CN102604923A - Reusable intelligent biocatalyst (including immobilized enzyme and stress carrier) and its application in organic synthesis - Google Patents
Reusable intelligent biocatalyst (including immobilized enzyme and stress carrier) and its application in organic synthesis Download PDFInfo
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- CN102604923A CN102604923A CN2011100263094A CN201110026309A CN102604923A CN 102604923 A CN102604923 A CN 102604923A CN 2011100263094 A CN2011100263094 A CN 2011100263094A CN 201110026309 A CN201110026309 A CN 201110026309A CN 102604923 A CN102604923 A CN 102604923A
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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
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Abstract
The invention discloses a category of stress carriers of immobilized enzyme catalysts. Under different external stimulations, the carrier materials can reversibly realize dissolution and precipitation in water or other solvents. A carrier immobilized biocatalyst and the carrier show a consistent dissolution-precipitation phenomenon in water and other solvents. When the immobilized catalyst finishes reaction in a homogeneous system, by properly changing the pH value, the temperature, and the ionic strength of the reaction system or adding one chemical substance, the immobilized catalyst can dissolve out of the system in the form of precipitation, and the product is in the solution, thus realizing separation of the product and the catalyst. A recovered enzyme can be re-dissolved in a buffer solution for recycling, thus realizing recycling of the enzyme. Employment of the above technical method can be effectively combine the advantages of homogeneous catalysis and heterogeneous catalysis, and effectively avoid their respective disadvantages.
Description
Technical field
Reagent of the present invention uses the preparation and the application in important chemical is synthetic thereof of the intelligent catalysis agent that carrier carries out enzyme obtaining after the immobilization.
Background technology
Along with the growing interest to environment protection, when synthesis of chemicals, it is very urgent developing environment amenable compound method.Under this demand, enzyme is as catalyzer, because its gentle reaction conditions, excellent solid, zone, chemo-selective, very effective method when making it become synthetic important industrial chemical.Adopt the biocatalysis process, can fundamentally optimize fine chemicals like the synthetic route of medicine, agricultural chemicals, spices and perfume, foodstuff additive and care products.In the application process of enzyme, people also notice some deficiency of enzyme, as: the catalytic activity of enzyme is not high, the less stable of enzyme, can not reuse, be not easy to separation and purification.To the above-mentioned deficiency of enzyme, utilize the biomolecules engineering, can effectively improve the aspect such as pH stability, thermostability, catalytic activity of enzyme; Utilize enzyme immobilization technology, can effectively overcome enzyme and in use be difficult for reusing and isolating shortcoming, effectively reduced production cost.Immobilized enzyme had both kept the catalytic activity of enzyme, had overcome the deficiency of resolvase, very easily from system, separated; For continuous production; Be very useful, some experimental result shows through immobilization, can improve enzyme catalytic efficiency (, increase stability etc.Present method is that enzyme is fixed on the water-fast carrier, is reflected in the nonhomogeneous system and carries out.Exist some insufficient in the heterogeneous reaction system, as: the space conformation that may influence enzyme during owing to the combining of enzyme and carrier reduces the catalytic activity of enzyme; Because enzyme and reaction substrate in heterogeneous system, inevitably can make the interaction speed between the enzyme-to-substrate reduce, thereby the speed and the production efficiency of reaction have been influenced.
Summary of the invention
Deficiency to above-mentioned existing enzyme immobilization; The present invention introduced the enzyme that will have catalysis with have based on poly-N-isopropyl-acrylic amide (PNIPAAm) polymkeric substance and other stress function the polymkeric substance effect obtain the intelligence biological catalyzer, be applied to fine chemicals synthetic method subsequently.
In the present invention, enzyme catalyst be fixed on stress carrier on.These solid support materials, under different external stimuluss, reversible be implemented in the water or the dissolving in other solvents with separate out.Carrier immobilized biological catalyst and carrier have shown the phenomenon of consistent dissolving-separate out in water and in other solvents.When immobilized catalyst is accomplished reaction in homogeneous system after; PH value, temperature, ionic strength that can be through the appropriate change reaction system or add certain chemical substance; Make immobilized catalyst from system, separate out with precipitation forms; Product then in solution, thereby realized separating of product and catalyzer.The enzyme that reclaims can be dissolved in the buffered soln again and recycle, and has realized the recycling of enzyme.Using above-mentioned technological method, can effectively combine the advantage of homogeneous catalysis and heterogeneous catalysis, avoided shortcoming separately effectively, is the novel method that a kind of utmost point has using value.
