CN101113433B - A preparation process of chitosan microsphere immobilized lipase - Google Patents
A preparation process of chitosan microsphere immobilized lipase Download PDFInfo
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- CN101113433B CN101113433B CN2007100698206A CN200710069820A CN101113433B CN 101113433 B CN101113433 B CN 101113433B CN 2007100698206 A CN2007100698206 A CN 2007100698206A CN 200710069820 A CN200710069820 A CN 200710069820A CN 101113433 B CN101113433 B CN 101113433B
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- 229920001661 Chitosan Polymers 0.000 title claims abstract description 71
- 239000004367 Lipase Substances 0.000 title claims abstract description 67
- 102000004882 Lipase Human genes 0.000 title claims abstract description 64
- 108090001060 Lipase Proteins 0.000 title claims abstract description 64
- 235000019421 lipase Nutrition 0.000 title claims abstract description 64
- 239000004005 microsphere Substances 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 31
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims abstract description 42
- 238000003756 stirring Methods 0.000 claims abstract description 33
- 101710098556 Lipase A Proteins 0.000 claims abstract description 22
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- 102100026001 Lysosomal acid lipase/cholesteryl ester hydrolase Human genes 0.000 claims abstract description 22
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims abstract description 17
- 101710098554 Lipase B Proteins 0.000 claims abstract description 9
- 238000004132 cross linking Methods 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 45
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- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 19
- DCPMPXBYPZGNDC-UHFFFAOYSA-N hydron;methanediimine;chloride Chemical compound Cl.N=C=N DCPMPXBYPZGNDC-UHFFFAOYSA-N 0.000 claims description 19
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 15
- 239000008367 deionised water Substances 0.000 claims description 14
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- 238000005303 weighing Methods 0.000 claims description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 10
- 239000000872 buffer Substances 0.000 claims description 10
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 10
- 239000002953 phosphate buffered saline Substances 0.000 claims description 10
- 230000002378 acidificating effect Effects 0.000 claims description 7
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- 239000012530 fluid Substances 0.000 claims description 7
- 239000004971 Cross linker Substances 0.000 claims description 5
- 230000004913 activation Effects 0.000 claims description 5
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- 238000004321 preservation Methods 0.000 claims description 5
- 238000001291 vacuum drying Methods 0.000 claims description 5
- NWGKJDSIEKMTRX-AAZCQSIUSA-N Sorbitan monooleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O NWGKJDSIEKMTRX-AAZCQSIUSA-N 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 2
- SPOMEWBVWWDQBC-UHFFFAOYSA-K tripotassium;dihydrogen phosphate;hydrogen phosphate Chemical compound [K+].[K+].[K+].OP(O)([O-])=O.OP([O-])([O-])=O SPOMEWBVWWDQBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000012065 filter cake Substances 0.000 claims 1
- 108010093096 Immobilized Enzymes Proteins 0.000 abstract description 28
- 230000000694 effects Effects 0.000 abstract description 14
- 102000004190 Enzymes Human genes 0.000 abstract description 11
- 108090000790 Enzymes Proteins 0.000 abstract description 11
- 238000011084 recovery Methods 0.000 abstract description 11
- 239000008363 phosphate buffer Substances 0.000 abstract 2
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 abstract 1
- 238000004064 recycling Methods 0.000 abstract 1
- 229940040461 lipase Drugs 0.000 description 45
- LVGKNOAMLMIIKO-UHFFFAOYSA-N Elaidinsaeure-aethylester Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC LVGKNOAMLMIIKO-UHFFFAOYSA-N 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 11
- LVGKNOAMLMIIKO-QXMHVHEDSA-N ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 11
- 229940093471 ethyl oleate Drugs 0.000 description 11
- 229940088598 enzyme Drugs 0.000 description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 8
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- 238000001035 drying Methods 0.000 description 6
- 241000222175 Diutina rugosa Species 0.000 description 5
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 3
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 3
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 3
- 239000005642 Oleic acid Substances 0.000 description 3
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 229960004756 ethanol Drugs 0.000 description 3
