Marine Enzymes – Production and Applications 14.3 Examples of Marine Enzymes 419
Part B | 14.3
internal shell of cephalopods (squids and octopuses).
Most of these organisms live in the habitat of the marine ecosystem, and it stands to reason that the chitin
hydrolytic enzyme (chitinase) is abundant in the marine environment. Chitinolytic activity was reported in
several marine bacteria (pathogens) that attack living
arthropods or degrades the chitin-rich biomass derived
from their exoskeleton. Chitinase is used for the production of single cell proteins for animal and aquaculture
feed, for isolation of fungal protoplasts, and for the
preparation of chitooligosaccharides. Chitinases are not
widely used at commercial scale due to their high cost
but they have interesting potential applications in pest
control and preparation of medicines. Chitinase genes
can be used to develop chitinase-producing transgenic
plants that are resistant to insects and fungi. The cell
walls of some human pathogens including fungi, protozoa, and helminths are composed of chitin, which could
be hydrolyzed by microbial chitinases and thus, chitinase may be used for the treatment of these microbial
infections.
A chitinase C encoding gene from the marine isolate Salinivibrio costicola 5SM-1 strain was cloned
in the pGEM7Zf(C/ vector and overexpressed in E.
coli [14.46]. The protein was identified as an exochitinase that hydrolyzes colloidal chitin and produces
chitobiose as the major hydrolysis product. One catalytic domain and two chitin binding domains were
identified by amino acid sequence alignment. The complete loss of chitinase activity by mutation of three
conserved amino acid residues at the putative catalytic
site confirmed the location of the catalytic domain. Another chitinase C enzyme was reported from the marine
Alteromonas sp. strain O-7, and the catalytic domain
was predicted by a similar approach [14.47]. The same
research group also isolated the novel cold-adapted
chitinase B from this microorganism [14.48]. Gohel
et al. used the Plackett–Burman design to optimize media composition for chitinase production by the marine
strain of Pantoea dispersa [14.49]. In this optimized
media chitinase production increases by 4:21 fold in
comparison to the basal medium; also a 3:95-fold increase of endochitinase production and a 2:31-fold
increase of chitobiase production was reported in this
medium. In another study, the impact of process parameters was studied on extracellular chitinase production
by a Beauveria bassiana strain in solid-state fermentation using wheat bran [14.50]. The chitinolytic marine
fungus was isolated from marine sediments from the
south coast of India and characterized as a spore-forming alkaliphilic fungus. The initial moisture content
significantly affected the microbial growth rate and enzyme production. Maximum chitinase production was
achieved after the third day of fermentation using the
media containing wheat bran to a seawater ratio of 5 W 3
(w=v). Chitinase production by this fungus was also
optimized in solid-state fermentation using chitin-rich
prawn waste as nutrient [14.51]. The antifungal activity
of chitinase was reported from the marine Streptomyces
sp. DA11 isolated from a sponge from the South China
Sea [14.52]. Amino acid sequence alignment identified the 34 kDa protein as chitinase C. The antifungal
activity was confirmed against Aspergillus niger and
Candida albicans with a zone of inhibition of 10:98 ˙
0:49 mm and 10:48 ˙ 0:45 mm, respectively.
Agarases
Agarase is an important enzyme distributed in a wide
range of marine organisms. This enzyme has potential
application in the food, pharmaceutical, and cosmetics industries. It can also be used in microbiology
and molecular biology research. Two ˇ-agarase genes
(agaA and agaB) were isolated from the marine Pseudoalteromonas sp.CY24 [14.53, 54]. The agaB gene
was cloned and overexpressed in E. coli and the
50:8 kDa protein was characterized. This enzyme degrades agarose to generate neoagarooctaose and neoagarodecaose as the major products but cannot hydrolyze
carrageenan, alginate, or chitosan. The substrate binding site and the mechanism of hydrolysis of this enzyme
were predicted by analysis of the enzyme kinetics,
the degradation pattern of different oligosaccharides,
and
1 H-NMR analysis. The enzyme has a large substrate binding cleft and it appears to be structurally and
functionally different from other known agarases. It hydrolyzes glycosidic bonds with inversion of anomeric
configuration, in contrast to other known agarases that
retain the anomeric configuration. On the other hand,
the agaA gene encodes a 48:4 kDa protein corresponding to a catalytic domain of the glycosyl hydrolase family 16 and a carbohydrate-binding module type 13. It is
an endo-type agarase that hydrolyzes ˇ-1,4-linkages of
agarose to yield neoagarotetraose and neoagarohexaose
as the end products. Another ˇ-agarase producing
gene (agrP) was isolated from the marine Pseudoalteromonas sp. AG4 and was cloned and overexpressed
in E. coli [14.55]. Sequence similarity suggests that this
enzyme is a member of the glycosyl hydrolase family
16. It produces neoagarotetraose and neoagarohexaose
from agar and additional neoagarobiose by hydrolysis
of agarose. A ˇ-agarase gene from the agarolytic marine bacterium Agarivorans sp. HZ105 showed 98:6%
Part B | 14.3
internal shell of cephalopods (squids and octopuses).
