Part F | 30.3
734 Part F Marine Derived Metabolites
A new four-step mechanism for the hydrolysis
of glycosidic bonds involving oxidation-eliminationaddition-reduction reactions has been reported for
family 4 [30.14] of glycoside hydrolases. Members
of this family require divalent metal ion and a
NAD
C cofactor for activity. Also, ˛-1,4-glucan lyases,
cleaving glycosidic linkages in starch and glycogen,
and polysaccharide lyases which are involved in the
degradation of glycosaminoglycans and pectin, belong to the class of enzymes that employ elimination
chemistry.
30.2.2 Natural and Engineered Methods
for Synthesis
Two general protocols are in use for the utilization of
glycoside hydrolases in the synthesis of glycosidic linkage: the reverse hydrolysis procedure and the kinetic
approach. The former, starting with free monosaccharide and an acceptor, is reported as an efficient and
cost-effective methodology for just a few enzymes and
as a poor-yielding procedure for others [30.15]. The
alternative kinetic approach is based on the use of
a glycosyl donor which produces an intermediate glycosyl enzyme. In presence of a nucleophile other than
water, this ester intermediate is resolved into products that as new glycosides could still be substrates
for the enzyme, hence the reaction must be carefully
monitored to achieve good yield. Both these systems
could be considered unnatural due to the natural hydrolytic nature of glycosyl hydrolases. However, they
can be called natural in comparison to the ad hoc
produced mutant enzymes, known as glycosynthases,
introduced several years ago as an elegant approach to
solve the back-hydrolysis problem encountered using
wild type biocatalysts. This topic has been extensively
reviewed, and readers are referred to reports on this
technology [30.16–18]. Synthesis of oligosaccharides
by this engineered methodology can always be considered as an emerging alternative for a possible scale-up
of oligosaccharide production for each single example
of a newly isolated biocatalyst transformed in the glycosynthase version.
A stereochemical outcome of a glycosidase-based
reaction includes natural absolute anomeric selectivity
derived from the chemistry of the reaction mechanism.
However, regioselectivity and other aspects are dependent on the nature of the enzyme towards the donor(s)
and product(s) formed. Depending on the structure of
the active site, the biocatalyst can accommodate certain
sugar acceptor(s) and promote regiospecific formation
of glycosidic linkages. The impressive variety of available glycosidases clearly indicates that the potential
biodiversity of these enzymes (and also of the possibly derived glycosynthases) is still largely unexplored
and that potential applications of these biocatalysts will
increase in the near future starting from the knowledge
of wild-type biodiversity.
30.3 The Marine Ecosystem as a Source for New Glycoside Hydrolases
The importance of the marine environment as a source
of different and interesting glycosyl hydrolases can
be found in the literature as overall results of several research projects conducted in different nations
in the world from the beginning of the 1960s. Attention was focused mainly on the hydrolytic selectivity
of these enzymes, since efforts of the structural identification of carbohydrate molecules then faced the
complexity of oligosaccharide structures, before the
flourishing development of instrumental analysis that
started in the 1980s. Various potent activities were
found in marine organisms such as Charonia lampas,
Turbo cornutus, and others [30.19–21]. However, the
interest in these enzymes also led to a raised interest in the study of phylogenetic position and feeding
habits of various molluscs [30.22]. The sea still continues to furnish glycosyl hydrolases, which can be used
in different fields: (i) to solve the structural complexity
of new oligosaccharides obtained purely by improved
chromatographic techniques, (ii) to help in the treatment of waste material for the recovery of important
value-added molecules, and (iii) to allow synthesis of
glycosidic bonds.
