Immobilization and treatment of Streptococcusfaecalis for the continuous conversion of arginine into citruHine Gilles Cottenceau, Michel Dherbomez, Bernard Lubochinsky and Franqois Letellier Laboratoire de Biochimie Cellulaire, Equipe de Biotechnologie, Institut Universitaire de Technologie, La Rochelle, France
Citrulline is one of the steps of the arginine dihydrolase system of" Streptococcus faecalis. We have shown that the bacteria, immobilized in polyacrylamide gel and treated with Cetyl trimethyl ammonium bromide (CTAB) or heat, were able to convert arginine to citrulline. Used continuously in a column reactor, the entrapped cells have a stable enzymatic activity for at least 30 days at 45°C.
Keywords:Immobilization; Streptococcusfaecalis; citrulline; biosynthesis; arginine deiminase; continuous conversion; thermal treatment
Introduction A number of microorganisms have the arginine dihydrolase system, which includes the three enzymes arginine deiminase (ADase; E.C.3.5.3.6), ornithine transcarbamylase (OTCase; E.C.2.1.3.3), and carbamate kinase (E.C.2.7.2.1), which catalyse the three subsequent reactions arginine + H20 ~ citrulline + NH3; citrulline + Pi ~ ornithine + carbamyl phosphate; carbamyl phosphate + ADP ~ ATP + CO2 + NH3. This system has been studied in Streptococcus faecalis, among others, by Akamatsu et al., Knivett, 2,3 Oginsky et al.,4.5 and Petrack et al. 6 To have an accumulation of citrulline, it is necessary to stop the OTCase activity. To achieve this a number of treatments have already been used successfully: treatments with acetone, 3,4 with CTAB, 7 and by freezing the cell suspension. 8 We have shown in our study that a treatment with heat would give good results. The immobilization of intact cells in order to use one or several of their enzymatic activities is a technique which has been largely developed in the last few years. 9-11 The immobilization of S. faecalis in a poly-
acrylamide gel was studied by Franks, 8 who showed that a system capable of converting arginine by the way of arginine dihydrolase could be obtained. Ajinomoto patents 12,13 and the work of Yoshida ~4 deal with the production of citrulline by fermentation. The continuous bioconversion of arginine into citrulline by immobilized P. putida was studied by Yamamoto et a1.15 In this work, we have studied the capability of immobilized S. faecalis to achieve this continuous bioconversion, as well as the performances of the obtained system.
Materials and methods Organisms and culture conditions
The strain utilized, Streptococcus faecalis, A T C C 8043, was cultured for 16 h at 37°C, in aerobiosis, in a medium containing (per liter of deionized water): yeast extract (Difco), 5 g; bactocasitone (Difco), 2.5 g; Larginine, HCI, 5 g; glucose, 5 g; Na2HPO4, 0.9 g; NaC1, 0.5 g; MgSO4 • 7H20, 0.5 g; MnSO4 • H20, 0.2 g; FeSO4 • 7H20, 0.002 g. P r e p a r a t i o n o f cells
Address reprint requests to Dr. Cottenceau at the Laboratoire de Biochimie Cellulaire, Equipe de Biotechnologie, Institut Universitaire de Technologie, rue de Roux, 17026 La Rochelle, France Received 13 October 1989; revised 25 May 1989
The cells were harvested by centrifuging the culture medium (20 min, 4000 g), then washed three times by resuspending the pellet in 15 times its volume of 50 mM phosphate buffer at p H 7.
© 1990 Butterworth Publishers
Enzyme Microb. Technol., 1990, vol. 12, May
355
Papers Immobilization Unless otherwise stated, the cells were immobilized immediately after washing by inclusion in a polyacrylamide gel by the method described by Chibata et al.16" 0.3 g of cells (wet weight) were suspended in 3.7 ml 50 mM phosphate buffer at pH 7, then 700 mg of acrylamide, 80 mg NN'-bisacrylamide (BIS), 0.5 ml a 5% dimethyl-amino-propionitrile solution in water, and 0.5 ml a 2.5% potassium persulfate solution in water were sequentially added. The polymerization time was 15 min at 37°C. The gel, once constituted, was washed twice with 10 ml phosphate buffer then crushed. The particles were again washed three times with 50 ml phosphate buffer at pH 7.
