Fully automated chip‐based negative mode nanoelectrospray mass spectrometry of fructooligosaccharides produced by heterologously expressed levansucrase from Pseudomonassyringae pv. tomato DC3000

Abstract

Pseudomonas syringae pathovars possess multiple levansucrases with still unclear specific roles for bacteria. We have cloned and expressed three levansucrase genes, lsc1, lsc2 and lsc3, from P. syringae DC3000 in Escherichia coli. Levansucrases synthesize a high molecular weight fructan polymer, levan, from sucrose and in the case of some levansucrases, fructooligosaccharides (FOS) with potential prebiotic effects are also produced. The ability of purified Lsc3 protein of DC3000 to synthesize FOS was tested using prolonged incubation time and varied concentrations of sugar substrates. Thin-layer chromatography (TLC) analysis of reaction products disclosed formation of FOS from both sucrose and raffinose, revealing a new catalytic property for P. syringae levansucrases. In order to analyze Lsc3-produced FOS in underivatized form, we optimized a novel method recently introduced in carbohydrate research, based on fully automated chip-based nanoelectrospray ionization (nanoESI) high-capacity ion trap mass spectrometry (HCT-MS). Uding chip-based nanoESI MS in negative ion mode, FOS, with degrees of polymerization up to five, were detected in reaction mixtures of Lsc3 with sucrose and raffinose. For confirmation, further structural analysis by tandem mass spectrometry (MS/MS) employing collision-induced dissociation at low energies was performed. To validate the method, commercial inulin-derived FOS preparations Orafti P95 and Orafti Synergy1, which are currently used as prebiotics, were used as controls. By chip-based nanoESI HCT-MS, similar FOS distribution was observed in these reference mixtures. Thereby, the obtained data allowed us to postulate that FOS produced by the Lsc3 protein of P. syringae DC3000 may be prebiotic as well.

Levansucrases of a Pseudomonas syringae pathovar as catalysts for the synthesis of potentially prebiotic oligo- and polysaccharides

“…Levansucrases of Pst (Lsc2, Lsc3) and Pca (LscA) produce from sucrose aside of levan also FOS with DP 3-7 [74,84,85,87,89]. Polymerizing properties of two studied levansucrases of Pst, Lsc2 and Lsc3 were reported to be similar that agrees with their very high (95%) sequence identity [85,89].…”

“…The protein was also characterized as highly stable during long-term storage, retaining $50% of its activity after 140 days of incubation at 378C [90]. Importantly, Lsc3 could perfectly react with its substrates even in distilled water [87]. Taking into account a high catalytic turnover and stress tolerance, the Lsc3 is a good candidate for biotechnological applications.…”

“…The main conditions largely affecting the spectrum and yield of polymerization products of Lsc3 and Lsc2 proteins were substrate concentration and reaction temperature [84,87,94], but the amount of added catalyst had also an effect [94]. It is well known that all levansucrases possess two distinct activities: substrate (sucrose) cleavage and transfructosylation reactions [46,82,85].…”

“…The Lsc2 and Lsc3 of Pst have been produced as heterologous proteins in E coli and after the purification thoroughly biochemically characterized [74,77,84,85,[87][88][89][90]. Table 1 reviews some biochemical parameters of above-mentioned levansucrases.…”

Qualitative and quantitative analysis of gallic acid in Alchemilla vulgaris, Allium ursinum, Acorus calamus and Solidago virga-aurea by chip-electrospray ionization mass spectrometry and high performance liquid chromatography

“…Mass spectrometry was conducted on a high capacity ion trap instrument (HCT MS) from Bruker Daltonics, Bremen, Germany, in laboratory coupled [14][15][16] with the NanoMate robot for automatic infusion of samples by chip-electrospray (NanoMate TM ) from Advion Biosciences, Norfolk, UK. HCT MS instrument was controlled and manipulated through the Esquire Control 6.1.512 software and experimental data were processed via Data Analysis 3.4.179.…”