3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
retaining glycanase operating by Ping-Pong mechanism. This
is supported by the most of works published to this date 3,7,8 .
On the other hand, the kinetic data of XET isolated from suspension-culture
poplar cells are consistent with the sequential
mechanism 9 .
1/v (nmol -1 .min)
1/v (nmol-1.min)
1/v (nmol -1 .min)
750
500
250
0
0 0.5 1 1.5 2 2.5
1800
1200
600
1200
1/XG (g -1 .L)
0
0 0.5 1 1.5 2 2.5
800
400
1/XG (g -1 .L)
0
0 0.5 1 1.5 2 2.5
1/XG (g -1 .L)
Fig. 2. Plot of 1/v = f(1/xG) at different concentrations of
nonasaccharide (A), octasaccharide (b) and heptasaccharide
(C): 5 µM (�), 10 µM (♦), 20 µM (▲), 30 µM (�), 50 µM (�)
and 200 µM (◊)
Kinetic analysis was performed at both pH optima of
XET (Figure 1 A, B) using radioactive alditols of XG octasaccharide
(XLXGol + XXLGol).
While K M values for mixture of XLXGol + XXLGol
were very similar at both pH optima (123 µM at pH 5.8
and 137 µM at pH 8.8, respectively) for XG they differed
(0.565 g dm –3 at pH 5.8 and 2.42 g dm –3 at pH 8.8, respectively).
A strong inhibition with higher concentrations of XG
was observed especially at pH 8.8 where the linearity of reaction
in dependence on XG concentration was limited. From
A
B
C
s634
Table I
Kinetic parameters calculated from nonlinear regression
Acceptor Parameter values ± S.D.
substrate KMXG [g dm –3 ] KMXGO [µmol dm –3 ]
nonasaccharide 0.854 ± 0.21 42.199 ± 6.546
octasaccharide 0.589 ± 0.22 92.396 ± 10.120
heptasaccharide 0.755 ± 0.08 117.612 ± 5.588
this reason the further study of kinetic parameters was carried
out only at acidic pH using reducing XGOs.
The Lineweaver-Burk plots for XET showed parallel
lines at higher concentrations of reducing XGOs with DP 7, 8
and 9 as acceptor substrates (Fig. 2. A, B, C).
The kinetic parameters K M were determined by measuring
initial transfer rates of reducing XGOs with DP 7, 8
and 9 into XG (Table I). The data concluded in Table I show
that the K M values for acceptor substrates increase with their
decreasing DP. On the other hand the lowest K M value for
donor substrate was calculated for octasaccharide as an
acceptor.
Conclusions
The kinetic study analyzed at both pH optima of XET (pH
5.8 and 8.8, respectively) indicate that the affinity of enzyme
to acceptor substrate (mixture of XLXGol + XXLGol) is
independent on pH unlike the K M values for XG, where significant
differences can be seen.
The relationship between the K M values and DP of
acceptor substrates shows the decrease of enzyme affinity to
reducing XGOs with their decreasing DP. As a consequence,
the best acceptor substrate seems to be the nonasaccharide.
The comparison of K M values for octasaccharides indicates
that parsley XET has a higher affinity for reducing
oligosaccharide than to its alditol.
In all cases excepting the kinetic analysis using low concentrations
of XGOs, the Lineweaver-Burk plots for XET
showed parallel lines. These results indicated that the enzyme
catalyzed the reaction utilizing a Ping-Pong (Bi Bi) mechanism
rather than a sequential one. Hence, the non-parallel
lines at lower concentrations of XGOs can be explained by
the stronger influence of side reactions like interpolymeric
transglycosylation and inhibitions at such concentrations of
acceptor substrate.
This research was supported by the Slovak Grant Agencies
VEGA No. 2/6133/26, APVV No. LPP/0177/06 and by
grant No. II/2/2005 from the Slovak Academy of Sciences to
Centre of Excellence GLYCOBIOS.
REFEREnCES
1. Garajová S., Flodrová D. Ait-Mohand F., Farkaš V.,
Stratilová E.: Biologia 63, 313 (2008).
2. Sulová Z., Lednická M., Farkaš V.: Anal. Biochem. 229,
80 (1995).