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Purification and characterization of galactose-induced pectinases from the exo-1 mutant strain of Neurospora crassa
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V.All rights reserved.
787
Purification and characterisation of galactose-induced pectinases from the exo-1 mutant strain of Neurospora crassa L.B. Crotti, J.A. Jorge, H.F. Terenzi and M.L.T.M. Polizeli Departamento de Biologia - Faculdade de Filosofia, CiEncias e Letras de Ribeir~o Preto, Universidade de S~o Paulo, Av. Bandeirantes 3900, 14040-901 - Ribeir~o Preto, S~o Paulo, Brasil Abstract Pectinases produced by the exo-1 mutant of N. crassa in galactose plus glucose supplemented medium, were separated by ion-exchange chromatography into two pools. Pool I contained pectate and pectin lyases, and variable polygalacturonase activity. Pool 2 contained polygalacturonase activity only. Gel filtration indicated a MWapp of 80 kDa (higher than those of separate enzymes) for all activities in the first pool, suggesting a complex. Polygalacturonase, pectin and pectate lyases were purified 39-fold, 22-fold and 33fold, respectively. Optima of temperature and pH were 45~ and 5.5 for polygalacturonase activity and 50~ and 9.5 for lyase activities. Km and Vmax values for polygalacturonase were 0.023 mg polypectate/ml and 2.08 ~moles (reducing sugar)/min/mg protein.
1. INTRODUCTION Previous studies from our laboratory [1,2] demonstrate that the filamentous fungus Neurospora crassa produces pectic enzymes as effectively as other hydrolases such as
cellulases [3], amylases [4], and xylanases [5]. The production of polygalacturonase was studied using the mutant strain exo-1. This interesting strain exhibits a rather exaggerated synthesis and secretion of several exoenzymes, among others amylase and invertase [6]. We demonstrated that this strain, when cultivated in the presence of pectin as the sole carbon source, secretes five to six times more than the wild type a glucose-repressible endopolygalacturonase [2]. Interestingly, the production of polygalacturonase was also induced by galactose, four times more efficiently than by pectin. The inducing effect of galactose, different of that of pectin, was not counteracted by glucose. Thus, we decided to investigate in more detail the effect of galactose as inducer of pectolytic activities and to biochemically characterise the pectolytic complex produced by the N. crassa exo-1 strain in the presence of galactose and glucose. 2. METHODS Culture conditions: The exo-1 strain was cultivated in two-stages: (I) pre-cultivation for 24 hours in Vogel's medium [7] supplemented with 2% glucose, and (II) transfer of the mycelial
788 mass to fresh medium supplemented with 2% glucose plus 2% galactose or other carbon sources, for 48 or 72 hours, according with the experiment, at 30oc, with agitation.
Enzymatic assays: Polygalacturonase was assayed: (a) by measuring the amount of reducing sugar released from sodium polypectate as a substrate. An enzyme unit is the amount which releases reducing sugar at an initial rate of l~mol/min at 30oc, using galacturonic acid as the standard [8]. Co) By the decrease in relative viscosity of a 0.2% pectin solution using an Ostwald viscometer. One activity unit was expressed as a percentage (50%) change in viscosity [9]. Lyase activities were measured by the increase in A232 nm of the unsaturated products of degradation of pectin or sodium polypectate. One activity unit was the amount of enzyme which released 1 lxmol of unsaturated product per minute [10]. Protein was determined by the Lowry method using bovine serum albumin as standard [11 ]. Separation of pectic enzymes: The crude filtrate was precipitated with 2 volumes of ethanol for 2 hours at -20oc and then centrifuged at 15,900g for 10 minutes. The precipitate was dissolved in 10 ml of Tris-HC1 buffer 10mM, pH 7,5 (buffer A) and applied to a DEAEcellulose column (1,6 x 20cm) equilibrated and eluted with buffer A. The flow-through protein was dialysed against 10raM sodium acetate buffer, pH 5,0 (buffer B) and applied to a CM-cellulose column (1,6 x 25cm). The column was eluted with a NaC1 gradient (0 500mM) in buffer B. Fractions (10ml) were collected at a flow rate of 33.5 ml/h. Determination of molecular mass of pectic enzymes: The molecular mass were determined by gel filtration in a Sepharose CL-6B column (1,8 x 88cm) equilibrated and eluted with TrisHC150 mM, pH 7,5 buffer, plus 100 mM KC1. Fractions (3,3 ml) were collected at a flow rate of 10 ml/h. Molecular mass markers were: tyroglobulin (660 kDa); apoferritin (440 kDa); 13amylase (200 kDa); alcohol dehydrogenase (150 kDa); bovine serum albumin (66 kDa) and