CN1844382A - Method for preparing immobilized lipase attapulgite clay - Google Patents
Method for preparing immobilized lipase attapulgite clay Download PDFInfo
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Abstract
This invention provides a process for preparing immobilized lipase attapulgite, belonging to the technological field of attapulgite utilization and lipase immobilization. The invention utilizes attapulgite and lipase as raw material and comprises the following steps: attapulgite treatment, lipase immobilization by adsorption, using the absorption of lipase as indication, taking regard of the enzyme activity of immobilized lipase, achieving the ideal process of attapulgite treatement and lipase immobilizatin. The invention has beneficial effects of realizeing improved activity, multiple utilization, high utilization ratio and broader range of application of lipase, laying the foundation for lipase immobilization technology. Furthermoer, the invention enlarges the application range of attapulgite, increases the added value of attapulgite and has good guiding effects to use attapulgite as enzyme vectors.
Description
Technical field
A kind of preparation method of immobilized lipase attapulgite clay belongs to the utilization of attapulgite, and the lipase immobilization technical field.
Background technology
Attapulgite is a kind of Shuifu County's zeopan clay mineral that contains, have the unique layer chain crystalline structure and bar-shaped, the fibrous crystals form of very tiny (about 0.01 μ m * 1 μ m), be a kind of natural sorbent material, can be used as sorbent material, catalyzer, carrier etc.China's attapulgite resource is very abundant, and in Mingguang City, China Anhui, ground reserves such as Xuyi Jiangsu, the six directions are abundant, and only the existing attapulgite reserves in Xuyi County account for 70% of China's attapulgite total amount more than 6,700 ten thousand tons.Attapulgite crystalline theoretical chemistry formula is: Mg
5Si
8O
20(OH)
2(OH
2)
44H
2O.Enzyme is compared with chemical catalysis as a kind of biological catalyst, has reaction conditions gentleness, biodegradability, power consumption is low, ingredient requirement is low, the final product quality advantages of higher.The chemical nature of enzyme is a protein, and its catalysis characteristics strictness depends on proteinic three-dimensional structure and substrate and protein active group functional localization accurately.Resolvase survive usually temperature, pH value narrow range, sex change and inactivation are difficult to reuse easily, can not realize operate continuously.For modern industry, resolvase is not a kind of ideal catalyzer, thereby has limited the application of enzyme significantly.Enzyme is simplified purifying technique after immobilization, be easy to realize successive reaction and automatic production, realizes reusing, and increases operation rate, and reduces cost etc.Lipase is a kind of as enzyme, because it also can keep its activity highly under such as disadvantageous conditions such as higher relatively temperature, organic solvents, huge utility value is arranged in organic synthesis, compare with other enzyme, the substrate specificity of lipase is not too strict comparatively speaking, so lipase is widely used.The application of lipase comprise the production of production, chiral intermediate of improvement, the foodstuff additive of fat hydrolysis, flavour of food products and fragrance and medicament production production, the degreasing of leather silk spinning, greasy modification such as low, make an addition to washing composition and makeup are medium.Therefore, lipase has great potential as commercial catalysts.Utilize the attapulgite resource of China's abundant, adopt absorption method to carry out lipase immobilization, technology is simple, and cost is low.Up to now, the research to silicate clay abroad mainly sets foot in lamellated silicate clay---bentonitic research and development, there are tens of the applicability patents about layered silicate in the U.S., and has occupied the suitable market share, and its industrialization has also had considerable scale.The various silicate clay of external employing carries out immobilization to enzyme, though domestic have the relevant immobilized research to enzyme, but adopts silicate less as the research of enzyme carrier, does not also see the report of heavy industrialization.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of immobilized lipase attapulgite clay adopts attapulgite to carry out lipase immobilization and belongs to pioneering research.Attapulgite has bigger specific surface area as a kind of fibrous silicate than layered silicate, so, utilizing the attapulgite resource of China's abundant, the research and development immobilized lipase attapulgite clay is to improve the utility value and the utilization ratio of existing resource.