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- ZNZYKNKBJPZETN-WELNAUFTSA-N Dialdehyde 11678 Chemical compound N1C2=CC=CC=C2C2=C1[C@H](C[C@H](/C(=C/O)C(=O)OC)[C@@H](C=C)C=O)NCC2 ZNZYKNKBJPZETN-WELNAUFTSA-N 0.000 description 1
- 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 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 108050006759 Pancreatic lipases Proteins 0.000 description 1
- 102000019280 Pancreatic lipases Human genes 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 241000698291 Rugosa Species 0.000 description 1
- 241000594182 Sarcophaga sigma Species 0.000 description 1
- OGBUMNBNEWYMNJ-UHFFFAOYSA-N batilol Chemical class CCCCCCCCCCCCCCCCCCOCC(O)CO OGBUMNBNEWYMNJ-UHFFFAOYSA-N 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
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- 239000008103 glucose Substances 0.000 description 1
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Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
一种壳聚糖微球固定化脂肪酶的制备工艺：(1)将假丝酵母脂肪酶溶于磷酸盐缓冲液a中，以壳聚糖微球为固定化载体，采用戊二醛交联，制备得戊二醛交联固定化的脂肪酶A；(2)在用pH值为6.5～7.5磷酸盐缓冲液b配制的0.70～0.80mg/mL假丝酵母脂肪酶溶液中，加入固定化的脂肪酶A(终浓度为0.005～0.0055g/ml)，搅拌均匀后，再加入乙基[3-(二甲胺基)丙基]碳二亚胺盐酸盐溶液，搅拌，使其充分固定，处理后得壳聚糖微球固定化脂肪酶B。利用本发明方法制备壳聚糖微球固定化酶，提高了酶蛋白的结合率和活力回收率高，固定化酶活力、比活力强，可循环使用次数达6次。A preparation process of chitosan microsphere immobilized lipase: (1) dissolving Candida lipase in phosphate buffer a, using chitosan microsphere as immobilized carrier, cross-linking with glutaraldehyde , to prepare glutaraldehyde crosslinked and immobilized lipase A; (2) in the 0.70～0.80mg/mL Candida lipase solution prepared with pH value of 6.5～7.5 phosphate buffer b, add immobilized lipase A (final concentration is 0.005 ~ 0.0055g/ml), after stirring evenly, then add ethyl [3-(dimethylamino) propyl] carbodiimide hydrochloride solution, stirring, to make it fully After being fixed and treated, chitosan microspheres immobilized lipase B were obtained. The chitosan microsphere immobilized enzyme is prepared by the method of the invention, which improves the binding rate of the enzyme protein and has high recovery rate of activity, the activity and specific activity of the immobilized enzyme are strong, and the times of recycling can reach 6 times.
Description
(1) technical field
The invention belongs to technical field of biochemical industry, relate to the preparation technology of chitin microspheric immobilized lipase.
(2) background technology
Chitosan is a kind of N-deacetylation glucose polymer, and the source is abundant, and low price is the most important derivative of chitin.Chitosan has nontoxicity, good gelation, bio-compatibility, protein affinity and metal ion-chelant characteristic, is good fixed enzyme vector.The immobilized enzyme kind that with the chitosan is carrier has a lot, as stomach en-, glucose oxidase, L-Asnase, 'beta '-mannase etc.With the tegument glycan is that carrier immobilized enzyme major defect is: because of dissolving under the meta-acid condition, make immobilized enzyme be difficult to reclaim, wherein major part is Powdered, the carrier current difference, and surface-area is little, limits its application.Therefore present domestic and international research mainly concentrates on the preparation chitosan microball as fixed enzyme vector, and this carrier has characteristics such as absorption property is strong, and the acid-fast alkali-proof performance is good, thereby has overcome above-mentioned defective, is the good carrier of enzyme immobilization.
Lipase is as biological catalyst catalytic reaction condition gentleness, and product is of light color, and by product is few and have specificity, is beneficial to the fat products of producing high-quality mono-glycerides, lipid acid, glycerine and other kinds, is widely used industrial.Especially in recent years the application in grease production, to production environmental protection fuel, the friendly type of built environment society solves the serious day by day energy problem of China, and important practical sense and economic benefit are arranged.Therefore but the price of lipase is higher, and when reacting with the free-fat enzyme, lipase can only use once and can not reclaim, and causes the product cost higher and cause later separation step difficulty, has influenced lipase in industrial concrete application.By lipase is carried out immobilization, immobilized enzyme is when keeping efficient, single-minded and gentle enzymic catalytic reaction characteristic, also present package stability height, Separation and Recovery easily, can be repeatedly used, operate continuously and controlled, series of advantages such as technology is easy, improved productivity effect greatly.
