Biocatalysts from Aplysia 30.5 Other Enzymatic Activities of Interest Present in Aplysia sp. 741
Part F | 30.5
acetone the half-life was 8:5 h). The K M and V max values for pNP-ˇ-Man were determined to be 2:4 mM and
50:3 U mg
1 , respectively. From the body of these and
other results, ˇ-D-mannosidase from Aplysia fasciata
was judged to possess a very good catalytic efficiency,
in fact the k cat =K M obtained is 4800 min
1 mM
1 ,
which is higher than those of analogous enzymes from
other species. This enzyme is also extremely specific
for pNP-ˇ-Man, a useful and needed characteristic
when using a glycosydase for structural determination purposes. Self-transfer of a ˇ-mannosyl unit was
observed when pNP-ˇ-man is treated with the ˇ-Dmannosidase, in fact the most abundant disaccharidic
product obtained (1015% yield) possessed the ˇ-1,4
intermannosydic linkage as established by the analysis
of mono and two-dimensional NMR spectra of products (pNP-ˇ-Man-(1,4)-ˇ-Man). The transmannosylation was also studied using pNP-ˇ-Man as donor and onitrophenyl ˛-D-2-deoxy-N-acetyl glucopyranoside as
heteroacceptor, the molar ratio used was 1 W 3. Two
regioisomers (12% global yield) due to the ˇ-mannosylation of the heteroacceptor were formed in 85 W
15 ratio (by high-performance liquid chromatography, HPLC). The products were the ˇ-1,4 and the ˇ1,6 derivatives as assessed by two-dimensional NMR
spectroscopy.
As stated above, the production of mannooligosaccharides from mannan is based on endo-ˇ-1,4-mannanase (EC 3.2.1.78) as biocatalyst. ˇ-mannanases
have already been established as useful biocatalysts in
various industrial processes [30.60]. Aplysia kurodai
was considered to use two mannan-degrading enzymes,
namely a ˇ-mannanase and a ˇ-mannosidase, for the
complete depolymerization of mannan from seaweeds
for nutritional purposes [30.61]. The ˇ-mannanase
acted on the linear structure of mannans, producing
heterooligosaccharide residues. It could degrade mannooligosaccharides larger than mannotetraose; from
M5 (mannopentaose) it produced M3 and M2, and from
M6 it produced M3, suggesting that the size of substrate
binding site is similar to the size of mannnopentaosemannohexaose, and splitting of the central mannosidic
linkage of the mannohexaose unit occurred [30.62]. ˇmannosidase and ˇ-mannanase acted on linear mannan, degrading it to mannose in a more efficient
manner [30.61]. Unfortunately, no results have been
reported on these interesting enzymes about their possible transmannosylation properties on heteroacceptors.
A bacterial expression system for this ˇ-mannanase
was reported to acquire information about the primary
structure and other characteristic properties. The recombinant protein showed a broad pH optimum in the
acidic range, as did native protein; however, heat stability was lower, indicating that the stability is derived
from an appropriate folding and/or some posttranslational modifications in Aplysia cells [30.63].
Aplysia kurodai also contains two alginate lyase
isozymes [30.64] that could be useful in the determination of fine structures of natural polymers and in
the bio-production of functional monosaccharides and
oligosaccharides from alginate. To improve the knowledge of primary structure and catalytic residues of
marine alginate lyases, cloning complementary DNA
(cDNA) encoding an alginate lyase from Aplysia kurodai and assessing catalytically important residues by
site-directed mutagenesis were also described [30.65].
Two ˇ-1,3-glucanases with the molecular masses of
36 kDa and 33 kDa were also isolated from the digestive fluid of Aplysia kurodai. They act as endolytic (EC
3.2.1.6, AkLam36) and exolytic enzymes (EC 3.2.1.58,
AkLam33) degrading laminarin and related oligosaccharides to laminaritriose, laminaribiose, and glucose
in the first case, while producing glucose directly from
polymer laminarin in the second case. Although both
these catalysts acted on laminaribiose, they were capable of degrading the disaccharide via transglycosylation
reaction with laminaritriose. Both enzymes belong to
the glycosyl hydrolase family 16 like other molluscan ˇ-1,3-glucanases. According to the authors it is
still unclear whether the transglycosylation is a physiologically important event in the metabolism since the
transglycosylation reaction requires a relatively high
concentration of laminaribiose [30.66].
Other enzymes related to chitin could be expected to
be present in organisms of the Aplysia species. It was,
in fact, long known in the literature that chitosan-based
radulae and jaws of the opisthobranch Aplysia sp. give
very strong chitosan color tests [30.67].
