CN104388451A - Synthesis of thermostable xylanase gene (Xyl11M) and expression of gene in pichia pastoris - Google Patents
Synthesis of thermostable xylanase gene (Xyl11M) and expression of gene in pichia pastoris Download PDFInfo
- Publication number
- CN104388451A CN104388451A CN201410758941.1A CN201410758941A CN104388451A CN 104388451 A CN104388451 A CN 104388451A CN 201410758941 A CN201410758941 A CN 201410758941A CN 104388451 A CN104388451 A CN 104388451A
- Authority
- CN
- China
- Prior art keywords
- xyl11
- gene
- expression
- activity
- enzyme
- 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
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses synthesis of a thermostable xylanase gene (Xyl11M) and expression of the gene in pichia pastoris, and relates to a method for expression, activity determination and enzymatic property analysis of the thermostable beta-1 4-endoxylanase gene (Xyl11M) in the pichia pastoris. The thermostable xylanase gene is named as Xyl11M and has a nucleotide sequence of SEQ ID NO:1 and an amino acid sequence of SEQ ID NO:2, and coded protein is Xyl11M; the activity of prepared recombinase is 157 U/mL; the optimal reaction temperature is 70 DEG C and is kept for 60 minutes, and the activity of residual enzyme is 98% which shows that the thermostability of the enzyme is very good; the optimal pH value is 6.0 and the enzyme is relatively stable when the pH value is 5.0-8.0; the influence of metal ions on the enzyme activity is relatively small. As a thermostable enzyme preparation, the xylanase has relatively high industrialized application potentials and economic values.
Description
Technical field
The present invention relates to a kind of heat resistant xylanase (Xyl11
m) synthetic of gene and the expression in pichia spp thereof, belong to technical field of bioengineering.
Background technology
Inscribe β-Isosorbide-5-Nitrae-D-zytase (endo-β-Isosorbide-5-Nitrae-D-xylanase, EC 3.2.1.8) is called for short zytase.It is from the internal random cutting β-Isosorbide-5-Nitrae glycosidic link of xylan backbone, and xylan degrading being become oligomeric xylose, xylo-bioses and a small amount of wood sugar, is effectively utilize the indispensable biological catalyst of hemicellulose class resource.Based on sequence analysis and the hydrophobic cluster analysis of enzyme catalysis territory primary structure, most zytase ownership glycoside hydrolase 10 family and 11 families.Different from other family, 11 families are only containing zytase, the feature such as (<30kDa) and alkaline iso-electric point that has that substrate specificity is high, molecular weight is low, the single catalytic domain that space structure is formed primarily of two beta-pleated sheets and an alpha-helix, partly holds shape in the right hand.Zytase has important using value at industrial circles such as papermaking, food, feed, weavings.Because the technique such as association with pulp bleaching, feed granulating all needs comparatively high temps, therefore the research and development of heat resistant xylanase receives the favor of numerous investigator.
Found the microorganism of many Neng Chan 11 families heat resistant xylanase at present, wherein many heat resistant xylanase genes have been cloned and have expressed in suitable host.According to the Preference of codon, host cell can with different velocity composite protein.The tRNA content that in the gene of coded protein, some codon is corresponding is few (namely belonging to rare codon), so resultant quantity is less, host controls the resultant velocity of protein with this.Therefore according to the Preference of codon, the codon optimized expression amount that effectively can improve foreign protein is carried out to foreign protein encoding gene.Pichia spp (Pichia pastoris) expression system is the eukaryotic expression system that development in recent years is got up, its not only have intestinal bacteria breeding fast, be easy to cultivation, inheritance stability, simple to operate and be easy to the procaryotic features such as suitability for industrialized production, also there is eukaryotic gene expression regulation and protein modified function, as glycosylation, protein phosphorylation etc., avoid product and form inclusion body, the problems such as active reduction.Based on above reason, pichia spp is one of good system of expressed xylanase.
