Identification and Characterization of a ß-N-Acetylhexosaminidase with a Biosynthetic Activity from the Marine Bacterium Paraglaciecola hydrolytica S66T.

ß-N-Acetylhexosaminidases are glycoside hydrolases (GHs) acting on N-acetylated carbohydrates and glycoproteins with the release of N-acetylhexosamines. Members of the family GH20 have been reported to catalyze the transfer of N-acetylglucosamine (GlcNAc) to an acceptor, i.e., the reverse of hydrolysis, thus representing an alternative to chemical oligosaccharide synthesis. Two putative GH20 ß-N-acetylhexosaminidases, PhNah20A and PhNah20B, encoded by the marine bacterium Paraglaciecola hydrolytica S66T, are distantly related to previously characterized enzymes. Remarkably, PhNah20A was located by phylogenetic analysis outside clusters of other studied ß-N-acetylhexosaminidases, in a unique position between bacterial and eukaryotic enzymes. We successfully produced recombinant PhNah20A showing optimum activity at pH 6.0 and 50 °C, hydrolysis of GlcNAc ß-1,4 and ß-1,3 linkages in chitobiose (GlcNAc)2 and GlcNAc-1,3-ß-Gal-1,4-ß-Glc (LNT2), a human milk oligosaccharide core structure. The kinetic parameters of PhNah20A for p-nitrophenyl-GlcNAc and p-nitrophenyl-GalNAc were highly similar: kcat/KM being 341 and 344 mM-1 s-1, respectively. PhNah20A was unstable in dilute solution, but retained full activity in the presence of 0.5% bovine serum albumin (BSA). PhNah20A catalyzed the formation of LNT2, the non-reducing trisaccharide ß-Gal-1,4-ß-Glc-1,1-ß-GlcNAc, and in low amounts the ß-1,2- or ß-1,3-linked trisaccharide ß-Gal-1,4(ß-GlcNAc)-1,x-Glc by a transglycosylation of lactose using 2-methyl-(1,2-dideoxy-α-d-glucopyrano)-oxazoline (NAG-oxazoline) as the donor. PhNah20A is the first characterized member of a distinct subgroup within GH20 ß-N-acetylhexosaminidases.

Resolution and signal-to-noise in analysis of carbohydrate isomers by graphitised carbon chromatography with charged aerosol detection.

The effects of co-eluents and additives on separation and signal-to-noise ratio in analyses of monosaccharides by graphitised carbon chromatography (GCC) in combination with charged aerosol detection were studied. Design of experiments was used to model and predict the elution of two monosaccharide isomers, galactose and glucose and the corresponding amine at varying isocratic conditions, including concentration of water-soluble co-eluent, flow and temperature. The study confirmed the well-known order of eluent strength of the co-eluents investigated but showed that the eluent strength of MeOH was significantly lower than that of ACN, and at co-eluent concentrations ≥5% (v/v) IPA approached that of THF. Addition of NH3 increased retention and improved both peak shape and separation but the detector response decreased with increasing NH3 concentration lowering the signal-to-noise ratio. The best combination of response, retention and separation was obtained at 0.1% NH3. Increasing column temperature in the range of 15-50°C resulted in decreased retention times and resolution. The corresponding Van't Hoff correlations showed negative adsorption enthalpies indicating an exothermic adsorption process driven by a decrease in entropy minimising the surface energy of the system. Isocratic elution with MeOH as co-eluent offered limited possibilities for optimisation of resolution due to the opposite effects of changes in co-eluent concentration and changes in flow rate. Elution with acetonitrile as co-eluent showed possibilities for optimisation of the resolution within the range of flow rates of 0.6-0.95mL/min and co-eluent concentrations of 0.1-0.3%, with the highest resolution predicted at 0.1% acetonitrile and a flow rate of 0.81mL/min. Saccharides in the size range DP1-4, including amino, acetamido, and deoxy sugars, were separated using a binary gradient method. Higher retention was observed for increasing degree of polymerisation (DP) and N-acetylated saccharides were retained longer than non-substituted saccharides of corresponding DP. Partial resolution of two lacto-N-tetrasaccharide positional isomers was obtained.

Elution strategies for reversed-phase high-performance liquid chromatography analysis of sucrose alkanoate regioisomers with charged aerosol detection.

