CN103421762A - Immobilized enzyme and preparation method thereof - Google Patents
Immobilized enzyme and preparation method thereof Download PDFInfo
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- CN103421762A CN103421762A CN2012101597966A CN201210159796A CN103421762A CN 103421762 A CN103421762 A CN 103421762A CN 2012101597966 A CN2012101597966 A CN 2012101597966A CN 201210159796 A CN201210159796 A CN 201210159796A CN 103421762 A CN103421762 A CN 103421762A
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Abstract
The invention discloses an immobilized enzyme and a preparation method thereof. The preparation method comprises following steps: (1) contacting a carbon material having an adsorption function with an enzyme-contained liquid medium to perform adsorption, thus obtaining a enzyme-adsorbed carbon material, namely an enzyme-carbon material; (2) contacting the enzyme-carbon material with a gelatin-contained liquid medium to form an enzyme-carbon material coated with gelatin; and (3) crosslinking the gelatin in the enzyme-carbon material coated with the gelatin by utilization of a crosslinking agent to obtain a gelatin-coated carbon material immobilized enzyme. By utilization of the preparation method, the activity recovery rate of the enzyme can be increased. The heat stability and operation stability of the immobilized enzyme are improved.
Description
Technical field
The present invention relates to a kind of immobilized enzyme and preparation method thereof, especially improved immobilized enzyme of stability and preparation method thereof.
Background technology
Enzyme can be used as a kind of catalyzer, and enzymic catalytic reaction has high efficiency, specificity, selectivity, reaction conditions temperature and the characteristics such as pollution-free.Yet, because enzyme itself is a kind of protein, it is subject to the impact of reaction conditions and causes its inactivation when unbound state.For example, under the condition of strong acid, highly basic, high ion concentration, high temperature, low temperature or organic solvent, sex change may occur in enzyme, thereby causes its inactivation.At present, enzyme during as catalyzer, because its utilization ratio is low, cost is high, has therefore limited its application in industrial circle greatly.
CA 2277371 discloses a kind of method of enzyme immobilization: the tripoli upholder that will have surface hydroxyl is incubated with first aqueous solution containing many aldehyde, makes subsequently to contact and finally remove upholder containing second aqueous solution of enzyme with the carrier after modification from solution.
EP 133531 has described a kind of method of enzyme immobilization: (a) introduce the aqueous medium that comprises enzyme and polymine and (b) glutaraldehyde and chitosan added to aqueous medium and shift out subsequently cross-linking products from liquid medium.
US 4,888, and 285 disclose a kind of enzyme immobilization method: by organic solvent with the aminosilane derivatives reaction by the silica gel modification, then under glutaraldehyde, tannic acid and chitosan exist, the amination upholder obtained is connected with enzyme.
Above method all easily causes the inactivation of enzyme, and the activity recovery of enzyme is lower.
Select suitable solid support material by being fixed of enzyme with being fixed enzyme, thereby can realize the sharp separation of enzyme and reuse, and can strengthen the stability of enzyme by the microenvironment of controlling immobilized enzyme.For example, the Chinese patent application that application number is 02117614.0 discloses a kind of method of fixed lipase catalyzed synthesis of fatty acid low carbon alcohol ester, step wherein comprises: fixation support inert support film or yarn fabric are first with fixing agent activation altogether, fixation support and common fixing agent are mixed to (W: V) in the mass volume ratio ratio of 1: 1～1: 3, room temperature is dried, lipase solution is mixed with the fixation support after activation in the ratio of 1000～30000 units/gram carriers, dries stand-by.But, because the adsorptive power of carrier is lower, and being coated, the stability of above-mentioned immobilized enzyme is still good not.
At present, the solid support material of immobilized enzyme (for example catalase) is mainly and comprises organic materials and inorganic materials.Organic materials comprises chitosan, Mierocrystalline cellulose, modified biological material etc.; Inorganic materials comprises aluminum oxide, aluminium hydroxide etc.Porous material, owing to having balanced and adjustable aperture, high-specific surface area and the characteristics such as porosity and channel architecture, is considered to the desirable solid support material of immobilized enzyme.Carbon material is representative porous material, and it has very high specific surface area and porosity, thereby possesses stronger adsorptive power; In addition, this carbon material does not have toxicity, physical strength good, cheap.Therefore, carbon material is the sorbing material that immobilized enzyme is good.Document (Qi Wang for example, et al.Enzyme and Microbial Technology, 49 (2011), 11 – 16) a kind of method of immobilized enzyme is disclosed, take carbon nanotube as the carrier adsorption papoid, adopt bionical silicification at carbon nanotube-papoid bioconjugates Coated by Silica thin layer, thus being fixed papoid.It is compared with resolvase and uncoated carbon nanotube-papoid bioconjugates, has demonstrated higher activity and stability in strict pH, temperature environment.But repeat catalyzed reaction 7 times, its vigor only keeps 70% of original vigor, apart from industrial applications, also has a certain distance.Therefore, although carbon material is stronger to the adsorptivity of enzyme molecule, met the demand of high adsorption capacity, still there is the bottleneck of high-leakage rate and immobilized enzyme less stable in the method for simple physical adsorption.
Summary of the invention
In order to overcome the defect of prior art, the present inventor has carried out further investigation repeatedly.
One object of the present invention is to provide a kind of preparation method of immobilized enzyme, and it can improve the activity recovery of enzyme, and improves the thermostability of enzyme.Further, preparation method of the present invention can also improve the operational stability of enzyme.
Another object of the present invention is to provide a kind of immobilized enzyme, and its thermostability improves.Further, immobilized enzyme of the present invention can also improve the operational stability of enzyme.
The present inventor is surprisingly found out that following technical scheme can realize above-mentioned purpose.
A kind of preparation method of immobilized enzyme, it comprises the steps:
(1) will there is adsorbing carbon material and contact to be adsorbed with the liquid medium that contains enzyme, thereby obtain the carbon material that absorption has enzyme, i.e. enzyme-carbon material;
(2) this enzyme-carbon material is contacted with the liquid medium containing gelatin, coat the enzyme-carbon material of gelatin to form surface;
(3) by linking agent, surface is coated gelatin in the enzyme-carbon material of gelatin carry out crosslinked, to obtain gelatin carbon coated material immobilized enzyme.
According to the preparation method of immobilized enzyme of the present invention, preferably, specifically comprise the steps:
(1) will have adsorbing carbon material be placed in contain enzyme liquid medium to be adsorbed, remove supernatant liquor, thereby obtain absorption, the carbon material of enzyme, i.e. enzyme-carbon material are arranged;
(2) this enzyme-carbon material is mixed to form mixed solution with the liquid medium containing gelatin, and make enzyme-carbon material that surface coats gelatin;
(3) above-mentioned mixed solution is mixed to carry out crosslinking reaction with linking agent;
(4) the mixture lyophilize optionally, reaction obtained.
According to the preparation method of immobilized enzyme of the present invention, preferably, the adsorption temp of step (1) is 0 ° of C ~ 25 ° C, and adsorption time is 0.5 ~ 4h.
