CN102229970A - Method for producing dextran and fructose by recombinant enzyme process - Google Patents
Method for producing dextran and fructose by recombinant enzyme process Download PDFInfo
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- CN102229970A CN102229970A CN2011101323054A CN201110132305A CN102229970A CN 102229970 A CN102229970 A CN 102229970A CN 2011101323054 A CN2011101323054 A CN 2011101323054A CN 201110132305 A CN201110132305 A CN 201110132305A CN 102229970 A CN102229970 A CN 102229970A
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Abstract
The invention discloses a method for producing dextran and fructose by a recombinant enzyme process. The method comprises the following steps of: (1) culturing free recombinant dextransucrase by using a genetically engineered bacterial strain BL21 (DE3)/pET28-dexYG; and (2) catalytically synthesizing dextran and fructose with the recombinant dextransucrase. The invention provides a method for preparing recombinant dextransucrase by an IPTG (isopropyl beta-D-1-thiogalactopyranoside)/lactose combined induction method through using genetically engineered bacterial strain BL21 (DE3)/pET28-dexYG. The method greatly reduces the enzyme preparation cost in the single IPTG induction method and is favorable for large-scale production. Besides, by use of the intermittent vibratory enzyme catalysis method, the dextran and fructose are catalytically synthesized in one step, so as to increase the conversion efficiency, achieve the effects of multiple purposes of one enzyme and fructose recovery, reduce the cost and solve the problem of environmental pollution due to fructose as the byproduct of the dextran production process in the prior art.
Description
Technical field
The present invention relates to low-cost the recombinate method of dextransucrase and the method for enzyme catalysis production biological polyoses and monose thus of prepare of a kind of combined induction, be particularly related to the enzymatic process that the reorganization dextransucrase adopts the gap vibration, the method for synthetic dextran of one-step catalytic and fructose.
Background technology
Dextransucrase (Dextransucrase, EC 2.4.1.5) be a kind of by leuconostoc mesentroides (
Leuconstoc mesenteriodes) glucanotransferase (Glucosyltransferases) that produces, constitute by 1250 to 1600 different amino acid, molecular weight is about 170KDa, and it is synthetic at polyose medicament, a plurality of fields such as preparation of medicinal fructose, chromatography separation media and protective foods all have important application.The catalysis of this enzyme is to be substrate with sucrose, D-glucosyl group catalysis in the sucrose molecules is shifted forming D-glucosyl-Enzyme, and is discharged fructose, and its result generates two class staple products: 1. the different molecular weight dextran is synthesized in catalysis; 2. prepare high-purity medicinal fructose.
Dextran (dextran) is the polymkeric substance that is formed by some gluconate dehydratases, is mainly connected by α (1-6) glycosidic bond.Because of it has multiple advantages such as safe, nontoxic, good biocompatibility, many aspects such as medicine, food have been widely used in.Clinical application has three kinds of specifications: macrodex, Dextran 40 and Dextran 20 are that a kind of good Q volume of blood replenishes medicine, also are simultaneously the important source material of producing the hematopoietic Iron Dextran.The dextran of molecular weight about 70,000 is one of good plasma substitute of generally acknowledging at present, and the Q volume of blood of increasing effect is arranged, and clinically is mainly used in the treatment hemorrhagic shock; Molecular weight 40,000 and about 20,000 dextran have the microcirculation improvement effect, and be main by removing erythrocyte aggregation, reduces effect such as blood viscosity and microcirculation improvement also has certain replenishment of blood content effect simultaneously.
Fructose is a kind of levorotatory hexose, faster the human body metabolism than glucose, absorbed by body easily, and do not rely on Regular Insulin, very little to blood sugar influence, be applicable to patient's supplementing energy of glucose metabolism and hepatic insufficiency, it also has the breeding of the beneficial bacteria of promotion simultaneously, improve intestinal function and metabolism, characteristics such as unlikely carious tooth, so fructose is the desirable sweeting agent of diabetics, adiposis patient, infant foods.Take in fructose before the motion and can not cause hypoglycemia, and if the common carbohydrate of taking in rapid absorption in ultra-long time exercise back will cause this type of hypoglycemia.In pharmacopeia is all listed fructose by Europe, the United States, day and China, and as oral or injection, particularly glucose metabolism obstacle person (diabetics) is suitable for.
