CN1564865A - Enzyme immobilization method using silica gel or complex silica gel carrier - Google Patents

Enzyme immobilization method using silica gel or complex silica gel carrier Download PDF

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CN1564865A
CN1564865A CNA028195426A CN02819542A CN1564865A CN 1564865 A CN1564865 A CN 1564865A CN A028195426 A CNA028195426 A CN A028195426A CN 02819542 A CN02819542 A CN 02819542A CN 1564865 A CN1564865 A CN 1564865A
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carrier
silica gel
enzyme
compound
formula
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金正优
申相圭
黄鹤渊
崔相镕
尹钟赞
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CKD Bio Corp
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres

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Abstract

The present invention relates to a method for immobilizing D-aminoacid oxidase (DAOD) or glutaryl-7-aminocephalosporanic acid acylase (G1-7-ACA acylase) on silicagel or composite silicagel carrier, and the immobilized enzyme prepared according to the present invention is superior in initial activity and activity half life.

Description

Use the enzyme fixing means of silica gel or compound silica gel carrier
Technical field
The present invention relates to D-amino-acid oxidase (DAOD) or glutaryl-7-amino-cephalosporanic acid acyltransferase (G1-7-ACA acyltransferase) are fixed on method on silica gel or the compound silica gel carrier.
Background technology
Enzyme refers to finish the protein of katalysis in biochemical reaction, and general owing to can not be used under organic solvent or the high temperature in instability after the extraction.Therefore, enzyme reaction is undertaken by simple batch process usually, wherein substrate and lyoenzyme is mixed also cultivation at normal temperatures.But, in order to reclaim organized enzyme, must from reaction mixture, separate enzyme, but this is extremely difficult task technically.Generally use the method for protein (Progress in Chemical Industry, 25 (9), 596 (1985)) that from reaction mixture, separates enzyme and pollution by pH change or thermal treatment, but this method is uneconomical owing to the forfeiture of organized enzyme after reacting.
In order to solve described problem, such method is studied, wherein the catalytic activity of enzyme can be by with the enzyme physical restriction or be positioned certain space and repeatedly present, and representational example is an immobilized enzyme.For example, korean patent application 1987-13473 discloses by form the method for Schiff's base between the amino of amino on the carrier surface and enzyme molecule through the condensation reaction of a polyfunctional crosslinking agent.On the other hand, korean patent application 1991-123834 discloses the G1-7-ACA acyltransferase has been fixed on resin Doulite A7 with glutaraldehyde amino (glutaraldehydroaminogroup) and the method on the Doulite A568.But exist because the little problem that is difficult to obtain the high activity immobilization enzyme of carrier surface area by the carrier that described method obtains.On the other hand, korean patent application 1996-703112 discloses DAOD and G1-7-ACA acyltransferase has been fixed therein method on the organosiloxane polymer of having introduced glutaraldehyde amino.But, under the situation of reusing the immobilized enzyme for preparing by described method, organosiloxane polymer, carrier, breaking also can't be used as carrier, causes filterableness to reduce, thereby can not reuse.
As above as seen, be used as under the situation of carrier at organic substance, it shows the good advantage of fixed character of easy control microstructure and enzyme, but shows the shortcoming owing to carrier itself expands or reduction changes the specific characteristic of carrier under the situation of life-time service.On the contrary, when inorganic substance were used as carrier, it showed good thermotolerance, weather resistance, chemical resistant properties, and known to existing silanol to have good fixed character on the carrier surface in a large number.Therefore, the present inventor has developed and has used silica gel or compound silica gel, inorganic materials, and as carrier, the fixing method of DAOD and G1-7-ACA acyltransferase.
The object of the present invention is to provide the preparation method of immobilized enzyme, by this method, even make enzyme retentive activity still and under long-term reusable situation by being fixed on silica gel or the compound silica gel carrier.Another object of the present invention provides by what described preparation method obtained and is fixed on enzyme on the carrier.
Summary of the invention
The present invention relates to D-amino-acid oxidase (DAOD) or glutaryl-7-amino-cephalosporanic acid acyltransferase (G1-7-ACA acyltransferase) are fixed on method on silica gel or the compound silica gel carrier.DAOD is the tunning of the mutant of Trigonopsis variabilis, and the G1-7-ACA acyltransferase is the tunning of intestinal bacteria (E coli) recombinant chou.
