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Enzyme-substrate specific interactions: in situ assessments under pressure
Trends in High Pressure Bioscience and Biotechnology R. Hayashi (editor) 9 2002 Elsevier Science B.V. All rights reserved.
189
Enzyme-substrate specific interactions: in situ assessments under pressure A. Fern,~dez Garcia, P. Butz, R. Lindauer & B. Tauscher Institute for Chemistry and Biology, Federal Research Centre for Nutrition, Haid-und-Neustr. 9, 76131 Karlsrtthe, Germany Peroxidase (POD) enzyme was irrevers~ly inactivated (>90%) after 2 minutes at 500 MPa/25 ~ in the presence of guaiacol. POD reaction with ferulic acid or with catechol monoethyl ether as substrates is not influenced by pressure up to 500MPa (25 ~ POD reaction with guaiacol or with vanillin as substrates is strongly inhibited (reversibly) by pressures above 300MPa (25 ~ Polyphenoloxidase (PPO) reaction with chlorogenic and caffeic acid as substrates is not influenced by pressure up to 500MPa (25 ~ PPO reaction with pyrocatechol as substrate is strongly inhibited (reversibly) by pressures above 300MPa (25 and 40 ~ 1. INTRODUCTION
To avoid quality loss during and after high pressure treatment of fruits and vegetables, if possible, enzyme reactions should be inhibited during pressure build-up and enzymes should be irreversibly inactivated before storage [ 1, 2]. This is however often not feasible with pressure alone and high temperatures (not desirable for food treatment) must be applied additionally. Would it be possible to selectively and irreversibly inactivate undesired enzymatic activity at lower temperatures and pressures within minutes? Assessment of two of the most temperature- and high pressure- resistant enzymes-peroxidase (POD) and polyphenoloxidase (PPO)- measuring activities with a variety ofsubstrates in situ using a high pressure optical cell has been conducted. 2. MATERIALS AND METHODS Horseradish peroxidase (POD) [Boehringer Mannheim] and tyrosinase (PPO) [Sigma] were tested in excess of substrates. In situ measurements took place in an optical cell U102 [High Pressure Research Centre, Polish Academy of Sciences] for 700 MPa placed in a spectrophotometer [Phamacia Biochorm 4060]. Opposite shapphire windows allowed the light transmission from 200 to 800 nm. A high pressure intensifier hand pump external to the system was coupled to the optical cell with a high pressure capillary tube. Stable temperature was reached with a themaostat [Haake F3] connected to the system. Post-pressure measurements were performed in the multiple cell sample holder of the [Pharmacia] spectrophotometer after treatments in a high pressure device as described [3]. 3. RESULTS AND DISCUSSION The development of a product of the reaction from PPO and pyrocatechol was followed at 300
190 nm (Fig. 1). Pressures of 500MPa have nearly stopped the reaction, but after pressure release normal rate was reestablished. Other PPO substrates tested were chlorogenic and caffeic acid. No remarkable differences were observed in situ under pressure (results not shown). Different substrates with a similar structure to guaiacol - catechol-monoalkyl-ether - were tested (Fig. 2). So far, a reduction in the activity of POD when pressurised with vanillin as substrate was observed, but after pressure release, the normal rate was restored. So the common structure of these substrates might be determinant on the variation of K~ under high pressure.
120 9 ~100 m 80. --> 60. t~ 40~ 200-
100 80 .~ 60 t~
-$ 40~ 0" 20 0
~- --
0.1MPa
0.1MPa IOOMPa 300MPa 500MPa
100MPa 300MPa 500MPa
Treatment (2 min, 25 ~
Treatment (2 min)
Fig. 2" Relative rate of the reaction of peroxidase with different substrates. 9 ferulic acid [] catechol monoethyl ether vanillin
Fig. 1" Relative rate of the reaction of polyphenoloxidase with pyrocatechol. 9 25 ~ [] 40 ~
When POD was pressurised with guaiacol as substrate, pressure effects on the reaction were rapidly detected, and as soon as pressure was built up, a deceleration was evident (Fig. 3).
~-300iPa
.9 .== o, o2~ ~OOM~ ~ . r
.Y
X
0
500 MPa
400 M~a
0'04 I 0,035 ~
.
.jim,,,--
2oo t,ga 200M~a
..................................................................................
Trre clive (10 rrin)
Fig. 3" Development of product at 435 nm of the reaction ofpemxidase and guaiacol in dependence of high pressure intensity.
191 And measurements of activity after pressure release show that the inactivation of POD was dependent on the concentration ofsubstmte, in this case, on guaiacol concentration in the pressurised mixture, as reported in Fig. 4.
