CN105861527A - Expression and application of intracellular high-temperature xylanase gene and protein thereof - Google Patents
Expression and application of intracellular high-temperature xylanase gene and protein thereof Download PDFInfo
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Abstract
The invention belongs to the fields of biological engineering and industrial enzyme preparation, and relates to expression and application of an intracellular high-temperature xylanase gene xyn10B and a protein Xyn10B thereof. The intracellular high-temperature xylanase gene xyn10B is derived from caldicellulosiruptor kronotskyensis. The Xyn10B has the advantages of high reaction temperature, good thermal stability and the like, the optimal reaction temperature is 70 DEG C, and the optimal pH is 6.0. After the Xyn10B is subjected to heat treatment for 6 h at the temperature of 65 DEG C, the enzyme activity can be still kept at 100%. The Xyn10B can directly act on a natural substrate, and can significantly improve the degradation rate of natural lignocellulose biomass through synergistic effect with commercial cellulase. The Xyn10B as a novel enzyme preparation can be widely applied in the fields of feeds, food, energy sources and the like, and has the potential industrial application value.
Description
Technical field
The present invention relates to bioengineering and biomass economy field, in particular it relates to a kind of intracellular high-temperature xylanase gene and the expression of albumen thereof and application.
Background technology:
Lignocellulosic is living beings the abundantest on the earth, is considered as a valuable source of bio-fuel production owing to it is with low cost.Lignocellulose raw material is mainly made up of lignin, cellulose and hemicellulose.Wherein, the participation of the degradable needs multiple hydrolysis of hemicellulose enzyme of hemicellulose and jointly complete.Zytase is as main hemicellulase, it is possible to the β-Isosorbide-5-Nitrae in random hydrolysis hemicellulose key component xylan backbone-glycosidic bond produces wood sugar and oligosaccharide.Thermophilic xylanase addicted to compared with middle temperature enzyme, has more obvious advantage: high temperature enzyme can store at room temperature and will not inactivate with other in the degraded of living beings；The risk of microbial contamination significantly reduces；Reduce cost of transportation, reduce the loss of product in cooling procedure；Increase the flexibility of living beings refining.At present, the zytase of business application need to have high Rate activity, high-fire resistance, the pH value adaptability of wide scope and substrate specificity widely.Therefore one is found can be of great significance with the zytase tool that the high stability of hydrolyzing biomass and high enzyme are lived under extreme industrial environment.
Research shows that the zytase of heat resistance is many from high temperature anaerobic bacterium.The anaerobic type pyrolysis cellulose bacillus Caldicellulosiruptor kronotskyensis having been found that can grow in the environment of 45-82 DEG C, its growth optimum temperature is 70 DEG C, and can decompose and utilize (Margarita L. et al., 2008) such as cellulose, pectin, starch and xylans.The high-temperature xylanase of C.kronotskyensis secretion has appreciable prospects for commercial application.But, the xylanolytic enzyme system complicated components that this bacterium produces, isolated and purified difficulty, its growth conditions is harsh in addition, and stand density is low, is not suitable for industrialization large-scale production.Therefore use technique for gene engineering that the important glycoside hydrolase gene of this type of thermophilic microorganism carries out the focus of allos height efficient expression always research.The examining order of this bacterial strain at present is complete, find its genome exists multiple potential xylanase gene by Gene correlation, we therefrom have selected the endoxylanase gene deriving from GH10 family, it is that preferred Host Strains constructs C.kronotskyensis xylanase gene xyn10B recombination engineering with Escherichia coli BL21 (DE3), is successfully realized the efficient heterogenous expression of zytase Xyn10B.This zytase has good heat endurance, and it is alive at high temperature to have efficient enzyme, can be applicable to industrialized production.
Summary of the invention
Goal of the invention:
It is an object of the invention to provide a kind of high-temperature xylanase gene xyn10B and recombinant vector thereof and recombination engineering, and the expression of this kind of enzyme and application.Its goal of the invention includes:
1. providing a kind of high-temperature xylanase deriving from pyrolysis CELLULOLYTIC BACTERIUM Caldicellulosiruptor kronotskyensis that can be applied to industry, its amino acid sequence is as shown in SEQ ID NO:2.
2. providing the encoding gene of above-mentioned high-temperature xylanase, its gene order is as shown in SEQ ID NO:1.
