Optimization of glutaraldehyde activation of a support for enzyme immobilization

371

Journal of Molecular Catalysis. 3 (1977178) 371384 0 Elsevier Sequoia S-A_, Lausanne - Printed in the Netherlands

OPTIMIZATION SUPPORT FOR

OF GLUTARALDEHYDE ACTJYATION ENZYME IMMOBILIZATION

OF A

P- MONSAN Laboratoire de GEnie Biochimique, Rangueil. Toulouse (France) (Received

Insfifut

NafionaZ des Sciences

AppZiqu&es.

Avenue

de

April5,1977)

Summary The influence of several parameters (support porosity, glutaraldehyde concentration, time of action, pH) on the activation reaction of an amine porous silica (Spherosil) with glutaraldehyde has been studied_ Glutaraldehyde binding onto the support was followed by measuring the carbon content of the activated silica_ We established comparisons between the quantity of glutaraldehyde retained on the support after activation and the capacity of the activated silica to bind trypsin We have defined the optimal conditions for Spherosil activation and prepared derivatives with high enzymatic activity_ Our results are in agreement with a reaction mechanism with glutaraldehyde in a polymeric form resulting from aldol condensation, rather than in a monomeric form_

Introduction A variety of methods for enzyme immobilization has been described during recent years [l - 3]_ We have investigated the possibility of using, as an enzyme carrier, a porous silica manufactured by Rhcne-Poulenc, SpherosilThis silica may be functionalized via silane coupling, and one of the most suitable methods for large scale enzyme immobilization and reactor use, is amination with y-amino propyl trimethoxysilane, followed by glutaraldehyde activation_ This method has been widely used, particularly by Weetall’s group [4] _ Nevertheless, it seemed necessary to carry out a systematic study of the effect of the activation conditions with a view to optimizing the use of glutaraldehyde. Furthermore, we intended to compare the mechanism we recently proposed to describe the reaction of glutaraldehyde with protein amine groups [5], with the results concerning the activation of an amine support with glutaraldehyde. We have reported that the attainment of stable products after glutaraldehyde reaction may involve polymeric forms resulting

373 natant is sucked up after silica beads have settled down- Excess glutaraldehyde is washed off by repeated contact of the support with 20 ml of the above pyrophosphate buffer over a period of one hour at 25 “C_ The activated Spherosil is washed until the supernataut does not show a reaction with Schiff’s reagent for aIdehyde detection. The support is then washed three times with 20 ml of buffer solution in which immobilization will take place (if this buffer is not pyrophosphate buffer). The effect of the pH on glutaraldehyde activation was studied using the following 0.05 M buffers: hydrochloric acid-potassium chloride, pH 1-O; sodium citrate-citric acid, pH 3.0; monosodium phosphate-sodium citrate, pH 5.0; monosodium phosphate-disodium phosphate, pH ‘7.0; sodium pyrophosphate-hydrochloric acid, pH 9.0; sodium carbonate-sodium bicarbonate, pH 10.5. Carbon content and nitrogen content determination For the silica carbon content and nitrogen content determinations, excess glutaraldehyde is washed off as previously described. Samples are then washed with 20 ml of distilled water once during 15 h and four times during 1 h, at 25 “C, and finally oven dried. Carbon content determinations were undertaken at the Rhone-PouIenc Research Center in Decines- After sample combustion in a microanaIysis unit, the produced COz is measured by coulometric means in a carbon-type Schoeps cell packed with barium perchlorate. An automatic nitrogen analyser, ANA Mod. 1300 (Carlo Erba), was used for nitrogen content determinations. Nitrogen oxides from sampIe combustion are reduced on chromium and copper oxide beds. The resulting molecular nitrogen is detected with a catharometer and the corresponding peak is automatically integrated_ Trypsin immobilization We used a commercial hog pancreas trypsin preparation (Sigma ChemCo., type IX)_ A 100 mg sample of amine-SpherosiI activated with glutaraldehyde is roller-mixed with 20 ml of a 2 g 1-l trypsin solution in O-05 M pyrophosphate buffer, pH 8-6 for 2 h at 4 “C- The support is then washed at 4 “C, four times over 30 min with 20 ml of the same buffer, once during 15 h with 20 ml of a 1 M NaCl solution in 10-a N hydrochloric acid and four times during 30 min with 20 ml of a 10e3 N hydrochIoric acid solution_ The amount of immobihzed protein is determined by measuring the protein content of the trypsin solution, before and after reaction and successive washes, by the Lowry method. Activity measurements are performed using DL-lysine methyl ester as substrate. L-Iysine resulting from trypsin action is continuously neutralized by a 0.1 N sodium hydroxide addition with an automatic pH Stat device, Tacussel TAT 4_ The pH set point is pH 5.5 and the temperature 30 “C. The assay mixture contains 1 ml of trypsin solution (or a suspension of immobi-

