Proteomic analysis and characterization of a novel glucansucrase from a newly isolated strain of Leuconostoc mesenteroides AA1

Abstract

PurposeGlucansucrase is an industrially important enzyme that is produced by different species of bacteria like Leuconostoc, Streptococcus and Acetobacter. Among them Leuconostoc mesenteroides is used for the production of glucansucrase that catalyzes the synthesis of a commercially important long chain glucose polymer known as glucan on industrial scale.Due to its increasing demand in various biotechnological industries, isolation of new strains of Leuconostoc mesenteroides for the production of glucansucrase and glucan is of valuable importance.MethodsNew strain of Leuconostoc mesenteroides AA1 was isolated and various biochemical conditions and aspects regarding extracellular glucansucrase activity have been optimized. Glucansucrase was purified to homogeneity using conventional chromatographic techniques. Amino acid analysis and NTerminal sequence of the purified enzyme was also performed. Results Glucansucrase [EC 2.4.1.5] was purified to homogeneity with an overall purification factor of 162.5 times, with a specific activity of 2692 DSU/mg. The native protein showed a molecular mass of 177,000 Da by SDS PAGE. The enzyme exhibited optimum catalytic activity at 35°C and pH 5.0 with 0.1 M citrate phosphate buffer. The kinetic constants Km and Vmax were estimated as 69.88 mM and 61.75 DSU/ml/hr, respectively. The amino acid composition of the enzyme showed that the enzyme is rich in both the basic and polar/hydrophilic amino acid and is less rich in acidic amino acids. The first six amino acid residues at the Nterminal end are D-S-T-N-T-V. Conclusions This novel extracellular glucansucrase is significant for industrial perspective and is therefore suggested that this strain can be used for the production of glucansucrase and high molecular weight glucan on a large scale. Abbreviations: DSU, Dextransucrase Unit; SDS PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; PVDF, polyvinylidene difloride; HPLC, high performance liquid chromatography TrEMBL UniProtKB Accession #: P85080 E.C. 2.4.1.5 (glucosyltransferase)