CN100392078C - Acid trehalosease and preparation process thereof - Google Patents
Acid trehalosease and preparation process thereof Download PDFInfo
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- CN100392078C CN100392078C CNB2005100574083A CN200510057408A CN100392078C CN 100392078 C CN100392078 C CN 100392078C CN B2005100574083 A CNB2005100574083 A CN B2005100574083A CN 200510057408 A CN200510057408 A CN 200510057408A CN 100392078 C CN100392078 C CN 100392078C
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Abstract
The present invention discloses an acid trehalase extracted from the Metarhizium anisopliae strains CQMa102 of entomogenous fungi and a preparation method thereof. The method comprises the following steps: the spores of Metarhizium anisopliae strains CQMa102 are activated, microorganisms capable of producing the trehalase are induced and cultured in a special induction and culture medium to form the trehalase, and the formed trehalase is purified. The prepared zymoprotein is a glycoprotein having the isoelectric point of 4.7, the molecular weight of 170 kDa, the Trehazolin as a specificity inhibitor, and the IC50 of 1.3*10<-8>M. The zymoprotein performs an important function of using the competitiveness of trehalose in the hemolymph of insects in the process of infecting the insects by Metarhizium anisopliae; meanwhile, the purified trehalase can be used for qualitatively and quantitatively detecting the content of trehalose in industry and other fields.
Description
Technical field
The present invention relates to a kind of acid trehalosease, particularly a kind of entomogenous fungi acid trehalosease and preparation method thereof.
Background technology
Known, trehalose (alpha-D-glucose glycosides (1,1)-and the alpha-D-glucose glycosides) be non-reducing disaccharide, be main blood sugar in the insect hemolymph, its content is up to 20～100mM, account for more than 90% of blood sugar total amount, these insects comprise Asiatic migrotory locust (Locusta migratoria) (Elbein, A.D; 1974).Trehalose has the important physical effect in the insect vital movement; can be summarized as following 4 points: (1) is as the intravital energy storage of insect; (2) avoiding at low temperatures as freezeproof protectant, insect blood solidifies; (3) prevent hemolymph protein denaturation under situations such as heat is emergent as protein stabiliser, (4) regulate insect's food-taking as the feedback regulation factor of searching for food.The exhaustion of trehalose may play lethal effect to insect in the hemolymph.
Metarhizium anisopliae (Metarhizium anisopliae) is a kind of important entomogenous fungi, extensively applies to the biological control of Agricultural pests.Infect entomogenous fungi and to penetrate body wall and entered the insect haemocoele before insect death, trehalose abundant in the insect hemolymph has constituted the important nutrition source of fungi potential.Yet concerning many fungies such as Metarhizium anisopliae, trehalose is non-perviousness substrate, and must be outside corresponding born of the same parents being hydrolyzed into glucose under the condition that exists of acid trehalosease could be utilized by fungi.We studies show that the Asiatic migrotory locust green stiff fungus infection after, content of trehalose reduces in the blood; In addition, Isozyme Analysis shows and to occur stronger acid trehalosease activity in the hemolymph, and this is indicating acid trehalosease after entomogenous fungi is invaded insect hemolymph, plays an important role competing nutritive substance and make in the insect pathogenic course with the host.Yet for the entomogenous fungi acid trehalosease, correlative study both domestic and external does not appear in the newspapers.The separation and purification trehalase is studied its biochemical characteristic, this for further research its to the effect of fungi in the insect pathogenesis, to improving the pathogenic efficient of entomogenous fungi sterilant, important significance for theories is arranged.
Summary of the invention
The object of the present invention is to provide a kind of entomogenous fungi acid trehalosease.
Another object of the present invention is to provide the preparation method of above-mentioned enzyme.
The object of the present invention is achieved like this: a kind of acid trehalosease, and its trehalose of degrading specifically is a glucose, and has following physics-chem characteristic:
(1) effect: specificity degraded trehalose is a glucose;
(2) molecular weight: on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis, be about 170kDa;
(3) iso-electric point: on isoelectrofocusing gel electrophoresis (IEF) iso-electric point electrophoresis, be about 4.7;
(4) optimum temperuture and best pH: trehalase is the specificity substrate of this zymoprotein, and its optimal reactive temperature is 30 ± 0.2 ℃, and best pH is 5.5 ± 0.2;
(5) thermostability: to not influence of enzymic activity, when incubation temperature was 50 ℃, enzymic activity is linear to descend this zymoprotein along with incubation time prolongs at 40 ℃ of incubation 240min, and when temperature surpassed 60 ℃, enzymic activity was lost rapidly; Therefore be lower than in temperature that this zymoprotein is stable under 40 ℃ of conditions;
(6) inhibitor: Trehazolin is the specific inhibitor of this zymoprotein, its IC
50Be 1.3 * 10
-8M;
(7) enzyme kinetics characteristic: the Michaelis-Menton constant K of this zymoprotein
mBe 2.3mM, maximum enzyme velocity V
MaxBe 0.412mmol min
-1Mg
-1Zymoprotein;
Wherein said enzyme is to extract from preserving number is Metarhizium anisopliae (Metarhiziumanisopliae) the CQM a102 strain of CGMCC No. 0877.
Another object of the present invention, above-mentioned acid trehalosease makes like this: at first activate above-mentioned Metarhizium anisopliae bacterial strain CQMA102 spore, and the microorganism that inducing culture can produce described enzyme in nutritional medium to be forming described enzyme, and purifying in addition.
In order to improve sensitivity and specificity, in above-mentioned separation and purification process, the present invention detects according to crude enzyme liquid acid trehalosease isozyme, determines the iso-electric point of purifying target protein; The enzyme work that records with the enzyme biopsy serves as to indicate the Metarhizium anisopliae acid trehalosease to natural expression to carry out separation and purification.
