Marine Sponges – Molecular Biology and Biotechnology 8.6 Metagenomic Strategies for Natural Product Discovery 235
Part A | 8.6
this approach two proteases have been identified from
the aforementioned Haliclona simulans library [8.255],
while a zinc-dependent metalloprotease has also been
isolated from a deep-sea sediment metagenomic library
using this screen [8.256]. Interestingly, this enzyme also
displayed an ability to hydrolyze both fibrin and azocasein, thereby raising the possibility of its utility as
a potential therapeutic agent in the treatment of thrombosis. A novel subtilisin-like serine protease has also recently been cloned from an Antarctic coastal sediment
metagenomic library [8.257]. This protease displayed
good thermostability with an optimum temperature of
60
ı C with approximately 73% of its activity being retained after incubation at 50
ı C for 2 h.
The fluorogenic analog of chitin 4-methylumbelliferyl ˇ-D-N,N
0 -diacetylchitobioside (MUF-diNAG)
can also been successfully employed in function-based
screens to identify chitinases in marine metagenomic
libraries; it has been successfully employed to identify
two cloned chitinase genes from coastal seawater
metagenomic samples [8.242].
Metagenomic clones displaying cellulase activity
can be detected by using colorimetric assays involving
a cellulosic substrate. An example is the dye Congo red,
which interacts with intact ˇ-D-glucans and can thus
be used to screen for ˇ-D-glucan-hydrolase activity.
Cellulolytic metagenomic clones can be identified on
carboxymethylcellulose containing Luria Bertani (LB)
and subsequently staining with Congo red. Metagenomic clones exhibiting activity can be visualized due
to the formation of a yellow halo, which is due to
the formation of the dye-glucan complex (Fig. 8.6).
While a large number of cellulases have to date been
isolated from metagenomic libraries constructed from
different terrestrial and in particular mammalian gut
microbiomes [8.258], to the authors’ knowledge there
are no reports to date of cellulases being isolated
from marine metagenomic libraries. This is surprising,
given the recent increased interest in this family of enzymes from a bioenergy perspective, particularly with
respect to production strategies involving lignocellulose containing plant material/biomass, coupled with
the fact that a number of cellulases have been isolated from marine bacteria, including a Marinobacter
strain associated with the marine sponge Dendrilla nigra. Therefore, it appears highly likely that marine
sponge metagenomic libraries do contain large numbers of novel cellulase genes; a fact reinforced by the
recent cloning of a novel cellulase from a metagenomic library constructed from the gut microflora of
abalone [8.259].
Laccases are a family of blue multicopper oxidases,
which are mostly known from fungi but which have
been found in all domains of life. Bound copper atoms
catalyze the oxidation of aromatic compounds while
reducing molecular oxygen to water. Fungal laccases
are known to play a key role in lignin degradation,
while bacterial laccases are involved in the production
of spore coat pigment production for UV production
and in copper homeostasis. Laccases possess significant
biotechnological potential, including degradation and
detoxification of industrial dyes, particularly azo dyes
which are used extensively in the textile industry and
are thus present in many wastewater discharges. Laccases are known to degrade azo dyes and thus constitute
a promising, environmentally friendly method for the
treatment of azo dye containing waste streams. These
discharges are typically alkaline in nature and contain
high concentrations of salts such as NaCl, which limits
the use of fungal laccases. Thus, there is an increased
interest in new laccases from marine sources, particularly of bacterial origin, given that bacterial laccases
have been shown to possess excellent activity under alkaline conditions and to be very salt tolerant [8.260].
A number of functional screens are thus potentially
available for the detection of laccase activity in marine metagenomic libraries. These include the polyphenol dye Remazol Brilliant Blue R (RBBR), guiacol
and 2
0 ,2
0 -azino-bis(3-ethylbenzothiazoline-6-sulphonic
acid) (ABTS). RBBR is typically incorporated into
standard growth media such as LB at a final concentration of 0:04 w=v %, together with 250 M copper chloride (CuCl 2 ). The copper chloride is added as laccases
require copper ions to work and activity is indicated
by a clear or brown halo around the colony displaying
the activity. Guaicol C 6 H 4 .OH/.OCH 3 / is a colorless
natural organic compound which when oxidized by
laccases produces an amber color. The inclusion of
guaicol at levels of around (0:01 w=v %) in standard
bacterial growth media coupled with the addition of
copper chloride (250 M) ensures the ready detection
of laccase activity. ABTS can also be employed to
functionally screen for laccase activity. ABTS is an aromatic compound, which can be used to determine the
antioxidant capacities of foodstuffs. When ABTS is incorporated to standard bacterial growth at a concentration of 1 mM, together with copper chloride (250 M),
laccase activity can be detected by the appearance
of a green-blue color. Finally, syringaldazine [8.N,N
0 -
bis (3,5-dimethoxy-4-hydroxybenzylidene) hydrazine]
is also a good a good substrate that can be used to
detect laccase activity. The incorporation of syringal-
Part A | 8.6
this approach two proteases have been identified from
the aforementioned Haliclona simulans library [8.255],
while a zinc-dependent metalloprotease has also been
isolated from a deep-sea sediment metagenomic library
using this screen [8.256]. Interestingly, this enzyme also
displayed an ability to hydrolyze both fibrin and azocasein, thereby raising the possibility of its utility as
a potential therapeutic agent in the treatment of thrombosis. A novel subtilisin-like serine protease has also recently been cloned from an Antarctic coastal sediment
metagenomic library [8.257]. This protease displayed
good thermostability with an optimum temperature of
60
ı C with approximately 73% of its activity being retained after incubation at 50
ı C for 2 h.
