134

IMMOBILIZATION TECHNIQUES

[10]

Groups on the Protein. Polyisonitrile-nylon (50 rag) is suspended in 1 ml of cold 0.1 M Tris.HC1 buffer pH 7.0. A cold solution of enzyme in the same buffer (2-10 mg in I ml) is added followed by 0.1 ml of acetaldehyde, pipetted from a precooled pipette. The reaction mixture is left stirring overnight at 4 °, washed, and resuspended in water (4 ml) as described above. Maximum recoveries of immobilized enzymic activity (30-40% of the amount added to the reaction mixture) are obtained by both methods with 4-5 mg of enzyme per 100 mg of support. Coupling o] Enzymes to Polyaminoaryl-Nylon. Polyaminoaryl-nylon (100 rag) is suspended in cold 0.2 M HC1 (7 ml) and aqueous sodium nitrite (25 mg in 1 ml) added dropwise. The reaction mixture is stirred for 30 rain over ice; the red-brown diazotized polyaminoaryl-nylon is separated on a suction filter, washed with cold water, cold 0.1 M phosphate pH 8, and resuspended in 6 ml of the same buffer. A cold aqueous solution of enzyme (5-15 mg in 2-3 ml) is then added to the magnetically stirred suspension of diazotized support, and the reaction mixture is left stirring overnight a~ 4 °. The insoluble enzyme derivative is separated by filtration, washed with water, 1 M KC1, 0.1 M in NaHCOs, and water, resuspended in water (5 ml), and stored at 4 °.

[10] C o v a l e n t C o u p l i n g M e t h o d s for Inorganic Support Materials

By HOWARDH. WEETALL Choosing the Carrier Inorganic support materials have been shown to be excellent carriers for immobilized enzymes.1-s The charaoteristics of these inorganic support materials are extremely important and play an definitive role in the choice of the proper carrier for the enzyme to be immobilized. The carrier must meet several important criteria. Eaton has presented 1H. 2H. , H. 4H. 5H. e R. TR. s C.

H. Weetall, Science 166, 615 (1969). H. Weetall and L. S. Hersh, Biochim. Biophys. Acta 185, 464 (1969). H. Weetall, Nature (London) 223, 959 (1969). H. Weetall and N. B. Havewala, Biotechnol. Bioeng. Syrup. 3, 241 (1972). H. Weetall, Anal. Chem. 48, 602A (1974). A. Messing, Process Biochem. 1974, Nov. (1974). A. Messing and H. R. Stinson, Mol. Cell. Biochem. 4, 217 (1974). C. Q. Chin and F. Wold, Anal. Biochem. 61, 379 (1974).

[10]

135

COVALENT COUPLING METHODS

TABLE I DURABILITY TEST RESULTS FOR SUPPORTS

Static test

Dynamic test

(rag/m= per 16 hr)

(mg/m 2 per day)

Material description

TiO~ ZrO2 AlcOa Al~O3-SiO2 CPGa-ZrO~coAT CPG

1% NaOH 5 % HC1

0.2 0.2 0.6-0.8 1.85 1.3-2.0 3.06

0.8 1.1 2.0 3.65 0.2 0.08

pH 4.5

pH 7.0

pH 8.2

0.05 0.05 ND" 0.004 0.004 0.01 0.056-0.086 0.01 0.01 0.08-0.1 0.02-0.05 0.06 0.03 0.7 0.7-0.9 0.02 0.5 0.3

° Not done. CPG, controlled-poreglass. what he terms a "Decision Tree. ''9 Answering the questions Eaton poses permits one to choose the proper carrier for enzyme immobilization.

