CN102321597B - Dextran dextrinase for preparing dextran - Google Patents

Abstract

The invention relates to a dextran dextrinase for preparing dextran. The dextran dextrinase for preparing dextran can be separated and purified from Gluconobacter oxydans capable of synthesizing dextran, and the molecular weight of the dextran dextrinase by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is 62kD; the active substrate is maltodextrin, and the inhibitor is Fe<2+>, Zn<2+> and Cu<2+>; and the optimal pH value for enzyme reaction is 4-10. Compared with the dextran synthesized by using maltodextrin as the substrate and the leuconastoc mesenteroidas dextran, thedextran dextrinase provided by the invention only contains alpha-1,6-glycosidic bond and alpha-1,4-glycosidic bond, and the structure alpha-1,4- branch chain, which is very rare in leuconastoc mesenteroidas dextran, appears in the structure of the dextran dextrinase. In addition, compared with the dextran synthesized by using G.oxydans ATCC 11894, the ratio of branch chains in the dextran synthesized by using the dextran dextrinase provided by the invention is higher (12%), and the dextran synthesized by using the dextran dextrinase provided by the invention has the characteristic of low viscosity. Due to the characteristics, the dextran dextrinase can have wide application prospects in the field of food.

Description

A kind of dextran dextrinase for preparing dextran

Technical field

The invention belongs to dextran technology for producing field, be specifically related to a kind of dextran dextrinase of separation and purification in bacillus of oxidizing glucose (Gluconobacter oxydans) born of the same parents, and preparation method thereof with main zymologic property.

Background technology

Dextran (Dextran) has another name called dextran, is a kind of high molecular polymer that is formed by glucose unit dehydration, and its structure has diversity, is generally defined as a kind of complete in polysaccharide that α-D-Glucopyranose monomer constitutes.Dextran is some bacteriogenic a kind of exocellular polysaccharide, it is the extracellular products that is generated by the enzyme catalysis of secretion property, as the microbial polysaccharide of finding the earliest, dextran is first kind of Microbial exopolysaccharides that can be used for food of U.S. FDA (Food and Drug Administration) approval, also is the microbial polysaccharide of first suitability for industrialized production in the world.Dextran is widely used in a plurality of fields such as medicine, food, stratographic analysis because of advantages such as it is safe, nontoxic, good biocompatibilities.

The production of dextran includes following two kinds of methods: microorganism direct fermentation and enzymic synthesis method, and on the industrial production mainly based on direct fermentation.But during direct fermentation production dextran, the product molecular size is difficult to control, and fermentation back thalline is difficult to separate with product, and impurity such as the nitrogen of introducing in the production, chlorine cause the dextran quality low, and clinical side reaction is many.Induce isolated glucan synthase to prepare dextran can to overcome these deficiencies and utilize, therefore, from distinctive microorganism, to make up by Protocols in Molecular Biology and the full new enzyme source of the dextran synthetic enzyme of screening is one of present research emphasis.

Certain endonuclease capable that Hehre and Hamilton etc. find to derive among the G.oxydans under study for action utilizes maltodextrin and the synthetic dextran of starch partial hydrolysate, and this kind of enzyme is named as dextran dextrinase (DDase).But this enzyme catalysis forms and only contains α-1,6 and α-1, the dextran of 4-glycosidic link, this dextran is compared with the dextran that utilizes the Leuconostoc mesenteroides preparation, viscosity and calorific value are lower, and the characteristics with retentiveness, toughness as low calorie foodstuff additive, are widely used in food service industry.Studies show that using DDase is a good dextran route of synthesis.This kind product also has many potential Application Areass to demand exploitation urgently, as new food additive, starch base sweeting agent, lower fat food substitute etc.

Recent two decades comes, and people begin slowly to pay close attention to this special transglycosylase, respectively the characteristic of this enzyme have been carried out deep research, comprises that influence produces the synthetic etc. of the substrate specificity of the purifying of the environmental factors of enzyme, enzyme, enzyme and glycosyl derivatives.But, up to the present, rarely have report about the research of bacillus of oxidizing glucose dextran dextrinase separating and purifying technology method and zymologic property thereof, and molecular weight does not appear in the newspapers more less than 100 Dextran 10 dextrinase.

Summary of the invention

The dextran dextrinase that the purpose of this invention is to provide a kind of separation and purification in bacillus of oxidizing glucose (Gluconobacter oxydans) born of the same parents, and preparation method thereof and main zymologic property, to remedy the prior art deficiency.

