CN104388409A - F85-20 protein, coding gene thereof and application of F85-20 protein as beta-glucosidase - Google Patents
F85-20 protein, coding gene thereof and application of F85-20 protein as beta-glucosidase Download PDFInfo
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Abstract
The invention discloses an F85-20 protein, a coding gene thereof and an application of the F85-20 protein as beta-glucosidase. The invention provides the protein shown in the following 1) or 2): 1) a protein formed by amino acid residues shown in a sequence 2 in a sequence table; 2) a protein which is obtained by carrying out substitution and/or deletion and/or addition of one or more amino acid residues on an amino acid residue sequence of the sequence 2 in the sequence table, has the same functions as the protein shown in 1) and is derived from the protein shown in 1). Experiments prove that new beta-glucosidase F85-20 can still retain 80% of activity after being incubated at 50 DEG C for two hours under the conditions that p-nitrophenol glucoside (pNPG) is taken as the substrate and the pH value is 6.0, and the optimum temperature is 65 DEG C; new beta-glucosidase F85-20 can retain more than 70% of residual activity in a pH value range of 4.0-7.5 after being incubated in buffer solutions with different pH values for 24 hours at 4 DEG C.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of F85-20 albumen and encoding gene thereof and the application as beta-glucosidase thereof.
Background technology
According to statistics, annual global plant reaches 5,000,000,000 tons by photosynthesis synthon class material.Mierocrystalline cellulose (Cellulose) is the important component of plant cell wall, is the abundantest renewable resources of occurring in nature.The enzyme of cellulase (Cellulase) to be a class can be by cellulose hydrolysis glucose oligosaccharide and glucose, a lot of nature microorganism is all containing cellulase.The industry such as weaving, papermaking, food and biofuel can be widely used in due to cellulase, attract the research interest of more and more scholar in recent years.But in the process of cellulase hydrolysis, the activity of a large amount of cellobiose of generation and oligosaccharides meeting strongly inhibited cellulase, causes the sharply decline of cellulose hydrolysis efficiency, the reduction of whole production efficiency.Beta-glucosidase efficient catalytic cellobiose and oligosaccharides can be converted into glucose, therefore adds in cellulase by this enzyme, can remove Product inhibiton, makes cellulase recover efficient, thus ensures that whole hydrolytic process is stablized continuously.
In industry, cellulosic hydrolysis temperature is generally 50 DEG C, seldom has enzyme to keep more long catalytic effect and stability at such a temperature.Which limits the high-efficient development of biofuel industry, is also the important factor that restriction biofuel reduces production cost.In order to make enzyme reach higher thermotolerance and thermostability, a lot of scholar screens new enzyme from thermophilic microorganism, but they seldom can be useful in industrial condition.Occurring in nature, uncultured microorganisms accounts for 99% of all microorganisms.The research of major part screening beta-glucosidase is still confined in the microorganism of educable 1%.Over nearly 10 years, along with the development of sequencing technologies and High Throughput Screening Assay, people begin one's study the resource contained in uncultured microorganisms gradually, what adopt is technique of metagenome ripe day by day, and found the enzyme in a large number with outstanding character, describe the bright prospects that technique of metagenome excavates new enzyme.
Screen in the research of new enzyme at technique of metagenome, screening efficiency from physical environments such as soil to animal digestive tract, then increases progressively successively to the reactor of domestication.This is relevant to the intensity of taming and selective pressure.In physical environment, such as forest soil, needs the several years, and microorganism could by biomass degradation, in animal digestive tract, and the some skies of this reaction needed, but hydrolysis efficiency is still not high, the reason that Here it is herbivore is heavy; And for the reactor of domestication, the transformation efficiency of 90% within three days, can be reached.This research screens beta-glucosidase from domestication's reactor, ensure that its high efficiency.
Summary of the invention
An object of the present invention is to provide a kind of albumen.
Albumen provided by the invention, F85-20 is following 1) or 2) protein:
1) protein be made up of the amino-acid residue shown in sequence in sequence table 2;
2) by 1) shown in the amino acid residue sequence of protein through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have identical function by 1) derivative protein.
The replacement of above-mentioned one or several amino-acid residue of process and/or disappearance and/or be added to the replacement and/or disappearance and/or interpolation that are no more than 10 amino-acid residues.
The DNA molecular of above-mentioned albumen of encoding also is the scope of protection of the invention.
