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Studies on the morphology of immobilized catalase
B41
The Chemical Engineering Journal, 55 (1994) B41-B45
Studies on the morphology of immobilized catalase P.T. Vasudevan*t and R .H . Weilandtt Department of Chemical Engineering, Clarkson University, Potsdam, NY 13676 (USA) (Received June 10, 1993)
Abstract Scanning electron microscopy studies were carried out on bovine liver and Aspergillus catalase immobilized on a non-porous glass support during various stages of immobilization and reaction . Differences are reported in the morphology of the immobilized Aspergillus catalase compared with the bovine liver catalase .
1 . Introduction The work reported here was carried out as part of a larger study of the deactivation of bovine liver and Aspergillus catalase immobilized on a nonporous glass support . One of the objectives was to study the morphology of the immobilized enzyme during both immobilization and various stages of reaction . Both catalases were examined using scanning microscopy.
2. Experimental details Catalase was immobilized by the method of Malikkides and Weiland [11 with one change in the silanization step . The procedure was as follows : 50 ml of distilled water and 50 ml of 48% hydrofluoric acid were added to 75 g of glass beads (841-1000 µm from Ferro Corporation, Jackson, Mississippi) . After 1 h, the hydrofluoric acid was decanted and the beads washed with distilled water . Sodium hydroxide solution (1 ON) was added to the glass beads and the contents heated to 80 °C in a water bath for 1 h . The beads were washed with distilled water and dried at 80 °C . y-aminopropyltriethoxy silane in acetone (2% v v - ') was added to the glass beads and the contents were left in an oven at 45 °C for 24 h . The beads were resilanized after washing with water (different from ref . 1) and then stored in a refrigerator. In order to carry out the resilanization, y-aminopropyltriethoxy silane in acetone (2% v v - ') "Author to whom correspondence should be addressed . tPresent address : University of New Hampshire, Durham, NH, 03824, USA . "Present address : University of Newcastle, Newcastle, NSW, Australia 2308 .
was added to the glass beads and the contents were left in an oven at 45 °C for 24 h . The stability and the activity of the immobilized enzyme were enhanced as a result of resilanization . The beads were now ready from immobilization . The first step in immobilization was to mix the beads with a 2 .5% v-1) (v aqueous solution of glutaraldehyde for 2 h at room temperature. After this, the beads were washed with distilled water . To immobilize the enzyme (bovine liver catalase from Sigma, having an activity of approximately 2500 IU mg - ' of protein), the glass beads were immersed in a citrate phosphate buffer solution of pH 7 .0 containing the enzyme (150 000 IU ml -1) . After 5 h, the immobilized enzyme was washed with the buffer solution and stored in a refrigerator . The immobilization procedure for Aspergillus catalase was similar to the procedure outlined above . The only difference was in the immobilization step ; in this case, the glass beads were directly immersed in the enzyme suspension (3 .2 M ammonium sulfate, pH 6 .0, as obtained from Sigma Chemicals (Sigma C3515)) for 5 h at room temperature . The immobilized enzyme was thoroughly washed with the buffer solution and refrigerated . Scanning electron microscopy was used to obtain images of the support and enzyme during different stages of immobilization . Samples were examined either directly or after extensive washing with citrate phosphate buffer, and were gold sputtered for about 4 min before they were placed in the sample holder of the microscope (manufactured by International Scientific Instruments) .
0923-0467/94/$07.00 © 1994 Elsevier Science S .A. All rights reserved SSDI 0923-0467(94)06057-B
B 42
P. T. Vasudevan, R.H. Weiland / Morphology of immobilized catalase
3 . Results and discussion As a basis of comparison, Fig . 1 shows a micrograph of the original glass beads used for immobilization of the enzyme . The first step in the immobilization procedure was treatment of the glass beads with hydrofluoric acid followed by silanization . A reduction in the size of the glass beads was observed as a results of treatment with hydrofluoric acid. The glass beads were typically reduced to 600-800 ,a in size (a size decrease of about 20%), and the surface appeared to be rough and cracked due to hydrofluoric acid etching . Comparison of Figs . 1 and 2 shows a striking difference in the morphology of the non-porous beads before and after pre-treatment with hydrofluoric acid . After prolonged washing with distilled water, a considerable amount of the surface debris was lost .
