Detoxification of
gluten immunogenic
epitopes is a promising strategy for the treatment of
celiac disease. Our previous studies have shown that these
epitopes can be degraded in vitro by
subtilisin enzymes derived from Rothia mucilaginosa, a natural microbial colonizer of the oral cavity. The challenge is that the
enzyme is not optimally active under acidic conditions as encountered in the stomach. We therefore aimed to protect and maintain
subtilisin-A enzyme activity by exploring two
pharmaceutical modification techniques: PEGylation and Polylactic
glycolic acid (PLGA) microencapsulation. PEGylation of
subtilisin-A (Sub-A) was performed by attaching
methoxypolyethylene glycol (
mPEG, 5 kDa). The PEGylation protected
subtilisin-A from
autolysis at neutral pH. The PEGylated Sub-A (Sub-A-
mPEG) was further encapsulated by PLGA. The microencapsulated Sub-A-
mPEG-PLGA showed significantly increased protection against
acid exposure in vitro. In vivo,
gluten immunogenic
epitopes were decreased by 60% in the stomach of mice fed with chow containing Sub-A-
mPEG-PLGA (0.2 mg Sub-A/g chow) (n = 9) compared to 31.9% in mice fed with chow containing unmodified Sub-A (n = 9). These results show that the developed
pharmaceutical modification can protect Sub-A from auto-digestion as well as from
acid inactivation, thus rendering the
enzyme more effective for applications in vivo.