With chitosan microball is that the research of carrier immobilized lipase receives much concern in recent years.People such as Tien-Chieh Hung utilize the chitosan magnetic micro-sphere immobilized lipase, and enzyme activity reaches 13.8U/g; Shao-Hua Chiou prepares immobilized lipase by two kinds of chitosan microballs, and the enzyme rate of recovery alive is respectively 75% and 85%; People such as Wu Qianqian have studied chitosan absorption and glutaraldehyde cross-linking to the lipase immobilization condition, the De Shi rizolipase is fixed, and this immobilized enzyme is used for synthesizing of ester; People such as Chen Sheng are that raw material extracts chitosan with the crab shell, make linking agent with glutaraldehyde, and alkaline lipase is fixed on the chitosan; People such as Peng Lifeng have studied chitosan coat top condition of immobilized porcine pancreatic lipase again after film forming on the Mierocrystalline cellulose filter paper.
China adopts single method to fix at present mostly, as absorption method or the single immobilized lipase of crosslinking, because the irregularity of support shapes, immobilized enzyme activity recovery generally lower (36%-50%) and immobilized enzyme have limited the application of immobilized lipase to weak points such as the avidity of substrate are low.At this, we are fixation support with the chitosan microball, successively utilize hydroxyl and amino on the chitosan molecule, adopt glutaraldehyde and twice compound fixed fat enzyme of carbodiimide, improve the immobilized enzyme vigor.
(3) summary of the invention
The object of the present invention is to provide a kind of chitin microspheric immobilized lipase preparation technology.
For reaching the object of the invention, the technical solution used in the present invention is as follows:
A kind of preparation technology of chitin microspheric immobilized lipase is characterized in that described technology carries out as follows:
(1) prepares immobilized lipase A with glutaraldehyde as cross linker: lipase from candida sp is dissolved in phosphate buffered saline buffer
Among a be carrier, adopt glutaraldehyde cross-linking, be prepared into the immobilized lipase A of glutaraldehyde cross-linking with the chitosan microball;
(2) activate immobilized lipase A and prepare immobilized lipase B with ethyl [3-(dimethylin) propyl group] carbodiimide hydrochloride: in the 0.70～0.80mg/mL lipase from candida sp solution that with the pH value is 6.5～7.5 phosphate buffered saline buffer b preparation, add immobilized lipase A, the final concentration of described immobilized lipase A is 0.005～0.0055g/ml, after stirring, add ethyl [3-(dimethylin) propyl group] carbodiimide hydrochloride solution again, stirred 3～8 hours, make it fully fixing, use deionized water rinsing, filter, filtration cakes torrefaction gets chitin microspheric immobilized lipase B.
Step of the present invention (1) is: be in the lipase from candida sp solution of 0.75mg/mL of 6.5～7.5 phosphate buffered saline buffer a preparation with the pH value, add chitosan microball, the final concentration of described chitosan microball is 0.005～0.0055g/ml, after stirring, add glutaraldehyde solution again, the final concentration that makes glutaraldehyde is 2.0%～2.5% (g/ml), the continuous stirring after 5～7 o'clock, make it fully fixing, use deionized water rinsing, filter, filtration cakes torrefaction promptly gets described chitin microspheric immobilized lipase A.
Chitosan microball described in the step of the present invention (1) has the particle diameter of the microballoon more than 80% at 50～200nm.
