CN101113433A - Process for preparing chitosan microsphere immobilized lipolytic enzyme - Google Patents
Process for preparing chitosan microsphere immobilized lipolytic enzyme Download PDFInfo
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- CN101113433A CN101113433A CNA2007100698206A CN200710069820A CN101113433A CN 101113433 A CN101113433 A CN 101113433A CN A2007100698206 A CNA2007100698206 A CN A2007100698206A CN 200710069820 A CN200710069820 A CN 200710069820A CN 101113433 A CN101113433 A CN 101113433A
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Abstract
A preparation technique of chitosan microspheres immobilization lipase comprises the steps that: (1) lipase from candida SP is dissolved in phosphate buffer solution by taking chitosan microspheres as an immobilization carrier and adopting glutaraldehyde cross-linking, lipase A is obtained. (2) candida SP lipase solution of 0.70-0.80mg/mL prepared by phosphate buffer solution b with a pH value of 6.5-7.5 is added with immobilization lipase A (final concentration is 0.005-0.0055g/ml) and after being stirred, ethyl (3-dimethylamino propyl) carbodiimide hydrochloride solution is added, the mixture is then stirred to be fully immobilized, chitosan microspheres immobilization lipase B is obtained after the treatment of the mixture. The chitosan microspheres immobilization lipase prepared by the preparation technology of the invention improves apoenzyme combination rate and activity recovery, has powerful immobilization lipase activity and specific activity and the immobilization lipase can be recycled for six 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 13.8 U/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) prepare immobilized lipase A with glutaraldehyde as cross linker: lipase from candida sp is dissolved among the phosphate buffered saline buffer a with the chitosan microball is carrier, adopt glutaraldehyde cross-linking, is prepared into the immobilized lipase A of glutaraldehyde cross-linking;
(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 pH 7.2, 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 technology of a chitin microspheric immobilized lipase is characterized in that described technology carries out as follows:
(1) prepare immobilized lipase A with glutaraldehyde as cross linker: lipase from candida sp is dissolved among the phosphate buffered saline buffer a, is fixation support with the chitosan microball, adopts glutaraldehyde cross-linking, is prepared into the immobilized lipase A of glutaraldehyde cross-linking;
(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.
2. the preparation technology 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 chitin microspheric immobilized lipase A.
3. the preparation technology 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 technology 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, consider 0 ℃ of vacuum-drying of cake, obtain chitosan microball.
5. the preparation technology of chitin microspheric immobilized lipase as claimed in claim 1 or 2 is characterized in that described phosphate buffered saline buffer a 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
6. as the preparation technology 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 technology 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 technology of chitin microspheric immobilized lipase as described in the claim 2, it is characterized in that described preparation technology 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 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 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, 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.
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| CN102422848A (en) * | 2011-10-12 | 2012-04-25 | 江南大学 | Preparation method of immobilized glucose oxidase with muscle strengthening function in production process of bread |
| CN102533710A (en) * | 2011-12-31 | 2012-07-04 | 浙江工业大学 | Method for performing crosslinking immobilized modification on lipase Novozyme 435 by using glutaraldehyde |
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| CN102422848A (en) * | 2011-10-12 | 2012-04-25 | 江南大学 | Preparation method of immobilized glucose oxidase with muscle strengthening function in production process of bread |
| CN102533710A (en) * | 2011-12-31 | 2012-07-04 | 浙江工业大学 | Method for performing crosslinking immobilized modification on lipase Novozyme 435 by using glutaraldehyde |
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| 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 |
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| WO2022151828A1 (en) * | 2021-01-15 | 2022-07-21 | 百瑞全球有限公司 | Membrane-shaped immobilized cell, polypeptide, oligopeptide or protein, and preparation method therefor |
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