Dextran dextrinase of the present invention is that separation and purification obtains from the bacillus of oxidizing glucose that can synthesize dextran, and its SDS-PAGE electrophoresis molecular weight size is 62kD.

Above-mentioned bacillus of oxidizing glucose can be selected Gluconobacter oxydans DSM 2003 bacterium for use.

Above-mentioned dextran dextrinase, its effect substrate is maltodextrin.

Above-mentioned dextran dextrinase, its inhibitor are Fe ²⁺, Zn ²⁺Or Cu ²⁺

Above-mentioned dextran dextrinase, its suitableeest enzyme reaction pH scope is 4-10.

Above-mentioned dextran dextrinase, its suitableeest enzyme reaction temperature is 15-55 ℃; The temperature-stable interval is lower than 30 ℃; The pH stable region is 4.5-9.5; Michaelis-Menton constant K _mBe 0.63mmol/L; Maximum speed of reaction V _MaxBe 7.48 μ mol/ (mLmin).

Above-mentioned dextran dextrinase is that combined utilization ion exchange chromatography and gel-filtration chromatography purifying from the fermentation crude enzyme liquid of bacillus of oxidizing glucose obtain.

The application of dextran dextrinase of the present invention in the preparation dextran.

Dextran dextrinase of the present invention, to be the dextran that synthesizes of substrate with maltodextrin compare with the prepared dextran of leuconostoc mesentroides for it, only contain α-1,6-glycosidic link and α-1, the 4-glycosidic link, and having occurred on the leuconostoc mesentroides dextran in the dextran structure of preparation is rarely found α-1, this structure of 4-side chain.In addition, than the dextran that G.oxydans ATCC 11894 is synthesized, the ratio of side chain higher (12%) in the dextran that this enzyme synthesized, and have the low characteristics of viscosity.These characteristics will make it have more wide application prospect at field of food.

Description of drawings

Fig. 1: the electrophorogram of dextran dextrinase purge process of the present invention; Wherein swimming lane 1 is crude enzyme liquid, 2 is membrane filtration liquid, and 3 is Q Sepharose Fast Flow solution, and 4 is ANX Sepharose Fast Flow solution, 5 is Sephadex 75PG solution, and 6 is standard molecular weight albumen (18.4,25,35,45,66.4,116kD);

Fig. 2: the gel permeation chromatography figure of dextran dextrinase of the present invention;

Fig. 3: the suitableeest enzyme of dextran dextrinase of the present invention temperature alive and temperature stability synoptic diagram;

Fig. 4: the enzyme of dextran dextrinase of the present invention is lived the transformation period;

Fig. 5: the optimum pH of dextran dextrinase of the present invention and pH stability synoptic diagram;

Fig. 6: the synoptic diagram of various ion pair dextran dextrinase influences;

Fig. 7: concentration of substrate is to the figure that influences of dextran dextrinase speed of reaction;

Fig. 8: dextran dextrinase enzymatic reaction lyogel permeation chromatography figure of the present invention.

Embodiment

Below in conjunction with embodiment the purification step of enzyme of the present invention, the character of enzyme are described in detail.

Embodiment 1: the purification procedures of dextran dextrinase

Enzyme of the present invention is separation and purification from the bacillus of oxidizing glucose that can synthesize dextran, wherein a kind of is bacillus of oxidizing glucose DSM 2003, and this bacterial strain is open (oxidizing glucose acidfast bacilli DSM 2003 films are identified in conjunction with the purifying of ethanol dehydrogenase and 2010 31 13 phases of volume of property research Food science) in the literature.This bacterial strain is preserved in Food Science and Engineering institute of Chinese Marine University at present.

(1) yeast culture

Bacillus of oxidizing glucose DSM 2003 bacterial classification inoculations in seed culture medium, after 30 ℃ of constant temperature shaking tables are cultivated 24h, are inserted product enzyme substratum by inoculum size 10% (v/v), and 30 ℃ of constant temperature shaking tables are cultivated 60h.

Seed culture medium: sorbyl alcohol 80g/L, yeast powder 20g/L, KH ₂PO ₄0.6g/L, MgSO ₄7H ₂O0.5g/L.