Immobilized Enzyme Decision Tree A. Does the pore morphology permit entry of the enzyme? B. Can the enzyme be immobilized on the support? C. Is the immobilized enzyme durable in (1) acid, (2) base, (3) high salt? D. Can the material be conveniently handled? E. Does the carrier have compression strength? F. Is the maximum enzyme loading adequate for the system? G. What is the maximum tolerable pressure drop? H. How is the above affected by particle size, flow rate, and particle shape? I. What is the operational half-life of the system? J. How is the half-life affected by temperature, pH, and other conditions? K. Under what conditions can the derivative be stored? By answering the above questions, one can determine whether one has the best carrier for the enzyme of interest. Studies in our labor~tdries have shown that inorganic supports, including porous glass and porous ceramics, have extremely different physical characteristics. Table I gives the results of durability tests on several different support materials. One can see from these data ,that under some conditions the ceramic 9D. L. Eaton, in "Immobilized Biochemicals and Affinity Chromatography" (R. Bruce Dunlap, ed.), pp. 241-258. Plenum, New York, 1974.

136

IMMOBILIZATION TECHNIQUES

[10]

T A B L E II COMPARISON OF SURFACE AREA TO PORE VOLUME FOR A CONTROLLED-PoRE INORGANIC SUPPORT

Pore diameter (/~)

Surface area s (m~/g)

Surface area b (m~/g)

75 125 175 240 370 700 1250 2000

249 149 107 78 50 27 15 9

356 214 153 111 72 38 21 13

° The pore volume for these calculations was taken at 0.70 ml/g. b The pore volume for these calculations was taken at 1.0 ml/g.

carriers are more durable than the glass. Thus, if one intends to operate an immobilized enzyme system at alkaline pH values, a ceramic, such as Ti02, might be the carrier of choice rather than controlled-pore glass (CPG). Another major faotor in choosing the carrier is the pore diameter relative to surface area (Table II). There will always be an optimal pore diameter for an enzyme. The relationship between pore diameter and surface area is an inverse relationship. Therefore, one always should choose the lowest possible pore diameter giving the greatest possible surface area and greater enzyme loading. This relationship is shown very nicely in Fig. 1 with amyloglucosidase covalently coupled to porous glass. It is immediately obvious that the optimum pore diameter is 300/~. Anything less excludes the enzyme. Porous glass can be prepared at any desired pore diameter over the range of 30/~ to 2000 )~. The pore diameter available on the ceramic carriers developed at Coming I° are not quite as broad, but they fall within the useful range (Table III). The choice of ~,articlesize also has a major impact on the activity of an immobilized ~ azyme. The larger the particle,the greater the effect of diffusion control (Fig. 2). Although the data indicate that the smallest particle size is usually the best choice, one must also consider pressure drop vs particle size. The larger the particle,the lower the pressure drop across a bed of immobilized enzymes (Fig. 3). lo

R. A. Messing, Res. Dev. 25, 32 (1974).

[10]

COVALENT COUPLING METHODS

137

I00

2 Jooc

A

b-

,?

l,--

\

W .J

I

o

i, ,

0

W

,o o~

J J J I

I

,,

,,,,l

IO0

I

IOOO

l

i

a I III

3ooo

PORE DIAMETER(~) 1~0. 1. Relationship of pore size to surface area and enzymic activity. 0 , Surface area date; X, enzymic activity results. TABLE III P H Y S I C A L P A R A M E T E R S OF P O R O U S C E R A M I C S

Composition SiO~ TiO2 SiO, ZrO2 SiO2

75 % 25 % 90 % 10 % 100 %

SiO2 84.3 % ZrO2 15.7% SiO~ Alz03 TiO2 MgO Al2Oa TiO2 TiO2 TiOz

75 % 25 % 98 % 2% 100 % 100 % 100 % 100 %

Pore diameter range (.~)

Pore diameter, average (~)

Pore volume (ml/g)

205-875

465

0.76

185-700

435

O. 76

185-700 110-575

435 235

0.76 1.30

205-575

435

0.89

205-500

410

0.53

150-250 220--400 300-590 725-985

175 350 420 855

0.60 0.45 0.40 0.22

138

[10]

IMMOBILIZATION TECHNIQUES

A 2000 0

_>

t..) I 0 0 0 )N Z W

I

I

200

I

400

600

I 800

AVE. PARTICLE DIAMETER (/~m)

l~o. 2. Typical results expected for a diffusion-restricted material. Enzyme activity increases as the particle size and diffusion path decrease in size or length.

