Cellulase location in Cellvibriofulvus BJORNBERG^
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Institirte of Biochemistry, University of Uppsrila, Uppsala, Sweden Accepted August 22, 1974 firlr.irs. Can. J . Microbiol. 21: 51-57. BERG,B. 1975. Cellulase locat~onin Cell~~ibrio The location of cellulase in C.firlivis depends on thecarbon source for growth and the age of the culture. When cells were grown on glucose or cellobiose all CMC-hydrolyzing enzyme was cell-bound but only part of the activity was located on the cell surface. Treatment of cells with EDTA, lysozyme, and detergents and subsequent fractionation experiments showed that cellulase was also located in the periplasm and bound to a membrane fraction. Growth on cellulose gave cell-free cellulase active against CMC. The enzyme was repressed by glucose but formed at a constant differential rate on cellobiose and amylose. This rate was 8-10 times lower than on cellulose and possible reasons for this are discussed.
BERG,B. 1975. Cellulase location in Cellvihriojirlvrrs. Can. J. Microbiol. 21: 51-57. rs de la source de carbone pour la croissance La localisation de la cellulase chez C . f i r l ~ ~depend et de I'ige de la culture. Lorsque les cellules se sont developpees sur le glucose ou la cellobiose tous les enzymes hydrolisant le CMC sont attaches a la cellule mais seulement une partie de l'activite est localiste sur la surface cellulaire. Le traitement des cellules avec I'EDTA, le lysozyme et des detergents et des fractionations subsequentes montrent que la cellulase est aussi localisee dans le pkriplasme et attachee a une fraction de membrane. La croissance sur la cellulose donne une activite acellulaire de cellulase sur le CMC. L'enzyme est repress6 par le glucose mals forme un taux constant diffkrentiel sur la cellobiose et I'amylose. Ce taux est 8 a 10 fois plus faible que sur la cellulose et nous y discutons les raisons possibles de ce phenomene. [Traduit par le journal]
a
Introduction Cellulose is completely insoluble and cellulolytic microorganisms must therefore either form cell-free cellulases or have such enzymes located on the outside of the cells. It is often difficult t o show whether an enzyme found in a culture medium is secreted by growing cells o r passively released as a result of cell lysis (10). The terminology used in describing the location of enzymes in microbial cultures is confusing as the words exoenzymes, extracellular, and cell-free are used rather ambiguously. In this paper the term cell-free is used for enzyme not associated with the cells and cell-bound for enzyme associated with the cells. As will be shown in this paper the cell-bound cellulase in Celluibrio fiilr~is is only partly bound to the cell surface. It is therefore also necessary to distinguish between surface-bound and total cell-bound enzyme. The study of cellulolytic enzymes is con~plicated as they occur in solutions in the form of components of different specificities which are difficult t o separate. It is, besides, impossible to obtain a high sensitivity in assays with cellulose as substrate and it is iecessary-to rely on measurements with carboxvmethvl-cellulose (CMC). 'Received May 24, 1973. 2Present address: Department of Microbiology, Agricultural College, Uppsala 7, Sweden.
Pse~idomonas fl~iorescens var. cell~ilosae has been found t o -have cell-bound and cell-free cellulase in varying proportions depending on the carbon source for growth (14). We have reported earlier that the cellulase of C. fitluus is cell-bound when the bacteria grow on soluble carbon sources whereas cultures on cellulose mainly contain cell-free cellulase (2). The enzyme may be cell-free because of cell lysis in relatively old cultures but its release may also be caused by the carbon source for growth. We have compared the CMC-hydrolyzing activity of C. fulu~is grown on cellobiose and cellulose and studied the release of enzyme from cell suspensions treated in various ways. The results will be discussed in relation to ultrastructural observations presented in a preceding paper (1). The culture solution was shown to exhibit activity towards both C M C and Avicel (2). AS the assay for Avicel-degrading enzyme did not appear sensitive enough for the intended studies only the location of the CMC-hydrolyzing enzyme has been investigated.
Materials and Methods Bacterial Strain The strain used was Cellvibrio frrlolrs National Collection of Industrial Bacteria (NCIB) 8634 obtained from Torry Research Station, Aberdeen, Scotland.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/16/14 For personal use only.