Technical scheme: being to be raw material with attapulgite and lipase, is the carrier of lipase with the attapulgite, adopts absorption method that lipase is carried out immobilization.At first, attapulgite is handled; Adopt the method for concussion absorption to carry out the fixing of lipase then, whole adsorption process is carried out in phosphoric acid buffer, is index with the adsorptive capacity of lipase, takes all factors into consideration the enzyme of immobilized lipase and lives, and determines the processing condition that attapulgite is handled.
One, the processing of attapulgite
The processing of attapulgite was divided into for three steps: acid treatment, calcium chloride are handled and aluminum chloride-sodium-hydroxide treatment.
1, acid treatment
Attapulgite often contains a certain amount of montmorillonite, illite, quartz, feldspar and organic carbon, sometimes also contain calcite, rhombspar, halloysite, pearl pottery stone, a diaspore, gibbsite and pyrophyllite, also contain each metal ion species, so need to adopt the impurity in the acid treatment removal attapulgite, in addition, different acid treatment conditions exerts an influence to the pore passage structure of attapulgite, the technology of back is had a direct impact, thereby influence the fixed effect of lipase.
The influence of acid concentration
Various impurity silicate and metal ion are to the susceptibility difference of acid, different acid concentrations causes the stripping of various impurity and metal ion, and acid concentration is crossed conference and is caused attapulgite crystalline structure generation essential change, cause structure to be caved in, so acid concentration is very crucial to the influence of the pore passage structure of attapulgite.
The influence of acid treatment time
Acid treatment deficiency of time then removal of impurities is insufficient, and the long waste that causes the energy and resource of treatment time, so be necessary to inquire into the acid treatment time.
Suitable acid treatment condition is: add 1-6mol/l hydrochloric acid in attapulgite, the mass volume ratio of attapulgite and hydrochloric acid is counted 1: 10 with g/ml, in 50 ℃ of stir process 0.5-4 hours, centrifugal, after extremely detecting no chlorion with deionized water wash then with the 0.1mol/l silver nitrate solution, centrifuge dehydration, oven dry is pulverized.
2, calcium chloride is handled
Because the throwing out of calcium chloride makes the attapulgite viscosity in aqueous solution increase, mobile decline adds Ca
2+After, also make whole attapulgite-water system become a heavy-gravity flocculence suspensoid.If solid substance concentration is lower, add The addition of C aCl
2, then can form the puffy deposition, make attapulgite be short texture, help the carrying out of subsequent technique.
Take by weighing the attapulgite after acid treatment, add the CaCl of 1mol/l
2Solution, attapulgite and CaCl
2The mass volume ratio of solution is counted 1: 2.5 with g/ml, and is centrifugal at a slow speed in 55 ℃ of stirrings 1 hour, in order to avoid attapulgite is piled into imporosity, or natural subsidence, separate, after extremely usefulness 0.1mol/l silver nitrate solution detects no chlorion with deionized water wash then, dehydration, oven dry is pulverized.
3, aluminum chloride-sodium-hydroxide treatment
Attapulgite itself is a kind of silicate that contains Shuifu County's magnalium, contain hydroxyl structures such as a large amount of magnesium hydroxyls, silicon hydroxyl and aluminium hydroxyl, obtain Al (OH) x-attapulgite through aluminum chloride and sodium-hydroxide treatment, it is a kind of extension to attapulgite itself, form the attapulgite that the aluminium hydroxyl covers, this structure helps the absorption of high molecular weight protein more, promptly more helps lipase immobilization.
The AlCl that adds 1mol/l in the attapulgite after calcium chloride is handled
3Solution, the mass volume ratio of attapulgite and aluminum trichloride solution is counted 1: 4 with g/ml, stirs, and the NaOH that drips 0.5mol/l transfers pH to 7.0, stirred 2 hours in 55 ℃ then, stir and finish the back suction filtration, washing is to not having chlorion, centrifuge dehydration, oven dry, pulverize, sieve, obtain Al (OH) x-attapulgite.
