Marine Enzymes – Production and Applications 14.3 Examples of Marine Enzymes 421
Part B | 14.3
cell immobilization were also optimized for repeated
batch fermentation of the alkaline protease by T. turnirae. Ceramic support, the different sizes of broken
pumice stone, and silicone foam enhanced enzyme production by more than 200%, when compared to free
cells. Electron micrograph evident T. turnirae colonization in porous support matrices and five repeated
batch fermentation was achieved without significant decrease in the enzyme production. When the same strain
was immobilized in calcium alginate beads maximum
protease activity was achieved at 3 w=v % sodium alginate and 3% CaCl 2 concentrations with a 1 W 2 ratio
of cell and alginate. Protease production was independent of bead size but decreased significantly when the
beads were treated with glutaraldehyde. The immobilized cells were used for 8 cycles, each lasting 72h.
Protease production increased with an increased number of fermentation cycles and was maximized at the
fourth cycle when the enzyme activity reached about
3:5 times more than that of the first cycle.
14.3.3 Halogenating Enzymes
Halogenated molecules are widely distributed in the
environment and their existence is not only related
to human industrial processes but also to natural biotic as well as abiotic processes. To date almost 5000
organohalogens are known. Most of them contain chlorine or bromine rather than fluorine or iodine [14.68].
With respect to the relatively high concentration of
bromide found in the marine environment, brominated
compounds are found quite often and occur in a larger
number than in freshwater samples [14.69, 70].
While establishing a halogen–carbon bond, several
properties of the compound are changed. The following two effects are the most important: a) the introduction of halogen increases the molecule’s thermal
and oxidative activity, and b) increases the biological
membrane permeability [14.71]. The halogenated compounds currently manufactured by the industry can be
discriminated into bulk chemicals and higher valueadded pharmaceutical compounds, where it is estimated
that 20% of all pharmaceutical small drugs and approximately 30% of all active compounds in agrochemistry
are halogenated [14.71]. Considering the problems of
chemical halogenation, such as low yield, lack of sensitivity, and the use of harmful/toxic chemicals, there is
an increasing demand for less toxic and highly specific
halogenation processes. The latter is of particular interest for the production of halogenated pharmaceuticals.
However, halogenated compounds are also of interest
as intermediates in organic synthesis. Since they can
be used as reactant within palladium-catalyzed coupling reactions, novel synthetic routes are accessible for
manufacturing of organic fine chemicals.
The formulation of natural halogenated products
occurs via abiotic and biotic processes. Particular enzymes are responsible for the biogenous halogenation
process. However, halogenation can be accomplished
by several types of enzymes featuring distinct substrate
specificity. One can roughly distinguish the enzymes
as haloperoxidases, flavin-dependent halogenases, nonheme iron halogenases, and the fluorinase/chlorinase
system.
Haloperoxidases are versatile enzymes for transformation of many substrates. Most prominent is
the chloroperoxidase from Caldariomyces fumago.
Whereas the enzyme is more or less even unspecific
with regard to the substrate, it can be beneficially applied to numerous biotranformations. Reactions such
as epoxidations [14.72, 73], enatioselective sulfoxidations [14.74], N-oxidation of arylamines [14.75], hydroxylation of side chains of aromatic systems [14.76],
oxidation of indole to oxyindole [14.77], or oxidation
of primary alcohols to their corresponding aldehydes [14.78] have been reported. Since the expression
of the enzyme is accompanied by the production of
a black pigment, the efficient supply of the biocatalyst, and thus biotechnological application, is hampered. However, Yazbik and Ansorge-Schumacher et al.
recently developed a novel purification strategy enabling an efficient supply of the enzyme for technical
applications [14.79].
Regioselective halogenation can be accomplished
by the application of flavin-dependent halogenases.
However, halogenation of positions 4, 5, 6, and 7 of
tryptophan-derived rings of natural products, as well
as halogenation of tyrosines at the ortho position,and
mono and di-chlorination of pyrroles are the preferred
reactions catalyzed by such enzymes [14.80, 81]. Interestingly, similarly to haloperoxidases, the flavin-dependent halogenase uses hypochlorite for halogenation.
However, unlike haloperoxidases, the generated hypohalous acid cannot leave the active site and can only
react with substrates at the active site, resulting in selective halogenation reactions. Halogenation of inactivated
carbon atoms can be accomplished by non-heme iron,
˛-ketoglutarate and O 2 -dependent halogenases, which
use a radical mechanism [14.82]. Up until now, the application of such enzymes for biocatalytic processes (in
vitro) is still hampered due to the restricted substrate
spectrum, as well as the required cofactor recycling.