Summary of the invention
The object of this invention is to provide and a kind ofly derive from the thermophilic heat-resisting β-Isosorbide-5-Nitrae-endo-xylanase (Xyl11 splitting spore bacterium
m) heterogenous expression, determination of activity and characterization analysis method, be high expression and the industrialization production based theoretical of this enzyme.
Technical scheme of the present invention: carry out sequence optimisation according to pichia spp codon preference by being derived from the thermophilic heat-resisting β-Isosorbide-5-Nitrae-endo-xylanase xyl11 catalytic domain splitting spore bacterium, after optimizing, unnamed gene is xyl11
m, its nucleotides sequence is classified as SEQ ID NO:1, and the protein of coding is Xyl11
m, its aminoacid sequence is SEQ ID NO:2; This optimized gene is carried out secreting, expressing in pichia spp (Pichia pastoris) GS115, and determination of activity and characterization analysis are carried out to expression product.
Described xyl11 is that one derives from the thermophilic heat resistant xylanase gene (GenBank accession number AY795559) splitting spore bacterium (Thermobifida fusca NTU22), its thermostability is very good, has the ideal behavior being suitable for industrial application.
The activity determination method of described recombined xylanase:
In 25mL tool plug test tube A and B, respectively add with pH 6.0, citric acid-Na
2hPO
4the mass concentration of buffer is the birch xylan solution 2.4mL of 0.5%, and 50 DEG C of preheating 10min add the enzyme liquid that 0.1mL suitably dilutes, 50 DEG C of accurate response 15min in A pipe; Respectively add 2.5mL 3,5-dinitrosalicylic acid (DNS) reagent immediately, add the enzyme liquid of 0.1mL deactivation in B pipe again, A and B pipe all boils 7min; Respectively add deionized water 5mL after cooling, shake up; 540nm sentences B pipe for blank mensuration A pipe absorbance, and finds corresponding reducing sugar (in wood sugar) content from xylose standard curve and be converted to unit of enzyme activity.Unit of enzyme activity defines: under this condition determination, be defined as 1 unit of xylanase activity (U) with the per minute enzyme amount produced needed for 1 μm of ol reducing sugar.
Described xyl11
mheterogenous expression, determination of activity and analysis:
(1) recombinant plasmid pUCm-T-xyl11
mstructure: contrast pichia spp codon preference table is optimized deriving from the thermophilic rare codon split in the heat resistant xylanase catalytic domain gene of spore bacterium, and adding EcoR I, Not I site respectively at its catalytic domain gene two ends, this unnamed gene is xyl11
m, synthetic rear clone is to pUCm-T plasmid, and Transformed E .coli JM109, blue hickie screening positive transformant, serves Hai Shenggong order-checking, obtain recombinant plasmid pUCm-T-xyl11
m.
(2) structure of recombinant expression plasmid: by recombinant plasmid pUCm-T-xyl11 obtained above
mcarry out EcoR I and Not I double digestion, rubber tapping is reclaimed product and is connected with the expression plasmid pPIC9K through same double digestion, Transformed E .coli DH5 α competent cell, serves Hai Shenggong order-checking, obtain recombinant plasmid pPIC9K-xyl11 after PCR Screening and Identification
m(Fig. 1).
(3) GS115/xyl11
mthe mensuration of the structure of recon, expression and enzymic activity: check order correct pPIC9K-xyl11
mplasmid, through Sal I linearizing, carries out electricity according to Pichia anomala expression handbook and turns, screens, obtain the recon GS115/xyl11 of high copy
m; Abduction delivering is carried out according to the normal process on handbook; Recombined xylanase is measured active by the DNS method improved.
Described recombined xylanase characterization analysis:
(1) optimal reactive temperature of enzyme and thermostability: (50 ~ 80 DEG C) measure enzymic activity at different temperatures, with enzyme activity soprano for 100%; Be incubated 0 ~ 60min under enzyme liquid being placed in differing temps, measure residual enzymic activities, with the enzymic activity of untreated enzyme liquid (0min) for 100% by standard method.When remnant enzyme activity reaches more than 85%, be namely defined as stable.