Produce the enzyme substratum: grape sugar 17.67g/L, maltose 30g/L, Tryptones 12.20g/L, yeast powder 13.53g/L, ammonium nitrate 15g/L, copper sulfate 0.01g/L, zinc sulfate 0.01g/L, sodium-chlor 0.01g/L, initial pH 6.0.

(2) preparation of acellular crude enzyme liquid

Centrifugal fermented liquid is collected thalline, adds the 50mmol/LTris-HCl damping fluid of the pH 7.4 of 3 times of volumes in the thalline, and ultrasonic disruption is adopted 90 times (the ultrasonic disruption condition: every broken 5s is 5s at interval, power 300W) in resuspended back.Again 4 ℃ down centrifugal (10000r/min, 20min), supernatant liquor is acellular crude enzyme liquid;

(3) Q Sepharose Fast Flow sepharose anion-exchange chromatography

With the abundant balance HiTrap Q of the 50mmol/L Tris-HCl damping fluid FF ion exchange column (5mL, GE company) of pH 7.4, last sample absorption.Use the 50mmol/L Tris-HCl damping fluid (containing 1mol/LNaCl) of pH 7.4 to carry out stepwise elution then, elution speed is 2mL/min, and fraction collection is surveyed enzyme work and carried out the protein electrophoresis analysis the solution in the collection tube;

(4) HiPrep Desalting Sephadex desalination

With the abundant balance HiPrep 26/10 Desalting gel desalting column of the 50mmol/L Tris-HCl damping fluid of pH 7.4 (53mL, GE company), last sample absorption.50mmol/L Tris-HCl damping fluid with pH 7.4 carries out the damping fluid displacement then, and elution speed is the 2mL/min fraction collection, and the solution in the collection tube is surveyed enzyme work and carried out the protein electrophoresis analysis;

(5) ANX Sepharose Fast Flow sepharose anion-exchange chromatography

With the abundant balance HiTrap ANX of the 50mmol/L Tris-HCl FF ion exchange column (1mL, GE company) of pH 7.4, last sample absorption.Use the 50mmol/L Tris-HCl damping fluid (containing 1mol/L NaCl) of pH 7.4 to carry out stepwise elution then, elution speed is 2mL/min, and fraction collection is surveyed enzyme work and carried out the protein electrophoresis analysis the solution in the collection tube;

(6) Sephadex 75PG gel permeation chromatography

With the abundant balance HiLoad 16/60 Superdex 75PG gel column (120mL, GE company) of the 50mmol/L Tris-HCl damping fluid (containing 100mmol/L NaCl) of pH 7.4, the enzyme concentrated solution of last sample 2mL.Use 50mmol/L Tris-HCl damping fluid (the containing 100mmol/L NaCl) wash-out of pH 7.4 then, elution speed 0.5mL/min, fraction collection is surveyed enzyme work and is carried out the protein electrophoresis analysis solution in the collection tube.

Measured with the enzyme activity of last step sample respectively and carried out SDS-PAGE.Purification result sees Table 1, and crude enzyme liquid is behind a few step purifying, and specific activity is brought up to 112.5U/mg from 0.6U/mg, and the purifying multiple is 187.5 times.Protein electrophoresis result (Fig. 1) shows that the dextran dextrinase behind the purifying is a band at electrophoresis, and molecular weight is about 62kD.

Table 1: the purification step of dextran dextrinase and result

Embodiment 2: the zymologic property of dextran dextrinase of the present invention

(1) molecular weight determination of dextran dextrinase

Because the relative mobility of protein is directly proportional with the logarithm of molecular weight, therefore can measure the molecular weight of target protein by SDS-PAGE.According to the relative mobility of standard molecular weight albumen and target protein among the SDS-PAGE, the molecular weight size that calculates DDase is about 62kD.The molecular weight of albumen also can calculate by the elution volume of gel chromatography.As shown in Figure 2, the molecular weight that calculates DDase according to Sephacryl S-200 high resolution gel chromatography elution volume is about 52kDa.The result of comprehensive SDS-PAGE and gel chromatography shows that DDase exists with monomeric form.

(2) optimum temperuture of dextran dextrinase and thermal stability determination

DDase behind the purifying carries out enzyme assay under different temperature (10,20,25,30,35,40,45,50,60 ℃), the result as shown in Figure 3.The optimum temperuture of DDase is 35 ℃, when being lower than 20 ℃ or when being higher than 45 ℃, enzymic activity significantly reduces, and only is about 50% under the optimum temperuture.

