Part B | 14.3
420 Part B Tools and Methods in Marine Biotechnology
sequence identity with a ˇ-agarase (agaB) from Vibrio sp. JT0107 [14.56]. The enzyme was overexpressed
in E. coli and identified as an endo-type ˇ-agarase.
It can hydrolyze neoagarooligosaccharides with a degree of oligomerization above four and produce neoagarotetraose as a dominant end product. Another endotype ˇ-agarase was reported from marine Agarivorans
sp. LQ48 [14.57]. This enzyme showed 73% sequence
similarity with the ˇ-agarase (AgaB) isolated from the
marine Pseudoalteromonas sp. CY24. Like AgaB this
enzyme also hydrolyzes ˇ-1,4-linkages of agarose and
produces neoagarotetraose and neoagarohexaose as the
main products. Notably, this agarase retains 95% of its
initial activity after 1 h incubation at any pH between
3:0 and 11:0 – such a wide range of pH-stability has
rarely been observed in any other agarase.
14.3.2 Proteases
Proteolytic enzymes account for above 60% of the
global enzyme market [14.58], and there is no doubt
that proteases are one of the most widely studied marine
enzymes. They are vastly used in the food processing, detergent,leather, and pharmaceutical industries,
and in biotechnological research. They have potential
applications in bioremediation and waste management.
The increased demand for industrial proteases over
its supply from plant and animal sources led to an
increased interest in microbial proteases. 70% of industrial proteases come from microbial sources. Alkaline
and neutral proteases are obtained mainly from bacterial sources, mostly from different species of Bacillus,
while fungi are a well-known source of acid proteases.
Marine microbial proteases gained attention for their
extremophilic properties and stability in the presence
of a broad range of chemicals. They possess almost all
the characteristics desired for various biotechnological
applications of proteases.
A novel alkaline serine protease was reported from
the marine fungus Engyodontium album BTMFS10
strain isolated from marine sediment from the west
coast of India [14.59, 60]. The extracellular enzyme
was produced by solid-state fermentation and the process parameters of protease production were optimized.
The enzyme was active over a broad range of pH values (612) and temperature ranges (1565
ı C) and
retained its activity in the presence of hydrocarbons,
natural oils, surfactants, and organic solvents. The optimum activity of the purified enzyme at a high temperature (60
ı C) and pH value (pH 11) suggest its
potential application in detergent industry. Sana et al.
reported another alkaline serine protease from a marine -proteobacterium isolated from the intertidal zone
of the Bay of Bengal [14.61]. The enzyme was tolerant to salt, solvent, detergent, and bleaching agents,
and showed potential for the detergent industry and dry
washing. A protease encoding gene was isolated from
Engyodontium album, which encodes a protein with
96% sequence similarity to proteinase R of Tritirarchium album [14.62]. A homology comparison of the
amino acid sequence revealed that this protease belongs
to the subtilase family of serine protease. A comparative homology modeling suggested a broad substrate
specificity of the enzyme. The enzyme structure was reported to be stabilized by two disulfide bonds and more
than two Ca
2C binding sites, which are assumed to contribute to the thermostability of the enzyme.
Surface-displaying recombinant alkaline proteases
were produced on a Yarrowia lipolytica cell surface
by overexpressing a marine-derived alkaline protease
gene [14.63]. This surface-displaying protease can hydrolyze various proteins for the production of bioactive
peptides. The peptides have potential pharmaceutical
applications due to their angiotensin-converting enzyme (ACE) inhibitory activity and antioxidant activity.
Peptides with high ACE inhibitory activity have wellknown potential application in the treatment of hypertension and other bioactive peptides can be used as
clinical nutrient supplements.
The production of a thermostable alkaline protease
by a marine Bacillus was optimized and scaled up
in a stirred tank bioreactor using cheap and readily
available substrates [14.64]. The authors studied the effects of environmental factors, carbon sources, nitrogen
sources, and fermentation parameters on the protease
production kinetics. Enzyme production increased with
prolonged fermentation time and faster agitation rate.
Maximum protease production was achieved after 40h
fermentation at 42
ı C with an aeration rate of 1:5 vvm
and constant agitation at 400 rpm in a soybean–casein
medium (pH 9:6). Protease production by the marine bacterium Teredinobacter turnirae was optimized
for solid-state fermentation as well by immobilization on solid matrixes [14.65–67]. Media composition,
initial pH, inoculum concentration, and the effect of
different salts and carbon sources were studied to optimize protease production by solid-state fermentation
in a soybean-based medium. The maximum protease
production was obtained using 1 w=v % soybean concentration, initial pH 7:34, and a 2:5 v=v % inoculum
level. The material and particle size of the immobilizing medium, and the method and conditions for
420 Part B Tools and Methods in Marine Biotechnology
sequence identity with a ˇ-agarase (agaB) from Vibrio sp. JT0107 [14.56]. The enzyme was overexpressed
in E. coli and identified as an endo-type ˇ-agarase.