374

TABLE 2 Effect of amine-Spherosil

specific area on glutaraldehyde fixation

Specific area Cm2 g-I)

Carbon content variation after activation (W)

Number of gram-atoms of carbon per gramatom of nitrogen

6 37 120 180 445

O-76 2.41 5-62 7.32 10.48

10.50 12.56 12.00 11.73 11.49

Experimental conditions: amine-Spherosil of various specific areas: 100 mg; glutaraldehyde concentration: IO%, in 0.05 i#fpyrophosphate buffer, pH 8-6; volume of the glutaraldehyde solution: 20 ml; activation for 15 h at 25 “C.

lized trypsin in 1 ml of 10-s N hydrochloric acid) and 9 ml of DL-lysine methyl ester. The final concentration of DL-lysine methyl ester solution is 0.6 M in the case of soluble trypsin assay and l-5 M in the case of immobilized trypsin assay. One unit of enzyme activity corresponds to the production of one micromole of L-lysine per minute under the conditions of assay. The soluble trypsin specific activity is 50 U mg-‘_

Results (i) Effect of amine-Spherosil specific area Amine-Spherosil samples of various specific areas, ranging from 6 to 445 m2 g-’ were activated for 15 h at 25 “C with a 10% glutaraldehyde solution in O-05 M pyrophosphate buffer, pH 8.6. After washing off excess reactant, fixation of glutaraldehyde on the support was followed by measuring its carbon content. Figure 1 shows that the carbon content of unactivated Spherosil increases in a nearly linear way with specific area- After support activation, the higher the specific area, the higher the amount of silica-bound glutaraldehydeThe number of gram-atoms of carbon statistically bound per amine gr~upinitiallypresentonSpherosilmaybe determined from the variation of the carbon content and the initial nitrogen content of the silica. It can be seen from Table 2 that, ;J1 each case, this number is greater than five. But the gluta.raIdehyde molecule contains five carbon atoms_ This result is thus inconsistent with a reaction mechanism involving glutaraldehyde under its monomeric form. Furthermore, the calculated number of gram-atoms of carbon bound per gram-atom of nitrogen may only be undervalued because we considered that all the Spherosil amine groups had reacted with glutaraldehydeIf we assume that after glutaraldehyde reaction it remains one aldehyde group

375

TABLE

3

Effect of amine-Spherosil

specific area on trypsin immobilization

Specific area (m’ g-l)

Amount of immobilized trypsin (mg/g silica)

Activity (U/g silica)

Specific activity (U/mg immobihzed trypsin)

6 37 120 180 445

32.9 120-l 106.9 90.5 11.0

398 416 352 196 90

12.10 3.46 3-29 2.17 8.18

Experimental conditions: amine_Spherosil of various specific areas, activated with a 10% glutaraldehyde solution in 0.05 M pyrophosphate buffer, pH 8.6, during 15 h at 25 “C: 100 mg; trypsin concentration: 2 g 1-l in the same pyrophosphate buffer; volume of the trypsin solution= 20 ml; reaction for 2 h at 4 “C_

