Part B | 14.5
422 Part B Tools and Methods in Marine Biotechnology
A kinetic study of a Streptomyces sp. derived and
recombinantly expressed flavin-dependent tryptophan5-halogenase (including a cofactor regeneration) was
also reported by Muffler et al. [14.83]. The study was
accomplished due to the fact that halogenated tryptophan may serve as a potential serotonin precursor. By
application of the FDH/formiate-system for cofactor
regeneration, the space–time yield of the halogenation was improved by a factor of 1:6. Furthermore,
Muffler et al. applied a genetic algorithm approach to
optimize the enzyme’s performance and the conditions
for the biocatalytic process, whereby the yield of the
halogenated product improved significantly from 3:5 to
65% [14.84].
Fluorinase and chlorinase have a commonality in
that they mediate nucleophilic reactions of their respective halide ions to the C-5
0 carbon of S-adenosyl-Lmethionine (SAM) [14.85]. Biological fluorination is of
tremendous interest, since it might help to overcome the
problems associated with chemical fluorination. However, more basic research is needed to characterize the
potential of these types of enzymes.
14.4 Molecular Biology
Marine-derived enzymes are inevitable in modern
molecular biology research. Polymerase chain reaction
(PCR) is a technique for continuous in vitro DNA synthesis by use of DNA polymerases. It is widely used
for gene amplification, site directed mutagenesis, DNA
sequencing, and diagnostic purposes. The reaction mixture needs to be treated at high temperature, which
causes inactivation of the DNA polymerase [14.86].
It is essential to add fresh enzymes at each reaction
cycle, and it was not possible to use the technique
widely before discovery of the thermostable DNA polymerase. Today, two marine-derived thermostable DNA
polymerases are at the center of this technique. Vent
polymerase (New England Biolab) from Thermococcus
litoralis and Pfu polymerase (Stratagene) from Pyrococcus furiosus are widely used DNA polymerases for
this purpose. These enzymes show a better performance
than the previously used Taq polymerase; they have
a lower error rate than the Taq polymerase and also
have a proofreading ability due to 3
0 –5
0 exonuclease
activity [14.87–89]. DNA ligase from Thermal thermophillus is another important marine enzyme used
in molecular biology research [14.90, 91]. Marine microorganisms are also an important source of restriction
endonucleases. AspMD1 is an isoschizomer of Sau3AI
that was purified from the marine isolate Alcaligenes
sp. MD1 [14.92]. A novel restriction enzyme DmaI
was isolated from the marine bacterium Deleya marina
IAM 14 114 [14.93]. This enzyme is moderately thermostable and recognizes the hexanucleotide sequence
5
0 -CAGCTG-3
0 to produce the blunt-ended digestion
product. Several other restriction endonucleases, including DpaI, AgeI, HjaI, Hac1, Hsa1, and Hag1 have
been isolated from marine bacteria. All these enzymes
are potential tools for double-stranded DNA digestion.
DNA topoisomerases play important roles in replication, transcription and gene expression by controlling
DNA topology. Several DNA topoisomerases have been
isolated from marine bacteria, including Sulfolobus acidocalcarius, Desulfurococcus amylolyticus, Thermus
acidophilum, Fervidobacterium islandicum, Thermus
maritima, and Methanopyrus kandleri. [14.94]. Some
of these are characterized for their potential application
in biotechnological research.
