BIOTECHNOLOGY AND BIOENGINEERING, VOL. XVII’I, PAGES 1405-1412 (1976)
Immobilization of Tyrosinase within Polyacrylamide Gels JULIAN G. SCHILLER and C. C. LIU,* Department of Chemical and Petroleum Engineering, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
Summary The enzyme tyrosinase (E.C. 1.14.18.1)has been immobilized in a polyacrylamide gel and intermittently assayed for enzyme act,ivity over a period of 19 days using phenol as the substrate. The results of these studies indicate that the immobilized enzyme could be incorporated into a system to detect phenol and related compounds that are found in industrial effluents and as surface water contaminants.
INTRODUCTION The enzyme tyrosinase (E.C. 1.14.18.1) catalyzes the orthohydroxylation of phenols to catechols as well as the dehydrogenation of catachols t o o-quinones.‘ Tyrosinase has previously been immobilized* by covalent attachment t o diethylaminoethyl cellulose to elucidate the possibility of implanting the immobilized enzyme in the blood stream of patients with Parkinson’s disease. Under these conditions the enzyme would convert L-tyrosine to 3,4 dihydroxyphenylalinne which is used as a therapeutic agent during the treatment of Parkinson’s disease. The enzyme has also been trapped in liquid membrane emulsion^.^ The emulsion droplets, when dispersed in aqueous solutions of phenol, were able t o retain sufficient enzyme activity t o allow oxidation of the entering phenol with subsequent accumulation of the reaction products within the droplets. This system has the potential to be used as a pollution control device. During the course of our studies, the enzyme was immobilized in a polyacrylamide gel matrix that was cast around a thin platinum grid. The enzyme gels were then examined to determine appropriate *To whom correspondence should be addressed.
1405 @ 1976 by John Wiley & Sons, Inc.
1406
SCHILLER AND LIU
enzyme loadings, substrate concentrations, enzyme stability, and the ability of the immobilized enzyme to be reused. The results will be useful in examining the potential of immobilized tyrosinase for use in a system that could monitor the level of phenols in industrial effluents and in surface waters as well as for biomedical applications.
METHODS Immobilization Technique The enzyme tyrosinase was immobilized in polyacrylamide gels essentially as described by Hicks and Upkide4 to yield gels that contained 8.2 g (monomer plus crosslinking agent) of polymer per 100 ml of gel. Solutions of the enzyme acrylamide, bisacrylamide, riboflavin, and persulfate were all prepared in 0.1M potassium phosphate buffer, p H 6.5. Basket-weave platinum screening (0.11 X 1.5 X 1.5 cm) was placed inside an all glass mold (0.12 X 2.0 X 7.0 cm) which was then filled with deoxygenated gel solution. Polymerization was initiated by placing the mold within 5 cm of a 15 W fluorescent bulb. Thirty minutes after polymerization appeared complete, the enzyme grids were removed from the mold and stored in 0.25M potassium phosphate buffer, p H 6.5 a t 6°C. After 19 days of storage there was no observable change in the physical appearance or mechanical stability of these enzyme grids. All enzyme grids contained 230-250 mg of gel per grid. The amount of enzyme in all grids was 135-145 enzyme units (approximately 0.40 mg) unless otherwise noted. The results of all assays were normalized to a mean value of 240 mg of gel per grid. After assays and prior to storage, all enzyme grids were washed in 10 ml of stirred 0.25M potassium phosphate buffer (pH 6.5) for 15 min at room temperature.
Enzyme Assays
Enzyme grids Assays were performed at 22°C by suspending the enzyme grid in 10 ml of 0.25M potassium phosphate buffer, p H 6.5 containing 0.02M potassium ferricyanide5 and the appropriate concentration of phenol. The reaction medium was magnetically stirred and oxyOz/min) for 10 min prior to and during the genated (40 cc of 100~o course of the assay. At 5 min intervals after immersion of the grid, 1.0 ml aliquots were roemoved from the incubation medium and their absorbance a t 4200 A was determined in a 1.00 cm cell in a Cary
;
IMMOBILIZED TYROSINASE
1407
Model 14 dual beam spectrophotometer. The sample was returned to the reaction mixture after the assay (elapsed time of 20-30 sec).