Above-mentioned DNA molecular is following 1)-5) in arbitrary described DNA molecular:
1) coding region is sequence 1 in sequence table;
2) coding region be in sequence table sequence 1 from 5 ' end 19-1404 position Nucleotide;
3) coding region be in sequence table sequence 1 from 5 ' end 11-1404 position Nucleotide;
4) under strict conditions with 1) or 2) or 3) hybridize and encode there is the DNA molecular of identical function albumen;
5) with 1) or 2) or 3) there is more than 90% homology and encode there is the DNA molecular of identical function albumen.
Above-mentioned stringent condition can be with 0.1 × SSPE (or 0.1 × SSC), and the solution of 0.1%SDS, hybridizes at 65 DEG C and wash film in DNA or RNA hybrid experiment.
Expression cassette containing above-mentioned DNA molecular, recombinant vectors, recombinant bacterium, transgenic cell line or recombinant bacterium are also the scope of protection of the invention.
In aforesaid method, described expression vector is pET-28a carrier, and described recombinant vectors is that sequence in sequence table 1 is inserted from 5 ' end 11-1404 position Nucleotide the carrier obtained between the Nco I of pET-28a carrier and HindIII restriction enzyme site at embodiments of the invention;
Above-mentioned recombinant vectors is the recombinant vectors obtained by above-mentioned DNA molecular insertion expression vector.
Above-mentioned recombinant bacterium is that described recombinant vectors is imported the recombinant bacterium obtained in object bacterium.
Described object bacterium is intestinal bacteria, is specially BL21DE3.
Above-mentioned albumen is also being the scope of protection of the invention as the application in beta-glucosidase or cellulase or xylosidase.
Above-mentioned albumen or above-mentioned DNA molecular or above-mentioned expression cassette, recombinant vectors, recombinant bacterium, transgenic cell line or recombinant bacterium are also the scope of protection of the invention preparing the application in beta-glucosidase or cellulase or xylosidase.
The zymetology feature of described beta-glucosidase is specially: optimum pH is 6.0, and optimum temperuture is 65 DEG C.
Another object of the present invention is to provide a kind of method preparing beta-glucosidase or cellulase or xylosidase.
Method of the present invention is the above-mentioned recombinant bacterium that ferments, and namely obtains beta-glucosidase or cellulase or xylosidase.
In aforesaid method, described fermentation is induce bottom fermentation to cultivate at IPTG.
The application of above-mentioned albumen in degradation biological matter is also the scope of protection of the invention, and described biomass are corn cob.
Experiment of the present invention proves, the present invention finds new beta-glucosidase F85-20, with p-NP glucoside (pNPG) for substrate, under pH 6.0 condition, optimum temperuture is 65 DEG C, at 50 DEG C of incubations after 2 hours, still can retain the vigor of 80%; At 4 DEG C, after hatching 24 hours in different pH damping fluid, within the scope of pH 4.0-7.5, all can retain the residual activity more than 70%.
Accompanying drawing explanation
Fig. 1 is the prediction to F85-20 three-dimensional structure and avtive spot
Fig. 2 is F85-20 abduction delivering SDS-PAGE collection of illustrative plates
Fig. 3 is the impact of temperature on F85-20 activity
Fig. 4 is the temperature stability of F85-20
Fig. 5 is optimal pH and the pH stability of F85-20
Fig. 6 is that F175-3 and F85-20 studies cellulosic hydrolysate
Fig. 7 is the hydrolysate curve of F175-3 and F85-20 and F175-3 to corn cob.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The clone of embodiment 1, F85-20 gene
In early-stage Study, utilize the grand genomic library of Fosmid vector construction dry fermentation sludge system.ORF is predicted through order-checking and softberry, and NCBI and Pfam database annotation.Obtain F85-20 gene, the nucleotides sequence of this gene is classified as in sequence table that sequence 1 is from 5 ' end 19-1404 position Nucleotide, and protein designations of its coding is F85-20, its aminoacid sequence be in sequence table sequence 2 from N ' end 1-462 amino acids.Through comparison, F85-20 albumen has 60% the highest similarity with the beta-glucosidase from bacterium UASB270, predicts that it is beta-glucosidase.
Embodiment 2, F85-20 are as the application of beta-glucosidase
1, the structure of recombinant vectors
With the sequence 1 of synthetic for template, be primer with F85-20F and F85-20R, adopt the PrimeStar high-fidelity enzyme of TAKARA company to carry out pcr amplification, obtain the PCR primer of 1413bp, its nucleotides sequence is classified as sequence 1.