The numerous natural products or the polymkeric substance of synthetic; When temperature, pH value, ionic strength change; Corresponding change also can take place in its conformation; These high molecular polymers all are widely used aspect biological, medical as intelligent material, both have been applied in drug delivery system, the cell adhesion medium, can be applied in the function and the genetic expression aspect of control enzyme again.In these numerous intelligent materials, poly-N-isopropyl-acrylic amide (PNIPAAm) is a kind of polymkeric substance that dissolves-separate out at the hot reversible of aqueous phase, and its critical solution temperature is low to be 32 ℃.When the temperature of heating surpassed 32 ℃, this polymkeric substance will be separated out from aqueous phase and formed deposition, and when temperature was lower than 32 ℃, deposition can be dissolved in aqueous phase again, and this separating out-dissolved process is slap-up.
Through changing the critical solution temperature that to adjust this polymkeric substance with the kind of N-sec.-propyl-acrylamide copolymer.For example: in the mixed solvent of THF-toluene, add N-sec.-propyl-acrylic amide and N-acryloxy-succsinic acid imide at 40: 1, add with mol ratio
Diisopropyl azodicarboxylate(AIBN) bring out polyreaction.After reaction finishes, add normal hexane polymkeric substance is separated out, vacuum-drying.The critical solution temperature of the polymkeric substance that obtains is about 33 ℃, contains an activatory ester group group in its structure, and the reactive behavior of this ester group group is higher than the primary amine in the protein molecule far away.
Embodiment
Below further specify through concrete embodiment, but these embodiment are not construed as limiting the invention.
Embodiment: the immobilization of 1 carbonyl reductase
At phosphate buffer solution (100mM; PH 8.0) in, will derive under carbonyl reductase and the polymkeric substance room temperature of Sporobolomyces salmonicolor (SSCR) effect 2 hours, add NaCl solution after; White precipitate is separated out, and the deposition that obtains is with the NaCl solution washing of same concentrations.If do not add NaCl solution, when reaction soln was heated above 33 ℃ (LCST), same adularescent deposition was separated out.
The protein concentration and the reactive behavior of immobilized enzyme of separating and the enzyme in reaction soln are all measured.The combination of proteins productive rate is meant the proteinic quantity ratio that is immobilized protein and initial adding.The combination productive rate of enzyme is meant the reactive behavior and the initial proteins react specific activity that adds that is immobilized enzyme.Relative reactivity is the reactive behavior of immobilized enzyme and the reactivity ratio of resolvase.Experiment shows that the combination of proteins productive rate is 53%, and the relative reactivity of immobilized enzyme is 72%, and the combination productive rate of enzyme is 38%.
Embodiment: the immobilization of 2D-Hexose phosphate dehydrogenase (GDH)
In phosphate buffer solution (100mM, pH 8.0), (can be with GDH with NADP
+Be reduced to NADPH, NADPH is the carbonyl reduction enzyme cofactor) with the effect of gathering under the NIPAAm room temperature 2 hours, add NaCl buffered soln after, white precipitate is separated out, the deposition that obtains is with the NaCl solution washing of same concentrations.If do not add NaCl solution, when reaction soln was heated above 33 ℃ (LCST), same adularescent deposition was separated out.
The protein concentration and the reactive behavior of immobilized enzyme of separating and the enzyme in reaction soln are all measured.The combination of proteins productive rate is meant the proteinic quantity ratio that is immobilized protein and initial adding.The combination productive rate of enzyme is meant the reactive behavior and the initial proteins react specific activity that adds that is immobilized enzyme.Relative reactivity is the reactive behavior of immobilized enzyme and the reactivity ratio of resolvase.Experimental result shows that the combination of proteins productive rate is 47%, and the relative reactivity of immobilized enzyme is 75%, and the combination productive rate of enzyme is 35%.