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Standard determination o f ADase activity Unless otherwise stated, the measurement of ADase activity was performed according to Yamamoto et al.15: I g of crushed gel, or 1 ml of cell suspension containing the appropriate quantity of cells (wet weight), was incubated in 15 ml 10 mM arginine solution in 50 mM phosphate buffer at pH 7 and 37°C, with shaking (60 rev rain-l). The enzymatic reaction was stopped by addition of trichloroacetic acid, 0.25 M final concentration. The precipitate was removed by centrifugation (10 min, 5,000g). The quantity of e-citrulline in the supernatant was measured according to Archibald's method 17, improved by Boyde et al. ~8
50
0,2
0.4 mM
0.6 of
0.8
CTAB
Figure 1 Influence of substrate and cell concentration on the optimum CTAB. Gel at 0.3 g of wet cell g 1of gel, activity of 1.5 g of gel in presence of variable concentration of CTAB with 5 (O) or 10 (©) mM of arginine; (11) activity of 1.5 g of gel containing 0.052 g of cells g 1; (r~) activity of 3.0 g of gel containing 0.026 g of cells g-l; activity measured with variable concentration of CTAB
Results and discussion
Study o f the conditions o f immobilization on the ADase activity The effect of acrylamide and BIS concentration in the gels on the ADase activity has been studied in the presence of 0.62 m u of CTAB. For mechanical reasons, we have chosen a concentration in acrylamide of 120 mg g i of gel; with this concentration, the ADase activity is maximum for a concentration in BIS of 14 mg g-1 of gel. The gel entrapment of the cells obtained is stable; successive washings do not reduce the residual enzymatic activity of immobilized bacteria. A part of the gel (1 g) was washed successively with 50 ml phosphate buffer before addition of a new quantity of substrate and measure of the residual activity; the seventh washing does not give a result significantly different from the first.
Action o f CTAB on the ADase activity o f the immobilized cells The increase of ADase activity due to the treatment with CTAB is rather slow, because of the diffusion barrier constituted by the gel (about 10 min are necessary to obtain the maximum activity) and this action is irreversible. On the other hand, neither the concentration in substrate nor the cell concentration in the gel have an 356
Enzyme Microb. Technol., 1990, vol. 12, May
influence on the optimum concentration of CTAB, as shown in Figure 1. For a gel of given constitution, the optimal concentration of CTAB depends on the amount of cells compared with the volume of reactive medium. We have made a series of manipulations with variable amounts of cells immobilized; the activity of the gel + cells systems obtained has been measured in the presence of variable concentrations of CTAB with the aim of determining the optimal concentration. Figure 2 gives a synthesis of the results of these experiments; it represents a graph of the relation (optimal concentration of CTAB)
= fct (cell concentration in the reactive medium) where fct = abbreviated form of "function o f " The concentration in CTAB necessary for saturating the system increases in a rather linear way up to a cell concentration of about 6 mg of cells (wet weight) ml -~ of reactive medium, corresponding to a concentration of CTAB of 0.53 mM. Beyond that point, we noticed a change in the slope and a much faster increase of the optimal concentration in CTAB. Figure 3 gives the graph of the relation (maximal specific activity of the gel + cells systems)
= fct (cell concentration in the gel)
Citrulline production by S. faecalis: G. Cottenceau et al. / 1.0.
tration beyond which a new kinetics of diffusion inside the gel + cells system would take place.
/
A i E
/ 0.8 ¸
rn
i
i
,< t-
/
d
The effect of pH on the reaction rate is given in Figure 4. The optimum pH of entrapped cells shifted by one unit to the alkaline side compared with that of intact cells; on the other hand, the presence of CTAB has no effect. The influence of the temperature of reaction on the ADase activity of the intact cells in the presence of CTAB and of immobilized cells with and without CTAB, is summed up in Figure 5. We notice a 20°C increase in the optimum temperature with immobilized cells and a twofold increase in relative activity if CTAB treatment is omitted.