As being used for carrier of the present invention, silica gel or compound silica gel are preferred.As described compound silica gel, can enumerate TiO 2Compound silica gel, Al 2O 3Compound silica gel and ZrO 2Compound silica gel.For described carrier, the aperture is preferably in the scope of 100-1000 .In the aperture is under 1000 or the higher situation, and the enzymic activity of every volume carrier is because carrier surface area is little and low, and is under 100 or the littler situation in the aperture, because the aperture can not fully be fixed on enzyme in the hole less than the enzyme size.The particle diameter of preferred vector is the 40-80 order.
In order enzyme to be fixed on the described carrier first-selected the needs by introducing amino and activated carrier to its surface.Following reaction formula 1 representative is used for the method for activated carrier.That is to say that the carrier that can make following formula 1 by the silanol that exists on the carrier surface and the silane coupled reaction between the aminoalkyl group silane derivative and the aminoalkyl group silane derivative reaction of following formula 2 obtain wherein to have introduced the activated carrier of amino following formula 3.
[reaction formula 1]
Figure A0281954200061
Wherein, R represent methylidene or ethyl; M, n and o represent the integer of 0-3 independently; And carrier is represented silica gel or compound silica gel.
As the aminoalkyl group silane derivative of described formula 2, can enumerate beta-aminoethyl-gamma-amino propyl group methyl dimethoxysilane, aminopropyl methyldiethoxysilane, gamma-amino propyl trimethoxy silicane and γ-An Jibingjisanyiyangjiguiwan.
The activation method of carrier can following detailed explanation.That is to say, the silica gel or the compound silica gel of formula 1 are added in the organic solvent, the aminoalkyl group silane derivative of adding formula 2, and under refluxing, carry out silane coupled reaction, preferably under 100-110 ℃, carry out, to form the water insoluble carrier of wherein having introduced amino formula 3.The amount of described aminoalkyl group silane derivative is preferably the 1-20 weight % of carrier, is preferably 2-15 weight %.
On the other hand, can react and immobilized enzyme by making the activated carrier and the linking agent that as above obtain.Following reaction formula 2 has shown the fixing means of enzyme.That is to say, make and wherein introduced amino carrier (formula 3) and linking agent (formula 4) reaction, obtain the compound of following formula 5, make it then and have amino enzyme (formula 6) reaction, so that enzyme is fixed on the carrier with aldehyde radical.
[reaction formula 2]
Wherein, m, n and o represent the integer of 0-3 independently; Carrier is represented silica gel or compound silica gel; And enzyme is represented DAOD or G1-7-ACA acyltransferase.
As the linking agent of formula 4, can use polyacetals such as glutaraldehyde, dialdehyde starch and succinic aldehyde, wherein preferred glutaraldehyde.
Detailed enzyme fixing means is as follows.At first, make the linking agent reaction of the carrier of wherein having introduced amino formula 3 and formula 4, form the compound of formula 5.The amount of linking agent is preferably the 20-50 weight % of carrier.Reacting on 0-30 ℃ under agitation carried out 0.5-4 hour.Make the compound of formula 5 and the enzyme reaction of formula 6 then, so that enzyme is fixed on the carrier.Preferred enzyme solution concentration is 30-70U/mL.React on 10-30 ℃ and carried out 0.5-15 hour, preferably under agitation carried out 1-7 hour.The pH value of reaction soln and temperature should be controlled in the scope that does not cause the enzymic activity reduction.
The amount of preferred immobilized enzyme is the 50-200U/kg carrier.After the crosslinking reaction, preferably there are not removable enzyme of complete bonded and excessive linking agent by eliminating with suitable damping fluid thorough washing enzyme fixed carrier.
In addition, the invention provides the enzyme that is fixed on the carrier, described enzyme can be by following formula 7 representatives.
Wherein, carrier is represented silica gel or compound silica gel; And enzyme is represented DAOD or G1-7-ACA acyltransferase.
Embodiment
Further describe the present invention by the following examples.Following embodiment specific explanations the present invention, scope of the present invention is not subjected to the restriction of embodiment.
Embodiment 1: the activation of silica gel
100g silica gel is added in the 1000mL toluene, and mixture was stirred 30 minutes.Add 10g beta-aminoethyl-gamma-amino propyl group methyl dimethoxysilane, and, filter and obtain activated silica gel 110 ℃ of stirrings 3 hours that reflux down.
Embodiment 2:DAOD's is fixing
To add in the 200mL 1M phosphate buffered saline buffer (pH 7.5) as activatory 40-80 order silica gel (50g) as described in the embodiment 1, and slowly add the 20g glutaraldehyde.Described reaction mixture was stirred 2 hours down in 20 ℃, and filter.Add the aqueous solution (150mL) that contains DAOD (50U/mL), stirred 3 hours down, filter being fixed DAOD in 10 ℃.
Fixing of embodiment 3:G1-7-ACA acyltransferase
