A xylosidase gene, labeled as BH1068 in genome of Bacillus halodurans C-125, was successfully cloned and overexpressed in Escherichia coli JM109. The purified enzyme was thoroughly characterized and its xylosidase function was unambiguously confirmed. It has maximum activities in neutral condition and is stable over a wide range of pH (4.5-9.0). The enzyme has a broad temperature optimal (35 degrees C-45 degrees C) and is quite stable at temperature up to 45 degrees C. The unique pH and temperature profiles of the enzyme should allow a wide range of xylanolytic operational conditions. With high specific activity of 174 mU/mg protein for its artificial substrate (p-nitrophenyl-beta-xylose) and low xylose inhibition (inhibitor constant Ki = 300 mmol/L), this enzyme is among the most active and high tolerant bacterial xylosidase to xylose inhibition. Its high synergy with commercial xylanase has been demonstrated with beechwood xylan hydrolysis, achieving a hydrolysis yield of 40%. Its neutral pH optimal and high tolerance to product inhibition complements well with its fungal counterparts that are only optimal at acidic pH and susceptible to xylose inhibition. In conclusion, this enzyme has high potential in the saccharification of xylan and xylan-containing polysaccharides.
Amino Acid Sequence ; Bacillus ; classification ; enzymology ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Hydrolysis ; Molecular Sequence Data ; Recombinant Proteins ; genetics ; metabolism ; Substrate Specificity ; Xylose ; metabolism ; Xylosidases ; genetics ; metabolism

Background: The quality of a vaccine depends strongly on the effects of the adjuvants applied simultaneously with the antigen in the vaccine. The adjuvants enhance the protective effect of the vaccine against a viral challenge. Conversely, oil-type adjuvants leave oil residue inside the bodies of the injected animals that can produce a local reaction in the muscle. The longterm immunogenicity of mice after vaccination was examined. ISA206 or ISA15 oil adjuvants maintained the best immunity, protective capability, and safety among the oil adjuvants in the experimental group. Objectives: This study screened the adjuvant composites aimed at enhancing foot-andmouth disease (FMD) immunity. The C-type lectin or toll-like receptor (TLR) agonist showed the most improved protection rate. Methods: Experimental vaccines were fabricated by mixing various known oil adjuvants and composites that can act as immunogenic adjuvants (gel, saponin, and other components) and examined the enhancement effect on the vaccine. Results: The water in oil (W/O) and water in oil in water (W/O/W) adjuvants showed better immune effects than the oil in water (O/W) adjuvants, which have a small volume of oil component. The W/O type left the largest amount of oil residue, followed by W/O/W and O/W types. In the mouse model, intramuscular inoculation showed a better protection rate than subcutaneous inoculation. Moreover, the protective effect was particularly weak in the case of inoculation in fatty tissue. The initial immune reaction and persistence of long-term immunity were also confirmed in an immune reaction on pigs. Conclusions The new experimental vaccine with immunostimulants produces improved immune responses and safety in pigs than general oil-adjuvanted vaccines.

Foot-and-mouth disease (FMD) is an acute epidemic that spreads rapidly among cattle and pigs. In 2014, in Korea, despite enforced vaccination, the type O Southeast Asia (SEA) topotype viruses (Mya-98 lineage) infected mainly cattle and pigs simultaneously, thereby causing enormous damage. If a vaccine that is completely protective against this FMD virus is developed and used, it can become a very important preventive measure in Asia, which is where this type of virus mainly circulates. The SEA topotype has been steadily evolving and transforming into new variations since it became epidemic in Asia. Therefore, it became necessary to develop a new vaccine that could provide protection against the FMD virus strain that was responsible for the 2014–2015 outbreak in Korea. This study aimed to develop a vaccine that would provide complete protection against the SEA topotype FMD virus to control sporadic FMD outbreaks, which occur despite the enforcement of vaccination, and to completely prevent virus shedding, thereby preventing the virus from spreading. The vaccine candidate virus developed in this study showed low pathogenicity and can be distinguished from the wild-type FMD virus strain. The developed vaccine was able to protect mice from SEA and Middle East–South Asia topotype virus strains and induced high titers of antibodies against both virus strains in pigs, thereby confirming the sufficiency of its protective function. In particular, the results of the SEA topotype virus challenge test in pigs revealed that perfect immunity was created in the vaccinated pigs, without virus shedding and viremia.
Animals ; Antibodies ; Asia ; Asia, Southeastern ; Cattle ; Disease Outbreaks ; Foot-and-Mouth Disease ; Korea ; Mice ; Swine ; Vaccination ; Viremia ; Virulence ; Virus Shedding