CN103205414B - Preparation method of glass immobilized enzyme with biological activity - Google Patents
Preparation method of glass immobilized enzyme with biological activity Download PDFInfo
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- CN103205414B CN103205414B CN201310145212.4A CN201310145212A CN103205414B CN 103205414 B CN103205414 B CN 103205414B CN 201310145212 A CN201310145212 A CN 201310145212A CN 103205414 B CN103205414 B CN 103205414B
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Abstract
The invention relates to a preparation method of glass immobilized enzyme with a biological activity. The method comprises the following steps of: preparing a microbial emulsion by lactobacillus salivarius and an MRS culture medium; mixing the microbial emulsion, a silicon source, a phosphorus source, calcium salt and MO salt to sol; hydrothermally aging to form gel; drying to obtain xerogel; thermally treating to obtain a porous glass carrier material with biological activity. Enzyme in the duct is directly bonded and fixed with surface by the characteristics of biological activity and compatibility of the porous glass carrier material with biological activity, excellent surface chemical performance, adjustable multi-pore structure and enzyme molecular size matched so as to obtain a porous glass immobilized enzyme biological catalyst with biological activity. The method has the characteristics of high enzyme load, high enzyme activity, good thermal and chemical stabilities, repeated use, no pollution to products and the like, and can be used to the fields of fine chemical industry, food industry, biological chemicals, wastewater treatment, medicines and the like.
Description
(1) technical field
The preparation method who the present invention relates to a kind of bioactivity glass immobilized enzyme, belongs to technical field of biological materials.
(2) background technology
Enzyme immobilization new technology is in fine chemicals industry, foodstuffs industry, chemical analysis, wastewater treatment, and especially the aspect such as chipal compounds, biosensor and medicine has broad application prospects.The solid support material that enzyme immobilization is used is natural macromolecular material at first, such as resin, gel and fiber etc., but the shortcomings such as these materials have that reusability is low, cleaning difficulty, physical strength are low, thermostability and poor chemical stability, developed into afterwards and adopted inorganic materials and matrix material etc., particularly conventionally to have enzyme charge capacity high for porous inorganic carrier material, enzyme immobilization initial activity is higher, thermostability and chemical stability are good, physical strength is high, cheap, the advantage such as reusable, thereby receive much attention, for example, Luo Wen etc. be take Bio-Glas as carrier, adopt covalent method to fix lipase, contrasted the optimal reactive temperature of immobilized enzyme and resolvase, pH value and thermostability, and take prepared immobilized enzyme as catalyzer, in micro-aqueous systems, utilize rapeseed oil biodiesel synthesis, investigated quantity of solvent, system water-content, the impact of the factors such as methyl alcohol on immobilized enzyme catalysis performance, studied the operational stability of immobilized enzyme, the use split-phase methods such as Wang Baokang and completion method have been made two kinds of porous glass beads carriers, by physisorphtion, e and diazonium method, fixed α-amylase respectively, to their fixed effect relatively after, chosen e and carried out more detailed research, determined the character of best rigid condition, best applications condition and the immobilized enzyme of covalent coupling α-amylase, and studied porous glass beads manufacturing conditions on the impact of immobilized enzyme and porous glass beads the immobilization effect to catalase and L-Glutamic decarboxylase, patent document CN101451133B (200910044820.X) discloses a kind of preparation method who uses the immobilized enzyme biocatalyst of silicon-based mesoporous molecular sieve SBA-15, the method be take silicon-based mesoporous molecular sieve SBA-15 as carrier, horseradish peroxidase is fixed object, absorption method is process for fixation, the characteristic of utilizing the molecular dimension of the attribute of mesopore molecular sieve and the aperture of SBA-15 and horseradish peroxidase to match, realizes enzyme immobilization in the duct of silicon-based mesoporous molecular sieve SBA-15 and outside surface, patent document CN101423829A (200810160777.9) discloses adding calcium fly ash micropore glass carrier of a kind of immobilized enzyme and preparation method thereof, take polyurethane foamed material as matrix, dipping adding calcium fly ash slurry, after oven dry, heat-treat the adding calcium fly ash porous glass matrix that being fixed enzyme is used.
Above-mentioned document utilization porous silicate glass or borosilicate glass or the inorganic materials such as siliceous, need carry enzyme immobilization by finishing, although there are the advantages such as recyclablely reuse, production cost is low, but because of these material biological activitys and consistency poor, carrier structure is the defect such as easy-regulating, bonding enzyme ability not, make its enzymic activity and enzyme catalysis Efficiency Decreasing, and the easy desorption of enzyme, leakage and inactivation; In addition, finishing easily produces and pollutes or toxic side effect product with material, also has a strong impact on its catalytic performance and application.
