CN102321597A - Dextran dextrinase for preparing dextran - Google Patents
Dextran dextrinase for preparing dextran Download PDFInfo
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- CN102321597A CN102321597A CN201110300991A CN201110300991A CN102321597A CN 102321597 A CN102321597 A CN 102321597A CN 201110300991 A CN201110300991 A CN 201110300991A CN 201110300991 A CN201110300991 A CN 201110300991A CN 102321597 A CN102321597 A CN 102321597A
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- dextran
- dextrinase
- dextran dextrinase
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- expex
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- 108010012023 Dextrin dextranase Proteins 0.000 title claims abstract description 70
- 229920002307 Dextran Polymers 0.000 title abstract description 12
- 239000000758 substrate Substances 0.000 claims abstract description 11
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims abstract description 8
- 229920002774 Maltodextrin Polymers 0.000 claims abstract description 7
- 239000005913 Maltodextrin Substances 0.000 claims abstract description 7
- 238000006911 enzymatic reaction Methods 0.000 claims abstract description 7
- 229940035034 maltodextrin Drugs 0.000 claims abstract description 7
- 108090000790 Enzymes Proteins 0.000 claims description 51
- 102000004190 Enzymes Human genes 0.000 claims description 51
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 14
- 239000008103 glucose Substances 0.000 claims description 13
- 239000007788 liquid Substances 0.000 claims description 13
- 230000001590 oxidative effect Effects 0.000 claims description 13
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 12
- 230000000694 effects Effects 0.000 claims description 11
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- 238000000746 purification Methods 0.000 claims description 9
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- 230000004151 fermentation Effects 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- 238000000926 separation method Methods 0.000 claims description 5
- 241001460542 Gluconobacter oxydans DSM 2003 Species 0.000 claims description 2
- 238000004255 ion exchange chromatography Methods 0.000 claims description 2
- 239000002512 suppressor factor Substances 0.000 claims description 2
- 235000013305 food Nutrition 0.000 abstract description 8
- 241000589232 Gluconobacter oxydans Species 0.000 abstract description 6
- 239000003112 inhibitor Substances 0.000 abstract 1
- 230000002194 synthesizing effect Effects 0.000 abstract 1
- 238000013016 damping Methods 0.000 description 10
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- 238000006243 chemical reaction Methods 0.000 description 8
- 238000010828 elution Methods 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000011160 research Methods 0.000 description 5
- 229920002684 Sepharose Polymers 0.000 description 4
- 230000003197 catalytic effect Effects 0.000 description 4
- 150000004676 glycans Chemical class 0.000 description 4
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- 230000009466 transformation Effects 0.000 description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 229920005654 Sephadex Polymers 0.000 description 3
- 239000012507 Sephadex™ Substances 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
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- 239000010949 copper Substances 0.000 description 3
- 238000011033 desalting Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000011701 zinc Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000192132 Leuconostoc Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 239000012564 Q sepharose fast flow resin Substances 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 238000005571 anion exchange chromatography Methods 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
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- 239000001963 growth medium Substances 0.000 description 2
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- 238000011218 seed culture Methods 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- MEIRRNXMZYDVDW-MQQKCMAXSA-N (2E,4E)-2,4-hexadien-1-ol Chemical compound C\C=C\C=C\CO MEIRRNXMZYDVDW-MQQKCMAXSA-N 0.000 description 1
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- 241000024287 Areas Species 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 229920002444 Exopolysaccharide Polymers 0.000 description 1
- 241000192130 Leuconostoc mesenteroides Species 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 239000012506 Sephacryl® Substances 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-UHFFFAOYSA-N alpha-D-glucopyranose Natural products OCC1OC(O)C(O)C(O)C1O WQZGKKKJIJFFOK-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-DVKNGEFBSA-N alpha-D-glucose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-DVKNGEFBSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
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- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 108010052221 glucan synthase Proteins 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
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- 238000009776 industrial production Methods 0.000 description 1
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- 239000002054 inoculum Substances 0.000 description 1
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- 238000005374 membrane filtration Methods 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
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- 239000000178 monomer Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000003534 oscillatory effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
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- 238000001308 synthesis method Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 108010046845 tryptones Proteins 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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- Enzymes And Modification Thereof (AREA)
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, the dextran 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
Technical field
The invention belongs to Expex 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
Expex (Dextran) has another name called VISOSE, is a kind of high molecular polymer that is formed by glucose unit dehydration, and its structure has variety, is generally defined as a kind of complete in polysaccharide that α-D-Glucopyranose monomer constitutes.Expex is some bacteriogenic a kind of exocellular polysaccharide; It is the extracellular products that generates by the secreted enzyme catalysis; As the microbial polysaccharide of finding the earliest; Expex 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.Expex 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 Expex includes following two kinds of methods: mikrobe direct fermentation and enzymic synthesis method, and be main with direct fermentation mainly on the industrial production.But when direct fermentation was produced VISOSE, the product molecular size was 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 Expex quality low, and clinical side reaction is many.Induce isolated glucan synthase to prepare Expex can to overcome these deficiencies and utilize, therefore, from distinctive mikrobe, to make up and the full new enzyme source of the Expex synthetic enzyme of screening is one of present research emphasis through Protocols in Molecular Biology.