Above-mentioned Metarhizium anisopliae bacterial strain CQMA102 spore activates used substratum by 10g/l glucose, the 2.5g/l peptone, and the 5.0g/l yeast extract, 20g/l agar is formed, and its pH value transfers to 6.0; The carbon source that described inducing culture adopts is a Zulkovsky starch, can effectively induce the trehalose isozyme to produce, the nitrogenous source that adopts is an ammonium sulfate, can effectively reduce albumen kind and content in the nutrient solution, avoid the disadvantageous effect of foreign protein to purifying, reduced the stability influence of the secretion of proteolytic enzyme simultaneously, be beneficial to the target protein separation and purification target protein; And, collect crude enzyme liquid before the upper prop according to iso-electric point, the molecular weight characteristics of acid trehalosease, adopt two step ion exchange chromatographies, a step sieve chromatography and step narrow pH range isoelectrofocusing separation and purification to go out described trehalase.
The substratum of above-mentioned inducing culture is by Zulkovsky starch 10 ± 0.5g/l, ammonium sulfate 2 ± 0.2g/l, Repone K 1 ± 0.1g/l, five water magnesium sulfates, 0.5 ± 0.05g/l, potassium primary phosphate 1 ± 0.1g/l, 2-(N-morpholine)-ethylsulfonic acid 10 ± 0.5g/l, trace element: 0.1% iron vitriol and 0.3% 5 water zinc sulphate, 10 ± 0.5ml/l form, and adjust pH is 6.00～6.10; The chromatography column that described ion exchange chromatography adopts is respectively High Q Sepharoes anion column and 25Q Sepharoes anion column (Bio-Rad); Described molecular sieve chromatography is Sepharcyl-200sHR (Pharmacia); The pH value scope that the isoelectrofocusing offset plate that described narrow pH range isoelectrofocusing is adopted produces for the peace Pharmacia is 4.0～5.0 offset plate.
In described preparation method, it is to carry out isoelectric focusing electrophoresis earlier that described enzyme is carried out the isozyme detection, adopts gel-colored the detection then; Wherein gel-colored, be after adopting the rubber cover method to isoelectrofocusing in the gel acid trehalosease carry out activity and develop the color, A liquid is by 0.1M pH5.0 citrate buffer solution 10ml, 1000U glucose oxidase 50 μ l, 2500U/ml peroxidase 50 μ l, 25mg/ml 3-amino-9-ethyl-carbazole acetone soln is formed, and B liquid is 2% agar; And with A liquid 2ml, trehalose 100mg, B liquid 12ml mixing.
Specifically, the preparation method of above-mentioned acid trehalosease, the activation of described Metarhizium anisopliae bacterial strain CQMa102: Metarhizium anisopliae (Metarhizium anisopliae) CQMa102 conidium is inoculated on the 1/4SDA plate culture medium and activates, bacterial strain 7～the 14d that under 26～28 ℃ of dark conditions, grows, until producing spore, in 4 ℃ of refrigerators, preserve standby then;
Described product enzyme induction is cultivated: collect CQMa102 bacterial strain conidium on the 1/4SDA plate culture medium, and be mixed with 3 * 10
7～4 * 10
7Spore/ml bacteria suspension; Every 100ml liquid nutrient medium inoculation 3 * 10
7～4 * 10
7Spore, 150rpm shaking culture 72h on open shaking table, culture temperature is 26～27 ℃;
Described separation and purification: after inducing culture finishes, remove the mycelia in the nutrient solution, collect that filtrate is dialysed, centrifugal, suction filtration, and to regulate its specific conductivity be 0.6～0.7ms/cm
2Upper prop before crude enzyme liquid; Described chromatographic step is all carried out on Duo-flow high pressure zone analysis system (Bio-Rad); Described ion exchange column adopts the acid trehalosease of the linear gradient elution of bound of 0.0～0.5M sodium-chlor, collects to contain the trehalase active part; To the trehalase active part ultrafiltration and concentration of collecting, carry out sieve chromatography again, to the trehalase liquid of gained behind the sieve chromatography behind ultrafiltration and concentration, carrying out narrow PH scope isoelectric focusing electrophoresis again separates, after electrophoresis finishes, the band that will contain the trehalase active part scales off and changes in the dialysis tubing, zymoprotein is washed out again, and promptly gets the acid trehalosease of purifying.
Say that more specifically after the collection of crude enzyme liquid to be purified was meant that cultivation finishes before the upper prop in the purifying, nutrient solution was removed mycelia with filtered through gauze, collection filtrate is packed in the dialysis tubing, the molecular retention amount is 5000, with the deionized water of 10 times of volumes at 4 ℃ of dialysis 30h, during change water 4～5 times; With hydrochloric acid the dialyzate pH value is adjusted to 5.8 then, solution is 13, and 500rpm, 4 ℃ of centrifugal 20min handle with three metafiltration paper suction filtrations then, and regulating specific conductivity with sodium-chlor at last is 0.6～0.7ms/cm
2, obtain the preceding crude enzyme liquid to be purified of upper prop.
