CN105734069A - A high-temperature alpha-L-arabinfuranosidease gene, a high-temperature acetylxylan esterase gene, and protein expression and applications of the genes - Google Patents
A high-temperature alpha-L-arabinfuranosidease gene, a high-temperature acetylxylan esterase gene, and protein expression and applications of the genes Download PDFInfo
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Abstract
The invention provides a high-temperature alpha-L-arabinfuranosidease gene abf51A and a high-temperature acetylxylan esterase gene axeA which are derived from caldicellulosiruptor lactoaceticus 6A, and protein expression and applications of the genes. High-temperature alpha-L-arabinfuranosidease abf51A and high-temperature acetylxylan esterase axeA are advantaged by high optimum reaction temperatures, good thermal stability, and the like, and can effectively degrade natural xylan side chains at 65-85 DEG C with pH being 4.5-6.5 to produce arabinose, acetic acid and a small amount of xylose. The two enzymes can effectively eliminate steric hindrance of side chains to xylanase, improve biodegradability of the lignocellulose resource, can be widely used as novel enzyme preparations for feed, food, energy, and other fields, and have potential industrial application value.
Description
Technical field
The invention belongs to genetic engineering and biomass economy field, be specifically related to a kind of high temperature α-l-arabfuranglycosidase gene and a kind of high temperature acetyl xylan esterase gene and protein expression thereof and application.
Background technology:
Hemicellulose as one of the main component of plant cell wall, is one of Renewable resource the abundantest on the earth.Along with becoming increasingly conspicuous of energy problem, the hemicellulose utilization of resources is worth and is subject to people's attention gradually, and the research of its Biodegradation Mechanism is more and more seemed significant.It is xylan in the one that hemicellulose apoplexy due to endogenous wind is the abundantest; by xylopyranose with β-1; 4-glycosidic bond is formed by connecting chain; and the group (Scheller such as connection acetyl group generally all in various degree, aralino, glucuronic acid and ferulic acid ester group; H.V., P.Ulvskov.2010).The complexity of this structure causes that the participation of the multiple hydrolytic enzyme of degradable needs of xylan completes jointly, it includes backbone hydrolysis enzyme endo-xylanase (EC3.2.1.8, endo-β-1, 4-xylanase), xylobiase (EC3.2.1.37, β-xylosidase) and side-chain hydrolysis enzyme arabinofuranosidase (EC3.2.1.55, α-L-arabinofuranosidase, AFase), glycuronidase (EC3.2.1.131, α-glucuronidase) and acetyl xylan esterase (EC3.1.1.72, acetylxylanesterase, Axe) etc..Side-chain radical on xylan chain is to affect one of its factor being hydrolyzed, and the composition of these molecular weights half-and-half cellulosic biological degradability has important impact (Dodd, D., I.K.Cann.2009).The existence of side-chain radical limits the contact of main chain digestive enzyme xylanase and main chain, adds sterically hindered.Therefore, backbone hydrolysis enzyme endoxylanase and xylosidase cannot be degradable by xylan.In these side-chain hydrolysis enzymes, α-l-arabfuranglycosidase can from the non-reducing end hydrolyzing alpha-L-arabinose of arabinofuranosidase glucosides xylan；Acetyl xylan esterase can remove the O-acyl substituent of C-2 and C-3 in xylose residues.Both at the crucial side-chain hydrolysis enzyme (Juturu, V., J.C.Wu.2012) that xylan class hemicellulose is thoroughly degraded.
Arabinofuranosidase can be connected in the arabinose on xylose main chain with α-1,3, α-1,2 or α-1,5 key or oligomeric arabinan is collateral by catalyzing hydrolysis.In the process of degradation of hemicellulose, α-l-arabfuranglycosidase can work in coordination with other hemicellulases, carries the degradation efficiency of hemicellulose.Similarity according to aminoacid sequence and the architectural feature in proteins carry active center, α-l-arabfuranglycosidase is classified in glycoside hydrolase Families (GlycosideHydrolaseFamilies, GHs) the 3rd, 43,51,54,62 and 127 family (Dodd, D., I.K.Cann.2009).And the α-l-arabfuranglycosidase of GH51 family mainly adopts the acid base catalyzed reaction of retention mechanism to carry out the hydrolysis of catalysis glycosidic bond.Esterase, because the difference of substrate specificity, is divided into acetylesterase and feruloyl esterase.Acetyl xylan esterase is as a kind of accessibility enzyme in ligocellulose degradation, and the effect in natural microbial is to remove the O-acetyl group on C-2 or C-3 of xylan chain D-xylose residues.Similarity according to aminoacid sequence, esterase is divided into 16 carbohydrate esterase families (CarbohydrateEsteraseFamily, CE).Wherein acetyl xylan esterase is usually located at the 1st, 2,3,4,5,6,7 and 12 carbohydrate esterase family.
