Paul Colonna

The gelation and crystallisation of amylopectin

Abstract A range of physical and chemical techniques, including viscometry, rheological measurements, dilatometry, turbidity measurements, X-ray diffraction, and differential scanning calorimetry, has been used to study the gelation of amylopectin. Gels form on cooling concentrated aqueous solutions to 1°. The development of gel stiffness is closely related to the association of amylopectin chains, as monitored by dilatometry and differential scanning calorimetry. X-Ray diffraction studies suggest that intermolecular association involves a crystallisation process. The association of amylopectin chains in the gel is substantial and is thermo-reversible at temperatures below 100°. Heterogenous acid hydrolysis of the gel followed by examination of the residue by gel-permeation chromatography showed that the associated regions contained branched fragments, the individual chains of which had a d.p. of 15. The combined data suggest that the amylopectin molecules associate by crystallisation of the branches with d.p. 15 to form a network.

Online determination of structural properties and observation of deviations from power law behavior.

Synthetic amyloses, pullulans, phytoglycogen, rabbit liver glycogen, oyster glycogen, and dextrans were studied using high-performance size-exclusion chromatography combined with multiangle laser light scattering (MALLS) and online quasi-elastic light scattering (QELS), which provided the RH distributions up to 65 nm. Different structural parameters were extracted from entire molar mass distributions, including the slope of the log-log plot of R H(i) versus M(i)and the rho(i )= R(Gi)/R(Hi)ratio. This approach enabled to observe deviations from the De Gennes scaling law concept. Evidences that the power laws do not obey the general universality were furnished by the observation of strong deviations in the relation between radii and molar masses for the branched polysaccharides, a decrease of rho-parameter with molar mass toward values much lower than theoretically expected, and the fact that relation between rho-parameter and apparent segment density did not show the expected power law decrease with an exponent of 1/3. The universality of scaling behavior seems no longer to be realized if structural heterogeneity governs the system.

In depth study of a new highly efficient raw starch hydrolyzing α-amylase from Rhizomucor sp.

A new α-amylase from Rhizomucor sp. (RA) was studied in detail due to its very efficient hydrolysis of raw starch granules at low temperature (32 °C). RA contains a starch binding domain (SBD) connected to the core amylase catalytic domain by a O-glycosylated linker. The mode of degradation of native maize starch granules and, in particular, the changes in the starch structure during the hydrolysis, was monitored for hydrolysis of raw starch at concentrations varying between 0.1 and 31%. RA was compared to porcine pancreatic α-amylase (PPA), which has been widely studied either on resistant starch or as a model enzyme in solid starch hydrolysis studies. RA is particularly efficient on native maize starch and release glucose only. The hydrolysis rate reaches 75% for a 31% starch solution and is complete at 0.1% starch concentration. The final hydrolysis rate was dependent on both starch concentration and enzyme amount applied. RA is also very efficient in hydrolyzing the crystalline domains in the maize starch granule. The major A-type crystalline structure is more rapidly degraded than amorphous domains in the first stages of hydrolysis. This is in agreement with the observed preferential hydrolysis of amylopectin, the starch constituent that forms the backbone of the crystalline part of the granule. Amylose-lipid complexes present in most cereal starches are degraded in a second stage, yielding amylose fragments that then reassociate into B-type crystalline structures, forming the final resistant fraction.

In situ tracking of enzymatic breakdown of starch granules by synchrotron UV fluorescence microscopy.

Synchrotron UV fluorescence microscopy was used for the first time to visualize the adsorption and diffusion of an enzyme while degrading a solid substrate. The degradation pathway of single starch granules by two amylases, optimized for biofuel production and industrial starch hydrolysis, was followed by tryptophan fluorescence (excitation at 280 nm, emission filter at 300-400 nm) and visible light imaging. Thus, both the adsorption of enzyme onto starch granules at 283 nm resolution and the resulting morphological changes were recorded at different stages of hydrolysis. It is the first time that amylases were localized on starch without staining or adding a fluorescent probe at such high resolution. This technique presents a very high potential for imaging proteins in complex systems. Its sensitivity was demonstrated by the detection of GBSS (the granular bound starch synthase) at high recording times, GBSS being present at very low levels in maize starch granules.