100

_

200

360

900

Fig. 1. Effect of specific area on Spherosil carbon content before ( -_) and after (o--O) glutaraldehyde activation. Experimental conditions-. amine-Spherosil of various specific areas: 100 mg; glutaraldehyde concentration- 10% in 0.05 M pyrophosphate buffer. pH S-6; volume of the glutaraldehyde solution: 20 ml; activation for 15 h at 25 “C per five carbon immobilization

atoms, ranges

the density

of available

reactive

groups

for enzyme

from 1 X lO*O to 10 X 1020 aldehyde groups per gram

of activated Spherosil. The activated support has been reacted with a 2 g 1-l trypsin solution in O-05 M pyrophosphate buffer, pH 8.6, for 2 h at 4 “C. After elimination of

376

unreacted enzyme, the amount of bound protein and the activity of immobilized trypsin were determined. The efficiency of the reaction of trypsin couphng onto activated amine-Spherosil depends greatly on carrier porosity (Table 3) The optimal specific area is about 50 m2 g-l. The activity of trypsinSpherosil derivatives is lower for low specific areas as, to a greater extent, for high specific areas. Nevertheless, the specific activity of immobilized trypsin varies in an opposite way: the specific activity is higher for extreme specific areas than for intermediate specific areas. The yield of the immobilization reaction, defined as the ratio of immobilized trypsin specific activity to soluble trypsin specific activity, ranges from 4.3% (180 m2 g-l) to 24.2% (6 m2 g-l) (Table 3)_ (ii) Effect ofglufaraldehyde concentration AmineSpherosil(37 m2 g-l) was activated with various gktaraldehyde solutions in O-05 M pyrophosphate buffer, pH 8.6. The glutaraldehyde concentration varied from O_l% to 20% (Fig- 2). After activation, the support carbon content rapidly increases with glutaraldehyde concentration, and a constant value is obtained for 10% and 20% gIutaraIdehyde_ For glutaraIdehyde concentration ranging from O-l% to lo%, there is a linear relationship relating the activated Spherosil carbon content to the Iogarithm of glutaraIdehyde concentration:

lunclctwotcd

_-_-----------L-

glutaroIdRhydo 5

10

Spherosrt

.

concrntrotron 15

r 5-m1 20

Fig_ 2_ Effect of ghztaraldehyde concentration on Spherosil carbon content before (- -)

and after (-0) activation. Experimental conditions: amine-Spherosil (37 m2 g-I): 100 mg; various gIutaraldehyde concentrations in O-05 M pyrophosphate buffer, pH 8.6; volume of the glutaraIdehyde solution: 20 ml; activation for 2 h at 25 “C

377

carbon content = 1.927 + 0.395 log (glutaraldehyde

concentration).

Whei the glutaraldehyde concentration is higher than IO%, the carbon content is nearly constant and equal to 2_31%_ The number of gram-atoms of carbon remaining on the carrier after activation may be calculated from the carbon content variation and related to the initial nitrogen content (Table 4)_ Except for the 0.1% glutaraldehyde concentration, the number of gram-atoms of carbon per gram-atom of nitrogen is higher than five. TABLE 4 Effect of glutaraldehyde concentration on the reaction with amineSpherosil Glutaraldehyde concentration (%) O-1 : 10 20

Carhon content variation after activation (%)

Number of gramatoms of carbon per gram-atom of nitrogen

0.81 1.20 1.46 l-59 1.58

4.19 6.25 7.62 8.31 8.25

Experimental conditions: amine-Spherosil(37 RI* g-l): 100 mg; various glutaraldehyde concentrations in 0.05 M pyrophosphate buffer, pH S-6; volume of the gIutaraldehyde solution: 20 ml; activation for 2 h at 25 “C. Trypsin was immobilized onto amine-Spherosil activated with glutaraldehyde solutions of various concentrations, as described above_ Using activity as the criterion, the optimal glutaraldehyde concentration is equal to 2%