Important composition composition as hemicellulose degrading enzymes system, α-l-arabfuranglycosidase and acetyl xylan esterase synergism together with endo-xylanase may participate in the recycling of agricultural product castoff work garbage such as Caulis et Folium Oryzae, Testa Tritici, bagasse, produce to can be used for fermentation and generate monosaccharide and other side-products of alcohol fuel, therefore they have good application prospect (Collins in agricultural, industrial sector, T., C.Gerday, etal.2005).
Pyrolysis CELLULOLYTIC BACTERIUM Caldicellulosiruptorlactoaceticus6A separates strictly anaerobic energy one step obtained to utilize the gram positive bacteria of biomass from Iceland's alkalescence hot spring, and optimum temperature is 68 DEG C, growth temperature can be 50-78 DEG C.Optimum pH, close to 7.0, tolerates pH5.8-8.2 (Mladenovska, Z., I.M.Mathrani, etal.1995).It can Direct Resolution and utilize cellulose, hemicellulose, pectin, starch and xylan etc..The high temperature enzymes activity simultaneously secreted is high, high temperature resistant, and good stability has considerable prospects for commercial application (Mladenovska, Z., I.M.Mathrani, etal.1995).But the xylanolytic enzyme system complicated components that this bacterium produces, separates purification difficult, and its growth conditions is harsh, and stand density is low, is not suitable for industrialization large-scale production in addition.Therefore adopt technique for gene engineering that the important glycoside hydrolase gene of this type of thermophilic microorganism carries out allos height efficient expression and be always up the focus of research.The present invention is with pET-28b for preferred expression carrier, C.lactoaceticus α-l-arabfuranglycosidase gene abf51A and acetyl xylan esterase gene axeA recombination engineering is constructed respectively with EscherichiacoliRosetta (DE3) for preferred Host Strains, it is successfully realized the efficient heterogenous expression of α-l-arabfuranglycosidase Abf51A and acetyl xylan esterase AxeA, this invention will lay the foundation for this high temperature α-l-arabfuranglycosidase Abf51A of industrialized utilization from now on and acetyl xylan esterase AxeA degraded natural wood polysaccharide and lignocellulosic material.
Summary of the invention
Goal of the invention:
The present invention provides a kind of high temperature α-l-arabfuranglycosidase gene abf51A and a kind of acetyl xylan esterase axeA and recombinant vector thereof and recombination engineering, and the expression of two kinds of enzymes and application.Its goal of the invention includes:
1. the present invention provides a kind of high temperature α-l-arabfuranglycosidase Abf51A deriving from pyrolysis CELLULOLYTIC BACTERIUM Caldicellulosiruptorlactoaceticus6A, and its gene order is such as shown in SEQIDNO:1, and its aminoacid sequence is such as shown in SEQIDNO:2.
2. the present invention also provides for a kind of high temperature acetyl xylan esterase AxeA deriving from C.lactoaceticus6A, and its gene order is such as shown in SEQIDNO:3, and its aminoacid sequence is such as shown in SEQIDNO:4.
3. the present invention provides the recombinant expression carrier comprising above-mentioned α-l-arabfuranglycosidase gene and acetyl xylan esterase gene respectively, refers to coli expression carrier, lactic acid bacteria expression vectors, bacillus subtilis expression vector, yeast expression vector, filamentous fungi expression vector etc..The α-l-arabfuranglycosidase gene of the present invention and acetyl xylan esterase gene are connected acquisition recombinant expression carrier respectively with linear plasmid fragment.The most preferred embodiment as the present invention, it is preferably and respectively the α-l-arabfuranglycosidase gene of the present invention is connected with linear plasmid pET-28b with acetyl xylan esterase gene, respectively obtain recombinant expression plasmid pET-28b-abf51A and pET-28b-axeA.
null4. the present invention provides the recombinant bacterium comprising above-mentioned α-l-arabfuranglycosidase Abf51A and acetyl xylan esterase AxeA respectively，Described bacterial strain is that escherichia coli are (such as EscherichiacoliBL21 (DE3)、E.coliTop10、E.coliRosetta (DE3) etc.)、Lactic acid bacteria (such as Lactococcuslactis etc.)、Yeast is (such as Pichiapastoris、Saccharomycescerevisiae etc.)、Bacillus subtilis (such as BacillussubtilisBS168 etc.) and filamentous fungi are (such as Trichodermareesei、Aspergillusniger etc.)，It is preferably E. coli Rosetta (DE3).
5. the present invention provides the method preparing above-mentioned α-l-arabfuranglycosidase Abf51A and acetyl xylan esterase AxeA.
6. the present invention provides the above-mentioned high temperature α-l-arabfuranglycosidase Abf51A and acetyl xylan esterase AxeA synergistic application on enzymolysis natural wood polysaccharide and lignocellulosic material, it is preferable that itself and xylanase are in hydrolysis Herba bromi japonici xylan, Caulis Sacchari sinensis xylan, Corncob Xylan and the application in preparing xylose and arabinose thereof.