F85-20F:5 '-CATG
cCATGGaGATACAAATGAATAAACAT-3 ' comprises Nco I restriction enzyme site
F85-20R:5 '-CCC
aAGCTTgGCATAAAGGGCCTCC-3 ' comprises Hind III digestion site
The system of above-mentioned pcr amplification is as follows:
PCR program is as follows:
94 DEG C of 1min denaturations
98℃ 10s
68℃ 90s
Get back to 2,24 circulations
72℃ 5min
End
By above-mentioned PCR primer Nco I and Hind III double digestion, the digestion products obtained connects with the pET-28a carrier cut through same enzyme (Novagen pET-28a DNA Cat.No.69864-3), obtains recombinant vectors.
Through order-checking, this recombinant vectors is that sequence in sequence table 1 is inserted from 5 ' end 11-1404 position Nucleotide the carrier obtained between the Nco I of pET-28a carrier and Hind III digestion site, albumen shown in expressed sequence 2, by this recombinant vectors called after pET-28a-F85-20.
In sequence table in the DNA molecular shown in sequence 1 from 5 ' end 19-1404 position Nucleotide be F85-20 gene;
In sequence table in albumen shown in sequence 2 from N ' end 1-462 amino acids be F85-20 albumen.
2, the expression and purification of albumen
1) express
Above-mentioned recombinant vectors pET-28a-F85-20 is converted in BL21DE3 competent cell, 37 DEG C of overnight incubation in the LB flat board containing 50 μ g/mL kantlex, obtain single bacterium colony, bacterium colony PCR identifies, primer is F85-20F and F85-20R, what obtain 1389bp is positive recombinant bacterium, called after DE3/pET-28a-F85-20.
Adopting uses the same method proceeds to BL21DE3 competent cell by empty carrier pET-28a, obtains DE3/pET-28a.
Mono-for DE3/pET-28a-F85-20 colony inoculation is contained 37 DEG C of overnight incubation in the LB substratum of 50 μ g/mL kantlex to 100mL, when OD600nm reaches 0.4, add the IPTG (Isopropyl β-D-1-Thiogalactopyranoside) that final concentration is 1mM and induce four hours.Get 1mL bacterium liquid, collected after centrifugation thalline, be suspended from 200 μ L 1 × sds gel sample loading buffers, boil 10min, 25 DEG C of high speed centrifugation 1min, get supernatant liquor, applied sample amount 20 μ L.Take DE3/pET-28a as contrast.
10%SDS polyacrylamide gel electrophoresis, coomassie brilliant blue staining detects protein expression, result as shown in Figure 2,1 is DE3/pET-28a-F85-20 for DE3/pET-28a, 2-3, as can be seen from the figure, DE3/pET-28a-F85-20 was through the induction of four hours, and F175-3 success abduction delivering, target protein size is 53.2KD.
2), purifying
Collect DE3/pET-28a-F85-20 thalline, with PBS damping fluid (NaCl 137mM, KCl 2.7mM, Na
2hPO
410mM, KH
2pO
42mM, pH 7.4) wash thalline three times, resuspended, multigelation three broken walls.Centrifugal, collect supernatant, adopt nickel column packing purifying protein.First washings (NaH
2pO
450mM, NaCl 300mM, imidazole20mM, pH 8.0) wash three times, then elutriant (NaH
2pO
450mM, NaCl 300mM, imidazole 250mM, pH 8.0) wash-out three times, washing and elution requirement are 500g, 2min, albumen F85-20 for the purpose of the elutriant collecting three times of column volumes.
Target protein F85-20 is carried out SDS-PAGE detection, and obtaining size is 53.2KD, illustrates that purifying obtains target protein F85-20.
3, the functional verification of target protein F85-20 and enzyme Characteristics Detection
The mensuration of activity of beta-glucosidase: reaction system: 20 μ L 25mM pNPG solution+20 μ L 100mM sodium-acetate buffers.5 μ L enzyme liquid add in the reaction system of 50 DEG C of preheatings, incubation 5min, add 50 μ L 1M Na subsequently
2cO
3solution.Light absorption value is measured in 410nm place.
The enzyme (U) alive of a unit is defined as the amount (μm ol) that an enzyme molecule per minute hydrolysis pNPG discharges p-nitrophenyl (pNP).
1) condition determination of optimal pH:
Be different pH by buffer exchange, measure enzyme and live.The preparation of damping fluid: Sodium phosphate dibasic-citric acid (3.5-8.0), Tris-HCl (8.0-10.0), Sodium phosphate dibasic-sodium hydroxide (10.0-12.0).
As shown in Figure 5, target protein F85-20's result can degrade pNPG, be beta-glucosidase, and its optimal pH is 6.0.
2) mensuration of optimum temperuture:
By enzyme reaction system in advance at 20-90 DEG C of preheating 5min, then add enzyme and react.Reaction is pH 6.0.