(3) summary of the invention
In order to solve the problem of inorganic carrier material immobilized enzyme prior art existence, the invention provides a kind of preparation method of bioactivity glass immobilized enzyme, be a kind of based on SiO
2-CaO-P
2o
5the bioactivity glass that-MO (M is divalence Zn, Mg or trivalent Fe) forms system carries out the direct immobilization technology of enzyme.
Technical scheme of the present invention is as follows:
A preparation method for bioactivity glass immobilized enzyme, step is as follows:
(1) prepare microorganism emulsion
1～5g lactobacillus salivarius microorganism powder is dissolved in the MRS culture medium solution of 50～100ml, under the condition that is 4～7, cultivates emulsification 12～24 hours at 32 ℃～58 ℃, pH value, making lactic-acid bacteria cells concentration in solution is 7 * 10
6individual/ml～10 * 10
6individual/ml, forms emulsion A.
(2) prepare bioactivity glass precursor sol
According to SiO
2-CaO-P
2o
5-MO system biological activity glass chemical constitution molar percentage: 50%SiO
2, 4%P
2o
5(46-x) %CaO, x%MO(M is divalence Zn, Mg or trivalent Fe), x=5～15%, in proportion the precursor material tetraethoxy of silicon source, phosphorus source, calcium source and MO, triethyl phosphate, calcium salt, M metal-salt are added to respectively in order in the emulsion A that step (1) makes, tetraethoxy is fully hydrolyzed, forms bioactivity glass precursor sol B.
(3) sol B of step (2) is packed in the container of sealing in 60～80oC hydrothermal aging, 12～72h, obtain wet gel, then wet gel is placed in to 60～100 ℃ of dry 12～24h, obtain xerogel.
(4) xerogel is ground after in 600～800oC thermal treatment, 2～5h, obtain porous bioglass solid support material.
(5) get and treat immobilized enzyme, by the HAc-NaAc buffer preparation of pH4.5, become the enzyme solution of 0.5～2mg/ml, the enzyme solution of getting 1～5ml joins in the bioactivity glass carrier of 50～100mg of step (4), under the constant temperature of 25oC, vibrate 2～12 hours, then under 4oC, standing 10～18h carries out enzyme immobilization.
(6) take out enzyme immobilization sample, use refrigerated centrifuge centrifugation, obtain porous bioglass immobilized enzyme.
Porous bioglass immobilized enzyme prepared by the present invention is the biological catalyst with excellent properties, in the HAc-NaAc buffered soln that porous bioglass immobilized enzyme need be 4.5 in pH value, store for future use under 4oC.
According to the present invention, preferred, described in step (1), lactobacillus salivarius is CCTCC NO:M2010374.
MRS culture medium solution in step (1) is conventional lactic acid bacteria culture solution, consists of:
10.0 grams of casein peptones, 10.0 grams of beef leaching things, 5.0 grams of yeast extracts, 5.0 grams of glucose, 5.0 grams of sodium acetates, 2.0 grams of citric acid diamines, 1.0 grams of tween-80s, 2.0 grams of dipotassium hydrogen phosphates, 0.2 gram of magnesium sulfate heptahydrate, seven 0.05 gram of water manganous sulfate, 20.0 grams, calcium carbonate, 20.0 grams, agar, 1.0 liters of distilled water, pH6.8.
According to the present invention, preferred, SiO described in step (2)
2-CaO-P
2o
5-MO system biological activity glass chemical constitution: 50mol SiO
2, 4mol P
2o
5, 31～41mol CaO, 5～15mol MO(M is divalence Zn, Mg or trivalent Fe); In proportion by precursor material tetraethoxy 11.2ml, triethyl phosphate 1.36ml, calcium metal-salt 7g～10g, M metal-salt 1g～12g add to respectively in emulsion A in order, and tetraethoxy is fully hydrolyzed, and form bioactivity glass precursor sol B.
According to the present invention, preferred, described in step (2), calcium salt is selected from four water-calcium nitrate, and M metal-salt is selected from zinc nitrate hexahydrate, magnesium nitrate hexahydrate or nine water iron nitrates.
According to the present invention, preferred, described in step (5), enzyme is selected from glucose oxidase, amylase, trypsinase, lipase.Described in further preferred step (5), enzyme is selected from β-D-Glucose oxydo-reductase, α-amylase, lipase LVK-H100 or trypsinase EC3.4.21.4.