3.1 . Bovine liver catalase Following treatment with glutaraldehyde, the enzyme was immobilized onto the support . The soluble enzyme (Sigma CIO) had an activity of 2500 units per mg of protein, while the immobilized enzyme had an activity of 21 IU per gram of support, activity being defined as the rate of decomposition of hydrogen peroxide in µmoles per minutes per gram of dried catalyst at pH 7 .0 and a temperature of 25 °C, and at a peroxide concentration of 0 .01 M. The protein content of the pre-immobilized catalase was about 80% ; the remaining 20% was sodium citrate . The smooth uniform particles seen in the micrograph (Fig . 3) have an approximate size of 7-10 ,a and are believed to be the enzyme . The carbohydrate content of the catalase was less than 1%, and the particles observed in the micrograph are believed to be entirely protein . These particles could be observed on virtually the entire surface of the glass support . It is unclear of the ridges or cracks seen in the picture (and caused by HF etching) contain any enzyme since even higher magnifications did not permit a better resolution of these cracks . The micrograph was taken after immobilization and extensive washing of the beads with buffer . After immobilization, the beads were stored under refrigeration . Deactivation runs were then carried out in a CSTR using hydrogen peroxide with concentrations ranging from 0 .01 M to 1 .0 M . Figure 4 shows a micrograph of the immobilized enzyme at the end of the deactivation experiment and prior to cleaning with buffer solution . The concentration of the peroxide was 1 .0 M during this experiment . By comparison with Fig . 3, it can be seen that the
Fig. 1 . Plain glass bead, magnification 0 .036 KX. - 100 g .
Fig. 2 . Glass bead after etching and silanization, magnification 0.075 EX 100 µ.
Fig. 3 . Immobilized bovine liver catalase, magnification 0 .56 IC (on right) . - 10 µ.
B 43
P.T. Vasudevan, R.H. Weiland / Morphology of immobilized catalase TABLE 1 . Comparison of parameters : immobilized catalase Enzyme
Bovine CIO Bovine C40
Aspergillus
Activity (IU g - ')
Km (M)
K; (M)
20.9 34.9 30.6
0 .035±0.004 0 .032±0.002 0 .087±0.002
0.23±0.03 0.26±0.01 0.56±0.02
(p .mol min - ' g - ')
kd (X104
95±5 .6 148.3±3.7 295±5 .0
6.3±0.14 6.8±0.25 0.46±0.022
Vmax
S - ')
C40 were about the same, even though bovine C40 has a much higher initial activity . The morphology appeared to be similar to bovine C10 before and after a deactivation run .
3.2. Aspergil us catalase
Fig . 4 . Immobilized catalase after reaction, magnification KX. 10 µ.
2.16
surface of the bead appears to be similar before and after reaction . The effect of cleaning the surface of the beads with buffer solution was examined, since it is well known that washing restores some enzyme activity . The surface of the beads was examined at the end of a deactivation run, and after prolonged washing with citrate phosphate buffer . No major differences were observed as a result of the washing step . Microscopic studies were also conducted on immobilized bovine liver catalase (Sigma C40), an enzyme with higher purity . The activity of the soluble catalase was about five times higher than bovine C 10 . As seen in Table 1, the activity of the immobilized enzyme is around 35 IU per gram of support . The ratio of the activities of immobilized C40 catalase to immobilized C10 catalase is about 1 :7, this is quite a bit lower than the corresponding ratio for the soluble catalases . The activity referred to here is the initial activity of the enzyme prior to a deactivation experiment . During the process of immobilized of the enzyme to the support, some activity is lost . In the case of bovine C40, this loss appears to be higher. However, it is interesting to note from independent deactivation studies carried out by the authors [21, that the deactivation rates for bovine C 10 and bovine
One intriguing and well known problem, is the lower susceptibility ofAspergillus catalase to attack by hydrogen peroxide [3, 41 . Deactivation studies carried out independently by the authors [21, confirmed that even though immobilized bovine and Aspergillus catalases showed comparable activities for hydrogen peroxide decomposition, the deactivation rate for Aspergillus catalase was an order of magnitude lower . Table 1 compares the values of various parameters for bovine and Aspergillus catalase immobilized on non-porous glass beads obtained in that study . Figure 5 is a micrograph of immobilized Aspergillus catalase (SIgma C3515) . The activity of the soluble enzyme was about 7000 units per mg of protein while the activity of the immobilized enzyme was about 30 IU per gram of support . The carbohydrate content of the enzyme was less than 1%, and the enzyme is therefore entirely protein . The particles appear to be more closely packed and smaller in size when compared with immobilized
Fig .
