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The uptake system for citrate is induced in Bacillus subtilis W23 by growth in the presence of citrate and only membrane vesicles isolated from these cells show energy-dependent citrate uptake. Citrate transport in membrane vesicles is strictly dependent on the presence of divalent cations such as Mg", Mn", Zn2+, Ba2+, Be2+, Ca2+, Cu2+, Co2+ or Ni2+. The initial rate of citrate transport increases with the divalent cation concentration up to a maximum. The maximum initial rate of citrate uptake is reached with 2 mM Mg2+.The cations form stable chelates with citrate. The metal citrate complex is the transported solute. This is demonstrated for citrate uptake in the presence of Ca2+. Membrane vesicles from citrate-grown cells accumulate Ca2+ and citrate only if both solutes are present. Citrate and Ca2+ are accumulated in equimolar quantities. The uptake of Ca2+ but not of citrate is inhibited by Mg2+.Uptake of the metal-citrate complex is inhibited by the uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone and in the presence of K + ions by valinomycin and nigericin. The inhibitory effects correlate with the effects observed on the components of the proton-motive force, indicating that the proton-motive force is a driving force for metal-citrate transport. The number of protons (n) symported with the metal-citrate complex has been determined under different experimental conditions from the steady state levels of citrate accumulation, the electrical potential and pH gradient. This number varies from 1 at pH 4.7 to 2 at pH 8.0. This latter system has been studied more extensively in whole cells of B. suhtilis [5 -71. In order to avoid metabolism of the substrate a non-metabolizable citrate analogue 2-fluoro-L-erythro-citrate was used [7] or the studies were performed with an aconitase-negative strain of B. subtilis [5]. These studies indicate that uptake of citrate is mediated by an inducible transport system with a rather low affinity (Kapp 2300 -550 pM) [5,6]. Uptake of citrate appears to depend on the presence of divalent metals, preferably Mg2+.Since citrate is trivalent negatively charged at neutral pH (pK1 = 3.14, pK2 = 4.77, pK3 = 6.39) and forms a stable complex with divalent cations it was of interest to investigate in membrane vesicles the properties of the citrate transport system and to study the role of the proton-motive force in this translocation process. MATERIALS AND METHODS Cell Growth and Preparation of Membrane VesiclesBacillus subtilis W23 was grown at 37°C with vigorous aeration in the following media: 0.8 % trypton (Difco), 0.5 % NaCI, 25 mM KC1 and 10 mM sodium citrate, or 1.4%. 7 HzO, 0.5 mg/l FeS04, 0.1 ml/l Vishniac solution supKzHP04, 0.6 % KHzPO4, 0.2 (NH4)2SO4, 0.025 % MgS04 plemented with either 25 mM sodium citrate and 10 mM MgS04 or 1.5 % glucose.Logarithmically growing cells were harvested at an absorbance at 660 nm of 0.7-0.9. Membrane vesicles were prepared as described by Bisschop and Konings [8]. Transport AssaysTransport experiments were performed at 25 "C in a final ...

Granular potato starch and amylopectin potato starch were methylated in aqueous suspension to molar substitutions (MS) up to 0.29. A method was developed to determine the MS of both branched and linear regions. After exhaustive digestion of the methylated starches with -amylase, the highly branched fraction with a degree of polymerisation (DP) > 8 was separated from the linear oligomers by selective precipitation of the former in methanol. The substitution levels of branched and linear regions were determined. It was found that methylation takes place preferably at the branched regions of amylopectin and that amylose is higher substituted than linear regions of amylopectin. The distribution of methyl substituents in trimers and tetramers was determined by FABMS and compared to the outcome of a statistically random distribution. The results provided evidence for heterogeneous substituent distributions. Quanti®cation of the degree of heterogeneity of the branched and linear regions showed a much larger deviation from random distribution in the linear regions. #

The hydrolysis of starch to low‐molecular‐weight products (normally characterised by their dextrose equivalent (DE), which is directly related to the number‐average molecular mass) was studied at different temperatures. Amylopectin potato starch, lacking amylose, was selected because of its low tendency towards retrogradation at lower temperatures. Bacillus licheniformis α‐amylase was added to 10% [w/w] gelatinised starch solutions. The hydrolysis experiments were done at 50, 70, and 90°C. Samples were taken at defined DE values and these were analysed with respect to their saccharide composition. At the same DE the oligosaccharide composition depended on the hydrolysis temperature. This implies that at the same net number of bonds hydrolysed by the enzyme, the saccharide composition was different. The hydrolysis temperature also influenced the initial overall molecular‐weight distribution. Higher temperatures led to a more homogenous molecular weight distribution. Similar effects were observed for α‐amylases from other microbial sources such as Bacillus amyloliquefaciens and Bacillus stearothermophilus. Varying the pH (5.1, 6.2, and 7.6) at 70°C did not significantly influence the saccharide composition obtained during B. licheniformis α‐amylase hydrolysis. The underlying mechanisms for B. licheniformis α‐amylase were studied using pure linear oligosaccharides, ranging from maltotriose to maltoheptaose as substrates. Activation energies for the hydrolysis of individual oligosaccharides were calculated from Arrhenius plots at 60, 70, 80, and 90°C. Oligosaccharides with a degree of polymerisation exceeding that of the substrate could be detected. The contribution of these oligosaccharides increased as the degree of polymerisation of the substrate decreased and the temperature of hydrolysis increased. The product specificity decreased with increasing temperature of hydrolysis, which led to a more equal distribution between the possible products formed. Calculations with the subsite map as determined for the closely related α‐amylase from B. amyloliquefaciens reconfirmed this finding of a decreased substrate specificity with increased temperature of hydrolysis. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 344–355, 1999.

Granular potato starch and amylopectin potato starch were methylated in aqueous suspension with dimethyl sulfate to molar substitution (MS) up to 0.29. The percentage of amorphous starch compared with crystalline domains increased with increasing MS. Prolonged treatment of these methylated starches with hydrochloric acid below the swelling temperature resulted in the release of D-glucose and small D-glucose-oligomers from the amorphous domains. The granular structure was maintained during the acidic treatment, indicating that the crystalline lamellae were less affected by acid. The amorphous domains contained on average about twice as many substituents per glucose unit as the remaining crystalline network. The distributions of methyl substituents in trimers and tetramers, released from amorphous domains and prepared from crystalline fractions, were determined by FABMS and compared to the outcome of a statistically random distribution. Quantification of the degree of heterogeneity of the thus-obtained trimers and tetramers showed a much larger deviation from random substitution in the crystalline fractions compared with the amorphous ones. These results are in agreement with our previous study that describes substitution patterns in branched and linear regions of methylated starch granules.