Part B | 14.6
424 Part B Tools and Methods in Marine Biotechnology
prospective traits such as high activity even at low temperatures, as well as activity in a broad range of pH
values (e.g., [14.99, 100]). Hence the hydrolysis may
be performed at low temperatures and thermal enzyme
deactivation is minimized. Another approach is the socalled electroextraction, a technique which so far has
not been applied for recovery of intracellular enzymes
from marine microorganisms, although its potential has
been proven, mainly for yeasts (e.g., [14.101]). Electroextraction is based on electro-induced changes in the
cell envelope, which result in a release of intracellular
proteins without formation of debris.
The diluted enzyme is subsequently concentrated by
means of precipitation (e.g., with ammonium sulfate)
or ultrafiltration. The unspecific concentration of proteins is followed by chromatographic steps, whereby
the enzyme is separated from the impurities according to its charge, size, or hydrophobicity, or due to
specific groups of the target enzyme. Analog separation principles are provided by membrane adsorbers.
Such devices feature some advantages in comparison
to conventional column chromatography. These can be
summarized as follows: lower manufacturing costs, no
diffusion-controlled exchange kinetics, easier handling,
and up-scaling [14.102]. A membrane adsorber is based
on a stationary, modified microporous matrix allowing high fluxes and maximization of the mass transfer,
thus limiting diffusive transport phenomena as they
occur in column chromatography can be minimized.
Additionally, the compressibility can be neglected if
the module is applied under moderate pressure. Membrane adsorbers are commercially available and can
be purchased as ion-exchangers with acidic (e.g., sulfonic acid, or carboxylic acid), basic (e.g., quaternary
ammonium or diethylamine), or chelating membranes,
which are applicable for immobilized metal affinity
chromatography. One example for a successful application of a cation-exchange membrane adsorber for
recovery of a marine enzyme was reported by Muffler and Ulber [14.103]. They established a fed-batch
process for optimizing the enzyme expression of the
intracellular produced sulfite oxidase by Sulfitobacter
pontiacus. The supernatant obtained after ultrasonication of the suspended biomass and centrifugation of the
cell debris was directly applied to a cation-exchange
membrane adsorber. Thus the specific activity within
the pooled eluate fraction was increased about a factor
of 8 compared to the crude extract. A further increase
about a factor of 2:6 of the specific activity was finally
achieved by ultrafiltration.
If applicable, affinity chromatography is the method
of choice, because this technique relies on the highly
specific binding interactions of the target enzyme and
the stationary matrix. However, the target enzyme must
feature a characteristic subunit or subgroup which can
be used for binding to an affinity matrix. A successful application of this technique necessitates that such
binding-groups are absent in the co-expressed proteins. Therefore, affinity chromatography is usually
applied with recombinant enzymes rather than native
enzymes, because the relevant groups, such as, for
example, a histidine tag, a chitin binding tag, or maltose binding tag are linked to the native sequence of
the protein, thus allowing a subsequent gentle purification of the recombinant protein. For instance, Kang
et al. expressed an epoxide hydrolase of Sphingophyxis
alaskensis origin in E. coli and purified the heterologously expressed protein by metal affinity chromatography via his-tag [14.104]. Another binding method
was applied by Han et al., who expressed a chitinase with antifungal activity from marine Streptomyces
sp.DA11 by E. coli and purified the chitinase, showing an antifungal activity by 80% ammonium sulfate,
affinity binding to chitin, and diethylaminoethyl-cellulose anion-exchange chromatography [14.52]. However, affinity chromatography had already been successfully used for purification of native marine proteins.
Bertau et al., for example, reported the purification
and characterization of an ˛-L-fucosidase by application of the glycosidase inhibitor analogue 6-aminodeoxymannojirimycin as ligand for the saccharolytic
enzyme [14.105].