Above-mentioned the first step ion chromatography is 20ml High Q Sepharoes anion column 10mM 2-(N-morpholine)-ethylsulfonic acid pH5.8, and electricity is led 0.6～0.7ms/cm
2After the level pad balance, with above-mentioned crude enzyme liquid upper prop, last column flow rate is 3.0ml/min, all is flushed away (280nm no longer include absorption value) with the flushing of 3.0ml/min flow velocity until all unconjugated albumen with level pad after upper prop finishes; Adopt the acid trehalosease of the linear gradient elution of bound of 200ml 0.0～0.50M sodium-chlor, collection tube is set to the 2ml/ pipe, and collection contains the trehalase active part and is used for next step purifying;
The above-mentioned second step ion chromatography is meant that the enzyme liquid of collecting behind the High Q Sepharoes anion chromatography packs in the dialysis tubing, and in about 2L 15mM acetate buffer solution pH5.0, electricity is led 1.00～1.05ms/cm under 4 ℃ of conditions
2Middle dialysis 24h changes damping fluid therebetween 3 times; Fully the enzyme liquid after the dialysis again with 15mM acetate buffer solution pH5.0, electricity is led 1.00～1.05ms/cm
2After the equal-volume mixing, with 15mM acetate buffer solution balance mistake, last column flow rate is 1.0ml/min earlier for last 2ml 25Q Sepharoes anion column, this chromatography column; After finishing, upper prop all is flushed away (280nm no longer includes absorption value) with the flushing of 1.0ml/min flow velocity until all unconjugated albumen with level pad; Adopt the acid trehalosease of the linear gradient elution of bound of 100ml 0.0～0.50M sodium-chlor, collection tube is set to the 1ml/ pipe, and collection contains the trehalase active part and is used for next step purifying;
Above-mentioned sieve chromatography is meant sample that 25 Q Sepharoes negatively charged ion wash-outs obtain through 0.5ml ultra-filtration centrifuge tube Millipore, and the 10kDa ultrafiltration and concentration carries out sieve chromatography then to the 1ml volume; 2.6 * 60cm molecular sieve chromatography Sepharcyl-200s HR (Pharmacia) uses 15mM acetate buffer solution pH5.0 in advance, electricity is led 1.05ms/cm
2Fully after the balance, by sample 1ml on the last sample ring, acid trehalosease washes out with same acetate buffer solution then, and flow velocity is set to 0.5ml/min, and collected volume is set to the 1.0ml/ pipe, and collection contains the trehalase active part and is used for next step purifying;
Gained trehalase liquid was through 0.5ml ultra-filtration centrifuge tube Millipore after above-mentioned narrow pH range isoelectrofocusing separation was meant sieve chromatography, 10kDa ultrafiltration and concentration volume is to 0.5ml, be that 4.0～5.0 prefabricated isoelectrofocusing offset plates (Pharmacia) carry out electrophoretic separation with pH value scope then, after electrophoresis finishes, to contain trehalase band alive scales off, change in the dialysis tubing, the electricity consumption type of elution washes out zymoprotein.
The present invention has at first set up fast and convenient acid trehalosease detection architecture, is beneficial to the purifying process is monitored; Only make initial purifying enzyme liquid with culturing filtrate simultaneously, its albumen all produces justacrine to external for the fungi metabolism, and initial protein content is very low, and kind is less relatively, is beneficial to purifying.Adopt 20ml High Q anionresin as the purifying the first step, upper prop pH is set to 5.8 can removes most of foreign protein and other macromolecular substance such as the starch that exists in the nutrient solution simultaneously by rich fast long-pending target protein.Second step of purifying adopts 2ml 25Q anionresin and upper prop pH is set to 5.0, near the target protein iso-electric point, has improved the resolving power of chromatography.The 3rd step was adopted sieve chromatography, difference according to the molecular weight size has been carried out further separation to acid trehalosease, the 4th step was adopted the prefabricated isoelectrofocusing offset plate of narrow pH range (Pharmacia, pH4.0～5.0) separate, the albumen that iso-electric point can be differed very little separates, thereby makes acid trehalosease further be able to purifying.
The acid trehalosease that adopts separation purification method of the present invention to obtain, can the degrade specifically trehalose be glucose, have active high, the high characteristics of the single-minded life of substrate, can extensively apply to the qualitative and quantitative detection of content of trehalose in the scientific research, can avoid the interference of other disaccharides.
Description of drawings
Fig. 1: filtrate concentrates back acid trehalosease Isozyme Analysis;
Fig. 2: 20ml High Q Sepharoes (Bio-Rad) anion chromatography flows out figure;
Fig. 3: 2ml 25 Q Sepharoes (Bio-Rad) anion chromatographies flow out figure;
Fig. 4: (Sepharcyl-200s HR Parmacheica) flows out figure to 2.6 * 60cm sieve chromatography;
Fig. 5: different purification phase SDS-PAGE Solargentums dye detection;
Fig. 6: the acid marine alga enzyme IEF silver behind the purifying dyes detection;
Fig. 7: inhibitor Trehazolin is to the active influence of the acid trehalosease of purifying;
Fig. 8: temperature is to the active influence of acid trehalosease;
Fig. 9 .:pH is to the active influence of acid trehalosease;
Figure 10.: the thermostability of acid trehalosease;
Figure 11 .Lineweaver-Burk equation is measured the acid trehalosease K of purifying
mAnd V
Max
In Fig. 5, the 1st, 25Q eluting peak 2; The 2nd, molecular sieve eluting peak 1; The 3rd, the acid trehalosease of purifying; M is the molecular weight of albumen standard;
In Fig. 6, the 1st, crude enzyme liquid; The 2nd, the acid trehalosease of purifying.
Embodiment
The invention will be further described below in conjunction with drawings and Examples, but the present invention is not limited to this.
The activation of Metarhizium anisopliae bacterial strain CQMa102 spore
This routine Metarhizium anisopliae bacterial strain CQMa102 (Metarhizium anisopliae var.acridumCQMa102) is that the separation and Culture purifying obtains in the bamboo locust bombys batryticatus body of falling ill, by University Of Chongqing's genetically engineered center separation and purification, the bacterial strain that China Institute of Micro-biology preserves at bacterial classification preservation center, deposit number CGMCC No.0877, its conidium is stored in 30% glycerine solution, preserves in-80 ℃ of Ultralow Temperature Freezers are medium-term and long-term.Metarhizium anisopliae (Metarhiziumanisopliae) the CQMa102 inoculation of-80 ℃ of preservations is activated on the 1/4SDA plate culture medium, used substratum is by 10g/l glucose, 2.5g/l peptone, 5.0g/l yeast extract, 20g/l agar is formed, and its pH value is transferred to 6.0, the bacterial strain 7～14d that grows under 26～28 ℃ of dark conditions, until producing spore, in 4 ℃ of refrigerators, preserve standby then.
