular dynamics simulation.Mainly comprise the following steps:
First we carry out pre-treatment to space structure, use the pdb2gmx order in GROMACS to add the hydrogen atom of all disappearances, and export a .gro file that comprises all atom information.The form of this order is:
pdb2gmx-f11bs.pdb-o11bs.gro-p11bs.top-ignh-ter
It is AoXyn11A and AoXyn11A that second step utilizes editconf and genbox order in GROMACS
n9Hspace structure add the suitable aqueous solution and simulate their motions in the aqueous solution.Wherein solvent adopts TIP3P water model, for the system of simulation, adds the water molecule layer of 1.5am in solute periphery.Its detailed process is first to use the size of editconf command definition box, then with genbox order, reads in the structured file of GROMACS, and reads in the size of water box, and output file comprises minute subfile and water box.Genbox changes original top file simultaneously, makes it contain water molecules.The form of this order is:
Editconf-f1lbs.gro-o1lbs_box.gro-bt?cubic-d1.5
Genbox-cp1lbs_box.gro-cs-p1lbs.top-o1lbs_water.gro
The electric charge of cation equilibrium system for the 3rd step, command format is:
grompp-f?em.mdp-c1lbs_water_ion.gro-p1lbs.top-o?minimize_water.tpr
genion-s?minimize_water.tpr-o1lbs_water_ion.gro-p1lbs.top-pname?Na+-np10-random-g?trp_ion.log
genion-s?minimize_water.tpr-o1lbs_water_ion.gro-p1lbs.top-nname?CL--nn1-random-g?trp_ion.log
And need manually the atom number in the 1lbs_water_ion.gro obtaining and 1lbs.top file to be modified, match with the metal ion adding.
The 4th step utilizes respectively grompp and two orders of mdmn to carry out energy minimization processing.First retrain solute, by method of steepest descent, optimize 800 steps, then optimize 1200 steps by method of conjugate gradient.Then go to carry out 800 step method of steepest descent optimizations after constraint, 1200 step method of conjugate gradient are optimized again.Command format is:
grompp-f?emstl.mdp-c1lbs_water_ion.gro-p1lbs.top-o?minimize_water.tpr
mdrun-s?minimize_water.tpr-o?minimize_water.trr-c?minimize_water.gro-e?minimize_water.edr-g?minimize_water.log
grompp-f?emst.mdp-c?minimize_water.gro-p1lbs.top-o?minimize_water.tpr
mdrun-s?minimize_water.tpr-o?minimize_water.trr-c?minimize_water2.gro-e?minimize_water.edr-g?minimize_water.log
The 5th step molecular dynamics simulation is divided into two steps, first carries out the MD simulation of the constraint solute of 20ps, and now analog temperature is progressively elevated to 300K from OK; Then carry out simulating without constraint constant temperature MD of 500ps.Command format is:
grompp-f?pr.mdp-c?minimize_water2.gro-p1lbs.top-o?minimize_water1.tpr
mdrun-s?minimize_water1.tpr-o?minimize_water1.trr-c?minimize_water1.gro-e?minimize_water1.edr-g?minimize_water1.log
grompp-f?full.mdp-c?minimize_water1.gro-p1lbs.top-o?minimize_water2.tpr
mdrun-s?minimize_water2.tpr-o?minimize_water2.trr-c?minimize_water3.gro-e?minimize_water2.edr-g?minimize_water2.log
Finally obtain its total energy, RMSD value and each amino acid whose B-factor value.Command format is:
g_rms-s?minimize_water1.tpr-f?minimize_water1.trr-o?rms.xvg
xmgrace-nxy?rms.xvg
g_rmsf-s?minimize_water2.tpr-f?minimize_water2.trr-b400-e600-o?rmsf.xvg-oq1.pdb
xmgrace-nxy?rmsf.xvg
g_energy-f?minimize_water2-o?energy.xvg
xmgrace-nxy?energy.xvg
Embodiment 3GS115/Aoxyn11A
n9Hthe structure of recon, expression and enzyme assay
With Sac I to pPIC9K
m-Aoxyn11A
n9Hcarry out linearizing, according to Pichia anomala expression handbook, carry out electricity and transform, screen, obtain the pichia spp recon GS115/Aoxyn11A of high copy
n9H; 1.0% methanol induction 96h for this project bacterium, centrifuged supernatant is recombination mutation zytase crude enzyme liquid, and the zymologic property of this enzyme and protoenzyme is compared: in the situation that 0.5% beech xylan substrate, reaction times be 15min, AoXyn11A
n9Hoptimum temperuture by 55 ℃ of protoenzyme, rise to 60 ℃, its transformation period at 60 ℃, the 1min by protoenzyme extended to 18min.