Technical scheme:
For achieving the above object, the present invention is by the following technical solutions:
(1) structure of above-mentioned high temperature α-l-arabfuranglycosidase gene abf51A and acetyl xylan esterase gene axeA engineering bacteria
Extract the genome of pyrolysis CELLULOLYTIC BACTERIUM C.lactoaceticus6A, separately design primer amplification α-l-arabfuranglycosidase gene abf51A and acetyl xylan esterase gene axeA.The genetic fragment obtained and plasmid EK/LICDNA connect transformed host cell after processing, and 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, bacillus subtilis expression vector, yeast expression vector, filamentous fungi expression vector etc., it is preferably and respectively the α-l-arabfuranglycosidase gene of the present invention is connected with linear plasmid pET-28b with acetyl xylan esterase gene, respectively obtain recombinant expression plasmid pET-28b-abf51A and pET-28b-axeA.Extract recombiant plasmid, transformed host cell, it is thus achieved that α-l-arabfuranglycosidase gene abf51A and acetyl xylan esterase gene axeA recombinant bacterium.Described bacterial strain is escherichia coli (such as EscherichiacoliBL21 (DE3), E.coliTop10, E.coliRosetta (DE3) etc.), lactic acid bacteria (such as Lactococcuslactis etc.), yeast (such as Pichiapastoris, Saccharomycescerevisiae etc.), bacillus subtilis (such as BacillussubtilisBS168 etc.) and filamentous fungi (such as Trichodermareesei, Aspergillusniger etc.), it is preferred to E. coli Rosetta (DE3).
(2) preparation of above-mentioned high temperature α-l-arabfuranglycosidase Abf51A and acetyl xylan esterase AxeA
It is amplified the recombinant bacterium containing above-mentioned α-l-arabfuranglycosidase gene abf51A and acetyl xylan esterase gene axeA cultivating, and abduction delivering.Then pass through ultrasonication, recombiant protein is easily separated by heat inactivation, Ni-NAT affinity chromatograph, and is further purified by Superdex200 gel chromatography and obtains restructuring α-l-arabfuranglycosidase Abf51A and acetyl xylan esterase AxeA.By the expression of SDS-PAGE electrophoresis detection destination protein and purification situation, and measure the concentration of purifying protein, analyze the zymologic property of restructuring α-l-arabfuranglycosidase Abf51A and acetyl xylan esterase AxeA.
(3) the above-mentioned high temperature α-l-arabfuranglycosidase Abf51A and acetyl xylan esterase AxeA application on degraded natural wood polysaccharide and lignocellulosic material
Weighing a certain amount of pretreatment natural wood polysaccharide and lignocellulosic material joins in appropriate pH6.5 citrate buffer solution, be separately added into a certain amount of enzyme liquid by following experiment packet, matched group adds the citrate buffer solution of the pH6.5 of equivalent.Vibration hydrolysis under 75 DEG C of conditions after reactant liquor is mixed.All samples is boiled centrifugal by reaction after terminating, take the composition of all experimental group natural wood polysaccharide of supernatant high-efficient liquid phase chromatogram technique analysis and lignocellulosic material hydrolyzed solution.
Table 1 experiment packet
Beneficial effect: compared with prior art, α-l-arabfuranglycosidase Abf51A optimal reactive temperature provided by the invention is 80 DEG C, optimum pH is 5.5, and has the activity of more than 55% within the scope of temperature is 60-85 DEG C, pH be have within the scope of 4.5-6.5 more than 60% activity.Abf51A retains the activity of 57% after hatching 12h at 80 DEG C.Acetyl xylan esterase AxeA optimum temperature is 70 DEG C, and optimum pH is 6.5, and has the activity of more than 50% within the scope of temperature is 40-75 DEG C.This enzyme has high heat stability, remains to retain the activity of 84% after hatching 12h at 65,70 and 75 DEG C.Sterically hindered when the α-l-arabfuranglycosidase Abf51A and acetyl xylan esterase AxeA in this source can effectively eliminate side-chain radical to xylanase effect, improves the biological degradability of lignocellulosic sources.Be applicable to 65-85 DEG C, pH4.5-6.5 when degraded to natural wood polysaccharide and lignocellulosic material, there is the characteristics such as enzymatic activity height, easy purification, optimal reactive temperature height, Heat stability is good, be expected in multiple industrial processes and obtain extensive use.
Accompanying drawing explanation
Fig. 1 recombinates the α-l-arabfuranglycosidase Abf51A and acetyl xylan esterase AxeA expression in escherichia coli Rosetta (DE3).
Wherein: (A) Abf51ASuperdex200 gel chromatography collection of illustrative plates；(B) Abf51ASDS-PAGE analyzes；(C) AxeASuperdex200 gel chromatography collection of illustrative plates；(D) AxeASDS-PAGE analyzes.
Recombinate α-l-arabfuranglycosidase Abf51A and acetyl xylan esterase AxeA zymetology of Fig. 2 characterizes.
