CN101050455A - Immobilization lipase, and preparation method - Google Patents
Immobilization lipase, and preparation method Download PDFInfo
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- CN101050455A CN101050455A CN 200710064928 CN200710064928A CN101050455A CN 101050455 A CN101050455 A CN 101050455A CN 200710064928 CN200710064928 CN 200710064928 CN 200710064928 A CN200710064928 A CN 200710064928A CN 101050455 A CN101050455 A CN 101050455A
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Abstract
This invention discloses a method for preparing immobilized lipase. The method comprises: dissolving konjak glucomannan into water to obtain konjak glucomannan sol, adding lipase, mixing uniformly, spreading to obtain a thin film, and drying to obtain immobilized lipase. Ester exchange experiment shows that the immobilized lipase has high catalytic activity and usage stability, and the catalytic conversion rate is higher than 90%. The immobilized lipase can be used repeatedly, and soaking the immobilized lipase in grease overnight can largely improve the usage stability and catalytic effect of the immobilized lipase.
Description
Technical field
The present invention relates to a kind of immobilized lipase and preparation method thereof.
Background technology
Lipase (lipase, EC3.1.1.3) full name is a glycerine trigalloyl ester hydrolase, its basic function is that the catalyzing glycerol ester is hydrolyzed to glycerine and lipid acid, can be on oil-water interface synthetic, the transesterify of the hydrolysis of catalysis ester or alcoholysis, ester, lactone is synthetic, polypeptide is synthetic, the reactions such as fractionation of the synthetic and chipal compounds of high molecular polymer.Natural lipase exists poor stability, easy inactivation, can not reuse, and sneaks into product after the reaction, causes problems such as product separation, purification difficult, and the best approach that addresses these problems is exactly to carry out the immobilization of enzyme.Lipase immobilization carrier commonly used has diatomite, DEAE-Sephadex, aluminum oxide, silica gel, silk, sintered glass, kaolin, wilkinite, ion exchange resin, CM-Mierocrystalline cellulose, DEAE-Mierocrystalline cellulose, collagen, polyacrylamide, polyurethane foam particle etc.The lipase immobilization method mainly contains absorption method, entrapping method, covalent attachment method and crosslinking four big classes.
Rhizoma amorphophalli glucomannan (konjac glucomannan, KGM) be a kind of polymer non-ionic type polysaccharide, form with 1: 1.78 or 1.79 ratio by D-glucose (Glu) and D-seminose (Man), Glu and Man are by β-1,4 glycosidic links connect, and there is a side chain in every about 68 saccharide residues on main chain, and length is 3～4 saccharide residues, be connected with β-1,3 glycosidic link on C (3) position of Man.KGM has biodegradable, its water-sol has performances such as viscosity height, thickening, gelling and easy film forming, be often used as the Perserving materials of foodstuff additive and fruit and food, important use also arranged in fields such as biomaterial, chemical industry, environmental protection and oil drillings.With KGM is the more existing patent reports of carrier immobilized microorganism cells and enzyme.Patent CN 891097104 is a carrier covalent attachment method immobilization cyclodextrin glucanotransferase with KGM; Patent ZL 99113707.8 usefulness carrageenins and konjac polysaccharide gum blend entrapping method fixation of microbe carrier, and the immobilized microorganism that makes is used for the preparation of Nucleotide; Patent CN 1053089 is a carrier with the konjac polysaccharide, covalent modification method fixation of microbial cell and enzyme; In addition, also have some reports, and do not appear in the newspapers in being used for fixing lipase aspect about KGM fixation glucose amylase, proteolytic ferment, cerevisiae and beta-galactosidase enzymes etc.
Summary of the invention
The purpose of this invention is to provide a kind of immobilized lipase and preparation method thereof.
The preparation method of immobilized lipase provided by the present invention is to be carrier with the Rhizoma amorphophalli glucomannan, with lipase immobilization.
Described step with lipase immobilization comprises Rhizoma amorphophalli glucomannan water-soluble, behind the formation konjak portuguese gansu polyose sugar sol, adds lipase, and mixing spreads out into film, the fatty enzyme membrane of being fixed of drying.
Described step with lipase immobilization comprises that also the pH value of aqueous solution scope with alkali lye adjusting Rhizoma amorphophalli glucomannan is 6.0-13.0, makes described konjak portuguese gansu polyose sugar sol deacetylation form gel; The pH value scope of described konjak portuguese gansu polyose sugar sol deacetylation is preferably 8.0-12.0.