carbonic anhydrase (29 kDa). Urea-SDS-PAGE (7%) was carried out according to Swank and Munkres [12]. Molecular mass markers were: myosin (205 kDa); [3-galactosidase (116 kDa); phosphorylase b (97 kDa); bovine serum albumin (66 kDa), ovalbumin (45 kDa) and carbonic anhydrase (29 kDa). Determination of neutral carbohydrate: Total neutral carbohydrate in protein samples was estimated by the phenol/sulphuric acid method of Dubois [13] using mannose as standard. Chromatographic characterisation of hydrolysis products: Hydrolysis products from sodium polypectate were analysed by thin-layer chromatography on silica gel G-60, using ethyl acetate / acetic acid / formic acid / water (9:3:1:4, by volume) as the mobile phase system. Sugars were detected with 0,2% orcinol in sulphuric acid-methanol (10:90ml) [14]. 3. RESULTS AND DISCUSSION. The effects of galactose and pectin as inducers of polygalacturonase activity in the exo-1 N. crassa strain is shown in figure 1. Both substances were efficient inducers, but in the presence of galactose the enzyme production was about four-fold higher than with pectin. A remarkable difference between induction with pectin or with galactose, was that the former was severely repressed by glucose, whereas galactose induction was not repressed by addition of glucose.
789
.~ > .i
1,0
ci.
t~
0,8
19 U) t-
0,6
/ / / / / /
/
/
/
/
. , . . , / / ,
./ ,
./ /
'
/ /
/
/
/
.• ,\/• ?
2
,/
/
/
/
I
,,
,,
/
/
/
/
/
/
/.,
/
~
i
i
0,4
i
I:~ i. 0
/
0,2
.
.
0,0
no carbon pect pect + glu
gal + glu
gal
Figure 1. Effect of 1% (w/v) pectin (pect), 1% galactose (gal), and of the simultaneous presence of 2% glucose (glu) on the production of extracellular polygalacturonase activity. Two-stages cultures were prepared as described under methods. Polygalacturonase was assayed in the culture filtrate as reducing sugar-releasing activity using sodium polypectate as a substrate. These results prompted us to examine the characteristics of the extracellular pectolytic enzymes secreted in medium supplemented with glucose and galactose. Figure 2 shows the profile of elution of pectolytic activities recovered from the flow-through of a DEAEcellulose column chromatographed on a CMC-cellulose column. 0,35
~~ NaCI0~'_ l:: 12
0,30
~"
0,25
v
19
10
t~ >,, m
E
8
0,20
t~ C
0,15
"~
2
I
im
6
0,10
19
I~ 0 Q.
0,05
0
0
20
40
60
9
80
0,00 100
fractionnumber Figure 2. CM-cellulose chromatography of pectolytic enzymes. The activity peaks of the flow-through of a DEAE-cellulose chromatography was applied to a CM-cellulose column. The column was eluted with a NaC1 (0-0.5M) continuous gradient at a flow rate of 34 ml/h. 10 ml fractions were collected and assayed for pectolytic activities Symbols: (O) pectate lyase; ($) polygalacturonase (reducing sugar-releasing activity); (x) protein. Other details in Methods.
790 Polygalacturonase activity eluted into two main fractions, the first coeluting with pectate lyase (and pectin lyase, not shown) activities, and the second free of other activities. When the first peak was rechromatographed under the same conditions, identical result was obtained. The distribution of polygalacturonase in the two peaks varied with the experiment. In other cases, lyases and polygalacturonase activities separated completely into two peaks, one containing the two lyases and the other containing polygalacturonase activity only. Interestingly, at this stage of separation all pectolytic activities had reached a considerable degree of purification: polygalacturonase was purified about 39-fold, while pectate and pectin lyases were purified 33-fold and 22-fold, respectively. Gel filtration of the peak showing associated lyases and polygalacturonase activity (Figure 3A) gave a single activity peak eluting with a MWapp of approximately 79.4 kDa, suggesting the existence of a multienzyme complex. On the other hand, the same peak run under denaturing urea-SDS-PAGE (Figure 3B) was resolved into two bands, one of pectate/pectin lyase activity (MWapp 56.2 kDa) and a second band with polygalacturonase activity (MWapp 44.7 kDa). 6,5 6,0 r
E _~
5,4
5,2 2 3 5,0
5,0
pectin lyases 4,8
4,5
pectate/pectin lyase + polygalacturonase
0
~
B
5,5
(D
O E
1
A 1 2
4,0
4,6
3,5 4,4 310
i
1,6
,
i
i
1,8 VeNo
i
2,0
,
i
2,2
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
Rf
Figure 3. (A) Determination of molecular mass of pectic enzymes by gel filtration in Sepharose 6B. Molecular mass markers:l- tyroglobulin, 2- apoferritin, 3- 13-amylase, 4alcohol dehydrogenase, 5- bovine serum albumin, 6- carbonic anhydrase. (B) SDS-PAGE of pectolytic activities. Molecular mass markers: 1- myosin, 2- 13-galactosidase, 3- phosphorylase b, 4- bovine serum albumin, 5- ovalbumin, 6- carbonic anhydrase.
