sci超声波对酶活的稳定性

Process Biochemistry 35(2000)1037–1043

www.elsevier.com /locate /procbio

The stability of enzymes after sonication

zbek a, *,Kutlu O . U lgen b Belma O

Department of Chemical Engineering , YıldızTechnical Uni 6ersity , S ¸is ¸li Campus , S ¸is ¸li 80270, Istanbul , Turkey

Bog ˘azic ¸i Uni 6ersity , Department of Chemical Engineering , Bebek 80815, Istanbul , Turkey

Received 15October 1999; received in revised form 24December 1999; accepted 29January 2000

Abstract

The effects of operating conditions of sonication on the stability of some commercially puriﬁedenzyme preparations were

investigated. Buffered solutions of six enzymes, alcohol dehydrogenase (ADH),malate dehydrogenase (MDH),glucose-6-phos-phate dehydrogenase (G6PDH),L -lactic dehydrogenase (LDH),alkaline phosphatase (AP)and b -galactosidase (b G)were soniﬁedover a range of power outputs up to 40W. The enzymes had variable stabilities with complete stability for AP, and over 70%inactivation for G6PDH. Some inactivation models were tested for an understanding of the relation between soniﬁcationintensity and enzyme stability. Sonication processing times also affected the inactivation rate of ADH and MDH. The stability of soniﬁedADH was decreased with time when compared with unsoniﬁedcontrols. Increasing the viscosity of process ﬂuidwith glycerol gave 39%inactivation of ADH, while the control showed 15%inactivation for the operational conditions. The forces involved in the ﬂuidmust therefore have a signiﬁcantrole to play in the inactivation process. 2000Elsevier Science Ltd. All rights reserved.

Keywords :Enzyme stability; Inactivation; Acoustic cavitation; Wave duty cycle

1. Introduction

The majority of enzymes produced by microorgan-isms are intracellular and some of these have been successfully produced on an industrial scale. The ex-ploitation of intracellular microbial products for indus-trial use, mainly in food and medicine, is continuing to increase with the new developments in genetic engineer-ing and recombinant DNA technology. In order to isolate intracellular microbial products, it is necessary to use methods that are capable of breaking down the cell wall, but at the same time not to cause inactivation of the biological substances. Sonication, homogenisa-tion, bead milling and nebulisation are some of the processing methods available for cell disruption and protein release from cells [1–3].Among these effective methods, sonication offers many advantages [3,4],it has low operating costs and it does not require sophisti-cated equipment or extensive technical training. It has the easy cleaning in place feature. However, inactiva-tion of released products by ultrasonication can be

*Corresponding author. Fax:+90-212-2244968.

zbek) E -mail address :[email protected](B.O

caused by mechanical effects, i.e. shear stress developed

by eddies arising from shock waves.

Many studies on cell disruption have been carried out using the aforementioned methods and a range of microorganisms [4–9].Different models are proposed for cell disintegration as well as for the fast release of intracellular protein, either native or recombinant. The enzyme release process is mostly reported to follow ﬁrst-orderkinetics [5,7,10].However, the rate of release of an enzyme depends on its location within the cell [11].For example, alkaline phosphatase is located in cell membrane and therefore released slowly, while L -lactic dehydrogenase and alcohol dehydrogenase are mostly located in the cytoplasm and their release during cell disruption is instantaneous [5,6].Therefore, it is likely that they are highly affected by the disruption method.

The severity of the disruption conditions can have an impact on the yield of active protein /enzyme recovered in the process. In the present study, the effect of processes on maximal recovery efﬁciencyof active en-zyme was investigated using one of the recently pre-ferred mechanical disruption methods, i.e. ultrasonication. Disruption with ultrasonic energy at

