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The
A. vinelandii
alginates have higher contents of L-guluronic acid and
sufficiently long G-blocks to enable gelation with calcium. Currently (2013)
industrial production of alginates using genetically optimized strains (alginate
degrading lyases and acetylases inactivated) of
A. vinelandii
is seriously
considered.
Bacterial alginates differ from algal alginates by containing O-acetyl groups at
some of the M residues (figure 8).
Figure 7. Bacterial alginates often contain O-acetyl groups on O2 and/or O3
(acetic acid esterified to hydroxyls)
1.2.7. Determination of
composition and
sequence
in
alginates
Complex carbohydrates are often studied by first hydrolysing the glycosidic
linkages (in hot acid) to obtain the corresponding free monosaccharides. They
can easily be separated, identified and quantified by GC (after some
derivatization) or HPLC. Unfortunately, the conditions for complete acid
hydrolysis also lead to severe destruction of free mannuronic and guluronic
acids.
In the late 1970’s non-destructive NMR methods for studying the composition
of alginates were established. These methods give not only F
M
and F
G
, but
also sequence parameters such as those given in the table above. It is used
routinely today and is still the state-of-the-art method.
Before alginates can be studied by NMR they must be subjected to partial
hydrolysis (DP
→
ca. 50). This is done in order to reduce the viscosity
because NMR is conducted at fairly high concentrations (ca. 10 mg/ml). At the
same time, a DP of about 50 is high enough to minimize ‘end effects’
interfering in the calculation of sequence parameters.
Cations binding to alginate (e.g. Ca
++
) should also be removed to prevent
gelling or aggregation. For this purpose a chelating agent such as TTHA is
added. Finally, NMR is conducted at hight T to enhance the mobility of the
chains, which in turn gives sharper peaks.
Before continuing, let us go through a brief NMR course (or consult a
textbook).
O
O OH
COOH
O
O
OH OH
COOH
O
O
H
3
C