Free enzyme in solution The enzyme in solution (1.4 mg/ml) was assayed as described6 using 0.001M L-tyrosine as the substrate. Absorbancy readings a t 2800 were taken a t 2 min intervals.
Materials Acrylamide, bisacrylamide, riboflavin, and potassium persulfate were obtained from Bio-Rad Laboratories, Richmond, California. Mushroom tyrosinase was obtained from Worthington Biochemical Corporation, Freehold, New Jersey. All other chemicals were of reagent grade, supplied by Fisher Scientific, Pittsburgh, Pennsylvania.
RESULTS Tyrosinase was immobilized in polyacrylamide gels that were cast around thin pieces of platinum screen. The immobilized enzyme was then characterized in a number of ways. First, the response of the immobilized enzyme to different phenol concentrations was examined. In this study, identical amounts of enzyme (135-145 enzyme units) were immobilized in the enzyme grids. Subsequently, the reaction rates of these enzyme grids for various substrate concentrations were measured. Figure 1 shows the experimental results of this study. The results demonstrate that enzyme grids containing identical amounts of enzyme respond with
wR 0.8 c 0.8 a
P
1.0 r
0 t t l-
/
: a
t0.6 z
Q
m 0.4 0 v)
m
a 0.2 0
0
5
TIME
10 MINUTES
15
Fig. 1. Response of immobilized tyrosinase to different phenol concentrations.
0,1 0 - 3 ~ ;0, 1 0 - 4 ~ ;0, 10-6.
SCHILLER AND LIU
1408
increased reaction rates to increased concentrations of phenol. I n Figure 1, only the response to substrate concentration in the range of t o l k 5 M are presented; a lower substrate concentration of 10-6M was also employed to yield a low but detectable response. The response of different amounts of immobilized enzyme loadings to a fixed phenol concentration was also evaluated. Three different enzyme loadings, 140, 70, and 14 enzyme units, were used in this study to cover a tenfold difference in immobilized enzyme concentration. Figure 2 presents the experimental results. The data presented were ,obtained at a phenol concentration of l k 3 M and the results demonstrate that enzyme grids containing increased amounts of enzyme respond to the same concentration of phenol with increased reaction rates. For other phenol concentrations, this observation also holds. The feasibility of reusing the immobilized enzyme over a reasonable period of time was also examined. Two experimental studies were then carried out. I n the first experiment, 10-3M phenol was used and the reaction rates for this enzyme grid a t day 1, day 9, and day 19 were examined. Figure 3 shows the experimental results of this study. As expected, the enzyme activity decreased over this 19 day period of time. However, 47% of enzyme activity is maintained after 19 days compared to that of day 1. Furthermore, the degradation of the enzyme activity is always more severe between day 1 and day 9 compared to between day 9 and day 19. I n the second experiment four consecutive assays were carried out with the same enzyme grid a t 20 min intervals. Figures 4 and 5 show the experimental results of these consecutive uses of a single enzyme grid in a phenol concentration of lW3M and 10-5M, respec1.04
-
0
~ 0 . 8 0
50.6 W
V
z Q 0.4 m
-
-
0: 0
0
Fig. 2.
5 TIME
10 MINUTES
15
Activity of enzyme grids containing different amounts of enzyme. 0 , 140 enzyme units; A,70 enzyme units; 14 enzyme units.
+,
1409
IMMOBILIZED TYROSINASE
TIME
MINUTES
Fig. 3. Performance characteristics of repeated use of an enzyme grid over a period of 19 days. 0, day 1; 0 , day 9; 0,day 19. 1.0 r
a
g 0.8 N .. 9
2 0.6 -
0
5
TIME
10
15
MINUTES
Fig. 4. Performance characteristics of consecutive refuse of an enzyme grid in a phenol substrate concentration of 10-3M. 0, first use; 0 , second use, 0, third use; fourth use.