Chitosan microball described in the step of the present invention (1) prepares as follows: taking by weighing chitosan, to be dissolved in volume fraction be 2% acetic acid solution, make massfraction and be 2.5% chitosan acidic sol, under agitation, described chitosan acidic sol is slowly joined in the mixed solution of whiteruss and Arlacel-80, the add-on of described whiteruss is the 80mL/g chitosan, the add-on of described Arlacel-80 is the 5mL/g chitosan, stirring makes colloidal sol drop homodisperse, adding volume fraction then is 25% dialdehyde solution, and the add-on of described glutaraldehyde solution is the 10mL/g chitosan, stirs, transfer pH to 9～10, in 70 ℃ of water bath heat preservations 2～4 hours, leave standstill cooling after, discard oil reservoir, use sherwood oil more successively, acetone, dehydrated alcohol embathes, after the filtration, consider 0 ℃ of vacuum-drying of cake, obtain chitosan microball.
Phosphate buffered saline buffer a of the present invention or phosphate buffered saline buffer b independently are Na separately
2HPO
4-KH
2PO
4, Na
2HPO
4-NaH
2PO
4Or K
2HPO
4-KH
2PO
4
The concentration of the glutaraldehyde solution that adds described in the step of the present invention (1) is 25% (g/ml).
The concentration of ethyl [3-(dimethylin) propyl group] the carbodiimide hydrochloride solution that adds described in the step of the present invention (2) is 0.2% (g/ml).
Preparation technology of the present invention specifically carries out as follows:
A) with the pH value be 7 Na
2HPO
4-KH
2PO
4In lipase from candida sp (Candida Rugosa) solution of the 0.75mg/mL of damping fluid preparation, add chitosan microball, the final concentration of described chitosan microball is 0.005～0.0055g/ml, after stirring, the glutaraldehyde solution that adds 25% (g/ml) again, the final concentration that makes glutaraldehyde is 2.2% (g/ml), continues to stir 5～7 o'clock, makes it fully fixing, use deionized water rinsing, filter, filtration cakes torrefaction promptly gets described chitin microspheric immobilized lipase A;
B) activate immobilized lipase A and prepare immobilized lipase B with ethyl [3-(dimethylin) propyl group] carbodiimide hydrochloride: at the Na that with the pH value is 7
2HPO
4-KH
2PO
4In lipase from candida sp (Candida Rugosa) solution of 0.70～0.80mg/mL of damping fluid preparation, add immobilized lipase A, the final concentration of described immobilized lipase A is 0.005～0.0055g/ml, after stirring, add ethyl [3-(dimethylin) propyl group] carbodiimide hydrochloride solution again, vibration 6h, make it fully fixing, use deionized water rinsing, filter, filtration cakes torrefaction gets chitin microspheric immobilized lipase B.
Beneficial effect of the present invention is mainly reflected in: 1) utilize chitosan microball hydroxyl and amino twice immobilized lipase successively, the combination rate and the activity recovery (62.5%) of zymoprotein have been improved, the immobilized enzyme vigor reaches the 835U/g carrier, reaches 34.9U/mg albumen than vigor.2) the chitin microspheric immobilized enzyme that utilizes the inventive method to make prepares ethyl oleate, and behind the reaction 8h, the ethyl oleate transformation efficiency reaches 68.4%.Chitin microspheric immobilized enzyme recycles number of times and reaches 6 times, has reduced the reaction cost of resolvase.
(4) embodiment
Below with specific embodiment technical scheme of the present invention is described, but protection scope of the present invention is not limited thereto.Lipase is that rugosa.1 enzyme activity unit of lipase from candida sp Candida is defined as at 37 ℃ among the embodiment, during pH7.2, and the amount of 1 micromole's lipid acid in the hydrolysis sweet oil in 1 minute.What we used is the thick enzyme of the commercial enzyme type VII of U.S. sigma company.
The preparation of embodiment 1. chitosan microballs
Taking by weighing the 1g chitosan, to be dissolved in volume fraction be 2% acetic acid solution, makes massfraction and be 2.5% chitosan acidic sol.Under agitation, above-mentioned colloidal sol is slowly joined in the mixed solution of 80mL whiteruss and 5mLSpan-80, stir 20min, make colloidal sol drop homodisperse, adding the 10mL volume fraction then is 25% glutaraldehyde solution, stir 10min after, with the NaOH solution of 1mol/L its pH is transferred to 9, in 70 ℃ of water bath heat preservation 3h, after leaving standstill cooling, discard the upper strata oil reservoir, embathe with sherwood oil, acetone, dehydrated alcohol successively again, 50 ℃ of vacuum-dryings obtain chitosan microball.