Wherein: the optimum temperature of (A) Abf51A；(B) optimum temperature of AxeA；(C) optimum pH of Abf51A；(D) optimum pH of AxeA；(E) heat stability of Abf51A；(F) heat stability of AxeA.
Fig. 3 recombinates α-l-arabfuranglycosidase Abf51A and acetyl xylan esterase AxeA and xylanase Xyn10A synergetic hydrolysis Herba bromi japonici xylan.
Wherein: (A) hydrolyzate HPLC analyzes (0-17min)；(B) hydrolyzate HPLC analyzes (13.5-17min)；
Fig. 4 recombinates α-l-arabfuranglycosidase Abf51A and acetyl xylan esterase AxeA and xylanase Xyn10A synergetic hydrolysis Caulis Sacchari sinensis xylan.
Wherein: (A) hydrolyzate HPLC analyzes (0-17min)；(B) hydrolyzate HPLC analyzes (13.5-17min)；
Fig. 5 recombinates α-l-arabfuranglycosidase Abf51A and acetyl xylan esterase AxeA and xylanase Xyn10A synergetic hydrolysis Corncob Xylan.
Wherein: (A) hydrolyzate HPLC analyzes (0-17min)；(B) hydrolyzate HPLC analyzes (13.5-17min)；
Detailed description of the invention
Embodiment 1: the structure of high temperature α-l-arabfuranglycosidase gene abf51A and acetyl xylan esterase gene axeA engineering bacteria
The extraction of 1.1 genomic DNAs
Extracting C.lactoaceticus6A bacterial genomes by bacterial genomes DNA extraction kit, obtain genomic DNA ,-20 DEG C frozen standby.
1.2 design of primers
According to delivering C.lactoaceticus6A genomic information, it was predicted that α-l-arabfuranglycosidase and acetyl xylan esterase gene, design following 2 pairs of primers:
Primer for expanding α-l-arabfuranglycosidase gene is as follows:
abf51A-F5’-GCCGCGCGGCAGCATGAAAAAAGCAAAAGTCATCTAC-3’
abf51A-R5’-GCGGCCGCAAGCGTTTAATTTTCTTTCTTCTTTAACCTG-3’
Primer for expanding acetyl xylan esterase gene is as follows:
axeA-F5’-GCCGCGCGGCAGCATGATACCACTTTGGGAAAATC-3’
axeA-R5’-GCGGCCGCAAGCGTTTAAACATTATATCCTATAAACC-3’
The pcr amplification of 1.3 genes
With genomic DNA for template, carry out gene PCR amplification respectively.Reaction takes 5 μ l product after terminating and carries out agarose gel electrophoresis detection, and purpose band reclaims test kit with plain agar sugar gel DNA and reclaims.
The process of 1.4 α-l-arabfuranglycosidase gene abf51A and acetyl xylan esterase gene axeA be connected
After α-l-arabfuranglycosidase genetic fragment and acetyl xylan esterase genetic fragment being processed with T4DNAPolymerase, it is connected 15min with plasmid pET-28bEK/LICDNA room temperature.
1.5 recombinant plasmid transformed E. coli Top10
Take connection product respectively and join in 100 μ lE.coliTop10 competent cells, ice bath 30min.Then 42 DEG C of heat shock 60s, immediately ice bath 2min.Being separately added into 500 μ lLB fluid mediums, at 37 DEG C, 200rpm cultivates 1h.Take on the bacterium solution 200 μ l LB flat board coated containing 50 μ g/ml kanamycin respectively, cultivate 12-16h in 37 DEG C.
The qualification of 1.6 recombiant plasmid and extraction
Picking individual colonies incubated overnight carries out bacterium colony PCR qualification respectively, reaction takes 5 μ l product after terminating and carries out agarose gel electrophoresis detection, determining positive colony, order-checking also extracts plasmid with the little extraction reagent kit of plasmid, it is thus achieved that recombinant expressed sub-pET-28b-abf51A and pET-28b-axeA.
1.7 recombinant plasmid transformed E. coli Rosetta (DE3)
In recombinant plasmid transformed E. coli Rosetta (DE3) competent cell, on the LB flat board containing 50 μ g/ml kanamycin, the single bacterium colony of 12-16h acquisition will be cultivated for 37 DEG C respectively.3-5 monoclonal of picking contains overnight incubation in 50 μ g/ml kanamycin LB fluid mediums in 5ml, strain is preserved with glycerol, obtain with pET-28b for carrier respectively, the α-l-arabfuranglycosidase built with E.coliRosetta (DE3) for Host Strains and acetyl xylan esterase engineering bacteria.