I:~_

Ic~-~

E..o

,

!~l/l~! 0

5

/

/ I I I

IO 15 20 25 ~IO 35 ml/cm 2 / mln

Fro. 3. Experimental results for controlled-pore glass (CPG) showing the effect of particle size distribution on pressure drop and flow rate. All these parameters must be considered when choosing the inorganic support for enzyme immobilization.

E n z y m e Immobilization

Immobilization by adsorption techniques is covered in this volume [11]. W e concern ourselves here only with covalent attachment methods. Immobilization by covalent attachment to inorganic supports involves reactions that are similar to the covalent attachment of enzymes

[10]

COVALENT COUPLING METHODS

139

to organic supports. Only methods with which this author has had personal experience are presented (see also this volume [32], [54], [55] ). Preparation o/Carrier. Generally an acid wash (5% HN03) at 80 °90 ° for 60 min followed by rinsing with distilled water will hydrate and clean the carrier surface. Silane Coupling Techniques

Alkylamine Coupling Aqueous Silanization. This method of silanization appears to couple a monolayer of silane across the carrier surface. The organic solvent techniques give higher amine loadings. However, experience has shown that greater carrier durability with slightly lower enzyme loadings are achieved by aqueous silanization (Fig. 4). To 1 g of clean inorganic support material is added 18 ml of distilled water plus 2 ml of ~,-aminopropyltriethoxysilane (10% v/v). This compound is commercially available from Union Carbide, as are the other silanes. After addition of the silane solution, the pH is adjusted to between pH 3 and 4 with 6 N HC1. The neutralization of the basic silane solution may cause heating. After pH adjustment, place the reactants in a 75 ° water bath for 2 hr. Remove from bath, filter on a Biichner funnel, and wash with 20 ml of distilled water. Dry in a 115 ° oven for at least 4 hr. The product may be stored at this point for later use. I 0 I R (CHe)~ Si(OCH2CH3)3 ,'- H O - S i - O I 0 I HO--Si--O I 0 0

OH

O - - S i - - O -- Si(CHe ) n R 0

0

-O-Si-O0

WHERE R REPRESENTS AN ORGANIC FUNCTIONAL GROUP

Si(CHe)n R 0

- O - S l - O--Si(CH2)n R 0

OH

FIG. 4. Silanization of the surface of porous glass.

140

IMMOBILIZATION TECHNIQUES

[10]

Organic Silanization. This technique will give much higher loadings of alkylamine than the aqueous method. However, the silane appears to be somewhat patchy, and it is not quite as durable. To 1 g of clean porous carrier add 50 ml of a 10% solution of 7" aminopropyltriethoxysilane in toluene (v/v). Reflux overnight. Filter on a Biichner funnel, and wash with toluene followed by acetone. Air dry before placing in an oven overnight at 115% The product may be stored at this point. Another organic silanization method is an evaporative technique that lays down a more even silane layer. To 1 g of porous inorganic support material, add 25 ml of a 1% solution of ~-aminopropyltriethoxysilane in acetone (v/v). Evaporate to dryness and then heat at 115 ° overnight. The product may be stored at this point. Derivatives of Alkylamines and Coupling Techniques

Alkylamine Coupling Carbonyl Derivative. The technique using glutaraldehyde is simple, gentle, and rapid. It is important to remove all excess glutaraldehyde before adding the enzyme; otherwise cross-linking will occur. The crosslinked enzyme will decrease activity recovery by blocking pores and preventing passage of larger molecules {Fig. 5). To 1 g of alkylamine carrier, add 25ml of a 2.5% solution of glutaraldehyde in 0.05 M Na2HPO4 buffer adjusted to pH 7.0. Allow the reaction to continue for at least 60 minutes. During this time one should observe some sort of color change either to a magenta or tan. Wash exhaustively with distilled water on a Biichner funnel. To the activated carrier add the enzyme in as small a volume as possible at a minimum of a 1% concentration, if possible using the same buffer as that used in the activation step. The quantity of enzyme added should be between 50 and 100 mg per gram of carrier. Allow 2-4 hr for the reaction. Wash with distilled water. Soaking in 6 M urea has been used successfully for removing adsorbed enzyme without inactivating the coupled enzyme in most cases. However, before subjecting the entire preparation to a urea soak, it is advisible I