At present domestic production dextran technology mainly adopt anaerobically fermenting, ethanol to pinch to wash, hydrochloric acid hydrolysis, ethanol division etc.This technique ethanol consumption is big, production environment is abominable, thalline and dextran are twined mutually and thalline can not be removed in the fermenting process, impurity such as assorted nitrogen, chlorion, sulfated ash have been introduced in the production again, cause its product quality indicator not reach Japan, America and Europe's standards of pharmacopoeia, can't enter the world market, lack competitive power; And the high medicinal dextran of foreign matter content causes clinical supersensitivity side reaction many.Domestic the dextran process study is only limited in traditional zymotic technology filtering fermentation liquor, the improvement of unit operations such as hydrolysis, classification division can not be improved the quality of products in essence from technology.
The inventor cloned the complete genome that has obtained this enzyme from leuconostoc mesentroides in 2005
DexYG(GenBank No. DQ345760).With this gene reorganization dextransucrase intestinal bacteria that can efficiently express that have been fundamental construction, this genetic engineering bacterium has obtained patent (ZL 200710134765.4) then.The present invention starts with from genetic engineering bacterium BL21 (the DE3)/pET28-dexYG that has independent intellectual property right, obtain the method that lower-cost IPTG and lactose combined induction prepare the dextransucrase of recombinating, and utilize the reorganization dextransucrase to adopt the enzymatic process of gap vibration, single stage method is produced dextran and fructose, has significantly improved transformation efficiency.Realize an enzyme how with, reclaim fructose, reduced cost, improve the quality of products, and solved in the dextran original production process by product fructose the pollution problem of environment.
Summary of the invention
The objective of the invention is to use the free reorganization of lower-cost IPTG and lactose combined induction method preparation dextransucrase, then with this recombinase basis, begin to adopt one step of the enzymatic process preparation dextran and the fructose of gap vibration by substrate sucrose, this production method replaces the technology of being produced dextran by intestines mould shape leukonid, avoid the thalline that occurs in the production process along with bacterium, eliminate foreign protein, improve the quality of products; Realize an enzyme how with, reclaim fructose, reduced cost, and solved in the dextran original production process by product fructose the pollution problem of environment.
The method of reorganization enzyme method for preparing dextral glycoanhydride of the present invention and fructose comprises the steps:
(1) preparation of free reorganization dextransucrase: engineering strain BL21 (DE3)/pET28-dexYG is transferred in the fresh LB substratum that contains 40-60 μ g/mL kantlex, and in the 35-40 ℃ of following 15-18h that cultivates, again it is inoculated into to produce among the enzyme substratum B and cultivates, work as OD in 35-40 ℃ of bottom fermentation
600 During=2.5-4.0, adopting IPTG and lactose combined induction to IPTG final concentration is that 0.07-0.12 mmol/L, lactose final concentration are 2.0-3.0g/L, and inducing temperature is 20-30 ℃, and induction time is 4-6h; Liquid after the cultivation adds the pH5.4 acetate buffer solution behind 0-5 ℃ of centrifugal thalline, ultrasonication, and the centrifuging and taking supernatant liquor promptly gets the dextransucrase liquid of recombinating;
(2) with synthetic dextran of reorganization dextransucrase catalysis and fructose: adopt following step:
(a) use pH5.4 acetate-sodium acetate buffer dissolving 10-15g sucrose of the 5mmol/L of 50mL to prepare the enzyme substrates reaction solution; Behind the reorganization dextransucrase liquid 10-15 mL that wherein adds enzyme 70-170u/ml alive, pH5.4 acetate-sodium acetate buffer constant volume of using 5mmol/L is to 100mL again;
(b) down adopt vibrator that reaction solution is carried out 120n/min vibration 5h during the reaction beginning at 25-30 ℃, standing and reacting 5h then, the reaction that hockets according to this finishes catalyzed reaction behind the 22-25h;
(c) reaction solution after the catalysis is carried out the alcohol precipitation washing, vacuum-drying is weighed, obtain high molecular dextran, precipitation solution is removed macromole impurity through centrifuging, obtain fructose through ultrafiltration removal small molecules oligose, filtrate through vacuum concentration, spraying drying again.
Substratum B is used for inducing of colibacillary fermentation and enzyme in the step described in the present invention (1), wherein contains glycerine, peptone and substratum component of inorganic salts commonly used, and it can adopt following prescription:
The composition of substratum B: glycerine 5g/L; Peptone (trypyone) 10 g/L; Saltpetre 10 g/L; MgSO
40.1 mmol/L; NH
4Cl 1 g/L; Na
2HPO
47H
2O 12.8 g/L or Na
2HPO
412H
2O 17.1 g/L; KH
2PO
43g/L.