According to the preparation method of immobilized enzyme of the present invention, preferably, its of meeting the following conditions, two or three:
Condition 1: in step (1), enzyme is 5 ~ 200mg/g with the mass ratio with adsorbing carbon material, and in unit " mg/g ", " mg " is the mass fraction with adsorbing carbon material for the mass fraction of enzyme, denominator " g ";
Condition 2: in step (2), the mass percentage concentration that contains gelatin in the liquid medium of gelatin is 1wt% ~ 5wt%;
Condition 3: in step (3), linking agent is used with the form of the liquid medium containing linking agent, and the concentration expressed in percentage by volume that contains linking agent in the liquid medium of linking agent is 0.2vt% ~ 1.0vt%.
According to the preparation method of immobilized enzyme of the present invention, preferably,
A, describedly there is adsorbing carbon material and comprise carbon nanotube, gac or surface modification carbon material; And/or
B, described enzyme comprise catalase and/or glucose oxidase.
According to the preparation method of immobilized enzyme of the present invention, preferably,
In step (1), adsorption temp is 0 ~ 10 ° of C, and adsorption time is 0.3 ~ 6h, and enzyme is 50 ~ 100mg/g with having adsorbing carbon material mass ratio; With
In step (2), the mass percentage concentration that contains gelatin in the liquid medium of gelatin is 1 ~ 3wt%; With
In step (3), linking agent is used with the form of the liquid medium containing linking agent, and the concentration expressed in percentage by volume that contains linking agent in the liquid medium of linking agent is 0.5 ~ 0.8vt%.
A kind of immobilized enzyme, it prepares by above-mentioned preparation method.
A kind of immobilized enzyme, it has following structure:
The core part, it comprises that absorption has the carbon material of enzyme; With
Shell, it covers on the surface of described core part, comprises crosslinked gelatin coating.
According to immobilized enzyme of the present invention, preferably,
A, described carbon material comprise carbon nanotube, gac or surface modification carbon material; And/or
B, described enzyme comprise catalase and/or glucose oxidase.
According to immobilized enzyme of the present invention, preferably, the thickness of shell is 0.1～100 μ m.
Gelatin possesses good sol-gel character, can realize the effective embedding to the enzyme molecule, still, the immobilized enzyme that adopts traditional sol-gel method to prepare, its activity recovery is low and mechanical property is poor.Have adsorbing carbon material and can effectively improve the charge capacity of enzyme as the absorption carrier of enzyme, again because carbon material has the characteristics such as specific surface area is large, thereby the catalyzed reaction efficiency that can improve enzyme improves the activity recovery of enzyme.Yet immobilized enzyme prepared by the carbon material absorption method exists the enzyme leakage rate high, the defects such as poor stability have limited in industrial application.The present invention selects to have adsorbing carbon material and has effectively improved the charge capacity of enzyme as the absorption carrier of enzyme, and improved the activity recovery of enzyme, adopt gelatin to be coated at the carbon material surface that has adsorbed enzyme as coating material simultaneously, and utilize linking agent to carry out chemically crosslinked, thereby realize the effective coating to enzyme.Therefore, preparation method of the present invention has realized the dual immobilization of absorption, embedding to enzyme, has improved the activity recovery of enzyme, and has improved the stability of immobilized enzyme.Immobilized enzyme of the present invention is under the synergy of carbon material and gelatin, and its thermostability and operational stability are improved.
In addition, preparation method's cost of the present invention is lower and be easy to realize suitability for industrialized production.
The accompanying drawing explanation
Fig. 1 is the SEM figure of the immobilized enzyme of embodiment 1 preparation.
Fig. 2 is the immobilized enzyme thermostability curve of embodiment 1 preparation.
Fig. 3 is the immobilized enzyme stable operation linearity curve of embodiment 1 preparation.
Embodiment
Below in conjunction with accompanying drawing and specific embodiment, the present invention is further illustrated, but protection scope of the present invention is not limited to this.
In the present invention, there is adsorbing carbon material and be preferably porous carbon materials.Described " porous " refers in carbon material the hole pattern that comprises connection, and this hole pattern can be opening, clearance space or other passages.As preferably, whole volumes of porous carbon materials all comprise the hole pattern of connection.
In the present invention, so-called " granularity " or " size " refer to the maximum length of material cross-section.So-called " having adsorption " refers to that carbon material has and is adsorbed on carbon material by enzyme and by the ability of enzyme immobilization.Term " on " be illustrated in wherein or in the above unless specifically stated otherwise.
In the present invention, the vigor of enzyme, also referred to as the activity of enzyme, refers to the ability of the certain chemical reaction of enzyme catalysis.The size of enzyme activity is under certain condition available, and the speed of a certain chemical reaction of enzyme catalysis means.The vigor of enzyme can mean by U, and under certain condition, transforming the required enzyme amount of 1 micromole's substrate in 1 minute is a unit of activity (U).Usually, temperature is 25 degree, the optimum condition that other condition negates are answered.The vigor of enzyme also can adopt than vigor and mean, i.e. every milligram of enzyme activity that zymoprotein has, and unit is U/mg.The higher enzyme of specific activity is purer.
<immobilized enzyme >
Gelatin carbon coated material immobilized enzyme of the present invention (being designated hereinafter simply as immobilized enzyme) comprises core part and shell.Shell covers on the surface of core part.Core of the present invention partly comprises that absorption has the carbon material of enzyme.Shell of the present invention comprises crosslinked gelatin coating.
The shape of immobilized enzyme of the present invention is not particularly limited, and can be shape arbitrarily, for example spherical, elliposoidal, cylindrical, rectangle, taper, polyhedron or their distortion.The present invention also is not intended to limit the size of immobilized enzyme, for example, can be 0.01 ~ 16000 μ m.In order to improve the operational stability of immobilized enzyme, immobilized enzyme is preferably dimensioned to be 0.06～10000 μ m, more preferably 270～6000 μ m.
In the present invention, described core part can form by having adsorbing carbon material adsorptive enzyme.The shape of core part can be shape arbitrarily, for example spherical, elliposoidal, cylindrical, rectangle, taper, polyhedron or their distortion.The shape of core part depends primarily on the shape with adsorbing carbon material.The size of core part of the present invention does not have special requirement yet, is for example 0.01 ~ 16000 μ m, and in order to improve the operability of adsorption process, immobilized enzyme is preferably dimensioned to be 0.06～10000 μ m, more preferably 260～5000 μ m.
Carbon material of the present invention has adsorption, and it is preferably the carbon material with vesicular structure.In the present invention, the enzyme molecule can mainly be distributed in the inside in the hole of carbon material, on the surface of carbon material, also can have part enzyme molecule, as long as these enzyme molecules do not come off from the surface of carbon material.Preferred 50wt% at least, more preferably 70wt% at least, most preferably at least the enzyme molecule of 90wt% is present in the inside in the hole of carbon material.Like this, can further improve the operational stability of enzyme.
In the present invention, the surface that described shell covers described core part by gelatin forms.Preferably, by the gelatin of core part surface, undertaken crosslinked.Can improve the thermostability of immobilized enzyme like this.Shell of the present invention can cover the core part surface fully, also can partly cover the core part surface.From the angle of the further operational stability of improving immobilized enzyme and thermostability, consider, preferably at least 60%, more preferably at least 80%, most preferably the surface of at least 98% core part is covered by gelatin coating.Shell thickness of the present invention is not particularly limited, and still, from the angle of further raising adsorption efficiency and immobilization effect, considers, and its thickness is preferably 0.1～100 μ m, more preferably 5～50 μ m, more preferably 10～20 μ m.In the situation that the gelatin coating of core part surface is carried out crosslinked, the degree of crosslinking of gelatin can be at least 5%, is preferably at least 10%, more preferably at least 20%.
