3D imaging of enzymes working in situ.

Today, development of slowly digestible food with positive health impact and production of biofuels is a matter of intense research. The latter is achieved via enzymatic hydrolysis of starch or biomass such as lignocellulose. Free label imaging, using UV autofluorescence, provides a great tool to follow one single enzyme when acting on a non-UV-fluorescent substrate. In this article, we report synchrotron DUV fluorescence in 3-dimensional imaging to visualize in situ the diffusion of enzymes on solid substrate. The degradation pathway of single starch granules by two amylases optimized for biofuel production and industrial starch hydrolysis was followed by tryptophan autofluorescence (excitation at 280 nm, emission filter at 350 nm). The new setup has been specially designed and developed for a 3D representation of the enzyme-substrate interaction during hydrolysis. Thus, this tool is particularly effective for improving knowledge and understanding of enzymatic hydrolysis of solid substrates such as starch and lignocellulosic biomass. It could open up the way to new routes in the field of green chemistry and sustainable development, that is, in biotechnology, biorefining, or biofuels.

Structural, physicochemical, and pasting properties of starches from potato plants with repressed r1-gene.

The aim of this work was to investigate the effect on starch molecular and physicochemical properties of down regulation of the R1 protein in potato (Solanum tuberosum L. cv. "Dianella") tubers. Most prominent is a 90% suppression of the phosphate content in the isolated potato tuber starch. The amylopectin chain length distribution profile as determined by HPAEC/PAD was not affected, but the amylose content was increased in the most down-regulated plants. The pasting properties of the transgenic starch revealed a pronounced decrease in peak viscosity and increased setback viscosity as measured using a rapid Visco analyzer. The starch gels displayed an increased hardness and stickiness with a maximum at 1.7 nmol of Glc-6P mg-1 of starch compared to the control lines. At very low phosphate levels (1.4 nmol of Glc-6P mg-1 of starch), the gel hardness was decreased as a result of increased gel brittleness. The increase in gel brittleness is believed to be an effect of an increased proportion of free amylopectin blocklets in the starch as determined by SEC/RI. The possible links between the structural and physicochemical parameters are discussed.

Phosphorylated alpha(1-->4)Glucans as substrate for potato starch-branching enzyme I

The possible involvement of potato (Solanum tuberosum L.) starch-branching enzyme I (PSBE-I) in the in vivo synthesis of phosphorylated amylopectin was investigated in in vitro experiments with isolated PSBE-I using 33P-labeled phosphorylated and 3H end-labeled nonphosphorylated alpha(1-->4)glucans as the substrates. From these radiolabeled substrates PSBE-I was shown to catalyze the formation of dual-labeled (3H/33P) phosphorylated branched polysaccharides with an average degree of polymerization of 80 to 85. The relatively high molecular mass indicated that the product was the result of multiple chain-transfer reactions. The presence of alpha(1-->6) branch points was documented by isoamylase treatment and anion-exchange chromatography. Although the initial steps of the in vivo mechanism responsible for phosphorylation of potato starch remains elusive, the present study demonstrates that the enzyme machinery available in potato has the ability to incorporate phosphorylated alpha(1-->4)glucans into neutral polysaccharides in an interchain catalytic reaction. Potato mini tubers synthesized phosphorylated starch from exogenously supplied 33PO43- and [U-14C]Glc at rates 4 times higher than those previously obtained using tubers from fully grown potato plants. This system was more reproducible compared with soil-grown tubers and was therefore used for preparation of 33P-labeled phosphorylated alpha(1-->4)glucan chains.

Alpha-glucan binding of potato-tuber starch-branching enzyme I as determined by tryptophan fluorescence quenching, affinity electrophoresis and steady-state kinetics.

The affinity of potato tuber starch-branching enzyme-I (PSBE-I) for various linear malto-oligosaccharides, cyclodextrins, (CDs) and macromolecular alpha-glucans was investigated by alpha-glucan induced fluorescence quenching of intrinsic PSBE-I tryptophan residues and by affinity electrophoresis. alpha-Glucan binding was characterised by distinct shifts towards shorter wavelengths of the PSBE-I fluorescence emission spectrum and by concomitant reductions in fluorescence intensity. The magnitudes of both the maximum shift in emission spectrum and reduction in fluorescence intensity were dependent on the alpha-glucan ligands used. Maximum Kd for a range of linear malto-oligosaccharides analysed was 0.13 mM as found at a degree of polymerisation (DP) of 13. Large differences in dissociation constants were measured for CDs with DP 6 (alpha-CD, 6.0 mM), DP 7 (beta-CD, 0.25 mM) and DP 8 (gamma-CD, 0.67 microM). The high-molecular-mass alpha-glucans amylose and amylopectin, both substrates for PSBE-I, showed apparent affinities of 0.018 and 0.066 mg/ml, respectively. Small linear and cyclic oligosaccharides competed with amylopectin in the affinity electrophoresis system and they were also competitive inhibitors for PSBE-I activity. The affinities for oligosaccharides as measured by competition were, however, about 10-fold lower than as measured by fluorescence quenching suggesting the existence of a separate oligosaccharide binding site on PSBE-I. Affinity electrophoresis revealed multiform heterogeneity in the enzyme preparation with respect to alpha-glucan interaction.