1038zbek , K . O . U lgen /Process Biochemistry 35(2000)1037–1043B . O

sufﬁcientlyhigh acoustic power occurs due to the phe-nomenon of cavitation. However, cavitation causes

chemical effects such as formation of free radicals that can be harmful to labile molecules released from the cells [10].In order to study the stability (inactivationkinetics) of the biological substances, six commercial enzyme preparations from different microbial and mammalian sources were chosen:alcohol dehydroge-nase (ADH),malate dehydrogenase (MDH),glucose-6-phosphate dehydrogenase (G6PDH),L -lactic dehydrogenase (LDH),alkaline phosphatase (AP)and b -galactosidase (b G). The dependence of enzyme stabil-ity on power, wave duty cycle, temperature, viscosity and processing time was determined. The process parameters of ultrasonication were optimised, and the correlation equations between enzyme activity and op-erating variables derived. 2. Materials and methods

2. 1. Sonication of the enzymes in the disrupter

The ultrasonication experiments were carried out at

20kHz on a Branson 450Soniﬁerequipped with a horn of 9mm diameter. The tip of the horn was immersed about 1cm into 10ml solution to be processed. The acoustic power was controlled manually between 7and 40W. The ultrasonic energy was pulsed using the Duty Cycle Control in order to reduce the formation of free radicals. In pulsed mode, ultrasonic vibrations are transmitted to the solution at a rate of one pulse per second. The solution was processed with the sonication horn for 2×30s (with30s still time between the operations) unless otherwise stated. The temperature inside the solutions was intermittently checked and the temperature difference was kept below 5°Cby the use of an outer ice jacket. The treated solutions were analysed by the described techniques to assess the de-gree of stability.

2. 2. Enzymes used

Puriﬁedenzymes were obtained from Sigma Chemi-cal Co. (Munich,Germany):ADH from Bakers Yeast (ProductNo. A7011; EC 1.1.1.1), MDH from Porcine heart (mitochondrial)(ProductNo. M2634; EC 1.1.1.37), G6PDH from Bakers Yeast, (ProductNo. G7750; EC 1.1.1.49), LDH from Rabbit muscle (ProductNo. L2500; EC 1.1.1.27), AP from Bovine intestinal mucosa (ProductNo. P6774; EC 3.1.3.1) and b G from Escherichia coli (ProductNo. G2513; EC 3.2.1.23).

Enzyme solutions were prepared by suspending them in 20mM phosphate buffer at pH 7.5. These enzyme solutions contained approximately 1100–7500U /l de-pending on the enzyme tested.

Typically, a 10ml enzyme solution was soniﬁedin the disrupter. Once the samples had been soniﬁed,they were immediately stored on ice. Activities were then determined according to the assays given elsewhere [3,12–14].At least three measurements were made for each condition and the data recorded below is an average of these data. An original sample was kept without soniﬁcationas a control activity measurement.

3. Computational work

The software package MATLAB 5.0was used for numerical calculations. The parameters were evaluated by the non-linear least-squares method of Marquardt–Levenberg until minimal error was achieved between experimental and calculated values. The residual (SSR)is deﬁnedas the sum of the squares of the differences between experimental and calculated data, given by

N SSR =%d

(C obs m −C calc m )

m =1where m is observation number and N d is total number of observations. The estimated variance of the error (populationvariance) is calculated by the SSR at its minimum divided by its degrees of freedom:|2:s 2(SSR)min /(m −p )

where p is the number of parameters and s 2is the variance. The standard error, |, (theestimated standard deviation) is calculated by taking the square root of the estimated variance of the error.

4. Results and discussion

The enzymes were suspended in 20mM phosphate buffer at pH 7.5and were ultrasonicated under various process conditions. The acoustic power (W),processing time (s),wave duty cycle (%),viscosity (cps)and tem-perature (°C)were chosen as the key elements in inﬂu-encing the enzyme stability during ultrasonication. Enzyme activities prior to ultrasonication were also determined and used as controls. In calculations, A max was denoted as the enzyme activity without disruption, i.e. prior to sonication. This value was then considered as 100%activity. Activity at any operational condition (A ) was then recorded in terms of percentages of the undisrupted control.

4. 1. The effect of temperature during the sonication process

The violent collapse of cavitation bubbles in an ultra-sonic ﬁeldcauses extremely high local temperatures [9].The temperature change of ultrasonicates (10ml solu-

zbek , K . O . U lgen /Process Biochemistry 35(2000)1037–1043B . O 1039

tions) was investigated at two different wave duty cy-cles, i.e. 10and 90%,and at various power outputs (i.e.