/ / /
0,6 /
E s
j~ O
0,4-
Y
•
0.2-
i
i
i
i
3
4
5
6
7
reactive
medium
mg
wet
cells
/
ml
Properties of the ADase of immobilized bacteria in the presence of CTAB
Figure 2 O p t i m a l concentration in CTAB c o m p a r e d with the cell concentration of the reactive m e d i u m
Determination of the best thermal treatment of the gel + cells system for the development of the ADase activity A thermal treatment of the gel + cells system made it possible to reach higher ADase activity than that obtained by CTAB treatment. We have looked for the time-temperature couple to be applied to the immobilized cells to reach the highest ADase activity. For this purpose, we have heated the gel + cells system at different temperatures and measured the evolution of their ADase activity according to the time. The obtained maxima are shown in Table 1. From these results, it appears that the optimum duration of heating (fct a) and maximum specific activity
20 ¸
J::
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.>_ (0 ,o
Q. ~9
100
10-
,o
=o
mg
wet
cells
6'o /
g
a'o
80
,'0o
of g e l 60
Figure 3 Specific activity versus cell concentration in the gel. (11) C T A B concentration < 0 . 5 3 m M ; (rT) CTAB concentration > 0.53
mM rr
We notice that, here again, the points divide into two groups: below and beyond 0.53 mM of CTAB, the specific activity decreases linearly with the increase of the cell concentration because of the increasing difficulty of the diffusion of both substrate and reaction products inside the particles of the gel; but we also notice that on both sides of this concentration, the specific activity takes significantly different values for the same cell concentration. This may indicate that, around 0.53 mM of CTAB, there is a transition concen-
40-
20-
. 5
, 6
. 7
. 8
, 9
pH Figure 4 Effect of pH on the A D a s e activity of i m m o b i l i z e d bacteria. (11) Activity of 45 m g of intact cells (wet weight), measured with 0 . 3 m M of C T A B at various p H s ; (El) activity of i m m o b i l i z e d cells at various p H s ; ( T ) activity of i m m o b i l i z e d cells with 0 . 3 m M of C T A B at various pHs; activity measured at 3 7 ° C
Enzyme Microb. Technol., 1990, vol. 12, May
357
Papers The general equation of the second function (fct b) is
300
Log (Ao
/v,, /
\
.> u 0~ > i~
1 0C
¢"
..,.j..~~ .....
50'
/
... \
J
i
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Temperature
At)
-kT ° = p
=
(2)
Ao = specific limit activity that the immobilized bacteria can develop with heat treatment (/ZM ml J h i g J) At = m a x i m u m specific activity reached by the immobilized bacteria after a treatment at a temperature of T°C ([,ZM m1-1 h i g 1) T° = temperature o f treatment in °C From the values s h o w n in Table 1, we calculated the m a x i m u m specific activity of the system, Ao = 85 [LM ml 1 h 1 g - ] which depends on our conditions. This value reported in equation (1) allows us to plot the graph of this relation (Figure 6), in which we notice that the m a x i m u m specific activity is obtained within 1% for a temperature of 52°C, corresponding by equation (1) to a treatment of 1370 min. For practical reasons, w e have c h o s e n for the following manipulations a specific activity of 75 /L~Mml -I h -j g-I, which is obtained with a temperature of 66°C and a duration of treatment of 100 min.
A
20(~
-
70
°C
Figure 5 Effect of the temperature of reaction on the ADase activity of immobilized bacteria. (11) Activity of 45 mg of intact cells (wet weight), with 0.3 mM o f CTAB, at various temperatures; activity of immobilized cells with no CTAB (T), with 0.15 mM CTAB (V), w i t h 0.3 mM CTAB (rq), at various temperatures; (i) = relative activity compared with the one of the free cells at
Properties of the ADase of the immobilized b a c t e r i a after heating The influence of the temperature of reaction on the A D a s e activity of the immobilized and heated bacteria
37°C
Table 1
Ao-1
Temp. of treatment
Duration of treatment according to the temperature Optimum time of treatment
(°C) 77 73.5 70 66.5 63
Ao-lO
(min)
Max. specif activity a reached (/zM ml 1 h 1 g 1)
11 28 46 100 155
36.5 55 65 76.5 79
a Specific activity = /zM of citrulline formed ml ~ of reactive medium h 1, g 1 of immobilized cells (wet weight)
Table 2 Ao-IOG
,
i
,
i
55
60
65
70
Figure 6
Temp.
Citrulline in the effluent
(ml h 1)
(/~M ml 1)
24 30 36 42 36 42 48 54 48 54 60 66
10 10 9.0 8.2 10 9.7 9.0 8.1 10 9.7 8.9 8.0
% of conversion
°C
Graph of the relation (Ao - At) =
-kT ° + P
(fct b) are logarithmic functions of the temperature of treatment. With the linear regression method, w e have found that the first equation of the function (fct a) is: log tps = - 0 . 0 8 1 4 T ° + 9.1479
(1)
tps= optimum duration of treatment (s) T ° = temperature of treatment (°C)
358
Flow rate
z
75
(°C)
Temperature
Effect of feeding rate on the formation of citrulline
Enzyme Microb. Technol., 1990, vol. 12, May
35 35 35 35 45 45 45 45 55 55 55 55
100 100 90 82
100 97 90 81 100 97 89 80
Citrulfine production by S. faecalis: G. Cottenceau et al. / 150J f
.Z~j
~111~
co
.... o .....