(Table 5). For higher glutaraldehyde concentrations, the activity of immobilized trypsin derivatives is somewhat lower. The specific activity of immobilized trypsin is fairly constant for glutaraldehyde concentrations between 0.02% and 2%_ Also, the samples activated with a O-02% glutaraldehyde concentration exhibit 52.1% of the activity of the sample activated with a 100 times greater glutaraldehyde concentration, namely, 2%. (iii) Effect of time of giutaraldehyde activation We investigated the effect of contact time on the carbon content of aminespherosil activated with a 10% glutaraldehyde solution in O-05 M pyrophosphate buffer, pH 8_6The reaction of glutaraldehyde seems to involve a two step mechanism (Table 6). The reactant first very quickly reacts with support amine groups: the carbon content is rapidly increased during the first half-hour of agitation_ Then, in a second step, the carbon content increases at a much lower rate_ The number of gram-atoms of carbon per gram-atom of nitrogen is here, too, higher than five, even after just 15 min contact.

378

When bonding trypsin onto amine-Spherosil activated for various lengths of time with gIutara.ldehyde, the immobiked enzyme derivatives have nearly the same activity, whatever contact time is considered (Table 7). The amount of trypsin Iinked to the support rapidly increases and is nearly constant for agitation times longer than 30 min. The shorter the time (15 min), the higher the specific activity of the immobilized trypsin. (iv) Effect

of pN on glutarbldehyde

acfivation

Amine-Spherosil was activated with 10% glutaraldehyde solutions at different pH values for 3 h at 25 “C. L TABLE Effect

5 of ghrtaraldehyde

concentration

Glutaraldehyde concentration

Amount of immobilized

(m2 g-l)

trJTpsin (mg/g silica)

0.02 0.1 0.5 2 5 10

66 92 121 126 138 134

on trypsin

immobilization

Activity (U/g silica)

Specific activity (U/mg immobilized trypsin)

148 204 264 284 280 262

2.24 2-22 2.18 2.25 2.03 1.96

Experimentel conditions: amine-Spherosil(37 m2 g-l). activated with glutaraldehyde at various concentrations in O-05 M pyrophosphate buffer. pH S-6: 100 mg: trypsin concenbuffer; volume of the trypsin solution: 20 ml; tration: 2 g 1-l in the same pyrophosphate reaction for 2 h at 4 “C_ TABLE Effect Time (b)

0.25 0.50 0.75 1 1.5 2 3 15.5

6 of time on glutaraldehyde

fixation

onto

Carbon content variation after activation

amine-Spherosil

(%)

Number of gramatoms of carbon per gram-atom of nitrogen

1.55 1.38 1.74 174 l-72 l-78 1.96 2.48

8.06 8.75 9.06 9.06 8-94 9.25 10-19 12.94

Experimental conditions: amineSpherosil(37 tration: 10% in O-05 M pyrophosphate buffer, tion: 20 ml; activation for 2 h at 25 “C.

m2 g-I): 100 mg; glutaraldehyde concenpH 8.6; volume of the glutaraldehyde solu-

379 TABLE Effect

7 of the time of glutaraldehyde

Time

activation

on trypsin

immobilization

(h)

Amount of immobilked trypsin (mg/g silica)

Activity (U/g silica)

Specific activity (U/mg immobilized trypsin)

O-25 o-5 1 l-5 2 16

120 138 140 148 136 155

280 290 308 296 286

2-33 2.10 2.20 2.00 2-10

312

2-02

Experimental conditions: amine-Spherosil(37 m2 g-l), activated with a 10% glutaraldehyde solution in 0.05 M pyro hosphate buffer, pH 8.6, for various times at 4 “C: 100 mg; trypsin concentration: 2 g I-R m the same pyrophosphate buffer; volume of the trypsin solution: 20 ml; reaction for 2 h at 4 “C. Figure 3 shows that the carbon content of silica slowly increases when activated at a pH ranging from pH 1 to pH 9. For higher pH values the amount of glutaraldehyde bound to the carrier is sharply increased, owing to

carbon

content

( X)

