Skory Christopher D, Côté Gregory L
Renewable Product Technology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 North University St., Peoria, IL, 61604, USA.
Appl Microbiol Biotechnol. 2015 Dec;99(23):10001-10. doi: 10.1007/s00253-015-6854-0. Epub 2015 Aug 4.
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 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.
我们利用乳酸乳球菌中的乳链菌肽控制基因表达系统，表达了来自肠系膜明串珠菌的一种葡聚糖蔗糖酶DsrI，该酶可催化由蔗糖形成水不溶性葡聚糖。这些聚合物具有生产可生物降解凝胶、纤维和薄膜的潜力。我们使用几种不同的背景载体、信号肽、菌株、诱导条件和生物反应器参数优化了DsrI的生产，以增加细胞外积累。该酶的最佳生产利用了高拷贝质粒pMSP3535H3，其含有乳链菌肽免疫基因、乳酸乳球菌LM0230，以及维持在pH 6.0以稳定该酶的生物反应器。我们能够通过使用乳酸抑制剂血红素并通过用阴离子交换树脂连续去除乳酸来显著改善生长，但酶产量低于对照。在优化条件下，重组酶在培养基中的积累量约为380 mg/L，比天然肠系膜明串珠菌菌株高出150倍以上。文中还包括利用该酶的葡聚糖结合结构域纯化DsrI的方法。
