CN1285403A - Preparation of chitin microspheric immobilized papain - Google Patents
Preparation of chitin microspheric immobilized papain Download PDFInfo
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- CN1285403A CN1285403A CN 00117351 CN00117351A CN1285403A CN 1285403 A CN1285403 A CN 1285403A CN 00117351 CN00117351 CN 00117351 CN 00117351 A CN00117351 A CN 00117351A CN 1285403 A CN1285403 A CN 1285403A
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Abstract
The present invention uses chitosan as raw material to prepare chitosan microsphere, adds the chitosan microsphere to papain solution, and adopts the process of adsorption-crosslinking reaction so as to obtain chitosan microsphere immobilized enzyme. Said invented product features uniform form, large surface area and high enzyme activity recovery.
Description
Invention relates to biological technical field, relates in particular to the preparation of chitin microspheric immobilized papain.
Chitin has another name called chitin, chitin, and formal name used at school is poly--2-acetylaminohydroxyphenylarsonic acid 2-deoxidation-D-β-1,4-glucose.Chitin is very abundant in the occurring in nature resource, is mainly derived from the organisms such as arthropods, mollusk, annelid, protozoon, coelenterates, marine alga and fungi, and annual natural growing amount on earth just reaches more than billions of tons.Chitosan has another name called chitosan, soluble chitin, and formal name used at school is poly--2-amino-2-deoxidation-D-β-1,4-glucose, and it is a most important derivative in the chitin.In recent years, chitosan has caused a lot of scholars' attention as the research of fixation support.Because the amino in the chitosan molecule is very high to the avidity of range protein, can be effectively applied in the immobilization technology of enzyme.But as if being carrier immobilized enzyme with the tegument glycan, its major defect is: dissolve 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 it and uses the particularly application in reactor.Therefore present domestic and international research mainly concentrates on the preparation chitosan microball as fixed enzyme vector, this carrier has excellent property: have characteristics such as the acid-fast alkali-proof performance is good, the narrow distribution microspheroidal of strong, the single dispersion of absorption property, thereby having overcome above-mentioned defective, is the good carrier of enzyme immobilization.Papoid (Papain, EC 3.4.22.2) is a kind of thiol proteinase, be by papaya latex through refinement and a proteinoid lytic enzyme, be widely used in preventing that tenderization, the swelling agent of cake, the woolen of the cold muddiness of beer, meat is shrinkproof, day chemical industry such as preparation, toothpaste, cold cream and the washing composition of protein hydrolyzate and assist digestion medicine etc.In order to improve the service efficiency of papoid, reduce production costs, the researchist has carried out a lot of researchs to the immobilization of papoid both at home and abroad, has set up many process for fixation.But research mainly concentrates on nylon, bagasse fibre element, chitin or chitosan etc. as enzyme immobilization carrier, adopt absorption method or crosslinking immobilized papain, because the activity recovery generally lower (36%～51%) of the irregularity of support shapes, immobilized enzyme and immobilized enzyme have limited the application of immobilized papain to weak points such as the avidity of substrate are low.With the chitosan microball is fixed enzyme vector, does not see that with absorption-crosslinking fixed papain report is arranged.
The objective of the invention is to further develop the using value of the using value of chitosan, the application potential of widening this good enzyme immobilization carrier of chitosan microball, the service efficiency that improves papoid, the production cost that reduces immobilized papain, raising papoid and provide a class novel immobilized papain, be i.e. chitin microspheric immobilized papain.
The present invention is raw material with the chitosan, with acetum with chitosan dissolve fully the chitosan acidic sol, the ratio of chitosan and acetum is 2～5: 45～100 (weight and volume ratios), under agitation condition, the chitosan acidic sol is splashed in the dispersion medium turbine oil then, the volume ratio of chitosan acidic sol and turbine oil is 1: 2～5, after treating the colloidal sol homodisperse, splash into and be not less than 25% glutaraldehyde solution, the volume ratio of chitosan acidic sol and 25% glutaraldehyde solution is 20～30: 1, stir after 10～20 minutes, with NaOH solution its pH is transferred to 9～10 again, in 60～70 ℃ of water bath heat preservations 3～5 hours, after leaving standstill cooling, discard the upper strata oil reservoir, adding earlier sherwood oil (I) in the solid substance of lower floor dissolves each other with unnecessary turbine oil, discard the upper strata again, adding acetone then in lower floor's solid substance dissolves each other with the turbine oil that is left, discard the upper strata again, take out with dehydrated alcohol then that to wash solid substance clean fully to turbine oil, vacuum-drying, cross 60～100 mesh sieves, take out eccysis with distilled water and remove sherwood oil (I), acetone and dehydrated alcohol, and vacuum-drying, i.e. carrier one chitosan microball of being fixed enzyme.Then chitosan microball was soaked in the phosphoric acid buffer of pH7.2 12～24 hours, drain.It is in the papoid solution of 1～2.5mg/mL that chitosan microball after draining is added concentration, the ratio of chitosan microball and papoid solution is 0.5～1: 0.6～2 (weight and volume ratio), in 4～40 ℃ absorption 4～24 hours after, the glutaraldehyde solution of adding 0.3～1.5%, making the glutaraldehyde solution and the final volume ratio of total solution is 0.2～1.0: 100, crosslinked 1～5 hour in 4～40 ℃, with the unnecessary glutaraldehyde solution of the phosphoric acid buffer flush away of pH7.2 and blot, promptly make chitin microspheric immobilized papain again.
