A. Esterases from Sponge Metagenomes
Many antibacterials and bioactive substances with anticancer and other properties
have bene found in sponges. Most of them are produced by the microorganisms
living in a symbiotic relationship within the sponges. Since the majority of these
symbiotes are VBNC bacteria, a metagenomics analysis of sponges is believed to
be a useful avenue for identifying these substances’ biosynthetic pathways. New
enzyme genes may also be identified from the symbiotes through the metagenomics
analysis of sponges (Okamura et al. 2010).
Recently, genes for EstHE1, an esterase with a molecular weight of around
25,000, were isolated from the metagenomic library for the Japanese sponge Hytios
erecta (Okamura et al. 2010). A homology search showed that the enzyme
belonged to the SGNH hydrolase superfamily, consisting of serine (Ser), glycine
(Gly), asparagine (Asn), and histidine (His) as catalyzing amino acid residues.
Recombinant EstHE1 expressed in E. coli hydrolyzed fatty acid q-nitrophenyl
esters with two to four carbon atoms, but not fatty acid esters with a greater number
of carbon atoms. From this, it was determined to be an esterase that breaks down
short-chain fatty acid esters, rather than a lipase that breaks down fatty acid esters
with 10 carbon atoms or more. In addition, it had an optimal temperature of 40 °C,
and while it showed 50% or more of maximum activity at a range of 25–55 °C,
activity dropped to 58% when cultured for 12 h at 40 °C. This indicates that is not a
thermophilic enzyme, but a mesophilic enzyme that operates in a normal temperature range (Selvin et al. 2012).
At the same time, the enzyme tolerance to high concentrations of salt: activity
dropped to 55% up to an NaCl concentration of 1.9 M, but slowly increased at
higher concentrations, eventually reaching 62% at 3.8 M. This is thought to relate
to the sponge’s living environment, namely seawater containing approximately
0.6 M NaCl. This property of EstHE1 is something that had not been obtained in
previous genetic engineering improvements of esterases, showing that metagenomics analysis is an effect means of obtaining genes for enzymes with new
characteristics
B. Esterase from Deep-Sea Mud Metagenomes
Recently, a recombinant EstF enzyme was produced by cloning the esterase gene
estF from a South China Sea deep-sea mud metagenomics library and using E. coli
as an expression system (Fu et al. 2011). The enzyme showed its strongest
decomposition performance with p-nitrophenyl butyrate (C4), but did not work on
fatty acid esters with 10 or more carbon atoms. EstF is a cold-adapted enzyme that
exhibits high levels of activity even at low temperatures. For example, it has shown
roughly 20% of its maximum activity (50 °C) even at a reaction temperature of 0 °
C. This property shows the enzyme to a product of adaptation to the
low-temperature deep-sea environment (Schmeisser et al. 2007).
EstF is also an alkaline enzyme that exhibits maximum activity at a pH of 9.0,
which drops sharply at a pH below 7. Its catalytic site consists of 331 residues, and
11.4 Development of Biocatalysts from Microorganisms
403
Many antibacterials and bioactive substances with anticancer and other properties
have bene found in sponges. Most of them are produced by the microorganisms
living in a symbiotic relationship within the sponges. Since the majority of these
symbiotes are VBNC bacteria, a metagenomics analysis of sponges is believed to
be a useful avenue for identifying these substances’ biosynthetic pathways. New
enzyme genes may also be identified from the symbiotes through the metagenomics
analysis of sponges (Okamura et al. 2010).
Recently, genes for EstHE1, an esterase with a molecular weight of around
25,000, were isolated from the metagenomic library for the Japanese sponge Hytios
erecta (Okamura et al. 2010). A homology search showed that the enzyme
belonged to the SGNH hydrolase superfamily, consisting of serine (Ser), glycine
(Gly), asparagine (Asn), and histidine (His) as catalyzing amino acid residues.
Recombinant EstHE1 expressed in E. coli hydrolyzed fatty acid q-nitrophenyl
esters with two to four carbon atoms, but not fatty acid esters with a greater number
of carbon atoms. From this, it was determined to be an esterase that breaks down
short-chain fatty acid esters, rather than a lipase that breaks down fatty acid esters
with 10 carbon atoms or more. In addition, it had an optimal temperature of 40 °C,
and while it showed 50% or more of maximum activity at a range of 25–55 °C,
activity dropped to 58% when cultured for 12 h at 40 °C. This indicates that is not a
thermophilic enzyme, but a mesophilic enzyme that operates in a normal temperature range (Selvin et al. 2012).
At the same time, the enzyme tolerance to high concentrations of salt: activity
dropped to 55% up to an NaCl concentration of 1.9 M, but slowly increased at
higher concentrations, eventually reaching 62% at 3.8 M. This is thought to relate
to the sponge’s living environment, namely seawater containing approximately
0.6 M NaCl. This property of EstHE1 is something that had not been obtained in
previous genetic engineering improvements of esterases, showing that metagenomics analysis is an effect means of obtaining genes for enzymes with new
characteristics
B. Esterase from Deep-Sea Mud Metagenomes
Recently, a recombinant EstF enzyme was produced by cloning the esterase gene
estF from a South China Sea deep-sea mud metagenomics library and using E. coli
as an expression system (Fu et al. 2011). The enzyme showed its strongest
decomposition performance with p-nitrophenyl butyrate (C4), but did not work on
fatty acid esters with 10 or more carbon atoms. EstF is a cold-adapted enzyme that
exhibits high levels of activity even at low temperatures. For example, it has shown
roughly 20% of its maximum activity (50 °C) even at a reaction temperature of 0 °
C. This property shows the enzyme to a product of adaptation to the
low-temperature deep-sea environment (Schmeisser et al. 2007).
EstF is also an alkaline enzyme that exhibits maximum activity at a pH of 9.0,
which drops sharply at a pH below 7. Its catalytic site consists of 331 residues, and
11.4 Development of Biocatalysts from Microorganisms
403