Embodiment 2. glutaraldehyde as cross linker prepare immobilized lipase
Take by weighing the 0.50g chitosan microball, add the Na of pH=7
2HPO
4-KH
2PO
4Lipase from candida sp (Candida Rugosa) the solution 100mL of the 0.75mg/mL of damping fluid preparation makes chitosan microball final concentration 0.005g/ml, stirs 1h under the room temperature.The glutaraldehyde solution 11.1mL that then adds 25% concentration, making the glutaraldehyde final concentration is 2.5%g/ml, continues to stir 6h, full cross-linked, washes repeatedly 3 times with deionized water, filters, and is drying to obtain immobilized lipase.This immobilized enzyme vigor reaches the 473U/g carrier, reaches 22.1U/mg albumen than vigor, and activity recovery reaches 38.1%.
The activation of embodiment 3. usefulness ethyls [3-(dimethylin) propyl group] carbodiimide hydrochloride prepares immobilized lipase once more
The glutaraldehyde fixed chitosan microball lipase that takes by weighing in the 0.50g step 2 is dipped in the lipase liquid (pH=7) of 100mL volume 0.75mg/mL, and making glutaraldehyde fixed chitosan microball lipase concentration is 0.005g/ml, stirs 1h.Add 25mL ethyl [3-(dimethylin) propyl group] carbodiimide hydrochloride solution (0.2%) then, making ethyl [3-(dimethylin) propyl group] carbodiimide hydrochloride concentration is 0.04%g/ml, continue vibration 6h, make it fully fixing, wash repeatedly 3 times, filter with deionized water, be drying to obtain chitin microspheric immobilized lipase, the immobilized enzyme vigor reaches the 813U/g carrier, reaches 35.3U/mg albumen than vigor, and activity recovery reaches 60.2%.
Embodiment 4. immobilized enzyme are applied to the synthetic of ethyl oleate
Take by weighing 3g oleic acid and join in the 10ml scale test tube, add entry 3mL and ethanol 3mL, in reaction system, add chitin microspheric immobilized enzyme 0.4g afterwards, 47 ℃ of temperature of reaction, behind the reaction 8h, the ethyl oleate transformation efficiency reaches 68.4%.Chitin microspheric immobilized enzyme recycles number of times and reaches 6 times, and the ethyl oleate transformation efficiency reduces to 32% from 68.4%.
The preparation of embodiment 5. chitosan microballs
Taking by weighing the 1g chitosan, to be dissolved in volume fraction be 2% acetic acid solution, makes massfraction and be 2.5% chitosan acidic sol.Under agitation, above-mentioned colloidal sol is slowly joined in the mixed solution of 80mL whiteruss and 5mLSpan-80, stir 20min, make colloidal sol drop homodisperse, adding the 10mL volume fraction then is 25% glutaraldehyde solution, stir 10min after, with the NaOH solution of 1mol/L its pH is transferred to 10, in 70 ℃ of water bath heat preservation 3h, after leaving standstill cooling, discard the upper strata oil reservoir, embathe with sherwood oil, acetone, dehydrated alcohol successively again, 50 ℃ of vacuum-dryings obtain chitosan microball.
Embodiment 6. glutaraldehyde as cross linker prepare immobilized lipase
Take by weighing the 0.525g chitosan microball, add the Na of pH=6.5
2HPO
4-NaH
2PO
4Lipase from candida sp (Candida Rugosa) the solution 100mL of the 0.75mg/mL of damping fluid preparation makes chitosan microball final concentration 0.00525g/ml, stirs 1h under the room temperature.The glutaraldehyde solution 8.7mL that then adds 25% concentration, making the glutaraldehyde final concentration is 2.0%g/ml, continues to stir 5h, full cross-linked, washes repeatedly 3 times with deionized water, filters, and is drying to obtain immobilized lipase.This immobilized enzyme vigor reaches the 468U/g carrier, reaches 20.2U/mg albumen than vigor, and activity recovery reaches 36.4%.