5 . Immobilized Aspergillus catalase, magnification 7.0 KX. 1 µ.
B44
P. T. Vasudevan, R.H. Weiland / Morphology of immobilized catalase
bovine catalase (Fig. 3). As was the case with bovine catalase, comparison of micrographs before and after a deactivation experiment showed no apparent difference in the morphology . It is not clear if the closely knit structure of immobilized Aspergillus catalase has any effect on the observed lower deactivation rate of the enzyme . Micrographs also independently confirmed that fact that there was no apparent loss of the enzyme during reaction due to attrition.
3.3. Comparison Independent studies on catalase deactivation [2] have indicated that the deactivation rate of Aspergillus catalase in order of magnitude lower than that of catalase from bovine liver . The values for the deactivation rate constant are shown in Table 1 . Scanning electron micrographs of Aspergillus catalase show a denser and close surface packing of the enzyme on the surface of the non-porous glass, compared with bovine liver catalase . The value of Vma . (295 for Aspergillus catalase vs . 95 for bovine catalase (Sigma C10)) appears to correlate with the morphology (close packing in the case of Aspergillus catalase) of the enzyme on the surface of the support, and the initial activities of the soluble enzyme . Gruft et al. [5] studied the properties of Aspergillus and bovine catalase in the soluble form . They found that the molecular weight of Aspergillus catalase was 323 000 compared with 240 000 for bovine catalase . They also determined the amino acid composition of Aspergillus catalase . Table 2 compares the amino acid composition ofAspergillus catalase with bovine catalase . The amino acid comTABLE 2 . Comparison of amino acid compositions Amino acid
Aspergillus catalase (Residue per mol)
Bovine liver catalase (residue per mol)
Asp Glu Pro Gly Ala Val Met Ileu Leu Tyr Phe Lys His Arg Cys
451 349 266 302 363 245 72 146 257 149 245 230 99 180 68
294 197 166 155 154 141 41 79 154 86 132 116 89 130 58
position for Aspergillus catalase is reported by Gruft et al ., whereas the amino acid composition for bovine catalase is reproduced from the work of Schroeder et al. [6] . It is clear from Table 2 that the amino acid composition for the two catalases differ considerably. Gruft et al. used purified samples of Aspergillus catalase in their experiments, and found that the enzyme contained only trace amounts of lipids and carbohydrates . The lipid and carbohydrate content of bovine catalase used in Schroeder et al.'s work is not known . However, it is clear from Table 2 that the ratios of the amino acids Glycine, alanine and lysine in the two catalase (Aspergillus vs . bovine) are much higher compared with the other amino acids. Graft et al. also found that the qualitative change in the visible spectra of the two catalase was the same, which led the authors to conclude that the active site forAspergillus catalase is probably a porphyrin bound to the protein, similar to other catalases . Present information on immobilization seems to indicate that, most often, free amino groups do not participate in the catalytic activity of the enzyme molecule (for example, the observation that with glutaraldehyde, it is almost exclusively the free amino groups that take part in the cross-linking reaction) . Considering the fact that lysine, alanine and glycine are present in much higher quantities in Aspergillus catalase, it is quite likely that immobilization of the enzyme following treatment of the silanized support with glutaraldehyde, leads to a more stable enzyme . This probably accounts for the different morphology of the fungal enzyme .