The alkali lye of described adjusting pH value can be Ca (OH)
2, Na
2CO
3Or Na
3PO
4Solution is preferably Ca (OH)
2Solution.
The addition of described lipase can be the 10-100KLU/g carrier, is preferably 30KLU/g carrier (KLU refers to thousand lipase unit).
The water-soluble mass percent concentration of described Rhizoma amorphophalli glucomannan can be 1-10%, is preferably 1-5%.
Also can be added with emulsifying agent in the described konjak portuguese gansu polyose sugar soln.
The addition of described emulsifying agent is the 0-0.01g/g carrier.
Described emulsifying agent can be SP-60, stearic acid monoglycerides or hot certain herbaceous plants with big flowers acid glyceride, is preferably SP-60.
The thickness of described immobilized lipase enzyme membrane is 3-5mm.
The immobilized lipase of above-mentioned method preparation also belongs to protection scope of the present invention.
The principle of the inventive method is: behind the KGM water absorption and swelling, the colloidal sol that can form high viscosity and have the good filming performance can be used as the carrier of fixation of microbial cell or enzyme, especially as the carrier of nonaqueous phase reaction enzymes.In the nonaqueous phase reaction system, consider and to have the catalysis resistance between lipase and the reaction substrate because of immobilization causes, so the present invention adds emulsifying agent to improve the greasy reaction efficiency of enzyme catalysis in immobilization process.PH value (pH6.0-13.0) with alkali lye adjusting fixation support system makes KGM colloidal sol deacetylation form water-fast gel, ensures the maximum performance of lipase activity simultaneously.
Cheap, the support material wide material sources of the solid support material KGM of method of the present invention, low price; Become glue, film forming and water resistance good, make water-proof film easily, the auxiliary material of required interpolation is few, is the good carrier of lipase film immobilization.Method immobilization technology of the present invention is simple to operate, does not need special equipment, is fit to the heavy industrialization operation.
Immobilized lipase of the present invention has with the incomparable advantage of other carrier, and the waterproof membrane that KGM forms can also be as enzymatic reaction interface, contact interface and separating interface except playing immobilization role.By the heterogeneous film katalaze enzyme reactor that film constituted that is fixed with lipase, the catalysis, the product that integrate enzyme separate, are separated and catalyst recovery, have long-range prospect in the industrial applications of lipase.
The transesterification reaction experiment shows that immobilized lipase of the present invention has catalysis activity and stability in use preferably, and the transformation efficiency of its catalyzed reaction can reach more than 90%.Immobilized lipase can repeated multiple times uses, and before using enzyme soaked and react after processing such as regenerate can improve the stability in use and the catalytic effect of immobilized lipase greatly.
Embodiment
Method among the following embodiment if no special instructions, is ordinary method.
Below in conjunction with embodiment content of the present invention is further described:
The preparation of embodiment 1, immobilized lipase enzyme membrane and enzyme thereof are lived and are identified
1, the preparation of immobilized lipase enzyme membrane
(purity is 99% to take by weighing the smart powder of 10g Rhizoma amorphophalli glucomannan (KGM), available from luxuriant processing of farm products limited liability company in the Chongqing City) and 0.06g emulsifying agent (SP-60, Beijing chemical reagent company limited), add 700ml water, fully stirring and dissolving obtains colloidal sol, with saturated Ca (OH)
2Solution is regulated pH value to 11.0; abundant heated and stirred in 80 ℃ water-bath then; treat to obtain gel behind abundant swelling and the deacetylation; with its taking-up; leave standstill cooling; lipase (the Denmark Novozymes Company that adds 3ml (100KLU/ml) then; Lipozyme TL 100L; vigor is 100KLU/g) solution is (with lipase liquid 0.025mol/L; the pH value is that the dilution of 7.5 phosphoric acid buffer obtains), stir, it is thick with glass stick mixture to be spread out into 4mm; blowing at room temperature is dry, and obtaining thickness is the immobilized lipase enzyme membrane of 0.5mm.
2, the vitality test of immobilized lipase
Adopt polyvinyl alcohol-sweet oil emulsion process to measure the vigor of the immobilized lipase of above-mentioned preparation, concrete steps are as follows:
A. olive oil emulsion preparation: the polyvinyl alcohol solution with 4% mixes with 3: 1 (v/v) with sweet oil, magnetic stirring apparatus high-speed stirring 4min, leave standstill 5min after, high-speed stirring 4min until the surperficial no oil droplet of emulsion, preserves in 4 ℃ of refrigerators again.Carry out high-speed stirring before the use, it was 1 week that the emulsion of preparation is used validity period.
