CN104673857A - Application of glucose-tolerant cello-oligosaccharide enzyme - Google Patents
Application of glucose-tolerant cello-oligosaccharide enzyme Download PDFInfo
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- CN104673857A CN104673857A CN201510080021.3A CN201510080021A CN104673857A CN 104673857 A CN104673857 A CN 104673857A CN 201510080021 A CN201510080021 A CN 201510080021A CN 104673857 A CN104673857 A CN 104673857A
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Abstract
The invention relates to an application of a glucose-tolerant cello-oligosaccharide enzyme, and discloses an application of glucose-tolerant cello-oligosaccharide enzyme CHU-2268 in degraded cellulose. An amino acid sequence of the glucose-tolerant cello-oligosaccharide enzyme CHU-2268 is shown in SEQ ID NO.2. The protein with cello-oligosaccharide activity is obtained; the problems that a Harderian bacterium is long in growth cycle, an enzyme component is relatively complicated, and relatively low in yield, and cellulose cannot be directly separated and purified easily are effectively solved. The enzyme is capable of degrading cellose, also has relatively high degrading capability on cello-oligosaccharide, also has relatively high glucose tolerance, and has relatively great application potential in industries such as biological energy sources.
Description
Technical field
The present invention relates to and derive from the application of Kazakhstan addicted to the cell-oligosaccharide enzyme of a kind of glucose-tolerant of fiber bacterium (Cytophaga hutchinsonii), belong to technical field of bioengineering.
Background technology
Mierocrystalline cellulose is connected to form cellulose chain by D-Glucose residue by β-Isosorbide-5-Nitrae glycosidic link, and cellulose chain forms water-fast cellulose bundle by hydrogen bond association again.The primary biomass that Mierocrystalline cellulose is formed by photosynthesis as plant, is the main moiety of plant cell wall, becomes renewable resources the abundantest on the earth.In the society that current energy resource is day by day exhausted, make full use of these biomass resources, convert it into as the utilizable energy of the mankind and various Chemicals etc., the Sustainable development for human society is significant.
Due to the crystalline structure of its high-sequential, Mierocrystalline cellulose is not easy to be degraded very much.In physical environment, Mierocrystalline cellulose is mainly by cellulose degraded that microorganism produces.The cellulase that microorganism produces mainly comprises Mierocrystalline cellulose restriction endonuclease (endo-glucannase), Mierocrystalline cellulose excision enzyme (exo-glucanase) and beta-glucosidase (β-glucosidase).Wherein Mierocrystalline cellulose restriction endonuclease comprises typical restriction endonuclease and Progressive symmetric erythrokeratodermia restriction endonuclease, the cellulose chain of noncrystalline domain can be attacked, produce shorter sugar chain, and more attack site for excision enzyme provides, Progressive symmetric erythrokeratodermia restriction endonuclease can continuingly act on sugar chain and produce cell-oligosaccharide or cellobiose after opening the β of noncrystalline domain-Isosorbide-5-Nitrae glycosidic link.Mierocrystalline cellulose excision enzyme comprises cellobiohydrolase and cell-oligosaccharide enzyme, can continuingly act on cellulose chain and produce cellobiose or glucose.Beta-glucosidase then can the product of hydrolysis fiber disaccharide-hydrolysing enzymes, and cellobiose, produces glucose for substrate and then utilized by microbial metabolism.Some microorganisms then can produce cell-oligosaccharide enzyme (cellodextrinase), and hydrolysis fiber oligosaccharides generates glucose.
Fungi produces cellulase and mostly is extracellular enzyme, has than being easier to the features such as separation and purification, and present industrial fibre element enzyme mostly is fungal cellulase.And bacteria cellulose enzyme is easier carries out heterogenous expression and purifying in the escherichia expression system of present comparative maturity, have very large application space, the excavation thus for High Efficient Bacteria cellulase seems particularly important.
