Description
Presentation Blocks: 03-24-2018 - Saturday - 11:00 AM - 12:15 PM

Title: Stability Assessment of a Gluten-degrading Subtilisin Derived From Oral Microbes

Authors:

Guoxian Wei (Presenter)
Boston University

Eva Helmerhorst, Boston University
Ghassan Darwish, Boston University

Abstract:

Objectives: Celiac disease is a chronic immune-mediated inflammation of the duodenum, triggered by gluten contained in wheat, barley and rye. Our previous studies have shown that Rothia, Gram-positive oral bacteria, produce subtilisin enzymes which have the ability to degrade and detoxify gluten in vitro. The aim of this study is to assess the properties, stability and characteristics of subtilisin-A in solution.

Methods: Subtilisin-A (1 mg/ml) was dissolved in various solutions including deionized water, 50% DMSO, sodium carbonate buffer (pH 10), dilute HCl (pH 1.5), or PMSF (1 µg/ml), and incubated on ice for 1 h. The effect of exposure to various temperatures from 0°C to 100°C, and incubation time from 0h to 72h on enzyme activity were also assessed. Protein stability was analyzed by SDS-PAGE, and enzyme activity was determined using a paranitroanilide-derivatized enzyme substrate (Suc-AAPF-pNA) and in a casein zymogram. Autolysis of Sub-A was analyzed by RP-HPLC using a C18 column.

Results: Subtilisin-A dissolved at pH 1.5 showed a band of 27 kDa, while it only showed low molecular weight bands (<10 kDa) when dissolved at pH 7.0. The activity of the enzyme were high in 50% DMSO and at pH10, while inhibited in the presence of PMSF and at pH1.5. The enzyme activity decreased at increasing temperatures, where activity was completely lost at >60°C. The activity decreased more than 4 fold after 6 h incubation at 37°C. Prolonged incubation time and increased temperature led to the disappearance of the low MW bands in SDS-PAGE, and significantly reduced cleavage activity towards Suc-AAPF-pNA. The separation of autolyzed fractions of Sub-A was achieved by RP-HPLC, and SDS-PAGE analysis showed evidence for various small molecular weight species in the auto-digest.

Conclusions: Unless dissolved under conditions preventing proteolysis, subtilisin-A undergoes significant autolysis. These study results provide the basis for further investigations to stabilize the enzyme including pharmaceutical modification.These studies were funded by NIH/NIAID grant AI101067, the BU Ignition Award, and the BU CTSI Award.

Schedule
poster

Poster Session

11:00 am–12:15 pm Mar 24 (US - Eastern)

CC, Hall B/C