15–40W) with processing time 2×30s. The results are summarised in Table 1. At 27W and 90%duty cycle (DC),the temperature increased from ambient (23.7°C)to 39.5°Cin 2×30s. Therefore, strict control over the temperature was needed, maintained by the use of an ice jacket. At 27W and 90%DC with an ice jacket, the temperature difference was only 4.4°C.If the processing time was increased further, i.e. 3×30s, the tempera-ture difference (D T ) also increased to 36°Cat 27W and 90%DC. However, on ice, D T remained at 4.9°C.At 10%wave duty cycle, the temperature differences were smaller than those obtained at 90%wave duty cycle, e.g. at comparable outputs, 7.1°versus 20.5°C.

Table 1

Temperature change during the sonication process without using an ice jacket

Acoustic power (W)Duty cycle (%)Time (s)D T (°C)15102x303.632102x307.127902x3015.840

2x30

20.5

Fig. 1. (a)Percent activities of enzymes at various acoustic powers

(W)at duty cycle 90%.(b)Percent activities of enzymes at various acoustic powers (W)at duty cycle 10%

Pulsing the ultrasonic energy retarded the rate of tem-perature increase.

4. 2. The effect of acoustic power on enzyme stability

In order to investigate the effect of ultrasonic inten-sity, the enzyme solutions were sonicated at different acoustic powers ranging from 7to 40W for 2×30s at wave duty cycles of 10and 90%,respectively. Data shown in Fig. 1a,b indicate the relative activity (A /A max ) versus acoustic power proﬁlesfor six commercial enzyme preparations. Apart from AP, all enzymes showed some inactivation with increasing power. The proﬁleand the degree of degradation was dependent on the enzyme (seeFig. 1a,b). The order of stability throughout the power range tested at duty cycle 90%(Fig.1a) was as follows:

AP \LDH \b G \G6PDH \MDH \ADH

For ﬁrst-orderdependence of activity on power, the activity versus acoustic power proﬁlesin a semilogarith-mic plot were expected to yield straight lines. However, simple ﬁrst-orderkinetics were clearly not applicable to the inactivation of the enzymes at 90%DC. The linear regression coefﬁcients,R 2, are 0.7803, 0.8089, 0.9740, 0.9693and 0.9378for G6PDH, LDH, b G, MDH and ADH, respectively. The deviation from ﬁrst-orderde-pendence was less pronounced at 10%DC and the linear regression coefﬁcients,R 2, were above 0.9436. The presence of two types of enzyme subunits has been reported for ADH and some other enzymes [6].Thus, a two-component, ﬁrst-orderinactivation model was used to ﬁtthe data at both 10and 90%wave duty cycles. The following activity–powerexpression given by Skerker and Clark [7]was used:A /A max =a 1exp(−k 1D P ) +a 2exp(−k 2D P )

(1)

where k 1D , and k 2D , are the degradation coefﬁcientsdependent on the acoustic power output (W−1and W −2, respectively), and a 1and a 2are the initial frac-tions of enzyme components 1and 2, respectively. The parameters in Eq. (1),a 1, a 2, k 1D , and k 2D , were esti-mated using the Marquardt–Levenbergoptimisation routine of the MATLAB 5.0software. However, the program either estimated negative values or resulted in inconsistent values.

For some enzymes, it has been reported that the single-step inactivation leads to a ﬁnalstate that does exhibit some residual activity [15].

E k D

E h 1

In this reaction, E and E 1are enzyme states of different speciﬁcactivities and are homogeneous, h 1is the ratio of the speciﬁcactivity of the ﬁnalstate to the initial state, and k D is the degradation coefﬁcient(W−1).