Part B | 14.3
cell immobilization were also optimized for repeated
batch fermentation of the alkaline protease by T. turnirae. Ceramic support, the different sizes of broken
pumice stone, and silicone foam enhanced enzyme production by more than 200%, when compared to free
cells. Electron micrograph evident T. turnirae colonization in porous support matrices and five repeated
batch fermentation was achieved without significant decrease in the enzyme production. When the same strain
was immobilized in calcium alginate beads maximum
protease activity was achieved at 3 w=v % sodium alginate and 3% CaCl 2 concentrations with a 1 W 2 ratio
of cell and alginate. Protease production was independent of bead size but decreased significantly when the
beads were treated with glutaraldehyde. The immobilized cells were used for 8 cycles, each lasting 72h.
Protease production increased with an increased number of fermentation cycles and was maximized at the
fourth cycle when the enzyme activity reached about
3:5 times more than that of the first cycle.
14.3.3 Halogenating Enzymes
Halogenated molecules are widely distributed in the
environment and their existence is not only related
to human industrial processes but also to natural biotic as well as abiotic processes. To date almost 5000
organohalogens are known. Most of them contain chlorine or bromine rather than fluorine or iodine [14.68].
With respect to the relatively high concentration of
bromide found in the marine environment, brominated
compounds are found quite often and occur in a larger
number than in freshwater samples [14.69, 70].
While establishing a halogen–carbon bond, several
properties of the compound are changed. The following two effects are the most important: a) the introduction of halogen increases the molecule’s thermal
and oxidative activity, and b) increases the biological
membrane permeability [14.71]. The halogenated compounds currently manufactured by the industry can be
discriminated into bulk chemicals and higher valueadded pharmaceutical compounds, where it is estimated
that 20% of all pharmaceutical small drugs and approximately 30% of all active compounds in agrochemistry
are halogenated [14.71]. Considering the problems of
chemical halogenation, such as low yield, lack of sensitivity, and the use of harmful/toxic chemicals, there is
an increasing demand for less toxic and highly specific
halogenation processes. The latter is of particular interest for the production of halogenated pharmaceuticals.
However, halogenated compounds are also of interest
as intermediates in organic synthesis. Since they can
be used as reactant within palladium-catalyzed coupling reactions, novel synthetic routes are accessible for
manufacturing of organic fine chemicals.
The formulation of natural halogenated products
occurs via abiotic and biotic processes. Particular enzymes are responsible for the biogenous halogenation
process. However, halogenation can be accomplished
by several types of enzymes featuring distinct substrate
specificity. One can roughly distinguish the enzymes
as haloperoxidases, flavin-dependent halogenases, nonheme iron halogenases, and the fluorinase/chlorinase
system.
Haloperoxidases are versatile enzymes for transformation of many substrates. Most prominent is
the chloroperoxidase from Caldariomyces fumago.
Whereas the enzyme is more or less even unspecific
with regard to the substrate, it can be beneficially applied to numerous biotranformations. Reactions such
as epoxidations [14.72, 73], enatioselective sulfoxidations [14.74], N-oxidation of arylamines [14.75], hydroxylation of side chains of aromatic systems [14.76],
oxidation of indole to oxyindole [14.77], or oxidation
of primary alcohols to their corresponding aldehydes [14.78] have been reported. Since the expression
of the enzyme is accompanied by the production of
a black pigment, the efficient supply of the biocatalyst, and thus biotechnological application, is hampered. However, Yazbik and Ansorge-Schumacher et al.
recently developed a novel purification strategy enabling an efficient supply of the enzyme for technical
applications [14.79].
Regioselective halogenation can be accomplished
by the application of flavin-dependent halogenases.
However, halogenation of positions 4, 5, 6, and 7 of
tryptophan-derived rings of natural products, as well
as halogenation of tyrosines at the ortho position,and
mono and di-chlorination of pyrroles are the preferred
reactions catalyzed by such enzymes [14.80, 81]. Interestingly, similarly to haloperoxidases, the flavin-dependent halogenase uses hypochlorite for halogenation.
However, unlike haloperoxidases, the generated hypohalous acid cannot leave the active site and can only
react with substrates at the active site, resulting in selective halogenation reactions. Halogenation of inactivated
carbon atoms can be accomplished by non-heme iron,
˛-ketoglutarate and O 2 -dependent halogenases, which
use a radical mechanism [14.82]. Up until now, the application of such enzymes for biocatalytic processes (in
vitro) is still hampered due to the restricted substrate
spectrum, as well as the required cofactor recycling.