Two, lipase immobilization
Lipase claims the triacylglycerol Acyl-hydrolase again, is meant the enzyme of synthetic, the transesterification and the decomposition of trig lyceride that catalysis higher fatty acid and glycerol form.The optimal pH of testing used free-fat enzyme is 7-7.5; General all is to carry out under lower temperature by the absorption method immobilized enzyme, and to guarantee the activity of resolvase, still in order to improve the raising adsorption temp that adsorption rate can be suitable, temperature selects 10 ℃-30 ℃ to be advisable usually; Directly influence the amount of carrier-bound enzyme to the variation of enzyme amount, the enzyme that also can influence immobilized enzyme is simultaneously lived; Adsorption time reached adsorption equilibrium in 4 hours substantially.So it is as follows to carry out the processing condition of lipase immobilization:
Get pH and be 7.3 5mg/ml lipase phosphoric acid buffer liquor, add Al (OH) x-attapulgite then, the mass volume ratio of Al (OH) x-attapulgite and lipase phosphoric acid buffer liquor is counted 1: 100 with g/ml, in 25 ℃ of concussion absorption 3.5 hours, centrifugation, washing, normal temperature vacuum-drying, being fixed lipase attapulgite clay.
The mensuration of enzyme adsorptive capacity and calculating: adopt Bradford method (is standard substance with the bovine serum albumin), measure behind the attapulgite adsorption of immobilization concentration of lipase in the residual lipase solution and washings, calculate the unit adsorptive capacity of lipase on attapulgite.
The enzyme activity location survey is fixed
The enzyme activity unit definition: 1g solid enzyme powder, at 40 ℃, under the condition of pH=7.5, hydrolysed fat, per minute discharge the 1umol free fatty acids, are defined as a lipase activity unit of force (u/g).
The preparation of sweet oil emulsion: the 40g polyvinyl alcohol is dissolved in the 1000ml water fully, gets the 150ml polyvinyl alcohol solution, adds the 50ml sweet oil, ultrasonication emulsification.Again emulsification or stirring before each the use.
Enzyme activity determination: 4.00ml sweet oil emulsion and 5ml phosphoric acid buffer, mixing, in 40 ℃ of water bath with thermostatic control preheating 10min, then, add the 0.1g immobilized enzyme, accurate stirring reaction 15min adds 95% ethanol 7.5ml termination reaction in 40 ℃ of water-baths, with the titration of 0.05mol/l standard solution of sodium hydroxide, be indicating liquid with phenolphthalein.
The character of immobilized lipase
The optimal pH of immobilized enzyme and optimum temperuture and resolvase are compared, and the result shows that the optimal pH scope of immobilized enzyme is wide than resolvase, and immobilized enzyme descends to the susceptibility of temperature, and the temperature specific ionization enzyme of thermally denature has improved 5 ℃-10 ℃.
The reusable number of times of immobilized enzyme is measured, and the result shows that when using below 3 times, no considerable change slightly descends when reusing 3-4 time, obviously descends but enzyme is alive when use surpasses more than 5 times.
Beneficial effect of the present invention: the present invention adopts the attapulgite through certain art breading to carry out lipase immobilization, belongs to pioneering research.
Adopt attapulgite to carry out the fixing of lipase, can improve the lipase performance, utilization ratio and range of application, enlarge the range of application of attapulgite, increase the added value of attapulgite, simultaneously, for attapulgite has played good guiding function as the application of enzyme carrier.
Set up the specific embodiment that attapulgite is handled, and, realized the immobilized purpose of lipase attapulgite clay the mensuration of immobilized lipase enzyme performance.
The heat resistance of immobilized lipase obviously improves than resolvase, and the adaptation of pH is also strengthened than resolvase, and experiment shows to hydrolysis of Olive Oil to reuse immobilized lipase, can realize the repeated use of enzyme, improves the utilization ratio and the range of application of enzyme.
The present invention has practical significance to range of application and the added value that increases attapulgite, for the lipase immobilization technology is laid a good foundation.