The characteristic contrast accompanying drawing of chitin microspheric immobilized papain is illustrated.Accompanying drawing 1 is the electromicroscopic photograph (338 *) of chitin microspheric immobilized papain, and photo shows that chitin microspheric immobilized papain is the reddish-brown microballoon of particle size range at 42～70 μ m.Accompanying drawing 2 is to the influence of enzyme amount to the enzyme immobilization effect, and this figure shows: in preparation process, if constant other condition, when every gram chitosan microball be 1.92mg to the enzyme amount time, the enzyme activity of immobilized enzyme reaches the 36.31U/g carrier, and the enzymatic activity recovery of immobilized enzyme reaches 65.35% for the highest; Accompanying drawing 3 is the influence of adsorption time to the enzyme immobilization effect, and as can be seen from the figure: when adsorption time reaches 12 hours, the enzyme activity of immobilized enzyme and enzymatic activity recovery all reach maximum value, are respectively 38.00U/g carrier and 65.70%; Accompanying drawing 4 is the influence of adsorption temp to the enzyme immobilization effect, and this figure shows: when adsorption temp was 4～8 ℃, the enzyme activity of immobilized enzyme and enzymatic activity recovery were respectively 36.73U/g carrier and 63.45%, all reached maximum value; Accompanying drawing 5 is the influence of glutaraldehyde concentration to the enzyme immobilization effect, and this figure shows: when the ultimate density of glutaraldehyde solution was 0.5%, the enzyme activity of immobilized enzyme and enzymatic activity recovery all reached maximum value, were respectively 38.58U/g carrier and 66.74%; Accompanying drawing 6 is the influence of crosslinking time to the enzyme immobilization effect, and this figure shows: when the glutaraldehyde cross-linking time is 3 hours, and the enzyme activity of immobilized enzyme and enzymatic activity recovery all reach maximum value, is respectively 38.90U/g carrier and 67.25%; Accompanying drawing 7 is the influence of crosslinking temperature to the enzyme immobilization effect, and this figure shows: when the glutaraldehyde cross-linking temperature was 4～8 ℃, the enzyme activity of immobilized enzyme and enzymatic activity recovery all reached maximum value, were respectively 36.73U/g carrier and 63.45%.In sum, prepare chitin microspheric immobilized papain under above optimal fixation condition, its enzyme activity is 36～39U/g carrier, on average reaches the 38.49U/g carrier, and enzymatic activity recovery is 62～68%, on average reaches 66.60%.Accompanying drawing 8 is the Michaelis-Menton constant of solution enzyme and immobilized enzyme, shows among the figure: the apparent Michaelis-Menton constant K of chitin microspheric immobilized papain
m App(casein) is 0.055%; Accompanying drawing 9 is the optimum pH of solution enzyme and immobilized enzyme, shows among the figure: the optimal pH of chitin microspheric immobilized papain is 8.0; The pH stability of accompanying drawing 10 solution enzymes and immobilized enzyme shows among the figure: the pH stability of chitin microspheric immobilized papain is about 7.0; Accompanying drawing 11 is the thermostability of solution enzyme and immobilized enzyme, shows among the figure: the thermostability of chitin microspheric immobilized papain is in 15～35 ℃ of scopes; The operation transformation period of chitin microspheric immobilized papain is 25d.
The present invention makes full use of the superperformance of this fixed enzyme vector of chitosan microball, big, the good adsorption performance of shape homogeneous, surface-area etc. particularly, with absorption-crosslinking immobilized papain, thereby provide the good premise condition for the invention chitin microspheric immobilized papain.Characteristics of the present invention: chitin microspheric immobilized papain shape homogeneous, surface-area is big; Enzymatic activity recovery on average reaches 66.6%; Compare with the solution enzyme, immobilized enzyme has improved 7 times with first-class to the avidity of substrate (casein).