100 ~-
--
80
I--I
60 !4o
20 -~ x,iv.
o' 1,12
0,90
0,68
0,46
0,23 9~
Concentration (mM)
Control
- 4 , - - 5 0 0 M P a , 25~ .
.
.
2"
.
Fig. 4: Relative rate of the reaction ofguaiacol and peroxidase after pressure release in dependence of substrate (guaiacol) concentration. Following experiments evidenced that inactivation was only achieved when POD was pressurised in the presence ofH202 and guaiacol. Fig. 5 shows the results after pressure was released: effects of addition of different substrates indicate that the enzyme was inactivated but H202 was not limiting the reaction. POD itself was not inactivated at 25 ~ and 500MPa, and the rate was normal after pressure when guaiacol was used for testing enz3qne activity (results not shown, but confirmed by the literature).
0,25 ~
+ Guajacol
\ r-Jer
~
0,15
§
D
2-1 1 m i n
2 - 1 1 rain
o,1
ELI
i
"
f
2-8min
0,05 0
Time dri~
Fig. 5: Development of product at 435 nm of the reaction ofperoxidase and guaiacol after pressure release when adding different components, but not to the control (a cross in the diagramm). x control; E! control/plus guaiacol, plus fresh POD; A (POD, perydrol and guaiacol) 500MPa, 25 ~ 2 rain/plus guaiacol, plus fresh POD; ~ (POD and guaiacol) 500MPa, 25 ~ perydrol.
2 min/plus guaiacol
192 Effects of guaiacol on the structure of peroxidase under pressure are under investigation. Further experiments are required to determine the possibility of inactivating POD in a mixture of substrates. Existence of other substrates showing a similar effect is being also examined. 4. CONCLUSIONS The influence of pressure on the reaction rate during pressure build-up and at end pressure is different for different substrates: substrate specific pressure dependence of Kin and Vm~x. Peroxidase (POD), one of the highly pressure resistant enzymes, was irreversibly inactivated ( >90 % ) after 2 minutes at 500MPa/25 °C in the presence of guaiacol. 5. ACKNOWLEDGEMENTS FAIR project 98-5031 made possible this investigation.
REFERENCES 1. C. Balny and P. Masson, Food Reviews International, 9 (1993) 611. 2. M. Hendrickx, L. Ludikhuyze, I. Van den Broeck and C. Weemaes, Trends Food Sci. Technol. 9 (1998) 197. 3. P. Butz, W.-D. Koller, B. Tauscher and S. Wolf, Lebensm-Wiss u Technol. 27 (1994) 463.
189
Enzyme-substrate specific interactions: in situ assessments under pressure A. Fern,~dez Garcia, P. Butz, R. Lindauer & B. Tauscher Institute for Chemistry and Biology, Federal Research Centre for Nutrition, Haid-und-Neustr. 9, 76131 Karlsrtthe, Germany Peroxidase (POD) enzyme was irrevers~ly inactivated (>90%) after 2 minutes at 500 MPa/25 ~ in the presence of guaiacol. POD reaction with ferulic acid or with catechol monoethyl ether as substrates is not influenced by pressure up to 500MPa (25 ~ POD reaction with guaiacol or with vanillin as substrates is strongly inhibited (reversibly) by pressures above 300MPa (25 ~ Polyphenoloxidase (PPO) reaction with chlorogenic and caffeic acid as substrates is not influenced by pressure up to 500MPa (25 ~ PPO reaction with pyrocatechol as substrate is strongly inhibited (reversibly) by pressures above 300MPa (25 and 40 ~ 1. INTRODUCTION
To avoid quality loss during and after high pressure treatment of fruits and vegetables, if possible, enzyme reactions should be inhibited during pressure build-up and enzymes should be irreversibly inactivated before storage [ 1, 2]. This is however often not feasible with pressure alone and high temperatures (not desirable for food treatment) must be applied additionally. Would it be possible to selectively and irreversibly inactivate undesired enzymatic activity at lower temperatures and pressures within minutes? Assessment of two of the most temperature- and high pressure- resistant enzymes-peroxidase (POD) and polyphenoloxidase (PPO)- measuring activities with a variety ofsubstrates in situ using a high pressure optical cell has been conducted. 2. MATERIALS AND METHODS Horseradish peroxidase (POD) [Boehringer Mannheim] and tyrosinase (PPO) [Sigma] were tested in excess of substrates. In situ measurements took place in an optical cell U102 [High Pressure Research Centre, Polish Academy of Sciences] for 700 MPa placed in a spectrophotometer [Phamacia Biochorm 4060]. Opposite shapphire windows allowed the light transmission from 200 to 800 nm. A high pressure intensifier hand pump external to the system was coupled to the optical cell with a high pressure capillary tube. Stable temperature was reached with a themaostat [Haake F3] connected to the system. Post-pressure measurements were performed in the multiple cell sample holder of the [Pharmacia] spectrophotometer after treatments in a high pressure device as described [3]. 3. RESULTS AND DISCUSSION The development of a product of the reaction from PPO and pyrocatechol was followed at 300
190 nm (Fig. 1). Pressures of 500MPa have nearly stopped the reaction, but after pressure release normal rate was reestablished. Other PPO substrates tested were chlorogenic and caffeic acid. No remarkable differences were observed in situ under pressure (results not shown). Different substrates with a similar structure to guaiacol - catechol-monoalkyl-ether - were tested (Fig. 2). So far, a reduction in the activity of POD when pressurised with vanillin as substrate was observed, but after pressure release, the normal rate was restored. So the common structure of these substrates might be determinant on the variation of K~ under high pressure.
