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Levansucrase (EC 2.4.1.10.) of Zymomonas mobilis 113S can perform the polymerisation of fructose moiety from raffinose to levan concomitantly with a release of non-catabolised melibiose into the medium. The kinetic parameters of the levansucrase-catalysed reaction provide even higher reaction velocities on raffinose as compared to sucrose, particularly at low substrate concentrations. A decreased value in the number of the average molecular mass (M n = 1693 kDa), an increased intrinsic viscosity (g = 49.47 cm 3 /g), and a diminished Huggin's constant (K ' = 0.67) are intrinsic to the levan synthesis from raffinose, indicating certain structural peculiarities compared to a polysaccharide obtained from sucrose (M n = 1851 kDa, [g] = 42.47 cm 3 /g, K' = 1.21).

Abstract:The amino acid composition of sequences and structural attributes (α-helices, β-sheets) of C-and N-terminal fragments (50 amino acids) were compared to annotated (SWISS-PROT/ TrEMBL) type I (20 sequences) and type III (22 sequences) secreted proteins of Gramnegative bacteria. The discriminant analysis together with the stepwise forward and backward selection of variables revealed the frequencies of the residues Arg, Glu, Gly, Ile, Met, Pro, Ser, Tyr, Val as a set of strong (1-P< 0.001) predictor variables to discriminate between the sequences of type I and type III secreted proteins with a cross-validated accuracy of 98.6-100 % . The internal and external validity of discriminant analysis was confirmed by multiple (15 repeats) test-retest procedures using a randomly split original set of proteins; this validation method demonstrated an accuracy of 100 % for 191 non-selected (retest) sequences. The discriminant analysis was also applied using selected variables from the propensities for β-sheets and polarity of C-terminal fragments. This approach produced the next highest and comparable cross-validated classification accuracy for randomly selected and retest proteins (85.4-86.0 % and 82.4-84.5 %, respectively). The proposed sets of predictor variables could be used to assess the compatibility between secretion substrates and secretion pathways of Gram-negative bacteria by means of discriminant analysis.

Abstract:The Fourier transform (FT) method was applied to specify the distribution of 14 predefined groups of amino acids (64 residues) at both termini of annotated type III and type I secreted proteins from proteobacteria. Type I proteins displayed a higher occurrence of significant periodicities at both C-and N-termini, indicating potent features to discriminate between secretion types, particularly by the use of variables selected from the full periodicity profiles at 19 orders of FT. The Fishers linear discriminant analysis, together with the stepwise selection of variables throughout equal pairs of combinations for all predefined groups of residues, revealed the C-terminal harmonics of aromatic (HFWY) and aliphatic (VLIA) residues as a set of strong predictor variables to classify both types of secreted proteins with an accuracy of 100% for original grouped cases and 96.4% for cross-validated grouped cases. The prediction accuracy of proposed discriminant function was estimated by repeated kfold cross-validation procedures where the original data set was randomly divided into k subsets, with one of the k-subsets serving as the test set and the remaining data forming the training set. The average error rate computed across all k-trials and repeats did not exceed that of leave-one-out procedure. The proposed set of predictor variables could be used to assess the compatibility between secretion pathways and secretion substrates of proteobacteria by means of discriminant analysis.

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