52
CAN. J. MICROBIOL. VOL. 21, 1975
Salts Mediulii and C~lltiuatiorr The salts medium was that described earlier (2). The bacteria were cultivated at 28" either in 2.5-liter Fernbach flasks on a rotary shaker or in a 9-liter Biotec Fermentor with automatic pH control (Biotec F L 110, Biotec AB, Box 16152, Stockholm 16, Sweden). When cells were cultivated on soluble carbon sources, at a concentration of 0.5%, growth was followed in an Eppendorf photometer at a wavelength of 546 nm. On 0.5% cellulose, growth was followed in the Biotec Fermentor by measuring the acid used in titration to maintain the p H at 7.3. Haruesting and Storage of Cells When grown on soluble carbon sources, bacteria were harvested by centrifugation in the late logarithmic growth phase at an absorbance value of about 1.1. This corresponds to about 1.1 mg cell protein per milliliter. Cultures on cellulose were harvested when titration with acid had almost stopped and the cellulose was nearly used up. Remaining cellulose was allowed to settle in a glass cylinder for about an hour, then resuspended several times in salts medium. The cell-containing supernatants were combined, centrifuged, and washed several times. If not used immediately the cells were stored at -20" without loss of cellulase activity. Carbon Sources The soluble carbon sources used have been described in an earlier paper (2). The cellulose, a slightly acidhydrolyzed pulp cellulose free of reducing sugar, and the C M C solution were prepared as described earlier (2).
of Somogyi (1 3). When assays were run on cell-containing samples, controls in buffer were always run as the cells ~ ~ s u a l lreleased y some reducing sugar, especially during prolonged incubation. The cells or cell remnants were removed by filtering through a hard filter paper (Munktell 20 H) before measuring the color in a spectrophotometer. The enzyme units are as defined earlier (2). Determination of Protein Bacterial growth and protein content of suspensions and solutions were measured by the method of Lowry el al. (5) with serum albumin as standard. Sonication of Cells Cells were sonicated in a Raytheon D F 101 sonicator (Raytheon Manuf. Co., Waltham, Mass. U.S.A.) at 20 kcycles and 8" for 20 min. Slrock Treatme~rt Osmotic shock treatment was carried out as described by Nossal and Heppel (8). Spheroplast Formation and Clremical Treatnre~rts Spheroplasts were prepared by adding 0.5 mg lysozyme/ml to a suspension of cells in 0.05 M tris(hydroxymethyl)aminomethane (Tris)-HC1 buffer, pH 8.0, with 0.005 M ethylenediaminetetraacetic acid (EDTA) and 10% sucrose. The suspension was stirred gently for about 10 min at room temperature. Suspensions of washed cells and cell remnants were treated at room temperature for various times with EDTA, polymyxin B, or Triton X-100 as described earlier (1).
Assay of CMC-hydrolyzi?rg and Auicel-degrading E~rzynie The CMC-hydrolyzing activity was determined accordResults ing to Miller et a / . (6) and performed as described earlier (2). The assay for Avicel-degrading activity was per- Studies of Growing Cultures formed as described earlier (2) by the colorimetric method Treatment of C .f~illi~is cells with
the non-ionic
FIG. 1. Bacterial growth on 0.5% cellobiose and formation of CMC-hydrolyzing enzyme. Growth was followed by measuring the protein content of culture samples (m), CMC-hydrolyzing enzyme on the surface of intact cells ( 0 )was determined on washed cell samples. CMC-hydrolyzing enzyme determined in cell lysates after treatment with Triton-EDTA (A).
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/16/14 For personal use only.
BERG: CELLULASE LOCATION IN
0.5 1.0 Protein ( m g / m l ) F I G .2. Differential plot of cell~ilaseforniation on 0.5% glucose. Symbols as in Fig. 1 .
detergent Triton X-100 caused rapid cell lysis and partial dissolution of remaining cell walls (1). When suspensions of bacteria were assayed for CMC-hydrolyzing enzyme before and after such treatment, large increases in activity were observed with cells harvested from cultures growing on cellobiose (Fig. 1A). A marked difference in the shapes of the differential plot curves was obtained (Fig. IB). The formation of CMC-hydrolyzing enzyme was apparently not repressed by cellobiose as seen from the straight line obtained. The location of the enzyme in the cells was, however, affected by the sugar con-
A
I
I 5
10 Time (hl
15
C.