Example:
The preparation of chitosan microball.Take by weighing the 4g chitosan and be dissolved in 2% the acetum, make 4% chitosan acidic sol.In beaker, add turbine oil, stir the chitosan acidic sol of slow adding 4% down, stirred 15 minutes, make colloidal sol drop homodisperse, splash into 25% glutaraldehyde solution 0.25mL then, stir after 10 minutes, its pH is transferred to 9～10, in 60～70 ℃ of water bath heat preservations 3 hours with the NaOH solution of 1mol/L, after leaving standstill cooling, discard the upper strata oil reservoir, use sherwood oil (I), acetone, dehydrated alcohol to take out and wash again successively, after the vacuum-drying, (80 order) sieves, take out with distilled water at last and wash, vacuum-drying, standby.
Chitosan microball was soaked 24 hours in the phosphoric acid buffer of 0.1mol/L pH7.2, drain.Take by weighing the 0.5g chitosan microball and place test tube, the papoid solution 0.6mL that adds 1.6mg/mL, in 4～8 ℃ of absorption 12 hours, and stir frequently, splash into 0.6% glutaraldehyde solution 3mL (the borate buffer preparation of 0.1mol/L pH8.0) then, make the ultimate density of glutaraldehyde solution reach 0.5%, in 4～8 ℃ after crosslinked 3 hours, with the unnecessary glutaraldehyde solution of the phosphoric acid buffer flush away of 0.1mol/L pH7.2, suck dry moisture promptly makes chitin microspheric immobilized papain.
Claims (7)
1. the preparation of chitin microspheric immobilized papain, it is characterized in that with the chitosan being raw material, with acetum with chitosan dissolve fully the chitosan acidic sol, under agitation condition, the chitosan acidic sol is splashed in the dispersion medium turbine oil then, after treating the colloidal sol homodisperse, splash into and be not less than 25% glutaraldehyde solution, stir after 10～20 minutes, with NaOH solution its pH is transferred to 9～10 again, in 60～70 ℃ of water bath heat preservations 3～5 hours, after leaving standstill cooling, discard the upper strata oil reservoir, adding earlier sherwood oil (I) in the solid substance of lower floor dissolves each other with unnecessary turbine oil, discard the upper strata, adding acetone then in lower floor's solid substance dissolves each other with the turbine oil that is left, discard the upper strata, take out with dehydrated alcohol then that to wash solid substance clean fully to turbine oil, vacuum-drying, cross 60～100 mesh sieves, take out eccysis with distilled water and remove sherwood oil (I), acetone and dehydrated alcohol, and vacuum-drying, i.e. carrier-the chitosan microball of being fixed enzyme, chitosan microball was soaked in the phosphoric acid buffer of pH7.2 12～24 hours, drain, it is in the papoid solution of 1～2.5mg/mL that chitosan microball after draining is added concentration, in 4～40 ℃ of absorption 4～24 hours, the glutaraldehyde solution of adding 0.3～1.5%, making the glutaraldehyde solution and the final volume ratio of total solution is 0.2～1.0: 100, crosslinked in 4～40 ℃, with the unnecessary glutaraldehyde solution of the phosphoric acid buffer flush away of pH7.2 and blot, promptly make chitin microspheric immobilized papain again.
2. according to the preparation of claims 1 said chitin microspheric immobilized papain, the ratio that it is characterized in that chitosan and acetum is that the ratio of chitosan and acetum is 2～5: 45～100 (weight and volume ratios).
3. according to the preparation of claims 1 said chitin microspheric immobilized papain, the ratio that it is characterized in that chitosan acidic sol and turbine oil is 1: 2～5 (volume ratios).
4. according to the preparation of claims 1 said chitin microspheric immobilized papain, it is characterized in that the chitosan acidic sol and the ratio of 25% the glutaraldehyde solution that splashes into are 20～30: 1 (volume ratio).
5. according to the preparation of claims 1 said chitin microspheric immobilized papain, it is characterized in that the ratio of chitosan microball and papoid solution is 0.5～1: 0.6～2 (weight and volume ratio).
6. according to the preparation of claims 1 said chitin microspheric immobilized papain, it is characterized in that when papoid was the 1.92mg/g chitosan microball to the enzyme amount, the enzyme activity of immobilized enzyme and the enzymatic activity recovery of immobilized enzyme were for the highest.
7. according to the preparation of claims 1 said chitin microspheric immobilized papain, it is characterized in that the enzyme activity of immobilized enzyme and enzymatic activity recovery all reach maximum value when the ultimate density of glutaraldehyde solution is 0.5%.