1040zbek , K . O . U lgen /Process Biochemistry 35(2000)1037–1043B . O

Table 2

Estimated parameters of the single-step model (Eq.(2))Enzyme

h 1

k D

SSR

Standard error (W−1) (|) b G (at90%0.01070.01450.00440.0271DC)

b G (at10%0.00560.00540.01410.0484DC)

MDH (at90%0.00600.02750.02890.0694DC)

MDH (at10%0.36560.02320.00050.0091DC)

ADH (at90%0.20180.03170.00140.0153DC)

ADH (at10%0.5666

0.0585

0.0009

0.0122

DC)

Table 3

Estimated parameters of quadratic ﬁtEnzyme

k D1

k D2

R 2Statistic

Standard (W−1) (W−2) error (|) LDH (at0.25700.03520.99260.021390%DC) LDH (at0.16550.01410.99720.005910%DC) G6PDH (at0.24810.04150.99130.036890%DC) G6PDH (at0.1677

0.0436

0.9913

0.0235

10%DC)

Fig. 2. Percent activities of ADH and MDH enzymes at various processing times (s).

The activity–powerrelationship [15]is then as follows:

A /A max =(1−h 1) exp(−k D P ) +h 1

(2)

Eq. (2)was used to model the inactivation data of enzymes at both wave duty cycles of 10and 90%.The parameters, h 1and k D, were estimated using the Mar-quardt–Levenbergoptimisation routine of the MATLAB 5.0software and are presented in Table 2. The data of

the enzymes b G, ADH and MDH resulted in meaning-ful values of these parameters. The degradation coefﬁ-cients of enzymes, k D , increased in the order of decreasing stability.

A single-step non-ﬁrst-ordermodel (Eq.(2))simu-lated very effectively the inactivation data of b G, MDH and ADH enzymes at both 10and 90%DC (Fig.1a,b). The data of LDH and G6PDH, on the other hand, produced quadratic ﬁts.After evaluation of the data of LDH enzyme, Eqs. (3)and (4)were derived for 90and 10%duty cycles, respectively.

A LDH (90%)=99.61+0.2570P −0.0352P 2(3)A LDH (10%)=100.42−0.1655P −0.0141P 2

(4)

Again, after evaluation of the data of G6PDH enzyme, Eqs. (5)and (6)were derived for 90and 10%duty cycles, respectively.

A G6PDH (90%)=101.48−0.2481P −0.0415P 2(5)A G6PDH (10%)=99.35+0.1677P −0.0436P 2

(6)

where A LDH and A G6PDH are residual enzyme activities (percentagevalues after disruption), P is the acoustic power output value (W),and k D1and k D2are the degradation coefﬁcientsdependent on the acoustic power output (W−1and W −2, respectively). The parameters, k D1and k D2, were estimated using the Marquardt–Levenbergoptimisation routine of MAT-LAB 5.0software and given in Table 3.

4. 3. The effect of processing time on ADH and MDH enzymes

The time of exposure to ultrasound is a critical parameter responsible for cell disruption as well as for enzyme stability. Since the soniﬁeris a constant-ampli-tude device, i.e. the oscillating frequency and the ampli-tude of oscillation are ﬁxed,the rate of energy input is ﬁxedat the chosen output and duty cycle control level. The rate of change in activity with time was investi-gated using two enzymes from different sources, i.e. microbial and mammalian. Fig. 2shows the activity versus time proﬁlesof ADH (fromBaker’syeast) and MDH (fromporcine heart mitochondria) under process conditions of 15W acoustic power and 10%wave duty cycle, as well as at 27W acoustic power and 90%wave duty cycle. Steady decreases in activities of ADH and MDH were observed with time of exposure (Fig.2). The following relationship was used to represent the data of ADH and MDH enzymes:A =A max −k 0·t

(7)

where k 0is the zero order inactivation coefﬁcient(s−1). The zero-order rate constants are given in Table 4. ADH was then used to investigate some of the soniﬁerparameters.

zbek , K . O . U lgen /Process Biochemistry 35(2000)1037–1043B . O

Table 4

The effect of processing time on inactivation of ADH and MDH enzymes 1041

Enzyme Duty cycle (%)and acoustic power (W)ADH 10and 15ADH 90and 27MDH 10and 15MDH

90and

4. 4. The effect of wa 6e duty cycle on the acti 6ity of the ADH enzyme

ADH enzyme in 10ml of 20mM phosphate buffer was subjected to sonication for 2×30s. The ultrasonic energy was pulsed using duty cycle control settings from 10to 90, which changes the duration of the pulse from 10to 90%of each second, respectively. At 10and 20%wave duty cycles, the power output recorded 15W, and from 30%DC onwards it recorded 27W. The data are shown in Fig. 3. No linear relationship was obtained from these results. The data were then ﬁttedto a single-step unimolecular non-ﬁrst-orderenzyme inactivation model. The h 1and k D values were esti-mated as 0.5913and 0.0365W −1, respectively. The residual (SSR)was 0.0067. Compared with the results of ADH obtained at 27W (seeTable 2), the parame-ters, h 1and the degradation coefﬁcient,k D , with respect to change in wave duty cycle have the same order of magnitude.