Claims (2)
1. the xylanase mutant AoXyn11A that a thermostability improves
n9H, its Nucleotide and aminoacid sequence are respectively SEQ ID NO:1 and SEQ ID NO:2.
2. the design of heat resistant xylanase mutant and expression method:
(1) simulation of the space structure of AoXyn11A: login protein structure database, through BLAST comparison, finds the sequence with AoXyn11A with high homology; This experimental selection has the EvXyn11 of very high thermostability
tS(Protein Data Bank accession number is 2VUL) is template, and both homologys are 66.49%, and using SWISS-MODEL server on-line Full is AoXyn11A virtual space structure; Use software Verify_3D to evaluate and optimize the structural models of homology modeling, obtain the higher space-filling model of accuracy;
(2) design and rational of the xylanase mutant that thermostability improves: by sequential analysis and prediction, N end the 9th amino acids of AoXyn11A is carried out to rite-directed mutagenesis, original l-asparagine is replaced with to Histidine, structurally introduce imidazole ring; Recycling molecular dynamics simulation software package carries out molecular dynamics simulation to zytase protoenzyme and mutant;
(3) containing mutator gene Aoxyn11A
n9Hthe structure of recombinant expression plasmid: design 2 primers according to the gene order (GenBank accession No.JQ326257) of above-mentioned analytical results and the upper Aoxyn11A of GenBank:
Xyn11-F
n9H: 5 '-
cTCGAGaAAAGATCCACACCCAGTAGCACGGGCTATCACAATGGCTA-3 ', contains Xho I restriction enzyme site;
Xyn11-R:5 '-
gCGGCCGCtCAATAAACAGTGATAGCAG-3 ', contains Not I restriction enzyme site;
The plasmid that contains Aoxyn11A of take is template, Xyn11-F
n9Hwith Xyn11-R be that primer carries out PCR, reaction conditions is: 94 ℃ of 5min; 94 ℃ of 30s, 51 ℃ of 30s, 72 ℃ of 58s, 30 circulations; 72 ℃ of 10min; This PCR product is carried out to nucleic acid electrophoresis and rubber tapping recovery, then connect the checking of pUCm-T carrier, order-checking, correct pUCm-T-Aoxyn11A checks order
n9Hwith carrier pPIC9K
m(patent applied for, the patent No. 201110410391.0) plasmid all carries out double digestion with Xho I and Not I, and the enzyme of recovery is cut product and connected under the effect of T4DNA ligase enzyme, obtains recombinant plasmid pPIC9K
m-Aoxyn11A
n9H, and recombinant expression plasmid is carried out to sequencing;
(4) GS115/Aoxyn11A
n9Hthe structure of recon, expression and enzyme assay: use Sac I to pPIC9K
m-Aoxyn11A
n9Hcarry out linearizing, according to Pichia anomala expression handbook, carry out electricity and transform, screen, obtain the pichia spp recon GS115/Aoxyn11A of high copy
n9H; 1.0% methanol induction 96h for this project bacterium, centrifuged supernatant is recombination mutation zytase crude enzyme liquid, and the zymologic property of this enzyme and protoenzyme is compared: in the situation that 0.5% beech xylan substrate, reaction times be 15min, reAoXyn11A
n9Hoptimum temperuture by 55 ℃ of protoenzyme, rise to 60 ℃; Under 60 ℃ of thermal treatments, reAoXyn11A
n9Htransformation period by the 1min of protoenzyme, extend to 18min.
