CN101525607B - Preparation method of high-temperature xylanase - Google Patents
Preparation method of high-temperature xylanase Download PDFInfo
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- CN101525607B CN101525607B CN2009100943404A CN200910094340A CN101525607B CN 101525607 B CN101525607 B CN 101525607B CN 2009100943404 A CN2009100943404 A CN 2009100943404A CN 200910094340 A CN200910094340 A CN 200910094340A CN 101525607 B CN101525607 B CN 101525607B
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Abstract
The invention relates to the field of biotechnology. A preparation method of high-temperature xylanase comprises the following steps: (1) preparation of crude enzyme: inoculating Thermobifida halotolerans YIM 90462<T> into liquid seed culture medium with inoculum size by 5 to 10 percent of volume, shaking culturing, transferring the Thermobifida halotolerans YIM 90462<T> into liquid fermentation medium by 5 to 10 percent of volume and obtaining crude enzyme; (2) concentrating the volume of the crude enzyme to 1/6 of original volume thereof by an ultra-filtration membrane package with molecular weight cut-off of 10 KDa; (3) adding ammonium sulfate into the concentrated enzyme to lead the saturation thereof to be 50 percent, obtaining precipitates by centrifugation and dissolving the precipitates in buffer solution; (4) taking supernatant fluid from the obtained liquid by centrifugation, adding the supernatant fluid into a Butyl-Sepharose chromatography column which is balanced by Tris-HCl buffer solution containing 0.3M of ammonium sulfate and having pH of 8.0; (5) taking supernatant fluid from the obtained liquid by centrifugation, adding the supernatant fluid into a Phenyl-Sepharose chromatography column which is balanced by Tris-HCl buffer solution containing 0.3M of ammonium sulfate and having pH of 8.0 and eluting the supernatant fluid with Tris-HCl buffer solution which does not contain ammonium sulfate and has pH of 8.0; (6) adding the supernatant fluid into Q-Sepharose chromatography column which is balanced by Tris-HCl buffer solution having pH of 8.0, collecting eluting peaks successively according to retention time, and remaining active components of the xylanase; and finally obtaining the xylanase with the activation of 573U/ml and purification yield of 9 percent.
Description
Technical field
The present invention relates to biological technical field.
Background technology
Xylan is a kind of hemicellulose, says it is second largest natural polysaccharide from content.Xylan class hemicellulose is the carbohydrate that a class is renewable and demand urgently developing, the wood sugar that after biological degradation, is produced and a small amount of other monose, can produce various leavened prods as basic carbon source, comprise organic acid, amino acid, single cell protein, sugar alcohol, industrial enzyme, solvent or fuel alcohol.The zytase owner will comprise two kinds of enzymes, and a kind of is β-1, and 4-zytase, another kind are beta-xylanases, and the two acting in conjunction can become monose with xylan degrading.Zytase has vital role in bio-transformation, food, medicine, the energy, papermaking, fodder industry.The microorganism that can produce zytase has a variety of: fungi, bacterium, actinomycetes all can produce zytase etc.According to domestic and international research report, the actinomycetes that produce zytase mainly contain Streptomyces lividans, Thermobifidafusca etc.; Bacterium mainly contains Bacillus subtilis, Pseudomonas fluorescents etc.Produce the zytase fungi and mainly contain Aspergillusniger, Aspergillus nidulans, Aspergillus phoenicis, Trichoderma reesei, Trichoderma koningii.
The zytase physico-chemical property of institute's microorganisms of (genus) not of the same race has nothing in common with each other, and molecular weight all has distribution in 9500-243000 dalton, iso-electric point at 3.6-10.3.Generally about 50 ℃, optimum pH is followed its microbe-derived difference and difference to enzyme optimum temperuture alive, and majority is pH 4.0-6.0, and the microorganism that produces alkalescent xylanase is less.
The capacity variance of the production by biological zytase of (genus) not of the same race is also very big.Compare with the xylanase activity that every milliliter of nutrient solution was produced in the liquid fermenting in batches.In the foreign literature report, the Bacillus sp.XE of people such as Samain E report produce the highest vigor of zytase be 380IU/ml (1997, Journal of Biotechnology 58,71-78); The high yield enzyme activity of the Bacillus circulans AB 16 of people such as Dhillon A report be 55IU/ml (2000, Process Biochemistry, 35,849-856); The highest enzyme of the Thermoactinomyces thalphilus subgroup C. of people such as kohli U report live for 42IU/ml (2001, Enzyme and Microbial Technology 28,606-610).In the domestic report, it is the highest by Bacillus pumilus A30 generation Xylanase activity that people such as Qu Yinbo report, is 431IU/ml (2000, Chinese biological chemistry and molecular biosciences journal).