+,
Fig. 5 . Performance characteristics of consecutive reuse of an enzyme grid in a phenol substrate concentration of 10-5M. A, first use; A , second use; third use; 0, fourth use.
m,
S C A I L I ~ E IAN11 ~ LIU
1410
tively. Tliv eiizyiiie grid w a s was1ic.d bet\+ecn assays as dcacribcd. Thc experimental results show that a small dcgrce of dcgradation of thc enzymc activity is observed. This inactivation of the enzyme is morr apparent at highcr substrate concentrations (e.g., 10-3M phcnol) and has little or no cffcct on thc enzymc activity a t a lowcr substratr concentration. This obscrvation is, in principle, correct, sincr it is kno\i n that phrnol substratr \z ill inactivatc the tyrosinasc. An expcriment was also carried out to determine the pcriod of timc during which an unused cnzyme grid may be stored. The results of this study diffrr from those prrsented in Figure 3. I n Pigurc 3, the results n cre obtained from a singlc grid u hich n as uscd throughout the 10 day period of time. In this experiment, thrce identical cnzymc grids were prepared, one was used for assay a t day 1 while thc other unuscd grids \\ere stored in buffer a t 6°C. On day 9, onc of the unuscd grids \\as then assaycd and the remaining grid was assayed on day 19. Figure G reprcscnts thr exprrimcntal rcsults uhich show t h r stability of the cnzyme grid stored over diffcrcnt lengths of timc. Consistent \iith our previous observation, enzyme activity dccreascs as the storage pcriod increascs. Howcvcr, the unuscd grids show highcr cnzymc activities compared to used grids for an idrntical amount of storage time. This result is internally consistrnt $I ith thr study prescntcd in Figure 4. For comparative purposes, the frec enzyme in solution (445 mzymc units/ml in 0.25Ail potassium phosphate, p H 6.5) was also stored a t 6°C and tested for activity over a period of 19 days. Approxiniatcly 30% of the enzyme activity was lost over this 19 day period of timc. 1.0
-
0.6
-
6
9 b4
w
U
z 0.4
Q
-
m
TIME
MINUTES
Fig. 6 . St:thility of immobilized tyrosinase stored ovet different periods of time. 0, stored 1 day; 0 , stored 9 days, 0, stored 19 days.
IMMOBILIZED TYROSINASE
1411
-
For practical applications, this immobilized enzyme grid may have to be stored a t ambient temperature, 22"C, instead of the more ideal 6°C. Thus, a study on the stability of the immobilized enzyme stored a t 22°C was undertaken. In this study, five epzyme grids were prepared and used. One grid was assayed in the usual manner on the first day. Two enzyme grids were then stored at 22°C in 0.25M potassium phosphate buffer containing 0.01% sodium azide to inhibit the growth of microorganisms. The remaining grids were then assayed after five and 13 days of storage a t 22°C. The results indicate preservation of 43% and 22% of the initial enzyme activity after 13 days of storage at 22°C with NaN3 and without NaN3, respectively. This compares to 70y0retention of activity when the grids are stored a t 6°C. The buffer solution containing the enzyme grids stored without sodium azide showed definite signs of microbial growth after 13 days at 22°C. This could account for the lower enzyme activity found in this preparation after 13 days. However, if a sufficient amount of enzyme was immobilized initially, sufficient enzyme activity will be maintained to carry out the reaction even if the immobilized enzyme is stored a t 22°C. Consequently, the enzyme ,grid should be able to function and last for a reasonable period of time.
DISCUSSION The enzyme tyrosinase may be reproducibly immobilized in acrylamide gels and subject to a simple assay procedure. It was demonstrated that the enzyme is as stable in the acrylamide gel matrix as it is in free form over a period of 19 days, even though a fraction of the enzyme activity was lost in both instances. It was also shown that the enzyme can be stored at ambient temperature, 22"C, over a period of 13 days to maintain 43% of the initial (day 1) activity, while 70y0of the initial activity is maintained when stored at 6°C. This observation indicates that immobilized tyrosinase can be practically used at ambient temperature over a reasonable period of time. Although the enzyme shows degrees of inactivation at high substrate concentrations, e.g., 10-3M phenol, little or no effect on the enzyme activity is observed a t a lower substrate concentration, e.g., 10-5M phenol, which would approximate those concentrations of phenols detectable as pollutants. By utilizing our detection method, concentrations of phenol as low as 10-6M (100 ppb) are detectable using a 1.0 cm light path to
1412
SCHILLER AND LIU
follow the enzymatic reaction. The use of a 10.0 cm cell would immediately decrease this limit to 10 ppb. The present standard chemical method of analysis for phenols and related compounds indicate that a sensitivity of 1 ppb is obtainable. The physical configuration of our enzyme involved a basket-weave platinum screen which was similar to those used in our previous studies on glucose oxidase.s The rationale of this arrangement is based upon: 1) the ease of handling the immobilized enzyme in this matrix and 2) the configuration can be used directly as a n enzyme electrode in which the platinum screen serves as the electron pathway to a n external circuit. One realizes that the oxidation of phenol using tyrosinase as a catalyst is in fact a coupled redox reaction. Using proper electrochemical measurement techniques, one may be able to measure phenol concentration directly. In our laboratory, we are engaging in this development but the description of this phase of the work is beyond the scope of this study and shall be presented a t a later time. The technical assistance of Carol A. Parente in the earlier phase of this work is appreciated. Also, the support of this work by Lewis Research Center-NASA, under the research grant NSG-3002 is gratefully acknowledged. The discussion and comments by Dr. J. Stuart Fordyce of Lewis Research Center are also appreciated.