Certain endonuclease capable that Hehre and Hamilton etc. find to derive among the G.oxydans under study for action utilizes maltodextrin and the synthetic Expex 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 Expex of 4-glycosidic link, this Expex is compared with the Expex 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.Research shows that using DDase is a good Expex route of synthesis.This kind product also has many potential Application Areass to demand exploitation urgently, like new food additive, starch-based sweeting agent, lower fat food substitute or the like.
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 substrate specificity and glycosyl derivatives synthetic or the like of the purifying of the environmental factors of enzyme, enzyme, enzyme.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 with 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 Expex, 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 a maltodextrin.
Above-mentioned dextran dextrinase, its suppressor factor are Fe
2+, Zn
2+Or Cu
2+
Above-mentioned dextran dextrinase, its righttest enzyme reaction pH scope is 4-10.
Above-mentioned dextran dextrinase, its righttest 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 response speed 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 Expex.
Dextran dextrinase of the present invention; It uses maltodextrin to compare with the prepared Expex of leuconostoc mesentroides as the Expex that substrate synthesized; Only contain α-1,6-glycosidic link and α-1, the 4-glycosidic link; And having occurred on the leuconostoc mesentroides Expex in the Expex structure of preparation is rarely found α-1, this structure of 4-side chain.In addition, than 11894 synthetic Expexs of G.oxydans ATCC, the ratio higher (12%) of side chain in this enzyme institute synthetic Expex, 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 a 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 righttest 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 pair ion 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 carried out detailed description.
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 Expex; Wherein a kind of is bacillus of oxidizing glucose DSM 2003, this bacterial strain open in document (oxidizing glucose acidfast bacilli DSM 2003 films combine the purifying of ethanol dehydrogenase to identify and 2010 31 13 phases of volume of property research Food science).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, SANMALT-S 30g/L, Tryptones 12.20g/L, yeast powder 13.53g/L, an 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, in thalline, adds the 50mmol/LTris-HCl damping fluid of the pH 7.4 of 3 times of volumes, and ultrasonic disruption is adopted 90 times (the ultrasonic disruption condition: every broken 5s is 5s at interval, power 300W) in resuspended back.Centrifugal under 4 ℃ again (10000r/min, 20min), supernatant 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 appearance 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 appearance 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 appearance 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 liquid concentrator of last appearance 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 is seen 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 on electrophoresis, 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 proteinic relative mobility is directly proportional with the logarithm of molecular weight, therefore can measure the molecular weight of target protein through 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.Proteic molecular weight also can calculate through 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 is 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 is merely about 50% under the optimum temperuture.
The temperature stability analysis of enzyme is meant with enzyme that after certain temperature (10,20,25,30,35,40,45,50,60 ℃) is incubated 3h down measure remnant enzyme activity by standard method, the result is as shown in Figure 3.DDase is in that stability is preferably arranged below 30 ℃, and when temperature was higher than 45 ℃, pure enzyme is almost whole inactivations in 3h.
Enzyme is lived, and also the active transformation period of available enzyme representes that the enzyme of DDase is lived the transformation period under 30,35 and 40 ℃ of conditions of high spot reviews, and is as shown in Figure 4 to stability: under 30 ℃, the enzyme of the DDase transformation period alive can reach 17.66h, is higher than 35 and 40 ℃ far away.