4 . Conclusions Scanning electron microscopy of immobilized catalase has shown that the morphology of immobilized Aspergillus catalase is different to that of immobilized bovine liver catalase . The immobilized Aspergillus catalase appears to be much smaller and more closely packed than bovine liver catalase . The close surface packing and surface coverage appears to correlate with the variations of Vm,,. from enzyme source to enzyme source . It is believed that the higher proportion of amino acids in the fungal catalase leads to a more stable binding of the enzyme to the support . Since deactivation takes place at the molecular level, any changes in the morphology of the enzyme during the actual deactivation process will not show up on a scanning electron micrograph . However, the micrographs do show that there is
P.T. Vasudevan, R .H. Weiland / Morphology of immobilized catalase
no apparent loss of the enzyme during reaction with hydrogen peroxide as a result of mechanical agitation or attrition.
Acknowledgements Financial support of this work by the National Science Foundation, under grand no . CPE-8314344, is gratefully acknowledged .
B45
2 P.T. Vasudevan and R .H. Weiland, Blotechnot Bioeng ., 36 (1990) 783-789 . 3 R.E. Altomare, P.F. Greenfield and J.R . Kittrell, Biotechnol. Bioeng ., 16 (1974) 1675-1680. 4 N.M. Tai and P.F. Greenfield, Biotechnol. Bioeng., 23 (1981) 805-821 . 5 H. Gruft, R. Ruck and J . Traynor, Can. J. Biochem., 56 (1978) 916-919 . 6 WA . Schroeder, A . Saha, W.D. Fenninger and J .T. Cua, Biochim . Biophys . Acta, 58 (1962) 611-613 .
Appendix A : Nomenclature References 1 C .O . Mallikkides and R .H. Weiland, Biotechnol. Bioeng., 24 (1982) 2419-2439 .
kd Km K; Vma„
deactivation constant (s - ') Michaelis constant (M) inhibition constant (M) maximum reaction rate (µ mol min - ' g - ')
The Chemical Engineering Journal, 55 (1994) B41-B45
Studies on the morphology of immobilized catalase P.T. Vasudevan*t and R .H . Weilandtt Department of Chemical Engineering, Clarkson University, Potsdam, NY 13676 (USA) (Received June 10, 1993)
Abstract Scanning electron microscopy studies were carried out on bovine liver and Aspergillus catalase immobilized on a non-porous glass support during various stages of immobilization and reaction . Differences are reported in the morphology of the immobilized Aspergillus catalase compared with the bovine liver catalase .
1 . Introduction The work reported here was carried out as part of a larger study of the deactivation of bovine liver and Aspergillus catalase immobilized on a nonporous glass support . One of the objectives was to study the morphology of the immobilized enzyme during both immobilization and various stages of reaction . Both catalases were examined using scanning microscopy.
2. Experimental details Catalase was immobilized by the method of Malikkides and Weiland [11 with one change in the silanization step . The procedure was as follows : 50 ml of distilled water and 50 ml of 48% hydrofluoric acid were added to 75 g of glass beads (841-1000 µm from Ferro Corporation, Jackson, Mississippi) . After 1 h, the hydrofluoric acid was decanted and the beads washed with distilled water . Sodium hydroxide solution (1 ON) was added to the glass beads and the contents heated to 80 °C in a water bath for 1 h . The beads were washed with distilled water and dried at 80 °C . y-aminopropyltriethoxy silane in acetone (2% v v - ') was added to the glass beads and the contents were left in an oven at 45 °C for 24 h . The beads were resilanized after washing with water (different from ref . 1) and then stored in a refrigerator. In order to carry out the resilanization, y-aminopropyltriethoxy silane in acetone (2% v v - ') "Author to whom correspondence should be addressed . tPresent address : University of New Hampshire, Durham, NH, 03824, USA . "Present address : University of Newcastle, Newcastle, NSW, Australia 2308 .