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| CN 00117351 CN1285403A (en) | 2000-08-11 | 2000-08-11 | Preparation of chitin microspheric immobilized papain |
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| CN 00117351 CN1285403A (en) | 2000-08-11 | 2000-08-11 | Preparation of chitin microspheric immobilized papain |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1320111C (en) * | 2004-08-09 | 2007-06-06 | 王美岭 | Process for preparing papain |
| CN1320124C (en) * | 2005-09-15 | 2007-06-06 | 武汉大学 | Preparation of low-molecular weight chitoglycan or chitooligose |
| CN1321175C (en) * | 2004-01-13 | 2007-06-13 | 中国科学院动物研究所 | Microcarrier for large-scale culture cell |
| CN101260397B (en) * | 2008-04-28 | 2010-07-07 | 浙江省农业科学院 | Application of immobilized proteinase in preparing cream protein peptide |
| CN101914514A (en) * | 2010-04-06 | 2010-12-15 | 福建师范大学 | Method for preparing immobilized alpha-amylase by using spherical tannin as carrier |
| CN102181422A (en) * | 2011-03-08 | 2011-09-14 | 天津大学 | Macropore carrier 'synchronization method' covalent crosslinking-immobilized papain polymer and method |
| CN102234376A (en) * | 2011-03-07 | 2011-11-09 | 江西中医学院 | Preparation and application of tubulin ligand affinity material |
| CN107254460A (en) * | 2017-06-23 | 2017-10-17 | 广东海洋大学 | A kind of carboxymethyl chitosan/sodium alginate composite sponge immobilized papain and its application |
| CN108740997A (en) * | 2018-07-25 | 2018-11-06 | 华中农业大学 | A kind of preparation method of protease chitosan microball |
| RU2712690C1 (en) * | 2019-03-07 | 2020-01-30 | федеральное государственное бюджетное образовательное учреждение высшего образования "Воронежский государственный университет" (ФГБОУ ВО "ВГУ") | Method of producing papain preparation in gel based on food chitosan and chitosan succinate |
| EP3755376A4 (en) * | 2018-02-23 | 2021-12-01 | Mucpharm Pty Ltd | Formulations containing mucin-affecting proteases |
-
2000
- 2000-08-11 CN CN 00117351 patent/CN1285403A/en active Pending
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1321175C (en) * | 2004-01-13 | 2007-06-13 | 中国科学院动物研究所 | Microcarrier for large-scale culture cell |
| CN1320111C (en) * | 2004-08-09 | 2007-06-06 | 王美岭 | Process for preparing papain |
| CN1320124C (en) * | 2005-09-15 | 2007-06-06 | 武汉大学 | Preparation of low-molecular weight chitoglycan or chitooligose |
| CN101260397B (en) * | 2008-04-28 | 2010-07-07 | 浙江省农业科学院 | Application of immobilized proteinase in preparing cream protein peptide |
| CN101914514A (en) * | 2010-04-06 | 2010-12-15 | 福建师范大学 | Method for preparing immobilized alpha-amylase by using spherical tannin as carrier |
| CN102234376A (en) * | 2011-03-07 | 2011-11-09 | 江西中医学院 | Preparation and application of tubulin ligand affinity material |
| CN102234376B (en) * | 2011-03-07 | 2013-01-02 | 江西中医学院 | Preparation and application of tubulin ligand affinity material |
| CN102181422A (en) * | 2011-03-08 | 2011-09-14 | 天津大学 | Macropore carrier 'synchronization method' covalent crosslinking-immobilized papain polymer and method |
| CN102181422B (en) * | 2011-03-08 | 2013-01-09 | 天津大学 | Macropore carrier 'synchronization method' covalent crosslinking-immobilized papain polymer and method |
| CN107254460A (en) * | 2017-06-23 | 2017-10-17 | 广东海洋大学 | A kind of carboxymethyl chitosan/sodium alginate composite sponge immobilized papain and its application |
| CN107254460B (en) * | 2017-06-23 | 2021-01-12 | 广东海洋大学 | Carboxymethyl chitosan/sodium alginate composite sponge immobilized papain and application thereof |
| EP3755376A4 (en) * | 2018-02-23 | 2021-12-01 | Mucpharm Pty Ltd | Formulations containing mucin-affecting proteases |
| CN108740997A (en) * | 2018-07-25 | 2018-11-06 | 华中农业大学 | A kind of preparation method of protease chitosan microball |
| CN108740997B (en) * | 2018-07-25 | 2021-11-23 | 华中农业大学 | Preparation method of protease chitosan microspheres |
| RU2712690C1 (en) * | 2019-03-07 | 2020-01-30 | федеральное государственное бюджетное образовательное учреждение высшего образования "Воронежский государственный университет" (ФГБОУ ВО "ВГУ") | Method of producing papain preparation in gel based on food chitosan and chitosan succinate |
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