"S..'..~." --. ......
'~"':"'"L~
100-
>
50n-
T 1
i 2
i 3
I 4
5
Time
in
6
22
= 24
23
T 25
hours
Figure 7 Stability of the A D a s e activity in regard to the operational temperature. I m m o b i l i z e d cells heated in a p h o s p h a t e buffer (66°C, 100 rain) then at different t e m p e r a t u r e s during variable times; (C]) 70°C; (0) 65°C; (A) 60°C; (11) 55°C; (O) 50°C; (T) 45°C
has been studied; the same optimum as for the system treated with C T A B is obtained: 70°C. The kinetic constants of the A D a s e , as it works in our system, are Km = 1.18 /ZM ml -], g m a x = 1.25 /zM ml- 1 20- I min. The stability of the A D a s e activity has been tested; our results are s h o w n in Figure 7.
Continuous conversion of arginine into citrulline The continuous c o n v e r s i o n of arginine into citrulline was studied with a reactor consisting of a glass column of 16 m m of interior diameter with a thermostating jacket heated by water circulation, the substrate being fed from the bottom. The effect of the feeding rate on the formation o f citrulline was studied and the results are s h o w n in Table 2. To achieve it w e have fed the reactor at different feeding rates with a 10 mM arginine solution in 0.05 M phosphate buffer at pH 7; the reactor was filled up with 8 g o f heated gel + cells s y s t e m (66°C, 100 min) and heated at different temperatures. The operational stability o f the A D a s e activity of the reactor was tested at two different temperatures, 45°C and 55°C (see results in Figure 8). The activity of the reactor was stable over 30 days at 45°C for a feeding rate of 50 ml h -1. Moreover, the stability o f the activity is not affected by a treatment of the reactor with sodium azide at least up to a concentration o f 0.2%.
10-
A v
O) A
.*E--
r 5
i 10
i 15 Time
t 20 in
r 25
, 30
days
Figure 8 Operational stability of A D a s e activity of i m m o b i l i z e d and heated bacteria. A 10-mM sterilized arginine solution in p h o s p h a t e buffer was passed t h r o u g h the c o l u m n at a flow rate of 50 ml h -1, the c o l u m n being filled up with 8 g of heated gel + cells system; ( 0 ) operational t e m p e r a t u r e 45°C; (O) operational t e m p e r a t u r e 55°C; 1, 2, 3, 4, 5: a s o d i u m azide solution of respectively 0.02%, 0.04%, 0.06%, 0.08%, 0.010% was passed t h r o u g h the reactor for 1 h
Enzyme Microb. Technol., 1990, vol. 12, May
359
Papers Conclusion
Streptococcus faecalis, strain ATCC 8043, immobilized in a polyacrylamide gel, is a potent catalyst for the continuous production of L-citrulline from L-arginine. As it does for a large number of microorganisms, the polyacrylamide gel ensures an easy and very stable immobilization. Yet, as opposed to what happens with Pseudomonas putida, this immobilization does not sufficiently modify the cell, and particularly its membrane, to permit the accumulation of L-citrulline. Considering the two treatments used to modify the membrane, either with a surface active agent or with heat, the heat treatment is definitively more effective as it ensures a much better thermal stability. The maximum specific activity that we have reached after a heat treatment of the immobilized cells was 85/ZM m1-1 h -1 g-l.
References 1 2 3 4 6 7 8 9 10 11 12 13 14 15 16
Acknowledgements We thank Mrs. M. Quillet for technical assistance and Mrs. G. Weyland for linguistic advice.