53
FULVUS
centration and during the active growth only about one fourth was found o n the cell surface. A similarly formed curve was obtained with cells growing on amylose. Cells growing on glucose had very little cellulase activity until the sugar concentration became low (less than 0.04%) (2). Both surface-bound and total cellulase activities increased simultaneously (Fig. 2). N o CMC-hydrolyzing enzyme could be detected in the cell-free medium of cultures growing on glucose, cellobiose, or amylose before the stationary phase of growth when enzyme was released as a result of cell lysis. When 0.6% C M C was included in the medium with 0.5% cellobiose as carbon source, cellulase activity was already detected in the medium during the active growth phase (Fig. 3). The C M C used had a degree of substitution (DS) of 0.4 and could not be used as a carbon source (2), and no effect on the growth rate or cell yield was noted. The presence of the C M C decreased the ratio between surface-bound and total enzyme activity and about one-fifth of the activity occurred cell-free at the beginning of the stationary growth phase. Cultures grown on acid-hydrolyzed cellulose free from reducing sugar (2) formed at least 10 times as much CMC-hydrolyzing enzyme as did cultures on cellobiose or any other soluble carbon source investigated. As the bacteria grow within the cellulose fibers it is not possible to
6
I
0.5
1.0 1.5 Protein (mglrnl)
I
FIG.3. Bacterial growth on 0.5z, cellobiose in presence of 0.69, C M C (DS, 0.4; DP, 300) and formation of CMC-hydrolyzing enzyme. Symbols as in Fig. I. Cell-free CMC-hydrolyzing enzyme (0) was, in addition, measured on centrifuged culture samples.
CAN. 1. MICROBIOL.
VOL. 21,
1975
TABLE 1 CMC-hydrolyzing and Avicel-degrading enzyme formed on some carbon sources CMC-hydrolyzing enzyme, units/mg protein in culture
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/16/14 For personal use only.
Carbon source, 0.5%
Cell-bound"
Cell-freeb
Avicel-degrading enzyme, units/mg protein in culture Cell-bound"
Cell-freeb
ODetermined o n washed and sonicated cell suspensions. bActivity in media expressed per milligram cell protein formed. CCells harvested in the late logarithmic growth phase. C e l l s harvested from stationary phase cultures.
TABLE 2 Release of CMC-hydrolyzing enzyme (enzyme activity, unitslrnl) from cells cultivated on cellobiose"
Treatment Control (no treatment) EDTA ( 5 x M, 15 min) Lysozyrne and EDTA (0.5 mg/ml and M resp. in 10% sucrose, 15 min) Polymyxin (0.2 mg/ml, 10% sucrose, 20 min) Triton X-100 (2 mg/ml, 20 min) Triton X-100 and EDTA (concn. as above)
Cell suspension
Supernatant after treatment
Washings of cellsb
Pellet after washingb
41
0.1
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/16/14 For personal use only.
Institirte of Biochemistry, University of Uppsrila, Uppsala, Sweden Accepted August 22, 1974 firlr.irs. Can. J . Microbiol. 21: 51-57. BERG,B. 1975. Cellulase locat~onin Cell~~ibrio The location of cellulase in C.firlivis depends on thecarbon source for growth and the age of the culture. When cells were grown on glucose or cellobiose all CMC-hydrolyzing enzyme was cell-bound but only part of the activity was located on the cell surface. Treatment of cells with EDTA, lysozyme, and detergents and subsequent fractionation experiments showed that cellulase was also located in the periplasm and bound to a membrane fraction. Growth on cellulose gave cell-free cellulase active against CMC. The enzyme was repressed by glucose but formed at a constant differential rate on cellobiose and amylose. This rate was 8-10 times lower than on cellulose and possible reasons for this are discussed.
BERG,B. 1975. Cellulase location in Cellvihriojirlvrrs. Can. J. Microbiol. 21: 51-57. rs de la source de carbone pour la croissance La localisation de la cellulase chez C . f i r l ~ ~depend et de I'ige de la culture. Lorsque les cellules se sont developpees sur le glucose ou la cellobiose tous les enzymes hydrolisant le CMC sont attaches a la cellule mais seulement une partie de l'activite est localiste sur la surface cellulaire. Le traitement des cellules avec I'EDTA, le lysozyme et des detergents et des fractionations subsequentes montrent que la cellulase est aussi localisee dans le pkriplasme et attachee a une fraction de membrane. La croissance sur la cellulose donne une activite acellulaire de cellulase sur le CMC. L'enzyme est repress6 par le glucose mals forme un taux constant diffkrentiel sur la cellobiose et I'amylose. Ce taux est 8 a 10 fois plus faible que sur la cellulose et nous y discutons les raisons possibles de ce phenomene. [Traduit par le journal]
a
Introduction Cellulose is completely insoluble and cellulolytic microorganisms must therefore either form cell-free cellulases or have such enzymes located on the outside of the cells. It is often difficult t o show whether an enzyme found in a culture medium is secreted by growing cells o r passively released as a result of cell lysis (10). The terminology used in describing the location of enzymes in microbial cultures is confusing as the words exoenzymes, extracellular, and cell-free are used rather ambiguously. In this paper the term cell-free is used for enzyme not associated with the cells and cell-bound for enzyme associated with the cells. As will be shown in this paper the cell-bound cellulase in Celluibrio fiilr~is is only partly bound to the cell surface. It is therefore also necessary to distinguish between surface-bound and total cell-bound enzyme. The study of cellulolytic enzymes is con~plicated as they occur in solutions in the form of components of different specificities which are difficult t o separate. It is, besides, impossible to obtain a high sensitivity in assays with cellulose as substrate and it is iecessary-to rely on measurements with carboxvmethvl-cellulose (CMC). 'Received May 24, 1973. 2Present address: Department of Microbiology, Agricultural College, Uppsala 7, Sweden.