References 1. H. W. Duckworth and J. E. Coleman, J . Biol. Chem., 235, 1613 (1970). 2. J. R. Wykes, P. Dunnil, and M. D. Lilly, Nature, New Biology, 230, 187 (1971). 3. S. W. May, and N. N. Li, in Enzyme Engineering, vol. 2, E. K. Pye and L. B. Wingard, Eds., Plenum Press, New York, 1974, p. 78. 4. G. P. Hicks and S. J. Updike, Anal. Chem., 38, 726 (1966). 5. A. S. Sussman, Arch. Biochem. Biophys., 95, 407 (1961). 6. Worthington Enzyme Manual, Worthington Biochemical Corporation, Freehold, New Jersey, 1972, p. 39. 7. Standard Methods for the Examination of Water and Wastewater Including Bottom Sediments and Sludges 12th ed., Boyd Printing Co., Inc., Albany, New York, 1965. 8. E. J. Lahoda, C. C. Liu, and L. B. Wingard, Biotechnol. Bioeng., 17, 413 (1975).
Accepted for Publication May 11, 1976
Immobilization of Tyrosinase within Polyacrylamide Gels JULIAN G. SCHILLER and C. C. LIU,* Department of Chemical and Petroleum Engineering, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
Summary The enzyme tyrosinase (E.C. 1.14.18.1)has been immobilized in a polyacrylamide gel and intermittently assayed for enzyme act,ivity over a period of 19 days using phenol as the substrate. The results of these studies indicate that the immobilized enzyme could be incorporated into a system to detect phenol and related compounds that are found in industrial effluents and as surface water contaminants.
INTRODUCTION The enzyme tyrosinase (E.C. 1.14.18.1) catalyzes the orthohydroxylation of phenols to catechols as well as the dehydrogenation of catachols t o o-quinones.‘ Tyrosinase has previously been immobilized* by covalent attachment t o diethylaminoethyl cellulose to elucidate the possibility of implanting the immobilized enzyme in the blood stream of patients with Parkinson’s disease. Under these conditions the enzyme would convert L-tyrosine to 3,4 dihydroxyphenylalinne which is used as a therapeutic agent during the treatment of Parkinson’s disease. The enzyme has also been trapped in liquid membrane emulsion^.^ The emulsion droplets, when dispersed in aqueous solutions of phenol, were able t o retain sufficient enzyme activity t o allow oxidation of the entering phenol with subsequent accumulation of the reaction products within the droplets. This system has the potential to be used as a pollution control device. During the course of our studies, the enzyme was immobilized in a polyacrylamide gel matrix that was cast around a thin platinum grid. The enzyme gels were then examined to determine appropriate *To whom correspondence should be addressed.
1405 @ 1976 by John Wiley & Sons, Inc.
1406
SCHILLER AND LIU
enzyme loadings, substrate concentrations, enzyme stability, and the ability of the immobilized enzyme to be reused. The results will be useful in examining the potential of immobilized tyrosinase for use in a system that could monitor the level of phenols in industrial effluents and in surface waters as well as for biomedical applications.