Embodiment 2: restructuring α-l-arabfuranglycosidase Abf51A and acetyl xylan esterase AxeA expression in escherichia coli
Recombinant bacterium E.coliRosetta (DE3) is inoculated in the 5ml LB fluid medium containing 50 μ g/ml kanamycin by inoculum concentration by 1% respectively, and 37 DEG C of 200rpm shaken cultivation are overnight.Being transferred to the 200ml LB fluid medium containing 50 μ g/ml kanamycin by same inoculum concentration, 37 DEG C of 200rpm shaken cultivation are to OD600When reaching about 0.4-0.6, add IPTG to final concentration 0.1mM, continue 37 DEG C of 200rpm shaken cultivation 4-6h.Cultivation terminates the centrifugal 15min of rear 4000g and collects thalline, adds 30mlBindingBuffer (50mMTris-HClpH7.5,300mMNaCl) resuspended collection thalline.After ultrasonication, 4 DEG C of centrifugal 15min gained supernatants of 10000g are crude enzyme liquid.
Embodiment 3: restructuring α-l-arabfuranglycosidase Abf51A and acetyl xylan esterase AxeA expression in trichoderma reesei
With C.lactoaceticus6A genome for template, design primer amplification respectively obtains α-l-arabfuranglycosidase gene abf51A and acetyl xylan esterase gene axeA, it is connected into carrier pSKCST, respectively obtains expression vector pSKCST-abf51A and pSKCST-axeA.To convert T.reesei protoplast after expression vector pSKCST-abf51A and pSKCST-axeA linearisation respectively, after transformant grows, extract transformant genomic DNA, PCR identifies transformant exogenous origin gene integrator situation.With sickle, the transformant bacterium colony on selectivity flat board is cut into the truffle that diameter is 0.3cm, transfers to EG after taking 5 pieces from each transformant and screen flat board, cultivate 2-3d for 28 DEG C.Timing is observed, and records growth diameter.Therefrom select the transformant that growth rate is fast, colony diameter is big.The transformant and the original strains that obtain screening line PDA inclined-plane respectively, cultivate 5-7d for 28 DEG C.After Sporulation, wash spore with 5ml sterilized water.Being inoculated in the triangular flask containing seed culture medium by spore suspension, 28 DEG C of 200rpm cultivate 48h.Centrifugal filtration thalline, is transferred to culture medium, inoculum concentration 10%, liquid amount 50ml/250ml shaking flask, and 28 DEG C of 200rpm cultivate 48h, timing sampling detection supernatant enzyme activity.
Embodiment 4: the purification of restructuring α-l-arabfuranglycosidase Abf51A and acetyl xylan esterase AxeA
Utilize recombinase C, N-end to contain histidine-tagged, by 50 DEG C of heat inactivations, Ni-NAT affinity chromatograph, recombiant protein is easily separated.50 DEG C of heating in water bath of crude enzyme liquid being processed 30min and removes thermally labile albumen, 4 DEG C of centrifugal 20min of 10000g take supernatant.After balancing 500 μ lNi-NAT affinity columns with 5mlBindingBuffer, supernatant is crossed post.First wash with 5mlBindingBuffer after crossing post, then with 500 μ lElutionBuffer (50mMTris-HClpH7.5,300mMNaCl, 150mM imidazoles) eluting, collect the sample liquid of eluting.With citrate buffer solution (50mM citrate pH6.0,150mMNaCl) pre-balance 30mlSuperdex200 sephadex column, then elution samples liquid is crossed post, detect when crest occurs and collect albumen.Eluent is all citrate buffer solution (50mM citrate pH6.0,150mMNaCl), and flow velocity is set to 7.5rpm.Finally adopt the super filter tube of 30KDa molecular cut off by sample concentration to 500 μ l.Take each sample 20 μ l operating period and add 4 μ l5 × protein electrophoresis sample-loading buffer, fully boil the centrifugal 15min of 15min, 10000g after mixing, take 10 μ l supernatants and carry out expression and the purification situation of SDS-PAGE electrophoresis detection destination protein.Result is as it is shown in figure 1, purified purity of protein is higher, and restructuring α-l-arabfuranglycosidase molecular weight is 70KDa, and acetyl xylan esterase molecular weight is 30KDa.After purification, α-l-arabfuranglycosidase concentration is 0.35mg/ml, and acetyl xylan esterase concentration is 0.63mg/ml.
Embodiment 5: the zymologic property of restructuring α-l-arabfuranglycosidase Abf51A and acetyl xylan esterase AxeA measures
The mensuration of 5.1 optimal reactive temperatures
The enzyme liquid that 5 μ l suitably dilute is added in the 95 μ l citrate buffer solution of pH5.5 containing 0.5mM substrate, under 40-100 DEG C of reaction condition, measures the vigor of restructuring α-l-arabfuranglycosidase Abf51A, response time 3min.The enzyme liquid that 5 μ l suitably dilute is added in the 95 μ l citrate buffer solution of pH6.5 containing 0.5mM substrate, measures the vigor of restructuring acetyl xylan esterase AxeA, response time 5min under 40-100 DEG C of reaction condition.Abf51A and AxeA optimum temperature is 80 DEG C and 70 DEG C, Abf51A and have within the scope of temperature is 60-85 DEG C more than 55% activity, AxeA has the activity (Fig. 2) of more than 50% within the scope of temperature is 40-75 DEG C.