To add in the 500mL 1M phosphate buffered saline buffer (pH 8.0) as activatory 40-80 order silica gel (30g) as described in the embodiment 1, and slowly add the 10g glutaraldehyde.Described reaction mixture was stirred 2 hours down in 20 ℃, and filter.Add the aqueous solution (100mL) that contains G1-7-ACA acyltransferase (40U/mL), stirred 3 hours down, filter being fixed G1-7-ACA acyltransferase in 10 ℃.
Experimental example 1: according to activity and the transformation period of pore size determination DAOD
-DAOD efficacy determinations
The 100mL substrate [is dissolved in 100mM KPO 4The 20mM cephalosporin solution (pH 8.0) of damping fluid] place the 100mL Erlenmeyer flask, use oxygen saturation, add 300mg DAOD and stirring.At this moment, measure the amount of the oxygen that consumes by DAOD, with definite DAOD activity, and at 25 ℃ of oxygen of deciding to be consumed with the D.O instrumentation down.On behalf of per minute, DAOD activity unit (U) produce the amount of the enzyme of 1 μ mole G1-7-ACA.
-the transformation period is measured
Add 1.2kU DAOD in the 1000mL cephalosporin solution (is that 78% cephalosporin is dissolved in the 0.1M phosphate buffered saline buffer (pH 7.2) with 20g purity) and stir.Temperature is remained on 25 ℃, and by adding 5%NH 4OH and the pH value is remained on 7.5.At this moment, introduce oxygen (0.1vvm, volume/liquor capacity/minute), and internal pressure is remained on 1kgf/cm 2Termination reaction when analyzing the conversion of cephalosporin according to HPLC and reach 95% to G1-7-ACA, and render a service and determine the transformation period by measuring DAOD.
Initial activity and the half life determination result of DAOD who depends on the silica gel aperture is as shown in table 1.
Table 1.
Aperture () Initial activity (U/g) The active transformation period (reaction times)
????10-100 ????50 ????82
????100-300 ????70 ????99
????300-500 ????66 ????105
????500-700 ????65 ????98
????700-1000 ????64 ????99
????1000-1500 ????42 ????71
????1500-2000 ????21 ????42
According to table 1, the initial activity of immobilization DAOD enzyme and transformation period are subjected to the influence in silica-gel carrier aperture widely as can be seen, and suitable aperture is 100-1000 .
Experimental example 2: the activity and the half life determination that depend on the G1-7-ACA acyltransferase in aperture
The efficacy determinations of-G1-7-ACA acyltransferase
The 50mL substrate [is dissolved in 5mM KPO 452mM G1-7-ACA solution (pH 8.0) in the damping fluid] and 150mg G1-7-ACA acyltransferase add in the 100mL reactor, and stir.By adding 0.1M NaOH the pH value is remained on 8.0-10, kept 15 minutes.At this moment, measure the amount of the NaOH consumed, determining the activity of G1-7-ACA acyltransferase, and temperature is remained on 37 ℃.On behalf of per minute, the activity unit of G1-7-ACA acyltransferase (U) produce the amount of the enzyme of 1 μ mol 7-ACA.
-the transformation period is measured
Add 4.8kU G1-7-ACA acyltransferase in the 1000mL G1-7-ACA solution (is that 95% G1-7-ACA is dissolved in the 0.1M phosphate buffered saline buffer (pH 8.0) with 25g purity) and stir.Under atmospheric pressure temperature is remained on 25 ℃, and by adding 5%NH 4OH and the pH value is remained 8.0.Termination reaction when the conversion of analyzing G1-7-ACA to 7-ACA according to HPLC reaches 95%, and determine the transformation period by the effectiveness of measuring the G1-7-ACA acyltransferase.
The initial activity and the half life determination result of G1-7-ACA acyltransferase who depends on the silica gel aperture is as shown in table 2.
Table 2.
Aperture () Initial activity (U/g) The active transformation period (reaction times)
????10-100 ????85 ????119
????100-300 ????118 ????153
????300-500 ????113 ????145
????500-700 ????110 ????144
????700-1000 ????108 ????139
????1000-1500 ????75 ????89
????1500-2000 ????50 ????67
As can be seen from Table 2, the initial activity of immobilization G1-7-ACA acyltransferase and transformation period are subjected to the influence in silica-gel carrier aperture widely, and suitable aperture is 100-1000 .
Experimental example 3: the activity and the half life determination that depend on the G1-7-ACA acyltransferase of silica gel kind
Method according to identical with embodiment 3 is fixed on G1-7-ACA on the compound silica gel that the aperture is 100-300 , and determines the initial activity and the transformation period of immobilization G1-7-ACA acyltransferases as experimental example 2.The result is as shown in table 3.
Table 3.
Initial activity (U/g) The active transformation period (reaction times)
Silica gel ????118 ????153
??TiO 2Compound silica gel ????115 ????154
??Al 2O 3Compound silica gel ????117 ????156
??ZrO 2Compound silica gel ????120 ????150
As can be seen from Table 3, even under the situation of using compound silica gel, initial activity and transformation period also are good.
Industrial applicibility
According to the enzyme that is fixed on the carrier of the present invention; activity is 64-70U/g in the situation of DAOD; be 108-118U/g in the situation of G1-7-ACA acyltransferase; the situation (table 1 and table 2 that korean patent application 1991-23834 is the 24th page) that is higher than prior art; in 98 times of the situation half-life of DAOD or larger; be 139 times or larger in the situation of G1-7-ACA acyltransferase, highly stable.

