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Table 2
L. mesenteroides FT045B dextransucrase activity, determined by Methods 1–3
Methoda
Description of the method
Experiment 1
(IUb/mL)
Experiment 2
(IUb/mL)
Experiment 3
(IUb/mL)
Mean and mean deviation
1
2
3
14C-Dextran precipitation and LSCc
1.93
1.10
1.94
2.04
1.10
2.02
1.98
1.11
1.98
1.98 0.04
1.10 0.00
1.98 0.03
Addition of 14C-dextran to filter paper squares with methanol washing
Precipitation of dextran, dehydration, and weighing
a
b
c
The assays were run at 30 °C.
IU = International Unit = 1.0
LSC = liquid scintillation counting.
l
mol of D-glucose incorporated into dextran/mL/min.
Method-6 is the measurement of the reducing value by DNS
after the removal of dextran by precipitation with two volumes
of ethanol. This method also gave a high IU value of 308 units
two additional times, followed by dehydration (Methods 1 and 3)
give the most accurate measurement of the amount of dextran
synthesized by dextransucrase, and, therefore, are the methods
to be used to obtain an accurate and useful activity of dextransu-
crase for assays and kinetic studies. These methods can be used
for any glucansucrase that synthesizes glucan from sucrose, such
as mutan, alternan, comb-dextrans, and so forth. We further con-
clude that the use of reducing value methods to determine the
activity of dextransucrase and related enzymes by measuring the
due to over-oxidation of the D-fructose and leucrose in the digest,
which is high but less than that of Method-5, due to the removal
of dextran, confirming the over-oxidation of dextran in Method-
5. This method, too, is completely invalid for assays and for kinetic
studies of dextransucrases, as it is 140 U greater than that of 168 U
for the direct methods for determining dextran.
Method-7 is also the measurement of the reducing value of the
sucrose/dextransucrase digest after the removal of dextran by pre-
cipitation with two volumes of ethanol. The reducing value used in
this method was the ferricyanide/arsenomolybdate method54 that
also gave a high IU of 353, with a relatively high mean deviation.
This also is due to the relatively high alkaline conditions required
for the reduction that give over-oxidation and is unacceptable for
assays and kinetic studies.
Method-8 is the measurement of the reducing value by the cop-
per bicinchoninate method55 after the removal of dextran by pre-
cipitation with two volumes of ethanol. This method gave an IU
of dextransucrase of 183, which is not nearly as high as that of
the two alkaline DNS methods and the ferricyanide/arsenomolyb-
date method. The copper bicinchoninate method is the most sensi-
tive of any of the known reducing value methods and gave 15 IU
greater than the direct measurement of Methods 1 and 3. This
putative reducing value of
unacceptable values due to over-oxidation of
D
-fructose gives highly inflated and
-fructose and other
D
reducing sugars in the digest, as well as dextran if it is allowed to
remain in the assay solution. This is especially so for DNS and the
ferricyanide/arsenomolybdate methods, both in the presence of
dextran and in its absence. The ferricyanide/arsenomolybdate
method gave an even higher value for dextransucrase, in the ab-
sence of dextran, than did the DNS method. Unfortunately, many
laboratories have found the relative ease of using the DNS method
for determining the activity of dextransucrases and related en-
zymes and apparently have not been aware of the problems of
over-oxidation by DNS, giving highly inflated assays and kinetics
and consequently incorrect measurements.
References
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D-fructose and
D
-glucose, and the reducing value of leucrose. The method does
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not give over-oxidation of oligosaccharides and polysaccharides
as shown in Figure 1 and is sensitive enough to give reducing val-
ues of polysaccharides that can be used in determining the D.P. and
the number-average molecular weights of polysaccharides.59–61
Method-9 utilizes an ‘enzyme kit’ obtained from Megazyme
that determines the amount of D-fructose. This method gave a rel-
atively low IU value for dextransucrase of 149 IU. This method is
similar to that of Method-4 that gave an IU of 151 with values of
19 and 17 IU, respectively, below the values obtained by Methods
1 and 3.
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Because all of the above-described assay methods involved the
dextransucrase preparation obtained from L. mesenteroides B-
512FMC, we also performed methods 1–3 with a second dextran-
sucrase produced by a L. mesenteroides strain FT045B obtained
from Brazil. The results are given in Table 2, which shows the same
relative results of 1.98 IU obtained by Methods 1 and 3 for this dex-
transucrase, and a lower value of 1.10 IU for Method-2, which is
consistent with the results obtained for B-512FMC dextransucrase.
4. Conclusions
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Chem. 1992, 11, 359–378.
It is concluded that the direct measurement of dextran, after
precipitation from the dextransucrase digest and re-precipitation
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Lang, C.; Paul, F.; Monsan, P. Enzyme Microb. Technol. 1993, 15, 77–85.