14.5 Downstream Processing of Marine Enzymes
Capturing and purification of a marine enzyme is a
very challenging task, which is commonly addressed
to a minor extent in comparison to the fermentation
process. But both steps are directly linked together,
since efficient downstream processing requires a minimum of the target compound otherwise every isolation
strategy fails. Furthermore, the fermentation medium
should contain only small amounts of supplements
which might interfere with the purification strategy of
the target enzyme. Thus, optimization of the cultivation process with regard to the yield of the product
should be accomplished prior to establishing a downstream technique. This optimization should also focus
on a minimization of by-product formation, as these
must be separated from the product. Moreover, the production of (unwanted) side products also affects the
422 Part B Tools and Methods in Marine Biotechnology
A kinetic study of a Streptomyces sp. derived and
recombinantly expressed flavin-dependent tryptophan5-halogenase (including a cofactor regeneration) was
also reported by Muffler et al. [14.83]. The study was
accomplished due to the fact that halogenated tryptophan may serve as a potential serotonin precursor. By
application of the FDH/formiate-system for cofactor
regeneration, the space–time yield of the halogenation was improved by a factor of 1:6. Furthermore,
Muffler et al. applied a genetic algorithm approach to
optimize the enzyme’s performance and the conditions
for the biocatalytic process, whereby the yield of the
halogenated product improved significantly from 3:5 to
65% [14.84].
Fluorinase and chlorinase have a commonality in
that they mediate nucleophilic reactions of their respective halide ions to the C-5
0 carbon of S-adenosyl-Lmethionine (SAM) [14.85]. Biological fluorination is of
tremendous interest, since it might help to overcome the
problems associated with chemical fluorination. However, more basic research is needed to characterize the
potential of these types of enzymes.
14.4 Molecular Biology
Marine-derived enzymes are inevitable in modern
molecular biology research. Polymerase chain reaction
(PCR) is a technique for continuous in vitro DNA synthesis by use of DNA polymerases. It is widely used
for gene amplification, site directed mutagenesis, DNA
sequencing, and diagnostic purposes. The reaction mixture needs to be treated at high temperature, which
causes inactivation of the DNA polymerase [14.86].
It is essential to add fresh enzymes at each reaction
cycle, and it was not possible to use the technique
widely before discovery of the thermostable DNA polymerase. Today, two marine-derived thermostable DNA
polymerases are at the center of this technique. Vent
polymerase (New England Biolab) from Thermococcus
litoralis and Pfu polymerase (Stratagene) from Pyrococcus furiosus are widely used DNA polymerases for
this purpose. These enzymes show a better performance
than the previously used Taq polymerase; they have
a lower error rate than the Taq polymerase and also
have a proofreading ability due to 3
0 –5
0 exonuclease
activity [14.87–89]. DNA ligase from Thermal thermophillus is another important marine enzyme used
in molecular biology research [14.90, 91]. Marine microorganisms are also an important source of restriction
endonucleases. AspMD1 is an isoschizomer of Sau3AI
that was purified from the marine isolate Alcaligenes
sp. MD1 [14.92]. A novel restriction enzyme DmaI
was isolated from the marine bacterium Deleya marina
IAM 14 114 [14.93]. This enzyme is moderately thermostable and recognizes the hexanucleotide sequence
5
0 -CAGCTG-3
0 to produce the blunt-ended digestion
product. Several other restriction endonucleases, including DpaI, AgeI, HjaI, Hac1, Hsa1, and Hag1 have
been isolated from marine bacteria. All these enzymes
are potential tools for double-stranded DNA digestion.
DNA topoisomerases play important roles in replication, transcription and gene expression by controlling
DNA topology. Several DNA topoisomerases have been
isolated from marine bacteria, including Sulfolobus acidocalcarius, Desulfurococcus amylolyticus, Thermus
acidophilum, Fervidobacterium islandicum, Thermus
maritima, and Methanopyrus kandleri. [14.94]. Some
of these are characterized for their potential application
in biotechnological research.
14.5 Downstream Processing of Marine Enzymes
Capturing and purification of a marine enzyme is a
very challenging task, which is commonly addressed
to a minor extent in comparison to the fermentation
process. But both steps are directly linked together,
since efficient downstream processing requires a minimum of the target compound otherwise every isolation
strategy fails. Furthermore, the fermentation medium
should contain only small amounts of supplements
which might interfere with the purification strategy of
the target enzyme. Thus, optimization of the cultivation process with regard to the yield of the product
should be accomplished prior to establishing a downstream technique. This optimization should also focus
on a minimization of by-product formation, as these
must be separated from the product. Moreover, the production of (unwanted) side products also affects the