3. provide the recombinant vector comprising above-mentioned high-temperature xylanase gene, including coli expression carrier, lactic acid bacteria expression vectors, hay bacillus expression vector, yeast expression vector, filamentous fungi expression vector etc..The high-temperature xylanase gene of the present invention is connected with linear plasmid fragment acquisition recombinant expression carrier.Recombinant expression plasmid pET-28b-xyn10B is as the most preferred embodiment of the present invention.
null4. the recombinant strain comprising above-mentioned high-temperature xylanase gene is provided，Described bacterial strain is that Escherichia coli are (such as Escherichia coli BL 21 (DE3)、E.coli Top10、E.coli Rosetta (DE3) etc.)、Lactic acid bacteria (such as Lactococcus lactis etc.)、Saccharomycete is (such as Pichia pastoris、Saccharomyces cerevisiae etc.)、Hay bacillus (such as Bacillus subtilis BS168 etc.) and filamentous fungi are (such as Trichoderma reesei、Aspergillus niger etc.)，It is preferably E. coli BL 21 (DE3).
5. a kind of method preparing above-mentioned high-temperature xylanase and the application on enzymolysis natural wood glycan and lignocellulosic material thereof are provided.
Technical scheme:
For achieving the above object, the present invention is by the following technical solutions:
(1) structure of above-mentioned high-temperature xylanase gene xyn10B engineering bacteria
1. the genome of pyrolysis CELLULOLYTIC BACTERIUM C.kronotskyensis is extracted, design primer carries out PCR amplification, and the xylanase gene fragment obtained and carrier XhoI and NdeI carry out double digestion, connects and is transformed in host cell, screening positive clone checks order, it is thus achieved that recombinant expression carrier.Described expression vector, refer to coli expression carrier, lactic acid bacteria expression vectors, hay bacillus expression vector, yeast expression vector, filamentous fungi expression vector etc., it is preferably the xylanase gene xyn10B by the present invention provides to be connected with linear plasmid pET-28b, obtains recombinant expression plasmid pET-28b-xyn10B.
2. recombinant plasmid, transformed host cell are extracted, it is thus achieved that containing xylanase gene xyn10B recombinant bacterium.Described bacterial strain is Escherichia coli (such as E.coli BL 21 (DE3), E.coli Top10, E.coli Rosetta (DE3) etc.), lactic acid bacteria (such as L.lactis etc.), saccharomycete (such as P.pastoris, S.cerevisiae etc.), hay bacillus (such as B.subtilis BS168 etc.) and filamentous fungi (such as T.reesei, A.niger etc.), preferably E. coli BL 21 (DE3).
(2) preparation of above-mentioned high-temperature xylanase Xyn10B
1. it is amplified the recombinant bacterium containing above-mentioned xylanase gene xyn10B cultivating, and abduction delivering.
2. collect thalline, separate chromatography counterweight histone by ultrasonication, Ni-NAT affinity chromatography and gel and carry out isolated and purified.
3. utilize expression and the purifying situation of SDS-PAGE electrophoresis detection destination protein, and measure the concentration of purifying protein.
(3) above-mentioned high-temperature xylanase Xyn10B zymologic property measures
1. zytase Xyn10B is carried out optimal pH, optimum temperature and thermal stability determination.
2. zytase Xyn10B is carried out specific enzyme activity determination.
(4) above-mentioned high temperature endoxylanase Xyn10B application on enzymolysis natural wood glycan and lignocellulosic material
Weigh a certain amount of pretreatment natural wood glycan and lignocellulosic material, add a certain amount of recombined xylanase Xyn10B enzyme liquid, in 65 DEG C, 80rpm vibration hydrolysis under the conditions of pH 6.0, sample at set intervals, centrifuging and taking supernatant P-hydroxybenzoic acid hydrazides (p-hydroxybenzoic acid hydrazide, PHBAH) method measures content of reducing sugar, with TLC qualitative analysis natural wood glycan and the composition of lignocellulosic material hydrolyzate.
(5) above-mentioned high temperature endoxylanase Xyn10B and cellulase application on synergetic hydrolysis natural wood glycan and lignocellulosic material.
It is separately added into a certain amount of enzyme liquid in the buffer solution of the natural wood glycan and lignocellulosic material that are mixed with pretreatment, the buffer solution of control group addition equivalent by following experiment.In 50 DEG C, 80rpm vibration hydrolysis under the conditions of pH 5.5 after reactant liquor is mixed, sample at set intervals, centrifuging and taking supernatant high performance liquid chromatography quantitative analysis all experimental group natural wood glycan and the composition of lignocellulosic material hydrolyzate.
Table 1 experiment packet
Accompanying drawing explanation
Fig. 1: A. high temperature endoxylanase Xyn10B gel chromatography collection of illustrative plates；After B.Superdex 200 gel chromatography, the SDS-PAGE of Xyn10B analyzes.