<enzyme >
Preparation in accordance with the present invention can be for the various enzymes of immobilization.In the present invention, unless specifically stated otherwise, term of the present invention " enzyme " can be the combination of a kind of enzyme or plurality of enzymes.Equally, unless specifically stated otherwise, " immobilized enzyme " of the present invention can be the combination of a kind of immobilized enzyme or multiple immobilized enzyme.In addition, term of the present invention " enzyme " also comprises enzyme variants, and term " immobilized enzyme " also comprises the immobilized enzyme variant.The example of enzyme variants comprises those disclosed enzyme variants such as EP251446, WO91/00345, EP 525610.
In the present invention, described enzyme is preferably the enzyme that can be had adsorbing carbon material and adsorb.Described enzyme includes but not limited to oxydo-reductase, transferring enzyme, lytic enzyme, lyase, isomerase and/or ligase enzyme.
The group that oxydo-reductase of the present invention preferably forms from following enzyme: peroxidase (EC 1.11.1), laccase (EC 1.10.3.2) and glucose oxidase (EC 1.1.3.4)].The example of preferred oxydo-reductase comprises the Gluzyme purchased from Novozymes A/S
TMEnzyme.
Peroxidase of the present invention comprises the peroxidase that derives from plant, bacterium or originated from fungus, or superoxide enzyme variants chemically modified or that protein engineering forms.The example of described peroxidase comprises from the peroxidase of Coprinus (Coprinus) or its variant.For example,, from peroxidase or its variant of Coprinus cinereus (C.cinereus).WO 93/24618, WO 95/10602 and WO98/15257 disclose above-mentioned enzyme, at this, it are incorporated herein as a reference.Peroxidase also is selected from the Guardzyme purchased from Novozymes A/S
TM.
The group that transferring enzyme of the present invention preferably forms from following enzyme: shift the transferring enzyme (EC2.1) of one-carbon-based group, the transferring enzyme (EC 2.2) that shifts the aldehydes or ketones residue, acyltransferase (EC 2.3), glycosyltransferase (EC 2.4), shift the aliphatic hydrocarbyl (alkyl) except methyl, the transferring enzyme (EC 2.5) of transfer aryl and the transferring enzyme (EC 2.6) of transfer nitrogen-containing group.Transferring enzyme is preferably transglutaminase (EC 2.3.2.13).The example of transglutaminase comprises WO96/06931 those disclosed transferring enzyme.
Lytic enzyme of the present invention is preferably from carboxylic ester hydrolase.Carboxylic ester hydrolase is selected from carbohydrase, peptase (proteolytic enzyme, EC 3.4), lipase (EC 3.1.1.3), 3-Phytase (EC 3.1.3.8), 6-phytase (EC 3.1.3.26) or other carbonylic hydrolase.
Carbohydrase of the present invention can be selected from Glycosylase (EC 3.2).Glycosylase (EC 3.2) is preferably from amylase, cellulase or mannase.Carbohydrase of the present invention includes but not limited to α-amylase (EC 3.2.1.1), beta-amylase (EC 3.2.1.2), chitoanase (EC 3.2.1.14), polygalacturonase (EC3.2.1.15), N,O-Diacetylmuramidase (EC 3.2.1.17), beta-glucosidase enzyme (EC 3.2.1.21), amylo-1，6-glucosidase (EC 3.2.1.33), alpha-galactosidase (EC 3.2.1.22), beta-galactosidase enzymes (EC 3.2.1.23), Galactanase (EC 3.2.1.89), xylan Isosorbide-5-Nitrae-xylobiase (EC 3.2.1.37), Schardinger dextrin inscribe-1,6-alpha-glucosidase (EC 3.2.1.41), sucrose alpha-glucosidase (EC 3.2.1.48), dextran inscribe-1,3-β-D-Polyglucosidase (EC 3.2.1.39), dextran Isosorbide-5-Nitrae-alpha-glucosidase (EC 3.2.1.3), inscribe-Isosorbide-5-Nitrae-beta-glucanase (cellulase, EC 3.2.1.4), inscribe-1,3 (4)-beta-glucanase (EC3.2.1.6), inscribe-Isosorbide-5-Nitrae-beta-glucanase (EC 3.2.1.8), dextranase (EC 3.2.1.11), dextran inscribe-1,3-alpha-glucosidase (EC 3.2.1.59), dextran Isosorbide-5-Nitrae-beta-glucosidase enzyme (EC 3.2.1.74), dextran inscribe-1,6-beta-glucosidase enzyme (EC 3.2.1.75), arabinan inscribe-1,5-α-L-arabinose glycosides enzyme (EC 3.2.1.99), Sumylact L (EC 3.2.1.108), chitosanase (EC 3.2.1.132), glucose isomerase (EC 5.3.1.9) and/or xylose isomerase (EC 5.3.1.5).The carbohydrase of the present invention group that also preferably freely following enzyme forms: purchased from the Alpha-Gal of Amyloglucosidase Novo
TM, Bio-Feed
TMAlpha, Bio-Feed
TMBeta, Bio-Feed
TMPlus, Bio-Feed
TMPlus, Novozyme
TM188, Celluclast
TM, Cellusoft
TM, Ceremyl
TM, Citrozym
TM, Denimax
TM, Dezyme
TM, Dextrozyme
TM, Finizym
TM, Fungamyl
TM, Gamanase
TM, Glucanex
TM, Lactozym
TM, Maltogenase
TM, Pentopan
TM, Pectinex
TM, Promozyme
TM, Pulpzyme
TM, Novamyl
TM, Termamyl
TM, or AMG
TM, or purchased from the Maltogenase of Novozymes A/S
TM, Sweetzyme
TMOr Aquazym
TM.
Amylase of the present invention (α-and/or beta-amylase) comprises amylase or variant chemically modified or that protein engineering forms that derives from bacterium or originated from fungus.Described amylase comprises the α-amylase from bacillus.Amylase variant of the present invention comprises WO 94/02597, WO 94/18314, WO 96/23873, the disclosed variant of WO 97/43424.Diastatic example of the present invention comprises the Natalase purchased from Novozymes A/S
TM, Stainzyme
TM, Duramyl
TM, Ter-mamyl
TM, Termamyl
TMUltra, Fungamyl
TM, BAN
TM, Rapidase
TM, or Purastar
TM, or purchased from the Purastar OxAm of Genencor International Inc.
TM.
Cellulase of the present invention comprises the cellulase that derives from bacterium or fungi, or variant chemically modified or that protein engineering forms.Described cellulase comprises the cellulase from bacillus, Rhodopseudomonas, Humicola, fusarium, Thielavia (Thielavia) or the branch mould genus of top spore (Acremonium); The fungal cellulase more preferably produced from Humicola insolens (Humicola insolens), thermophilic fungus destroyed wire (Myceliophthora thermophila) or sharp sickle spore (Fusarium oxysporum).These cellulases are at US 4,435,307, US 5,648,263, US 5,691,178, US5,776,757 and WO 89/09259 have been described in detail, be introduced into this paper at this.Be applicable to cellulase of the present invention and be preferably alkali cellulose enzyme or neutral cellulase.For example, EP 0495257, EP0531372, WO96/11262, WO 96/29397, disclosed those cellulases of WO 98/08940.Cellulase variants can be PCT/DK98/00299, WO 94/07998, EP 0531315, US5,457,046, US5,686,593, US 5,763,254, WO 95/24471, WO 98/12307 those disclosed cellulase variants.Cellulase of the present invention can also be selected from the group that following enzyme forms: purchased from the Celluzyme of Novozymes A/S
TM, Endolase
TM, Renozyme
TMOr Carezyme
TM, or purchased from the Clazinase of GenencorInternational Inc.