I

? CARRIER

o

. - O - S i ( C H l ) , NH, + (CH=)3 ~

(~--O-- Si (CH= )3 N-CH(CH =)sCHO

"-

(

o I

o'I

FIG. 5. Preparation of the aldehyde derivative from the alkylamine derivative.

[10]

COVALENT COUPLING METHODS I 0 CARRIER

--0I

I 0

$ (CH2)3NHz+

CI-C

141

-Cl

t

-0-

(CHzlsNCS 0 I

FIG. 6. Preparation of the isothioeyanatefrom the alkylamine derivative. to test a small batch first. Do not dry the sample of immobilized enzyme. Retain as a damp preparation, as recovered from the filter, in a sealed container in the refrigerator. Isothiocyanate Coupling. To 1 g of alkylamine carrier is added a 10% solution o f thiophosgene in chloroform (v/v). All work must be carried out under a hood because of the nauseous and toxic nature of the thiophosgene. The reactants are refluxed for at least 4 hr; generally 12-15 hr is best. Wash with dry chloroform and vacuum-dry in a desiccator. The derivative should be used as soon as possible for coupling to an enzyme {Fig. 6). The derivative is added slowly to a 1% enzyme solution, containing 50-100 mg of enzyme per gram of carrier, previously adjusted to pH 8.5-9.0. Monitor the pH and maintain until the pH stabilizes. Allow an additional 2 hr before washing and storing the final product. The reaction can also be carried out with an arylamine glass. The preparation of the arylamine will be presented below. Carbodiimide Coupling. This technique is useful if the enzyme must be coupled at an acidic pH value, as in the case of pepsin." It is preferable to use a carboxyl derivative, since there will be less chance of crosslinking to the carboxyl-activated carrier. However, coupling via carbodiimide to the alkylamine in many cases is an excellent coupling method (Fig. 7). To 1 g of alkylamine glass add 50 ml of 0.03 M H3P04 adjusted to pH 4.0. To this add 100-200 mg of a water-soluble carbodiimide. The carbodiimide we prefer to use is 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide, metho-p-toluene sulfonate. The enzyme, usually 50-100 mg per gram of carrier, is also added directly to the mixture. The reaction is continued overnight at 4 ° . The product is washed and stored until use.

Triazine Coupling. This reaction is relatively simple and deserves greater attention (Fig. 8). To 1 g of alkylamine carrier add 10 ml of benzene containing 0.2 ml of triethylamine and 0.3 g of 1,3-dichloro-5-methoxytriazine. Allow the mixture to react for 2-4 hr at 45°-55% Decant, wash with benzene, and dry 1~W. F. Line, A. Kwong, and H. H. Weetall, Biochim. Biophys. Acta 242, 194 (1971).

142

IMMOBILIZATION TECHNIQUES

[10]

I 0 R' I I H* R "' - - 0 - Si(CHI)3 NHt + N + + HOOC ! II 0 C I II N I R"

CARRIER

I 0

-0-

I4

0

NHR

ilCH=13NHCR °= + O=C 'I-H* I ? NHR"

"

'

FIO. 7. Preparation of an amide-linked ligand with the alkylamine derivative and a carbodiimide. R' and R" represent groups on the carbodiimide, e~;., cyclohexyl groups. R " represents the ligand having a free carboxyl group.

in an evaporator oven at 100 °. Coupling is in 0.05 M phosphate buffer pH 8.0 at 4 ° overnight using 50-100 mg of enzyme per gram of activated carrier.