The activation of embodiment 7. usefulness ethyls [3-(dimethylin) propyl group] carbodiimide hydrochloride prepares immobilized lipase once more
The glutaraldehyde fixed chitosan microball lipase that takes by weighing in the 0.525g step 2 is dipped in the lipase liquid (pH=6.5) of 100mL volume 0.70mg/mL, and making glutaraldehyde fixed chitosan microball lipase concentration is 0.00525g/ml, stirs 1h.Add 25mL ethyl [3-(dimethylin) propyl group] carbodiimide hydrochloride solution (0.2%) then, making ethyl [3-(dimethylin) propyl group] carbodiimide hydrochloride concentration is 0.04%g/ml, continue vibration 5h, make it fully fixing, wash repeatedly 3 times, filter with deionized water, be drying to obtain chitin microspheric immobilized lipase, the immobilized enzyme vigor reaches the 814U/g carrier, reaches 31.8U/mg albumen than vigor, and activity recovery reaches 61.2%.
Embodiment 8. immobilized enzyme are applied to the synthetic of ethyl oleate
Take by weighing 3g oleic acid and join in the 10ml scale test tube, add entry 3mL and ethanol 3mL, in reaction system, add chitin microspheric immobilized enzyme 0.40g afterwards, 47 ℃ of temperature of reaction, behind the reaction 8h, the ethyl oleate transformation efficiency reaches 67.1%.Chitin microspheric immobilized enzyme recycles number of times and reaches 6 times, and the ethyl oleate transformation efficiency reduces to 32.6% from 67.1%.
The preparation of embodiment 9. chitosan microballs
Taking by weighing the 1g chitosan, to be dissolved in volume fraction be 2% acetic acid solution, makes massfraction and be 2.5% chitosan acidic sol.Under agitation, above-mentioned colloidal sol is slowly joined in the mixed solution of 80mL whiteruss and 5mLSpan-80, stir 20min, make colloidal sol drop homodisperse, adding the 10mL volume fraction then is 25% glutaraldehyde solution, stir 10min after, with the NaOH solution of 1mol/L its pH is transferred to 9.5, in 70 ℃ of water bath heat preservation 3h, after leaving standstill cooling, discard the upper strata oil reservoir, embathe with sherwood oil, acetone, dehydrated alcohol successively again, 50 ℃ of vacuum-dryings obtain chitosan microball.
Embodiment 10. glutaraldehyde as cross linker prepare immobilized lipase
Take by weighing the 0.55g chitosan microball, add the K of pH=7.5
2HPO
4-KH
2PO
4Lipase from candida sp (Candida Rugosa) the solution 100mL of the 0.75mg/mL of damping fluid preparation makes under the chitosan microball final concentration 0.0055g/ml room temperature and stirs 1h.The glutaraldehyde solution 9.5mL that then adds 25% concentration, making the glutaraldehyde final concentration is 2.17%g/ml, continues to stir 7h, full cross-linked, washes repeatedly 3 times with deionized water, filters, and is drying to obtain immobilized lipase.This immobilized enzyme vigor reaches the 482U/g carrier, reaches 21.3U/mg albumen than vigor, and activity recovery reaches 37.9%.
The activation of embodiment 11. usefulness ethyls [3-(dimethylin) propyl group] carbodiimide hydrochloride prepares immobilized lipase once more
The glutaraldehyde fixed chitosan microball lipase that takes by weighing in the 0.55g step 2 is dipped in the lipase liquid (pH=7.5) of 100mL volume 0.80mg/mL, and making glutaraldehyde fixed chitosan microball lipase concentration is 0.0055g/ml, stirs 1h.Add 25mL ethyl [3-(dimethylin) propyl group] carbodiimide hydrochloride solution (0.2%) then, making ethyl [3-(dimethylin) propyl group] carbodiimide hydrochloride concentration is 0.04% (g/ml), continue vibration 7h, make it fully fixing, wash repeatedly 3 times, filter with deionized water, be drying to obtain chitin microspheric immobilized lipase, the immobilized enzyme vigor reaches the 835U/g carrier, reaches 34.9U/mg albumen than vigor, and activity recovery reaches 62.5%.