TM, or purchased from the Puradax HA of Kao Corporation
TMOr KAC-500 (B)
TM.
Mannase of the present invention (Mannanase) comprises the mannase that derives from bacterium or fungi, or the mannosans enzyme variants of chemically modified or genetic modification.Preferred mannase is derived from the genus bacillus of basophilic.Suitable mannase comprise purchased from Mannaway
TMMannase.
Proteolytic enzyme of the present invention can comprise the ease variants that the proteolytic enzyme that derives from animal, plant or microorganism or chemically modified or protein engineering form.Described proteolytic enzyme is selected from serine protease or metalloprotease.Described proteolytic enzyme is preferably alkaline microbial protease or trypsinase-sample proteolytic enzyme.The alkalescence microbial protease comprises subtilisin (subtilisin).Subtilisin comprises subtilisin Novo(WO 89/06279), subtilisin Carlsberg, subtilisin 309, subtilisin 147 or subtilisin 168.Trypsinase-sample proteolytic enzyme comprises trypsinase, fusarium (Fusarium) proteolytic enzyme (WO 89/06270).Ease variants of the present invention can be WO 98/20115, WO98/20116, WO 98/34946, the disclosed ease variants of WO 92/19729.Proteolytic enzyme of the present invention (peptase) specific examples comprises the Kannase purchased from Novozymes A/S
TM, Everlase
TM, Esperase
TM, Alcalase
TM, Neutrase
TM, Durazym
TM, Savinase
TM, Ovozyme
TM, Pyrase
TM, Pancreatic Trypsin NOVO (PTN), Bio-Feed
TMPro or Clear-Lens
TMPro proteolytic enzyme; Perhaps purchased from Genencor International Inc. or Gist-Brocades; Perhaps purchased from the Ronozyme of BASF or DSM NutritionalProducts
TMPro, Maxatase
TM, Maxacal
TM, Maxapem
TM, Opticlean
TM, Propease
TM, Purafect
TMOr Purafect Ox
TMProteolytic enzyme.
Lytic enzyme of the present invention is glucose isomerase and lipase more preferably.Optional glucose isomerase or glucose isomerase enzyme variants chemically modified or that protein engineering forms from deriving from bacterium or fungi of glucose isomerase.Lipase is selected from lipase or lipase Variant chemically modified or that protein engineering forms that derives from bacterium or fungi.Comprise mycocandida (Candida), Rhizomucor (Rhizomucor), antarctic candida (C.Antarctica), Man Hegen Mucor (R.miehei), Humicola (Humicola), Rhodopseudomonas (Pseudomonas) or bacillus (Bacillus) for bacterium or the fungi that obtains lipase.The example of Humicola (Humicola) comprises dredges cotton shape humicola lanuginosa (H.lanuginosa) or Humicola insolens (H.insolens).The example of Rhodopseudomonas (Pseudomonas) comprise Pseudomonas alcaligenes (P.alcaligenes), pseudomonas pseudoalcaligenes (P.pseudoalcaligenes), pseudomonas cepacia (P.cepacia) (EP331376), Pseudomonas stutzeri (P.stutzeri) (GB1,372,034), Pseudomonas fluorescens (P.fluorescens), Rhodopseudomonas bacterial classification bacterial strain SD 705(WO95/06720) or Wisconsin pseudomonas (P.wisconsinensis) (WO 96/12012).The example of bacillus (Bacillus) comprises subtilis (B.subtilis), bacstearothermophilus (B.stearothermophilus) or bacillus pumilus (B.pumilus).
In addition, lipase of the present invention also can be selected from penicillium cammenberti (Penicillium camembertii) lipase, geotrichum candidum lipase (Geotricum candidum), moral row rhizopus equinus (R.delemar) lipase, snow-white head mold (R.niveus) lipase and Rhizopus oryzae (R.oryzae) lipase.Lipase Variant of the present invention can also be those disclosed lipase Variant in EP 407225, EP 260105, WO 95/35381, WO 96/00292, WO95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079, WO97/07202, WO 92/05249, WO 94/01541.The group that lipase of the present invention more preferably forms from following enzyme: purchased from Lipex, the Lipoprime of Novozymes A/S
TM, Lipolase
TM, Lipolase
TMUltra, Lipozyme
TM, Palatase
TM, Novozym
TM435 or Lecitase
TMLipase; Perhaps purchased from the Lumafast of GenencorInternational Inc
TMLipase; Perhaps purchased from the Lipomax of DSM/GenencorInt.Inc.
TMPerhaps purchased from the bacillus bacterial classification lipase of Genencor enzymes.
The example of 3-Phytase of the present invention (EC 3.1.3.8), 6-phytase (EC 3.1.3.26) comprises the Bio-Feed purchased from Novozymes
TMThe Ronozyme of Phytase, DSM Nutritional Products
TMP, purchased from the Natuphos of BASF
TM, purchased from the Finase of AB Enzymes
TMOr purchased from the Phyzyme of Danisco
TMProduct.Other example comprises the phytase be disclosed in WO 98/28408, WO00/43503 and WO 03/066847.
In the present invention, lyase can be selected from pectate lyase.Pectate lyase comprises the pectate lyase that derives from bacterium or fungi; Perhaps chemically modified or pectate lyase variant genetic modification.Preferred pectate lyase is derived from the bacterial strain of bacillus, for example bacterial strain of subtilis.Preferred pectate lyase is derived from Bacillus licheniformis.
According to the present invention, in an embodiment, the group that the freely following enzyme of enzyme choosing of the present invention forms: lytic enzyme, at, oxydase transferring enzyme, reductase enzyme, hemicellulase, esterase, isomerase, polygalacturonase, Sumylact L, peroxidase, laccase, polygalacturonase, catalase, nitrilase or their mixture.In another embodiment, the group that the freely following enzyme of enzyme choosing of the present invention forms: proteolytic enzyme, amylase, lipase, Phospholipid hydrolase, esterase, mannonase cellulase and their mixture.In another embodiment, the group that the freely following enzyme of enzyme choosing of the present invention forms: proteolytic enzyme, lipase, Glycosylase, oxydo-reductase, oxydase, ketone isomerase and esterase.
In the preferred embodiment of the present invention, enzyme of the present invention is selected from catalase and/or glucose oxidase.
In the present invention, the liquid medium that contains enzyme is preferably the solution that contains enzyme.It can obtain the liquid medium that contains enzyme by this area usual manner, for example, by enzyme is dispersed in liquid medium and obtains.Liquid medium of the present invention is hydrophilic medium, is preferably aqueous medium.Other organic substance or the biological substance outside dezymotizing can be do not contained in aqueous medium, also other organic substance or biological substance can be contained.Other organic substance can be organic solvent.Organic solvent is preferably the organic solvent that does not reduce enzymic activity.Organic solvent is alcohols for example, comprises ethylene glycol, propylene glycol, polyoxyethylene glycol and/or polypropylene glycol.Other biological substance can be those biological substances residual in fermentation culture.