During fermentation culture, adopt IPTG and lactose combined induction legal system to be equipped with dextransucrase in the step described in the present invention (1), preferably work as OD
600 , adopt IPTG and lactose combined induction to best final concentration to be respectively 0.09mmol/L and 2.5g/L, 25 ℃ of inducing culture at=3.0 o'clock.
The enzyme of the preferred reorganization dextransucrase liquid that adds is lived and is 145-170u/ml in the step described in the present invention (2).
Genetic engineering bacterium BL21 (DE3)/pET28-dexYG bacterial classification inoculation, cultivation and enrichment among the present invention can be adopted following manner:
From the inclined-plane with transfering loop picking thalline, be transferred to the fresh LB substratum of 20mL (contain among that 50 μ g of card/mL), 37 ℃, 300r/min incubated overnight, incubation time is 16-18h.Again according to the bacterium liquid of 1% inoculum size inoculation incubated overnight fermentation culture in the substratum B.
Fermented liquid is in 37 ℃, 300r/min cultivation, and preceding 2h per hour gets fermented liquid 1mL, with 10 times of sterilized water dilutions, compares with substratum, measures OD
600 Every afterwards 1.5h gets fermented liquid 1mL, with 10 times of sterilized water dilutions, compares with substratum, measures OD
600 Work as OD
600 =3.0-4.0, fermentation culture finishes, and changes next step over to and induces the product enzyme.
The LB culture medium prescription is as follows:
LB flat-plate solid substratum is used for the screening of thalline, and the inclined-plane solid medium is used for thalline short-term (about 1 month) preservation, and the LB liquid nutrient medium is used for the incubated overnight of thalline.
The composition of LB substratum: Tryptones (bacto-trypyone) 10 g/L; Yeast extract (yeast extract) 5 g/L; NaCl 10 g/L; Distilled water 1000 mL are 7.0 with NaOH or the HCl adjust pH of 1 mol/L, the 0.1MPa 20min that sterilizes, with preceding adding kantlex to 50ug/mL.Solid medium also need add the agar of 1.5-2%, and the 0.1MPa 20min that sterilizes is to be cooled during to 50-60 ℃, adds kantlex to 50ug/mL, is cooled to inclined-plane or flat board.
Substratum B is used for inducing of colibacillary fermentation and enzyme, and it is as follows to fill a prescription:
The composition of substratum B: glycerine 5g/L; Peptone (trypyone) 10 g/L; Saltpetre 10 g/L; MgSO
40.1 mmol/L; NH
4Cl 1 g/L; Na
2HPO
47H
2O 12.8 g/L or Na
2HPO
412H
2O 17.1 g/L; KH
2PO
43g/L.
Before the preparation dextran, detect the vigor of prepared dextransucrase earlier, the mensuration of dextransucrase vigor adopts 3, and 5-edlefsen's reagent method is surveyed the amount (DNS method) of reducing sugar.Step is: get the 5mL fermented liquid, the centrifugal 10min of 8000r/min, get supernatant liquor 2ml (enzyme liquid) and 2ml substrate (0.2M NaAc 860mL, 0.2M HAc 140ml, sucrose 100g, pH5.4) mix, take out 0.5ml and add 0.375ml DNS reagent, in boiling water bath, heat 5min, treat its cool to room temperature after, add 5.375ml distilled water, shake up and put into refrigerator as blank.Remaining mixing solutions is cultivated 60min down for 25 ℃, takes out 0.5mL and adds 0.375ml DNS reagent, heats 5min in boiling water bath, treat its cool to room temperature after, add 5.375ml distilled water, shake up.Under the 520nm wavelength, survey its OD value.Cofabrication typical curve is found the fructose content of enzyme liquid from typical curve, calculates the enzyme activity of dextransucrase under various culture condition.The enzyme activity definition: under 35 ℃, per hour catalytic substrate sucrose produces the required enzyme amount of 0.1mg fructose and is defined as an enzyme activity unit (U).
The invention provides and a kind ofly utilize engineering strain BL21 (DE3)/pET28-dexYG and adopt IPTG and lactose combined induction legal system is equipped with free reorganization dextransucrase; this method significantly reduces system enzyme cost when being induced separately by IPTG, active high, helps large-scale production.Simultaneously, utilize the free reorganization dextransucrase that cost of the present invention is low, vigor is high, adopt the enzymatic process of gap vibration, synthetic dextran of a step catalysis and fructose, transformation efficiency height.Realize an enzyme how with, reclaim fructose, reduced cost, and solved in the dextran original production process by product fructose the pollution problem of environment.