METHODS Immobilization Technique The enzyme tyrosinase was immobilized in polyacrylamide gels essentially as described by Hicks and Upkide4 to yield gels that contained 8.2 g (monomer plus crosslinking agent) of polymer per 100 ml of gel. Solutions of the enzyme acrylamide, bisacrylamide, riboflavin, and persulfate were all prepared in 0.1M potassium phosphate buffer, p H 6.5. Basket-weave platinum screening (0.11 X 1.5 X 1.5 cm) was placed inside an all glass mold (0.12 X 2.0 X 7.0 cm) which was then filled with deoxygenated gel solution. Polymerization was initiated by placing the mold within 5 cm of a 15 W fluorescent bulb. Thirty minutes after polymerization appeared complete, the enzyme grids were removed from the mold and stored in 0.25M potassium phosphate buffer, p H 6.5 a t 6°C. After 19 days of storage there was no observable change in the physical appearance or mechanical stability of these enzyme grids. All enzyme grids contained 230-250 mg of gel per grid. The amount of enzyme in all grids was 135-145 enzyme units (approximately 0.40 mg) unless otherwise noted. The results of all assays were normalized to a mean value of 240 mg of gel per grid. After assays and prior to storage, all enzyme grids were washed in 10 ml of stirred 0.25M potassium phosphate buffer (pH 6.5) for 15 min at room temperature.
Enzyme Assays
Enzyme grids Assays were performed at 22°C by suspending the enzyme grid in 10 ml of 0.25M potassium phosphate buffer, p H 6.5 containing 0.02M potassium ferricyanide5 and the appropriate concentration of phenol. The reaction medium was magnetically stirred and oxyOz/min) for 10 min prior to and during the genated (40 cc of 100~o course of the assay. At 5 min intervals after immersion of the grid, 1.0 ml aliquots were roemoved from the incubation medium and their absorbance a t 4200 A was determined in a 1.00 cm cell in a Cary
;
IMMOBILIZED TYROSINASE
1407
Model 14 dual beam spectrophotometer. The sample was returned to the reaction mixture after the assay (elapsed time of 20-30 sec).
Free enzyme in solution The enzyme in solution (1.4 mg/ml) was assayed as described6 using 0.001M L-tyrosine as the substrate. Absorbancy readings a t 2800 were taken a t 2 min intervals.
Materials Acrylamide, bisacrylamide, riboflavin, and potassium persulfate were obtained from Bio-Rad Laboratories, Richmond, California. Mushroom tyrosinase was obtained from Worthington Biochemical Corporation, Freehold, New Jersey. All other chemicals were of reagent grade, supplied by Fisher Scientific, Pittsburgh, Pennsylvania.
RESULTS Tyrosinase was immobilized in polyacrylamide gels that were cast around thin pieces of platinum screen. The immobilized enzyme was then characterized in a number of ways. First, the response of the immobilized enzyme to different phenol concentrations was examined. In this study, identical amounts of enzyme (135-145 enzyme units) were immobilized in the enzyme grids. Subsequently, the reaction rates of these enzyme grids for various substrate concentrations were measured. Figure 1 shows the experimental results of this study. The results demonstrate that enzyme grids containing identical amounts of enzyme respond with
wR 0.8 c 0.8 a
P
1.0 r
0 t t l-
/
: a
t0.6 z
Q
m 0.4 0 v)
m
a 0.2 0
0
5
TIME
10 MINUTES
15
Fig. 1. Response of immobilized tyrosinase to different phenol concentrations.
0,1 0 - 3 ~ ;0, 1 0 - 4 ~ ;0, 10-6.