Cell-oligosaccharide can be changed into glucose by beta-glucosidase and cell-oligosaccharide enzyme, it is the important step that Mierocrystalline cellulose is thoroughly degraded, but its activity is subject to the feedback inhibition of its product glucose, therefore be difficult to accumulate glucose in a large number, be very limited in industrial application, find and there is the beta-glucosidase of glucose-tolerant characteristic and cell-oligosaccharide enzyme has important practical usage.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of application of cell-oligosaccharide enzyme of glucose-tolerant is provided.
The application of cell-oligosaccharide enzyme CHU_2268 in degraded cellulose for glucose-tolerant, the aminoacid sequence of the cell-oligosaccharide enzyme CHU_2268 of described glucose-tolerant is as shown in SEQ ID NO.2.
Preferably, the cell-oligosaccharide enzyme CHU_2268 degradation of fibers oligosaccharides of above-mentioned glucose-tolerant and cellobiose.
The cell-oligosaccharide enzyme CHU_2268 of glucose-tolerant is the glycoside hydrolase of a GH3 family, can degradation of fibers oligosaccharides and cellobiose, end product is all glucose, in the environment that glucose concn is higher, also can show relatively high vigor, cellulose conversion can be glucose with the acting in conjunction of Mierocrystalline cellulose restriction endonuclease by this enzyme.
Beneficial effect
The cell-oligosaccharide enzyme CHU_2268 ratio of glucose-tolerant of the present invention is easier to realize heterogenous expression in E. coli, obtain the albumen with cell-oligosaccharide enzymic activity, efficiently solve the Kazakhstan bacteria growing cycle long, enzyme component more complicated, output is lower, is not easy the problem of separation and purification.This enzyme cell-oligosaccharide comprising cellobiose of can degrading produces glucose, and has higher glucose tolerance, in the industry such as bioenergy, has larger application potential.
Accompanying drawing explanation
The cell-oligosaccharide enzyme CHU_2268SDS-polyacrylamide gel electrophoresis figure of Fig. 1, glucose-tolerant;
Wherein: the crude protein that after swimming lane 1, induction engineering bacteria, ultrasonication obtains; Cell-oligosaccharide enzyme CHU_2268 after purifying after swimming lane 2, purifying; Swimming lane M, marker, molecular weight is for shown in figure, and unit is KD;
Fig. 2, take pNPG as the optimal reaction graphic representation of the cell-oligosaccharide enzyme CHU_2268 of substrate;
Wherein: a, optimal reactive temperature curve, b, optimal reaction pH curve;
The electrophorogram of Fig. 3, cell-oligosaccharide enzyme CHU_2268 degraded pNPG and pNPC product;
Wherein: swimming lane M, marker, standard substance used are as diagram; Swimming lane 1, cell-oligosaccharide enzyme CHU_2268 degraded pNPG product; The pNPG that swimming lane 2, pNPG are hatched jointly with deactivation cell-oligosaccharide enzyme CHU_2268 contrasts; Swimming lane 3, cell-oligosaccharide enzyme CHU_2268 degraded pNPC product; The pNPC that swimming lane 4, pNPC are hatched jointly with deactivation cell-oligosaccharide enzyme CHU_2268 contrasts;
The electrophorogram of Fig. 4, cell-oligosaccharide enzyme CHU_2268 degradation of fibers disaccharides and cell-oligosaccharide product;
Wherein: swimming lane M, marker, standard substance used are as diagram; Swimming lane 1,3,5,7 are respectively cell-oligosaccharide enzyme CHU_2268 degradation of fibers disaccharides (G2), procellose (G3), the product of cellotetrose (G4) and cellopentaose (G5); Swimming lane 2,4,6,8 are respectively the G2 that corresponding oligosaccharides and deactivation cell-oligosaccharide enzyme CHU_2268 hatch jointly, the contrast of G3, G4 and G5;
Fig. 5, cell-oligosaccharide enzyme CHU_2268 are to the enzyme of the tolerance of glucose curve alive;
Embodiment
Below in conjunction with embodiment, technical solutions according to the invention are further elaborated, but institute of the present invention protection domain is not limited thereto.