30.5 Other Enzymatic Activities of Interest Present in Aplysia sp.
Other examples of interesting enzymes have been reported in the literature on Aplysia sp. This marine
organism, in fact, has proved to be useful as a model
in neurobiology and in other scientific sectors. Some of
the enzymes cited in the present section may be of interest for biocatalyst practitioners and it appears to be
Part F | 30.5
acetone the half-life was 8:5 h). The K M and V max values for pNP-ˇ-Man were determined to be 2:4 mM and
50:3 U mg
1 , respectively. From the body of these and
other results, ˇ-D-mannosidase from Aplysia fasciata
was judged to possess a very good catalytic efficiency,
in fact the k cat =K M obtained is 4800 min
1 mM
1 ,
which is higher than those of analogous enzymes from
other species. This enzyme is also extremely specific
for pNP-ˇ-Man, a useful and needed characteristic
when using a glycosydase for structural determination purposes. Self-transfer of a ˇ-mannosyl unit was
observed when pNP-ˇ-man is treated with the ˇ-Dmannosidase, in fact the most abundant disaccharidic
product obtained (1015% yield) possessed the ˇ-1,4
intermannosydic linkage as established by the analysis
of mono and two-dimensional NMR spectra of products (pNP-ˇ-Man-(1,4)-ˇ-Man). The transmannosylation was also studied using pNP-ˇ-Man as donor and onitrophenyl ˛-D-2-deoxy-N-acetyl glucopyranoside as
heteroacceptor, the molar ratio used was 1 W 3. Two
regioisomers (12% global yield) due to the ˇ-mannosylation of the heteroacceptor were formed in 85 W
15 ratio (by high-performance liquid chromatography, HPLC). The products were the ˇ-1,4 and the ˇ1,6 derivatives as assessed by two-dimensional NMR
spectroscopy.
As stated above, the production of mannooligosaccharides from mannan is based on endo-ˇ-1,4-mannanase (EC 3.2.1.78) as biocatalyst. ˇ-mannanases
have already been established as useful biocatalysts in
various industrial processes [30.60]. Aplysia kurodai
was considered to use two mannan-degrading enzymes,
namely a ˇ-mannanase and a ˇ-mannosidase, for the
complete depolymerization of mannan from seaweeds
for nutritional purposes [30.61]. The ˇ-mannanase
acted on the linear structure of mannans, producing
heterooligosaccharide residues. It could degrade mannooligosaccharides larger than mannotetraose; from
M5 (mannopentaose) it produced M3 and M2, and from
M6 it produced M3, suggesting that the size of substrate
binding site is similar to the size of mannnopentaosemannohexaose, and splitting of the central mannosidic
linkage of the mannohexaose unit occurred [30.62]. ˇmannosidase and ˇ-mannanase acted on linear mannan, degrading it to mannose in a more efficient
manner [30.61]. Unfortunately, no results have been
reported on these interesting enzymes about their possible transmannosylation properties on heteroacceptors.
A bacterial expression system for this ˇ-mannanase
was reported to acquire information about the primary
structure and other characteristic properties. The recombinant protein showed a broad pH optimum in the
acidic range, as did native protein; however, heat stability was lower, indicating that the stability is derived
from an appropriate folding and/or some posttranslational modifications in Aplysia cells [30.63].
Aplysia kurodai also contains two alginate lyase
isozymes [30.64] that could be useful in the determination of fine structures of natural polymers and in
the bio-production of functional monosaccharides and
oligosaccharides from alginate. To improve the knowledge of primary structure and catalytic residues of
marine alginate lyases, cloning complementary DNA
(cDNA) encoding an alginate lyase from Aplysia kurodai and assessing catalytically important residues by
site-directed mutagenesis were also described [30.65].
Two ˇ-1,3-glucanases with the molecular masses of
36 kDa and 33 kDa were also isolated from the digestive fluid of Aplysia kurodai. They act as endolytic (EC
3.2.1.6, AkLam36) and exolytic enzymes (EC 3.2.1.58,
AkLam33) degrading laminarin and related oligosaccharides to laminaritriose, laminaribiose, and glucose
in the first case, while producing glucose directly from
polymer laminarin in the second case. Although both
these catalysts acted on laminaribiose, they were capable of degrading the disaccharide via transglycosylation
reaction with laminaritriose. Both enzymes belong to
the glycosyl hydrolase family 16 like other molluscan ˇ-1,3-glucanases. According to the authors it is
still unclear whether the transglycosylation is a physiologically important event in the metabolism since the
transglycosylation reaction requires a relatively high
concentration of laminaribiose [30.66].
Other enzymes related to chitin could be expected to
be present in organisms of the Aplysia species. It was,
in fact, long known in the literature that chitosan-based
radulae and jaws of the opisthobranch Aplysia sp. give
very strong chitosan color tests [30.67].
30.5 Other Enzymatic Activities of Interest Present in Aplysia sp.
Other examples of interesting enzymes have been reported in the literature on Aplysia sp. This marine
organism, in fact, has proved to be useful as a model
in neurobiology and in other scientific sectors. Some of
the enzymes cited in the present section may be of interest for biocatalyst practitioners and it appears to be