The distribution of fucoidan hydrolases and other
glycosidases, for example, was only recently investigated in 33 species of marine invertebrates [30.23],
although the presence of cellulases has been assessed
in Littorina sp. [30.24] and in Telescopium telescopium [30.25] for a long time. It is of interest to note
that both marine microorganisms and invertebrates such
as Haliotis, Mizyhopecten yessoensis, and sea urchins
all contain fucoidan hydrolases and other important glycosyl hydrolases [30.26–29]. Fucoidan hydrolases are
important enzymes for structural identification of com-
734 Part F Marine Derived Metabolites
A new four-step mechanism for the hydrolysis
of glycosidic bonds involving oxidation-eliminationaddition-reduction reactions has been reported for
family 4 [30.14] of glycoside hydrolases. Members
of this family require divalent metal ion and a
NAD
C cofactor for activity. Also, ˛-1,4-glucan lyases,
cleaving glycosidic linkages in starch and glycogen,
and polysaccharide lyases which are involved in the
degradation of glycosaminoglycans and pectin, belong to the class of enzymes that employ elimination
chemistry.
30.2.2 Natural and Engineered Methods
for Synthesis
Two general protocols are in use for the utilization of
glycoside hydrolases in the synthesis of glycosidic linkage: the reverse hydrolysis procedure and the kinetic
approach. The former, starting with free monosaccharide and an acceptor, is reported as an efficient and
cost-effective methodology for just a few enzymes and
as a poor-yielding procedure for others [30.15]. The
alternative kinetic approach is based on the use of
a glycosyl donor which produces an intermediate glycosyl enzyme. In presence of a nucleophile other than
water, this ester intermediate is resolved into products that as new glycosides could still be substrates
for the enzyme, hence the reaction must be carefully
monitored to achieve good yield. Both these systems
could be considered unnatural due to the natural hydrolytic nature of glycosyl hydrolases. However, they
can be called natural in comparison to the ad hoc
produced mutant enzymes, known as glycosynthases,
introduced several years ago as an elegant approach to
solve the back-hydrolysis problem encountered using
wild type biocatalysts. This topic has been extensively
reviewed, and readers are referred to reports on this
technology [30.16–18]. Synthesis of oligosaccharides
by this engineered methodology can always be considered as an emerging alternative for a possible scale-up
of oligosaccharide production for each single example
of a newly isolated biocatalyst transformed in the glycosynthase version.
A stereochemical outcome of a glycosidase-based
reaction includes natural absolute anomeric selectivity
derived from the chemistry of the reaction mechanism.
However, regioselectivity and other aspects are dependent on the nature of the enzyme towards the donor(s)
and product(s) formed. Depending on the structure of
the active site, the biocatalyst can accommodate certain
sugar acceptor(s) and promote regiospecific formation
of glycosidic linkages. The impressive variety of available glycosidases clearly indicates that the potential
biodiversity of these enzymes (and also of the possibly derived glycosynthases) is still largely unexplored
and that potential applications of these biocatalysts will
increase in the near future starting from the knowledge
of wild-type biodiversity.
30.3 The Marine Ecosystem as a Source for New Glycoside Hydrolases
The importance of the marine environment as a source
of different and interesting glycosyl hydrolases can
be found in the literature as overall results of several research projects conducted in different nations
in the world from the beginning of the 1960s. Attention was focused mainly on the hydrolytic selectivity
of these enzymes, since efforts of the structural identification of carbohydrate molecules then faced the
complexity of oligosaccharide structures, before the
flourishing development of instrumental analysis that
started in the 1980s. Various potent activities were
found in marine organisms such as Charonia lampas,
Turbo cornutus, and others [30.19–21]. However, the
interest in these enzymes also led to a raised interest in the study of phylogenetic position and feeding
habits of various molluscs [30.22]. The sea still continues to furnish glycosyl hydrolases, which can be used
in different fields: (i) to solve the structural complexity
of new oligosaccharides obtained purely by improved
chromatographic techniques, (ii) to help in the treatment of waste material for the recovery of important
value-added molecules, and (iii) to allow synthesis of
glycosidic bonds.
The distribution of fucoidan hydrolases and other
glycosidases, for example, was only recently investigated in 33 species of marine invertebrates [30.23],
although the presence of cellulases has been assessed
in Littorina sp. [30.24] and in Telescopium telescopium [30.25] for a long time. It is of interest to note
that both marine microorganisms and invertebrates such
as Haliotis, Mizyhopecten yessoensis, and sea urchins
all contain fucoidan hydrolases and other important glycosyl hydrolases [30.26–29]. Fucoidan hydrolases are
important enzymes for structural identification of com-