Claims (14)

1. D-amino-acid oxidase or glutaryl-7-amino-cephalosporanic acid acyltransferase are fixed on the method on silica gel or the compound silica gel carrier.
2. method as claimed in claim 1, the aperture of wherein said carrier are 100-1000 .
3. method as claimed in claim 1, the particle diameter of wherein said carrier are the 40-80 order.
4. method as claimed in claim 1 is characterized in that making the silica gel of following formula 1 or the aminoalkyl group silane derivative of compound silica gel and following formula 2 to react, and forms the activated carrier of following formula 3, enzyme is fixed on this activated carrier then:
Figure A028195420002C1
(RO) 3Si——(CH 2) n(N) m(CH 2) o-NH 2????????????????????????????????????????????2
Wherein, R represent methylidene or ethyl; M, n and o represent the integer of 0-3 independently; And carrier is represented silica gel or compound silica gel.
5. method as claimed in claim 4 is characterized in that described aminoalkyl group silane derivative is at least a following component that is selected from: beta-aminoethyl-gamma-amino propyl group methyl dimethoxysilane, aminopropyl methyldiethoxysilane, gamma-amino propyl trimethoxy silicane and γ-An Jibingjisanyiyangjiguiwan.
6. as claim 4 method, the amount that it is characterized in that described aminoalkyl group silane derivative is the 1-20 weight % of carrier.
7. as each the method for claim 1-4, it is characterized in that described fixing following carrying out: the carrier that makes formula 3 reacts with the linking agent of formula 4, forms the compound of formula 5, makes the enzyme reaction of this compound and formula 6 then:
Figure A028195420003C1
OHC-A-CHO?????????????????????????????????????????????????????????????????????????4
Figure A028195420003C2
H 2N-enzyme 6
Wherein, m, n and o represent the integer of 0-3 independently; Carrier is represented silica gel or compound silica gel; And enzyme is represented DAOD or G1-7-ACA acyltransferase.
8. method as claimed in claim 7 is characterized in that described linking agent is polyacetals such as glutaraldehyde, dialdehyde starch and succinic aldehyde.
9. method as claimed in claim 7, the amount that it is characterized in that described linking agent are the 20-50 weight % of carrier.
10. method as claimed in claim 1 is characterized in that described enzyme is in the aqueous solution of 30-70U/mL in concentration.
11. method as claimed in claim 7 is characterized in that reacting on 0-30 ℃ and under agitation carrying out 2-4 hour of described activated carrier and described linking agent.
12. method as claimed in claim 7 is characterized in that carrying out in 0.5-15 hour by stirring in 10-30 ℃ with the reaction of enzyme.
13. method as claimed in claim 1, the amount that it is characterized in that immobilized enzyme is the 50-200U/kg carrier.
14. be fixed on the enzyme on the carrier, described enzyme can be represented by following formula 7:
Figure A028195420003C3
Wherein, carrier is represented silica gel or compound silica gel; And enzyme is represented DAOD or G1-7-ACA acyltransferase.
CNA028195426A 2001-10-06 2002-10-04 Enzyme immobilization method using silica gel or complex silica gel carrier Pending CN1564865A (en)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
CN100372931C (en) * 2006-04-18 2008-03-05 北京科技大学 Process for immobilization of glutaryl-7-amino cephalosporanic acid acylase

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KR100883206B1 (en) * 2007-02-05 2009-02-13 부경대학교 산학협력단 Support for immobilizing a biocatalyst comprising silica bead and use thereof
KR101068873B1 (en) * 2008-11-28 2011-09-30 연세대학교 산학협력단 Hydrogel entrapping biomarker-immobilized silica nanoparticles and method for preparing the same
KR101045567B1 (en) * 2008-12-17 2011-06-30 연세대학교 산학협력단 Hydrogel entrapping biomarker-immobilized magnetic nanoparticles and method for preparing the same

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US4683203A (en) * 1984-04-14 1987-07-28 Redco N.V. Immobilized enzymes, processes for preparing same, and use thereof
KR940005581A (en) * 1992-06-03 1994-03-21 리로이 휘테커 Angiotensin II Antagonist
DE4342770A1 (en) * 1993-12-15 1995-07-06 Boehringer Mannheim Gmbh Carrier-fixed enzymes

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100372931C (en) * 2006-04-18 2008-03-05 北京科技大学 Process for immobilization of glutaryl-7-amino cephalosporanic acid acylase

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