Embodiment: the co-immobilization of 3 carbonyl reductases and D-Hexose phosphate dehydrogenase
At phosphate buffer solution (100mM; PH 8.0) in, add the carbonyl reductase that derives from Sporobolomyces salmonicolor (SSCR), the D-Hexose phosphate dehydrogenase; Gather under the NIPAAm room temperature and act on 2 hours; After adding NaCl buffered soln, white precipitate is separated out, and the deposition that obtains is with the NaCl solution washing of same concentrations.If do not add NaCl solution, when reaction soln was heated above 33 ℃ (LCST), same adularescent deposition was separated out.
Embodiment: 4 immobilization carbonyl reductases are to the reduction of ketone
3,3-dimethyl--2-carbonyl ethyl n-butyrate detects the reactive behavior and the enantioselectivity of immobilized enzyme as substrate.3,3-dimethyl--2-carbonyl ethyl n-butyrate and immobilized enzyme are dissolved in the buffer solution of potassium phosphate, and cofactor NADPH is through D-glucose/D-Hexose phosphate dehydrogenase system regeneration.Immobilized SSCR enzyme is reused six times.Each reaction, substrate 3,3-dimethyl--2-carbonyl ethyl n-butyrate is under the reduction of immobilized enzyme, and the transformation efficiency with 100% obtains optically pure (R)-3,3-dimethyl--2-3-hydroxyethyl butyrate.
After using six times, through measuring after the NADPH oxidation at 340nm (ε=6.22mM
-1Cm
-1) the absorption spectrum intensity of locating confirms the reactive behavior of immobilization SSCR enzyme.The result shows, is 70% of initial enzymic activity in the activity of using enzyme after six times.The above results proof immobilization SSCR enzyme has excellent enantioselectivity, and can repeat repeatedly to use.
Embodiment: 5 immobilization carbonyl reductases and immobilization D-Hexose phosphate dehydrogenase are to the reduction of ketone
3,3-dimethyl--2-carbonyl ethyl n-butyrate detects the reactive behavior and the enantioselectivity of immobilization SSCR enzyme and fixation glucose desaturase as substrate.Experimental procedure is the same.Immobilization SSCR enzyme and fixation glucose desaturase are reused six times.Each reaction, substrate 3,3-dimethyl--2-carbonyl ethyl n-butyrate is under the effect of immobilization SSCR enzyme and fixation glucose desaturase, and the transformation efficiency with 100% obtains optically pure (R)-3,3-dimethyl--2-3-hydroxyethyl butyrate.
Because the existence of GDH, can't be through measuring after the NADPH oxidation reactive behavior of confirming immobilization SSCR enzyme in the absorption spectrum intensity at 340nm place.
Embodiment: 6 co-immobilization carbonyl reductases and D-Hexose phosphate dehydrogenase are to the reduction of ketone
3,3-dimethyl--2-carbonyl ethyl n-butyrate detects the reactive behavior and the enantioselectivity of SSCR enzyme and Hexose phosphate dehydrogenase co-immobilization enzyme as substrate.Experimental procedure is the same.The SSCR enzyme of co-immobilization and Hexose phosphate dehydrogenase are reused six times.Each reaction, substrate 3,3-dimethyl--2-carbonyl ethyl n-butyrate is under the effect of immobilization SSCR enzyme and fixation glucose desaturase, and the transformation efficiency with 100% obtains optically pure (R)-3,3-dimethyl--2-3-hydroxyethyl butyrate.
Because the existence of GDH, can't be through measuring after the NADPH oxidation reactive behavior of confirming immobilization SSCR enzyme in the absorption spectrum intensity at 340nm place.
In a word, be that the biological catalyst of carrier is at room temperature water-soluble with the polymkeric substance, when being heated above 32 ℃ or add salt,, from solution, separating out and form deposition like NaCl.Compare with resolvase, immobilized enzyme during 3-dimethyl--2-carbonyl ethyl n-butyrate, has shown outstanding reactive behavior and enantioselectivity at reduction substrate 3 equally.After recycling six times, immobilized enzyme does not significantly reduce aspect reactive behavior and enantioselectivity.