360
Enzyme Microb. Technol., 1990, vol. 12, May
17 18
Akamatsu, S. and Sekine, T. J. Biol. Chem. 1951,38, 349-354 Knivett, V. A. Bioehem. Prep 1953, 3, 104-107 Knivett, V. A. in Reports o f International Symposium o f CNRS: Biochimie comparde des acides aminds basiques Concarneau, 1959, pp. 243-260 Oginsky, E. L. and Gehrig, R. F. J. Biol. Chem. 1952, 198, 791-797 Petrack, B., Sullivan, L. and Ratner, R. Arch. Biochem. Biophys. 1957, 69, 186-197 Kakimoto, T., Shibatani, T., Nishirnura, T. and Chibata, I. Appl. Microbiol. 1971, 22, 992-999 Franks, N. E. Biochem. Biophys. Acta 1971, 252, 246-254 Fukui, S. and Tanaka, A. Ann. Rev. Microbiol. 1982, 36, 145172 Hallenbeck, P. C. Enzyme Microb. Technol. 1983, 5, 171-180 Mattiasson, B. Immobilized Cells and Organelles, Vols. 1, 2 CRC Press, Boca Raton, FL, 1983 Ajinimoto, International patent 1982 C 12P-013/10 C12R-001/13 Ajinomoto, International patent 1982 C12P-013/10 CI2R-001/ 15 Yoshida, H. Prog. lndust. Microbiol. 1986, 24, 131-143 Yamamoto, K., Sato, T., Tosa, T. and Chibata, I. Biotechnol. Bioeng. 1974, 16, 1589-1599 Chibata, I., Tosa, T. and Sato, T. Appl. Microbiol. 1974, 27, 878-885 Archibald, R. M. J. Biol. Chem. 1944, 156, 121-142 Boyde, T. R. C. and Rahmatullah, M. Anal. Biochem. 1980, 107, 424-431
Citrulline is one of the steps of the arginine dihydrolase system of" Streptococcus faecalis. We have shown that the bacteria, immobilized in polyacrylamide gel and treated with Cetyl trimethyl ammonium bromide (CTAB) or heat, were able to convert arginine to citrulline. Used continuously in a column reactor, the entrapped cells have a stable enzymatic activity for at least 30 days at 45°C.
Keywords:Immobilization; Streptococcusfaecalis; citrulline; biosynthesis; arginine deiminase; continuous conversion; thermal treatment
Introduction A number of microorganisms have the arginine dihydrolase system, which includes the three enzymes arginine deiminase (ADase; E.C.3.5.3.6), ornithine transcarbamylase (OTCase; E.C.2.1.3.3), and carbamate kinase (E.C.2.7.2.1), which catalyse the three subsequent reactions arginine + H20 ~ citrulline + NH3; citrulline + Pi ~ ornithine + carbamyl phosphate; carbamyl phosphate + ADP ~ ATP + CO2 + NH3. This system has been studied in Streptococcus faecalis, among others, by Akamatsu et al., Knivett, 2,3 Oginsky et al.,4.5 and Petrack et al. 6 To have an accumulation of citrulline, it is necessary to stop the OTCase activity. To achieve this a number of treatments have already been used successfully: treatments with acetone, 3,4 with CTAB, 7 and by freezing the cell suspension. 8 We have shown in our study that a treatment with heat would give good results. The immobilization of intact cells in order to use one or several of their enzymatic activities is a technique which has been largely developed in the last few years. 9-11 The immobilization of S. faecalis in a poly-
acrylamide gel was studied by Franks, 8 who showed that a system capable of converting arginine by the way of arginine dihydrolase could be obtained. Ajinomoto patents 12,13 and the work of Yoshida ~4 deal with the production of citrulline by fermentation. The continuous bioconversion of arginine into citrulline by immobilized P. putida was studied by Yamamoto et a1.15 In this work, we have studied the capability of immobilized S. faecalis to achieve this continuous bioconversion, as well as the performances of the obtained system.
Materials and methods Organisms and culture conditions
The strain utilized, Streptococcus faecalis, A T C C 8043, was cultured for 16 h at 37°C, in aerobiosis, in a medium containing (per liter of deionized water): yeast extract (Difco), 5 g; bactocasitone (Difco), 2.5 g; Larginine, HCI, 5 g; glucose, 5 g; Na2HPO4, 0.9 g; NaC1, 0.5 g; MgSO4 • 7H20, 0.5 g; MnSO4 • H20, 0.2 g; FeSO4 • 7H20, 0.002 g. P r e p a r a t i o n o f cells
Address reprint requests to Dr. Cottenceau at the Laboratoire de Biochimie Cellulaire, Equipe de Biotechnologie, Institut Universitaire de Technologie, rue de Roux, 17026 La Rochelle, France Received 13 October 1989; revised 25 May 1989
The cells were harvested by centrifuging the culture medium (20 min, 4000 g), then washed three times by resuspending the pellet in 15 times its volume of 50 mM phosphate buffer at p H 7.