Preparation of Arylamine Carrier The arylamine derivative is normally prepared by reaction of pnitrobenzoylchloride with the alkylamine derivative. The arylamine can be activated by the same processes used for organic arylamines. However, the most common use of arylamines is for coupling via azo linkage (Fig. 9). To 1 g of alkylamine carrier add 25-50 ml of chloroform containing 5% triethylamine (v/v) and 1 g of p-nitrobenzoylchloride. Reflux for at least 4 hr. Wash with chloroform, dry, and boil in a 5% solution of sodium dithionite in water. Dry and store in a dark place. CHtO H + II I

N'~N Cl

N'~N CI

* NH= -PROTEIN pH 8 - 9 ~ CH,O-I-N-..r-C, NyN CI

~-CH=Oy%C,

N-~

NH- PROTEIN

I~G. 8. Coupling through a substituted triazine.

[10]

COVALENT COUPLING METHODS I

O

O

CARRIER - O - S i (CH=)3 NH= + NO I O

I

143

'

,-O-;

CI

REFLUX

I

I

0 0 ( I II CARRIER/-O--Sli [CH, ], NHC " ~ O =

SODIUM DITHIONITE

O I

I

0

0 II CARRIER/--O- 4i (CH,)3 NHC-~,-NH, "

O I

Fro. 9. Preparation of the arylamine derivative from the alkylamine derivative.

Azo Coupling. To 1 g of arylamine carrier add 20 ml of 2 N HC1 and cool in an ice bath. To the cold preparation add 100 mg of solid NaN0s. Allow diazotization to continue at least 30 min. The carrier should take on a yellowish color. By testing with starch-iodine paper, one can tell whether any additional NAN02 is necessary during the diazotization procedure. After 30 min remove the derivative, then wash on a Bfichner funnel with ice cold water. The derivative is now ready for enzyme coupling {Fig. 10). The enzyme is dissolved in an appropriate buffer at pH 8.5. Usually 100 mg of enzyme per gram of carrier is a reasonable quantity of enzyme. The higher the enzyme concentration, the more efficient will be the coupling. A range of 0.5--2.0% enzyme solution is an excellent working concentration. For maximum coupling, allow the reaction to continue in

DIAZONIUM

CHLORIDE

OH

OH

PROTEIN

PROTEIN

F1o. I0. Coupling through azo linkage.

144

[10]

IMMOBILIZATION TECHNIQUES

FIG. 11. Preparation of the phenylhydrazine. the cold for at least 2 hr for proteases, 18 hr for enzymes not autolyzed. However, maximum coupling and recoverable activity may not be the same. One must determine optimum coupling time. Phenylhydrazine Coupling. This coupling technique is not very common but can be used for coupling through carbonyl groups (Fig. 11). One gram of arylamine carrier is diazotized as described above, washed with ice cold water followed by a wash with ice cold 1% sulfamic acid, followed again by ice cold water. The product is reduced by reaction with 10 ml of 1% sodium dithionite solution at pH 8.5 by refluxing. Refiuxing is continued with the addition of NaOH for pH maintenance as necessary for 60 min. The product is washed with water and is ready for immediate coupling under slightly alkaline conditions at 0 °.

Preparation of the Carboxy Derivative The earboxy derivative can be prepared by the reaction of the alkylamine carrier with succinie anhydride (Fig. 12). To 1 g of alkylamine carrier is added 25 ml of 0.05 M phosphate buffer adjusted to pH 6.0. To this is added 1.0 g of succinic anhydride. The preparation is stirred at room temperature with pH adjustment as necessary over the first few hours. Continue the reaction for at least 15 hr at room temperature. The product is washed and can be dried. Carbodiimide Coupling to Carboxyl Derivative. Carbodiimide coupling to the alkylamine carrier can cross-link; coupling to the carboxyl derivative cannot, if handled properly (Fig. 13). To 1 g of carboxylated derivative is added 200 mg of 3-(2-morpholinoethyl) carbodiimide previously dissolved in distilled water. The reacrants are adjusted to pH 10.0 and allowed to react at room temperature for 2 hr. Successful activation can also be achieved at pH 4.0. The product is washed with distilled water and added to the enzyme solution. Although I

o

CARRIER

-0-

%f

Si (CH~,), NH= * /

6 I

o -..~o

(

I

o~

o,

I""~"~)-O-Si(CH,),NHC(CH,)~,

COOH

0 I

FIo. 12. The covalent coupling of an a m i n e functional ligand with the derivative followedby reduction with sodiumborohydride.