was added to the glass beads and the contents were left in an oven at 45 °C for 24 h . The stability and the activity of the immobilized enzyme were enhanced as a result of resilanization . The beads were now ready from immobilization . The first step in immobilization was to mix the beads with a 2 .5% v-1) (v aqueous solution of glutaraldehyde for 2 h at room temperature. After this, the beads were washed with distilled water . To immobilize the enzyme (bovine liver catalase from Sigma, having an activity of approximately 2500 IU mg - ' of protein), the glass beads were immersed in a citrate phosphate buffer solution of pH 7 .0 containing the enzyme (150 000 IU ml -1) . After 5 h, the immobilized enzyme was washed with the buffer solution and stored in a refrigerator . The immobilization procedure for Aspergillus catalase was similar to the procedure outlined above . The only difference was in the immobilization step ; in this case, the glass beads were directly immersed in the enzyme suspension (3 .2 M ammonium sulfate, pH 6 .0, as obtained from Sigma Chemicals (Sigma C3515)) for 5 h at room temperature . The immobilized enzyme was thoroughly washed with the buffer solution and refrigerated . Scanning electron microscopy was used to obtain images of the support and enzyme during different stages of immobilization . Samples were examined either directly or after extensive washing with citrate phosphate buffer, and were gold sputtered for about 4 min before they were placed in the sample holder of the microscope (manufactured by International Scientific Instruments) .
0923-0467/94/$07.00 © 1994 Elsevier Science S .A. All rights reserved SSDI 0923-0467(94)06057-B
B 42
P. T. Vasudevan, R.H. Weiland / Morphology of immobilized catalase
3 . Results and discussion As a basis of comparison, Fig . 1 shows a micrograph of the original glass beads used for immobilization of the enzyme . The first step in the immobilization procedure was treatment of the glass beads with hydrofluoric acid followed by silanization . A reduction in the size of the glass beads was observed as a results of treatment with hydrofluoric acid. The glass beads were typically reduced to 600-800 ,a in size (a size decrease of about 20%), and the surface appeared to be rough and cracked due to hydrofluoric acid etching . Comparison of Figs . 1 and 2 shows a striking difference in the morphology of the non-porous beads before and after pre-treatment with hydrofluoric acid . After prolonged washing with distilled water, a considerable amount of the surface debris was lost .
3.1 . Bovine liver catalase Following treatment with glutaraldehyde, the enzyme was immobilized onto the support . The soluble enzyme (Sigma CIO) had an activity of 2500 units per mg of protein, while the immobilized enzyme had an activity of 21 IU per gram of support, activity being defined as the rate of decomposition of hydrogen peroxide in µmoles per minutes per gram of dried catalyst at pH 7 .0 and a temperature of 25 °C, and at a peroxide concentration of 0 .01 M. The protein content of the pre-immobilized catalase was about 80% ; the remaining 20% was sodium citrate . The smooth uniform particles seen in the micrograph (Fig . 3) have an approximate size of 7-10 ,a and are believed to be the enzyme . The carbohydrate content of the catalase was less than 1%, and the particles observed in the micrograph are believed to be entirely protein . These particles could be observed on virtually the entire surface of the glass support . It is unclear of the ridges or cracks seen in the picture (and caused by HF etching) contain any enzyme since even higher magnifications did not permit a better resolution of these cracks . The micrograph was taken after immobilization and extensive washing of the beads with buffer . After immobilization, the beads were stored under refrigeration . Deactivation runs were then carried out in a CSTR using hydrogen peroxide with concentrations ranging from 0 .01 M to 1 .0 M . Figure 4 shows a micrograph of the immobilized enzyme at the end of the deactivation experiment and prior to cleaning with buffer solution . The concentration of the peroxide was 1 .0 M during this experiment . By comparison with Fig . 3, it can be seen that the
Fig. 1 . Plain glass bead, magnification 0 .036 KX. - 100 g .
Fig. 2 . Glass bead after etching and silanization, magnification 0.075 EX 100 µ.
Fig. 3 . Immobilized bovine liver catalase, magnification 0 .56 IC (on right) . - 10 µ.
