Part B | 14.2
414 Part B Tools and Methods in Marine Biotechnology
drug from the sea was the peptide ziconotide, originally
derived from a tropical marine cone snail. The compound was approved in the United States in 2004 under
the trade name Prialt for the treatment of chronic pain
in spinal cord injury [14.2]. A second drug (ET-743)
from sea squirt origin was approved by the European
Union in October 2007 for the treatment of soft-tissue
sarcoma [14.2]. However, further promising candidates
are still in several stages of clinical trials.
Today, natural product research also focuses on the
enzyme systems that are responsible for the production
of bioactive secondary metabolites, such as, for example, polyketide synthases [14.3, 4], which catalyzes
the linkage of acyl-coenzyme A subunits via the generation of cyclic structures (e.g., bryostatin 1). Aside
from the natural product research some (novel) marine
enzymes, catalyzing a multitude of processes relevant
for biocatalytic processes in industrial biotechnology,
showing desired properties like high salt tolerance,
hyperthermostability, barophilicity and cold adaptivity
as mentioned before. Their performance results from
the adaptation of their host organisms during the evolutional development to the environmental surroundings. However, the rising demand for environmentallyfriendly and beneficial economical manufacturing of
bulk and fine chemicals requires the exploitation of
these enzymes for the specific and selective production of value-added products like chiral amines, alcohols, halogenated amino acids, as well as the enzymes
themselves in the case of, for example, thermostable
proteases or polymerases. Thus, marine microorganisms may contribute to expanding the number of (even
more) stable and selective biocatalysts for industrial
biotransformations. One major concern with establishing these enzymes in industrial-scale applications is
their sustainable access. With respect to the difficulties of enzyme expression or cultivation of the natural
expression system when transferred from its originally
habitat to laboratory conditions, the responsible gene or
even the gene cluster must be heterologously expressed.
This approach requires identification, isolation, characterization, and cloning of the responsible genes in
adequate host organisms (considering the codon usage
of marine microorganisms), as well as optimization of
enzyme stability and enzyme expression. In addition
to microorganisms such as bacteria or fungi, higher
organisms such as fish, prawns, crabs, snakes, plants,
and algae can also be used for tapping of marine enzymes [14.5]. In particular the latter one represents
a mentionable source for a number of halogenating
enzymes.
The following chapters give an overview about
handling and cultivation of the marine enzymes’ hosts
as well as expression of the biocatalysts. Moreover,
some examples of enzymes from marine origin that
are or may be of relevance for biotransformation processes are presented. In the main focus are saccharolytic enzymes such as amylases, cellulases, chitinases, agarases, as well as proteolytic enzymes, DNA
polymerases, and halogenating enzymes. Since the supply of the biocatalyst depends on the expression and
the applied downstream process, the final section of this
review covers current purification techniques for intracellularly and extracellularly expressed proteins.
14.2 Cultivation Techniques of Marine Bacteria
The first step in the supply of an enzyme is the cultivation of the naturally occurring marine producing organism, except for using a metagenome approach, which
is not addressed in this overview. Within the marine
ecosystem microorganisms such as bacteria or fungi
can be found in the free water column, in marine sediments, or in symbiotic associations (endosymbiontic
and surface associated) with higher marine organisms
even in the light and dark zones of the sea. Phototrophic
microorganisms like cyanobacteria and microalgae are,
however, primarily found within the zones affected by
the sunlight.
The transfer of marine organisms from their natural ecosystems and their reproduction under laboratory
conditions often fails, because a potential adaptation of
the organism to its natural habitat and its participation
in several symbiotic interactions with other organisms
is commonly not considered. By establishing as far as
possible similar environmental conditions to the marine
strain these problems may be overcome in laboratory
cultivations. Attention must be paid to the nutrient conditions for cultivation of the desired microorganism.
Since bacteria or fungi deriving from the free water column exhibit different nutrient requirements than such
organisms deriving from sediments or from the inner
tissue of invertebrates, no standard nutrient broth that
might be applied to support growth and production of
all microorganisms exists.