The temperature tolerance of different microorganisms product zytase is widely different.Because heat-resisting or fire resistant xylanase has many good qualities in industrial application, so more about the patent documentation report of this respect in recent years.Disclose a kind of thermostable xylanases as CN1143387, the zytase of introduction is having activity more than 80 ℃.CN00814240.8 discloses the method for the thermostability of improving G11 family zytase, and has expanded its pH stability boundary.Chinese patent application numbers 200710065854.8 discloses a kind of " method of producing heat resistant xylanase with genetically recombinant Pichia yeast ", be from aspergillus niger (Aspergillusnigervarniger strain N402), to isolate xylanase gene, in the eukaryotic expression system pichia spp, efficiently express.Used aspergillus niger Aspergillus nigervarniger strain N402 xylanase gene, adopted yeast expression vector PGAP Z alphaA and host strain Pichia pastoris (Pichia pastoris) SMD1168, GS115, constructed gene engineering yeast has the ability of producing fire resistant xylanase, constructed gene engineering yeast removes has the form of host strain SMD1168 and GS115, outside heredity and the physiological and biochemical property, also comprise aspergillus niger Aspergillus nigervarnigerstrain N402 xylanase gene, have the ability of producing fire resistant xylanase.
Industrial, zytase has been widely used in the bleaching of paper pulp.In bleaching process, need have zytase high temperature resistant, alkaline-resisting and that molecular weight is low.Though the microorganism that can produce zytase is a lot,, on industrial application, be subjected to considerable restraint because the zytase of most microorganisms is middle low temperature or slant acidity.Thereby, the exploitation and the research of zytase with these character is just seemed very necessary.
Summary of the invention
The object of the present invention is to provide a kind of method for preparing high-temperature xylanase by the generation of microbial strains fermented liquid, separation, purifying.
The preparation method of high-temperature xylanase of the present invention is made up of following steps:
One, the preparation of crude enzyme liquid: with Thermobifida halotolerans YIM 90462
TInoculum size with the volume percent of 5-10% inserts in the liquid seed culture medium, shaking culture is to exponential phase of growth under 37～45 ℃ condition, the bacterium liquid that will be in exponential phase of growth is transferred in liquid fermentation medium with the inoculum size of the volume percent of 5-10%, be cultured to initial stage stationary phase at 37～45 ℃, fermented liquid is centrifugal, get supernatant liquor, filter paper filtering gets crude enzyme liquid;
Three, be that the ultra-filtration membrane bag of 10KDa is concentrated into original 1/6 with the volume of crude enzyme liquid with molecular weight cut-off;
Three, the enzyme liquid after concentrating adds ammonium sulfate to 30% saturation ratio, and the centrifuging and taking supernatant liquor continues to add ammonium sulfate to 50% saturation ratio, and the centrifuging and taking precipitation is dissolved in the Tris-HCl damping fluid of pH8.0,0.3M ammonium sulfate;
Four, with step (three) gained liquid centrifuging and taking supernatant liquor, be added to the good Butyl-Sepharose chromatography column of Tris-HCl damping fluid balance of pH8.0,0.3M ammonium sulfate, to collect respectively by the priority of retention time through the peak, flow through 8-10 chromatography column volume until damping fluid, what remain with xylanase activity sees through peak enzyme liquid part;
Five, with step (four) gained liquid centrifuging and taking supernatant liquor, be added to the good Phenyl-Sepharose chromatography column of Tris-HCl damping fluid balance of pH8.0,0.3M ammonium sulfate, with the Tris-HCl buffer solution elution of no ammonium sulfate pH8.0, collect all elution peak components;
Six, with step (five) gained liquid centrifuging and taking supernatant liquor, be added to the good Q-Sepharose chromatography column of Tris-HCl damping fluid balance of pH8.0, with the Tris-HCl buffer solution elution that contains NaClpH8.0, at first make elutriant electricity dodar to 30mS/cm, elution peak is collected by the priority of retention time, flow through 5-10 chromatography column volume until damping fluid, remain with the part of xylanase activity, be product of the present invention.