Table i shows some biochemical properties of the pectolytic enzymes present in pool 1. The pectin lyase/pectate lyase activities (pool I) and polygalacturonase activity (pool II) were not significantly affected by NH4+, Na + and K + (0,25 - 2,5mM), while A13+, 13-mercaptoethanol, Hg 2+, EDTA, Ba 2+ and Zn +2 (2,5mM) inhibited 30-100% these activities. On the other hand, Ca2+, Mg 2+ and Mn 2+ at 2,5mM concentration activated 20-100% pectin/pectate lyases but Ca 2+ and Cu 2+ (2,5mM) inhibited polygalacturonase activity about 42 - 70%.
791 Viscosimetric assays and analysis of hydrolysis products by thin layer chromatography (TLC) were used to determine the mechanism of action of the polygalacturonase on sodium polypectate. The time required for 50% decrease in viscosity of a 2.0% (w/v) substrate solution at 45~ was approximately 105 min, at which time about 9% of total galacturonide bonds had been hydrolysed. The products of hydrolysis, analysed by TLC, demonstrated that oligogalacturonates accumulated initially, but the monomer was found after 24 h of reaction (not shown). These results suggested that the polygalacturonase of the mutant exo-1 of N.crassa induced by galactose in the medium exhibited a random mechanism of hydrolysis of sodium polypectate, suggesting that is was an endopolygacturonase.
Table 1 Kinetic constants and others intrinsic properties of pectolytic activities Parameters
Polygalacturonase
pectate/pectin lyases
reducing sugar
viscosity
pectin
pectate
KM (mg/ml)
0.023
n.d.
0.076
0.50
Vmax (U/min/mg protein)
2.08
n.d.
363.4
273.2
neutral carbohydrate
38.8%
38.0% (*)
optimal temperature
45~
45~
50~
500C
thermostability 60~ (T50 -min)
5
30
1.5
3
optimal pH
5.5
4.5
10
9
stability pH
5.0
4.0-5.5
9.5
10
(*) the sugar content of lyases was determined in a fraction containing both activities.
792
References
5 6 7 8 9 10 11 12 13 14
M.L.T.M. Polizeli, R.C.L.R. Pietro, J.A. Jorge and H.F. Terenzi, J. Gen. Microbiol., 136 (1990) 1463. M.L.T.M. Polizeli, J.A. Jorge and H.F. Terenzi, J. Gen. Microbiol., 137 (1991) 1815. B.M. Eberhart, R.S. Beck and K.M. Goolsby, J. Bacteriol., 130 (1977) 181. R.D. Sigmund, M.T. McNally, D.B. Lee and S.J. Free, Biochem. Genet., 23 (1975) 89. C. Mishra, S. Keskar and M. Rao, Appl. Environ. Microbiol., 48 (1984) 224. H.G. Gratzner and D.N. Sheehan, J. Bacteriol., 97 (1969) 544. H.J. Vogel, Am. Nat., 98 (1964) 435. G.L. Miller, Anal. Biochem., 31 (1959) 426. R. Tuttobello and P.J. Mill, Biochem. J., 79 (1961) 51. C.W. Nagel and M.M. Anderson, Arch. Biochem. Biophys., 112 (1965) 322. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. R.W. Swank and K.D. Munkres, Anal. Biochem., 39 (1971) 462. M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers and F. Smith, Anal. Chem., 28 (1956) 350. J.D. Fontana, M. Gebara, M. Blumel, H. Schneider, C.R. Mackenzie and K.G. Johnson, Methods Enzymol., 160 (1988) 560.