Technical characterstic of the present invention and excellent results:
The present invention is based on a kind of new year enzyme mechanism, biotechnology is combined with chemical synthesis, it is microorganism-sol-gel method, precursor material tetraethoxy, triethyl phosphate, metal-salt are mixed with lactobacillus salivarius cell solution, due to the rapping plate effect in tetraethoxy sol-gel process of lactobacillus salivarius cell, so, make sol particle more easily adsorb and deposit on lactobacillus salivarius cell.After thermal treatment, form and there is the porous bioglass solid support material of high surface curvature macropore (100～200nm) and mesoporous (2～50nm), thereby realize the bionical synthetic of porous bioglass.Lactobacillus salivarius cell is in bionical building-up process, by controlling MRS nutrient solution culture temperature and incubation time, regulate size, concentration and the shape of lactobacillus salivarius cell, not only played and formed high surface curvature macroporous structure hard template effect and make pore structure adjustable, and played the effect of formation meso-hole structure soft template, make the vesicular structure of bioactivity glass match and to prevent enzyme Molecular leak with enzyme molecular dimension, can make again substrate and product molecular energy free diffusing, thereby improve year enzyme immobilization performance of porous bioglass.Simultaneously, metal ion in bioactivity glass network structure has biological activity and consistency, the catalysis stabilization that enzyme molecule is had to the effect of combining closely and cofactor, be suitable for the Direct Bonding immobilization of enzyme molecule and carrier, do not need complicated finishing and modification, thereby improve linkage force, activity, the catalytic performance of enzyme and reuse number of times, significantly reducing costs, and can avoid product contamination and produce toxic side effect.
The present invention compared with the prior art, adopts microorganism-collosol and gel synthesis technique to prepare porous bioglass solid support material, utilizes bioactivity glass to have good biological activity and biocompatibility, and makes it have vesicular structure and pore structure is adjustable; Except having that inorganic materials carrier is former and having superiority, also there is metal ion as the cofactor of enzyme, can be directly and enzyme molecule immobilization and do not need complicated finishing and modification and the advantage such as have no side effect.This is a kind of good immobilized enzyme biocatalyst that has using value, and the using value to further raising enzyme immobilization technology, reduces costs, and promotes the field quality producies such as enzyme technology and biochemical industry and production efficiency and has very important significance.The present invention can be applicable to fine chemicals industry, foodstuffs industry, biochemical industry, wastewater treatment, medicine and other fields.
(4) accompanying drawing explanation
Fig. 1 is the synthetic TEM figure with macropore and mesoporous porous bioglass solid support material of embodiment 1;
Fig. 2 is synthetic nitrogen adsorption desorption isothermal curve (b) and the pore size distribution curve (c) with macropore and mesoporous porous bioglass solid support material of embodiment 1.
(5) embodiment
Below in conjunction with embodiment, the present invention will be further described, but be not limited to this.
In embodiment, lactobacillus salivarius used is CCTCC NO:M2010374; Glucose oxidase used is β-D-Glucose oxydo-reductase (EC1.1.3.4); Amylase is α-amylase (EC3.2.1.1.); Lipase is LVK-H100; Trypsinase is EC3.4.21.4, is commercial product.
Embodiment 1
Get lactobacillus salivarius 3g and be dissolved in 80ml MRS culture medium solution, under the condition that is 6, cultivate emulsification 17 hours 42 ℃ and pH value, in control solution, cell concn is 7.9 * 10
6individual/ml is emulsion A.
Be 50mol SiO in molar ratio
2-4mol P
2o
5the bioactivity glass of-31mol CaO-15mol ZnO forms, by precursor material tetraethoxy 11.2ml, triethyl phosphate 1.36ml, four water-calcium nitrate 7.32g, zinc nitrate hexahydrate 4.46g adds to respectively in emulsion A in order, often add a kind of raw material midfeather and stir 30min, form precursor sol solution B.