Most of these organisms live in the habitat of the marine ecosystem, and it stands to reason that the chitin
hydrolytic enzyme (chitinase) is abundant in the marine environment. Chitinolytic activity was reported in
several marine bacteria (pathogens) that attack living
arthropods or degrades the chitin-rich biomass derived
from their exoskeleton. Chitinase is used for the production of single cell proteins for animal and aquaculture
feed, for isolation of fungal protoplasts, and for the
preparation of chitooligosaccharides. Chitinases are not
widely used at commercial scale due to their high cost
but they have interesting potential applications in pest
control and preparation of medicines. Chitinase genes
can be used to develop chitinase-producing transgenic
plants that are resistant to insects and fungi. The cell
walls of some human pathogens including fungi, protozoa, and helminths are composed of chitin, which could
be hydrolyzed by microbial chitinases and thus, chitinase may be used for the treatment of these microbial
infections.
A chitinase C encoding gene from the marine isolate Salinivibrio costicola 5SM-1 strain was cloned
in the pGEM7Zf(C/ vector and overexpressed in E.
coli [14.46]. The protein was identified as an exochitinase that hydrolyzes colloidal chitin and produces
chitobiose as the major hydrolysis product. One catalytic domain and two chitin binding domains were
identified by amino acid sequence alignment. The complete loss of chitinase activity by mutation of three
conserved amino acid residues at the putative catalytic
site confirmed the location of the catalytic domain. Another chitinase C enzyme was reported from the marine
Alteromonas sp. strain O-7, and the catalytic domain
was predicted by a similar approach [14.47]. The same
research group also isolated the novel cold-adapted
chitinase B from this microorganism [14.48]. Gohel
et al. used the Plackett–Burman design to optimize media composition for chitinase production by the marine
strain of Pantoea dispersa [14.49]. In this optimized
media chitinase production increases by 4:21 fold in
comparison to the basal medium; also a 3:95-fold increase of endochitinase production and a 2:31-fold
increase of chitobiase production was reported in this
medium. In another study, the impact of process parameters was studied on extracellular chitinase production
by a Beauveria bassiana strain in solid-state fermentation using wheat bran [14.50]. The chitinolytic marine
fungus was isolated from marine sediments from the
south coast of India and characterized as a spore-forming alkaliphilic fungus. The initial moisture content
significantly affected the microbial growth rate and enzyme production. Maximum chitinase production was
achieved after the third day of fermentation using the
media containing wheat bran to a seawater ratio of 5 W 3
(w=v). Chitinase production by this fungus was also
optimized in solid-state fermentation using chitin-rich
prawn waste as nutrient [14.51]. The antifungal activity
of chitinase was reported from the marine Streptomyces
sp. DA11 isolated from a sponge from the South China
Sea [14.52]. Amino acid sequence alignment identified the 34 kDa protein as chitinase C. The antifungal
activity was confirmed against Aspergillus niger and
Candida albicans with a zone of inhibition of 10:98 ˙
0:49 mm and 10:48 ˙ 0:45 mm, respectively.
Agarases
Agarase is an important enzyme distributed in a wide
range of marine organisms. This enzyme has potential
application in the food, pharmaceutical, and cosmetics industries. It can also be used in microbiology
and molecular biology research. Two ˇ-agarase genes
(agaA and agaB) were isolated from the marine Pseudoalteromonas sp.CY24 [14.53, 54]. The agaB gene
was cloned and overexpressed in E. coli and the
50:8 kDa protein was characterized. This enzyme degrades agarose to generate neoagarooctaose and neoagarodecaose as the major products but cannot hydrolyze
carrageenan, alginate, or chitosan. The substrate binding site and the mechanism of hydrolysis of this enzyme
were predicted by analysis of the enzyme kinetics,
the degradation pattern of different oligosaccharides,
and
1 H-NMR analysis. The enzyme has a large substrate binding cleft and it appears to be structurally and
functionally different from other known agarases. It hydrolyzes glycosidic bonds with inversion of anomeric
configuration, in contrast to other known agarases that
retain the anomeric configuration. On the other hand,
the agaA gene encodes a 48:4 kDa protein corresponding to a catalytic domain of the glycosyl hydrolase family 16 and a carbohydrate-binding module type 13. It is
an endo-type agarase that hydrolyzes ˇ-1,4-linkages of
agarose to yield neoagarotetraose and neoagarohexaose
as the end products. Another ˇ-agarase producing
gene (agrP) was isolated from the marine Pseudoalteromonas sp. AG4 and was cloned and overexpressed
in E. coli [14.55]. Sequence similarity suggests that this
enzyme is a member of the glycosyl hydrolase family
16. It produces neoagarotetraose and neoagarohexaose
from agar and additional neoagarobiose by hydrolysis
of agarose. A ˇ-agarase gene from the agarolytic marine bacterium Agarivorans sp. HZ105 showed 98:6%