As shown in Figure 3, target protein F85-20's result can degrade pNPG, be beta-glucosidase, and its optimum temperuture is 65 DEG C.
3) temperature stability condition determination:
Enzyme is distinguished incubation 20min at various temperatures, 40min, 60min, 80min, 100min and 120min, subsequently at 65 DEG C, pH 6.0 measures residual enzyme and lives;
As shown in Figure 4, target protein F85-20's result can degrade pNPG, be beta-glucosidase, and it after 2 hours, still remains with the activity of 80% at 50 DEG C of incubations.
4) pH Stability Determination:
Enzyme is added in the damping fluid of corresponding pH, place 24h in 4 DEG C, finally at 65 DEG C, under pH 6.0 condition, measure remaining activity.
As shown in Figure 5, target protein F85-20's result can degrade pNPG, be beta-glucosidase, and they is at 4 DEG C, after hatching 24 hours, all can retain the residual activity more than 70% within the scope of pH 4.0-7.5 in different pH damping fluid.
5) mensuration of Michaelis-Menton constant:
Prepare the pNPG (p-NP glucoside) of 0-50mM, pNPX (p-NP xyloside) and pNPC (p-NP cellobioside) solution, measure the reactive kinetics parameters (Lineweaver-Burkplot) of enzyme.
The research of enzymolysis product adopts the method for thin-layer chromatography, and enzyme is after 50 DEG C with substrate reactions 4h, and sampling spot is on silica-gel plate, and chromatography poststaining observes degraded product.
As shown in Figure 6, mark product are glucose G1 to result, cellobiose G2, procellose G3 and cellotetrose G4.Swimming lane 1: negative control; Swimming lane 2:F85-20 results of hydrolysis; Swimming lane three: F175-3 results of hydrolysis; Swimming lane four: F85-20+F175-3 results of hydrolysis, result shows, as F85-20 independent role CMC, does not substantially have hydrolysate; When F175-3 independent role, product is glucose, cellobiose and a large amount of oligosaccharides; When both merge use, the amount of cellobiose obviously reduces, and the amount of glucose increases, and illustrates that F85-20 can remove the substrate suppression of F175-3, thus improves the output of glucose.
4, F85-20 promotes that F175-3 is to corn cob degradation experiment
The ratio of 200U/g biomass (corn cob) adds F175-3 (aminoacid sequence is sequence 3) and the F85-20 of purifying, and 50 DEG C of incubation 72h, liquid phase measures sugared content.
Separation condition:
Analytical column: Shim-pack ISA-07 (4.0mm × 25cm)
Moving phase: A liquid 0.1M boric acid (being adjusted to pH=8 with potassium hydroxide)
B liquid 0.4M boric acid (being adjusted to pH=9 with potassium hydroxide)
Gradient: A liquid 100%-B liquid 100%, 2%/min linear gradient wash-out
Flow: 0.6mL/min
Temperature: 65 DEG C
Derivatization conditions:
Derivative liquid: 1%L-arginine, 3% boric acid
Flow velocity: 0.5mL/min
Temperature of reaction: 150 DEG C
The results are shown in Figure 7, the hydrolysis efficiency of this enzyme is 11.7%, and after adding the F85-20 of above-mentioned 2 purifying of 50U/g biomass, hydrolysis efficiency is increased to 52.3%.Illustrate that F85-20 has significant promoter action to F175-3 hydrolysis of corncob.
5, the simulation of three-dimensional structure
In swiss-model, search the suitableeest template, finally adopt pymol software to modify image.Adopt Cluster W to carry out Multiple Sequence Alignment, obtain avtive spot, see Fig. 1.
6, metal ion (5mM) is on the impact of enzymic activity
Method: pNPG solution+20 μ L 100mM sodium-acetate buffer (pH 6.0) of 20 μ L 25mM, wherein adds each metal ion species that final concentration is 5mM.The F85-20 of above-mentioned 2 purifying of 5 μ L adds in the reaction system of 65 DEG C of preheatings, incubation 5min, adds 50 μ L 1M Na subsequently
2cO
3solution.Light absorption value is measured in 410nm place.
Table 1 is that metal ion (5mM) is on the impact of enzymic activity
Result is as shown in table 1, can find out, the activity influence of most of metal ion to F85-20 is little.Mn ion has the effect strengthening enzyme and live, and mercury ion and cupric ion have than stronger restraining effect enzyme is alive.