B. react titration: in the Erlenmeyer flask of 100ml, add the sweet oil emulsion of 4ml and the phosphoric acid buffer of 5ml pH value 7.0, in 40 ℃ of water-baths, be incubated 3～5 minutes then, add immobilized lipase, begin accurate timing, oscillatory reaction 15 minutes, add 10ml ethanol-acetone (volume ratio 3: 1) mixed solution, termination reaction.Blank then is to add immobilized lipase again after adding ethanol-acetone mixed solution, does not need insulation.Reaction solution after the enzyme effect and blank are used the NaOH solution titration of 0.05mol/L respectively, and the difference that consumes alkali lye according to both is calculated the vigor of lipase.A unit lipase activity is defined as: at 40 ℃, under the pH7.5 condition, per minute produces the enzyme amount that lipid acid consumed of 1 μ mol, and unit is U/g.
C. the calculating of lipase activity:
The NaOH volume that A-example reaction liquid consumes in the formula, ml;
The NaOH volume that the B-blank consumes, ml;
The volumetric molar concentration of C-NaOH solution, mol/L;
1000-NaOH amounts to into the reduction factor of micromole's lipid acid;
The extension rate of f-enzyme liquid;
The quality of used enzyme when w-measures, g;
The t-reaction times, min;
The vigor that records the self-control immobilized lipase according to the method described above is 51.26U/g.
3, the effect of immobilized lipase check
With the immobilized lipase of step 1 preparation, be used for greasy catalytic transesterification test.Concrete steps are: with the above-mentioned immobilized lipase enzyme membrane that obtains of 20g, place 100g grease (commercially available " remittance good fortune board " soybean oil is housed, three rivers are converged good fortune grain and oil group company and are produced) and the reaction flask of 9.5g methanol mixed substrate in, under 45 ℃, oscillatory reaction 24 hours.After reaction finishes, reacting final product is carried out centrifugal, rotary evaporation and the greasy transformation efficiency of gas Chromatographic Determination, the result shows that the transformation efficiency that the lipase-catalyzed grease of said fixingization generates Fatty acid methyl ester is 91%.
The immobilized lipase of step 1 preparation is applied to greasy transesterify experiment, and after using 10 times repeatedly, catalytic transformation efficiency still maintains about 90%; With without any blank that is treated to, use respectively through the lipase that the trimethyl carbinol soaks or grease soaks and carry out above-mentioned transesterify experiment, the result shows that after grease spent the night immersion, the catalysis activity of immobilized lipase obviously improved.
The preparation of embodiment 2, immobilized lipase enzyme membrane and enzyme thereof are lived and are identified
1, the preparation of immobilized lipase enzyme membrane
(purity is 99% to take by weighing the smart powder of 50g Rhizoma amorphophalli glucomannan (KGM), available from luxuriant processing of farm products limited liability company in the Chongqing City) and 0.4g emulsifying agent (hot certain herbaceous plants with big flowers acid glyceride is available from the auspicious continuous heavy rain chemical industry in Hangzhou company limited), add about 1L water, fully stirring and dissolving obtains colloidal sol, with the Na of 0.5mol/L
2CO
3Solution is regulated pH value to 9.0; abundant heated and stirred in 80 ℃ water-bath then; treat to obtain gel behind abundant swelling and the deacetylation; with its taking-up; leave standstill cooling; lipase (the Denmark Novozymes Company that adds 25ml (100KLU/ml); Lipozyme TL 100L; vigor is 100KLU/g) solution is (with lipase liquid 0.025mol/L; the pH value is that the dilution of 7.5 phosphoric acid buffer obtains), stir, with glass stick that the even 5mm in enzyme glue mixture stand is thick; blowing at room temperature is dry, and obtaining thickness is the immobilized lipase enzyme membrane of 0.6mm.
The vigor that records the immobilized lipase of above-mentioned preparation according to the described method of step 2 among the embodiment 1 is 49.92U/g.
