Rheological and physicochemical properties of starches from moist- and dry-type sweetpotatoes.

Although starch makes up from 50 to 70% of sweetpotato (SP) dry matter, its role in cooked texture is unknown. The purpose of this research was to characterize raw starches isolated from SP cultivars and experimental selections (C/S) with a wide range of textural properties when cooked and to investigate the relationship between textural properties of the cooked roots and characteristics of the isolated starches. Shear stress measured by uniaxial compression of cooked SP cylinders served as an objective measure of SP texture. Starches were isolated from C/S representing three SP texture types: moist (Jewel and Beauregard); intermediate (NC10-28 and NC2-26); and dry (NC6-30 and NC8-22). The following parameters of isolated starches were measured: amylose content by colorimetric and differential scanning calorimetric (DSC) methods; swelling power, solubility, gelatinization enthalpy (DeltaH), and pasting properties by Brabender amylograph (BA) and rapid viscoanalyzer (RVA). Pasting temperatures for SP C/S measured by BA and RVA were significantly correlated. Due to high shear degradation in RVA, RVA viscosities of starch suspensions decreased as much as 40% during cooking at 95 degrees C, whereas the BA viscosities changed little at this temperature. There were no statistically significant differences among the C/S for amylose or DeltaH. However, significant C/S differences in swelling power, solubility, and pasting properties were observed. Although differences in some rheological and physical properties were observed for C/S starches, shear stress was statistically correlated only with DSC onset temperature (r = 0.78), indicating that factors other than the properties measured on isolated starches are mainly responsible for the texture of cooked SP C/S.

Expression of Escherichia coli glycogen synthase in the tubers of transgenic potatoes (Solanum tuberosum) results in a highly branched starch.

A chimeric gene containing the patatin promoter and the transit-peptide region of the small-subunit carboxylase gene was utilized to direct expression of Escherichia coli glycogen synthase (glgA) to potato (Solanum tuberosum) tuber amyloplasts. Expression of the glgA gene product in tuber amyloplasts was between 0.007 and 0.028% of total protein in independent potato lines as determined by immunoblot analysis. Tubers from four transgenic potato lines were found to have a lowered specific gravity, a 30 to 50% reduction in the percentage of starch, and a decreased amylose/amylopectin ratio. Total soluble sugar content in these selected lines was increased by approximately 80%. Analysis of the starch from these potato lines also indicated a reduced phosphorous content. A very high degree of branching of the amylopectin fraction was detected by comparison of high and low molecular weight carbohydrate chains after debranching with isoamylase and corresponding high-performance liquid chromatography analysis of the products. Brabender viscoamylograph analysis and differential scanning calorimetry of the starches obtained from these transgenic potato lines also indicate a composition and structure much different from typical potato starch. Brabender analysis yielded very low stable paste viscosity values (about 30% of control values), whereas differential scanning calorimetry values indicated reduced enthalpy and gelatinization properties. The above parameters indicate a novel potato starch based on expression of the glgA E. coli gene product in transgenic potato.

Phosphotransbutyrylase from Clostridium acetobutylicum ATCC 824 and its role in acidogenesis.

Phosphotransbutyrylase (phosphate butyryltransferase [EC 2.3.1.19]) from Clostridium acetobutylicum ATCC 824 was purified approximately 200-fold to homogeneity with a yield of 13%. Steps used in the purification procedure were fractional precipitation with (NH4)2SO4, Phenyl Sepharose CL-4B chromatography, DEAE-Sephacel chromatography, high-pressure liquid chromatography with an anion-exchange column, and high-pressure liquid chromatography with a hydrophobic-interaction column. Gel filtration and denaturing gel electrophoresis data were consistent with a native enzyme having eight 31,000-molecular-weight subunits. Within the physiological range of pH 5.5 to 7, the enzyme was very sensitive to pH change in the butyryl phosphate-forming direction and showed virtually no activity below pH 6. This finding indicates that a change in internal pH may be one important factor in the regulation of the enzyme. The enzyme was less sensitive to pH change in the reverse direction. The enzyme could use a number of substrates in addition to butyryl coenzyme A (butyryl-CoA) but had the highest relative activity with butyryl-CoA, isovaleryl-CoA, and valeryl-CoA. The Km values at 30 degrees C and pH 8.0 for butyryl-CoA, phosphate, butyryl phosphate, and CoASH (reduced form of CoA) were 0.11, 14, 0.26, and 0.077 mM, respectively. Results of product inhibition studies were consistent with a random Bi Bi binding mechanism in which phosphate binds at more than one site.

Coenzyme A transferase from Clostridium acetobutylicum ATCC 824 and its role in the uptake of acids.

Coenzyme A (CoA) transferase from Clostridium acetobutylicum ATCC 824 was purified 81-fold to homogeneity. This enzyme was stable in the presence of 0.5 M ammonium sulfate and 20% (vol/vol) glycerol, whereas activity was rapidly lost in the absence of these stabilizers. The kinetic binding mechanism was Ping Pong Bi Bi, and the Km values at pH 7.5 and 30 degrees C for acetate, propionate, and butyrate were, respectively, 1,200, 1,000, and 660 mM, while the Km value for acetoacetyl-CoA ranged from about 7 to 56 microM, depending on the acid substrate. The Km values for butyrate and acetate were high relative to the intracellular concentrations of these species; consequently, in vivo enzyme activity is expected to be sensitive to changes in those concentrations. In addition to the carboxylic acids listed above, this CoA transferase was able to convert valerate, isobutyrate, and crotonate; however, the conversion of formate, n-caproate, and isovalerate was not detected. The acetate and butyrate conversion reactions in vitro were inhibited by physiological levels of acetone and butanol, and this may be another factor in the in vivo regulation of enzyme activity. The optimum pH of acetate conversion was broad, with at least 80% of maximal activity from pH 5.9 to greater than 7.8. The purified enzyme was a heterotetramer with subunit molecular weights of about 23,000 and 25,000.

Thiolase from Clostridium acetobutylicum ATCC 824 and Its Role in the Synthesis of Acids and Solvents.

Thiolase (acetyl-coenzyme A [CoA] acetyltransferase, E.C. 2.3.1.19) from Clostridium acetobutylicum ATCC 824 has been purified 70-fold to homogeneity. Unlike the thiolase in Clostridium pasteurianum, this thiolase has high relative activity throughout the physiological range of internal pH of 5.5 to 7.0, indicating that change in internal pH during acid production is not an important factor in the regulation of this thiolase. In the condensation direction, the thiolase is inhibited by micromolar levels of CoA, and this may be an important factor in modulating the net condensation of acetyl-CoA to acetoacetyl-CoA. Other cofactors and metabolites that were tested and shown to be inhibitors are ATP and butyryl-CoA. The native enzyme consists of four 44,000-molecular-weight subunits. The kinetic binding mechanism is ping-pong. The K(m) value for acetyl-CoA is 0.27 mM at 30 degrees C and pH 7.4. The K(m) values for sulfhydryl-CoA and acetoacetyl-CoA are, respectively, 0.0048 and 0.032 mM at 30 degrees C and pH 8.0. The active site apparently contains a sulfhydryl group, but unlike other thiolases, this thiolase is relatively stable in the presence of 5,5'-dithiobis(2-nitrobenzoic acid). Studies of thiolase specific activity under various types of continuous fermentations show that regulation of this enzyme at both the genetic and enzyme levels is important.