In the specific embodiment of the present invention, the liquid medium that contains enzyme of the present invention can be selected from and be dissolved in pure enzyme, the fermentation culture basically of aqueous medium or the enzyme concentrated solution obtained by the purification of fermentation nutrient solution.Purification process comprises ultrafiltration; Perhaps by protein precipitation, separation be dissolved in again other aqueous medium.Wherein, " pure enzyme basically " means that the purity of enzyme is very high, is term known in the art, and for example the purity of enzyme is more than 90wt%, more than 95wt% or more than 98wt%.In the preferred embodiment of the present invention, the liquid medium of the preferred self-contained catalase of the liquid medium that contains enzyme and/or glucose oxidase, the more preferably solution of self-contained catalase and/or glucose oxidase.
<carbon material >
In the present invention, as the carbon material of carrier, there is adsorption, its can adsorptive enzyme with being fixed.Preferably, there is adsorbing carbon material and be preferably porous carbon materials.
Carbon material of the present invention can be selected from following material: the carbide that is derived from the animals and plants materials such as brown coal, inferior brown coal, hard coal, coke, charcoal or coconut husk charcoal; The resultant of the fluoropolymer resin of heating carbon containing under rare gas element; Be derived from the porous carbon materials of fish scale; Or be derived from the carbon material of diene gas.Preferably, carbon material of the present invention is for take porous carbon materials prepared as raw material by coconut husk, resol, furane resin, permalon, polyimide, polypyrrole, polyacrylamide, polyvinylpyridine, polyacrylonitrile, pitch, gelatin or fish scale.The porous carbon materials that is derived from fish scale can be prepared by the embodiment 1-6 of CN102107863A.The porous carbon materials that is derived from gelatin can be prepared by the embodiment 1-3 of CN101880037A.The carbon material that is derived from diene gas can be prepared by the embodiment 1 of WO2008/082897.It can be prepared other porous carbon materials by the embodiment 1-10 of WO98/30496.At this, they are incorporated herein as a reference.Can be used for other carbon materials of the present invention can for CN101143719A, CN101012057A, GB-1383085, WO02/12380, US-3909449, US-4045368, WO01/19904, CN102112221A, CN101888970A, CN101683977A, CN1821182A disclosed those.
In a specific embodiment of the present invention, carbon material of the present invention is particulate porous carbon material.The particle of carbon material of the present invention is preferably dimensioned to be 0.01 ~ 16000 μ m, and in order to improve the operability of adsorption process, carbon material is preferably dimensioned to be 0.05～9000 μ m, more preferably 255～5000 μ m.The surface-area of this carbon material is 5-5000m
2/ g, for example, 200-4000m
2/ g, preferably 300-3000m
2/ g, more preferably 1000～2300m
2/ g.The pore size of this carbon material is 1nm-5 μ m, for example 2nm-1000nm, preferably 5-500nm, more preferably 5～30nm.
In another embodiment of the present invention, the particle of carbon material of the present invention is of a size of 50-1500 μ m, preferably 100-1000 μ m, more preferably 250-700 μ m.The surface-area of this carbon material is 5-5000m
2/ g, for example, 200-4000m
2/ g, preferably 300-3000m
2/ g, more preferably 1000～2300m
2/ g.The pore size of this carbon material is 1nm-5 μ m, for example 2nm-1000nm, preferably 5-500nm, more preferably 5～30nm.Can improve like this charge capacity of carbon material to enzyme.In another embodiment of the present invention, the granularity of the particle of particulate porous carbon material of the present invention is 100-600 μ m, 150-500 μ m more preferably, and more preferably 200-450 μ m, most preferably be 250-400 μ m.The surface-area of this particulate porous carbon material and aperture are as described in this section.
In another embodiment of the present invention, the present invention's carbon material used is preferably carbon nanotube, gac or surface modification carbon material.Because they possess stronger adsorptivity, thereby be preferred.
As preferably, the carbon material that the present invention uses is hole, fish scale Quito carbon material.Wherein, described porous is the multi-stage porous of the macropore that is continuous distribution, mesoporous and micropore, its BET specific surface 1000～2300m
2/ g, total hole volume 0.2～2.7cm
3/ g, mean pore size is 2～30nm.Being less than 2nm in Zhong， aperture, these holes is micropore; Aperture is mesoporous at 2～50nm; It is macropore that aperture is greater than 50nm.
In the present invention, the surface modification carbon material can carry out for surface the carbon material of hydrophilic modifying.For example, with the basically hydrophobic surface that has of organic moiety carbon coating material, prepare.Said process is known in the art.For example, JP 09000257-A, the disclosed method of JP 08126489-A, as long as will be used carbon material as respective carrier.For example, carbon material is carried out to acidification, then adopt surface treatment agent (for example N-phenyl-gamma-amino propyl trimethoxy silicane) to carry out coating processing.The surface treatment reagent that coating processing is used is also known in the art, and surface treatment reagent is preferably coupling agent.This coupling agent preferably has organic siloxane.Organo-siloxane of the present invention comprises mono amino, two amino, triamino or polyamino siloxanes.Suitable organo-siloxane is selected from aminoalkyl group trialkoxy silane, Diaminoalkyl trialkoxy silane, triamino alkyltrialkoxysilaneand, aminoaryl trialkoxy silane, diamino aryl trialkoxy silane, triamino aryl trialkoxy silane or polyamino aryl trialkoxy silane.
In above-mentioned organo-siloxane, the carbon atom of alkyl and alkoxyl group is 1 ~ 8, is preferably 1 ~ 5, more preferably 1 ~ 3.Alkyl is selected from methyl, ethyl, propyl group, butyl, amyl group, hexyl, heptyl, octyl group or their combination.Alkoxyl group is selected from methoxyl group, oxyethyl group, propoxy-, butoxy, pentyloxy, hexyloxy, heptan oxygen base, octyloxy or their combination.
In above-mentioned organo-siloxane, the carbonatoms of aryl is 6 ~ 12, more preferably 6 ~ 8.Aryl is selected from phenyl, benzyl, styroyl, hydrocinnamyl, benzene butyl, naphthyl, xenyl or their combination.
The concrete example of above-mentioned organo-siloxane comprises Y-aminopropyl trimethoxysilane, Y-aminopropyl triethoxysilane, N-(aminoethyl)-Y-aminopropyl trimethoxysilane, N-(aminoethyl)-Y-aminopropyl triethoxysilane, N-(aminoethyl)-Y-aminopropyl methyldiethoxysilane, phenylamino methyltrimethoxy silane, phenylamino Union carbide A-162, aminoethyl aminoethyl aminopropyl trimethoxysilane or N-phenyl-gamma-amino propyl trimethoxy silicane.
<gelatin >
Gelatin is the collagen partial hydrolysis in a kind of reticular tissue by animal (bone, skin, tendon etc.) and the protein that obtains has extremely good physical properties (as jelly power, wetting ability, high dispersion, low viscosity characteristics, dispersion stabilization) and chemical property (the side-chain radical reactive behavior is high).The gelatin no antigen, good biocompatibility, the damage to enzyme in immobilization process is less.