2, the effect of immobilized lipase check
With the immobilized lipase of step 1 preparation, be used for greasy catalytic transesterification test.Concrete steps are: with the immobilized lipase enzyme membrane of 100g step 1 preparation, place 200g grease (commercially available " remittance good fortune board " soybean oil is housed, three rivers are converged good fortune grain and oil group company and are produced) and the reaction flask of 20g methanol mixed substrate in, under 45 ℃, oscillatory reaction 24 hours.After reaction finishes, reacting final product is carried out centrifugal, rotary evaporation and the greasy transformation efficiency of gas Chromatographic Determination, the result shows that the transformation efficiency that the fixed lipase catalyzed grease of step 1 preparation generates Fatty acid methyl ester is 89%.
After the immobilized lipase of step 1 preparation was used for greasy transesterify experiment repeatedly for 10 times, enzymatic transformation efficiency was still about 90%; With without any blank that is treated to, be used for the transesterify experiment through the grease lipase that soaks that spends the night, its catalytic vigor obviously improves.
The preparation of embodiment 3, immobilized lipase enzyme membrane and enzyme thereof are lived and are identified
1, the preparation of immobilized lipase enzyme membrane
(purity is 99% to take by weighing the smart powder of 200g Rhizoma amorphophalli glucomannan (KGM), available from luxuriant processing of farm products limited liability company in the Chongqing City) and 1.0g emulsifying agent (stearic acid monoglycerides is available from Beijing chemical reagent company limited), add about 5L water, fully stirring and dissolving obtains colloidal sol, with the Na of 0.5mol/L
3PO
4Solution is regulated pH value to 8.0; abundant heated and stirred in 80 ℃ water-bath then; treat to obtain gel behind abundant swelling and the deacetylation; with its taking-up; leave standstill cooling; lipase (the Denmark Novozymes Company that adds 40ml (100KLU/ml); Lipozyme TL 100L; vigor is 100KLU/g) solution is (with lipase liquid 0.025mol/L; the pH value is that the dilution of 7.5 phosphoric acid buffer obtains), stir, with glass stick that the even 5mm in enzyme glue mixture stand is thick; blowing at room temperature is dry, and obtaining thickness is the immobilized lipase enzyme membrane of 0.6mm.
The vigor that records the immobilized lipase of above-mentioned preparation according to the described method of step 2 among the embodiment 1 is 51.25U/g.
2, the effect of immobilized lipase check
With the immobilized lipase of step 1 preparation, be used for greasy catalytic transesterification test.Concrete steps are: with the immobilized lipase enzyme membrane of 100g step 1 preparation, place 300g grease (commercially available " remittance good fortune board " soybean oil is housed, three rivers are converged good fortune grain and oil group company and are produced) and the reaction flask of 30g methanol mixed substrate in, under 45 ℃, oscillatory reaction 24 hours.After reaction finishes, reacting final product is carried out centrifugal, rotary evaporation and the greasy transformation efficiency of gas Chromatographic Determination, the result shows that the transformation efficiency that the fixed lipase catalyzed grease of step 1 preparation generates Fatty acid methyl ester is 87%.
After the immobilized lipase of step 1 preparation was used for greasy transesterify experiment repeatedly for 10 times, enzymatic transformation efficiency was still about 90%; With without any blank that is treated to, be used for the transesterify experiment through the grease lipase that soaks that spends the night, its catalytic vigor obviously improves.
Claims (10)
1, a kind of preparation method of immobilized lipase is to be carrier with the Rhizoma amorphophalli glucomannan, with lipase immobilization.
2, method according to claim 1 is characterized in that: described step with lipase immobilization comprises Rhizoma amorphophalli glucomannan water-soluble, behind the formation konjak portuguese gansu polyose sugar sol, adds lipase, and mixing spreads out into film, the fatty enzyme membrane of being fixed of drying.
3, method according to claim 2, it is characterized in that: described step with lipase immobilization comprises that also the pH value of aqueous solution scope with alkali lye adjusting Rhizoma amorphophalli glucomannan is 6.0-13.0, makes described konjak portuguese gansu polyose sugar sol deacetylation form gel; The pH value scope of described konjak portuguese gansu polyose sugar sol deacetylation is preferably 8.0-12.0.
4, method according to claim 3 is characterized in that: the alkali lye of described adjusting pH value is Ca (OH)
2, Na
2CO
3Or Na
3PO
4Solution is preferably Ca (OH)
2Solution.
5, method according to claim 4 is characterized in that: the addition of described lipase is the 10-100KLU/g carrier, is preferably the 30KLU/g carrier.