Produce the enzyme induction culture condition
Used substratum is by Zulkovsky starch 10 ± 0.5g/l, ammonium sulfate 2 ± 0.2g/l, Repone K 1 ± 0.1g/l, five water magnesium sulfates, 0.5 ± 0.05g/l, potassium primary phosphate 1 ± 0.1g/l, 2-(N-morpholine)-ethylsulfonic acid 10 ± 0.5g/l, trace element: 0.1% iron vitriol and 0.3% 5 water zinc sulphate, 10 ± 0.5ml/l form, and its pH is transferred to 6.00～6.10.Concrete compound method: take by weighing 3L substratum material in the 1L glass beaker, the about 1L deionized water of adding stirs back heating in microwave oven with glass rod and thoroughly dissolves Zulkovsky starch earlier.Treat then to regulate pH to 6.00～6.10 with sodium hydroxide after the substratum cooling.At last substratum is transferred to 3L with deionized water, divide to install to 250ml triangular flask, every bottle of 100ml nutrient solution.Medium sterilization is standby.
Collect CQMa102 bacterial strain conidium on the 1/4SDA plate culture medium, be mixed with 3 * 10 with the 0.05% tween 80 solution of sterilizing
7Spore/ml bacteria suspension; Every 100ml nutrient solution inoculation 3 * 10
7Spore.Nutrient solution is 150rpm shaking culture 72h on open shaking table, and culture temperature is 26～27 ℃.
The acid trehalosease isozyme detects
Isoelectric focusing electrophoresis adopts T=5% with reference to Wang Jiazheng (2001), and C=3% vertical panel polyacrylamide isoelectric focusing electrophoresis (PAGE-IEF) adopts wide pH scope amphotericeledrolyte (pH 3-9.5).Electrophoresis equipment is Mini-Proteanapparatus (Bio-Rad).Ability cathode electrophoresis liquid is to be 2mM NaOH solution; Anolyte is to be dissolved in disposing in the 1L distilled water by 0.84ml phosphoric acid forming.Contain 50% glycerine in the sample solution.Adopt the substep electrophoresis, earlier electrophoresis 1h under 100V voltage, then electrophoresis 2h under 250V voltage.
Gel-colored employing rubber cover method (overlay gel) is carried out activity colour developing (Manchenk, 1994) to acid trehalosease in the gel after the isoelectrofocusing.A liquid is by 0.1M pH5.0 citrate buffer solution 10ml, 1000U glucose oxidase 50 μ l, 2500U/ml peroxidase 50 μ l, 25mg/ml 3-amino-9-ethyl-carbazole acetone soln is formed, consumption 2ml, trehalose 100mg, B liquid is 2% agar (60 ℃), consumption 12ml evenly is poured over gel surface after mixing, and gel is placed on to be incubated under 37 ℃ up to reddish-brown dyeing band occurring then.Acetate with 7% is fixed gel.
The biopsy of acid trehalosease enzyme is surveyed
Determine that with the glucose content that enzyme and trehalose reaction discharge the trehalase enzyme is alive.Get the aqueous trehalose of certain volume enzyme liquid (x μ l) and 12.5 μ l 0.1M, in 20mM 2-(N-morpholine)-ethylsulfonic acid (pH5.5), 30 ℃ of following reaction 10min, reaction system cumulative volume 50 μ l.Reaction finishes the back earlier 100 ℃ of water-bath 5min termination reactions, and then ice bath 5min is to prevent the zymoprotein refolding.The glucose content that discharges after the enzymatic reaction is with improved determination of glucose oxidase (Trinder1969), add 150 μ l glucose assays reagent and [contain 0.5mM 4-aminoantipyrene (4-aminoantipryine), 20mM p-phenolsulfonate (p-hydroxybenzene sulphonate), 15,000U/L glucose oxidase and 10,000U/L peroxidase (horseradish) is at 27 ℃ of 30min that develop the color down.Glucose standard substance with a series of different concns are done typical curve, use Model 550 microplate reader (Bio-Rad), in 490nm photometry absorption value.Enzyme work is defined as: it is an enzyme activity unit (U) that per minute discharges the required enzyme amount of 1mmol glucose.
For ease of sample preparation, cultivate inoculation 3L nutrient solution for every batch in this example, to cultivate through 72h, gained filtrate is through after the dialysis treatment, and volume is about 2.7L.Owing to do not contain foreign protein in the substratum, albumen all derives from fungi self metabolism justacrine in the substratum in the nutrient solution, so total protein content is very low, has only about 20mg.Isozyme Analysis shows two isozyme bands, wherein a band pI (iso-electric point) is 4.7, active higher, and another band pI is in 8.4 left and right sides (see figure 1)s, activity a little less than, others experiments shows the acid trehalosease that the isozyme of pI 4.7 may work for fungi in locust dip-dye process.
The purifying of acid trehalosease
After cultivate finishing, nutrient solution is removed mycelia with filtered through gauze, collect filtrate and pack in the dialysis tubing (the molecular retention amount is 5000), with the deionized water of 10 times of volumes at 4 ℃ of 30h that dialyse, during change water 4～5 times.With HCl solution dialyzate pH value is adjusted to 5.8 then, solution is 13, and 500rpm, 4 ℃ of centrifugal 20min handle with three metafiltration paper suction filtrations then, and regulating specific conductivity with NaCl solution at last is 0.7ms/cm
2, obtain the preceding crude enzyme liquid of upper prop.
All chromatographies of the present invention all are to carry out on Duo-flow high pressure zone analysis system (Bio-Rad).The first step ion chromatography is that (pH5.8, electricity is led 0.7ms/cm to 20ml High Q Sepharoes anion column with 10mM MES
2) after the level pad balance, with above-mentioned crude enzyme liquid upper prop, last column flow rate is 3.0ml/min, gone up the back and washed with the 3.0ml/min flow velocity with level pad and all be flushed away (280nm no longer includes absorption value) until all unconjugated albumen.Adopt the acid trehalosease of 200ml0.0～linear gradient elution of bound of 0.5M NaCl solution, collection tube is set to the 2ml/ pipe, and collection contains the trehalase active part and is used for next step purifying.
