Dextranase, Mutanase, and Lipase Chewing Gum Enzymatic Biofilm Suppression

Objectives: Available methods to treat oral biofilm have many disadvantages and are not supportive of efforts to increase affordability and accessibility of dental care. Chemical methods of dental biofilm treatment cannot penetrate the extracellular polysaccharide matrix (EPS). To target this, we expressed lipase (Mycobacterium tuberculi), dextranase (Streptococcus mutans), and mutanase (Paenibacillus spp) in edible plant cells that do not require prohibitively expensive fermentation, purification, cold storage/transportation, or invasive surgical treatment. Dextranase and mutanase focus on glucans in the EPS but lack antimicrobial properties. Lipase possesses those antimicrobial properties. Our goal is to develop chewing gum containing these enzymes to achieve non-invasive, topical dental plaque reduction.
Methods: Lettuce plant powder from current generation of plants expressing dextranase, mutanase, and lipase were evaluated for respective enzyme activities and compared with activities of past generations grown in Penn greenhouse.
Results: Enzymes were expressed in lettuce, free of antibiotic resistance genes through chloroplast engineering developed in the Daniell lab. Plant biomass from T1 and subsequent generations (T2, T3) was assessed for lipase, dextranase, and mutanase activity by p-nitro-phenyl-butyrate, dextran, and mutan substrates, respectively. Lipase activity for T3 was 4204 ± 32.5 µmol/h/g DW compared to 749 µmol/h/g DW for the T2 generation. Dextranase activity for T3 was 138 ± 4.4 µmol/h/g DW compared to 60.68 µmol/h/g DW in the T2 generation. Mutanase activity for T2 was 386 ± 32.8 µmol/h/g DW compared to 33.68µmol/h/g DW in the T1 generation. Synergistic effects of lipase, dextranase, and mutanase using current materials with improved activities will be evaluated for anti-biofilm activities.
Conclusions: Increasing activity in crude extracts of enzyme in subsequent generations will lower future doses needed for anti-biofilm assays and eliminate need for fermentation, purification, and cold storage/transportation, increasing accessibility and affordability. Creation of chewing gum containing plant-derived dental plaque reducing enzymes presents a method to overcome challenges faced with chemical and mechanical methods of dental plaque reduction and provides a novel approach to dental care.