6, method according to claim 5 is characterized in that: the water-soluble mass percent concentration of described Rhizoma amorphophalli glucomannan is 1-10%, is preferably 1-5%.
7, method according to claim 6 is characterized in that: also be added with emulsifying agent in the described konjak portuguese gansu polyose sugar soln.
8, method according to claim 7 is characterized in that: the addition of described emulsifying agent is the 0-0.01g/g carrier.
9, method according to claim 8 is characterized in that: described emulsifying agent is SP-60, stearic acid monoglycerides or hot certain herbaceous plants with big flowers acid glyceride, is preferably SP-60; The thickness of described immobilized lipase enzyme membrane is 3-5mm.
10, the immobilized lipase of each described method preparation of claim 1-10.
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| CN200710064928A CN100582228C (en) | 2007-03-29 | 2007-03-29 | Immobilization lipase, and preparation method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109030602A (en) * | 2018-08-10 | 2018-12-18 | 上海应用技术大学 | A kind of electric potential type organophosphorus biosensor and preparation method thereof |
| CN109097351A (en) * | 2018-08-01 | 2018-12-28 | 浙江海洋大学 | A kind of preparation method of the novel immobilised enzymes for oil spilling reparation |
| CN109750083A (en) * | 2019-03-19 | 2019-05-14 | 大连大学 | The active measuring method of immobilized lipase in a kind of supercritical carbon dioxide |
| CN111758876A (en) * | 2020-07-01 | 2020-10-13 | 天津融信蓝海生物科技有限公司 | Quick-frozen dumpling grease antioxidant composition and preparation process thereof |
| CN114214300A (en) * | 2021-12-17 | 2022-03-22 | 安徽工业大学 | Immobilized enzyme material and preparation method and application thereof |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1053089A (en) * | 1989-12-30 | 1991-07-17 | 中国科学院成都生物研究所 | The novel method of a kind of konjac polysaccharide fixation of microbial cell and enzyme |
| AU8760898A (en) * | 1997-09-24 | 1999-04-12 | Enzymotec Ltd. | Surfactant-lipase complex immobilized on insoluble matrix |
| CN1086736C (en) * | 1999-05-17 | 2002-06-26 | 华东理工大学 | Microorganism carrier fixed with glue compounded with carragheen and konjak polysaccharide and use thereof |
| CN1275619A (en) * | 1999-06-01 | 2000-12-06 | 上海大众凌伟生化股份有限公司 | Carrageenan and glucomannan compounded immobilized cell carrier |
| JP2005261284A (en) * | 2004-03-18 | 2005-09-29 | Gunma Prefecture | Method for immobilizing enzyme and reactor using immobilized enzyme |
| CN1885022A (en) * | 2006-06-25 | 2006-12-27 | 襄樊学院 | Konjac glucomannan hydrogel immobilized enzyme electrode, preparation method and application thereof |
| CN1885024A (en) * | 2006-06-27 | 2006-12-27 | 上海应用技术学院 | Method for fixing enzyme in glucose sensor |
-
2007
- 2007-03-29 CN CN200710064928A patent/CN100582228C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109097351A (en) * | 2018-08-01 | 2018-12-28 | 浙江海洋大学 | A kind of preparation method of the novel immobilised enzymes for oil spilling reparation |
| CN109030602A (en) * | 2018-08-10 | 2018-12-18 | 上海应用技术大学 | A kind of electric potential type organophosphorus biosensor and preparation method thereof |
| CN109030602B (en) * | 2018-08-10 | 2019-12-03 | 上海应用技术大学 | A kind of electric potential type organophosphorus biosensor and preparation method thereof |
| CN109750083A (en) * | 2019-03-19 | 2019-05-14 | 大连大学 | The active measuring method of immobilized lipase in a kind of supercritical carbon dioxide |
| CN111758876A (en) * | 2020-07-01 | 2020-10-13 | 天津融信蓝海生物科技有限公司 | Quick-frozen dumpling grease antioxidant composition and preparation process thereof |
| CN114214300A (en) * | 2021-12-17 | 2022-03-22 | 安徽工业大学 | Immobilized enzyme material and preparation method and application thereof |
| CN114214300B (en) * | 2021-12-17 | 2024-03-12 | 安徽工业大学 | Immobilized enzyme material and preparation method and application thereof |
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| CN100582228C (en) | 2010-01-20 |
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