The fluorogenic analog of chitin 4-methylumbelliferyl ˇ-D-N,N
0 -diacetylchitobioside (MUF-diNAG)
can also been successfully employed in function-based
screens to identify chitinases in marine metagenomic
libraries; it has been successfully employed to identify
two cloned chitinase genes from coastal seawater
metagenomic samples [8.242].
Metagenomic clones displaying cellulase activity
can be detected by using colorimetric assays involving
a cellulosic substrate. An example is the dye Congo red,
which interacts with intact ˇ-D-glucans and can thus
be used to screen for ˇ-D-glucan-hydrolase activity.
Cellulolytic metagenomic clones can be identified on
carboxymethylcellulose containing Luria Bertani (LB)
and subsequently staining with Congo red. Metagenomic clones exhibiting activity can be visualized due
to the formation of a yellow halo, which is due to
the formation of the dye-glucan complex (Fig. 8.6).
While a large number of cellulases have to date been
isolated from metagenomic libraries constructed from
different terrestrial and in particular mammalian gut
microbiomes [8.258], to the authors’ knowledge there
are no reports to date of cellulases being isolated
from marine metagenomic libraries. This is surprising,
given the recent increased interest in this family of enzymes from a bioenergy perspective, particularly with
respect to production strategies involving lignocellulose containing plant material/biomass, coupled with
the fact that a number of cellulases have been isolated from marine bacteria, including a Marinobacter
strain associated with the marine sponge Dendrilla nigra. Therefore, it appears highly likely that marine
sponge metagenomic libraries do contain large numbers of novel cellulase genes; a fact reinforced by the
recent cloning of a novel cellulase from a metagenomic library constructed from the gut microflora of
abalone [8.259].
Laccases are a family of blue multicopper oxidases,
which are mostly known from fungi but which have
been found in all domains of life. Bound copper atoms
catalyze the oxidation of aromatic compounds while
reducing molecular oxygen to water. Fungal laccases
are known to play a key role in lignin degradation,
while bacterial laccases are involved in the production
of spore coat pigment production for UV production
and in copper homeostasis. Laccases possess significant
biotechnological potential, including degradation and
detoxification of industrial dyes, particularly azo dyes
which are used extensively in the textile industry and
are thus present in many wastewater discharges. Laccases are known to degrade azo dyes and thus constitute
a promising, environmentally friendly method for the
treatment of azo dye containing waste streams. These
discharges are typically alkaline in nature and contain
high concentrations of salts such as NaCl, which limits
the use of fungal laccases. Thus, there is an increased
interest in new laccases from marine sources, particularly of bacterial origin, given that bacterial laccases
have been shown to possess excellent activity under alkaline conditions and to be very salt tolerant [8.260].
A number of functional screens are thus potentially
available for the detection of laccase activity in marine metagenomic libraries. These include the polyphenol dye Remazol Brilliant Blue R (RBBR), guiacol
and 2
0 ,2
0 -azino-bis(3-ethylbenzothiazoline-6-sulphonic
acid) (ABTS). RBBR is typically incorporated into
standard growth media such as LB at a final concentration of 0:04 w=v %, together with 250 M copper chloride (CuCl 2 ). The copper chloride is added as laccases
require copper ions to work and activity is indicated
by a clear or brown halo around the colony displaying
the activity. Guaicol C 6 H 4 .OH/.OCH 3 / is a colorless
natural organic compound which when oxidized by
laccases produces an amber color. The inclusion of
guaicol at levels of around (0:01 w=v %) in standard
bacterial growth media coupled with the addition of
copper chloride (250 M) ensures the ready detection
of laccase activity. ABTS can also be employed to
functionally screen for laccase activity. ABTS is an aromatic compound, which can be used to determine the
antioxidant capacities of foodstuffs. When ABTS is incorporated to standard bacterial growth at a concentration of 1 mM, together with copper chloride (250 M),
laccase activity can be detected by the appearance
of a green-blue color. Finally, syringaldazine [8.N,N
0 -
bis (3,5-dimethoxy-4-hydroxybenzylidene) hydrazine]
is also a good a good substrate that can be used to
detect laccase activity. The incorporation of syringal-