Fig- 3. Effect of pH on Spherosil carbon content before () and after (u ) glutaraldehyde activation. Experimental conditions: amineSpherosil(37 m2 g-l): 100 mg; glutaraldehyde concentration: 10% in various 0.05 Jf buffers (see Materials and Method); volume oE the glutaraldebyde solution: 20 ml; activation for 3 h at 25 “C_

380 TAEkLE 8 Effect

of pH on glutamldehyde

PH

1-O 3-O 5-O 7.0 9.0 10-O 10.5

fixation

onto

amine-Spherosil

Carbon content variation after activation (%)

Number of gram-atoms of carbon per gram-atom of nitrogen

0.89 1.43 l-48 l-84 1.98 2.83 10.12

4-51 7-25 7-51 s-33 10.04 14-36 51.33

ExperimentaI conditions: amine-Spherosil(37 m2g-‘): 100 mg; glutaraldehyde eoncentration: 10% in various 0.05 M buffers (see Materials and Methods); volume of the glutaraIdehyde soiution: 20 ml; activation for 3 h at 25 “C. TABLE Effect

9 of the pH of activation

on trypsin

immobilization

PH

Amount of immobihzed trypsin (mg/g silica)

Activity (U1g silica)

Specific activity (U1mg immobilized trypsin)

1.0 3-o 5-O 7-o 8-6 10.5

60 71 90 114 166 152

172 196 240 296 348 218

2-87 2.76 2-67 2.60 2-10 1.43

Experimentaf conditions: amin+Spherosil(37 m2 g-‘) activated with a 10% glutaraidehyde solution in various 0.05 M buffers. for 3 h at 25 “C: 100 mg; trypsin concentration: 2g1-= in O-05 M pyrophosphate buffer, pH 8.6; reaction for 2 h at 4 “C

a polymerisation of glutaraldehyde resulting from aldof condensation. The number of gram-atoms of carbon per gram-atom of nitrogen is lower than five only when activation was performed at pH 1, For higher pH values, the number of gram-atoms of carbon increases up to 51-33 at pH 10.5. This, statistically, corresponds to about 10 glutaraldehyde molecules per amine group initiahy present on the carrier. lkypsin was immobilized on amine-Spherosil sampIes activated at various pH’s. Both enzymatic activity and the amount of immobilized trypsin show a maximum for pH 8-6 (Table 9) The specific activity of immobilized trypsin is lowered when the activation pH is increased, particuIarly at pH 10.5. Moreover, silica undergoes an important dissohxtion above pH 9171, and this restricts the support activation possibilities at high pH values.

381

Discussion We have investigated

the effect of the main parameters on the glutar-

aldehyde activation step of an amine porous silica, Spherosil, used for enzyme immobilization. The amine group density of the support is directly related to its spec’&c area- This may be why the amount of giutaraldehyde bound to amine-spherosil after activation (determined by following the carbon content variation of the carrier) is higher as the specific area increases- The number of carbon atoms retained on the support per nitrogen atom initially present is higher than five- This is inconsistent with a glutaraldehyde reaction mechanism involving this reactant as a monomer molecule containing five carbon atoms. On the other hand, this result may be consistent with the reaction mechanism we proposed for glutaraldehyde [5 ] involving polymeric forms resulting from aldol condensation- The size of these glutaraldehyde polymers depends on the medium conditions_ When trypsin is bound to activated amine-Spherosil of various specific areas, the most important parameter is not the density of the reactive groups, that is to say the amount of glutaraldehyde present after activation, but the support porosity_ In fact, there is an optimal specific area of about 50 m2 g-l_ This corresponds to a pore size of 600 A. The optimum is nearly the same for the amount of immobilized trypsin and for the catalytic activity. It can be seen that trypsin is bound to an important extent for a pore diameter greater than 150 A. This result may be compared with the observation made by Messing 18) in the case of glucose oxidase and catalase adsorption onto a porous carrier: for both enzymes, the optimal pore size is equal to twice the length of the enzyme molecule major axis. For higher specific areas, the pore size is not large enough to allow penetration of the enzyme and, thus, only the external area is available for immobilization: 11 mg of trypsin are then linked per g of Spherosil- For the 6 m2 g-I specific area support,trypsin freely gets inside the pores, and the limitation only comes from the available area. The specific activity of immobilized trypsin is higher for extreme specific areas, 6 m2 g-l and 445 m2 g-l_ Th*IS may be a consequence either of low diffusional limitations (the enzyme acts on bead surface or within very large pores), or of the minimum number of enzyme-support bonds (the enzyme is just bound at the surface either of the Spherosil bead or of the pore and the weak enzyme-enzyme interactions because of the low amount of immobilized trypsinThe amount of glutaraldehyde bound to activated amine-Spherosil increases with initial glutaraIdehyde concentration. Nevertheless, a constant value is obtained for 10% and 20% glutaraldehyde, probably because of a saturation of all the available amine functions_ Except for 0.1% glutaraldehyde the number of carbon atoms per nitrogen atom is here, also, greater than five. The optimai activity of immobilized trypsin is observed for about 2% glutaraldehyde and does not correspond to the constant level of glutaralde-