787
Purification and characterisation of galactose-induced pectinases from the exo-1 mutant strain of Neurospora crassa L.B. Crotti, J.A. Jorge, H.F. Terenzi and M.L.T.M. Polizeli Departamento de Biologia - Faculdade de Filosofia, CiEncias e Letras de Ribeir~o Preto, Universidade de S~o Paulo, Av. Bandeirantes 3900, 14040-901 - Ribeir~o Preto, S~o Paulo, Brasil Abstract Pectinases produced by the exo-1 mutant of N. crassa in galactose plus glucose supplemented medium, were separated by ion-exchange chromatography into two pools. Pool I contained pectate and pectin lyases, and variable polygalacturonase activity. Pool 2 contained polygalacturonase activity only. Gel filtration indicated a MWapp of 80 kDa (higher than those of separate enzymes) for all activities in the first pool, suggesting a complex. Polygalacturonase, pectin and pectate lyases were purified 39-fold, 22-fold and 33fold, respectively. Optima of temperature and pH were 45~ and 5.5 for polygalacturonase activity and 50~ and 9.5 for lyase activities. Km and Vmax values for polygalacturonase were 0.023 mg polypectate/ml and 2.08 ~moles (reducing sugar)/min/mg protein.
1. INTRODUCTION Previous studies from our laboratory [1,2] demonstrate that the filamentous fungus Neurospora crassa produces pectic enzymes as effectively as other hydrolases such as
cellulases [3], amylases [4], and xylanases [5]. The production of polygalacturonase was studied using the mutant strain exo-1. This interesting strain exhibits a rather exaggerated synthesis and secretion of several exoenzymes, among others amylase and invertase [6]. We demonstrated that this strain, when cultivated in the presence of pectin as the sole carbon source, secretes five to six times more than the wild type a glucose-repressible endopolygalacturonase [2]. Interestingly, the production of polygalacturonase was also induced by galactose, four times more efficiently than by pectin. The inducing effect of galactose, different of that of pectin, was not counteracted by glucose. Thus, we decided to investigate in more detail the effect of galactose as inducer of pectolytic activities and to biochemically characterise the pectolytic complex produced by the N. crassa exo-1 strain in the presence of galactose and glucose. 2. METHODS Culture conditions: The exo-1 strain was cultivated in two-stages: (I) pre-cultivation for 24 hours in Vogel's medium [7] supplemented with 2% glucose, and (II) transfer of the mycelial
788 mass to fresh medium supplemented with 2% glucose plus 2% galactose or other carbon sources, for 48 or 72 hours, according with the experiment, at 30oc, with agitation.
Enzymatic assays: Polygalacturonase was assayed: (a) by measuring the amount of reducing sugar released from sodium polypectate as a substrate. An enzyme unit is the amount which releases reducing sugar at an initial rate of l~mol/min at 30oc, using galacturonic acid as the standard [8]. Co) By the decrease in relative viscosity of a 0.2% pectin solution using an Ostwald viscometer. One activity unit was expressed as a percentage (50%) change in viscosity [9]. Lyase activities were measured by the increase in A232 nm of the unsaturated products of degradation of pectin or sodium polypectate. One activity unit was the amount of enzyme which released 1 lxmol of unsaturated product per minute [10]. Protein was determined by the Lowry method using bovine serum albumin as standard [11 ]. Separation of pectic enzymes: The crude filtrate was precipitated with 2 volumes of ethanol for 2 hours at -20oc and then centrifuged at 15,900g for 10 minutes. The precipitate was dissolved in 10 ml of Tris-HC1 buffer 10mM, pH 7,5 (buffer A) and applied to a DEAEcellulose column (1,6 x 20cm) equilibrated and eluted with buffer A. The flow-through protein was dialysed against 10raM sodium acetate buffer, pH 5,0 (buffer B) and applied to a CM-cellulose column (1,6 x 25cm). The column was eluted with a NaC1 gradient (0 500mM) in buffer B. Fractions (10ml) were collected at a flow rate of 33.5 ml/h. Determination of molecular mass of pectic enzymes: The molecular mass were determined by gel filtration in a Sepharose CL-6B column (1,8 x 88cm) equilibrated and eluted with TrisHC150 mM, pH 7,5 buffer, plus 100 mM KC1. Fractions (3,3 ml) were collected at a flow rate of 10 ml/h. Molecular mass markers were: tyroglobulin (660 kDa); apoferritin (440 kDa); 13amylase (200 kDa); alcohol dehydrogenase (150 kDa); bovine serum albumin (66 kDa) and carbonic anhydrase (29 kDa). Urea-SDS-PAGE (7%) was carried out according to Swank and Munkres [12]. Molecular mass markers were: myosin (205 kDa); [3-galactosidase (116 kDa); phosphorylase b (97 kDa); bovine serum albumin (66 kDa), ovalbumin (45 kDa) and carbonic anhydrase (29 kDa). Determination of neutral carbohydrate: Total neutral carbohydrate in protein samples was estimated by the phenol/sulphuric acid method of Dubois [13] using mannose as standard. Chromatographic characterisation of hydrolysis products: Hydrolysis products from sodium polypectate were analysed by thin-layer chromatography on silica gel G-60, using ethyl acetate / acetic acid / formic acid / water (9:3:1:4, by volume) as the mobile phase system. Sugars were detected with 0,2% orcinol in sulphuric acid-methanol (10:90ml) [14]. 