Kazakhstan is addicted to fiber bacterium purchased from American DSMZ, and bacterium numbering is ATCC 33406, this bacterial strain non-the application institute preservation strain.
Embodiment 1: the clonal expression of glucose-tolerant cell-oligosaccharide enzyme
1, Kazakhstan is addicted to the cultivation of fiber bacterium
Kazakhstan is PY6 substratum addicted to the culture condition of fiber bacterium, 30 DEG C, 160rpm shaking culture.It is as follows that PY6 substratum often rises component: glucose 4g, Tryptones 6g, yeast extract 0.5g, and water is settled to 1L, pH 7.0.
2, genomic extraction
By the bacterial cultures centrifugal (10 of cultured Kazakhstan addicted to fiber bacterium (Cytophaga hutchinsonii), 000 × g, 3min) collect thalline, with bacterial genomes DNA extraction kit (the bacterial genomes DNA extraction kit that TIANGEN company produces), genomic dna is extracted according to product description, for subsequent use.
3, goal gene clone
(1) design of primers
Kazakhstan completes addicted to the genome sequencing of fiber bacterium, and we select the gene chu_2268 being predicted as a beta-glucosidase of GH3 family as research object.
Gene sequences encode 758 amino acid, the aminoacid sequence of the glycoside hydrolase precursor protein of its coding is as shown in SEQ ID NO.2, by SignalP4.1 software analysis, 18 hydropathic amino acid that predicted protein N holds are stronger, contriver predicts that this section of aminoacid sequence is signal peptide, get rid of the maturation protein that this part hydrophobic sequence obtains glycoside hydrolase, its aminoacid sequence is as shown in SEQ ID NO.4, and the gene order of corresponding this glycoside hydrolase maturation protein of coding is as shown in SEQ ID NO.3.
According to the sequence characteristic of this gene, and sequence pair answers signal peptide sequence and the maturation protein sequence characteristic of expressing protein, inventor has devised the primer of two ends with restriction enzyme digestion sites:
Upstream primer: ACGAAC
gGATCCtCTTGTGAGAAAAAAACAGT, dashed part is BamH I restriction enzyme site.
Downstream primer: CGGACG
gTCGACtTACTCATTAAAATATATTTC, dashed part is Sal I restriction enzyme site.
Entrust Hua Da genome company to synthesize this pair of primers for cloning this glycoside hydrolase mature protein gene (aminoacid sequence of the cell-oligosaccharide enzyme maturation protein of this genes encoding), clone gene takes the method for pcr amplification.
(2) pcr amplification of goal gene and purifying
(purchased from Transegene company in 50 μ l TransStart FastPfu DNA Polymerase reaction systems, compound method is see reagent specification sheets), to extract the genomic dna of acquisition in step 2 for template, above-mentioned primer is as amplimer, increased by the method for PCR, PCR reaction conditions:
Denaturation 95 DEG C, 2 minutes; Thermal cycle conditions is 95 DEG C of sex change 20 seconds, anneals 20 seconds for 50 DEG C, and 72 DEG C extend 30 seconds, and 32 circulations are carried out in reaction; Finally 72 DEG C of insulation extensions 10 minutes.
Be 2220bp by the full length gene of pcr amplification, detect after confirmation obtains goal gene DNA cloning product with 1.0% agarose gel electrophoresis, use DNA gel to reclaim test kit (being purchased from Omega company) and cut glue and reclaim, purifying gained amplified production.
4, the structure of recombinant plasmid
The DNA fragmentation of above-mentioned purifying and carrier pMAL-c2x restriction enzyme BamH I and restriction enzyme Sal I (being purchased from Takara company) are carried out double digestion, and experimental implementation is see Takara product description.Enzyme carries out purifying with Cycle-pure kit (being purchased from Omega company) to digestion products after cutting.