SCHILLER AND LIU
1408
increased reaction rates to increased concentrations of phenol. I n Figure 1, only the response to substrate concentration in the range of t o l k 5 M are presented; a lower substrate concentration of 10-6M was also employed to yield a low but detectable response. The response of different amounts of immobilized enzyme loadings to a fixed phenol concentration was also evaluated. Three different enzyme loadings, 140, 70, and 14 enzyme units, were used in this study to cover a tenfold difference in immobilized enzyme concentration. Figure 2 presents the experimental results. The data presented were ,obtained at a phenol concentration of l k 3 M and the results demonstrate that enzyme grids containing increased amounts of enzyme respond to the same concentration of phenol with increased reaction rates. For other phenol concentrations, this observation also holds. The feasibility of reusing the immobilized enzyme over a reasonable period of time was also examined. Two experimental studies were then carried out. I n the first experiment, 10-3M phenol was used and the reaction rates for this enzyme grid a t day 1, day 9, and day 19 were examined. Figure 3 shows the experimental results of this study. As expected, the enzyme activity decreased over this 19 day period of time. However, 47% of enzyme activity is maintained after 19 days compared to that of day 1. Furthermore, the degradation of the enzyme activity is always more severe between day 1 and day 9 compared to between day 9 and day 19. I n the second experiment four consecutive assays were carried out with the same enzyme grid a t 20 min intervals. Figures 4 and 5 show the experimental results of these consecutive uses of a single enzyme grid in a phenol concentration of lW3M and 10-5M, respec1.04
-
0
~ 0 . 8 0
50.6 W
V
z Q 0.4 m
-
-
0: 0
0
Fig. 2.
5 TIME
10 MINUTES
15
Activity of enzyme grids containing different amounts of enzyme. 0 , 140 enzyme units; A,70 enzyme units; 14 enzyme units.
+,
1409
IMMOBILIZED TYROSINASE
TIME
MINUTES
Fig. 3. Performance characteristics of repeated use of an enzyme grid over a period of 19 days. 0, day 1; 0 , day 9; 0,day 19. 1.0 r
a
g 0.8 N .. 9
2 0.6 -
0
5
TIME
10
15
MINUTES
Fig. 4. Performance characteristics of consecutive refuse of an enzyme grid in a phenol substrate concentration of 10-3M. 0, first use; 0 , second use, 0, third use; fourth use.
+,
Fig. 5 . Performance characteristics of consecutive reuse of an enzyme grid in a phenol substrate concentration of 10-5M. A, first use; A , second use; third use; 0, fourth use.
m,
S C A I L I ~ E IAN11 ~ LIU
1410
tively. Tliv eiizyiiie grid w a s was1ic.d bet\+ecn assays as dcacribcd. Thc experimental results show that a small dcgrce of dcgradation of thc enzymc activity is observed. This inactivation of the enzyme is morr apparent at highcr substrate concentrations (e.g., 10-3M phcnol) and has little or no cffcct on thc enzymc activity a t a lowcr substratr concentration. This obscrvation is, in principle, correct, sincr it is kno\i n that phrnol substratr \z ill inactivatc the tyrosinasc. An expcriment was also carried out to determine the pcriod of timc during which an unused cnzyme grid may be stored. The results of this study diffrr from those prrsented in Figure 3. I n Pigurc 3, the results n cre obtained from a singlc grid u hich n as uscd throughout the 10 day period of time. In this experiment, thrce identical cnzymc grids were prepared, one was used for assay a t day 1 while thc other unuscd grids \\ere stored in buffer a t 6°C. On day 9, onc of the unuscd grids \\as then assaycd and the remaining grid was assayed on day 19. Figure G reprcscnts thr exprrimcntal rcsults uhich show t h r stability of the cnzyme grid stored over diffcrcnt lengths of timc. Consistent \iith our previous observation, enzyme activity dccreascs as the storage pcriod increascs. Howcvcr, the unuscd grids show highcr cnzymc activities compared to used grids for an idrntical amount of storage time. This result is internally consistrnt $I ith thr study prescntcd in Figure 4. For comparative purposes, the frec enzyme in solution (445 mzymc units/ml in 0.25Ail potassium phosphate, p H 6.5) was also stored a t 6°C and tested for activity over a period of 19 days. Approxiniatcly 30% of the enzyme activity was lost over this 19 day period of timc. 1.0
-
0.6
-
6
9 b4
w
U
z 0.4
Q
-
m
TIME
MINUTES
Fig. 6 . St:thility of immobilized tyrosinase stored ovet different periods of time. 0, stored 1 day; 0 , stored 9 days, 0, stored 19 days.