It can hydrolyze neoagarooligosaccharides with a degree of oligomerization above four and produce neoagarotetraose as a dominant end product. Another endotype ˇ-agarase was reported from marine Agarivorans
sp. LQ48 [14.57]. This enzyme showed 73% sequence
similarity with the ˇ-agarase (AgaB) isolated from the
marine Pseudoalteromonas sp. CY24. Like AgaB this
enzyme also hydrolyzes ˇ-1,4-linkages of agarose and
produces neoagarotetraose and neoagarohexaose as the
main products. Notably, this agarase retains 95% of its
initial activity after 1 h incubation at any pH between
3:0 and 11:0 – such a wide range of pH-stability has
rarely been observed in any other agarase.
14.3.2 Proteases
Proteolytic enzymes account for above 60% of the
global enzyme market [14.58], and there is no doubt
that proteases are one of the most widely studied marine
enzymes. They are vastly used in the food processing, detergent,leather, and pharmaceutical industries,
and in biotechnological research. They have potential
applications in bioremediation and waste management.
The increased demand for industrial proteases over
its supply from plant and animal sources led to an
increased interest in microbial proteases. 70% of industrial proteases come from microbial sources. Alkaline
and neutral proteases are obtained mainly from bacterial sources, mostly from different species of Bacillus,
while fungi are a well-known source of acid proteases.
Marine microbial proteases gained attention for their
extremophilic properties and stability in the presence
of a broad range of chemicals. They possess almost all
the characteristics desired for various biotechnological
applications of proteases.
A novel alkaline serine protease was reported from
the marine fungus Engyodontium album BTMFS10
strain isolated from marine sediment from the west
coast of India [14.59, 60]. The extracellular enzyme
was produced by solid-state fermentation and the process parameters of protease production were optimized.
The enzyme was active over a broad range of pH values (612) and temperature ranges (1565
ı C) and
retained its activity in the presence of hydrocarbons,
natural oils, surfactants, and organic solvents. The optimum activity of the purified enzyme at a high temperature (60
ı C) and pH value (pH 11) suggest its
potential application in detergent industry. Sana et al.
reported another alkaline serine protease from a marine -proteobacterium isolated from the intertidal zone
of the Bay of Bengal [14.61]. The enzyme was tolerant to salt, solvent, detergent, and bleaching agents,
and showed potential for the detergent industry and dry
washing. A protease encoding gene was isolated from
Engyodontium album, which encodes a protein with
96% sequence similarity to proteinase R of Tritirarchium album [14.62]. A homology comparison of the
amino acid sequence revealed that this protease belongs
to the subtilase family of serine protease. A comparative homology modeling suggested a broad substrate
specificity of the enzyme. The enzyme structure was reported to be stabilized by two disulfide bonds and more
than two Ca
2C binding sites, which are assumed to contribute to the thermostability of the enzyme.
Surface-displaying recombinant alkaline proteases
were produced on a Yarrowia lipolytica cell surface
by overexpressing a marine-derived alkaline protease
gene [14.63]. This surface-displaying protease can hydrolyze various proteins for the production of bioactive
peptides. The peptides have potential pharmaceutical
applications due to their angiotensin-converting enzyme (ACE) inhibitory activity and antioxidant activity.
Peptides with high ACE inhibitory activity have wellknown potential application in the treatment of hypertension and other bioactive peptides can be used as
clinical nutrient supplements.
The production of a thermostable alkaline protease
by a marine Bacillus was optimized and scaled up
in a stirred tank bioreactor using cheap and readily
available substrates [14.64]. The authors studied the effects of environmental factors, carbon sources, nitrogen
sources, and fermentation parameters on the protease
production kinetics. Enzyme production increased with
prolonged fermentation time and faster agitation rate.
Maximum protease production was achieved after 40h
fermentation at 42
ı C with an aeration rate of 1:5 vvm
and constant agitation at 400 rpm in a soybean–casein
medium (pH 9:6). Protease production by the marine bacterium Teredinobacter turnirae was optimized
for solid-state fermentation as well by immobilization on solid matrixes [14.65–67]. Media composition,
initial pH, inoculum concentration, and the effect of
different salts and carbon sources were studied to optimize protease production by solid-state fermentation
in a soybean-based medium. The maximum protease
production was obtained using 1 w=v % soybean concentration, initial pH 7:34, and a 2:5 v=v % inoculum
level. The material and particle size of the immobilizing medium, and the method and conditions for