3. RESULTS AND DISCUSSION. The effects of galactose and pectin as inducers of polygalacturonase activity in the exo-1 N. crassa strain is shown in figure 1. Both substances were efficient inducers, but in the presence of galactose the enzyme production was about four-fold higher than with pectin. A remarkable difference between induction with pectin or with galactose, was that the former was severely repressed by glucose, whereas galactose induction was not repressed by addition of glucose.
789
.~ > .i
1,0
ci.
t~
0,8
19 U) t-
0,6
/ / / / / /
/
/
/
/
. , . . , / / ,
./ ,
./ /
'
/ /
/
/
/
.• ,\/• ?
2
,/
/
/
/
I
,,
,,
/
/
/
/
/
/
/.,
/
~
i
i
0,4
i
I:~ i. 0
/
0,2
.
.
0,0
no carbon pect pect + glu
gal + glu
gal
Figure 1. Effect of 1% (w/v) pectin (pect), 1% galactose (gal), and of the simultaneous presence of 2% glucose (glu) on the production of extracellular polygalacturonase activity. Two-stages cultures were prepared as described under methods. Polygalacturonase was assayed in the culture filtrate as reducing sugar-releasing activity using sodium polypectate as a substrate. These results prompted us to examine the characteristics of the extracellular pectolytic enzymes secreted in medium supplemented with glucose and galactose. Figure 2 shows the profile of elution of pectolytic activities recovered from the flow-through of a DEAEcellulose column chromatographed on a CMC-cellulose column. 0,35
~~ NaCI0~'_ l:: 12
0,30
~"
0,25
v
19
10
t~ >,, m
E
8
0,20
t~ C
0,15
"~
2
I
im
6
0,10
19
I~ 0 Q.
0,05
0
0
20
40
60
9
80
0,00 100
fractionnumber Figure 2. CM-cellulose chromatography of pectolytic enzymes. The activity peaks of the flow-through of a DEAE-cellulose chromatography was applied to a CM-cellulose column. The column was eluted with a NaC1 (0-0.5M) continuous gradient at a flow rate of 34 ml/h. 10 ml fractions were collected and assayed for pectolytic activities Symbols: (O) pectate lyase; ($) polygalacturonase (reducing sugar-releasing activity); (x) protein. Other details in Methods.
790 Polygalacturonase activity eluted into two main fractions, the first coeluting with pectate lyase (and pectin lyase, not shown) activities, and the second free of other activities. When the first peak was rechromatographed under the same conditions, identical result was obtained. The distribution of polygalacturonase in the two peaks varied with the experiment. In other cases, lyases and polygalacturonase activities separated completely into two peaks, one containing the two lyases and the other containing polygalacturonase activity only. Interestingly, at this stage of separation all pectolytic activities had reached a considerable degree of purification: polygalacturonase was purified about 39-fold, while pectate and pectin lyases were purified 33-fold and 22-fold, respectively. Gel filtration of the peak showing associated lyases and polygalacturonase activity (Figure 3A) gave a single activity peak eluting with a MWapp of approximately 79.4 kDa, suggesting the existence of a multienzyme complex. On the other hand, the same peak run under denaturing urea-SDS-PAGE (Figure 3B) was resolved into two bands, one of pectate/pectin lyase activity (MWapp 56.2 kDa) and a second band with polygalacturonase activity (MWapp 44.7 kDa). 6,5 6,0 r
E _~
5,4
5,2 2 3 5,0
5,0
pectin lyases 4,8
4,5
pectate/pectin lyase + polygalacturonase
0
~
B
5,5
(D
O E
1
A 1 2
4,0
4,6
3,5 4,4 310
i
1,6
,
i
i
1,8 VeNo
i
2,0
,
i
2,2
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
Rf
Figure 3. (A) Determination of molecular mass of pectic enzymes by gel filtration in Sepharose 6B. Molecular mass markers:l- tyroglobulin, 2- apoferritin, 3- 13-amylase, 4alcohol dehydrogenase, 5- bovine serum albumin, 6- carbonic anhydrase. (B) SDS-PAGE of pectolytic activities. Molecular mass markers: 1- myosin, 2- 13-galactosidase, 3- phosphorylase b, 4- bovine serum albumin, 5- ovalbumin, 6- carbonic anhydrase.