The described Thermobifida halotolerans of step 1 of the present invention YIM 90462
TBe that salt tolerant high temperature bifidus bacterium (is published the sp.nov. in Thermobifida halotolerans, isolated from a salt mine sample, and emended description ofthe genus hermobifida Yang et al.Int J SystEvol Microbiol.2008; 58:1821-1825).Described liquid seed culture medium and liquid fermentation medium all can adopt existing substratum, but should contain the AVICEL that the quality volume percent is 0.1-2% (w/v) in liquid fermentation medium, only in this way could guarantee the generation of enzyme of the present invention.Be 2-3 days described exponential phase of growth, and described initial stage stationary phase is 5-7 days, is to produce the enzyme peak period when being cultured to initial stage stationary phase.Every milliliter of fermented liquid activity can reach 7U.
Related xylanase activity measuring method reference M.J.Bailey in the above-mentioned steps four, six, P.Biely, K.Poutanen, Journal of Biotechnology 23 (1992) 257-270.Concrete grammar is dissolved in the slow damping fluid of the different pH6.0 citric acid-SODIUM PHOSPHATE, MONOBASIC of 50ml for the birch xylan that accurately takes by weighing 0.50g.Getting above-mentioned substrate solution 80 μ l and be loaded in the Eppendorf pipe, add the suitable dilution enzyme liquid of 20 μ l, is contrast with the enzyme liquid of deactivation.70 ℃ of reaction 30min.After reaction finishes, add DNS rapidly and measure concentration of reduced sugar.The DNS method is measured concentration of reduced sugar reference Use ofdinitrosalicylic acid reagentfor determination of reducing sugar.ANALYTICAL CHEMISTRY.1 959.
The advantage of the high-temperature xylanase that is prepared by salt tolerant high temperature bifidus bacterium of the present invention is to have higher enzyme activity under 70-80 ℃, in papermaking, bio-transformation industry certain industrial application value is arranged; All all use a kind of damping fluid (pH8.0 50mMTris-HCl) in the separation and purification process, and the separation and purification process is simple.
High-temperature xylanase of the present invention has following zymologic property:
1, to produce the suitableeest enzyme of the high-temperature xylanase temperature of living be 80 ℃ to salt tolerant high temperature bifidus bacterium, and purification yield is more than 9%, every liter of purified pure enzyme of 1159U that obtains of fermented liquid.
2, high-temperature xylanase vigor between 4～70 ℃ of salt tolerant high temperature bifidus bacterium generation is comparatively stable, reaches 573.3U/ml, 70 ℃ of insulations 2 hours, also can keep the enzyme activity more than 80%, reaches 458.6 U/ml; When temperature surpassed 75 ℃, enzyme activity descended very fast, and it is nearly 80% to cause enzyme activity to descend at 75 ℃ of insulation 120min, reaches 1 14.6 U/ml; It should be noted that zytase at 90 ℃ of insulation 40min, enzyme is lived and still can be preserved originally 30%, reaches 171.99 U/ml, shows that salt tolerant high temperature bifidus bacterium produces zytase and belongs to the typical high temperature zytase.
3, salt tolerant high temperature bifidus bacterium high-temperature xylanase optimal reaction pH is 6.0, at pH less than 4 or greater than 10 o'clock, enzyme activity was lower.
4, it is stable that salt tolerant high temperature bifidus bacterium produces high-temperature xylanase enzyme activity between pH6.0～10.0, incubated overnight in the damping fluid of pH6.0～10.0, and its residual enzyme activity reaches 458.6 U/ml all more than 80%.
5, Mn
2+, K
+, Ba
2+, Fe
2+Salt tolerant high temperature bifidus bacterium high-temperature xylanase there is activation, Li
+, Cd
2+, Mg
2+, Zn
2+, Mn
2+, Ca
2+, Na
+, Pb
2+, Co
2+, Al
3+And Fe
3+Not obvious to the enzyme effect, Ni
2+, Cu
2+, Hg
2+Enzyme there is significant inhibitory effect.
6, denaturing agent SDS has the obvious suppression effect to salt tolerant high temperature bifidus bacterium high-temperature xylanase, the single-minded inhibitor phenylmethyl sulfonylfluoride of intercalating agent EDTA and serine protease (PMSF) produces high-temperature xylanase to salt tolerant high temperature bifidus bacterium does not have restraining effect, has shown that salt tolerant high temperature bifidus bacterium produces high-temperature xylanase and do not have dependency and its active centre not to contain Serine to metal ion.