The impact on Xyn10B activity of Fig. 2: A. difference pH；B. the different temperatures impact on Xyn10B activity.
The heat endurance of Fig. 3: Xyn10B.
Fig. 4: Xyn10B with cellulase synergistic hydrolysis of corncob product analysis.
Specific embodiment
Embodiment 1: high-temperature xylanase Xyn10B recombinant expression carrier pET-28b-xyn10B and the structure of recombination engineering
Extracting C.kronotskyensis bacterial genomes by bacterial genomes DNA extraction kit, obtain genomic DNA ,-20 DEG C frozen standby.According to the endoxylanase gene xyn10B nucleotide sequence of prediction, design following primer:
xyn10B-F:5'-GGAATTCCATATGAGCGAAGATTATTATG-3'
xyn10B-R:5'-CCGCTCGAGTAAAAAGTCAATTATTCTAAAAAATG-3'
Carrying out gene PCR amplification with genomic DNA for template, PCR primer is carried out agarose gel electrophoresis after terminating by reaction, and purpose band reclaims kit with DNA glue and reclaims.Genes of interest after recovery carries out 37 DEG C of double digestions with plasmid pET-28b restriction endonuclease XhoI and NdeI and overnight processes.Digestion products PCR primer reclaims kit and is purified, and 4 DEG C of connections of xyn10B and the pET-28b T4 DNA Ligase after being digested process 12 hours.Take and connect in the 100 μ l Top10 competent cells that product 5 μ l joins ice bath, ice bath 30min.Then 42 DEG C of heat shock 60s, ice bath 2min.Adding 200 μ l LB fluid nutrient mediums, at 37 DEG C, 200rpm cultivates 1 hour.Bacterium solution is all coated on the LB flat board containing 50 μ g/ml kanamycins, cultivate 12-16 hour in 37 DEG C.Picking individual colonies, in 450 μ l contain the LB fluid nutrient medium of 50 μ g/ml kanamycins, is cultivated 4-6 hour for 37 DEG C.Carrying out bacterium solution PCR, reaction takes 5 μ l product after terminating and carries out agarose gel electrophoresis detection, determines positive colony.The correct little extraction reagent kit of rear plasmid of order-checking extracts plasmid, it is thus achieved that recombinant expression plasmid pET-28b-xyn10B.
Recombinant expression plasmid pET-28b-xyn10B is transformed in E. coli BL21 (DE3) competent cell, on the LB flat board containing 50 μ g/ml kanamycins, cultivates the single bacterium colony of 12-16h acquisition for 37 DEG C.5 single bacterium colonies of picking are overnight incubation in 5ml contains 50 μ g/ml kanamycins LB fluid nutrient mediums, bacterial classification is preserved with glycerine, obtain with pET-28b as carrier, be the high-temperature xylanase Xyn10B engineering bacteria that Host Strains builds with E.coli BL21 (DE3).
Embodiment 2: high-temperature xylanase Xyn10B expression in Escherichia coli
Recombinant bacterial strain is transferred to 100ml containing the LB fluid nutrient medium of 50 μ g/ml kanamycins according to the inoculum concentration of 1%, when 37 DEG C of 200rpm shaken cultivation to OD600 reach about 0.6, adds IPTG to final concentration 0.1mM, continues shaken cultivation 4-6 hour.Cultivation terminates the centrifugal thalline of collecting of rear 4000rpm 15min, addition 3ml Binding Buffer (50mM Tris-HCl pH7.5,300mM NaCl) resuspended thalline.After Ultrasonic Cell Disruptor ultrasonication, 4 DEG C of 10000g are centrifuged 30min gained supernatant and are crude enzyme liquid.
Embodiment 3: recombinant high temperature zytase expression in Pichia pastoris
With C.kronotskyensis genome as template, design primer amplification obtains high-temperature xylanase gene xyn10B, is connected into carrier pPIC9.Connecting product to convert to Escherichia coli Top10 competent cell, coated plate is 37 DEG C of incubated overnight on the less salt LB solid medium containing ammonia benzyl.Determine positive colony, extract plasmid after order-checking is correct, it is thus achieved that recombinant expression plasmid pPIC9-xyn10B.Linearizing plasmid vector electricity converts and enters Pichia pastoris GS115 competence, cultivates 2-3d screening positive transformant for 30 DEG C on MD culture medium flat plate.Extract positive transformant gene, carry out PCR checking, further determine that positive conversion.Picking monoclonal is seeded in the shaking flask containing 100ml YPD.In shaking table 30 DEG C, 220rpm cultivates to OD600=5.Under room temperature, 3000g is centrifuged 5min and collects cell, with BMMY inducing culture re-suspended cell, puts into shaking table and continues to cultivate.Add methyl alcohol to final concentration of 0.5% to continue induction every 24h, after cultivating 72h continuously, measure xylanase activity in induction fermentation liquid supernatant.