(2) optimal pH of enzyme and pH stability: with different pH value (pH 3.0 ~ 9.0), 0.1mol/L citric acid-Na
2hPO
40.5% xylan solution of buffer, measures the activity of recombined xylanase, with enzyme activity soprano for 100% by standard method.Enzyme liquid is placed in respectively different pH buffer (pH 3.0 ~ 8.0 citric acid-Na
2hPO
4with pH 8.5-9.0Tris-HCl) in, at 40 DEG C of insulation 60min, measure residual enzymic activities, with the enzymic activity of untreated enzyme liquid for 100%.When remnant enzyme activity reaches more than 85%, be namely defined as stable.
(3) metal ion is on the impact of enzymic activity: be incubated 1h in 40 DEG C after being mixed with different metal ion or EDTA solution (final concentration is 2.0 mmol/L) by enzyme liquid and measure residual enzymic activities.100% is counted with the enzymic activity not adding metal ion and EDTA.
Beneficial effect of the present invention: the invention provides and a kind ofly derive from the thermophilic heat-resisting β-Isosorbide-5-Nitrae-endo-xylanase (Xyl11 splitting spore bacterium
m) heterogenous expression, determination of activity and characterization analysis method, its recombinase optimal reactive temperature is 70 DEG C, 60min is incubated at 70 DEG C, remnant enzyme activity is 98%, show that this enzyme heat stability is very good, have the ideal behavior being suitable for industrial application, the present invention is that theoretical basis has been established in the industrialization production of this enzyme, there are larger industrial applications potentiality and economic worth, also for theoretical basis has been established in the research of other heat resistant xylanase.
Accompanying drawing explanation
Fig. 1: recombinant plasmid pPIC9K-xyl11
mstructure 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 recombinant plasmid pUCm-T-xyl11
mstructure
For realizing deriving from the thermophilic high expression of heat resistant xylanase gene xyl11 in pichia spp splitting spore bacterium, xyl11 catalytic domain gene order (570bp) being analyzed, finds that the Preference of its codon and pichia spp have larger difference.Then contrast pichia spp codon preference table to be optimized the rare codon in xyl11 catalytic domain gene, and add EcoR I, Not I site respectively at its catalytic domain gene two ends, this unnamed gene is xyl11
m, synthetic rear clone is to pUCm-T plasmid, and Transformed E .coli JM109, blue hickie screening positive transformant, serves Hai Shenggong order-checking, obtain recombinant plasmid pUCm-T-xyl11
m.
The structure of embodiment 2 recombinant expression plasmid
By recombinant plasmid pUCm-T-xyl11 obtained above
mcarry out EcoR I and Not I double digestion, rubber tapping is reclaimed product and is connected with the expression plasmid pPIC9K through same double digestion, Transformed E .coli DH5 α competent cell, serves Hai Shenggong order-checking, obtain recombinant plasmid pPIC9K-xyl11 after PCR Screening and Identification
m.
Embodiment 3GS115/xyl11
mthe mensuration of the structure of recon, expression and zymologic property: pPIC9K-xyl11
mplasmid, through Sal I linearizing, carries out electricity according to Pichia anomala expression handbook and turns, screens, obtain the pichia spp recon GS115/xyl11 of high copy
m.This project bacterium 1.0% methanol induction 72h.Centrifuged supernatant is recombined xylanase crude enzyme liquid, and DNS method records recombined xylanase activity in crude enzyme liquid and reaches 157U/mL.The optimal reactive temperature of this enzyme is 70 DEG C, and after 70 DEG C of process 60min, activity of residual enzyme is 98%; Its optimal pH is 6.0, more stable in the scope of pH 5.0 ~ 8.0; Metal ion and EDTA less on its impact.
Claims (2)
1. one kind derives from the thermophilic heat-resisting β-Isosorbide-5-Nitrae-endo-xylanase (Xyl11 splitting spore bacterium (Thermobifida fusca)
m) optimized gene (xyl11
m), nucleotide sequence and the protein sequence of its correspondence are respectively: SEQ ID NO:1 and SEQ ID NO:2.