4. 5. The stability of ADH enzyme after disruption

Enzyme inactivation is, to some extent, reversible [16–18].In a further experiment, ADH was sonicated at different intensities from 7to 32W at 10%DC for 2×30s and the fractions assayed for enzyme activity. The ultrasonicates were then incubated at 20°Cfor 5h and activities determined periodically. No recovery in enzyme activity was observed. The activities continued to decline with incubation time in a ﬁrst-orderdecay (Fig.4a). The acoustic power appeared to increase the susceptibility of the ADH enzyme over the control. Fig. 4b shows the effect of the acoustic power in the range 0–32W at 10%DC on the inactivation rate constant, k I , of ADH enzyme. The inactivation rate constant dependent on time, k I , increased linearly with increasing power intensity in the 5h following the sonication process. As can be seen in Fig. 4b, a linear relationship was observed from these results:ln A =ln A max −k I ·t

(8)

The half-life of ADH enzyme was determined using these data and linear regression analysis (Table5). The half-life of ADH at 20°Cdecreased from 12.7to 8.1h at 32W and 10%wave duty cycle.

k 0(s−1) R 2Statistic Standard error (|) 0.28720.9980.00810.81930.9980.01730.20890.9950.01210.8267

0.997

0.0217

4. 6. The effect of the 6iscosity on the acti 6ity of the ADH enzyme

Another factor known to affect the inactivation of enzymes in high shear disruption medium is the viscos-

Fig. 3. Percent activity of ADH enzyme at various wave duty cycles (%).

Fig. 4. (a)ln(activityof ADH) enzyme at duty cycle 10%versus time (h).(b)Inactivation rate constant (k I , h −1) of ADH enzyme versus acoustic power (W)at duty cycle 10%.

1042zbek , K . O . U lgen /Process Biochemistry 35(2000)1037–1043B . O

Table 5

Half-life of the ADH enzyme at various acoustic power outputs Acoustic power (W)Inactivation rate constant, k I (h−1) 00.054670.0614120.0672150.068918.50.0731250.0792300.081832

0.0862

ity of the solution. Glycerol solutions at 10, 20, 30, 40and 50%were prepared in 20mM phosphate buffer at pH 7.5. These give viscosities [19]of 1.311–6.050cPoise. ADH was then added to the 20mM phosphate buffer containing 10–50%glycerol. The effect of glyc-erol on the enzyme activity was investigated both prior to and after the sonication process (at15W acoustic power, 10%duty cycle and 2×30s processing time), and it was shown that the enzyme activity reduced in proportion to glycerol concentration. The reduction in activity was calculated as a percentage reduction against an undisrupted control at the speciﬁedglycerol concentration. Increasing the viscosity of the process ﬂuidfrom 1to 6cps with glycerol gave 39%inactiva-tion of ADH, while the control showed 15%inactiva-tion for the operational conditions.

No linear relationship was obtained from these re-sults (Fig.5). The data were then ﬁttedto a single-step unimolecular non-ﬁrst-orderenzyme inactivation model (Eq.(2)).The h 1and k DV (inactivationrate constant dependent on viscosity) values were estimated as 0.5990and 0.4891(cps−1), respectively. The residual (SSR)and standard error (|) values were 0.0006and 0.0122, respectively.