Description of drawings
Fig. 1 is that salt tolerant high temperature bifidus bacterium produces the temperature of reaction of high-temperature xylanase and the relation curve of relative reactivity.
Fig. 2 is that salt tolerant high temperature bifidus bacterium produces the reaction pH of high-temperature xylanase and the relation curve of relative reactivity.
Fig. 3 is the thermostability curve that salt tolerant high temperature bifidus bacterium produces high-temperature xylanase.
Fig. 4 is the pH beta stability line that salt tolerant high temperature bifidus bacterium produces high-temperature xylanase.
Among above-mentioned Fig. 2:
◆ expression pH3.0,3.6,4.8 acetic acid-sodium-acetate buffer.
■ represents pH4,5,6,7,7.6 citric acids-phosphate sodium dihydrogen buffer solution.
▲ expression pH 7.5,8.5 Tris-HCl damping fluids.
● expression pH9,10 glycine-sodium hydrate buffer solution.
Embodiment
Embodiment 1,
One, the preparation of crude enzyme liquid: with Thermobifida halotolerans YIM 90462
TInsert in the liquid seed culture medium with inoculum size 10% (volume percent), shaking culture 36 hours is to exponential phase of growth under 50 ℃ condition, the bacterium liquid that will be in exponential phase of growth is transferred in liquid fermentation medium with inoculum size 10% (volume percent), be cultured to 5-7 days to initial stage stationary phase and be at 37～45 ℃ and produce the enzyme peak period, fermented liquid is centrifugal, get supernatant liquor, filter paper filtering gets crude enzyme liquid;
Two, be that the ultra-filtration membrane bag of 10KDa is concentrated into original 1/6 with the volume of crude enzyme liquid with molecular weight cut-off;
Three, the enzyme liquid after concentrating adds ammonium sulfate to 30% saturation ratio, and the centrifuging and taking supernatant liquor continues to add ammonium sulfate to 50% saturation ratio, and the centrifuging and taking precipitation is dissolved in the Tris-HCl damping fluid of pH8.0,0.3M ammonium sulfate;
Four, step (three) gained liquid centrifuging and taking supernatant liquor is added to the good Butyl-Sepharose chromatography column of Tris-HCl damping fluid balance of pH8.0,0.3M ammonium sulfate, to collect a pipe for per four milliliters by the priority of retention time through the peak, flow through 10 chromatography column volumes until damping fluid, according to the method among reference Journal of Biotechnology 23 (1992) 257-270 enzyme liquid in each collected pipe is carried out zytase and measure reserve glycanase enzyme liquid part;
Five, step (four) gained liquid centrifuging and taking supernatant liquor is added to the good Phenyl-Sepharose chromatography column of Tris-HCl damping fluid balance of pH8.0,0.3M ammonium sulfate,, collects all elution peak components with the Tris-HCl buffer solution elution of no ammonium sulfate pH8.0;
Six, step (five) gained liquid centrifuging and taking supernatant liquor is added to the good Q-Sepharose chromatography column of Tris-HCl damping fluid balance of pH8.0, with the Tris-HCl buffer solution elution that contains NaClpH8.0, at first make elutriant electricity dodar to 30mS/cm, elution peak is collected a pipe for per four milliliters by the priority of retention time, flow through 10 chromatography column volumes until damping fluid, remain with the part of xylanase activity, be product of the present invention.
By substrate specificity Journal of Sex Research to this product, different substrates are respectively birch xylan, carboxymethyl cellulose, PNPG (4-Nitrophenyl β-D-glucopyranoside) and AVICEL (Microcrystalline Cellulose), find that its xylanase activity is higher than its cellulase activity far away, can determine that this product is typical zytase.
In the substrate specificity test, cellulase activity measuring method reference Methods for Measuring CellulaseActivities.T.M.WOOD et al.METHODS IN ENZYMOLOGY, VOL.160.Xylanase activity reference Journal of Biotechnology 23 (1992) 257-270.
The composition of the described seed culture medium in step 1:
10 * a large amount of salts solution 100ml
50 * trace amount salts solution 20ml
Peptone 5g
Add water and be settled to 1L.
10 * a large amount of salts solution prescriptions above-mentioned are:
NaCl 15g
(NH
4)
2SO
4 31g
Na
2HPO
4 91g
Add water and be settled to 1L.
50 * trace amount salts solution prescription above-mentioned is:
EDTA 0.5g
MgSO
4 2.00g
ZnSO
4 0.08g
FeSO
4 0.2g
MnSO
4 0.15g
CaCl
2 0.2g
Add water and be settled to 200ml.