Embodiment 12. immobilized enzyme are applied to the synthetic of ethyl oleate
Take by weighing 3g oleic acid and join in the 10ml scale test tube, add entry 3mL and ethanol 3mL, in reaction system, add chitin microspheric immobilized enzyme 0.40g afterwards, 47 ℃ of temperature of reaction, behind the reaction 8h, the ethyl oleate transformation efficiency reaches 66.8%.Chitin microspheric immobilized enzyme recycles number of times and reaches 6 times, and the ethyl oleate transformation efficiency reduces to 34.2% from 66.8%.
Claims (8)
1. the preparation method of a chitin microspheric immobilized lipase is characterized in that described method carries out as follows:
(1) prepare immobilized lipase A with glutaraldehyde as cross linker: it is among 6.5～7.5 phosphate buffered saline buffer a that lipase from candida sp is dissolved in the pH value, with the chitosan microball is fixation support, adopt glutaraldehyde cross-linking, be prepared into the immobilized immobilized lipase A of glutaraldehyde cross-linking;
(2) the immobilized lipase A that makes with ethyl [3-(dimethylin) propyl group] carbodiimide hydrochloride activation step (1) prepares immobilized lipase B: in the 0.70～0.80mg/mL lipase from candida sp solution that with the pH value is 6.5～7.5 phosphate buffered saline buffer b preparation, add immobilized lipase A, the final concentration of described immobilized lipase A is 0.005～0.0055g/ml, after stirring, add ethyl [3-(dimethylin) propyl group] carbodiimide hydrochloride solution again, stirred 3～8 hours, make it fully fixing, use deionized water rinsing, filter, filtration cakes torrefaction gets immobilized lipase B, promptly described chitin microspheric immobilized lipase.
2. the preparation method of chitin microspheric immobilized lipase as claimed in claim 1, it is characterized in that described step (1) is: be in the lipase from candida sp solution of 0.75mg/mL of 6.5～7.5 phosphate buffered saline buffer a preparation with the pH value, add chitosan microball, the final concentration of described chitosan microball is 0.005～0.0055g/ml, after stirring, add glutaraldehyde solution again, the final concentration that makes glutaraldehyde is 2.0%～2.5%g/ml, continue to stir after 5～7 o'clock, make it fully fixing, use deionized water rinsing, filter, filtration cakes torrefaction promptly gets described immobilized lipase A.
3. the preparation method of chitin microspheric immobilized lipase as claimed in claim 1 or 2 is characterized in that the chitosan microball described in the described step (1) has the particle diameter of the microballoon more than 80% at 50～200nm.
4. the preparation method of chitin microspheric immobilized lipase as claimed in claim 1 or 2, it is characterized in that the chitosan microball described in the described step (1) prepares as follows: taking by weighing chitosan, to be dissolved in volume fraction be 2% acetic acid solution, make massfraction and be 2.5% chitosan acidic sol, under agitation, described chitosan acidic sol is slowly joined in the mixed solution of whiteruss and Arlacel-80, the add-on of described whiteruss is the 80mL/g chitosan, the add-on of described Arlacel-80 is the 5mL/g chitosan, stirring makes colloidal sol drop homodisperse, adding volume fraction then is 25% glutaraldehyde solution, the add-on of described glutaraldehyde solution is the 10mL/g chitosan, stir, transfer pH to 9～10, in 70 ℃ of water bath heat preservations 2～4 hours, after leaving standstill cooling, discard oil reservoir, use sherwood oil more successively, acetone, dehydrated alcohol embathes, after the filtration, 0 ℃ of vacuum-drying of filter cake obtains chitosan microball.
5. the preparation method of chitin microspheric immobilized lipase as claimed in claim 1 or 2 is characterized in that described phosphate buffered saline buffer a is Na
2HPO
4-KH
2PO
4, Na
2HPO
4-NaH
2PO
4Or K
2HPO
4-KH
2PO
4Described phosphate buffered saline buffer b is Na
2HPO
4-KH
2PO
4, Na
2HPO
4-NaH
2PO
4Or K
2HPO
4-KH
2PO
4
6. as the preparation method of chitin microspheric immobilized lipase as described in the claim 2, it is characterized in that the concentration of the glutaraldehyde solution that adds in the described step (1) is 25%g/ml.