Table i shows some biochemical properties of the pectolytic enzymes present in pool 1. The pectin lyase/pectate lyase activities (pool I) and polygalacturonase activity (pool II) were not significantly affected by NH4+, Na + and K + (0,25 - 2,5mM), while A13+, 13-mercaptoethanol, Hg 2+, EDTA, Ba 2+ and Zn +2 (2,5mM) inhibited 30-100% these activities. On the other hand, Ca2+, Mg 2+ and Mn 2+ at 2,5mM concentration activated 20-100% pectin/pectate lyases but Ca 2+ and Cu 2+ (2,5mM) inhibited polygalacturonase activity about 42 - 70%.
791 Viscosimetric assays and analysis of hydrolysis products by thin layer chromatography (TLC) were used to determine the mechanism of action of the polygalacturonase on sodium polypectate. The time required for 50% decrease in viscosity of a 2.0% (w/v) substrate solution at 45~ was approximately 105 min, at which time about 9% of total galacturonide bonds had been hydrolysed. The products of hydrolysis, analysed by TLC, demonstrated that oligogalacturonates accumulated initially, but the monomer was found after 24 h of reaction (not shown). These results suggested that the polygalacturonase of the mutant exo-1 of N.crassa induced by galactose in the medium exhibited a random mechanism of hydrolysis of sodium polypectate, suggesting that is was an endopolygacturonase.
Table 1 Kinetic constants and others intrinsic properties of pectolytic activities Parameters
Polygalacturonase
pectate/pectin lyases
reducing sugar
viscosity
pectin
pectate
KM (mg/ml)
0.023
n.d.
0.076
0.50
Vmax (U/min/mg protein)
2.08
n.d.
363.4
273.2
neutral carbohydrate
38.8%
38.0% (*)
optimal temperature
45~
45~
50~
500C
thermostability 60~ (T50 -min)
5
30
1.5
3
optimal pH
5.5
4.5
10
9
stability pH
5.0
4.0-5.5
9.5
10
(*) the sugar content of lyases was determined in a fraction containing both activities.
792
References
5 6 7 8 9 10 11 12 13 14
M.L.T.M. Polizeli, R.C.L.R. Pietro, J.A. Jorge and H.F. Terenzi, J. Gen. Microbiol., 136 (1990) 1463. M.L.T.M. Polizeli, J.A. Jorge and H.F. Terenzi, J. Gen. Microbiol., 137 (1991) 1815. B.M. Eberhart, R.S. Beck and K.M. Goolsby, J. Bacteriol., 130 (1977) 181. R.D. Sigmund, M.T. McNally, D.B. Lee and S.J. Free, Biochem. Genet., 23 (1975) 89. C. Mishra, S. Keskar and M. Rao, Appl. Environ. Microbiol., 48 (1984) 224. H.G. Gratzner and D.N. Sheehan, J. Bacteriol., 97 (1969) 544. H.J. Vogel, Am. Nat., 98 (1964) 435. G.L. Miller, Anal. Biochem., 31 (1959) 426. R. Tuttobello and P.J. Mill, Biochem. J., 79 (1961) 51. C.W. Nagel and M.M. Anderson, Arch. Biochem. Biophys., 112 (1965) 322. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. R.W. Swank and K.D. Munkres, Anal. Biochem., 39 (1971) 462. M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers and F. Smith, Anal. Chem., 28 (1956) 350. J.D. Fontana, M. Gebara, M. Blumel, H. Schneider, C.R. Mackenzie and K.G. Johnson, Methods Enzymol., 160 (1988) 560.