© 1990 Butterworth Publishers
Enzyme Microb. Technol., 1990, vol. 12, May
355
Papers Immobilization Unless otherwise stated, the cells were immobilized immediately after washing by inclusion in a polyacrylamide gel by the method described by Chibata et al.16" 0.3 g of cells (wet weight) were suspended in 3.7 ml 50 mM phosphate buffer at pH 7, then 700 mg of acrylamide, 80 mg NN'-bisacrylamide (BIS), 0.5 ml a 5% dimethyl-amino-propionitrile solution in water, and 0.5 ml a 2.5% potassium persulfate solution in water were sequentially added. The polymerization time was 15 min at 37°C. The gel, once constituted, was washed twice with 10 ml phosphate buffer then crushed. The particles were again washed three times with 50 ml phosphate buffer at pH 7.
lOOt
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go
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Standard determination o f ADase activity Unless otherwise stated, the measurement of ADase activity was performed according to Yamamoto et al.15: I g of crushed gel, or 1 ml of cell suspension containing the appropriate quantity of cells (wet weight), was incubated in 15 ml 10 mM arginine solution in 50 mM phosphate buffer at pH 7 and 37°C, with shaking (60 rev rain-l). The enzymatic reaction was stopped by addition of trichloroacetic acid, 0.25 M final concentration. The precipitate was removed by centrifugation (10 min, 5,000g). The quantity of e-citrulline in the supernatant was measured according to Archibald's method 17, improved by Boyde et al. ~8
50
0,2
0.4 mM
0.6 of
0.8
CTAB
Figure 1 Influence of substrate and cell concentration on the optimum CTAB. Gel at 0.3 g of wet cell g 1of gel, activity of 1.5 g of gel in presence of variable concentration of CTAB with 5 (O) or 10 (©) mM of arginine; (11) activity of 1.5 g of gel containing 0.052 g of cells g 1; (r~) activity of 3.0 g of gel containing 0.026 g of cells g-l; activity measured with variable concentration of CTAB
Results and discussion
Study o f the conditions o f immobilization on the ADase activity The effect of acrylamide and BIS concentration in the gels on the ADase activity has been studied in the presence of 0.62 m u of CTAB. For mechanical reasons, we have chosen a concentration in acrylamide of 120 mg g i of gel; with this concentration, the ADase activity is maximum for a concentration in BIS of 14 mg g-1 of gel. The gel entrapment of the cells obtained is stable; successive washings do not reduce the residual enzymatic activity of immobilized bacteria. A part of the gel (1 g) was washed successively with 50 ml phosphate buffer before addition of a new quantity of substrate and measure of the residual activity; the seventh washing does not give a result significantly different from the first.
Action o f CTAB on the ADase activity o f the immobilized cells The increase of ADase activity due to the treatment with CTAB is rather slow, because of the diffusion barrier constituted by the gel (about 10 min are necessary to obtain the maximum activity) and this action is irreversible. On the other hand, neither the concentration in substrate nor the cell concentration in the gel have an 356
Enzyme Microb. Technol., 1990, vol. 12, May
influence on the optimum concentration of CTAB, as shown in Figure 1. For a gel of given constitution, the optimal concentration of CTAB depends on the amount of cells compared with the volume of reactive medium. We have made a series of manipulations with variable amounts of cells immobilized; the activity of the gel + cells systems obtained has been measured in the presence of variable concentrations of CTAB with the aim of determining the optimal concentration. Figure 2 gives a synthesis of the results of these experiments; it represents a graph of the relation (optimal concentration of CTAB)
= fct (cell concentration in the reactive medium) where fct = abbreviated form of "function o f " The concentration in CTAB necessary for saturating the system increases in a rather linear way up to a cell concentration of about 6 mg of cells (wet weight) ml -~ of reactive medium, corresponding to a concentration of CTAB of 0.53 mM. Beyond that point, we noticed a change in the slope and a much faster increase of the optimal concentration in CTAB. Figure 3 gives the graph of the relation (maximal specific activity of the gel + cells systems)
= fct (cell concentration in the gel)
Citrulline production by S. faecalis: G. Cottenceau et al. / 1.0.
tration beyond which a new kinetics of diffusion inside the gel + cells system would take place.
/
A i E
/ 0.8 ¸
rn
i
i
,< t-
/
d
The effect of pH on the reaction rate is given in Figure 4. The optimum pH of entrapped cells shifted by one unit to the alkaline side compared with that of intact cells; on the other hand, the presence of CTAB has no effect. The influence of the temperature of reaction on the ADase activity of the intact cells in the presence of CTAB and of immobilized cells with and without CTAB, is summed up in Figure 5. We notice a 20°C increase in the optimum temperature with immobilized cells and a twofold increase in relative activity if CTAB treatment is omitted.