Embodiment
Embodiment 1:
(1) combined induction preparation reorganization dextransucrase fermentation
Engineering strain BL21 (DE3)/pET28-dexYG, being transferred to the fresh LB substratum of 20mL from the inclined-plane (contains among that 50 μ g of card/mL), 37 ℃, 300r/min incubated overnight, incubation time are 16h, are inoculated into fermentation culture among the substratum B according to 1% inoculum size again.Fermented liquid is per hour got fermented liquid 1mL in 37 ℃, 300r/min cultivation, with 10 times of sterilized water dilutions, measures OD
600
Adopt IPTG and lactose combined induction legal system to be equipped with dextransucrase, work as OD
600 =3.0 o'clock, adding IPTG was 0.09 mmol/L to final concentration, lactose final concentration 2.5g/L, and in 25 ℃, 300r/min inducing culture, induction time is 4h.Liquid after the fermentation adds the pH5.4 acetate buffer solution after 4 ℃, the centrifugal 15min of 12000r/min get thalline, ultrasonication 10min, and the centrifuging and taking supernatant liquor gets crude enzyme liquid, and enzyme work reaches 145～160U/mL, has reduced the system enzyme cost when being induced separately by IPTG.
(2) the reorganization dextransucrase of the above-mentioned combined induction of application adopts the enzymatic process of gap vibration, synthetic dextran of one-step catalytic and fructose, and step is:
1) enzyme substrates reaction solution preparation: use acetate-sodium acetate buffer (pH5.4) dissolving 14g sucrose of the 5mmol/L of 50mL, different volumes can be in this ratio preparation;
2) by substrate reactions liquid: after the work of above-mentioned 50mL enzyme substrates reaction solution adding enzyme is enzyme liquid 15 mL of 145～160 U/mL, use the same buffer constant volume to 100mL;
3) above-mentioned mixed solution 25 ℃, adopt the enzymatic process of gap vibration, can significantly improve transformation efficiency.Promptly when beginning reaction adopts vibrator that reaction solution is carried out 120n/min vibration 5h, standing and reacting 5h then, and the reaction that hockets according to this finishes catalyzed reaction behind the 22h;
4) reaction solution after the catalysis is carried out the alcohol precipitation washing, vacuum-drying is weighed, obtain high molecular dextran, precipitation solution is removed macromole impurity through centrifuging, obtain fructose through ultrafiltration removal small molecules oligose, filtrate through vacuum concentration, spraying drying again, molar yield reaches 90%.
Embodiment 2:
(1) combined induction preparation reorganization dextransucrase fermentation 2
Engineering strain BL21 (DE3)/pET28-dexYG, from the inclined-plane, be transferred in the fresh LB substratum of 20mL (containing that 50ug/mL of card), 37 ℃, 300r/min incubated overnight, incubation time are 16h, are inoculated into fermentation culture among the substratum B according to 1% inoculum size again.Fermented liquid is per hour got fermented liquid 1mL in 37 ℃, 300r/min cultivation, with 10 times of sterilized water dilutions, measures OD
600
Work as OD
600 =3.5 o'clock, adopting IPTG and lactose combined induction legal system to be equipped with dextransucrase to IPTG final concentration was that 0.12 mmol/L, lactose final concentration are 3.0g/L, and in 28 ℃, 300r/min inducing culture, induction time is 5h.Liquid after the fermentation adds the pH5.4 acetate buffer solution after 4 ℃, the centrifugal 15min of 12000r/min get thalline, ultrasonication 10min, and the centrifuging and taking supernatant liquor gets crude enzyme liquid, and enzyme work reaches 160～170U/mL.
(2) the reorganization dextransucrase of the above-mentioned combined induction of application adopts the enzymatic process of gap vibration, synthetic dextran of one-step catalytic and fructose, and step is:
1) enzyme substrates reaction solution preparation: use acetate-sodium acetate buffer (pH5.4) dissolving 14g sucrose of the 5mmol/L of 50mL, different volumes can be in this ratio preparation;
2) by substrate reactions liquid: after the work of above-mentioned 50mL enzyme substrates reaction solution adding enzyme is enzyme liquid 10 mL of 160～170U/mL, use the same buffer constant volume to 100mL;
3) above-mentioned mixed solution is at 30 ℃ of enzymatic process that adopt the gap vibration, promptly when beginning reaction adopts vibrator that reaction solution is carried out 120n/min vibration 5h, standing and reacting 5h then, and reaction according to this hockets, finish catalyzed reaction behind the 25h, significantly improved transformation efficiency.