The present invention's gelatin used is to take animal skin, bone, muscle or squama as the raw material preparation.These gelatin can be prepared by disclosed methods such as US6689556B2, CN102391790A, WO9421739A1, CN101735464A, CN100594798C.This is incorporated herein them as a reference.
In a specific embodiment of the present invention, the molecular weight of gelatin, between 1000Da ~ 200000Da, is preferably between 5000Da ~ 50000Da.In another embodiment of the present invention, the molecular weight of gelatin, between 500Da ~ 20000Da, is preferably between 100Da ~ 5000Da.Wherein, Da is dalton, means the unit of molecular weight.Those gelatin that gelatin of the present invention also can be used the table B of WO2006/128685 to enumerate.
<linking agent >
In the present invention, linking agent can be crosslinked compound to occur with gelatin.The group that the following linking agent of the optional freedom of linking agent of the present invention forms: multifunctional acid anhydrides, multifunctional lsothiocyanates, polyfunctional isocyanate, multifunctional aldehyde, multifunctional Organohalogen compounds, multifunctional azo-compound, multi-epoxy compounds, Biological cross-linker and their mixture.Biological cross-linker refers to the linking agent extracted from biomaterial, for example from cape jasmine and glossy privet, extracts and obtains Biological cross-linker.Preferably, linking agent of the present invention is selected from dicarboxylic anhydride, dialdehyde, two sense azo-compounds, vulcabond, diisothio-cyanate, diepoxide, Biological cross-linker and their mixture.More preferably, linking agent of the present invention is selected from suceinic aldehyde, glutaraldehyde, hexanedial, heptan dialdehyde, suberic aldehyde, azel aldehyde, decanedial, terephthalic aldehyde, two-diazo benzidine-2,2 '-disulfonic acid, poly-azetidine, cyanuryl chloride, diepoxide, toluylene group diisocyanate, hexamethylene diisocyanate, diacetyl oxide, diglycidyl ether, hot two imido acid methyl esters, genipin or glutamine transaminage (TG enzyme).
In the specific embodiment of the present invention, linking agent is preferably from glutaraldehyde, genipin, TG enzyme or their mixture, more preferably from genipin or TG enzyme.Can further improve the activity recovery of immobilized enzyme like this, and reduce the toxicity of product.
Genipin (Genipin) is the product of jasminoidin after the beta-glucoside enzymic hydrolysis, and its structural formula is:
Genipin can be by chemical process or microbe transformation method preparation.For example, extract Geniposide from cape jasmine, then use the beta-glucoside enzymic hydrolysis, then make with extracted with diethyl ether, vacuum concentration, recrystallization.
In the present invention, linking agent is preferably used with the form of the liquid medium containing linking agent.This liquid medium is preferably hydrophilic medium, more preferably aqueous medium.Liquid medium containing linking agent can contain other component, for example salt, carbohydrate.Other concrete component can be starch, dextran, wax, dextrin, polyvinylpyrrolidone sorbyl alcohol, polyoxyethylene glycol, metal silicate or metal ortho-silicate.
<preparation method >
Below will describe the preparation method of immobilized enzyme of the present invention in detail, it at least comprises the steps: (1) adsorption step; (2) encapsulation steps; (3) cross-linking step.Optionally, comprise (4) lyophilize step.Optionally, also comprise (5) cleaning step.
In the present invention, adsorption step (1) can comprise following process: will have adsorbing carbon material and contact to be adsorbed with the liquid medium that contains enzyme, thereby obtain the carbon material that absorption has enzyme.This absorption has the carbon material of enzyme can be referred to as enzyme-carbon material, core part or one-level immobilized enzyme.In adsorption step of the present invention, so-called " contact " method is not particularly limited, can be to be placed in by having adsorbing carbon material the liquid medium that contains enzyme, can be also that liquid medium sprinkling or the spray that will contain enzyme has on adsorbing carbon material.In order to improve adsorption efficiency, preferably, so-called " contact " is: will have adsorbing carbon material and be placed in the liquid medium that contains enzyme.More preferably, adsorption step of the present invention (1) is: will have adsorbing carbon material be placed in contain enzyme liquid medium to be adsorbed, remove supernatant liquor, thereby obtain absorption, the carbon material (being enzyme-carbon material) of enzyme is arranged.
The adsorption temp of adsorption step of the present invention (1) can be preferably 0 ~ 15 ° of C for 0 ° of C ~ 25 ° C, and more preferably 0～10 ° of C, most preferably be 4 ° of C.The vigor that can keep like this, enzyme.In adsorption step (1), adsorption time can be 0.3-6h, is preferably 0.5 ~ 4h, and more preferably 1 ~ 2h, most preferably be 1.5h.Like this, can improve the charge capacity of carbon material to enzyme, and keep the vigor of enzyme.In adsorption step (1), enzyme is 5 ~ 200mg/g with having adsorbing carbon material mass ratio, is preferably 10～150mg/g, more preferably 50 ~ 100mg/g; Wherein, the molecule " mg " in unit " mg/g " is the mass fraction with adsorbing carbon material for the mass fraction of enzyme, denominator " g ".Like this, can effectively utilize the loading capacity of carbon material, and make carbon material more firm to the absorption of enzyme.If this mass ratio is less than 5mg/g, the utilization ratio of the loading capacity of carbon material is lower; If this mass ratio is greater than 200mg/g, excessive enzyme breaks away from from carbon material.
In the present invention, encapsulation steps (2) can comprise following process: this enzyme-carbon material is contacted with the liquid medium containing gelatin, coat the enzyme-carbon material of gelatin to form surface.Enzyme-carbon material that this surface coats gelatin can be referred to as the secondary immobilized enzyme.In encapsulation steps of the present invention, so-called " contact " method is not particularly limited, and can be the liquid medium containing gelatin is sprayed or spray on enzyme-carbon material, can be also that enzyme-carbon material is placed in to the liquid medium containing gelatin.In order to improve covered effect, preferably, so-called " contact " is for to be placed in the liquid medium containing gelatin by enzyme-carbon material.In a specific embodiment of the present invention, encapsulation steps (2) is: this enzyme-carbon material is mixed to form mixed solution with the liquid medium containing gelatin, and make enzyme-carbon material that surface coats gelatin.
In encapsulation steps of the present invention (2), the liquid medium that contains gelatin is preferably gelatin solution.Mass percentage concentration containing gelatin in the liquid medium of gelatin is 1wt% ~ 5wt%, is preferably 1wt ~ 4wt%, and more preferably 1 ~ 3wt%, most preferably be 1wt%.The temperature of encapsulation steps (2) is not particularly limited, and can, for 0 ° of C ~ 30 ° C, be preferably 5 ~ 25 ° of C, more preferably 10～25 ° of C.Like this, can guarantee the even coating of gelatin.In encapsulation steps (2), the coating time can be 0.1 ~ 2h, is preferably 0.15 ~ 1h, and more preferably 0.2 ~ 0.8h, most preferably be 0.25 ~ 0.5h.Can guarantee that like this gelatin coating fully coats enzyme-carbon material.