Embodiment
Embodiment 1
Used attapulgite is the attapulgite raw ore that picks up from the Xuyi, and its composition and content composition see Table 1:
The main component of table 1 attapulgite and content
| Composition | MgO | Al 2O 3 | SiO 2 | SO 2 | K 2O | CaO | TiO 2 | MnO | Fe 2O 3 |
| Content (%) | 8.2 | 11.9 | 63.8 | 1.0 | 1.9 | 1.6 | 1.4 | 0.5 | 9.7 |
Used lipase enzyme is lived and is 5000u/g.
(1) acid treatment of attapulgite: add 300 milliliters of 2mol/l hydrochloric acid in the 30 gram attapulgites, centrifugal in 50 ℃ of stir process 3 hours, then with deionized water wash after detect no chlorion with the 0.1mol/l silver nitrate solution, centrifuge dehydration, oven dry, pulverizing.
(2) calcium chloride is handled: take by weighing the above-mentioned acid-treated attapulgite of 20g, add the CaCl of 1mol/l
2Solution 50ml stirred 1 hour in 55 ℃, and centrifugal at a slow speed (in order to avoid attapulgite is piled into imporosity) or leave standstill separates, then with deionized water wash after detect no chlorion with the 0.1mol/l silver nitrate solution, dehydration, oven dry, pulverizing.
(3) aluminum chloride and sodium-hydroxide treatment: the 10g attapulgite after calcium chloride is handled adds the AlCl of 40ml1mol/l
3, stirring, the NaOH that drips 0.5mol/l regulates pH to 7.0, then in 55 ℃ of stirrings 2 hours, stirs and finishes the back suction filtration, and washing is not to there being chlorion, and centrifuge dehydration is dried, and pulverizes, and sieves, and obtains Al (OH) x-attapulgite.
(4) lipase immobilization: get 20ml5mg/ml lipase phosphoric acid buffer liquor, pH is 7.3, adds 0.2gAl (OH) x-attapulgite then, in 25 ℃ of concussion absorption 3.5 hours, centrifugation, washing, normal temperature vacuum-drying, being fixed lipase attapulgite clay.Measure the enzyme content in raffinate and the washings, calculate the enzyme adsorptive capacity, simultaneously, measure the enzyme of immobilized enzyme and live.
The average unit adsorptive capacity of lipase on attapulgite and average enzyme are lived in the detection gained the finished product, the unit adsorptive capacity of lipase on attapulgite is 29mg/g (lipase of every gram attapulgite absorption is 29 milligrams), and enzyme is lived and is 78u/g (the enzyme work of every gram immobilized lipase attapulgite clay is 78 enzymes units alive).
Gained immobilized lipase enzyme between pH6.5-7.5 is lived all more satisfactory, and all effectively decomposition reactions of catalysis sweet oil still have more satisfactory enzyme to live when reusing 4 times when temperature is lower than 45 ℃.
Embodiment 2
The acid treatment of attapulgite: add 300 milliliters of 1mol/l hydrochloric acid in the 30 gram attapulgites, centrifugal in 50 ℃ of stir process 4 hours, then with deionized water wash after detect no chlorion with the 0.1mol/l silver nitrate solution, centrifuge dehydration, oven dry, pulverizing.
Used attapulgite raw material and other technologies are with embodiment 1.The average unit adsorptive capacity of lipase on attapulgite and average enzyme are lived close with the result of embodiment 1 in the products obtained therefrom.
Embodiment 3
The acid treatment of attapulgite: add 300 milliliters of 6mol/l hydrochloric acid in the 30 gram attapulgites, centrifugal in 50 ℃ of stir process 0.5 hour, then with deionized water wash after detect no chlorion with the 0.1mol/l silver nitrate solution, centrifuge dehydration, oven dry, pulverizing.
Used attapulgite raw material and other technologies are with embodiment 1.The average unit adsorptive capacity of lipase on attapulgite and average enzyme are lived close with the result of embodiment 1 in the products obtained therefrom.