Embodiment: the immobilization of 7 itrile group lytic enzymes
At phosphate buffer solution (100mM; PH 8.0) in, with effect under itrile group lytic enzyme bll6402 that derives from Bradyrhizobium japonicum USDA110 and the polymkeric substance room temperature 2 hours, behind the adding NaCl solution; White precipitate is separated out, and the deposition that obtains is with the NaCl solution washing of same concentrations.If do not add NaCl solution, when reaction soln was heated above 33 ℃ (LCST), same adularescent deposition was separated out.
The protein concentration and the reactive behavior of immobilized enzyme of separating and the enzyme in reaction soln are all measured.The combination of proteins productive rate is meant the proteinic quantity ratio that is immobilized protein and initial adding.The combination productive rate of enzyme is meant the reactive behavior and the initial proteins react specific activity that adds that is immobilized enzyme.Relative reactivity is the reactive behavior of immobilized enzyme and the reactivity ratio of free enzyme.Experimental result shows that the combination of proteins productive rate is 63%, and the relative reactivity of immobilized enzyme is 74%, and the combination productive rate of enzyme is 47%.
Claims (8)
- One type can be used for fixing enzyme catalyst stress carrier on.These solid support materials, under different external stimuluss, reversible be implemented in the water or the dissolving in other solvents with separate out.
- 2. carrier according to claim 1 with the intelligent catalysis agent that the back generates that interacts of carbonyl reductase and carrier, through changing ionic strength or solution temperature, can very easily obtain with solid form.
- 3. carrier according to claim 1 generates the intelligent catalysis agent with D-Hexose phosphate dehydrogenase and the carrier back that interacts, and through changing ionic strength or solution temperature, can very easily obtain with solid form.
- 4. carrier according to claim 1, with carbonyl reductase and D-Hexose phosphate dehydrogenase common with the carrier intelligent catalysis agent that the back generates that interacts, through change ionic strength or solution temperature, can very easily obtain with solid form.
- 5. carrier according to claim 1 with the intelligent catalysis agent that the back generates that interacts of itrile group lytic enzyme and carrier, through changing ionic strength or solution temperature, can very easily obtain with solid form.
- 6. like the said intelligent catalysis agent of claim 2, apply it to 3, in the reduction reaction of 3-dimethyl--2-carbonyl ethyl n-butyrate.
- 7. like claim 2,3 said intelligent catalysis agent, apply it to 3, in the reduction reaction of 3-dimethyl--2-carbonyl ethyl n-butyrate.
- 8. like the said intelligent catalysis agent of claim 4, apply it to 3, in the reduction reaction of 3-dimethyl--2-carbonyl ethyl n-butyrate.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08196274A (en) * | 1995-01-24 | 1996-08-06 | Japan Synthetic Rubber Co Ltd | Immobilized enzyme |
| CN101173020A (en) * | 2007-10-11 | 2008-05-07 | 同济大学 | Method for producing temperature sensing polymer gel microsphere |
| CN101200526A (en) * | 2007-11-29 | 2008-06-18 | 上海交通大学 | Multiple environment-responsive tri-block copolymer and preparation method thereof |
| CN101280297A (en) * | 2008-05-28 | 2008-10-08 | 华中科技大学 | Preparation of immobilized lipase |
-
2011
- 2011-01-25 CN CN2011100263094A patent/CN102604923A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08196274A (en) * | 1995-01-24 | 1996-08-06 | Japan Synthetic Rubber Co Ltd | Immobilized enzyme |
| CN101173020A (en) * | 2007-10-11 | 2008-05-07 | 同济大学 | Method for producing temperature sensing polymer gel microsphere |
| CN101200526A (en) * | 2007-11-29 | 2008-06-18 | 上海交通大学 | Multiple environment-responsive tri-block copolymer and preparation method thereof |
| CN101280297A (en) * | 2008-05-28 | 2008-10-08 | 华中科技大学 | Preparation of immobilized lipase |
Non-Patent Citations (1)
| Title |
|---|
| 周玉恒等: "Eudragit L-100固定球毛壳霉菌木聚糖酶酶学性质的研究", 《食品科学》, no. 03, 15 March 2007 (2007-03-15) * |
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Application publication date: 20120725 |