414 Part B Tools and Methods in Marine Biotechnology
drug from the sea was the peptide ziconotide, originally
derived from a tropical marine cone snail. The compound was approved in the United States in 2004 under
the trade name Prialt for the treatment of chronic pain
in spinal cord injury [14.2]. A second drug (ET-743)
from sea squirt origin was approved by the European
Union in October 2007 for the treatment of soft-tissue
sarcoma [14.2]. However, further promising candidates
are still in several stages of clinical trials.
Today, natural product research also focuses on the
enzyme systems that are responsible for the production
of bioactive secondary metabolites, such as, for example, polyketide synthases [14.3, 4], which catalyzes
the linkage of acyl-coenzyme A subunits via the generation of cyclic structures (e.g., bryostatin 1). Aside
from the natural product research some (novel) marine
enzymes, catalyzing a multitude of processes relevant
for biocatalytic processes in industrial biotechnology,
showing desired properties like high salt tolerance,
hyperthermostability, barophilicity and cold adaptivity
as mentioned before. Their performance results from
the adaptation of their host organisms during the evolutional development to the environmental surroundings. However, the rising demand for environmentallyfriendly and beneficial economical manufacturing of
bulk and fine chemicals requires the exploitation of
these enzymes for the specific and selective production of value-added products like chiral amines, alcohols, halogenated amino acids, as well as the enzymes
themselves in the case of, for example, thermostable
proteases or polymerases. Thus, marine microorganisms may contribute to expanding the number of (even
more) stable and selective biocatalysts for industrial
biotransformations. One major concern with establishing these enzymes in industrial-scale applications is
their sustainable access. With respect to the difficulties of enzyme expression or cultivation of the natural
expression system when transferred from its originally
habitat to laboratory conditions, the responsible gene or
even the gene cluster must be heterologously expressed.
This approach requires identification, isolation, characterization, and cloning of the responsible genes in
adequate host organisms (considering the codon usage
of marine microorganisms), as well as optimization of
enzyme stability and enzyme expression. In addition
to microorganisms such as bacteria or fungi, higher
organisms such as fish, prawns, crabs, snakes, plants,
and algae can also be used for tapping of marine enzymes [14.5]. In particular the latter one represents
a mentionable source for a number of halogenating
enzymes.
The following chapters give an overview about
handling and cultivation of the marine enzymes’ hosts
as well as expression of the biocatalysts. Moreover,
some examples of enzymes from marine origin that
are or may be of relevance for biotransformation processes are presented. In the main focus are saccharolytic enzymes such as amylases, cellulases, chitinases, agarases, as well as proteolytic enzymes, DNA
polymerases, and halogenating enzymes. Since the supply of the biocatalyst depends on the expression and
the applied downstream process, the final section of this
review covers current purification techniques for intracellularly and extracellularly expressed proteins.
14.2 Cultivation Techniques of Marine Bacteria
The first step in the supply of an enzyme is the cultivation of the naturally occurring marine producing organism, except for using a metagenome approach, which
is not addressed in this overview. Within the marine
ecosystem microorganisms such as bacteria or fungi
can be found in the free water column, in marine sediments, or in symbiotic associations (endosymbiontic
and surface associated) with higher marine organisms
even in the light and dark zones of the sea. Phototrophic
microorganisms like cyanobacteria and microalgae are,
however, primarily found within the zones affected by
the sunlight.
The transfer of marine organisms from their natural ecosystems and their reproduction under laboratory
conditions often fails, because a potential adaptation of
the organism to its natural habitat and its participation
in several symbiotic interactions with other organisms
is commonly not considered. By establishing as far as
possible similar environmental conditions to the marine
strain these problems may be overcome in laboratory
cultivations. Attention must be paid to the nutrient conditions for cultivation of the desired microorganism.
Since bacteria or fungi deriving from the free water column exhibit different nutrient requirements than such
organisms deriving from sediments or from the inner
tissue of invertebrates, no standard nutrient broth that
might be applied to support growth and production of
all microorganisms exists.