7, the enzymatic reaction kinetics parameter of F85-20
Prepare the pNPX of 0-50mM, pNPG and pNPC solution, substrate solution+20 μ L 100mM sodium-acetate buffer (pH 6.0) of 20 μ L different concns.The F85-20 of above-mentioned 2 purifying of 5 μ L adds in the reaction system of 60 DEG C of preheatings, incubation 5min, adds 50 μ L 1M Na subsequently
2cO
3solution.Light absorption value is measured in 410nm place.
Result is as shown in table 2, can find out, F85-20 is the highest to pNPG and pNPX affinity, but to the catalytic efficiency of pNPG far above pNPX, illustrates that this enzyme is a kind of beta-glucosidase.In addition, F85-20 also has greater activity to pNPC, and this enzyme substrate specificity is widely described, can degradation of fibers polysaccharide.But be the highest to the catalytic efficiency of glucoside bond.
Table 2 is the enzymatic reaction kinetics parameter of F85-20
8, the substrate specificity of F85-20
The different substrate solution+20 μ L 100mM sodium-acetate buffer (pH 6.0) of 20 μ L.The F85-20 of above-mentioned 2 purifying of 5 μ L adds in the reaction system of 65 DEG C of preheatings, incubation 5min, adds 50 μ L 1M Na subsequently
2cO
3solution.Light absorption value is measured in 410nm place.Substrate comprises beech xylan, CMC (Xylo-Mucine), pNPX (p-NP glucoside), pNPG (p-NP glucoside), pNPA (p-NP Arabinoside), MIC (Microcrystalline Cellulose) and filter paper (50mg).
Result is as shown in table 3, shows that F85-20 has substrate-function spectrum more widely, the highest to the activity of pNPG; In addition the activity of certain xylosidase and cellulase is also had.
Table 3 is the substrate specificity of F85-20
Claims (10)
1. an albumen is following 1) or 2) protein:
1) protein be made up of the amino-acid residue shown in sequence in sequence table 2;
2) by 1) shown in the amino acid residue sequence of protein through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have identical function by 1) derivative protein.
2. the DNA molecular of albumen described in coding claim 1.
3. DNA molecular according to claim 2, is characterized in that: described DNA molecular is following 1)-5) in arbitrary described DNA molecular:
1) coding region is sequence 1 in sequence table;
2) coding region be in sequence table sequence 1 from 5 ' end 19-1404 position Nucleotide;
3) coding region be in sequence table sequence 1 from 5 ' end 11-1404 position Nucleotide;
4) under strict conditions with 1) or 2) or 3) hybridize and encode there is the DNA molecular of identical function albumen;
5) with 1) or 2) or 3) there is more than 90% homology and encode there is the DNA molecular of identical function albumen.
4. the expression cassette containing DNA molecular described in Claims 2 or 3, recombinant vectors, recombinant bacterium, transgenic cell line or recombinant bacterium.
5. recombinant vectors according to claim 4, is characterized in that: the recombinant vectors that described recombinant vectors obtains for DNA molecular described in Claims 2 or 3 being inserted expression vector.
6. recombinant bacterium according to claim 4, is characterized in that: described recombinant bacterium is that described recombinant vectors is imported the recombinant bacterium obtained in object bacterium.
7. albumen according to claim 1 is as the application in beta-glucosidase or cellulase or xylosidase;
The zymetology feature of described beta-glucosidase is specially: optimum pH is 6.0, and optimum temperuture is 65 DEG C.
8. DNA molecular described in albumen according to claim 1 or Claims 2 or 3 or expression cassette according to claim 4, recombinant vectors, recombinant bacterium, transgenic cell line or recombinant bacterium are preparing the application in beta-glucosidase or cellulase or xylosidase.
9. preparing a method for beta-glucosidase or cellulase or xylosidase, is the recombinant bacterium according to claim 6 that ferments, and namely obtains beta-glucosidase or cellulase or xylosidase.
10. the application of albumen according to claim 1 in degradation biological matter, described biomass are corn cob.
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| CN109456954A (en) * | 2018-11-21 | 2019-03-12 | 中山大学 | The β-glucosidase mutants and its application that thermal stability improves |
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| CN103589702A (en) * | 2013-11-19 | 2014-02-19 | 南京市第一医院 | Application of heat-resistant beta-glucosidase and mutants thereof |
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| CN109355275A (en) * | 2018-11-21 | 2019-02-19 | 中山大学 | High thermal stability β-glucosidase mutants and its application |
| CN109456954A (en) * | 2018-11-21 | 2019-03-12 | 中山大学 | The β-glucosidase mutants and its application that thermal stability improves |
| CN109456954B (en) * | 2018-11-21 | 2021-08-20 | 中山大学 | Beta-glucosidase mutant with improved thermal stability and application thereof |
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