Precursor sol solution B is packed in the container of sealing in 70oC hydrothermal aging 48h, obtain wet gel, then, in 80 ℃ of dry 18h, obtain xerogel; After being ground, in 700oC thermal treatment 3h, obtain bioactivity glass solid support material, white powder; This material has vesicular structure, and macropore diameter is that 100～200nm(is shown in Fig. 1), mesoporous aperture is 2～20nm, sees Fig. 2 (b), (c).To treat that HAc-NaAc buffer preparation that immobilized glucose oxidase is 4.5 by pH value becomes the enzyme solution of 1mg/ml, then the enzyme solution of getting 3ml joins in the bioactivity glass carrier of 80mg, under the constant temperature of 25oC, vibrate 7 hours, then under 4oC, standing 14h carries out enzyme immobilization, obtains porous bioglass immobilized enzyme biocatalyst after centrifugation.After tested, the activity of its immobilized enzyme is 352U g
-1, its optimal reaction temperature is 50 ℃, specific ionization enzyme has improved 10 ℃, reuses after 7 times and still can retain 40% of former activity.
Embodiment 2
Get lactobacillus salivarius 1g and be dissolved in 50mlMRS culture medium solution, under the condition that is 7, cultivate emulsification 12 hours 32 ℃ and pH value, in control solution, cell concn is 7.0 * 10
6individual/ml is emulsion A.
Be 50mol SiO in molar ratio
2-4mol P
2o
5the bioactivity glass of-31mol CaO-15mol ZnO forms, by precursor material tetraethoxy 11.2ml, triethyl phosphate 1.36ml, four water-calcium nitrate 7.32g, zinc nitrate hexahydrate 4.46g adds to respectively in emulsion A in order, often add a kind of raw material midfeather and stir 30min, form precursor sol solution B.
Precursor sol solution B is packed in the container of sealing in 60oC hydrothermal aging 72h, obtain wet gel, then, in 60 ℃ of dry 24h, obtain xerogel; After being ground, in 600oC thermal treatment 5h, obtain bioactivity glass white powder; This powder is vesicular structure, and macropore diameter is 80～150nm, and mesoporous aperture is 4～25nm.To treat that HAc-NaAc buffer preparation that immobilized glucose oxidase is 4.5 by pH value becomes the enzyme solution of 0.5mg/ml, then the enzyme solution of getting 1ml joins in the bioactivity glass carrier of 50mg, under the constant temperature of 25oC, vibrate 2 hours, then be placed in standing 18h under 4oC and carry out enzyme immobilization, after centrifugation, obtain porous bioglass immobilized enzyme biocatalyst.After tested, the activity of its immobilized enzyme is 341U g
-1, its optimal reaction temperature is 48 ℃, specific ionization enzyme has improved 12 ℃, reuses after 7 times and still can retain 45% of former activity.
Embodiment 3
Get lactobacillus salivarius 5g and be dissolved in 100mlMRS culture medium solution, under the condition that is 4, cultivate emulsification 24 hours 58 ℃ and pH value, in control solution, cell concn is 10.0 * 10
6individual/ml is emulsion A.
Be 50mol SiO in molar ratio
2-4mol P
2o
5the bioactivity glass of-31mol CaO-15mol ZnO forms, by precursor material tetraethoxy 11.2ml, triethyl phosphate 1.36ml, four water-calcium nitrate 7.32g, zinc nitrate hexahydrate 4.46g adds to respectively in emulsion A in order, often add a kind of raw material midfeather and stir 30min, form precursor sol solution B.
Precursor sol solution B is packed in the container of sealing in 80oC hydrothermal aging 12h, obtain wet gel, then, in 100 ℃ of dry 12h, obtain xerogel; After being ground, in 800oC thermal treatment 2h, obtain bioactivity glass white powder; This powder is vesicular structure, and macropore diameter is 120～240nm, and mesoporous aperture is 5～30nm.To treat that HAc-NaAc buffer preparation that immobilized glucose oxidase is 4.5 by pH value becomes the enzyme solution of 2mg/ml, then the enzyme solution of getting 5ml joins in the bioactivity glass carrier of 100mg, under the constant temperature of 25oC, vibrate 12 hours, then be placed in standing 10h under 4oC and carry out enzyme immobilization, after centrifugation, obtain porous bioglass immobilized enzyme biocatalyst.After tested, the activity of its immobilized enzyme is 368U g
-1, its optimal reaction temperature is 52 ℃, specific ionization enzyme has improved 8 ℃, reuses after 7 times and still can retain 50% of former activity.
Embodiment 4
As described in Example 1, difference is to be 50mol SiO in molar ratio
2-4mol P
2o
5the bioactivity glass of-41mol CaO-5molMgO forms, and precursor material tetraethoxy, triethyl phosphate, four water-calcium nitrate, magnesium nitrate hexahydrate are added to respectively in emulsion A in order, and other conditions are with embodiment 1.The porous bioglass white powder obtaining, its macropore diameter is 80～260nm, mesoporous aperture is 2～12nm.The activity of its immobilized enzyme is 209U g
-1, its optimal reaction temperature is 55 ℃, specific ionization enzyme has improved 15 ℃, reuses after 7 times and still can retain 50% of former activity.
