The increasing prevalence of foodborne diseases caused by
Escherichia coli O157:H7 as well as its ability to form biofilms
poses major threats to public health worldwide. With increasing
concerns about the limitations of current disinfectant treatments,
phage-derived depolymerases may be used as promising
biocontrol agents. Therefore, in this study, the characterization,
purification, and application of a novel phage depolymerase,
Dpo10, specifically targeting the lipopolysaccharides
of E. coli O157, was performed. Dpo10, with a molecular
mass of 98 kDa, was predicted to possess pectate lyase
activity via genome analysis and considered to act as a receptor-
binding protein of the phage. We confirmed that the
purified Dpo10 showed O-polysaccharide degrading activity
only for the E. coli O157 strains by observing its opaque halo.
Dpo10 maintained stable enzymatic activities across a wide
range of temperature conditions under 55°C and mild basic
pH. Notably, Dpo10 did not inhibit bacterial growth but significantly
increased the complement-mediated serum lysis
of E. coli O157 by degrading its O-polysaccharides. Moreover,
Dpo10 inhibited the biofilm formation against E. coli O157
on abiotic polystyrene by 8-fold and stainless steel by 2.56 log
CFU/coupon. This inhibition was visually confirmed via fieldemission
scanning electron microscopy. Therefore, the novel
depolymerase from E. coli siphophage exhibits specific binding
and lytic activities on the lipopolysaccharide of E. coli O157
and may be used as a promising anti-biofilm agent against
the E. coli O157:H7 strain.
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died
and applied in processes targeted for industrial scale.
In this work, the cutinase gene tfu from Thermobifida fusca
was artificially synthesized according to codon usage bias of
Saccharomyces cerevisiae and investigated in Saccharomyces
cerevisiae. Using the α-factor signal peptide, the T. fusca cutinase
was successfully overexpressed and secreted with the
GAL1 expression system. To increase the cutinase level and
overcome some of the drawbacks of induction, four different
strong promoters (ADH1, HXT1, TEF1, and TDH3) were comparatively
evaluated for cutinase production. By comparison,
promoter TEF1 exhibited an outstanding property and significantly
increased the expression level. By fed-batch fermentation
with a constant feeding approach, the activity of cutinase
was increased to 29.7 U/ml. The result will contribute
to apply constitutive promoter TEF1 as a tool for targeted cutinase
production in S. cerevisiae cell factory.
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