In the present invention, cross-linking step (3) can comprise following process: by linking agent, surface is coated gelatin in the enzyme-carbon material of gelatin carry out crosslinked, to obtain gelatin carbon coated material immobilized enzyme (being the carrier immobilized enzyme of gelatin carbon coated).In the present invention, the mode that adds of linking agent is not particularly limited, for example, can be that linking agent is directly added to enzyme-carbon material and the mixed solution formed containing the liquid medium of gelatin, can be also enzyme-carbon material that surface is coated to gelatin adds in the liquid medium containing linking agent.For cost-saving, preferably, the mode that adds of linking agent is that linking agent is directly added to enzyme-carbon material and the mixed solution formed containing the liquid medium of gelatin (containing in the solution of enzyme-carbon material that surface coats gelatin).More preferably, cross-linking step of the present invention (3) is: to enzyme-carbon material and the mixed solution that the liquid medium that contains gelatin forms, add linking agent to carry out crosslinking reaction.
In cross-linking step of the present invention (3), linking agent can be used separately or use with the form of the liquid medium containing linking agent.Preferably, linking agent of the present invention is used with the form of the liquid medium containing linking agent.Concentration expressed in percentage by volume containing linking agent in the liquid medium of linking agent is 0.2vt% ~ 1.0vt%, is preferably 0.4 ~ 0.9vt%, and more preferably 0.5 ~ 0.8vt%, most preferably be 0.5vt%.The temperature of cross-linking step (3) is not particularly limited, and can, for 0 ° of C ~ 30 ° C, be preferably 5 ~ 25 ° of C, more preferably 10～25 ° of C.The vigor that can keep like this, enzyme.In cross-linking step (3), cross-linking reaction time can be 0.1 ~ 4.0h, is preferably 0.11 ~ 2.0h, and more preferably 0.12 ~ 1.5h, most preferably be 0.13 ~ 0.5h.Can guarantee that like this gelatin coating is full cross-linked, to improve the stability of enzyme.
Further, preparation method of the present invention can also comprise lyophilize step (4), is about to the cryodesiccated process of mixture that cross-linking step (3) obtains.Cryodesiccated temperature is not particularly limited, and can be for example 0～-20 ° of C, is preferably-5～-15 ° of C; The cryodesiccated time is 1～8 hour, more preferably 3～6 hours.Adopting freeze-drying further to process immobilized enzyme, can, in the upper drilling of shell (being gelatin coating), be the passage that provided of enzymatic reaction.
In addition, preparation method of the present invention can also comprise cleaning step (5),, after lyophilize, repeatedly cleans the process of immobilized enzyme (being the lyophilize product) with phosphate buffer solution.The not special restriction of the concentration of phosphate buffer solution, as long as it does not cause the vigor of enzyme to reduce.
<SEM test >
The metal spraying processing is carried out in immobilized enzyme (being product, the lower same) surface made, with HITACHI S-4700 type scanning electron microscopic observation and analytic sample surface appearance feature, acceleration voltage 10kV.
The measuring method of<enzyme activity >
Before carrying out catalyzed reaction, by immobilized enzyme, in 1mL phosphate buffer solution hatching 30min, ultrasonic dispersion treatment, obtain homodisperse immobilized enzyme solution.Get 10 μ L and join (50mM in the phosphate buffer solution that contains hydrogen peroxide, pH=7.0), stirring at low speed, separate rapidly immobilized enzyme after reaction 5min, clear liquid reads absorbancy and calculates the vigor of immobilized enzyme under the characteristic absorption wavelength of 240nm hydrogen peroxide.Immobilized enzyme is defined as at 30 ℃, and under the reaction conditions of pH=7.0, per minute decomposes the required immobilized enzyme amount of 1 μ M hydrogen peroxide.Its enzyme activity unit means with U/mg
Wherein: V means the volume (mL) of superoxol; C
0The starting point concentration (M) that means the front superoxol of reaction; C
eThe residual concentration (M) that means the rear superoxol of reaction; T means the reaction times (min); M means the quality (mg) of immobilized enzyme used.
The measuring method of the operational stability of<enzyme >
Immobilized enzyme carried out under suitable reaction condition to catalyzed reaction and measure enzyme activity, reclaim immobilized enzyme after reaction and repeatedly rinse with phosphate buffer solution, more again being placed in the substrate solution of newly getting, reusing repeatedly, surveying its residual enzyme and live.The enzyme activity for the first time of take is 100%, means the variation tendency of enzyme activity with relative activity.
The mensuration of the thermostability of<enzyme >
Respectively resolvase and immobilized enzyme are preserved to 2h under differing temps (15 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 50 ℃, 60 ℃) water bath with thermostatic control condition, then measure the remaining vigor of enzyme simultaneously.Take respectively the two initial live masterpiece is 100%, means the variation tendency of enzyme activity with relative activity.
Embodiment 1
By the 10mg activated carbon, in 1ml concentration, be fully to mix in 0.5mg/ml hydrogen peroxide enzyme solution (the phosphate buffer solution preparation that is 50mM by pH 7.0, concentration), under 4 ℃ of conditions, absorption is after 2 hours, remove supernatant liquor, and clean 3 times with phosphate buffer solution.Add the 1wt% gelatin solution of 150 μ l, fully mix, under room temperature, reaction adds crosslinked 10 minutes of the 0.5vt% glutaraldehyde solution of 15 μ l after 15 minutes.Mixing solutions being uniformly coated on culture dish, carrying out lyophilize and process 6 hours, obtain product---gelatin coats the activity carbon carrier immobilized catalase.
After product is rinsed to several times repeatedly, measure vigor and character that gelatin coats the activity carbon carrier immobilized catalase, as shown in Figure 1, as seen from the figure, gelatin coating is coated on the activity carbon carrier immobilized catalase wherein the SEM photo of its surface tissue.
After measured, it is 114.96 ± 8U/mg that gelatin coats activity carbon carrier immobilized catalase vigor, and activity recovery is 65.69%.
The measurement result of the thermostability of gelatin coating activity carbon carrier immobilized catalase is referring to Fig. 2.As seen from the figure, preserving vigor after 2 hours under 65 ℃ is still 57% of initial enzyme activity.Its thermostability improves more than 10% than the simple activated carbon that uses.Therefore, the gelatin coating wrapped up in activated carbon surface has played provide protection to the catalase of absorption, has alleviated to a certain extent the damage of temperature to enzyme.
The measurement result of the operational stability of gelatin coating activity carbon carrier immobilized catalase as shown in Figure 3.As seen from the figure, after 15 times are reused, gelatin coats the activity carbon carrier immobilized catalase still can keep 80% of original vigor, and the immobilized enzyme only adsorbed by activity carbon carrier keeps 50% of original vigor, and therefore after gelatin coats, operational stability improves greatly.
By the 10mg carbon nanotube, in 1ml concentration, be fully to mix in 0.5mg/ml hydrogen peroxide enzyme solution (the phosphate buffer solution preparation that is 50mM by pH 7.0, concentration), under 4 ℃ of conditions, absorption is after 2 hours, remove supernatant liquor, and clean 3 times with phosphate buffer solution.Add the 2wt% gelatin solution of 150 μ l, fully mix, under room temperature, reaction adds crosslinked 10 minutes of the 1.0vt% glutaraldehyde solution of 15 μ l after 15 minutes.Mixing solutions is uniformly coated on culture dish, carries out lyophilize and process 6 hours, obtain product---the carrier immobilized catalase of gelatin enveloped carbon nanometer tube.