Suppose that enzyme deactivation meets first order reaction kinetics, each formula is following:
λ-disintegration constant
E
t=E
0E
-λ tThe 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 is as shown in Figure 4.The enzymic activity of DDase is relatively more responsive to pH, and ph optimum 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 ph optimum.
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 is as shown in Figure 5.The result shows that DDase all can keep stability preferably between pH 4.5-9.5.
(4) metals 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 each item 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 beappreciated from fig. 7, be ordinate zou with the inverse 1/ [V] of enzyme ' s reaction speeding, the inverse 1/ [S] of substrate can obtain straight line for the X-coordinate mapping.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 DDase thus to substrate p-nitrophenyl-α-D-glycopyranoside (NPG)
mWith maximum response speed 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 is as shown in Figure 8, and its reaction process with acellular enzyme liquid is similar, and one and substrate different detection peak have 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 an Expex, this also explains the dextran dextrinase that is that purifying obtains.The result shows that dextran dextrinase of the present invention can be used for preparing Expex.
Claims (9)
1. a dextran dextrinase is that separation and purification obtains from the bacillus of oxidizing glucose that can synthesize Expex, and its SDS-PAGE electrophoresis molecular weight size is 62kD.
2. dextran dextrinase as claimed in claim 1 is characterized in that described bacillus of oxidizing glucose is Gluconobacter oxydans DSM 2003 bacterium.
3. dextran dextrinase as claimed in claim 1 is characterized in that described dextran dextrinase, and its effect substrate is a maltodextrin.
4. dextran dextrinase as claimed in claim 1, the suppressor factor that it is characterized in that described dextran dextrinase is Fe
2+, Zn
2+Or Cu
2+
5. dextran dextrinase as claimed in claim 1 is characterized in that the righttest enzyme reaction pH scope of described dextran dextrinase is 4～10.
6. dextran dextrinase as claimed in claim 1, the enzyme reaction temperature that it is characterized in that described dextran dextrinase is 15-55 ℃.
7. the working method of the described dextran dextrinase of claim 1 is that purifying obtains from the fermentation crude enzyme liquid of the bacillus of oxidizing glucose that can synthesize Expex.
8. working method as claimed in claim 7 is characterized in that it being that described fermentation crude enzyme liquid is prepared dextran dextrinase behind ion exchange chromatography and gel permeation chromatography.
9. the application of the described dextran dextrinase of claim 1 in the preparation Expex.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101487035A (en) * | 2009-02-25 | 2009-07-22 | 青岛生物能源与过程研究所 | Method for preparing dextran with Gluconobacter oxydans as strain |
| CN101709293A (en) * | 2009-12-16 | 2010-05-19 | 青岛生物能源与过程研究所 | Method for separating and purifying dextran dextrinase from gluconobater oxydans |
| CN101928698A (en) * | 2009-06-19 | 2010-12-29 | 青岛生物能源与过程研究所 | Method for quickly extracting and determining dextran dextrinase in cells of gluconobacter oxydans |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101487035A (en) * | 2009-02-25 | 2009-07-22 | 青岛生物能源与过程研究所 | Method for preparing dextran with Gluconobacter oxydans as strain |
| CN101928698A (en) * | 2009-06-19 | 2010-12-29 | 青岛生物能源与过程研究所 | Method for quickly extracting and determining dextran dextrinase in cells of gluconobacter oxydans |
| CN101709293A (en) * | 2009-12-16 | 2010-05-19 | 青岛生物能源与过程研究所 | Method for separating and purifying dextran dextrinase from gluconobater oxydans |
Non-Patent Citations (2)
| Title |
|---|
| 《微生物学通报》 20110820 王舒等 氧化葡萄糖杆菌合成右旋糖酐糊精酶的pH两阶段控制策略 1155-1159 1-6，9 第38卷, 第8期 * |
| 《生物加工过程》 20100531 王舒等 应用细胞透性化技术快速提取氧化葡萄糖杆菌胞内右旋糖酐糊精酶 35-39 1-9 第8卷, 第3期 * |
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