The mensuration of 5.2 optimal reaction pH
The enzyme liquid that 5 μ l suitably dilute is added separately in the 95 μ l citric acid containing 0.5mM substrate or phosphate buffer (pH4.0-8.5), restructuring α-l-arabfuranglycosidase Abf51A or acetyl xylan esterase AxeA vigor, response time difference 3 or 5min is measured under 80 DEG C of reaction conditions.The optimum pH of Abf51A and AxeA respectively 5.5 and 6.5, pH be have within the scope of 4.5-6.5 more than 60% activity (Fig. 2).
The mensuration of 5.3 heat stability
By recombinase Abf51A at 75,80,85 DEG C, process 0.5,1,2,4,6 and 12h.Recombinase AxeA, at 65,70,75 DEG C, processes 0.5,1,2,4,6 and 12h.Measure the two residual activity respectively.Result, as in figure 2 it is shown, Abf51A retains the activity of 63% after hatching 12h at 75 DEG C, retains the activity of 57%, and retains the activity of 57% after hatching 30min at 85 DEG C after hatching 12h at 80 DEG C；AxeA remains to retain the activity of 84% after hatching 12h at 65,70 and 75 DEG C.
Embodiment 6: restructuring α-l-arabfuranglycosidase Abf51A and acetyl xylan esterase AxeA and xylanase Xyn10A synergetic hydrolysis Herba bromi japonici xylan
Xylanase Xyn10A derives from pyrolysis CELLULOLYTIC BACTERIUM C.lactoaceticus6A equally, and optimum temperature and pH be 80 DEG C and 6.5 (X.J.Jiaetal.2014) respectively.Weighing 0.1g Herba bromi japonici xylan to join in 100 μ lpH6.5 citrate buffer solutions, be separately added into a certain amount of enzyme liquid by following experiment packet, matched group adds the citrate buffer solution of the pH6.5 of equivalent.After being mixed by reactant liquor, under 75 DEG C of conditions, 80rpm vibration is hydrolyzed 12h.All samples is boiled 10min inactivation after terminating by reaction, and 4000g is centrifuged 10min.Take the composition of the 10 μ l all experimental grouies of supernatant high-efficient liquid phase chromatogram technique analysis and matched group Herba bromi japonici xylan digest.
Table 2 experiment packet
Result as it is shown on figure 3, this restructuring α-l-arabfuranglycosidase Abf51A 75 DEG C, pH6.5 when, it is possible to the rapidly aralino on degraded Herba bromi japonici xylan side chain, generate a small amount of arabinose；With acetyl xylan esterase AxeA synergism, arabinose growing amount increases by 3.0%；With xylanase Xyn10A synergism, arabinose growing amount increases by 85.4%.This restructuring acetyl xylan esterase AxeA 75 DEG C, pH6.5 when, the acetyl group on Herba bromi japonici xylan side chain of degrading, generate a small amount of acetic acid；With α-l-arabfuranglycosidase Abf51A synergism, xylose growing amount increases by 33.5%, and with xylanase Xyn10A synergism, xylose growing amount increases ratio and is individually hydrolyzed increase by 68.9% with xylanase Xyn10A.When utilizing 3 kinds of enzyme hydrolysiss, xylose growing amount increases uses xylanase Xyn10A to be hydrolyzed increase by 139.9% than individually, and arabinose growing amount increases ratio and individually uses xylanase Xyn10A to be hydrolyzed increase by 138.8% simultaneously.
Embodiment 7: utilize restructuring α-l-arabfuranglycosidase Abf51A and acetyl xylan esterase AxeA and xylanase Xyn10A Synergistic degradation producing xylose oligomer from bagasse
From bagasse, extract xylan by alkali density method, prepare bagasse xylan suspension, 2h, 12000g centrifugal 10min is stirred at room temperature, takes supernatant and be solubility bagasse xylan.Diluting solubility bagasse xylan solution with the citrate buffer solution of pH6.5, making reaction system xylan mass concentration is 0.5%.Being separately added into a certain amount of enzyme liquid by following experiment packet, matched group adds the citrate buffer solution of the pH6.5 of equivalent.After being mixed by reactant liquor, under 75 DEG C of conditions, 80rpm vibration is hydrolyzed 12h.All samples is boiled 10min inactivation after terminating by reaction, and 4000rpm is centrifuged 10min.Take the composition of the 10 μ l all experimental grouies of supernatant high-efficient liquid phase chromatogram technique analysis and matched group bagasse xylan digest.