IMMOBILIZED TYROSINASE
1411
-
For practical applications, this immobilized enzyme grid may have to be stored a t ambient temperature, 22"C, instead of the more ideal 6°C. Thus, a study on the stability of the immobilized enzyme stored a t 22°C was undertaken. In this study, five epzyme grids were prepared and used. One grid was assayed in the usual manner on the first day. Two enzyme grids were then stored at 22°C in 0.25M potassium phosphate buffer containing 0.01% sodium azide to inhibit the growth of microorganisms. The remaining grids were then assayed after five and 13 days of storage a t 22°C. The results indicate preservation of 43% and 22% of the initial enzyme activity after 13 days of storage at 22°C with NaN3 and without NaN3, respectively. This compares to 70y0retention of activity when the grids are stored a t 6°C. The buffer solution containing the enzyme grids stored without sodium azide showed definite signs of microbial growth after 13 days at 22°C. This could account for the lower enzyme activity found in this preparation after 13 days. However, if a sufficient amount of enzyme was immobilized initially, sufficient enzyme activity will be maintained to carry out the reaction even if the immobilized enzyme is stored a t 22°C. Consequently, the enzyme ,grid should be able to function and last for a reasonable period of time.
DISCUSSION The enzyme tyrosinase may be reproducibly immobilized in acrylamide gels and subject to a simple assay procedure. It was demonstrated that the enzyme is as stable in the acrylamide gel matrix as it is in free form over a period of 19 days, even though a fraction of the enzyme activity was lost in both instances. It was also shown that the enzyme can be stored at ambient temperature, 22"C, over a period of 13 days to maintain 43% of the initial (day 1) activity, while 70y0of the initial activity is maintained when stored at 6°C. This observation indicates that immobilized tyrosinase can be practically used at ambient temperature over a reasonable period of time. Although the enzyme shows degrees of inactivation at high substrate concentrations, e.g., 10-3M phenol, little or no effect on the enzyme activity is observed a t a lower substrate concentration, e.g., 10-5M phenol, which would approximate those concentrations of phenols detectable as pollutants. By utilizing our detection method, concentrations of phenol as low as 10-6M (100 ppb) are detectable using a 1.0 cm light path to
1412
SCHILLER AND LIU
follow the enzymatic reaction. The use of a 10.0 cm cell would immediately decrease this limit to 10 ppb. The present standard chemical method of analysis for phenols and related compounds indicate that a sensitivity of 1 ppb is obtainable. The physical configuration of our enzyme involved a basket-weave platinum screen which was similar to those used in our previous studies on glucose oxidase.s The rationale of this arrangement is based upon: 1) the ease of handling the immobilized enzyme in this matrix and 2) the configuration can be used directly as a n enzyme electrode in which the platinum screen serves as the electron pathway to a n external circuit. One realizes that the oxidation of phenol using tyrosinase as a catalyst is in fact a coupled redox reaction. Using proper electrochemical measurement techniques, one may be able to measure phenol concentration directly. In our laboratory, we are engaging in this development but the description of this phase of the work is beyond the scope of this study and shall be presented a t a later time. The technical assistance of Carol A. Parente in the earlier phase of this work is appreciated. Also, the support of this work by Lewis Research Center-NASA, under the research grant NSG-3002 is gratefully acknowledged. The discussion and comments by Dr. J. Stuart Fordyce of Lewis Research Center are also appreciated.
References 1. H. W. Duckworth and J. E. Coleman, J . Biol. Chem., 235, 1613 (1970). 2. J. R. Wykes, P. Dunnil, and M. D. Lilly, Nature, New Biology, 230, 187 (1971). 3. S. W. May, and N. N. Li, in Enzyme Engineering, vol. 2, E. K. Pye and L. B. Wingard, Eds., Plenum Press, New York, 1974, p. 78. 4. G. P. Hicks and S. J. Updike, Anal. Chem., 38, 726 (1966). 5. A. S. Sussman, Arch. Biochem. Biophys., 95, 407 (1961). 6. Worthington Enzyme Manual, Worthington Biochemical Corporation, Freehold, New Jersey, 1972, p. 39. 7. Standard Methods for the Examination of Water and Wastewater Including Bottom Sediments and Sludges 12th ed., Boyd Printing Co., Inc., Albany, New York, 1965. 8. E. J. Lahoda, C. C. Liu, and L. B. Wingard, Biotechnol. Bioeng., 17, 413 (1975).
Accepted for Publication May 11, 1976