4. 7. Comparison of the energy efﬁcienciesof ca 6itation processes

The energy efﬁcienciesof cavitation processes (acous-tic versus hydrodynamic) were compared in order to see the energy requirements. The power consumption of the ultrasonic horn was between 7and 40W (J/s). The quantity of the suspension treated was 10ml. The energy requirement per millilitre of suspension is calcu-lated for the lowest (7W, 2×30s) and highest (40W, 8×30s) cases as follows. (7J /s) (60s) /(10ml) =42J /ml (requiredminimum energy) (40J /s) (240s) /(10ml) =960J /ml (requiredmaximum energy)

Half-life, t (h)R 2Statistic Standard error (|) 12.70.99330.008411.30.99580.007510.30.99490.008910.10.99180.01179.50.99480.00988.80.99230.01308.50.97830.02158.1

0.9838

0.0207

Compared with hydrodynamic cavitation [8]of 21J /ml, the acoustic cavitation (ultrasonication)equip-ment required higher amounts of energy in order to treat the same quantity of suspension.

5. Conclusions

An evaluation of the experimental and theoretical data showed that many process factors are involved in the inactivation of enzymes after disruption by soniﬁca-tion. The order of stability was obtained as AP (100%)\b G (85%)\LDH (81%)\MDH (66%)\ADH (64%)\G6PDH (52%)and AP (100%)\b G (55%)\LDH (54%)\MDH (33%)\ADH (44%)\G6PDH (29%)at acoustic powers 32W with 10%DC and 40W with 90%DC, respectively. As the acoustic power (ultrasonicintensity) increased, the loss of en-zyme activity also increased.

The inactivation mechanisms with each enzyme stud-ied were different, and was speciﬁcto the enzyme. Various kinetic parameters dependent on the soniﬁca-tion intensity and enzyme stability were estimated. The modelling studies showed that a single-step model with non-zero activity of the ﬁnalenzyme state accurately represented the inactivation data of enzymes b G, ADH and MDH. The data of LDH and G6PDH, on the other hand, resulted in quadratic ﬁts.

Fig. 5. Percent activity of ADH enzyme versus viscosity of the solution (cps).

zbek , K . O . U lgen /Process Biochemistry 35(2000)1037–1043B . O 1043

Sonication processing time also affected the relative

activities of the enzymes. There was a zero-order inacti-vation in acoustic cavitation for the enzymes ADH and MDH.

From the studies on the activity of ADH enzyme at various duty cycles, it was observed that 30%wave duty cycle was a threshhold value causing maximum degra-dation. A signiﬁcantdecrease in relative activity was not observed in the range 30–90%wave duty cycles (DC).

The effect of acoustic power output over the long-term stability for ADH enzyme after disruption was also investigated. This showed that ADH progressively lost its activity with time and that the decay rates were related to the operating acoustic power of the soniﬁer.The effect of the ﬂuidviscosity was investigated on ADH enzyme activity. Further deactivation of the en-zyme was observed in glycerol solutions with an in-crease in the viscosity. The physical nature of the ﬂuidand the forces generated within the ﬂuid,especially those associated with shear ﬁeld,are known to be of importance in generating conditions for inactivation. Finally, the energy requirements were calculated for both hydrodynamic cavitation and acoustic cavitation (ultrasonication)equipment. The maximum energy re-quirement for acoustic cavitation was approximately 45times higher than that for hydrodynamic cavitation in treating the same quantity of suspension. Ultrasonica-tion could be an uneconomical process for large-scale use due to this higher energy requirement. Enzyme degradation should also be compared and its potential kept in mind.

The forces involved in the soniﬁedﬂuidhave a signiﬁcantrole to play in the inactivation process. The most likely are those associated with the shear ﬁeldwithin the ﬂuidwhile soniﬁcationoccurs. The stability behaviour observed was different for each enzyme. This could be related to location in the cell (i.e.in the cell membrane or in the cytoplasm etc.), their molecular weights or their sources (i.e.microbial or mammalian). Thus, the operational parameters of the soniﬁershould be optimised speciﬁcallyfor each enzyme, as parame-ters such as time and intensity could cause degradation of the released enzyme during the disruption of cells.

Acknowledgements

This work was supported by the Yildiz Technical University Research Fund (ProjectNumber 98-A-07-

01-03) and the Bogazic ¸i University Research Fund (ProjectNumber 99HA502D).

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sci超声波对酶活的稳定性

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