Table 3 experiment packet
Result as shown in Figure 4, this restructuring α-l-arabfuranglycosidase Abf51A 75 DEG C, pH6.5 when, it is possible to the rapidly aralino on degraded Caulis Sacchari sinensis xylan side chain, generate a small amount of arabinose；With acetyl xylan esterase AxeA synergism, arabinose growing amount increases by 45.7% than individually with α-l-arabfuranglycosidase；With xylanase Xyn10A synergism, arabinose growing amount increases by 16.0% than individually with xylanase.This restructuring acetyl xylan esterase AxeA 75 DEG C, pH6.5 when, the acetyl group on Herba bromi japonici xylan side chain of degrading, generate a small amount of acetic acid；With xylanase Xyn10A synergism, xylose growing amount increases ratio and is individually hydrolyzed increase by 90.3% with xylanase Xyn10A.When utilizing 3 kinds of enzyme hydrolysiss, xylose growing amount increases uses xylanase Xyn10A to be hydrolyzed increase by 237.4% than individually, and arabinose growing amount increases ratio and individually uses xylanase Xyn10A to be hydrolyzed increase by 418% simultaneously.
Embodiment 8: utilize restructuring α-l-arabfuranglycosidase Abf51A and acetyl xylan esterase AxeA to produce xylose and arabinose with xylanase Xyn10A Synergistic degradation pretreatment corn cob
Weighing the 1.0g corn cob slag through high temperature steaming, join in 100 μ lpH6.5 citrate buffer solutions, be separately added into a certain amount of enzyme liquid by following experiment packet, matched group adds the citrate buffer solution of the pH6.5 of equivalent.After being mixed by reactant liquor, under 75 DEG C of conditions, 80rpm vibration is hydrolyzed 12h.All samples is boiled 10min inactivation after terminating by reaction, and 4000g is centrifuged 10min.Take monosaccharide component in the 10 μ l all experimental grouies of supernatant high-efficient liquid phase chromatogram technique analysis and matched group Corncob hydrolysate.
Table 4 experiment packet
Result is as it is shown in figure 5, the content of xylose and arabinose is all along with that reacts being deeply gradually increased in experimental group hydrolyzate.This restructuring α-l-arabfuranglycosidase Abf51A 75 DEG C, pH6.5 when, it is possible to the rapidly aralino on degrading maize core xylan side chain, generate a small amount of arabinose；With acetyl xylan esterase AxeA synergism, arabinose growing amount increases by 6.0% than individually with α-l-arabfuranglycosidase；With xylanase Xyn10A synergism, arabinose growing amount is than being individually hydrolyzed increase by 17.5% with xylanase Xyn10A.This restructuring acetyl xylan esterase AxeA 75 DEG C, pH6.5 when, the acetyl group on Herba bromi japonici xylan side chain of degrading, generate a small amount of acetic acid；With α-l-arabfuranglycosidase Abf51A synergism, xylose growing amount is than individually increasing by 197.1% with acetyl xylan esterase, and with xylanase Xyn10A synergism, xylose growing amount increases ratio and is individually hydrolyzed increase by 86.3% with xylanase Xyn10A.When utilizing 3 kinds of enzyme hydrolysiss, xylose growing amount increases uses xylanase Xyn10A to be hydrolyzed increase by 131.7% than individually, and arabinose growing amount increases ratio and individually uses xylanase Xyn10A to be hydrolyzed increase by 69.3% simultaneously.Corn cob can be degraded to oligomeric xylose, xylose and arabinose with xylanase cooperation by restructuring α-l-arabfuranglycosidase and acetyl xylan esterase fast and effectively.By controlled enzymatic hydrolysis time, enzyme liquid consumption and pretreatment corn cob content, it is possible to prepare the oligomeric xylose, xylose and the arabinose that meet the degree of polymerization and require.
Embodiment 9: utilize restructuring α-l-arabfuranglycosidase Abf51A and acetyl xylan esterase AxeA to produce oligomeric xylose with xylanase Xyn10A Synergistic degradation sweet potato waste
Take 5g destarched sweet potato meal and add in 100ml1%NaOH, in 85 DEG C of water-baths vibration 1h.Wash the granulated slag after process with water neutral to pH, put and baking oven is dried to constant weight.Weigh a certain amount of dry process granulated slag raw material, add citrate buffer solution (50mM citrate pH6.5,150mMNaCl) according to the ratio of solid-liquid ratio (g:ml) 1:10.Experimental group one adds a certain amount of xylanase Xyn10A, and experimental group two adds the mixed enzyme solution of a certain amount of restructuring α-l-arabfuranglycosidase Abf51A, acetyl xylan esterase AxeA and xylanase Xyn10A.By reactant liquor mix after under 75 DEG C of conditions 80rpm vibrate enzymolysis 24h.Hydrolyzed solution 4000g is centrifuged 10min after terminating by reaction.Take all experimental grouies of supernatant high-efficient liquid phase chromatogram technique analysis and matched group Corncob hydrolysate component.Experimental result shows, experimental group one enzymolysis solution is mainly composed of oligomeric xylose and xylose, and wherein xylooligosaccharide content is 82.4%, and Xylose Content is 17.6%, and sweet potato waste granulated slag degradation rate reaches 70.9%.Experimental group two enzymolysis solution is mainly composed of oligomeric xylose, xylose and arabinose, in enzymatic hydrolysate, xylooligosaccharide content is 27.4%, Xylose Content is 47.3%, arabinose content is 20.3%, sweet potato waste granulated slag degradation rate reaches 93.6%, hydrolytic degradation rate raising 32.0% more independent than this xylanase Xyn10A.Radix Ipomoeae granulated slag can be degraded to oligomeric xylose, xylose and arabinose with xylanase synergism by restructuring α-l-arabfuranglycosidase and acetyl xylan esterase fast and effectively.