The second step ion chromatography: the enzyme liquid of collecting behind the High Q anion chromatography is packed in the dialysis tubing, and (pH5.0, electricity is led 1.05ms/cm in 2L 15mM acetate buffer solution under 4 ℃ of conditions
2) middle dialysis 24h, change damping fluid therebetween 3 times.Fully (pH5.0, electricity is led 1.05ms/cm to the enzyme liquid after the dialysis with the 15mM acetate buffer solution again
2) with after the equal-volume mixing, last 2ml25 Q Sepharoes anion column (chromatography column is used 15mM acetate buffer solution balance in advance), last column flow rate is 1.0ml/min.After finishing, upper prop all is flushed away (280nm no longer includes absorption value) with the flushing of 1.0ml/min flow velocity until all unconjugated albumen with level pad.Adopt the acid trehalosease of the linear gradient elution of bound of 100ml 0.0～0.5M NaCl solution, collection tube is set to the 1ml/ pipe, and collection contains the trehalase active part and is used for next step purifying.
Sieve chromatography: (Millipore, 10KDa) ultrafiltration and concentration carries out sieve chromatography to the 1ml volume to the sample that 25 Q Sepharoes negatively charged ion wash-outs obtain then through the 0.5ml ultra-filtration centrifuge tube.2.6 (Sepharcyl-200s HR, (pH5.0, electricity is led 1.05ms/cm to * 60cm molecular sieve chromatography Pharmacia) to use the 15mM acetate buffer solution in advance
2) fully after the balance, by sample on the last sample ring, acid trehalosease washes out with same damping fluid, flow velocity is set to 0.5ml/min, and collected volume is set to the 1.0ml/ pipe, and collection contains the trehalase active part and is used for next step purifying.
The narrow pH range isoelectrofocusing separates: gained trehalase liquid is through 0.5ml ultra-filtration centrifuge tube (Millipore behind the sieve chromatography, 10kDa) the ultrafiltration and concentration volume is to 0.5ml, use the prefabricated isoelectrofocusing offset plate of narrow pH range (Pharmacia then, pH4.0～5.0) separate, by specification requires to carry out electrophoresis on leveling board electrophoresis apparatus (Beijing 6 1), after electrophoresis finishes, to contain trehalase band alive scales off, change in the dialysis tubing, the electricity consumption type of elution washes out zymoprotein, can obtain the acid trehalosease of purifying.
This example is in conjunction with the Isozyme Analysis data, at first select 20ml High Q Sepharoes (Bio-Rad) anion chromatography for use, this medium diameter is 50 μ m, flow velocity is very fast, can quickly the target protein in the sample be adsorbed onto on the medium, thereby reach the purpose of fast enriching target protein, upper prop pH is set to 5.8, it is in order to allow target protein well be adsorbed onto on the post that specific conductivity is transferred to 0.7, as shown in Figure 2, by this step chromatography, got rid of impurity such as most of foreign protein and starch in the filtrate, simultaneously target protein is concentrated into about about 30ml from 2700ml.From Fig. 2, we can find out that also the trehalase active part concentrates on 18～34 pipes (30ml).
Behind the first step anion-exchange chromatography, foreign protein and other interference factor reduce in the sample, further adopt 2ml25 Q Sepharoes anionresin, 25 Q Sepharoes negatively charged ion medium diameters are 25 μ m, because pillar is little, medium is thin, thereby resolving power is higher, adopt pH 5.0, specific conductivity 1.05 upper props, upper prop PH are very near acid trehalosease iso-electric point 4.7, but target protein can be adsorbed onto on the post, further reduce the absorption of non-differential protein, increase the resolving power of chromatography; Through wash-out, obtained elution peak preferably, the acid trehalosease active part occurs in the 2nd peak, and volume further drops to 3ml (referring to Fig. 3).
Behind the 25Q ion exchange chromatography, sample detects through SDS-PAGE, only contains several main protein bands, and molecular weight differs bigger, therefore further selects sieve chromatography for use, utilizes between the albumen molecular weight difference in size to separate.As shown in Figure 4, through behind the sieve chromatography, the acid trehalosease active part occurs in first elution peak, and the active part volume is collected as 10ml.
Behind the sieve chromatography, collect the acid trehalosease active part, detect through SDS-PAGE, only remaining 2 protein bands (silver dyes), article two, molecular weight of albumen is respectively 170KDa and 87KDa, but when sieve chromatography, in same peak, occur, infer that 87KDa albumen is a dimer under natural condition.
Further adopt the prefabricated isoelectrofocusing offset plate of narrow pH range (Pharmacia, pH4.0～5.0) to separate, obtain the pure product of acid trehalosease after separating, SDS-PAGE (referring to Fig. 5) and IEF (referring to Fig. 6) detect all has only a protein band.The acid trehalosease molecular weight of purifying is 170KDa, and iso-electric point (pI) is 4.7.Albumen biochemical characteristic to purifying has carried out identifying (Fig. 7-11, table 1).
Table 1. Metarhizium anisopliae acid trehalosease substrate specificity is analyzed
The acid trehalosease that this routine purifying is obtained carries out CHARACTERISTICS IDENTIFICATION
1, molecular weight and iso-electric point
The mensuration of molecular weight is to measure with 12% SDS-PAGE according to the method for Laemmli, and protein molecular weight Marker is lower molecular weight Marker (Pharmacia).Iso-electric point is measured on IEF-PAGE.