hyde pointed out above- For glutaraldehyde concentrations ranging from 0.02% to 2% the specific activity of immobilized trypsin is almost constant: the activity of insoluble derivatives is thus directly proportional to the amount of immobilized-enzyme_ For glutaraldehyde concentrations higher than 510, the specific activity of immobilized trypsin decreases, although the amount of fixed enzyme is slightly increased. This may be either due to a more important enzyme-enzyme interaction, or to a higher number of enzymesupport bonds per trypsin molecule resulting in a lowered catalytic efficiency_ Surprisingly, a glutaraldehyde concentration as low as 0.02% , i.e., 2.1 X 10-s M, allows binding of 66 mg of kypsin per gram of siiica. The activation of a limited number of amine functions thus seems to be sufficient for binding up to 48% of the maximal amount of trypsin which can be immobilized. The glutaraldehyde reaction mechanism with Spherosil amine groups may involve two steps. Glutaraldehyde firstly reacts very rapidly with the support: after 15 min contact, the carbon content of activated Spherosil is equal to 90% of the carbon content after one hour of activation- Beyond one and a half hours, glutaraldehyde continues to react with Spherosil, but at a decreased rate_ This may be a consequence of diffusion limitation of the reaction within silica pores, glutaraldehyde reacting with more and more hidden amine functions. A second hypothesis may also be considered, however, involving aldol condensation of free glutaraldehyde molecules with glutaraldehyde molecules still grafted onto silica- In fact, whatever contact time is considered, the number of carbon atoms per nitrogen atom of the activated support is always higher than five, and varies from 8-06 to 12.94 for 15 min and 15.5 h of contact, respectively. Trypsin immobilization seems to be directly related to the amount of glutaraldehyde bound to the support: the amount of immobilized enzyme and the activity of derivatives vary in the same way as Spherosil carbon content In consequence, the specific activity of immobilized trypsin is nearly constant for all activation times. The mechanism we proposed for glutaraldehyde reaction 151 may explain the effect of pH on the carbon content of activated Spherosil. For very low pH values (pH I), glutaraldehyde essentially reacts under its monomeric form: the number of carbon atoms fixed per nitrogen atom is then lower than five- On the other hand, for high pH values, particularly above pH 9, the amount of glutaraldehyde fixed onto silica sharply increases as a consequence of glutaraldehyde reaction in its polymeric form. So, the number of carbon atoms per nitrogen atom is equal to 51.33 after activation at pH 10.5. This corresponds to an average length of about ten residues for the “poIy glutaraldehyde” chains- However, the number of carbon atoms per nitrogen atom is higher than five from a pH value as low as 3, and this implies the presence of glutaraldehyde poIymers on the support at this pH value. But our observations [5] and the observations made by several authors [9 - 221, agree with the absence of any polymeric form in glutamldehyde solutions at pH 3 (which is approximately the pH of glutaraldehyde commercial solutions). Polymerisation of glutaraldehyde may thus be induced by an appro-

