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Study of the autohydrolysis of Rubus fruticosus cells cultured in suspension
Food Hydrocolloids Vol.5 no.1/2 pp .173-176, 1991
Study of the autohydrolysis of Rubus fruticosus cells cultured in suspension Juan Fernandez-Bolanos, Gerard Chambat and Jean-Paul Joseleau
Centre de Recherches sur les Macromolecules Vegetales (CER MA V-CN RS), BP 53 X 38041 Grenoble cedex, France Conditions under which cells of Rubu s fruticosus cultured in suspension exhibited autohydrolysis were determined. The fresh cells cultured in the medium of Heller containing glucose as a carbon source were washed and transferred into distilled water, then kept at 37°C under gentle agitation. A comparison was established with cells in which the enzymes were denaturated by boiling for 20 min. The release of polysaccharides by endogenous enzymes reached a maximum after 24 h . The same was true for the solubilized reducing sugar (1) (Figure 1). Autohydrolysis was studied at various stages of the culture cycle. Estimation of the total polysaccharides excreted showed that the proportion of the wall material solubilized by autolysis process by 10-, 18-, 24- and 30-day-old cells, respectively, was nearly the same-about 1.2% of the wall dry weightregardless of the growth state of the cells. Analysis of the extracted polysaccharide complexes showed an increase in the net content of neutral sugars 70 R~ d~c i n9~u ~a rs .
60
...
.s:
.."'"
..
. . .n
(as glu cose)
50
..
~
....s, 40 .... 0
'" '"
0
~
.. s,
c.
'" E '"
Cont rol (al
o
2 4 6 8 10 12
24
48 Time (hour s l
Fig. 1. Autohydrolytic activity of fresh cells from Rubus fruticosus in H 20 as a function of time . Boiled cells (a) were used as control (as reducing sugars) .
173
J.Fernandez-Bolanos, G.Chambat and J.-P.Joseleau
25 .------ - - - -- - - - - - - - - -- - - -- - - -------,
'" ., s,
e>, '0c.
20
CJ
:;; ~
»,
'"
15
'i'c: 0
c: "0
10
'" e '" 0
;::
.,c. s,
5
V>
E '"
0
Rha _10 d
~ 18 d
024 d
~30 d
Fig. 2. Neutral sugars analysis of the non -dializable polymers obt ained during autolysis of R ifruticosus cells of different ages (10, 18, 24 and 30 days). Cells were incubated for 24 h. The composition in neutral sugars after hydrolysis with 2 rnol/dm' TF A was analyzed from their alditol acetate deriv atives in gas-liquid chrom ato graph y. Results represent the mean values from three repeat expe riments for each of the above samples. (a) Samples treated with a-amylase revealed that a large percentage of glucose originate s of the starch.
(2) from 33% to 55% during the exponential growth then declining to 45% during the stationary phase (Figure 2) . The uronic acids (3) remai ned very much lower, not exceeding 6% . The most abu ndant sugars were arabinose and galactose which were in the same ratio as in the extracellular polysaccharides (Gal/Ara = 1.4) (4). Arabinogalactans were more abundantly released at the end of the expon ential growth and during the stationary phase (5) . However, fractionation by ultrafiltration (Figure 3) revealed that arabinogalactan oligosaccharides were excreted in significant amount by Ill-day-old cells. In other cells, the amount of xyloglucan constituents increased up to 30 days in such a way that the relative amount of arabinose, xylose, mannose, galactose and glucose became nearly the same in the fractions betwe en 5000 and 1000 mol. wt (Figure 3A) . These results are correlated with wall-bound enzyme activities in the cells during a culture cycle . The released monosaccharides (fraction <1000 mol. wt contained exclusively monomers) were mainly mannose and glucose (Figure 3B) . The present results indicate that arabinogalactans and xyloglucans are the main components appearing during autolysis, that they occur as oligo and polysaccharides, and that a mannose-rich polysaccharide could also serve as autolytic substrate. Since mannose-containing polysaccharides are structural components of primary walls of suspension-cultured Rubus [ruticosus , as shown by the isolation of a galactoglucomannan from the culture medium (6), they 174
Autohydrolysis of Rubus fruticosus cells
A
60
.