Embodiment 5
As described in Example 1, difference is to be 50molSiO in molar ratio
2-4mol P
2o
5-31mol CaO-10Fe
2o
3bioactivity glass form, precursor material tetraethoxy, triethyl phosphate, four water-calcium nitrate, nine water iron nitrates are added to respectively in emulsion A in order, other conditions are with embodiment 1.The porous bioglass white powder obtaining, its macropore diameter is 60～135nm, mesoporous aperture is 2～9nm.The activity of its immobilized enzyme is 186U g
-1, its optimal reaction temperature is 50 ℃, specific ionization enzyme has improved 10 ℃, reuses after 8 times and still can retain 40% of former activity.
Embodiment 6
As described in Example 1, difference is to treat that immobilized glucose oxidase is replaced by α-amylase, and other conditions are with embodiment 1.The activity of its immobilized enzyme is 359U g
-1, its optimal reaction temperature is 60 ℃, specific ionization enzyme has improved 12 ℃, reuses after 9 times and still can retain 54% of former activity.
Embodiment 7
As described in Example 1, difference is to treat that immobilized glucose oxidase is replaced by lipase, and other conditions are with embodiment 1.The activity of its immobilized enzyme is 388U g
-1, its optimal reaction temperature is 40 ℃, specific ionization enzyme has improved 8 ℃, reuses after 10 times and can retain 84% of former activity.
Embodiment 8
As described in Example 1, difference is to treat that immobilized glucose oxidase is replaced by trypsinase, and other conditions are with embodiment 1.The activity of its immobilized enzyme is 14.8U mg
-1, its optimal reaction temperature is 35 ℃, specific ionization enzyme has improved 5 ℃, reuses 8 times and still can retain 40% of former activity.
Claims (3)
1. a preparation method for bioactivity glass immobilized enzyme, step is as follows:
(1) prepare microorganism emulsion
1～5g lactobacillus salivarius microorganism powder is dissolved in the MRS culture medium solution of 50～100ml, under the condition that is 4～7, cultivates emulsification 12～24 hours at 32 ℃～58 ℃, pH value, making lactic-acid bacteria cells concentration in solution is 7 * 10
6individual/ml～10 * 10
6individual/ml, forms emulsion A;
(2) prepare bioactivity glass precursor sol
According to SiO
2-CaO-P
2o
5-MO system biological activity glass chemical constitution molar percentage: 50% SiO
2, 4% P
2o
5, (46-x) %CaO, x%MO, M is divalence Zn or Mg, x=5～15 %; In proportion the precursor material tetraethoxy of silicon source, phosphorus source, calcium source and MO, triethyl phosphate, calcium salt, M metal-salt are added to respectively in order in the emulsion A that step (1) makes, tetraethoxy is fully hydrolyzed, forms bioactivity glass precursor sol B;
Described tetraethoxy dosage is 11.2ml;
Described calcium salt is selected from four water-calcium nitrate, and M metal-salt is selected from zinc nitrate hexahydrate or magnesium nitrate hexahydrate;
(3) sol B of step (2) is packed in the container of sealing in 60～80 ℃ of hydrothermal aging 12～72 h, obtain wet gel, then wet gel is placed in to 60～100 ℃ of dry 12～24 h, obtain xerogel;
(4) xerogel is ground after in 600～800 ℃ of thermal treatment 2～5h, obtain porous bioglass solid support material;
(5) get and treat immobilized enzyme, by the HAc-NaAc buffer preparation of pH 4.5, become the enzyme solution of 0.5～2 mg/ml, the enzyme solution of getting 1～5 ml joins in the bioactivity glass carrier of 50～100mg of step (4), under the constant temperature of 25 ℃, vibrate 2～12 hours, then at 4 ℃, standing 10～18h carries out enzyme immobilization;
(6) take out enzyme immobilization sample, use refrigerated centrifuge centrifugation, obtain porous bioglass immobilized enzyme.
2. the preparation method of bioactivity glass immobilized enzyme according to claim 1, is characterized in that described in step (1), lactobacillus salivarius is CCTCC NO:M2010374.
3. the preparation method of bioactivity glass immobilized enzyme according to claim 1, is characterized in that described in step (5), enzyme is selected from glucose oxidase, amylase, trypsinase, lipase.
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