By the gene fragment of above-mentioned purifying and carrier segments mixing, at 16 DEG C, carry out spending the night ligation with Solution I (purchased from Takara company).Connection product is proceeded in E. coli DH5 α (purchased from Takara company), be coated with containing on the solid LB plate of 100 μ g/ml penbritins, 37 DEG C of quiescent culture, until there is single bacterium colony, selectivity picking list bacterium colony is 37 DEG C of shaking culture in the LB liquid medium containing 100 μ g/ml penbritins.Bacterium liquid PCR verifies in the recombinant plasmid of engineering bacteria containing object fragment.The recombinant plasmid pChu_2268 built entrusts Hua Da gene to carry out sequencing.Measure sequence and Kazakhstan bacterium protogene is compared, without base mistake or phase shift mutation, illustrate that this engineering bacteria can express the maturation protein CHU_2268 of aminoacid sequence described in SEQ 4.
5, the expression of recombinant plasmid in Host Strains
Recombinant plasmid containing object fragment is proceeded in Host Strains E.coli JM109 (purchased from Takara company), choose positive colony incubated overnight in the liquid nutrient medium containing penbritin, next day, 37 DEG C of concussions were cultured to OD with 2% inoculum size access fresh culture
600be between 0.4 to 0.6, add isopropylthio β-D-galactoside (IPTG), final concentration is 0.3mM, 18 DEG C, 80rpm low-speed oscillation cultivates 15 hours, collected by centrifugation thalline, the engineering bacteria of obtained IPTG induction, then ultrasonication, 12,000g, centrifugal 15min, gained supernatant carries out 12%SDS-polyacrylamide gel electrophoresis, shows the expression of target protein.After affinitive layer purification, take pNPG as substrate, with the output of 410nm place absorbance detection pNP, the pNPG recording the process of purifying gained albumen has a large amount of pNP to produce, and namely we have arrived the activated CHU_2268 of tool by heterogenous expression.
6, the expression method of other these cell-oligosaccharide enzymes
Except reaching except this cell-oligosaccharide enzyme with above-mentioned method table, other biological means can be taked to express.
Can pass through by the gene integration of this cell-oligosaccharide enzyme of coding on the karyomit(e) of host, or select other carriers or host cell, such as other bacteriums or fungi.After other host cell and carrier are loaded with the multifunctional cellulase gene that the present invention relates to, can express equally, produce multifunctional fibre fibroin involved in the present invention, such organism just becomes genetic engineering bacterium or the genetically engineered cell that can express, produce this multifunctional cellulase.
Embodiment 2: the purifying of glucose-tolerant cell-oligosaccharide enzyme
By the engineering bacteria collected by centrifugation thalline that above-mentioned IP TG induces, with sonication buffer (20mM Tris-HCl, 1mM EDTA-Na
2200mM NaCl, pH 7.4) resuspended, rear ultrasonication, the broken liquid of gained, the centrifugal 15min of 12,000g, obtains supernatant, be crude enzyme liquid, cross maltose binding protein affinity column (Amylose Resin), concrete operations, see NEB operational manual, can obtain the cell-oligosaccharide enzyme CHU_2268 of purifying.Purification result as shown in Figure 1.
Embodiment 3: the character of recombinant fiber oligosaccharidase
(1) optimal reactive temperature of cell-oligosaccharide enzyme and pH
The optimal reactive temperature of cell-oligosaccharide enzyme involved in the present invention and pH take pNPG as substrate reactions, detects after reaction 30min.The detection of optimal reaction pH is as follows: with 0.2mM pNPG for substrate, and damping fluid is the Na of different pH
2hPO
4/ KH
2pO
4(50mM), add appropriate enzyme, after 30 DEG C of reaction 30min, add 200 μ l Na
2cO
3(10%, w/v) termination reaction, represents the generation of pNP with 410nm place light absorption value.The detection of optimal reactive temperature is then under optimal reaction pH, reacts in differing temps.
After testing, cell-oligosaccharide enzyme optimal reaction pH involved in the present invention is 6.8, and optimal reactive temperature is 30 DEG C.Result as shown in Figure 2.
(2) cell-oligosaccharide enzyme is to the degraded of pNPG and pNPC
With pNPG or pNPC of 0.5% (w/v) for substrate, phosphoric acid buffer system (Na
2hPO
4/ KH
2pO
4, 50mM, pH 6.8), react different time at 30 DEG C, the light absorption value of the generation or detection 405nm place that detect product by the method for thin-layer chromatography represents the output of pNP.
