Recombinant Expression of Thermostable Endo-Arabinase in Vitro and in Planta for Generating Functional Oligosaccharides from Plant Cell Wall for Colon-Specific Health Benefits

Lignin-deficient(LD) plant fibers, e.g., sugar beet pulp and rice bran, are rich sources of functional cell wall polysaccharides. Used primarily as low-value animal feed, these established feedstock can be capitalized on for generation of value-added bioproducts, which will improve the economic viability and competitiveness of sugar beet and rice industry. The overall goal of this project is to develop an efficient enzymatic platform for generating functional oligosaccharides, specifically, feruloylated arabino-oligosaccharides (FAOs), from LD plant fibers. FAOs may be used in food and feed applications for healthful colon functioning through prebiotic, anti-inflammatory and mucosal immunomodulatory activities. FAOs of defined structure can be released from LD fibers through selectively cleaving the arabinan chain of cell wall polysaccharides by a key glycohydrolase - arabinase (ABN). In this study, a thermostable endo-1,5-a-L-ABN from Bacillus thermodenitrifican was expressed in yeast (Pichia pastoris) to produce significant quantity of enzyme for testing its activity of digesting plant fibers. Recombinant enzyme was secreted into culture media at a yield of -70 mg/L and showed a specific enzymatic activity of 350 U/mg. ABN was also expressed in planta (in tobacco) to assess its function in post-harvest modification of cell wall polysaccharides to facilitate in situ FAOs releasing. Transgenic tobacco accumulated -20 (Jg/gFW active ABN, and were not phenotypically different from wild-type plants. Finally, T84 epithelial cell culture was established to assay the anti-inflammatory activities of FAOs. Preliminary study indicated that FAOs isolated from rice bran significantly enhanced the expression of occludin, a biomarker of epithelial cells' immune response to FAOs.