383

priate microenvironment on the Spherosil surface, because of the presence of positively charged amine functions which increase the local pH valueHardy et al. 1121 recently proposed a glutaraldehyde reaction mechanism involving three glutaraldehyde molecules and one lysine moiecuie to lead to the formation of a derivative, the structure of which is similar to the structure of desmosine and isodesmosine. If such a mechanism may describe the reaction of glutaraldehyde with Spherosil amine groups, it may absolutely not describe the reaction of an enzyme with a glutaraldehyde activated supportAs a matter of fact, as previously described, excess glutaraldehyde is thoroughly washed off before enzyme coupling. The reaction of the enzyme molecule may only then involve glutaraldehyde residues bound to the carrierWhen trypsin is immobilized on amin+Spherosil activated at different pH values, the amount of bound enzyme is directly related to the amount of glutaraldehyde linked to the support_ Nevertheless, when Spherosil is activated in highly alkaline conditions (pH 10.5), the amount of immobilized trypsin is slightly lower than after activation at pH 8-6, although the amount of bound glutaraidehyde is largely more important_ This may be ascribed to the fact that the very high glutaraldehyde con_+nt .-_ of Spherosil after activation at pH -10.5 causes a decrease of the area availabIe for immobilization by plugging silica pores. Furthermore, the specific activity of trypsin immobilized on Spherosil activated at pH 10.5 is much lower than the specific activity of other trypsin derivatives. This may be the consequence of the higher number of enzyme-support bonds, resulting from the higher number of aldehyde groups available on the silica, which restricts the trypsin catalytic efficiencyThe optimal conditions for glutaraldehyde activation of amine-Spherosil are as follows: a porous silica of 37 m* g-l specific area is activated for 1 h at 25 “C with a 2% glutaraldehyde solution at pH 8.6_ The glutaraldehyde reaction mechanism is, from the carbon content variation of the support after activation, inconsistent with a reaction of glutaraldehyde under its five carbon atoms monomeric form_ On the other hand, our results may be explained by a mechanism involving gIutaraldehyde polymeric forms resulting from aldol condensation.

Acknowledgements We thank Messrs Meiller and Mirabel (Rhbne-Poulenc Research Centre, Croix de Bemy) for kindly giving Spherosil, Mr Martinaud (RhGne-Poulenc Research Center, Decines) for determination of the carbon content of activated Spherosil, and Mrs Suderie for technical assistance_

References 1 0. FL Zaborsky, Immobilized Enzymes, CRC Press. Cleveland, 1974_ 2 H_ & Weetall (ea.), Immobilized Enzymes, Antigens, Antibodies and Peptides, Marcel Dekker, New York, 1975.

384 3 4 5 6 7 8 9 10 11 12

G_ Durand and P- Monsan, Les Enzymes Immobilis&s, APRIA. Paris, 1974H. & Weetall, Cereal Foods World, 21 (1976) 58X- 587P_ Monsan, G_ Puzo and H_ Mazarguil, Biocbimie, 57 (1975) 1281- 1292. J_ M_ Navarro and P_ Monsan, Ann. Microbial. (Paris), 127B (1976) 295 - 307A_ M_ Filbert, in R_ A. Messing (ed.), Immobilized Enzymes for Industrial Reactors, Academic Ress. New York, 1975, pp_ 39 - 61. R A. Messing, Biotechnol- Bioeng., 16 (1974) 897 - 908P- M- Hardy. A- C. Nicholls and H_ N. Rydon, Chem_ Commun., (1969) 565 - 566. A. H. Kom, S. H. Feairheller and E. M. Filachione, J. Mol. Biol., 65 (1972) 525 - 529. E. B. Whipple and M_ Ruta, J. Org_ Chem., 39 (1974) 1666 - 1668_ P. M. Hardy, A_ C_ Nicholls and H_ N_ Rydon, J. Chem. Sot., 9 (1976) 958 - 962.