Pse~idomonas fl~iorescens var. cell~ilosae has been found t o -have cell-bound and cell-free cellulase in varying proportions depending on the carbon source for growth (14). We have reported earlier that the cellulase of C. fitluus is cell-bound when the bacteria grow on soluble carbon sources whereas cultures on cellulose mainly contain cell-free cellulase (2). The enzyme may be cell-free because of cell lysis in relatively old cultures but its release may also be caused by the carbon source for growth. We have compared the CMC-hydrolyzing activity of C. fulu~is grown on cellobiose and cellulose and studied the release of enzyme from cell suspensions treated in various ways. The results will be discussed in relation to ultrastructural observations presented in a preceding paper (1). The culture solution was shown to exhibit activity towards both C M C and Avicel (2). AS the assay for Avicel-degrading enzyme did not appear sensitive enough for the intended studies only the location of the CMC-hydrolyzing enzyme has been investigated.
Materials and Methods Bacterial Strain The strain used was Cellvibrio frrlolrs National Collection of Industrial Bacteria (NCIB) 8634 obtained from Torry Research Station, Aberdeen, Scotland.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/16/14 For personal use only.
52
CAN. J. MICROBIOL. VOL. 21, 1975
Salts Mediulii and C~lltiuatiorr The salts medium was that described earlier (2). The bacteria were cultivated at 28" either in 2.5-liter Fernbach flasks on a rotary shaker or in a 9-liter Biotec Fermentor with automatic pH control (Biotec F L 110, Biotec AB, Box 16152, Stockholm 16, Sweden). When cells were cultivated on soluble carbon sources, at a concentration of 0.5%, growth was followed in an Eppendorf photometer at a wavelength of 546 nm. On 0.5% cellulose, growth was followed in the Biotec Fermentor by measuring the acid used in titration to maintain the p H at 7.3. Haruesting and Storage of Cells When grown on soluble carbon sources, bacteria were harvested by centrifugation in the late logarithmic growth phase at an absorbance value of about 1.1. This corresponds to about 1.1 mg cell protein per milliliter. Cultures on cellulose were harvested when titration with acid had almost stopped and the cellulose was nearly used up. Remaining cellulose was allowed to settle in a glass cylinder for about an hour, then resuspended several times in salts medium. The cell-containing supernatants were combined, centrifuged, and washed several times. If not used immediately the cells were stored at -20" without loss of cellulase activity. Carbon Sources The soluble carbon sources used have been described in an earlier paper (2). The cellulose, a slightly acidhydrolyzed pulp cellulose free of reducing sugar, and the C M C solution were prepared as described earlier (2).
of Somogyi (1 3). When assays were run on cell-containing samples, controls in buffer were always run as the cells ~ ~ s u a l lreleased y some reducing sugar, especially during prolonged incubation. The cells or cell remnants were removed by filtering through a hard filter paper (Munktell 20 H) before measuring the color in a spectrophotometer. The enzyme units are as defined earlier (2). Determination of Protein Bacterial growth and protein content of suspensions and solutions were measured by the method of Lowry el al. (5) with serum albumin as standard. Sonication of Cells Cells were sonicated in a Raytheon D F 101 sonicator (Raytheon Manuf. Co., Waltham, Mass. U.S.A.) at 20 kcycles and 8" for 20 min. Slrock Treatme~rt Osmotic shock treatment was carried out as described by Nossal and Heppel (8). Spheroplast Formation and Clremical Treatnre~rts Spheroplasts were prepared by adding 0.5 mg lysozyme/ml to a suspension of cells in 0.05 M tris(hydroxymethyl)aminomethane (Tris)-HC1 buffer, pH 8.0, with 0.005 M ethylenediaminetetraacetic acid (EDTA) and 10% sucrose. The suspension was stirred gently for about 10 min at room temperature. Suspensions of washed cells and cell remnants were treated at room temperature for various times with EDTA, polymyxin B, or Triton X-100 as described earlier (1).