7. as the preparation method of chitin microspheric immobilized lipase as described in the claim 2, it is characterized in that the concentration of ethyl [3-(dimethylin) propyl group] the carbodiimide hydrochloride solution that adds in the described step (2) is 0.2%g/ml.
8. as the preparation method of chitin microspheric immobilized lipase as described in the claim 2, it is characterized in that described preparation method carries out as follows:
A) with the pH value be 7 Na
2HPO
4-KH
2PO
4In the lipase from candida sp solution of the 0.75mg/mL of damping fluid preparation, add chitosan microball, the final concentration of described chitosan microball is 0.005～0.0055g/ml, after stirring, the glutaraldehyde solution that adds 25%g/ml again, the final concentration that makes glutaraldehyde is 2.2%g/ml, continues to stir 5～7 o'clock, makes it fully fixing, use deionized water rinsing, filter, filtration cakes torrefaction promptly gets immobilized lipase A;
B) the immobilized lipase A that a) makes with ethyl [3-(dimethylin) propyl group] carbodiimide hydrochloride activation step prepares immobilized lipase B: at the Na that with the pH value is 7
2HPO
4-KH
2PO
4In the lipase from candida sp solution of 0.70～0.80mg/mL of damping fluid preparation, add immobilized lipase A, the final concentration of described immobilized lipase A is 0.005～0.0055g/ml, after stirring, adds ethyl [3-(dimethylin) propyl group] carbodiimide hydrochloride solution again, vibration 6h, make it fully fixing, use deionized water rinsing, filter, filtration cakes torrefaction gets immobilized lipase B, promptly described chitin microspheric immobilized lipase.
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| CN101643726B (en) * | 2008-08-05 | 2011-05-04 | 中国农业大学 | Biocompatible hollow polysaccharide microsphere and preparation method thereof |
| CN102422848B (en) * | 2011-10-12 | 2013-07-03 | 江南大学 | Preparation method of immobilized glucose oxidase with muscle strengthening function in production process of bread |
| CN102533710B (en) * | 2011-12-31 | 2014-06-11 | 浙江工业大学 | Method for performing crosslinking immobilized modification on lipase Novozyme 435 by using glutaraldehyde |
| ES2456771A1 (en) * | 2012-09-17 | 2014-04-23 | Instituto Nacional De Investigación Y Tecnología Agraria Y Alimentaria (Inia) | BIOCATALIZERS AND ITS APPLICATIONS |
| CN103074152A (en) * | 2012-12-26 | 2013-05-01 | 东北农业大学 | Method for preparing soybean oil through enzymatic hydrolysis of immobilized enzyme |
| CN103951837A (en) * | 2014-05-08 | 2014-07-30 | 南开大学 | Preparation method of chitosan hydrogel microspheres |
| CN106279727B (en) * | 2016-08-17 | 2018-10-02 | 倪协照 | A kind of preparation method of chitosan microball |
| CN107522781A (en) * | 2017-09-01 | 2017-12-29 | 浦江县欧立生物技术有限公司 | The method that collagen peptide is extracted from grass carp scales |
| CN107541539A (en) * | 2017-09-01 | 2018-01-05 | 浦江县欧立生物技术有限公司 | A kind of preparation method of ocean fish leftover protein powder |
| CN108946955A (en) * | 2018-06-26 | 2018-12-07 | 江苏新亿源环保科技有限公司 | A kind of preparation method of degreaser |
| CN111388739B (en) * | 2020-01-06 | 2021-11-16 | 太原理工大学 | Nano silicon dioxide/decomposition enzyme/polycaprolactone composite microsphere and preparation method and application thereof |
| CN114763543A (en) * | 2021-01-15 | 2022-07-19 | 百瑞全球有限公司 | Membranous immobilized cell, polypeptide, oligopeptide or protein and preparation method thereof |
| CN115141858B (en) * | 2022-07-12 | 2024-11-15 | 江苏斯盖环保科技有限公司 | A method for using microorganisms to fix carbon dioxide to produce organic products |
| CN117925570A (en) * | 2023-12-18 | 2024-04-26 | 蚌埠学院 | Method for extracting bioactive substances from placenta caprae seu ovis |
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