The composition of the said fermention medium in step 1:
Peptone 10g
Yeast 5g
NaCl 10g
AVICEL (Microcrystalline Cellulose sigma company) 1-20g
Add water and be settled to 1L.
Claims (1)
1. the preparation method of a high-temperature xylanase is characterized in that being made up of following steps:
One, the preparation of crude enzyme liquid: with Thermobifida halotolerans YIM 90462
TInoculum size with the volume percent of 5-10% inserts in the liquid seed culture medium, shaking culture is to exponential phase of growth under 37～45 ℃ condition, the bacterium liquid that will be in exponential phase of growth is transferred in liquid fermentation medium with the inoculum size of the volume percent of 5-10%, contain the AVICEL that the quality volume percent is 0.1-2% in the described liquid fermentation medium, be cultured to initial stage stationary phase at 37～45 ℃, fermented liquid is centrifugal, get supernatant liquor, filter paper filtering gets crude enzyme liquid;
Two, be that the ultra-filtration membrane bag of 10KDa is concentrated into original 1/6 with the volume of crude enzyme liquid with molecular weight cut-off;
Three, the enzyme liquid after concentrating adds ammonium sulfate to 30% saturation ratio, and the centrifuging and taking supernatant liquor continues to add ammonium sulfate to 50% saturation ratio, and the centrifuging and taking precipitation is dissolved in the Tris-HCl damping fluid of pH8.0,0.3M ammonium sulfate;
Four, with step (three) gained liquid centrifuging and taking supernatant liquor, be added to the good Butyl-Sepharose chromatography column of Tris-HCl damping fluid balance of pH8.0,0.3M ammonium sulfate, to collect respectively by the priority of retention time through the peak, flow through 8-10 chromatography column volume until damping fluid, what remain with xylanase activity sees through peak enzyme liquid part;
Five, with step (four) gained liquid centrifuging and taking supernatant liquor, be added to the good Phenyl-Sepharose chromatography column of Tris-HCl damping fluid balance of pH8.0,0.3M ammonium sulfate, with the Tris-HCl buffer solution elution of no ammonium sulfate pH8.0, collect all elution peak components;
Six, with step (five) gained liquid centrifuging and taking supernatant liquor, be added to the good Q-Sepharose chromatography column of Tris-HCl damping fluid balance of pH8.0, with the Tris-HCl buffer solution elution that contains NaClpH8.0, at first make elutriant electricity dodar to 30mS/cm, elution peak is collected by the priority of retention time, flow through 5-10 chromatography column volume until damping fluid, remain with the part of xylanase activity, be product of the present invention.
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| CN103243082A (en) * | 2013-04-07 | 2013-08-14 | 云南大学 | Bifunctional enzyme with catalytic activities of cellulose and xylan |
| CN107858310A (en) * | 2017-12-04 | 2018-03-30 | 云南大学 | The fragrant actinomyces of one plant of production and its application to promise Deng's ham flavouring |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1676605A (en) * | 2005-03-25 | 2005-10-05 | 武汉新华扬生物有限责任公司 | Method for producing bacterial xylanase |
| CN101012457A (en) * | 2007-01-29 | 2007-08-08 | 中国农业大学 | Method of preparing heat-proof xylanase, heat-proof beta-xylosidase or heat-proof beta-glucosidase |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1676605A (en) * | 2005-03-25 | 2005-10-05 | 武汉新华扬生物有限责任公司 | Method for producing bacterial xylanase |
| CN101012457A (en) * | 2007-01-29 | 2007-08-08 | 中国农业大学 | Method of preparing heat-proof xylanase, heat-proof beta-xylosidase or heat-proof beta-glucosidase |
Non-Patent Citations (3)
| Title |
|---|
| Ling-Ling Yang等.Thermobifida halotolerans sp. nov., isolated from a salt mine sample, and emended description of the genus Thermobifida.International Journal of Systematic and Evolutionary Microbiology.IUMS,2008,(58),1821–1825. * |
| Stephan Berens等.Purification and characterization of two different xylanases from the thermophilic actinomycete Microtetrasporaflexuosa SIIX.Antonie van Leeuwenhoek.KluwerAcademic Publishers,1996,(69),235-241. * |
| 江正强等.嗜热真菌棉毛状嗜热菌耐热木聚糖酶的性质及其对面包品质的改善.食品工业科技.2004,增刊141-146. * |
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