14.6 Conclusion
Marine microorganisms can be considered as a powerful resource of enzymes with special traits which are
often different from their terrestrial counterparts. In
particular, enzymes from extremophiles can be valuable in a broad variety of processes. Enzymes from
cryophilic microorganisms are often active at low temperatures and may contribute to, for example, energy
savings in laundry processes (cryophilic lipases or
proteases). Such enzymes may also contribute to increase the quality of food because processing can be
424 Part B Tools and Methods in Marine Biotechnology
prospective traits such as high activity even at low temperatures, as well as activity in a broad range of pH
values (e.g., [14.99, 100]). Hence the hydrolysis may
be performed at low temperatures and thermal enzyme
deactivation is minimized. Another approach is the socalled electroextraction, a technique which so far has
not been applied for recovery of intracellular enzymes
from marine microorganisms, although its potential has
been proven, mainly for yeasts (e.g., [14.101]). Electroextraction is based on electro-induced changes in the
cell envelope, which result in a release of intracellular
proteins without formation of debris.
The diluted enzyme is subsequently concentrated by
means of precipitation (e.g., with ammonium sulfate)
or ultrafiltration. The unspecific concentration of proteins is followed by chromatographic steps, whereby
the enzyme is separated from the impurities according to its charge, size, or hydrophobicity, or due to
specific groups of the target enzyme. Analog separation principles are provided by membrane adsorbers.
Such devices feature some advantages in comparison
to conventional column chromatography. These can be
summarized as follows: lower manufacturing costs, no
diffusion-controlled exchange kinetics, easier handling,
and up-scaling [14.102]. A membrane adsorber is based
on a stationary, modified microporous matrix allowing high fluxes and maximization of the mass transfer,
thus limiting diffusive transport phenomena as they
occur in column chromatography can be minimized.
Additionally, the compressibility can be neglected if
the module is applied under moderate pressure. Membrane adsorbers are commercially available and can
be purchased as ion-exchangers with acidic (e.g., sulfonic acid, or carboxylic acid), basic (e.g., quaternary
ammonium or diethylamine), or chelating membranes,
which are applicable for immobilized metal affinity
chromatography. One example for a successful application of a cation-exchange membrane adsorber for
recovery of a marine enzyme was reported by Muffler and Ulber [14.103]. They established a fed-batch
process for optimizing the enzyme expression of the
intracellular produced sulfite oxidase by Sulfitobacter
pontiacus. The supernatant obtained after ultrasonication of the suspended biomass and centrifugation of the
cell debris was directly applied to a cation-exchange
membrane adsorber. Thus the specific activity within
the pooled eluate fraction was increased about a factor
of 8 compared to the crude extract. A further increase
about a factor of 2:6 of the specific activity was finally
achieved by ultrafiltration.
If applicable, affinity chromatography is the method
of choice, because this technique relies on the highly
specific binding interactions of the target enzyme and
the stationary matrix. However, the target enzyme must
feature a characteristic subunit or subgroup which can
be used for binding to an affinity matrix. A successful application of this technique necessitates that such
binding-groups are absent in the co-expressed proteins. Therefore, affinity chromatography is usually
applied with recombinant enzymes rather than native
enzymes, because the relevant groups, such as, for
example, a histidine tag, a chitin binding tag, or maltose binding tag are linked to the native sequence of
the protein, thus allowing a subsequent gentle purification of the recombinant protein. For instance, Kang
et al. expressed an epoxide hydrolase of Sphingophyxis
alaskensis origin in E. coli and purified the heterologously expressed protein by metal affinity chromatography via his-tag [14.104]. Another binding method
was applied by Han et al., who expressed a chitinase with antifungal activity from marine Streptomyces
sp.DA11 by E. coli and purified the chitinase, showing an antifungal activity by 80% ammonium sulfate,
affinity binding to chitin, and diethylaminoethyl-cellulose anion-exchange chromatography [14.52]. However, affinity chromatography had already been successfully used for purification of native marine proteins.
Bertau et al., for example, reported the purification
and characterization of an ˛-L-fucosidase by application of the glycosidase inhibitor analogue 6-aminodeoxymannojirimycin as ligand for the saccharolytic
enzyme [14.105].
14.6 Conclusion
Marine microorganisms can be considered as a powerful resource of enzymes with special traits which are
often different from their terrestrial counterparts. In
particular, enzymes from extremophiles can be valuable in a broad variety of processes. Enzymes from
cryophilic microorganisms are often active at low temperatures and may contribute to, for example, energy
savings in laundry processes (cryophilic lipases or
proteases). Such enzymes may also contribute to increase the quality of food because processing can be