120 9 ~100 m 80. --> 60. t~ 40~ 200-
100 80 .~ 60 t~
-$ 40~ 0" 20 0
~- --
0.1MPa
0.1MPa IOOMPa 300MPa 500MPa
100MPa 300MPa 500MPa
Treatment (2 min, 25 ~
Treatment (2 min)
Fig. 2" Relative rate of the reaction of peroxidase with different substrates. 9 ferulic acid [] catechol monoethyl ether vanillin
Fig. 1" Relative rate of the reaction of polyphenoloxidase with pyrocatechol. 9 25 ~ [] 40 ~
When POD was pressurised with guaiacol as substrate, pressure effects on the reaction were rapidly detected, and as soon as pressure was built up, a deceleration was evident (Fig. 3).
~-300iPa
.9 .== o, o2~ ~OOM~ ~ . r
.Y
X
0
500 MPa
400 M~a
0'04 I 0,035 ~
.
.jim,,,--
2oo t,ga 200M~a
..................................................................................
Trre clive (10 rrin)
Fig. 3" Development of product at 435 nm of the reaction ofpemxidase and guaiacol in dependence of high pressure intensity.
191 And measurements of activity after pressure release show that the inactivation of POD was dependent on the concentration ofsubstmte, in this case, on guaiacol concentration in the pressurised mixture, as reported in Fig. 4.
100 ~-
--
80
I--I
60 !4o
20 -~ x,iv.
o' 1,12
0,90
0,68
0,46
0,23 9~
Concentration (mM)
Control
- 4 , - - 5 0 0 M P a , 25~ .
.
.
2"
.
Fig. 4: Relative rate of the reaction ofguaiacol and peroxidase after pressure release in dependence of substrate (guaiacol) concentration. Following experiments evidenced that inactivation was only achieved when POD was pressurised in the presence ofH202 and guaiacol. Fig. 5 shows the results after pressure was released: effects of addition of different substrates indicate that the enzyme was inactivated but H202 was not limiting the reaction. POD itself was not inactivated at 25 ~ and 500MPa, and the rate was normal after pressure when guaiacol was used for testing enz3qne activity (results not shown, but confirmed by the literature).
0,25 ~
+ Guajacol
\ r-Jer
~
0,15
§
D
2-1 1 m i n
2 - 1 1 rain
o,1
ELI
i
"
f
2-8min
0,05 0
Time dri~
Fig. 5: Development of product at 435 nm of the reaction ofperoxidase and guaiacol after pressure release when adding different components, but not to the control (a cross in the diagramm). x control; E! control/plus guaiacol, plus fresh POD; A (POD, perydrol and guaiacol) 500MPa, 25 ~ 2 rain/plus guaiacol, plus fresh POD; ~ (POD and guaiacol) 500MPa, 25 ~ perydrol.
2 min/plus guaiacol
192 Effects of guaiacol on the structure of peroxidase under pressure are under investigation. Further experiments are required to determine the possibility of inactivating POD in a mixture of substrates. Existence of other substrates showing a similar effect is being also examined. 4. CONCLUSIONS The influence of pressure on the reaction rate during pressure build-up and at end pressure is different for different substrates: substrate specific pressure dependence of Kin and Vm~x. Peroxidase (POD), one of the highly pressure resistant enzymes, was irreversibly inactivated ( >90 % ) after 2 minutes at 500MPa/25 °C in the presence of guaiacol. 5. ACKNOWLEDGEMENTS FAIR project 98-5031 made possible this investigation.
REFERENCES 1. C. Balny and P. Masson, Food Reviews International, 9 (1993) 611. 2. M. Hendrickx, L. Ludikhuyze, I. Van den Broeck and C. Weemaes, Trends Food Sci. Technol. 9 (1998) 197. 3. P. Butz, W.-D. Koller, B. Tauscher and S. Wolf, Lebensm-Wiss u Technol. 27 (1994) 463.