Production, Partial Purification and Kinetic Characterization of Dextransucrase from Leuconostoc mesenteroides in Relation to Dextran Problem in Sugarcane

Abstract

Dextransucrase was produced from Leuconostoc mesenteroides strain MTCC 107. Maximum enzyme production was observed after 60 h of incubation, with drop in pH from 7.0 at 0 h to 5.5 at 60 h. The crude enzyme was subjected to ammonium sulphate fractionation and DEAE-cellulose anion-exchange chromatography. Three isoforms of dextransucrase viz. DS-I, DS-II and DS-III, each with optimum pH 5.5 and optimum temperature 45 °C were identified. Isoforms DS-I and DS-III were found to be thermostable between 10 and 60 °C while isoform DS-II was thermostable between 10 and 50 °C. The Km values (for sucrose as substrate) of the isoforms DS-I, DS-II and DS-III at their optimum pH and temperature were 206.34, 492.45 and 108.00 mM, respectively. Corresponding Vmax values were 428.08, 505.58 and 410.85 μmol reducing sugars produced/min/mg protein, respectively. The pK a values of ionizing groups obtained by using Dixon and Marangoni methods suggested the participation of Asp/Glu residues and His residues in both substrate binding and catalysis. Lineweaver Burk plots obtained by varying concentrations of dextran (as substrate) at fixed sucrose concentration were found to be biphasic for all the three dextransucrase isoforms, suggesting negative cooperativity. Both the transferase and sucrase activities of the dextransucrase isoforms were markedly inhibited by sodium lauryl sulphate, barium chloride, EDTA, potassium chloride, manganese chloride and sodium metasilicate. These metal salts/chemicals when exogenously added to sugarcane juice effectively reduced the dextran formation during storage.