Embodiment 4: the purifying of recombinant high temperature zytase Xyn10B
Utilize recombinase to contain histidine-tagged, by Ni-NAT affinity chromatography, recombinant protein is purified.Elute, with the Elution Buffer (50mM Tris-HCl pH 7.5,300mM NaCl) containing 500mM imidazoles, the recombinant protein being combined with Ni-NAT and collect eluent.The bag filter that the eluent addition collected processed, in citrate buffer solution (pH6.0), dialysis 24h removes imidazoles and changes buffer solution.Enzyme liquid after removing imidazoles carries out sephadex chromatography, occurs collecting albumen during crest, and gel chromatography collection of illustrative plates is as shown in Figure 1A.The sample of isolated is put into bag filter, uses polyethylene glycol concentrating sample, by Bradford method at OD595Lower mensuration protein concentration is 0.645mg/ml.Take sample 5 μ l after purification and add 20 μ l 5 × protein electrophoresis sample-loading buffers, fully boil 10min, 13000rpm after mixing and be centrifuged 10min, take 8 μ l supernatants and carry out expression and the purifying situation of SDS-PAGE electrophoresis detection destination protein.Result is as shown in Figure 1B, it can be seen that single electrophoresis band, and molecular weight of albumen is 39KDa.
Embodiment 5: recombinant high temperature zytase Xyn10B zymologic property measures
5.1 optimal reaction pH and optimal reactive temperatures
The recombinant high temperature zytase Xyn10B purified measures the vigor of recombinant high temperature zytase under different pH, and recording optimal pH is 6.0, and under the reaction condition of 5.5-8.0, its vigor remains to reach more than 60% that the highest enzyme is lived, as shown in Figure 2 A.
The recombined xylanase purified measures its hydrolyzed xylan vigor under different temperatures (40-100 DEG C), and recording optimal reactive temperature is 70 DEG C, and under the reaction condition of 60-80 DEG C, remains to reach more than 85% that the highest enzyme is lived, as shown in Figure 2 B.
5.2 heat endurance
Recombinase Xyn10B, at 65,70,75 DEG C, processes 0,0.5,1,2,3,4 and 6 hours, then measures its residual enzyme by PHBAH method under its optimum reaction conditions and live.Recombinase is hatched at 65 DEG C 6 hours and can be remained stable, and when 70 DEG C, its half-life is 2h, and when 75 DEG C, its half-life is 5min, and result is as shown in Figure 3.
5.3 specific enzyme activity determinations
Xylanase activity unit definition: under given conditions (optimal reactive temperature and pH), the enzyme amount required for generation 1 μm ol wood sugar per minute is an enzyme activity unit (U).
Recombinase Xyn10B 70 DEG C, under conditions of pH 6.0, join in the beech xylans of 70 DEG C of preheating 5min or oat xylan solution and react 2min.Measure content of reducing sugar by PHBAH method, calculate the Xyn10B Rate activity (table 2) to different substrates.
Table 2 enzyme-specific is lived
| Substrate | Rate activity (IU/mg) |
| Beech xylan | 122.1±2.1 |
| Oat xylan | 118.6±5.4 |
Embodiment 6: recombinant high temperature zytase Xyn10B hydrolysis beech xylan and xylo-oligosaccharide
Taking restructuring crude enzyme liquid, be added separately in the substrate solution that 50 μ l contain 1% beech glycan or 2% xylo-oligosaccharide, at 65 DEG C, hydrolyze 4h under the conditions of pH 6.0, hydrolysate carries out thin-layer chromatography detection.Beech xylan hydrolysate is mainly xylobiose, Xylotetrose and a small amount of wood sugar.The final hydrolysate of xylo-oligosaccharide is mainly xylobiose and a small amount of wood sugar.
Embodiment 7: utilize recombined xylanase Xyn10B degraded bagasse xylan
Extracting xylan from bagasse by alkali density method, prepare bagasse xylan suspension with pH 6.0 citrate buffer solution, 13000rpm is centrifuged 10min, and centrifugal supernatant is solubility bagasse xylan.Add a certain amount of recombinant high temperature zytase Xyn10B, 600rpm vibration hydrolysis 12h under the conditions of pH is 6.0,65 DEG C.Sample is boiled 10min inactivation after terminating by reaction, and hydrolyzation sample 13000rpm is centrifuged 20min, and supernatant PHBAH method measures content of reducing sugar, analyzes the composition of product in bagasse xylan digest with TLC.Result shows, after enzymolysis 12h, enzymolysis liquid is mainly composed of wood sugar and xylobiose.Enzymolysis product wood oligose content is 87.5%, and xylobiose content is more than 60%, and Xylose Content is 2.1%, and xylan degrading rate reaches 80.0%.