The temperature stability analysis of enzyme refers to enzyme measure remnant enzyme activity by standard method after certain temperature (10,20,25,30,35,40,45,50,60 ℃) is incubated 3h down, and the result as shown in Figure 3.DDase is in that stability is arranged below 30 ℃ preferably, when temperature is higher than 45 ℃, and pure enzyme whole inactivations almost in 3h.

Enzyme live stability also the active transformation period of available enzyme represent that the enzyme of DDase is lived the transformation period under high spot reviews 30, the 35 and 40 ℃ of conditions, as shown in Figure 4: under 30 ℃, the enzyme of DDase transformation period of living can reach 17.66h, is higher than 35 and 40 ℃ far away.

Suppose that enzyme deactivation meets first order reaction kinetics, each formula is as follows:

$\frac{\mathrm{dE}}{\mathrm{dt}}=-\mathrm{\&lambda;E}$ E _t-remnant enzyme activity

$\mathrm{Ln}\left(E_t\right)=-\mathrm{\&lambda;t}+\mathrm{Ln}\left(E_0\right)\&DoubleRightArrow;{\mathrm{<figure-callout\; id="1"\; label="t"\; filenames="HDA0000097078210000011.png,HDA0000097078210000041.png"\; state="\{\{state\}\}">t</figure-callout>}}_{1/2}=\frac{\mathrm{<figure-callout\; id="2"\; label="Ln"\; filenames="HDA0000097078210000011.png,HDA0000097078210000012.png"\; state="\{\{state\}\}">Ln</figure-callout>}2}{\mathrm{\&lambda;}}$ E ₀-initial enzyme is lived

λ-disintegration constant

E _t=E ₀E ^{-λ t}The t-time

(3) mensuration of the optimum pH of dextran dextrinase

The method of DDase behind the purifying under different pH carried out enzyme assay, and the result as shown in Figure 4.The enzymic activity of DDase is relatively more responsive to pH, and optimal pH is 6.3.When pH is lower than 5.5 or when being higher than 7, enzymic activity significantly descends, and be lower than 50% of optimal pH.

The mensuration of pH stability is that the enzyme liquid of equivalent is placed 3h for 4 ℃ with the damping fluid of different pH, measures remnant enzyme activity by standard method during mensuration, and the result as shown in Figure 5.The result shows that DDase all can keep stability preferably between pH 4.5-9.5.

(4) metal ion is to the influence of enzymic activity

Do not investigate the various ion (Mn of 0.1mmol/L ²⁺, Zn ²⁺, Cu ²⁺, Ca ²⁺, Ba ²⁺, Mg ²⁺, Ni ²⁺, Co ²⁺, Fe ²⁺, Fe ³⁺) to the influence that enzyme is lived, the enzyme work of control group is decided to be 100%.As shown in Figure 6, Cu ²⁺And Zn ²⁺Enzyme work to DDase has had strong inhibitory effects, secondly is Fe ²⁺, Ni ²⁺, Co ²⁺, Mn ²⁺Ca ²⁺And Mg ²⁺Work has slight activation to enzyme.

(5) mensuration of enzyme kinetics parameter

(0-1.5mmol/L) as substrate, measure every reaction power mathematic(al) parameter (maximum initial reaction rate V of DDase with p-nitrophenyl-α-D-glycopyranoside (NPG) _Max, Michaelis-Menton constant K _m, catalytic constant K _CatWith catalytic efficiency K _Cat/ K _m).As can be seen from Figure 7, with the 1/[V reciprocal of enzyme ' s reaction speeding] be ordinate zou, the 1/[S reciprocal of substrate] for the X-coordinate mapping, can obtain straight line.Its transverse axis intercept is-1/K _m, vertical axis intercept is 1/V _Max(maximum reaction velocity), slope are K _m/ V _Max, obtain the Michaelis-Menton constant K of the substrate p-nitrophenyl-α of DDase-D-glycopyranoside (NPG) thus _mWith maximum speed of reaction V _MaxBe respectively 0.63mmol/L and 7.48 μ mol/ (mLmin).By V _Max=K _Cat[E ₀], can try to achieve catalytic constant K _CatWith catalytic efficiency K _Cat/ K _mBe respectively 523.6l/s, 832.6L/ (mmols).