SO
~
40
'0
..'"'" a .Z .. L.
::l
z
30 20 10 0 Rha
Fuc
Ara
~ lO d
Xyl §
Man
24 d
Gal 30 d
B
. '0
80 60
~
..'"'" . Z .. L.
40
~
::l
20
Z
0 Rha
Fuc
Ara
~ IO d
Xyl §
Man
24 d
Gal
Glc
30 d
Fig. 3. Changes in sugar composition of (A) oligosaccharide fraction (1000-5000 mol. wt) and (B) monosaccharide fractions «1000 mol. wt) released from autolytic activity of the cultured cells of Rfruticosus between day 10 and day 30 after inoculation. (a) Treatment of the fractions with oamylase led to a decrease of glucose content.
must be substrate of hydrolytic enzymes. It is interesting that, although some polysaccharides are quantitatively minor constituents of the wall, they might have important function during the autolytic process. 175
J .Fernandez-Bolanos, G.Chambat and J .·P.Joseleau
References 1. Somogyi, M. (1952) J. BioI. Chem . , 195, 19-23. 2. Albe rsheim,P., Nevins,D .l. , English,P.D . and Karr ,A . (1967) Carbohydr. Res. , 22, 453-456. 3. Blumenk rant z,N. and Asboe-Hanse n,G . (1973) A nalyt. Biochem. , 54, 484- 489. 4. Cartier,N . , Chambat ,G. and Joseleau ,J.P. (1987) Carbohydr. Res. , 168, 275-283. 5. Takeuchi.Y. and Komamine .A . (1980) Physiol. Plant, 50, 113- 118. 6. Cartier,N ., Charnbat.G. and Joseleau,J .P . (1988) Phytochem . , 27, 1361-1364 .
176
Study of the autohydrolysis of Rubus fruticosus cells cultured in suspension Juan Fernandez-Bolanos, Gerard Chambat and Jean-Paul Joseleau
Centre de Recherches sur les Macromolecules Vegetales (CER MA V-CN RS), BP 53 X 38041 Grenoble cedex, France Conditions under which cells of Rubu s fruticosus cultured in suspension exhibited autohydrolysis were determined. The fresh cells cultured in the medium of Heller containing glucose as a carbon source were washed and transferred into distilled water, then kept at 37°C under gentle agitation. A comparison was established with cells in which the enzymes were denaturated by boiling for 20 min. The release of polysaccharides by endogenous enzymes reached a maximum after 24 h . The same was true for the solubilized reducing sugar (1) (Figure 1). Autohydrolysis was studied at various stages of the culture cycle. Estimation of the total polysaccharides excreted showed that the proportion of the wall material solubilized by autolysis process by 10-, 18-, 24- and 30-day-old cells, respectively, was nearly the same-about 1.2% of the wall dry weightregardless of the growth state of the cells. Analysis of the extracted polysaccharide complexes showed an increase in the net content of neutral sugars 70 R~ d~c i n9~u ~a rs .
60
...
.s:
.."'"
..
. . .n
(as glu cose)
50
..
~
....s, 40 .... 0
'" '"
0
~
.. s,
c.
'" E '"
Cont rol (al
o
2 4 6 8 10 12
24
48 Time (hour s l
Fig. 1. Autohydrolytic activity of fresh cells from Rubus fruticosus in H 20 as a function of time . Boiled cells (a) were used as control (as reducing sugars) .
173
J.Fernandez-Bolanos, G.Chambat and J.-P.Joseleau
25 .------ - - - -- - - - - - - - - -- - - -- - - -------,
'" ., s,
e>, '0c.