Embodiment 8: utilize recombined xylanase Xyn10B and commercial fibres element enzyme synergetic hydrolysis corncob
Being pulverized by corncob with pulverizer, corncob slag running water rinses repeatedly until extracted liquid becomes colorless.Then use distilled water flushing 3 times, put into drying box and dry.Experimental group 1 adds 2 μ g recombined xylanase crude enzyme liquids；Experimental group 2 adds commercial fibres element enzyme Cellic CTec2 (Novi's letter) of final concentration 150 μ g/ml；Experimental group 3 adds 2 μ g recombined xylanases and the Cellic CTec2 of final concentration 150 μ g/ml；Experimental group 4 adds commercial fibres element enzyme (Su Kehan) of final concentration 150 μ g/ml；Experimental group 5 adds the cellulase (Su Kehan) of 2 μ g recombined xylanases and final concentration 150 μ g/ml.All samples is placed at 80 DEG C difference abstraction reaction liquid after vibration enzymolysis 48h, and centrifuging and taking supernatant PHBAH method measures content of reducing sugar, with the composition of monose in high-efficient liquid phase chromatogram technique analysis Corncob hydrolysate.Result shows, recombined xylanase Xyn10B and Cellic CTec2 synergetic hydrolysis corncob effect is best.As shown in Figure 4, after enzymolysis 48h, in experimental group 3 hydrolyzate, glucose yield increases by 32.8%；Glucose yield increases by 58.0%.
Claims (8)
1. a high-temperature xylanase gene xyn10B, it is characterised in that: its nucleotide sequence is as shown in SEQ ID NO:1；Derive from pyrolysis cellulose fruit juice bacillus (Caldicellulosiruptor kronotskyensis).
2. the high-temperature xylanase Xyn10B of the high-temperature xylanase gene expression described in claim 1, it is characterised in that: its amino acid sequence is as shown in SEQ ID NO:2.
3. the high-temperature xylanase Xyn10B described in claim 2, the amino acid in its amino acid sequence SEQ ID NO:2 through one or more amino acid residues replacement, lack and add the derived protein with xylanase activity of formation.
4. one kind for the method that expands high-temperature xylanase gene xyn10B as claimed in claim 1, it is characterised in that: used primer to for:
xyn10B-F:5'-GGAATTCCATATGAGCGAAGATTATTATG-3'
xyn10B-R:5'-CCGCTCGAGTAAAAAGTCAATTATTCTAAAAAATG-3' 。
5. a high-temperature xylanase recombinant vector and recombination engineering, it is characterised in that containing the xylanase gene xyn10B described in claim 1；The recombinant expression carrier of described high temperature endoxylanase gene, refers to coli expression carrier, lactic acid bacteria expression vectors, hay bacillus expression vector, yeast expression vector, filamentous fungi expression vector etc.；Described recombinant strain is that Escherichia coli are (such as Escherichia coli BL 21 (DE3), E.coli Top10, E.coli Rosetta (DE3) etc.), lactic acid bacteria (such as Lactococcus lactis etc.), saccharomycete is (such as Pichia pastoris, Saccharomyces cerevisiae etc.), hay bacillus (such as Bacillus subtilis BS168 etc.) and filamentous fungi are (such as Trichoderma reesei, Aspergillus niger etc.).
6. the method preparing high-temperature xylanase, it is characterised in that:
1) with the recombinant vector transformed host cell described in claim 5, recombinant bacterial strain is obtained；
2) cultivating the recombination engineering described in claim 5, Induced hyperthermia zytase is expressed；
3) reclaim and purify expressed high-temperature xylanase Xyn10B.
7. the application on enzymolysis natural wood glycan and lignocellulosic material of the high-temperature xylanase described in claim 2, the optimum temperature of its enzymolysis and pH are respectively 70 DEG C and 6.0, and can remain stable in 65 DEG C hatch 6 hours.
8. the high-temperature xylanase described in claim 2 and other hemicellulases or cellulase synergistic application on natural wood glycan and lignocellulosic material, natural wood glycan and lignocellulosic material hydrolysis result improve 10-60%.
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