aldehyde

[10]

COVALENT COUPLING METHODS

145

I

0 0 0 R ( I II II I CARRIER /--O-Si(CHe)aNHC (CH=)tC-OH + NIl 0 C I II N I

R"

RI

,°' I

oII

oII

/H+ I

O-Si(CH:)3 NHC (CHI) z C--O-C I 0 NH I I R"

FIG. 13. The preparation of the pseudourea of a carboxyl derivative by reaction with a carbodiimide. coupling will occur at pH 4.0-5.0, we have observed coupling at pH 9-10 also. The final product is washed and stored for use. Acid Chloride Coupling. The acid chloride can be prepared from the carboxyl derivative. However, it is not very stable and should be used soon after preparation (Fig. 14). To 1 g of carboxylated carrier add 50 ml of a 10% solution of thionylchloride in chloroform. The preparation is refluxed for 4-6 hr, filtered, and dried in a vacuum oven at 600-80 °. .Coupling is in a slightly alkaline solution. The activated carrier is added to the enzyme solution previously adjusted to pH 8.0-8.5 with the appropriate buffer. The pH must be maintained by the addition of NaOH solution. After complete addition of the derivative, permit the reaction to continue for 1-2 hr at room temperature. Wash and store at 4 ° until use.

N-Hydroxysuccinimide Ester. Active esters are rather easy to prepare. They can be stored for later use and coupled to enzymes by rather gentle procedures (Fig. 15).

~

_CH2CO0H SOCI=_ {--CH=COCI

~

-CH,COCl + N H , --PROTEIN pH8-9=

~--CH=CONH-PROTEIN

FxG. 14. Coupling through an acid chloride.

146

[10]

IMMOBILIZATION TECHNIQUES

o COOH ÷ HO.--

o R-N=C=N'R'"

o

C-O-

Fro. 15. The preparation of the N-hydroxysuccinimide ester.

To 1 g of carboxylated carrier, add 10 ml of dioxane. To this now add 200 mg of N-hydroxysuccinimide and 400 mg of N,N'-dicyclohexylcarbodiimide. The reaction is continued for 4 hr. Wash the derivative with dioxane followed by methanol. The product can be dried in an evacuated oven at 70°-80 ° for 1 hr. Store refrigerated in an amber bottle. Keep desiccated if possible.

Activation o] Inorganic Supports with ,/-Mercaptopropyltrimethoxysilane The preparation of an inorganic support having surface sulfhydryl groups may be useful for coupling through sulfhydryl linkages. To 1 g of cleaned porous carrier add 5 ml of a 10% aqueous solution (v/v) of ~-mercaptopropyltrimethoxysilane previously adjusted to pH 5.0 with 6 N HC1. The mixture is refluxed for 4 hr, then washed with distilled water. The product is heated in an oven at 120° for 4 hr.

Preparation o] Alkylhalide Silane Derivative To 1 g of clean porous glass is added 25 ml of a 10% solution of ~-chloropropyltriethoxysilane (v/v) in toluene. The mixture is refluxed for 4 hr, washed with toluene, and dried at 120° for an additional 4 hr. The dried product should be stored in a desiccator until use. Coupling can be accomplished by very slow addition of the alkyl chloride to an enzyme solution at pH 8-9. The pH should be maintained with NaOH. Coupling is carried out at 0 ° in an ice bath. Covalent Attachment to Inorganic Supports in the Absence of Silane Coupling Agents The silanol residues on the surface of glass and the metal oxide groups on ceramic surfaces appear to be capable of reaction with several organic activating groups. The activated carriers will react with enzymes, forming permanent linkages.