/ / /
0,6 /
E s
j~ O
0,4-
Y
•
0.2-
i
i
i
i
3
4
5
6
7
reactive
medium
mg
wet
cells
/
ml
Properties of the ADase of immobilized bacteria in the presence of CTAB
Figure 2 O p t i m a l concentration in CTAB c o m p a r e d with the cell concentration of the reactive m e d i u m
Determination of the best thermal treatment of the gel + cells system for the development of the ADase activity A thermal treatment of the gel + cells system made it possible to reach higher ADase activity than that obtained by CTAB treatment. We have looked for the time-temperature couple to be applied to the immobilized cells to reach the highest ADase activity. For this purpose, we have heated the gel + cells system at different temperatures and measured the evolution of their ADase activity according to the time. The obtained maxima are shown in Table 1. From these results, it appears that the optimum duration of heating (fct a) and maximum specific activity
20 ¸
J::
"-,,,~, O 15
.>_ (0 ,o
Q. ~9
100
10-
,o
=o
mg
wet
cells
6'o /
g
a'o
80
,'0o
of g e l 60
Figure 3 Specific activity versus cell concentration in the gel. (11) C T A B concentration < 0 . 5 3 m M ; (rT) CTAB concentration > 0.53
mM rr
We notice that, here again, the points divide into two groups: below and beyond 0.53 mM of CTAB, the specific activity decreases linearly with the increase of the cell concentration because of the increasing difficulty of the diffusion of both substrate and reaction products inside the particles of the gel; but we also notice that on both sides of this concentration, the specific activity takes significantly different values for the same cell concentration. This may indicate that, around 0.53 mM of CTAB, there is a transition concen-
40-
20-
. 5
, 6
. 7
. 8
, 9
pH Figure 4 Effect of pH on the A D a s e activity of i m m o b i l i z e d bacteria. (11) Activity of 45 m g of intact cells (wet weight), measured with 0 . 3 m M of C T A B at various p H s ; (El) activity of i m m o b i l i z e d cells at various p H s ; ( T ) activity of i m m o b i l i z e d cells with 0 . 3 m M of C T A B at various pHs; activity measured at 3 7 ° C
Enzyme Microb. Technol., 1990, vol. 12, May
357
Papers The general equation of the second function (fct b) is
300
Log (Ao
/v,, /
\
.> u 0~ > i~
1 0C
¢"
..,.j..~~ .....
50'
/
... \
J
i
3O
50
Temperature
At)
-kT ° = p
=
(2)
Ao = specific limit activity that the immobilized bacteria can develop with heat treatment (/ZM ml J h i g J) At = m a x i m u m specific activity reached by the immobilized bacteria after a treatment at a temperature of T°C ([,ZM m1-1 h i g 1) T° = temperature o f treatment in °C From the values s h o w n in Table 1, we calculated the m a x i m u m specific activity of the system, Ao = 85 [LM ml 1 h 1 g - ] which depends on our conditions. This value reported in equation (1) allows us to plot the graph of this relation (Figure 6), in which we notice that the m a x i m u m specific activity is obtained within 1% for a temperature of 52°C, corresponding by equation (1) to a treatment of 1370 min. For practical reasons, w e have c h o s e n for the following manipulations a specific activity of 75 /L~Mml -I h -j g-I, which is obtained with a temperature of 66°C and a duration of treatment of 100 min.
A
20(~
-
70
°C
Figure 5 Effect of the temperature of reaction on the ADase activity of immobilized bacteria. (11) Activity of 45 mg of intact cells (wet weight), with 0.3 mM o f CTAB, at various temperatures; activity of immobilized cells with no CTAB (T), with 0.15 mM CTAB (V), w i t h 0.3 mM CTAB (rq), at various temperatures; (i) = relative activity compared with the one of the free cells at
Properties of the ADase of the immobilized b a c t e r i a after heating The influence of the temperature of reaction on the A D a s e activity of the immobilized and heated bacteria
37°C
Table 1
Ao-1
Temp. of treatment
Duration of treatment according to the temperature Optimum time of treatment
(°C) 77 73.5 70 66.5 63
Ao-lO
(min)
Max. specif activity a reached (/zM ml 1 h 1 g 1)
11 28 46 100 155
36.5 55 65 76.5 79
a Specific activity = /zM of citrulline formed ml ~ of reactive medium h 1, g 1 of immobilized cells (wet weight)
Table 2 Ao-IOG
,
i
,
i
55
60
65
70
Figure 6
Temp.