20
CJ
:;; ~
»,
'"
15
'i'c: 0
c: "0
10
'" e '" 0
;::
.,c. s,
5
V>
E '"
0
Rha _10 d
~ 18 d
024 d
~30 d
Fig. 2. Neutral sugars analysis of the non -dializable polymers obt ained during autolysis of R ifruticosus cells of different ages (10, 18, 24 and 30 days). Cells were incubated for 24 h. The composition in neutral sugars after hydrolysis with 2 rnol/dm' TF A was analyzed from their alditol acetate deriv atives in gas-liquid chrom ato graph y. Results represent the mean values from three repeat expe riments for each of the above samples. (a) Samples treated with a-amylase revealed that a large percentage of glucose originate s of the starch.
(2) from 33% to 55% during the exponential growth then declining to 45% during the stationary phase (Figure 2) . The uronic acids (3) remai ned very much lower, not exceeding 6% . The most abu ndant sugars were arabinose and galactose which were in the same ratio as in the extracellular polysaccharides (Gal/Ara = 1.4) (4). Arabinogalactans were more abundantly released at the end of the expon ential growth and during the stationary phase (5) . However, fractionation by ultrafiltration (Figure 3) revealed that arabinogalactan oligosaccharides were excreted in significant amount by Ill-day-old cells. In other cells, the amount of xyloglucan constituents increased up to 30 days in such a way that the relative amount of arabinose, xylose, mannose, galactose and glucose became nearly the same in the fractions betwe en 5000 and 1000 mol. wt (Figure 3A) . These results are correlated with wall-bound enzyme activities in the cells during a culture cycle . The released monosaccharides (fraction <1000 mol. wt contained exclusively monomers) were mainly mannose and glucose (Figure 3B) . The present results indicate that arabinogalactans and xyloglucans are the main components appearing during autolysis, that they occur as oligo and polysaccharides, and that a mannose-rich polysaccharide could also serve as autolytic substrate. Since mannose-containing polysaccharides are structural components of primary walls of suspension-cultured Rubus [ruticosus , as shown by the isolation of a galactoglucomannan from the culture medium (6), they 174
Autohydrolysis of Rubus fruticosus cells
A
60
.
SO
~
40
'0
..'"'" a .Z .. L.
::l
z
30 20 10 0 Rha
Fuc
Ara
~ lO d
Xyl §
Man
24 d
Gal 30 d
B
. '0
80 60
~
..'"'" . Z .. L.
40
~
::l
20
Z
0 Rha
Fuc
Ara
~ IO d
Xyl §
Man
24 d
Gal
Glc
30 d
Fig. 3. Changes in sugar composition of (A) oligosaccharide fraction (1000-5000 mol. wt) and (B) monosaccharide fractions «1000 mol. wt) released from autolytic activity of the cultured cells of Rfruticosus between day 10 and day 30 after inoculation. (a) Treatment of the fractions with oamylase led to a decrease of glucose content.
must be substrate of hydrolytic enzymes. It is interesting that, although some polysaccharides are quantitatively minor constituents of the wall, they might have important function during the autolytic process. 175
J .Fernandez-Bolanos, G.Chambat and J .·P.Joseleau
References 1. Somogyi, M. (1952) J. BioI. Chem . , 195, 19-23. 2. Albe rsheim,P., Nevins,D .l. , English,P.D . and Karr ,A . (1967) Carbohydr. Res. , 22, 453-456. 3. Blumenk rant z,N. and Asboe-Hanse n,G . (1973) A nalyt. Biochem. , 54, 484- 489. 4. Cartier,N . , Chambat ,G. and Joseleau ,J.P. (1987) Carbohydr. Res. , 168, 275-283. 5. Takeuchi.Y. and Komamine .A . (1980) Physiol. Plant, 50, 113- 118. 6. Cartier,N ., Charnbat.G. and Joseleau,J .P . (1988) Phytochem . , 27, 1361-1364 .
176