Cyanogen Bromide Coupling to Porous Glass One gram of clean porous glass is suspended in 25 ml of distilled water. The pH is adjusted to 10-11 with NaOH, and 1.0 g of CNBr is very slowly added with maintenance of pH. The reaction temperature

[10]

COVALENT COUPLING METHODS

I

I

- O-Si-OH I 0

I

- O - S iI- O " C = N t- BrCN

~

-O- ISi-O~

0

~

0

, -o-=,-o..

,

-o- 4, - J

0

0

0

I

I

I

I

0 I

-O-Si-Q

i, o: c -

I

O I

0 II

- O - S i - O - C N H - PROTEIN • N + NH z-

-0-

CfNH

I

0 I

147

PROTEIN

" -O-

O

Si-OH I

O I

Fro. 16. Possible mechanism of CNBr activities of glass. should be kept between 15 ° and 20 ° by the addition of ice as necessary. The CNBr should be ground into small pieces before addition. Wear gloves and carry out the reaction in a hood (Fig. 16). After addition of the CNBr is complete, continue the reaction until the pH remains constant. Wash on a filter with cold distilled water and add to an enzyme solution previously adjusted to pH 9.0. Continue reaction in an ice bath for 2-4 hr. Wash and store. Several washes with 6 M urea will remove the adsorbed enzyme. This can be monitored by assay after each 30-min urea soak. When the activity levels off, the adsorbed activity has been completely removed.

Coupling to Porous Glass through a Bi]unctional Bis-Diazotized Reagent TM A solution of 0.01~ 4,4'-bis(2-methoxybenzene diazonium) chloride is prepared by suspension in distilled water. The diazonium salt can be purchased from J. T. Baker Company. The suspended agent is stirred until dissolved at room temperature. To 500 mg of porous glass add 2 ml of 0.01% coupling reagent. Allow the reagent to react with the carrier at room temperature for 20 rain. Decant and wash the glass with distilled water. To the activated glass now add 10 ml of a 1% enzyme solution (w/v) dissolved in 0.1 M phosphate buffer at pH 7.8. The reaction is continued at 37 ° for 3 hr (Fig. 17). The product is then allowed to react overnight at room temperature, washed with distilled water, then with " R . A. Messing and It. R. Stinson, Mol. Cell. Bioehem. 4 (3), 217 (1974).

148

IMMOBILIZATION TECHNIQUES OCHs

OCHs

[11]

/ ,

CIN=

N-NCl 4- HO- Si-O-I

0 I

oc.,.

oc,,

GIN=N ~ - O - - ! i - - O -

,l,Np * HCl 0 I

FI¢]. 17. Coupling through a bifunctional reagent.

0.5 M NaC1 solution followed again by water. The sample can be stored in water at 4 ° .

Choice of Coupling Techniques The choice of coupling technique should be based upon the characteristic of the enzyme to be immobilized. Enzymes with deactivate at pH values greater than pH 8 should be immobilized using techniques which are most effective at lower pH values, e.g., glutaraldehyde or, as in the case of pepsin, carbodiimide. Similarly, enzymes easily denatured at lower pH values should be immobilized with methods most useful at alkaline pH values. Other factors to be considered should include: groups in the active site that may be capable of binding to the activated carrier, coupling temperature, ionic strength, composition, and any other parameter that could denature the enzyme or interfere with the coupling reaction. In the case of proteases, one should always couple at low temperatures (00--4° ) for the shortest period of time to decrease the amount of autolysis that occurs. The simplest rule of thumb is to use a little common sense.

[11] A d s o r p t i o n a n d I n o r g a n i c B r i d g e F o r m a t i o n s

By RALPH

A. MESSING

Adsorption appears in the author's opinion to be the most economical procedure for immobilizing an enzyme on a carrier (See also this volume [3] on hydrophobic adsorption of enzymes and [51] on aminoacylase ad-

Covalent coupling methods for inorganic support materials.

134 IMMOBILIZATION TECHNIQUES [10] Groups on the Protein. Polyisonitrile-nylon (50 rag) is suspended in 1 ml of cold 0.1 M Tris.HC1 buffer pH 7.0...
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