B 43
P.T. Vasudevan, R.H. Weiland / Morphology of immobilized catalase TABLE 1 . Comparison of parameters : immobilized catalase Enzyme
Bovine CIO Bovine C40
Aspergillus
Activity (IU g - ')
Km (M)
K; (M)
20.9 34.9 30.6
0 .035±0.004 0 .032±0.002 0 .087±0.002
0.23±0.03 0.26±0.01 0.56±0.02
(p .mol min - ' g - ')
kd (X104
95±5 .6 148.3±3.7 295±5 .0
6.3±0.14 6.8±0.25 0.46±0.022
Vmax
S - ')
C40 were about the same, even though bovine C40 has a much higher initial activity . The morphology appeared to be similar to bovine C10 before and after a deactivation run .
3.2. Aspergil us catalase
Fig . 4 . Immobilized catalase after reaction, magnification KX. 10 µ.
2.16
surface of the bead appears to be similar before and after reaction . The effect of cleaning the surface of the beads with buffer solution was examined, since it is well known that washing restores some enzyme activity . The surface of the beads was examined at the end of a deactivation run, and after prolonged washing with citrate phosphate buffer . No major differences were observed as a result of the washing step . Microscopic studies were also conducted on immobilized bovine liver catalase (Sigma C40), an enzyme with higher purity . The activity of the soluble catalase was about five times higher than bovine C 10 . As seen in Table 1, the activity of the immobilized enzyme is around 35 IU per gram of support . The ratio of the activities of immobilized C40 catalase to immobilized C10 catalase is about 1 :7, this is quite a bit lower than the corresponding ratio for the soluble catalases . The activity referred to here is the initial activity of the enzyme prior to a deactivation experiment . During the process of immobilized of the enzyme to the support, some activity is lost . In the case of bovine C40, this loss appears to be higher. However, it is interesting to note from independent deactivation studies carried out by the authors [21, that the deactivation rates for bovine C 10 and bovine
One intriguing and well known problem, is the lower susceptibility ofAspergillus catalase to attack by hydrogen peroxide [3, 41 . Deactivation studies carried out independently by the authors [21, confirmed that even though immobilized bovine and Aspergillus catalases showed comparable activities for hydrogen peroxide decomposition, the deactivation rate for Aspergillus catalase was an order of magnitude lower . Table 1 compares the values of various parameters for bovine and Aspergillus catalase immobilized on non-porous glass beads obtained in that study . Figure 5 is a micrograph of immobilized Aspergillus catalase (SIgma C3515) . The activity of the soluble enzyme was about 7000 units per mg of protein while the activity of the immobilized enzyme was about 30 IU per gram of support . The carbohydrate content of the enzyme was less than 1%, and the enzyme is therefore entirely protein . The particles appear to be more closely packed and smaller in size when compared with immobilized
Fig .
5 . Immobilized Aspergillus catalase, magnification 7.0 KX. 1 µ.
B44
P. T. Vasudevan, R.H. Weiland / Morphology of immobilized catalase
bovine catalase (Fig. 3). As was the case with bovine catalase, comparison of micrographs before and after a deactivation experiment showed no apparent difference in the morphology . It is not clear if the closely knit structure of immobilized Aspergillus catalase has any effect on the observed lower deactivation rate of the enzyme . Micrographs also independently confirmed that fact that there was no apparent loss of the enzyme during reaction due to attrition.