Assay of CMC-hydrolyzi?rg and Auicel-degrading E~rzynie The CMC-hydrolyzing activity was determined accordResults ing to Miller et a / . (6) and performed as described earlier (2). The assay for Avicel-degrading activity was per- Studies of Growing Cultures formed as described earlier (2) by the colorimetric method Treatment of C .f~illi~is cells with
the non-ionic
FIG. 1. Bacterial growth on 0.5% cellobiose and formation of CMC-hydrolyzing enzyme. Growth was followed by measuring the protein content of culture samples (m), CMC-hydrolyzing enzyme on the surface of intact cells ( 0 )was determined on washed cell samples. CMC-hydrolyzing enzyme determined in cell lysates after treatment with Triton-EDTA (A).
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/16/14 For personal use only.
BERG: CELLULASE LOCATION IN
0.5 1.0 Protein ( m g / m l ) F I G .2. Differential plot of cell~ilaseforniation on 0.5% glucose. Symbols as in Fig. 1 .
detergent Triton X-100 caused rapid cell lysis and partial dissolution of remaining cell walls (1). When suspensions of bacteria were assayed for CMC-hydrolyzing enzyme before and after such treatment, large increases in activity were observed with cells harvested from cultures growing on cellobiose (Fig. 1A). A marked difference in the shapes of the differential plot curves was obtained (Fig. IB). The formation of CMC-hydrolyzing enzyme was apparently not repressed by cellobiose as seen from the straight line obtained. The location of the enzyme in the cells was, however, affected by the sugar con-
A
I
I 5
10 Time (hl
15
C.
53
FULVUS
centration and during the active growth only about one fourth was found o n the cell surface. A similarly formed curve was obtained with cells growing on amylose. Cells growing on glucose had very little cellulase activity until the sugar concentration became low (less than 0.04%) (2). Both surface-bound and total cellulase activities increased simultaneously (Fig. 2). N o CMC-hydrolyzing enzyme could be detected in the cell-free medium of cultures growing on glucose, cellobiose, or amylose before the stationary phase of growth when enzyme was released as a result of cell lysis. When 0.6% C M C was included in the medium with 0.5% cellobiose as carbon source, cellulase activity was already detected in the medium during the active growth phase (Fig. 3). The C M C used had a degree of substitution (DS) of 0.4 and could not be used as a carbon source (2), and no effect on the growth rate or cell yield was noted. The presence of the C M C decreased the ratio between surface-bound and total enzyme activity and about one-fifth of the activity occurred cell-free at the beginning of the stationary growth phase. Cultures grown on acid-hydrolyzed cellulose free from reducing sugar (2) formed at least 10 times as much CMC-hydrolyzing enzyme as did cultures on cellobiose or any other soluble carbon source investigated. As the bacteria grow within the cellulose fibers it is not possible to
6
I
0.5
1.0 1.5 Protein (mglrnl)
I
FIG.3. Bacterial growth on 0.5z, cellobiose in presence of 0.69, C M C (DS, 0.4; DP, 300) and formation of CMC-hydrolyzing enzyme. Symbols as in Fig. I. Cell-free CMC-hydrolyzing enzyme (0) was, in addition, measured on centrifuged culture samples.
CAN. 1. MICROBIOL.
VOL. 21,
1975
TABLE 1 CMC-hydrolyzing and Avicel-degrading enzyme formed on some carbon sources CMC-hydrolyzing enzyme, units/mg protein in culture
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/16/14 For personal use only.
Carbon source, 0.5%
Cell-bound"
Cell-freeb
Avicel-degrading enzyme, units/mg protein in culture Cell-bound"
Cell-freeb
ODetermined o n washed and sonicated cell suspensions. bActivity in media expressed per milligram cell protein formed. CCells harvested in the late logarithmic growth phase. C e l l s harvested from stationary phase cultures.
TABLE 2 Release of CMC-hydrolyzing enzyme (enzyme activity, unitslrnl) from cells cultivated on cellobiose"
Treatment Control (no treatment) EDTA ( 5 x M, 15 min) Lysozyrne and EDTA (0.5 mg/ml and M resp. in 10% sucrose, 15 min) Polymyxin (0.2 mg/ml, 10% sucrose, 20 min) Triton X-100 (2 mg/ml, 20 min) Triton X-100 and EDTA (concn. as above)
Cell suspension
Supernatant after treatment
Washings of cellsb
Pellet after washingb
41
0.1