(4) reaction solution after the catalysis is carried out the alcohol precipitation washing, vacuum-drying is weighed, obtain high molecular dextran, precipitation solution is removed macromole impurity through centrifuging, obtain fructose through ultrafiltration removal small molecules oligose, filtrate through vacuum concentration, spraying drying again.Molar yield reaches 95%.
Claims (4)
1. the method for enzyme method for preparing dextral glycoanhydride and fructose of recombinating is characterized in that: comprise the steps:
(1) preparation of free reorganization dextransucrase: engineering strain BL21 (DE3)/pET28-dexYG is transferred in the fresh LB substratum that contains 40-60 μ g/mL kantlex, and in the 35-40 ℃ of following 15-18h that cultivates, again it is inoculated into to produce among the enzyme substratum B and cultivates, work as OD in 35-40 ℃ of bottom fermentation
600 During=2.5-4.0, adopting IPTG and lactose combined induction to IPTG final concentration is that 0.07-0.12 mmol/L, lactose final concentration are 2.0-3.0g/L, and inducing temperature is 20-30 ℃, and induction time is 4-6h; Liquid after the cultivation adds the pH5.4 acetate buffer solution behind 0-5 ℃ of centrifugal thalline, ultrasonication, and the centrifuging and taking supernatant liquor promptly gets the dextransucrase liquid of recombinating;
(2) with synthetic dextran of reorganization dextransucrase catalysis and fructose: adopt following step:
(a) use pH5.4 acetate-sodium acetate buffer dissolving 10-15g sucrose of the 5mmol/L of 50mL to prepare the enzyme substrates reaction solution; Behind the reorganization dextransucrase liquid 10-15 mL that wherein adds enzyme 70-170u/ml alive, pH5.4 acetate-sodium acetate buffer constant volume of using 5mmol/L is to 100mL again;
(b) down adopt vibrator that reaction solution is carried out 120n/min vibration 5h during the reaction beginning at 25-30 ℃, standing and reacting 5h then, the reaction that hockets according to this finishes catalyzed reaction behind the 22-25h;
(c) reaction solution after the catalysis is carried out the alcohol precipitation washing, vacuum-drying is weighed, obtain high molecular dextran, precipitation solution is removed macromole impurity through centrifuging, obtain fructose through ultrafiltration removal small molecules oligose, filtrate through vacuum concentration, spraying drying again.
2. the method for reorganization enzyme method for preparing dextral glycoanhydride as claimed in claim 1 and fructose, it is characterized in that: substratum B is used for inducing of colibacillary fermentation and enzyme in the described step (1), wherein contains glycerine, peptone and substratum component of inorganic salts commonly used.
3. the method for reorganization enzyme method for preparing dextral glycoanhydride as claimed in claim 1 and fructose is characterized in that: during fermentation culture, work as OD in the described step (1)
600 =3.0 o'clock, adopt IPTG and lactose combined induction legal system to be equipped with dextransucrase, the final concentration that adds IPTG and lactose is respectively 0.09mmol/ and 2.5g/L, then in 25 ℃ of inducing culture.
4. the method for reorganization enzyme method for preparing dextral glycoanhydride as claimed in claim 1 and fructose is characterized in that: the reorganization dextransucrase liquid enzyme that adds in the described step (2) is lived and is 145-170u/ml.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105177086A (en) * | 2015-10-21 | 2015-12-23 | 合肥工业大学 | Process for preparing crystalline fructose from dextransucrase through one-step enzymatic method |
| CN107201332A (en) * | 2017-07-26 | 2017-09-26 | 合肥工业大学 | A kind of genetic engineering bacterium and its construction method and purposes for expressing heat resistant type Dextransucrase |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101363009A (en) * | 2007-10-17 | 2009-02-11 | 合肥工业大学 | Expression of dextran sucrase genetic engineering bacteria, construction method and use thereof |
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2011
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101363009A (en) * | 2007-10-17 | 2009-02-11 | 合肥工业大学 | Expression of dextran sucrase genetic engineering bacteria, construction method and use thereof |
Non-Patent Citations (1)
| Title |
|---|
| 张洪斌等: "重组大肠杆菌右旋糖酐蔗糖酶的表达条件优化", 《生物工程学报》, 25 December 2009 (2009-12-25), pages 2022 - 2028 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105177086A (en) * | 2015-10-21 | 2015-12-23 | 合肥工业大学 | Process for preparing crystalline fructose from dextransucrase through one-step enzymatic method |
| CN107201332A (en) * | 2017-07-26 | 2017-09-26 | 合肥工业大学 | A kind of genetic engineering bacterium and its construction method and purposes for expressing heat resistant type Dextransucrase |
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