2.Xyl11
mheterogenous expression, activity and zymologic property mensuration:
(1) recombinant plasmid pUCm-T-xyl11
mstructure: be optimized deriving from the thermophilic rare codon split in the heat resistant xylanase catalytic domain gene of spore bacterium with reference to pichia spp codon preference table, and adding EcoR I, Not I site respectively at its catalytic domain gene two ends, this unnamed gene is xyl11
m, synthetic rear clone is to pUCm-T plasmid, and Transformed E .coli JM109, blue hickie screening positive transformant, serves Hai Shenggong order-checking, obtain recombinant plasmid pUCm-T-xyl11
m;
(2) structure of recombinant expression plasmid: by recombinant plasmid pUCm-T-xyl11 obtained above
mcarry out EcoR I and Not I double digestion, rubber tapping is reclaimed product and is connected with the expression plasmid pPIC9K through same double digestion, Transformed E .coli DH5 α competent cell, serves Hai Shenggong order-checking, obtain recombinant plasmid pPIC9K-xyl11 after PCR Screening and Identification
m;
(3) GS115/xyl11
mthe mensuration of the structure of recon, expression and zymologic property: pPIC9K-xyl11
mplasmid, through Sal I linearizing, carries out electricity according to Pichia anomala expression handbook and turns, screens, obtain the pichia spp recon GS115/xyl11 of high copy
m; This project bacterium 1.0% methanol induction 72h, centrifuged supernatant is recombined xylanase crude enzyme liquid, and DNS method records recombined xylanase activity in crude enzyme liquid and reaches 157U/mL; The optimal reactive temperature of this enzyme is 70 DEG C, and after 70 DEG C of process 60min, activity of residual enzyme is 98%; Its optimal pH is 6.0, more stable in the scope of pH 5.0 ~ 8.0; Metal ion and EDTA less on its impact.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410758941.1A CN104388451A (en) | 2014-12-10 | 2014-12-10 | Synthesis of thermostable xylanase gene (Xyl11M) and expression of gene in pichia pastoris |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410758941.1A CN104388451A (en) | 2014-12-10 | 2014-12-10 | Synthesis of thermostable xylanase gene (Xyl11M) and expression of gene in pichia pastoris |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104388451A true CN104388451A (en) | 2015-03-04 |
Family
ID=52606408
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410758941.1A Pending CN104388451A (en) | 2014-12-10 | 2014-12-10 | Synthesis of thermostable xylanase gene (Xyl11M) and expression of gene in pichia pastoris |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104388451A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104694518A (en) * | 2015-03-27 | 2015-06-10 | 湖北博大高科生物技术有限公司 | Xylanase resisting heat, acid and alkali and preparation method and application thereof |
| RU2725475C1 (en) * | 2019-09-25 | 2020-07-02 | Федеральное государственное бюджетное учреждение "Государственный научно-исследовательский институт генетики и селекции промышленных микроорганизмов Национального исследовательского центра "Курчатовский институт" (НИЦ "Курчатовский институт" - ГосНИИ генетика) | Recombinant yeast strain pichia pastoris - producer of xylanase from pyromyces finnis |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103014040A (en) * | 2012-12-24 | 2013-04-03 | 江南大学 | Heterologous expression method of heat-resisting beta-1, 4-endo-xylanase (SyXyn11) gene |
-
2014
- 2014-12-10 CN CN201410758941.1A patent/CN104388451A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103014040A (en) * | 2012-12-24 | 2013-04-03 | 江南大学 | Heterologous expression method of heat-resisting beta-1, 4-endo-xylanase (SyXyn11) gene |
Non-Patent Citations (1)
| Title |
|---|
| CHENG.Y.F: "登录号：AY795559.1", 《GENEBANK》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104694518A (en) * | 2015-03-27 | 2015-06-10 | 湖北博大高科生物技术有限公司 | Xylanase resisting heat, acid and alkali and preparation method and application thereof |
| CN104694518B (en) * | 2015-03-27 | 2017-12-22 | 湖北博大高科生物技术有限公司 | A kind of zytase of thermal resistance and acid-alkali resistance and its production and use |