A broad range of elution strategies for RP-HPLC analysis of sucrose alkanoate regioisomers with CAD was systematically evaluated. The HPLC analyses were investigated using design-of-experiments methodology and analysed by analysis of variance (ANOVA) and regression modelling. Isocratic elutions, isocratic elutions with increased flow, and gradient elutions with step-down profiles and step-up profiles were performed and the chromatographic parameters of the different elution strategies were described by suitable variables. Based on peak resolutions general resolution deviation for multiple peaks (RDm) was developed for sample-independent evaluation of separation of any number of peaks in chromatographic analysis. Isocratic elutions of sucrose alkanoates showed similar relationships between eluent acetonitrile concentration and retention time for all regioisomers of sucrose caprate and sucrose laurate, as confirmed by evaluation of the curvatures using approximate second derivatives and Kendall rank correlation coefficients. Regression modelling and statistical analysis showed that acetonitrile concentration and flow rate were highly significant for both average adjusted retention time and RDm for sucrose laurate. For both responses the effect of changes in acetonitrile concentration was larger than the effect of changes in flow rate, over the ranges studied. Regression modelling of the step-down gradient profiles for the sucrose alkanoates showed that the eluent acetonitrile concentrations were the overall most significant variables for retention time and separation. The models for average adjusted retention time of sucrose caprate and sucrose laurate showed only a few differences in the significance levels of terms, while the models for RDm showed larger differences between the sucrose alkanoates, in both the number of terms and their significance. Efficiency evaluation of elution strategies, in terms of RDm and analysis time, showed that the best results were offered by step-down gradient elution for sucrose caprate and isocratic elution with increased flow for sucrose laurate. Step-down gradient elution of sucrose caprate offered improvements in separation at similar analysis time compared to isocratic elution, with the most efficient elutions achieved with elution profile acetonitrile concentrations at 32.5% and 25%, resulting in reduction of RDm by 13-38% and reduction of analysis time by 3-9%. For sucrose laurate, isocratic elution with increased flow showed improvements in separation and reductions in analysis time compared to isocratic elution, such as elution at 37% with flow 2.0mL/min resulting in reduction of analysis time by 34% and equal RDm, while elution at 35% with flow 2.0mL/min reduced RDm by 29% and analysis time by 6%, compared to isocratic elution at 38% acetonitrile with standard flow (1.0mL/min).

Design of experiments and multivariate analysis for evaluation of reversed-phase high-performance liquid chromatography with charged aerosol detection of sucrose caprate regioisomers.

The use of step-down gradient elution profiles to improve separation of sucrose caprate regioisomers was investigated as part of the development of a quantitative RP-HPLC analysis method with charged aerosol detection. The investigation was conducted using design-of-experiments methodology and evaluated by multivariate regression analysis. This approach was proven to be useful for systematic method development in HPLC analysis. The gradient elution profiles were described by four variables - two concentration variables and two duration variables. The regression analysis showed that the concentration variables had the most significant effects on retention times, both as individual terms and as part of variable interactions. All the regioisomers exhibited non-linear relationships between eluent acetonitrile concentration and retention time with similar curvatures. Kendall rank correlation coefficients confirmed that the curvatures of the regioisomer curves were highly dependent on each other. Charged aerosol detection provided a mass-sensitivity of 10-100 ng for the sucrose fatty acid ester regioisomers. Resolution deviation (RD) was defined as an aggregate objective function for evaluating the separation of three specific sucrose caprate regioisomers with similar elution properties substituted at positions 6-, 3- and 1'-, respectively. The investigation resulted in the development of elution strategies for separation and quantitative RP-HPLC analysis of regioisomers of sucrose caprate with all eight sucrose caprate regioisomers successfully identified. Thus, resolutions above the level of adequacy for quantification, R(s)≥1.0, were achieved for all regioisomers, both with isocratic and gradient elution strategies. For isocratic elutions, the best separation was achieved with eluent acetonitrile concentration 34%. Gradient elution resulted in a similar RD, but decreased the analysis time by 7-28%. For the gradient resulting in the most desirable combination of separation and analysis time, the R(s)-values ranged from 1.31 to 6.82, and the analysis time was 24 min.