caa a22 4500 605504687 CHVBK 20210128100617.0 cr unu---uuuuu 210128e20151201xx s 000 0 eng 10.1007/s00253-015-6854-0 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6854-0 Secreted expression of Leuconostoc mesenteroides glucansucrase in Lactococcus lactis for the production of insoluble glucans [Elektronische Daten] [Christopher Skory, Gregory Côté] We expressed a glucansucrase, DsrI, from Leuconostoc mesenteroides that catalyzes formation of water-insoluble glucans from sucrose using a nisin-controlled gene expression system in Lactococcus lactis. These polymers have potential for production of biodegradable gels, fibers, and films. We optimized production of DsrI 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 accumulated in the culture medium to approximately 380mg/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. Springer-Verlag Berlin Heidelberg (outside the USA), 2015 Glucansucrase nationallicence Dextransucrase nationallicence Dextran nationallicence Mutan nationallicence Insoluble polysaccharide nationallicence Skory Christopher Renewable Product Technology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 North University St., 61604, Peoria, IL, USA aut Côté Gregory Renewable Product Technology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 North University St., 61604, Peoria, IL, USA aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/23(2015-12-01), 10001-10010 0175-7598 99:23<10001 2015 99 253 https://doi.org/10.1007/s00253-015-6854-0 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6854-0 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Skory Christopher Renewable Product Technology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 North University St., 61604, Peoria, IL, USA aut NATIONALLICENCE 700 1- Côté Gregory Renewable Product Technology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 North University St., 61604, Peoria, IL, USA aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/23(2015-12-01), 10001-10010 0175-7598 99:23<10001 2015 99 253