| RU2725475C1 (en) * | 2019-09-25 | 2020-07-02 | Федеральное государственное бюджетное учреждение "Государственный научно-исследовательский институт генетики и селекции промышленных микроорганизмов Национального исследовательского центра "Курчатовский институт" (НИЦ "Курчатовский институт" - ГосНИИ генетика) | Recombinant yeast strain pichia pastoris - producer of xylanase from pyromyces finnis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Pei et al. | Thermoanaerobacterium thermosaccharolyticum β-glucosidase: a glucose-tolerant enzyme with high specific activity for cellobiose | |
| Ribeiro et al. | A novel thermostable xylanase GH10 from Malbranchea pulchella expressed in Aspergillus nidulans with potential applications in biotechnology | |
| Hu et al. | Cloning and enzymatic characterization of a xylanase gene from a soil-derived metagenomic library with an efficient approach | |
| Wang et al. | Characterization of a novel thermostable β-glucosidase from a metagenomic library of termite gut | |
| Geng et al. | Expression and characterization of a novel metagenome-derived cellulase Exo2b and its application to improve cellulase activity in Trichoderma reesei | |
| Campos et al. | Purification and characterization of a GH43 β-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteria | |
| Tan et al. | A bifunctional cellulase–xylanase of a new Chryseobacterium strain isolated from the dung of a straw‐fed cattle | |
| Yin et al. | Enhanced thermostability of a mesophilic xylanase by N‐terminal replacement designed by molecular dynamics simulation | |
| Zhang et al. | Cloning, expression, and characterization of an alkaline thermostable GH11 xylanase from Thermobifida halotolerans YIM 90462T | |
| García-Huante et al. | The thermophilic biomass-degrading fungus Thielavia terrestris Co3Bag1 produces a hyperthermophilic and thermostable β-1, 4-xylanase with exo-and endo-activity | |
| AU2017239832B2 (en) | Method for producing protein | |
| KR20150079676A (en) | Beta-glucosidase from magnaporthe grisea | |
| Sun et al. | Direct cloning, expression of a thermostable xylanase gene from the metagenomic DNA of cow dung compost and enzymatic production of xylooligosaccharides from corncob | |
| Basit et al. | Highly efficient degradation of xylan into xylose by a single enzyme | |
| Yin et al. | Expression and characteristics of two glucose-tolerant GH1 β-glucosidases from Actinomadura amylolytica YIM 77502T for promoting cellulose degradation | |
| JP2017532967A (en) | Compositions and methods related to beta-glucosidase | |
| Berto et al. | Functional characterization and comparative analysis of two heterologous endoglucanases from diverging subfamilies of glycosyl hydrolase family 45 | |
| Shrivastava et al. | Characterization, cloning and functional expression of novel xylanase from Thermomyces lanuginosus SS-8 isolated from self-heating plant wreckage material | |
| Li et al. | Functional expression and synergistic cooperation of xylan‐degrading enzymes from Hypocrea orientalis and Aspergillus niger | |
| Sheng et al. | Discovery and characterization of endo-xylanase and β-xylosidase from a highly xylanolytic bacterium in the hindgut of Holotrichia parallela larvae | |
| CN102227499A (en) | Cellulolytic polypeptides and their use in micro-organisms for production of solvents and fuels | |
| Zhao et al. | A novel thermophilic xylanase from Achaetomium sp. Xz-8 with high catalytic efficiency and application potentials in the brewing and other industries | |
| Zhang et al. | Cloning and expression of a Paecilomyces thermophila xylanase gene in E. coli and characterization of the recombinant xylanase | |
| Zhao et al. | Two family 11 xylanases from Achaetomium sp. Xz-8 with high catalytic efficiency and application potentials in the brewing industry | |
| CN102864160A (en) | Xylanase gene as well as expression protein and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20150304 |