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Cited By (8)
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|---|---|---|---|---|
| CN104388412A (en) * | 2014-12-10 | 2015-03-04 | 江南大学 | Method for improving heat stability of AoXyn11A through N-terminal replacement |
| CN104450652A (en) * | 2014-12-10 | 2015-03-25 | 江南大学 | Method for improving catalytic activity of aspergillususamiimannase (AuMan5A) |
| CN105441404A (en) * | 2015-12-08 | 2016-03-30 | 浙江科技学院 | Omega-transaminase mutant and encoding gene and preparation method thereof |
| CN105779491A (en) * | 2014-12-25 | 2016-07-20 | 中国石油天然气股份有限公司 | Thermophilic beta-1, 4-xylanase-His fusion protein, preparation method and application, and construction of genetic engineering bacteria thereof |
| EP3216864A4 (en) * | 2014-11-05 | 2018-05-02 | Toray Industries, Inc. | Endoxylanase mutant, enzyme composition for biomass decomposition, and method for producing sugar solution |
| CN110607291A (en) * | 2018-06-14 | 2019-12-24 | 青岛蔚蓝生物集团有限公司 | Heat-resistant xylanase mutant |
| CN115851670A (en) * | 2022-11-30 | 2023-03-28 | 山东龙昌动物保健品有限公司 | Xylanase mutant, compound preparation of xylanase mutant and eucommia ulmoides leaf extract and application of xylanase mutant and eucommia ulmoides leaf extract in feed additive |
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Cited By (13)
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|---|---|---|---|---|
| US10435728B2 (en) | 2014-11-05 | 2019-10-08 | Toray Industries, Inc. | Endoxylanase mutant, enzyme composition for biomass decomposition, and method of producing sugar solution |
| AU2015344324B2 (en) * | 2014-11-05 | 2021-09-30 | Toray Industries, Inc. | Endoxylanase mutant, enzyme composition for biomass decomposition, and method for producing sugar solution |
| EP3216864A4 (en) * | 2014-11-05 | 2018-05-02 | Toray Industries, Inc. | Endoxylanase mutant, enzyme composition for biomass decomposition, and method for producing sugar solution |
| CN104450652A (en) * | 2014-12-10 | 2015-03-25 | 江南大学 | Method for improving catalytic activity of aspergillususamiimannase (AuMan5A) |
| CN104388412A (en) * | 2014-12-10 | 2015-03-04 | 江南大学 | Method for improving heat stability of AoXyn11A through N-terminal replacement |
| CN105779491A (en) * | 2014-12-25 | 2016-07-20 | 中国石油天然气股份有限公司 | Thermophilic beta-1, 4-xylanase-His fusion protein, preparation method and application, and construction of genetic engineering bacteria thereof |
| CN105441404B (en) * | 2015-12-08 | 2018-09-11 | 浙江科技学院 | ω-transaminase mutant and its encoding gene and preparation method |
| CN105441404A (en) * | 2015-12-08 | 2016-03-30 | 浙江科技学院 | Omega-transaminase mutant and encoding gene and preparation method thereof |
| CN110607291A (en) * | 2018-06-14 | 2019-12-24 | 青岛蔚蓝生物集团有限公司 | Heat-resistant xylanase mutant |
| CN110607291B (en) * | 2018-06-14 | 2022-05-31 | 青岛蔚蓝生物集团有限公司 | Heat-resistant xylanase mutant |
| CN114921440B (en) * | 2022-05-16 | 2023-05-05 | 北京昕大洋科技发展有限公司 | Xylanase mutant and application thereof |
| CN115851670A (en) * | 2022-11-30 | 2023-03-28 | 山东龙昌动物保健品有限公司 | Xylanase mutant, compound preparation of xylanase mutant and eucommia ulmoides leaf extract and application of xylanase mutant and eucommia ulmoides leaf extract in feed additive |
| CN115851670B (en) * | 2022-11-30 | 2024-04-12 | 山东龙昌动物保健品股份有限公司 | Xylanase mutant, compound preparation of xylanase mutant and eucommia ulmoides leaf extract and application of xylanase mutant and eucommia ulmoides leaf extract in feed additive |
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