Citrulline in the effluent
(ml h 1)
(/~M ml 1)
24 30 36 42 36 42 48 54 48 54 60 66
10 10 9.0 8.2 10 9.7 9.0 8.1 10 9.7 8.9 8.0
% of conversion
°C
Graph of the relation (Ao - At) =
-kT ° + P
(fct b) are logarithmic functions of the temperature of treatment. With the linear regression method, w e have found that the first equation of the function (fct a) is: log tps = - 0 . 0 8 1 4 T ° + 9.1479
(1)
tps= optimum duration of treatment (s) T ° = temperature of treatment (°C)
358
Flow rate
z
75
(°C)
Temperature
Effect of feeding rate on the formation of citrulline
Enzyme Microb. Technol., 1990, vol. 12, May
35 35 35 35 45 45 45 45 55 55 55 55
100 100 90 82
100 97 90 81 100 97 89 80
Citrulfine production by S. faecalis: G. Cottenceau et al. / 150J f
.Z~j
~111~
co
.... o .....
"S..'..~." --. ......
'~"':"'"L~
100-
>
50n-
T 1
i 2
i 3
I 4
5
Time
in
6
22
= 24
23
T 25
hours
Figure 7 Stability of the A D a s e activity in regard to the operational temperature. I m m o b i l i z e d cells heated in a p h o s p h a t e buffer (66°C, 100 rain) then at different t e m p e r a t u r e s during variable times; (C]) 70°C; (0) 65°C; (A) 60°C; (11) 55°C; (O) 50°C; (T) 45°C
has been studied; the same optimum as for the system treated with C T A B is obtained: 70°C. The kinetic constants of the A D a s e , as it works in our system, are Km = 1.18 /ZM ml -], g m a x = 1.25 /zM ml- 1 20- I min. The stability of the A D a s e activity has been tested; our results are s h o w n in Figure 7.
Continuous conversion of arginine into citrulline The continuous c o n v e r s i o n of arginine into citrulline was studied with a reactor consisting of a glass column of 16 m m of interior diameter with a thermostating jacket heated by water circulation, the substrate being fed from the bottom. The effect of the feeding rate on the formation o f citrulline was studied and the results are s h o w n in Table 2. To achieve it w e have fed the reactor at different feeding rates with a 10 mM arginine solution in 0.05 M phosphate buffer at pH 7; the reactor was filled up with 8 g o f heated gel + cells s y s t e m (66°C, 100 min) and heated at different temperatures. The operational stability o f the A D a s e activity of the reactor was tested at two different temperatures, 45°C and 55°C (see results in Figure 8). The activity of the reactor was stable over 30 days at 45°C for a feeding rate of 50 ml h -1. Moreover, the stability o f the activity is not affected by a treatment of the reactor with sodium azide at least up to a concentration o f 0.2%.
10-
A v
O) A
.*E--
r 5
i 10
i 15 Time
t 20 in
r 25
, 30
days
Figure 8 Operational stability of A D a s e activity of i m m o b i l i z e d and heated bacteria. A 10-mM sterilized arginine solution in p h o s p h a t e buffer was passed t h r o u g h the c o l u m n at a flow rate of 50 ml h -1, the c o l u m n being filled up with 8 g of heated gel + cells system; ( 0 ) operational t e m p e r a t u r e 45°C; (O) operational t e m p e r a t u r e 55°C; 1, 2, 3, 4, 5: a s o d i u m azide solution of respectively 0.02%, 0.04%, 0.06%, 0.08%, 0.010% was passed t h r o u g h the reactor for 1 h
Enzyme Microb. Technol., 1990, vol. 12, May
359
Papers Conclusion
Streptococcus faecalis, strain ATCC 8043, immobilized in a polyacrylamide gel, is a potent catalyst for the continuous production of L-citrulline from L-arginine. As it does for a large number of microorganisms, the polyacrylamide gel ensures an easy and very stable immobilization. Yet, as opposed to what happens with Pseudomonas putida, this immobilization does not sufficiently modify the cell, and particularly its membrane, to permit the accumulation of L-citrulline. Considering the two treatments used to modify the membrane, either with a surface active agent or with heat, the heat treatment is definitively more effective as it ensures a much better thermal stability. The maximum specific activity that we have reached after a heat treatment of the immobilized cells was 85/ZM m1-1 h -1 g-l.
References 1 2 3 4 6 7 8 9 10 11 12 13 14 15 16
Acknowledgements We thank Mrs. M. Quillet for technical assistance and Mrs. G. Weyland for linguistic advice.
360
Enzyme Microb. Technol., 1990, vol. 12, May
17 18
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