|Skory, Christopher - Chris|
Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/15/2015
Publication Date: 8/4/2015
Publication URL: https://handle.nal.usda.gov/10113/62398
Citation: Skory, C.D., Cote, G.L. 2015. Secreted expression of Leuconostoc mesenteroides glucansucrase in Lactococcus lactis for the production of insoluble glucans. Applied Microbiology and Biotechnology. 99(23):10001–10010.
Interpretive Summary: Certain bacteria used in fermented food produce an enzyme called glucansucrase that are able to produce long polymers of glucose from cane or beet sugars. These polymers are typically water-soluble, but we identified a novel glucansucrase enzyme that produces a unique gel-like polymer that is insoluble in water. In this study, we developed an optimized expression system that is capable of producing large quantities of this enzyme for producing the insoluble polymer. These polymers have potential for production of biodegradable fibers and films that can be used in a broad number of consumer applications and provide the foundation for developing new eco-friendly materials derived from renewable agricultural materials.
Technical Abstract: We expressed a glucansucrase, DsrI, from Leuconostoc mesenteroides that catalyzes formation of water-insoluble glucans from sucrose in Lactococcus lactis using a nisin-controlled gene expression system. Production of DsrI was optimized using several different background vectors, signal peptides, strains, induction conditions, and bioreactor parameters to increase extracellular accumulation. Optimal production of the enzyme utilized a high-copy plasmid, pMSP3535H3, which contains a nisin immunity gene, L. lactis LM0230, and bioreactors maintained at pH 6.0 to stabilize the enzyme. We were able to significantly improve growth using the lactic acid inhibitor heme and by continuous removal of lactic acid with anion exchange resins, but enzyme production was less than the controls. The recombinant enzyme under optimized conditions was accumulated in the culture medium to approximately 380 mg/L, which was over 150-fold higher compared to the native L. mesenteroides strain. Methods are also included for purification of DsrI utilizing the glucan binding domain of the enzyme.