Claims (11)
1. a high temperature α-l-arabfuranglycosidase gene abf51A, its gene order is such as shown in SEQIDNO:1.
2. the high temperature α-l-arabfuranglycosidase Abf51A of the high temperature α-l-arabfuranglycosidase gene expression described in claim 1, its aminoacid sequence is such as shown in SEQIDNO:2.
3. a high temperature acetyl xylan esterase gene axeA, its gene order is such as shown in SEQIDNO:3.
4. the high temperature acetyl xylan esterase AxeA of the high temperature acetyl xylan esterase gene expression described in claim 3, its aminoacid sequence is such as shown in SEQIDNO:4.
5. the method for expanding the high temperature α-l-arabfuranglycosidase gene abf51A described in claim 1, it is characterised in that the primer used is:
abf51A-F5’-GCCGCGCGGCAGCATGAAAAAAGCAAAAGTCATCTAC-3’
abf51A-R5’-GCGGCCGCAAGCGTTTAATTTTCTTTCTTCTTTAACCTG-3’。
6. the method for expanding the high temperature acetyl xylan esterase gene axeA described in claim 3, it is characterised in that the primer used is:
axeA-F5’-GCCGCGCGGCAGCATGATACCACTTTGGGAAAATC-3’
axeA-R5’-GCGGCCGCAAGCGTTTAAACATTATATCCTATAAACC-3’。
7. a high temperature α-l-arabfuranglycosidase recombinant vector and recombination engineering, it is characterised in that containing the α-l-arabfuranglycosidase gene abf51A described in claim 1.
8. a high temperature acetyl xylan esterase recombinant vector and recombination engineering, it is characterised in that containing the acetyl xylan esterase gene axeA described in claim 3.
9. the application on enzymolysis natural wood polysaccharide and lignocellulosic material of the high temperature α-l-arabfuranglycosidase described in claim 2, the optimum temperature of its enzymolysis and pH respectively 80 DEG C and 5.5, in enzymatic hydrolysate, arabinose content is 10-18%, xylooligosaccharide content is 67-87%, and Xylose Content is 3-15%.
10. the application on enzymolysis natural wood polysaccharide and lignocellulosic material of the high temperature acetyl xylan esterase described in claim 4, the optimum temperature of its enzymolysis and pH respectively 70 DEG C and 6.5, in enzymatic hydrolysate, acetic acid content is 2-10%, and xylooligosaccharide content is 75-95%, and Xylose Content is 3-15%.
11. the high temperature α-l-arabfuranglycosidase described in claim 2 and the high temperature acetyl xylan esterase described in claim 4, the application on enzymolysis natural wood polysaccharide and lignocellulosic material is acted on xylanase coordinated enzymatic hydrolysis, in enzymatic hydrolysate, arabinose content is 10-25%, acetic acid content is 5-15%, xylooligosaccharide content is 5-45%, and Xylose Content is 40-75%.
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| CN106422963A (en) * | 2016-09-09 | 2017-02-22 | 南京工业大学 | Application of acetyl xylan esterase (AXE) as emulsifying agent |
| CN109402089A (en) * | 2018-11-06 | 2019-03-01 | 江苏大学 | A kind of thermostable type arabinofuranosidase and its application |
| CN109797178A (en) * | 2019-01-09 | 2019-05-24 | 中国农业科学院农产品加工研究所 | The preparation method of wood oligose |
| CN109868292A (en) * | 2019-01-09 | 2019-06-11 | 中国农业科学院农产品加工研究所 | The method that single enzymatic prepares ferulic acid and xylose |
| CN110157696A (en) * | 2019-05-05 | 2019-08-23 | 云南与诺生物工程有限责任公司 | α-l-arabfuranglycosidase and its encoding gene and application |
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| CN109402089A (en) * | 2018-11-06 | 2019-03-01 | 江苏大学 | A kind of thermostable type arabinofuranosidase and its application |
| CN109797178A (en) * | 2019-01-09 | 2019-05-24 | 中国农业科学院农产品加工研究所 | The preparation method of wood oligose |
| CN109868292A (en) * | 2019-01-09 | 2019-06-11 | 中国农业科学院农产品加工研究所 | The method that single enzymatic prepares ferulic acid and xylose |
| CN110157696A (en) * | 2019-05-05 | 2019-08-23 | 云南与诺生物工程有限责任公司 | α-l-arabfuranglycosidase and its encoding gene and application |
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