The suitable temperature of reaction of research immobilized enzyme, get 10 μ L immobilized enzyme artemia hatching solution (50mM in the 10mL superoxol, the phosphoric acid buffer preparation of pH7.0), respectively under (15 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 50 ℃, 60 ℃) water bath with thermostatic control condition, reacting 5min, separate rapidly immobilized enzyme and measure enzyme and live.Using respectively its separately maximum enzyme activity as 100%, mean the variation tendency of enzyme activity under condition of different temperatures with enzyme activity.Result is as shown in the table: the immobilized enzyme optimal temperature scope after coating has obtained widening to a certain degree than resolvase.
The relative activity of enzyme under table 1, differing temps
In addition, the mensuration of immobilized enzyme stability is shown, after reusing 10 times, the carrier immobilized catalase of gelatin enveloped carbon nanometer tube still can keep more than 70% of original vigor.
Embodiment 3
In the glucose oxidase solution that is 1mg/ml in 1ml concentration by the 15mg carbon nanotube (the phosphate buffer solution preparation that is 50mM by pH 7.0, concentration), fully mix, under 4 ℃ of conditions, absorption is after 4 hours, remove supernatant liquor, and clean 3 times with phosphate buffer solution.Add the 4wt% gelatin solution of 150 μ l, fully mix, at room temperature react the 0.8vt% glutaraldehyde solution that adds 15 μ l after 15 minutes crosslinked 10 minutes.Mixing solutions is uniformly coated on culture dish, carries out lyophilize and process 5 hours, obtain product---the carrier immobilized glucose oxidase of gelatin enveloped carbon nanometer tube.
Mensuration to immobilized enzyme glucose oxidase enzyme stability shows, preserve 2 hours under 65 ℃ of high temperature after vigor still be respectively more than 50% of initial enzyme activity; After reusing 10 times, the carrier immobilized glucose oxidase of gelatin enveloped carbon nanometer tube still can keep more than 75% of original vigor.
In the glucose oxidase solution that is 1mg/ml in 1ml concentration by the 15mg carboxylic carbon nano-tube (the phosphate buffer solution preparation that is 50mM by pH 7.0, concentration), fully mix, under 4 ℃ of conditions, absorption is after 4 hours, remove supernatant liquor, and clean 3 times with phosphate buffer solution.Add the 2wt% gelatin solution of 150 μ l, fully mix, at room temperature react the 0.5vt% glutaraldehyde solution that adds 15 μ l after 15 minutes crosslinked 10 minutes.Mixing solutions being uniformly coated on culture dish, carrying out lyophilize and process 5 hours, obtain product---gelatin coats the carrier immobilized glucose oxidase of carboxylic carbon nano-tube.
The mensuration of its stability shows, the vigor after 1 hour of preserving under 65 ℃ still is respectively more than 60% of initial enzyme activity; After reusing 15 times, gelatin coats the carrier immobilized glucose oxidase of carboxylic carbon nano-tube still can keep more than 65% of original vigor.
The present invention utilizes the good sol-gel character of gelatin, thereby processes and realize that dual fixing effect prepares the gelatin of excellent in stability and coat the activity carbon carrier immobilized enzyme with linking agent generation crosslinking reaction and by lyophilize.Immobilized enzyme prepared by this method is found still can show higher activity level through the enzyme activity test, has avoided damage and the sex change of enzyme molecule, thereby has kept the activity of enzyme.After repetitive operation for several times, immobilized enzyme still can keep higher activity, realizes good operational stability.Coat gelatin by the method by absorption, embedding and crosslinked combination at carbon material surface; can improve the adaptability of enzyme molecule to reaction system on the one hand; on the other hand, the coating of gelatin can be avoided the leakage of enzyme molecule effectively, and the protective enzyme molecule sustains damage in reaction process.
The present invention is not limited to above-mentioned embodiment, in the situation that do not deviate from flesh and blood of the present invention, any distortion it may occur to persons skilled in the art that, improvement, replacement all fall into scope of the present invention.
Claims (10)
1. the preparation method of an immobilized enzyme, it comprises the steps:
(1) will there is adsorbing carbon material and contact to be adsorbed with the liquid medium that contains enzyme, thereby obtain the carbon material that absorption has enzyme, i.e. enzyme-carbon material;
(2) this enzyme-carbon material is contacted with the liquid medium containing gelatin, coat the enzyme-carbon material of gelatin to form surface;
(3) by linking agent, surface is coated gelatin in the enzyme-carbon material of gelatin carry out crosslinked, to obtain gelatin carbon coated material immobilized enzyme.
2. preparation method according to claim 1, it specifically comprises the steps:
(1) will have adsorbing carbon material be placed in contain enzyme liquid medium to be adsorbed, remove supernatant liquor, thereby obtain absorption, the carbon material of enzyme, i.e. enzyme-carbon material are arranged;
(2) this enzyme-carbon material is mixed to form mixed solution with the liquid medium containing gelatin, and make enzyme-carbon material that surface coats gelatin;
(3) above-mentioned mixed solution is mixed to carry out crosslinking reaction with linking agent;
(4) mixture optionally, reaction obtained carries out lyophilize.
3. preparation method according to claim 1 and 2, wherein, the adsorption temp of step (1) is 0 ° of C ~ 25 ° C, adsorption time is 0.5 ~ 4h.
4. preparation method according to claim 1 and 2, its of meeting the following conditions, two or three:
Condition 1: in step (1), enzyme is 5 ~ 200mg/g with the mass ratio with adsorbing carbon material, and in unit " mg/g ", " mg " is the mass fraction with adsorbing carbon material for the mass fraction of enzyme, denominator " g ";
Condition 2: in step (2), the mass percentage concentration that contains gelatin in the liquid medium of gelatin is 1wt% ~ 5wt%;
Condition 3: in step (3), linking agent is used with the form of the liquid medium containing linking agent, and the concentration expressed in percentage by volume that contains linking agent in the liquid medium of linking agent is 0.2vt% ~ 1.0vt%.
5. preparation method according to claim 1 and 2, wherein,
A, describedly there is adsorbing carbon material and comprise carbon nanotube, gac or surface modification carbon material; And/or
B, described enzyme comprise catalase or glucose oxidase.
6. preparation method according to claim 1 and 2, wherein,
In step (1), adsorption temp is 0 ~ 25 ° of C, and adsorption time is 0.3 ~ 6h, and enzyme is 50 ~ 100mg/g with having adsorbing carbon material mass ratio; With
In step (2), the mass percentage concentration that contains gelatin in the liquid medium of gelatin is 1 ~ 3wt%; With
In step (3), linking agent is used with the form of the liquid medium containing linking agent, and the concentration expressed in percentage by volume that contains linking agent in the liquid medium of linking agent is 0.5 ~ 0.8vt%.
7. an immobilized enzyme, it prepares by the described preparation method of claim 1～6 any one.
8. an immobilized enzyme, it has following structure:
The core part, it comprises that absorption has the carbon material of enzyme; With
Shell, it covers on the surface of described core part, comprises crosslinked gelatin coating.
9. immobilized enzyme according to claim 8, wherein,
A, described carbon material comprise carbon nanotube, gac or surface modification carbon material; And/or
B, described enzyme comprise catalase and/or glucose oxidase.
10. immobilized enzyme according to claim 8 or claim 9, wherein, the thickness of shell is 0.1～100 μ m.
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