Embodiment 3: the application of dextran dextrinase of the present invention

The pure enzyme liquid of DDase (0.1U/mL) of 1mL the present invention preparation and the maltodextrin (being dissolved in (pH 6.5) in the 50mmol/L phosphoric acid buffer) of 7mL 0.1% are mixed, place 30 ℃ of shaking table oscillatory reactions, the enzymatic reaction process adopts gel chromatograph (GPC) to monitor.The result as shown in Figure 8, its reaction process to acellular enzyme liquid is similar, a detected peaks different with substrate has also appearred in the gel permeation chromatography figure of sample about 11.2min.Know that by gel permeation chromatography map analysis qualification result this product is dextran, this also illustrates the dextran dextrinase that is that purifying obtains.The result shows that dextran dextrinase of the present invention can be used for preparing dextran.

Claims (2)

1. dextran dextrinase, its SDS-PAGE electrophoresis molecular weight size is 62 kD; The molecular weight that Sephacryl S-200 high resolution gel chromatography elution volume calculates is 52 kDa;

The effect substrate is maltodextrin;

Inhibitor is Fe ²⁺, Zn ²⁺Or Cu ²⁺

The suitableeest enzyme reaction pH scope is 4～10;

Enzyme reaction temperature is 15-55 ℃;

Above-mentioned dextran dextrinase is that separation and purification obtains from the bacillus of oxidizing glucose that can synthesize dextran, and its preparation method is as follows:

(1) yeast culture

Bacillus of oxidizing glucose DSM 2003 bacterial classification inoculations in seed culture medium, after 30 ℃ of constant temperature shaking tables are cultivated 24 h, are inserted product enzyme substratum, 30 by inoculum size 10% ^OC constant temperature shaking table is cultivated 60 h;

Seed culture medium: sorbyl alcohol 80 g/L, yeast powder 20 g/L, KH ₂PO ₄0.6 g/L, MgSO ₄7H ₂O 0.5 g/L;

Produce the enzyme substratum: grape sugar 17.67 g/L, maltose 30 g/L, Tryptones 12.20 g/L, yeast powder 13.53 g/L, ammonium nitrate 15 g/L, copper sulfate 0.01 g/L, zinc sulfate 0.01 g/L, sodium-chlor 0.01 g/L, initial pH 6.0;

(2) preparation of acellular crude enzyme liquid

Centrifugal fermented liquid is collected thalline, adds the 50 mmol/L Tris-HCl damping fluids of the pH 7.4 of 3 times of volumes in the thalline, and ultrasonic disruption is adopted 90 times in resuspended back, and every broken 5 s are 5 s at interval, power 300 W; At 4 ℃ of following centrifugal 20 min of 10000 r/min, supernatant liquor is acellular crude enzyme liquid again;

(3) Q Sepharose Fast Flow sepharose anion-exchange chromatography

With the abundant balance HiTrap Q of the 50 mmol/L Tris-HCl damping fluids FF ion exchange column of pH 7.4, last sample absorption; 50 mmol/L Tris-HCl damping fluids with pH 7.4 carry out stepwise elution then, and elution speed is 2 mL/min, fraction collection;

(4) HiPrep Desalting Sephadex desalination

With sample absorption on the abundant balance HiPrep 26/10 Desalting gel desalting column of the 50 mmol/L Tris-HCl damping fluids of pH 7.4; 50 mmol/L Tris-HCl damping fluids with pH 7.4 carry out the damping fluid displacement then, and elution speed is 2 mL/min fraction collections, and the solution in the collection tube is surveyed enzyme work and carried out the protein electrophoresis analysis;

(5) ANX Sepharose Fast Flow sepharose anion-exchange chromatography

With sample absorption on the abundant balance HiTrap ANX of the 50 mmol/L Tris-HCl FF ion exchange column of pH 7.4,50 mmol/L Tris-HCl damping fluids with pH 7.4 carry out stepwise elution then, elution speed is 2 mL/min, fraction collection is surveyed enzyme work and is carried out the protein electrophoresis analysis the solution in the collection tube;

(6) Sephadex 75PG gel permeation chromatography

With the abundant balance HiLoad 16/60 Superdex 75PG gel column of the 50 mmol/L Tris-HCl damping fluids of pH 7.4, the enzyme concentrated solution of last sample 2 mL; Use the 50 mmol/L Tris-HCl buffer solution elution of pH 7.4 then, elution speed 0.5 mL/min, fraction collection is finished preparation.

2. the application of the described dextran dextrinase of claim 1 in the preparation dextran.

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