2, inhibitor trehazolin is to the active influence of trehalase
Trehazolin is the trehalase specific inhibitor, is at first from the culture of Micromonospora, to separate to obtain, and be the trehalose structure analogue.The trehazolin of 0-50ng/ml concentration gradient (final concentration) joins 1.5ml respectively and contains 12.5mM 2-(N-morpholine)-ethylsulfonic acid, pH5.5, and in the centrifuge tube of the acid trehalosease solution of 12.5mM trehalose and 25ng purifying, the reaction cumulative volume is 50 μ l; At 30 ℃ of water-bath 10min, 100 ℃ of deactivation 5min, ice bath 5min, reaction solution is changed in the 96 hole enzyme plates, and every hole adds 150 μ l Trinder and surveys sugared reagent, at 27 ℃ of reaction 30min, use Model 550 microplate reader (Bio-Rad), in 490nm photometry absorption value.Each gradient is done three times and is repeated, and establishes 8 gradients altogether; Establish a blank in experiment in addition, the trehalase of unconstrained dose and purifying is with the background correction value.
Conclusion: Trehazolin is the acid trehalosease specific inhibitor, its IC
50Be 1.3 * 10
-8M.
3, substrate specificity analysis
Measured the hydrolytic action of the trehalase of purifying respectively to 8 kinds of disaccharides.1.5ml contain 12.5mM 2-(N-morpholine)-ethylsulfonic acid, the trehalose that adds the 12.5mM final concentration in the centrifuge tube of the acid trehalosease solution of pH 5.5 and 25ng purifying respectively, cellobiose (glucose β 1-4 glucose), maltose (glucose α 1-4 glucose), sucrose (glucose α 1-2 β glucose), semi-lactosi (galactose1-4glucose), p-oil of mirbane-β-glucoside (pnp galactopyranoside), p-oil of mirbane-α-glucoside (pnp α-glucopyranoside), (pnp β-glucopyranoside), the reaction cumulative volume is 50 μ l to p-oil of mirbane-β galactoside.Each substrate is handled a blank is set, and does not promptly add the trehalase of purifying in above-mentioned reaction system, and all the other compositions are identical, with the background correction value.At 30 ℃ of water-bath 10min, 100 ℃ of deactivation 5min, ice bath 5min, reaction solution is changed in the 96 hole enzyme plates, and every hole adds 150 μ l glucose assays reagent, 27 ℃ of reaction 30min, use Model 550 microplate reader (Bio-Rad), in 490nm photometry absorption value.Every kind of substrate is done three times respectively and is repeated (comprising blank and the experiment processing).
Conclusion: trehalose is this proteolytic enzyme specificity substrate.
4, trehalase reaction optimum temperuture and pH measure
From 15～70 ℃ every 5 ℃ a thermograde is set, 1.5ml centrifuge tube adds the trehalose of 12.5mM final concentration respectively on ice, 12.5mM MES, the acid trehalosease of pH 5.5 and 25ng purifying, cumulative volume 50 μ l, the water-bath of putting into above-mentioned differing temps gradient then reacts 10min, 100 ℃ of deactivation 5min, ice bath 5min, reaction solution is changed in the 96 hole enzyme plates, and every hole adds 150 μ l Trinder and surveys sugared reagent, 27 ℃ of reaction 30min, use Model 550 microplate reader (Bio-Rad), in 490nm photometry absorption value.Each thermograde is done three times respectively and is repeated.
From pH3.0～9.0 every 0.5 pH a gradient is set, wherein 3.0～5.0 use acetate buffer solution, 5.5～6.5 use the MES damping fluid, 7.0～7.5 use the HEPES damping fluid, 8.0～9.0 usefulness Tris-HCl make damping fluid, 1.5ml centrifuge tube adds the different pH damping fluids that final concentration is 30mM, the acid trehalosease of the trehalose of 12.5mM final concentration and 25ng purifying, cumulative volume 50 μ l respectively on ice.At 30 ℃ of water-bath 10min, 100 ℃ of deactivation 5min, ice bath 5min, reaction solution is changed in the 96 hole enzyme plates, and every hole adds 150 μ l Trinder and surveys sugared reagent, 27 ℃ of reaction 30min, use Model 550 microplate reader (Bio-Rad), in 490nm photometry absorption value.Every kind of pH gradient is done three times respectively and is repeated.
Conclusion: trehalase reaction optimum temperuture is 30 ℃, and the optimal reaction pH value is 5.5.
5, acid trehalosease thermal stability determination
The acid trehalosease of purifying is distinguished water- bath 10min 40,50 under 60,70 ℃ of conditions, 30min, and 60min, 120min and 240min get 5 μ l (25ng) then and measure remaining enzyme work.Condition determination is: the trehalose of 12.5mM final concentration, 12.5mM MES, the acid trehalosease that pH5.5 and 25ng are heat treated, cumulative volume 50 μ l, at 30 ℃ of water-bath 10min, 100 ℃ of deactivation 5min, ice bath 5min changes reaction solution in the 96 hole enzyme plates over to, every hole adds 150 μ l Trinder and surveys sugared reagent, 27 ℃ of reaction 30min use Model 550 microplate reader (Bio-Rad), in 490nm photometry absorption value.Each is handled and does three repetitions respectively.
Conclusion: to not influence of enzymic activity, when incubation temperature was 50 ℃, enzymic activity is linear to descend this acid trehalosease along with incubation time prolongs at 40 ℃ of incubation 240min, and when temperature surpassed 60 ℃, enzymic activity was lost rapidly.