Claims (1)
1. the preparation method of an immobilized lipase attapulgite clay, it is characterized in that with attapulgite and lipase be raw material, with the attapulgite is the carrier of lipase, attapulgite is carried out acid treatment, calcium chloride processing and aluminum chloride-sodium-hydroxide treatment, adopt the method for absorption to carry out lipase immobilization then;
A) acid treatment of attapulgite: in attapulgite, add 1-6mol/l hydrochloric acid, the mass volume ratio of attapulgite and hydrochloric acid is counted 1: 10 with g/ml, in 50 ℃ of stir process 0.5-4 hours, centrifugal, after extremely detecting no chlorion with deionized water wash then with the 0.1mol/l silver nitrate solution, centrifuge dehydration, oven dry is pulverized;
B) calcium chloride is handled: take by weighing the attapulgite after acid treatment, add the CaCl of 1mol/l
2Solution, attapulgite and CaCl
2The mass volume ratio of solution is counted 1: 2.5 with g/ml, and is centrifugal at a slow speed in 55 ℃ of stirrings 1 hour, in order to avoid attapulgite is piled into imporosity, or natural subsidence, separate, after extremely usefulness 0.1mol/l silver nitrate solution detects no chlorion with deionized water wash then, dehydration, oven dry is pulverized;
C) aluminum chloride-sodium-hydroxide treatment: the AlCl that adds 1mol/l in the attapulgite after calcium chloride is handled
3Solution, the mass volume ratio of attapulgite and aluminum trichloride solution is counted 1: 4 with g/ml, stirs, and the NaOH that drips 0.5mol/l transfers pH to 7.0, stirred 2 hours in 55 ℃ then, stir and finish the back suction filtration, washing is to not having chlorion, centrifuge dehydration, oven dry, pulverize, sieve, obtain Al (OH) x-attapulgite;
D) lipase immobilization: get pH and be 7.3 5mg/ml lipase phosphoric acid buffer liquor, add Al (OH) x-attapulgite then, the mass volume ratio of Al (OH) x-attapulgite and lipase phosphoric acid buffer liquor is counted 1: 100 with g/ml, in 25 ℃ of concussion absorption 3.5 hours, centrifugation, washing, normal temperature vacuum-drying, being fixed lipase attapulgite clay.
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| CN102294221A (en) * | 2011-06-28 | 2011-12-28 | 淮阴工学院 | Preparation method of attapulgite-based adsorption drying agent suitable for high humidity environment |
| CN102382811A (en) * | 2011-11-08 | 2012-03-21 | 中国农业科学院油料作物研究所 | Preparation method of attapulgite immobilized enzyme for ester exchange reaction |
| CN102382811B (en) * | 2011-11-08 | 2013-06-05 | 中国农业科学院油料作物研究所 | Preparation method of attapulgite immobilized enzyme for ester exchange reaction |
| CN102952792A (en) * | 2012-11-12 | 2013-03-06 | 淮阴工学院 | Preparation method of beta-glucosidase immobilized by sodium alginate loaded with attapulgite |
| CN103045581A (en) * | 2012-12-21 | 2013-04-17 | 淮阴工学院 | Method for immobilizing myxococcus fulvus coated by chitosan loaded with attapulgite |
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| CN103303933A (en) * | 2013-05-31 | 2013-09-18 | 胡沂淮 | Attapulgite modification method and preparation method of attapulgite for fixing ligand protein A or protein G |
| CN103303933B (en) * | 2013-05-31 | 2016-01-27 | 胡沂淮 | The method of modifying of attapulgite and attapulgite fix the preparation method of ligand albumin A or Protein G |
| CN115386608A (en) * | 2022-09-02 | 2022-11-25 | 山东百阜福瑞达制药有限公司 | Method for efficiently preparing high-activity hyaluronic acid oligosaccharide composition by regulating and controlling enzyme activity in real time |
| CN115386608B (en) * | 2022-09-02 | 2023-11-10 | 山东百阜福瑞达制药有限公司 | Method for preparing high-activity hyaluronic acid oligosaccharide composition by regulating and controlling enzyme activity in real time |
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