Experiment detection shows, cell-oligosaccharide enzyme involved in the present invention can be degraded pNPG and pNPC, and end product be all pNP and glucose, is mainly pNPG and glucose at degraded pNPC initial stage product, to illustrate beyond CHU_2268 that butt formula is degraded pNPC.And have produce compared with the pNPG3 of long-chain in the initial reaction stage of degraded pNPC, illustrate that CHU_2268 also has transglycosylation.Result as shown in Figure 3.
(3) cell-oligosaccharide enzyme is to the degraded of cellobiose and cell-oligosaccharide
With the cellobiose of 1% (w/v) or cell-oligosaccharide for substrate, at 30 DEG C, react different time, detect the generation of reducing sugar by the method for thin-layer chromatography.
Cell-oligosaccharide enzyme involved in the present invention can the cell-oligosaccharide such as degradation of fibers disaccharides and procellose, cellotetrose and cellopentaose, and end product is all glucose.Result as shown in Figure 4.
(4) cell-oligosaccharide enzyme is to the tolerance of glucose
With the glucose tolerance of 0.2mM pNPG for substrate detection fibers oligosaccharidase, in phosphoric acid buffer system (Na
2hPO
4/ KH
2pO
4, 50mM, pH 6.8), react 30min at 30 DEG C, in reaction system, add the glucose of different concns, to react the generation that the light absorption value terminating rear 410nm place represents pNP.
Cell-oligosaccharide enzyme involved in the present invention (glucose concn is 100 times of substrate pNPG concentration) under the glucose condition of higher concentration still has about 40% enzyme activity.Result as shown in Figure 5.
As can be seen from the above results, cell-oligosaccharide enzyme involved in the present invention has higher tolerance to glucose, existing cell-oligosaccharide enzyme and beta-glucosidase can be solved to a certain extent by the problem of product feedback inhibition, the product glucose of accumulation higher concentration, has good application prospect at the energy and chemical field.
Claims (2)
1. the application of cell-oligosaccharide enzyme CHU_2268 in degraded cellulose for glucose-tolerant, the aminoacid sequence of the cell-oligosaccharide enzyme CHU_2268 of described glucose-tolerant is as shown in SEQ ID NO.2.
2. apply as claimed in claim 1, it is characterized in that, the cell-oligosaccharide enzyme CHU_2268 degradation of fibers oligosaccharides of glucose-tolerant and cellobiose.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002000858A2 (en) * | 2000-06-26 | 2002-01-03 | University Of Florida Research Foundation, Inc. | Methods and compositions for simultaneous saccharification and fermentation |
| CN102492710A (en) * | 2011-12-29 | 2012-06-13 | 山东大学 | Beta-glucosidase Cel1b, expression gene and application thereof |
| CN102888416A (en) * | 2012-10-12 | 2013-01-23 | 广西大学 | Beta-glycosidase gene for coding glycosyl hydrolase family and application thereof |
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2015
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002000858A2 (en) * | 2000-06-26 | 2002-01-03 | University Of Florida Research Foundation, Inc. | Methods and compositions for simultaneous saccharification and fermentation |
| CN102492710A (en) * | 2011-12-29 | 2012-06-13 | 山东大学 | Beta-glucosidase Cel1b, expression gene and application thereof |
| CN102888416A (en) * | 2012-10-12 | 2013-01-23 | 广西大学 | Beta-glycosidase gene for coding glycosyl hydrolase family and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| LIU C K: "Cellulose degradation system of Cytophaga hutchinsonii,", 《EDINBURGH RESEARCH ARCHIVE》 * |
| TANAKA T: "Creation of a cellooligosaccharide-assimilating Escherichia coli strain by displaying active beta-glucosidase on the cell surface via a novel anchor protein", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| XIE,G: "GenBank: ABG59531.1", 《NCBI》 * |
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Application publication date: 20150603 |