6, enzyme kinetics characteristic measurement
The trehalose final concentration is set to 0,0.1,0.2,0.3,0.4,0.5,0.6,0.8,1.0,1.25mM, the trehalose of different concns gradient respectively with the acid trehalosease of 25ng purifying at 30 ℃, react 10min (the reaction system cumulative volume is 50 μ l) under the pH5.5 condition, the reaction back 100 ℃ of deactivation 5min that finish, ice bath 5min, reaction solution is changed in the 96 hole enzyme plates, and every hole adds 150 μ l Trinder and surveys sugared reagent, 27 ℃ of reaction 30min, use Model550 microplate reader (Bio-Rad), in 490nm photometry absorption value.Each is handled and does three repetitions respectively.Meanwhile, configuration 0,0.2,0.4,0.6,0.8,1.0,1.2 1.6mM glucose normal gradients adds 50 μ l normal concentration glucose solutions in each enzyme plate hole, survey sugared reagent at 27 ℃ of reaction 30min with 150 μ l Trinder then, use Model 550 microplate reader (Bio-Rad), in 490nm photometry absorption value, production standard curve (n=3).Use the acid trehalosease K of Lineweaver-Burk Equation for Calculating purifying
mAnd V
Max
Conclusion: this acid trehalosease feature rice formula constant K
mBe 2.3mM, maximum enzyme velocity V
MaxBe 0.412mmolmin
-1Mg
-1Zymoprotein protein.
Claims (8)
1. acid trehalosease, its trehalose of degrading specifically is a glucose, and has following physics-chem characteristic:
(1) effect: specificity degraded trehalose is a glucose;
(2) molecular weight: be 170kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis;
(3) iso-electric point: on isoelectrofocusing gel electrophoresis iso-electric point electrophoresis is 4.7;
(4) optimum temperuture and best pH: trehalase is the specificity substrate of this zymoprotein, and its optimal reactive temperature is 30 ± 0.2 ℃, and best pH is 5.5 ± 0.2;
(5) thermostability: to not influence of enzymic activity, when incubation temperature was 50 ℃, enzymic activity is linear to descend this zymoprotein along with incubation time prolongs at 40 ℃ of incubation 240min, and when temperature surpassed 60 ℃, enzymic activity was lost rapidly;
(6) inhibitor: Trehazolin is the specific inhibitor of this zymoprotein, its IC
50Be 1.3 * 10
-8M;
(7) enzyme kinetics characteristic: the Michaelis-Menton constant K of this zymoprotein
mBe 2.3mM, maximum enzyme velocity V
MaxBe 0.412mmol min
-1Mg
-1
Wherein said enzyme is to extract from preserving number is the Metarhizium anisopliae CQM a102 bacterial strain of CGMCC No.0877.
2. method for preparing the described acid trehalosease of claim 1, described method comprises: activate described Metarhizium anisopliae CQMa102 bacterial strain spore, and the microorganism Metarhizium anisopliae CQM a102 that inducing culture can produce described enzyme in nutritional medium to be forming described enzyme, and purifying in addition.
3. the method for preparing acid trehalosease as claimed in claim 2 is characterized in that: in described preparation method, described enzyme is carried out isozyme detection and enzyme biopsy survey, to determine the iso-electric point of target protein, carry out purifying again.
4. the method for preparing acid trehalosease as claimed in claim 2, it is characterized in that: described Metarhizium anisopliae bacterial strain CQMa102 spore activates used substratum by 10g/l glucose, 2.5g/l peptone, 5.0g/l yeast extract, 20g/l agar is formed, and its pH value transfers to 6.0; The carbon source that described inducing culture adopts is a Zulkovsky starch, and the nitrogenous source of employing is an ammonium sulfate; Described purifying adopts collection, two step ion exchange chromatographies, a step sieve chromatography and the step narrow pH range isoelectrofocusing separation and purification of the preceding crude enzyme liquid of upper prop to go out described trehalase.
5. the method for preparing acid trehalosease as claimed in claim 4, it is characterized in that: the substratum of described inducing culture is by Zulkovsky starch 10 ± 0.5g/l, ammonium sulfate 2 ± 0.2g/l, Repone K 1 ± 0.1g/l, five water magnesium sulfates, 0.5 ± 0.05g/l, potassium primary phosphate 1 ± 0.1g/l, 2-(N-morpholine)-ethylsulfonic acid 10 ± 0.5g/l, trace element: 0.1% iron vitriol and 0.3% 5 water zinc sulphate, 10 ± 0.5ml/l form, and adjust pH is 6.00～6.10; The chromatography media that described ion exchange chromatography adopts is respectively High Q Sepharoes negatively charged ion medium and 25QSepharoes negatively charged ion medium; Described molecular sieve chromatography is Sepharcyl-200s HR; The pH value scope that the isoelectrofocusing offset plate that described narrow pH range isoelectrofocusing is adopted produces for the peace Pharmacia is 4.0～5.0 offset plate.
6. the method for preparing acid trehalosease as claimed in claim 3 is characterized in that: in described preparation method, it is to carry out isoelectric focusing electrophoresis earlier that described enzyme is carried out the isozyme detection, adopts gel-colored the detection then; Wherein gel-colored, be after adopting the rubber cover method to isoelectrofocusing in the gel acid trehalosease carry out activity and develop the color, A liquid is by 0.1M pH5.0 citrate buffer solution 10ml, 1000U glucose oxidase 50 μ l, 2500U/ml peroxidase 50 μ l, 25mg/ml 3-amino-9-ethyl-carbazole acetone soln is formed, and B liquid is 2% agar; And A liquid 2ml, trehalose 100mg, B liquid 12ml mixing be used for enzymic activity dyeing.
7. as claim 4 or the 5 described methods that prepare acid trehalosease, it is characterized in that:
The activation of Metarhizium anisopliae bacterial strain CQMa102: Metarhizium anisopliae (Metarhizium anisopliae) CQMa102 conidium is inoculated on the 1/4SDA plate culture medium and activates, bacterial strain 7～the 14d that under 26～28 ℃ of dark conditions, grows, until producing spore, in 4 ℃ of refrigerators, preserve standby then;
The product enzyme induction is cultivated: collect CQMa102 bacterial strain conidium on the 1/4SDA plate culture medium, and be mixed with 3 * 10
7～4 * 10
7Spore/ml bacteria suspension; Every 100ml liquid is induced culture medium inoculated 1ml, 150rpm shaking culture 72h on open shaking table, and culture temperature is 26～27 ℃;
Separation and purification: after inducing culture finishes, remove the mycelia in the nutrient solution, collection filtrate is dialysed, centrifugal, suction filtration, and crude enzyme liquid specific conductivity to be purified is 0.6～0.7ms/cm before the adjusting upper prop
2Chromatographic step is all carried out on Duo-flow high pressure zone analysis system Bio-Rad; Ion exchange column adopts the acid trehalosease of the linear gradient elution of bound of 0.0～0.5M sodium-chlor, collects to contain the trehalase active part; To the trehalase active part ultrafiltration and concentration of collecting, carry out sieve chromatography again, to the trehalase liquid of gained behind the sieve chromatography behind ultrafiltration and concentration, carrying out narrow PH scope isoelectric focusing electrophoresis again separates, after electrophoresis finishes, the band that will contain the trehalase active part scales off and changes in the dialysis tubing, zymoprotein is washed out again, and promptly gets the acid trehalosease of purifying.