3.3. Comparison Independent studies on catalase deactivation [2] have indicated that the deactivation rate of Aspergillus catalase in order of magnitude lower than that of catalase from bovine liver . The values for the deactivation rate constant are shown in Table 1 . Scanning electron micrographs of Aspergillus catalase show a denser and close surface packing of the enzyme on the surface of the non-porous glass, compared with bovine liver catalase . The value of Vma . (295 for Aspergillus catalase vs . 95 for bovine catalase (Sigma C10)) appears to correlate with the morphology (close packing in the case of Aspergillus catalase) of the enzyme on the surface of the support, and the initial activities of the soluble enzyme . Gruft et al. [5] studied the properties of Aspergillus and bovine catalase in the soluble form . They found that the molecular weight of Aspergillus catalase was 323 000 compared with 240 000 for bovine catalase . They also determined the amino acid composition of Aspergillus catalase . Table 2 compares the amino acid composition ofAspergillus catalase with bovine catalase . The amino acid comTABLE 2 . Comparison of amino acid compositions Amino acid
Aspergillus catalase (Residue per mol)
Bovine liver catalase (residue per mol)
Asp Glu Pro Gly Ala Val Met Ileu Leu Tyr Phe Lys His Arg Cys
451 349 266 302 363 245 72 146 257 149 245 230 99 180 68
294 197 166 155 154 141 41 79 154 86 132 116 89 130 58
position for Aspergillus catalase is reported by Gruft et al ., whereas the amino acid composition for bovine catalase is reproduced from the work of Schroeder et al. [6] . It is clear from Table 2 that the amino acid composition for the two catalases differ considerably. Gruft et al. used purified samples of Aspergillus catalase in their experiments, and found that the enzyme contained only trace amounts of lipids and carbohydrates . The lipid and carbohydrate content of bovine catalase used in Schroeder et al.'s work is not known . However, it is clear from Table 2 that the ratios of the amino acids Glycine, alanine and lysine in the two catalase (Aspergillus vs . bovine) are much higher compared with the other amino acids. Graft et al. also found that the qualitative change in the visible spectra of the two catalase was the same, which led the authors to conclude that the active site forAspergillus catalase is probably a porphyrin bound to the protein, similar to other catalases . Present information on immobilization seems to indicate that, most often, free amino groups do not participate in the catalytic activity of the enzyme molecule (for example, the observation that with glutaraldehyde, it is almost exclusively the free amino groups that take part in the cross-linking reaction) . Considering the fact that lysine, alanine and glycine are present in much higher quantities in Aspergillus catalase, it is quite likely that immobilization of the enzyme following treatment of the silanized support with glutaraldehyde, leads to a more stable enzyme . This probably accounts for the different morphology of the fungal enzyme .
4 . Conclusions Scanning electron microscopy of immobilized catalase has shown that the morphology of immobilized Aspergillus catalase is different to that of immobilized bovine liver catalase . The immobilized Aspergillus catalase appears to be much smaller and more closely packed than bovine liver catalase . The close surface packing and surface coverage appears to correlate with the variations of Vm,,. from enzyme source to enzyme source . It is believed that the higher proportion of amino acids in the fungal catalase leads to a more stable binding of the enzyme to the support . Since deactivation takes place at the molecular level, any changes in the morphology of the enzyme during the actual deactivation process will not show up on a scanning electron micrograph . However, the micrographs do show that there is
P.T. Vasudevan, R .H. Weiland / Morphology of immobilized catalase
no apparent loss of the enzyme during reaction with hydrogen peroxide as a result of mechanical agitation or attrition.
Acknowledgements Financial support of this work by the National Science Foundation, under grand no . CPE-8314344, is gratefully acknowledged .
B45
2 P.T. Vasudevan and R .H. Weiland, Blotechnot Bioeng ., 36 (1990) 783-789 . 3 R.E. Altomare, P.F. Greenfield and J.R . Kittrell, Biotechnol. Bioeng ., 16 (1974) 1675-1680. 4 N.M. Tai and P.F. Greenfield, Biotechnol. Bioeng., 23 (1981) 805-821 . 5 H. Gruft, R. Ruck and J . Traynor, Can. J. Biochem., 56 (1978) 916-919 . 6 WA . Schroeder, A . Saha, W.D. Fenninger and J .T. Cua, Biochim . Biophys . Acta, 58 (1962) 611-613 .
Appendix A : Nomenclature References 1 C .O . Mallikkides and R .H. Weiland, Biotechnol. Bioeng., 24 (1982) 2419-2439 .
kd Km K; Vma„
deactivation constant (s - ') Michaelis constant (M) inhibition constant (M) maximum reaction rate (µ mol min - ' g - ')