8. the method for preparing acid trehalosease as claimed in claim 5, it is characterized in that: after the collection of crude enzyme liquid is meant that cultivation finishes before the upper prop in the described purifying, nutrient solution is removed mycelia with filtered through gauze, collection filtrate is packed in the dialysis tubing, the molecular retention amount is 5000, with the deionized water of 10 times of volumes at 4 ℃ of dialysis 30h, during change water 4～5 times; With hydrochloric acid dialyzate pH value is adjusted to 5.8 then, solution is 13, and 500rpm, 4 ℃ of centrifugal 20min use three metafiltration paper suction filtrations then, and regulating specific conductivity with sodium-chlor again is 0.6～0.7ms/cm
2, obtain the preceding crude enzyme liquid to be purified of upper prop;
The first step ion chromatography of two step ion exchange chromatographies is 20ml High Q Sepharoes anion column 10mM 2-(N-morpholine)-ethylsulfonic acid pH5.8, and electricity is led 0.6～0.7ms/cm
2After the level pad balance, with above-mentioned crude enzyme liquid upper prop to be purified, last column flow rate is 3.0ml/min, all is flushed away until all unconjugated albumen with the flushing of 3.0ml/min flow velocity with level pad behind the upward intact post; Adopt the acid trehalosease of the linear gradient elution of bound of 200ml 0.0～0.50M sodium-chlor, collection tube is set to the 2ml/ pipe, and collection contains the trehalase active part and is used for next step purifying;
The second step ion chromatography is meant that the enzyme liquid of collecting behind the High Q Sepharoes anion chromatography packs in the dialysis tubing, and in 2L 15mM acetate buffer solution pH5.0, electricity is led 1.00～1.05ms/cm under 4 ℃ of conditions
2Middle dialysis 24h changes damping fluid therebetween 3 times; Fully the enzyme liquid after the dialysis again with 15mM acetate buffer solution pH5.0, electricity is led 1.00～1.05ms/cm
2After the equal-volume mixing, with 15mM acetate buffer solution balance mistake, last column flow rate is 1.0ml/min earlier for last 2ml 25 Q Sepharoes anion columns, this chromatography column; After finishing, upper prop all is flushed away until all unconjugated albumen with the flushing of 1.0ml/min flow velocity with level pad; Adopt the acid trehalosease of the linear gradient elution of bound of 100ml 0.0～0.50M sodium-chlor, collection tube is set to the 1ml/ pipe, and collection contains the trehalase active part and is used for next step purifying;
Described sieve chromatography is meant sample that 25 Q Sepharoes negatively charged ion wash-outs obtain through 0.5ml ultra-filtration centrifuge tube Millipore, and the 10kDa ultrafiltration and concentration carries out sieve chromatography then to the 1ml volume; 2.6 * 60cm molecular sieve chromatography Sepharcyl-200s HR Pharmacia uses 15mM acetate buffer solution pH5.0 in advance, electricity is led 1.05ms/cm
2Fully after the balance, by sample 1ml on the last sample ring, acid trehalosease washes out with same acetate buffer solution then, and flow velocity is set to 0.5ml/min, and collected volume is set to the 1.0ml/ pipe, and collection contains the trehalase active part and is used for next step purifying;
Gained trehalase liquid was through 0.5ml ultra-filtration centrifuge tube Millipore after described narrow pH range isoelectrofocusing separation was meant sieve chromatography, 10kDa ultrafiltration and concentration volume is to 0.5ml, be that 4.0～5.0 prefabricated isoelectrofocusing offset plate Pharmacia carry out electrophoretic separation with pH value scope then, after electrophoresis finishes, to contain trehalase band alive scales off, change in the dialysis tubing, the electricity consumption type of elution washes out zymoprotein.
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| CN102181472B (en) * | 2011-03-24 | 2013-05-29 | 重庆大学 | Fungus expression vector and construction and screening method thereof |
| CN103013955A (en) * | 2012-12-17 | 2013-04-03 | 福建农林大学 | Culture medium and method for producing trehalase by fermenting aschersonia placenta |
| CN103275999B (en) * | 2013-06-07 | 2014-08-20 | 江苏省农业科学院 | Apolygus lucorum membrane-bound trehalase, its coding sequence, vector and strain of sequence, and application of vector or strain |
| CN103333907B (en) * | 2013-06-07 | 2014-10-08 | 江苏省农业科学院 | Green plant bug water-soluble trehalase, coding sequence, vector, strain and application |
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| US5952212A (en) * | 1993-03-23 | 1999-09-14 | Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. | Protein tyrosine phosphatase |
| CN1542123A (en) * | 2003-11-07 | 2004-11-03 | 重庆大学 | Metarhizium anisopliae |
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| US5952212A (en) * | 1993-03-23 | 1999-09-14 | Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. | Protein tyrosine phosphatase |
| CN1180378A (en) * | 1996-02-20 | 1998-04-29 | 协和发酵工业株式会社 | Method for determining 1, 5 -anhydroglucitol |
| CN1542123A (en) * | 2003-11-07 | 2004-11-03 | 重庆大学 | Metarhizium anisopliae |
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