Takahiko Higasa

Comparison of degradation abilities of α- and β-amylases on raw starch granules.

The degradation abilities of α-amylase from Bacillus amyloliquefaciens and β-amylases from Bacillus cereus and soybean on raw starch granules from various botanical sources (potato, sweet potato, wheat, rice and corn) were examined by scanning electron microscopy. All the amylases showed different degradation patterns on starch granules. The α-amylase was more efficient than the β-amylases. α-Amylase showed both centrifugal and centripetal hydrolysis on corn, rice and wheat granules, but only centrifugal hydrolysis on potato granules. On the other hand, β-amylase moved very slowly on granules. The kinetic assays which explain the release of maltose were carried out at 12, 18 and 24 h. The rice granules were found to be the best substrate for enzymic hydrolysis by α and β-amylases. In addition, While Bacillus cereus β-amylase hydrolyzed corn granules efficiently at 45°C; soybean β-amylase was 60% less active than bacterial β-amylase at the same temperature.

Expression and accumulation of normal and modified soybean glycinins in potato tubers

Chimeric genes composed of the class I patatin promoter, A1aB1b preproglycinin cDNA and its modified versions (IV+4Met and V+4Met) containing four contiguous methionine codons and nopaline synthase gene terminator were inserted into the genome of potato by Agrobacterium-mediated transformation. Transcripts of the chimeric genes were detected specifically in tubers. Translational products were observed in parenchyma cells of the tubers of potato plants transformed with the normal and modified glycinin cDNAs. This distribution is consistent with the expression specificity of the patatin promoter. The expression levels of the normal and the modified glycinins were 0.2–1.0% of the total soluble tuber proteins. Both the normal and the modified glycinins expressed in the tubers were present as proglycinin trimers. These results indicate that the modifications in IV+4Met and V+4Met did not disrupt any stage in the biosynthetic process of glycinin in potato tubers.

Heat-induced Transparent Gel Formation of Bovine Serum Albumin

The formation of transparent gels by 6% bovine serum albumin (BSA), pH 7.5, was examined by one- and two-step heating methods. Heating of the BSA solutions at various NaCl concentrations produced transparent gels at 25-50mM NaCl and transparent sols at 0-20 mM NaCl (one-step heating method). The transparent sol obtained by heating without NaCl was reheated after mixing with various amounts of NaCl (two-step heating method I). The result was almost identical to that obtained by the one-step heating method. However, when the first heating was done with 10 mM NaCl, transparent gels were obtained over a wide range of NaCl concentrations with a second heating (two-step heating method II). Analyses of sols obtained at various NaCl concentrations by gel permeation chromatography and transmission electron microscopy showed the presence of linear polymers in the heated BSA sol (10 mM NaCl) and gel networks formed by the linear polymers (20 mM NaCl). The mechanism of transparent gel formation in BSA may be similar to that in ovalbumin.

Gelling Properties of Soybean β-Conglycinin Having Different Subunit Compositions

The effects of protein concentration, and heating temperature and time on the gelling properties of soybean β-conglycinin (7S globulins) lacking the α or α′ subunit were compared with those of 7S containing all three subunits (α, α′, and β) to determine whether each subunit contributes equally. In most of the conditions, the relative order of gel hardness was α′-lacking > 7S > α-lacking. From Fourier transform infrared studies, the secondary structure change after heating was very similar among the three samples; thus, the secondary structural change is not the reason for the differences in gel hardness. By using scanning electron microscopy, we observed differences in strand thickness and the density of the gel network among the three samples. These differences correlated well with the differences in gel hardness.

Heat-induced Transparent Gel from Hen Egg Lysozyme by a Two-step Heating Method.

The effects of ionic strength and dithiothreitol concentration, a reducing agent, on the heat-induced gelation of a lysozyme solution were examined at pH 7. Two different heating methods were used, one involving single heating, and the other, a two-step heating process in which a sample solution was heated twice under different medium conditions. With 7.5 mM dithiothreitol, a 5% lysozyme solution gave a transparent sol, a transparent gel, a translucent gel and a turbid gel after a single heating with 0, 40, 50, and 60mM NaCI, respectively. When the transparent sol obtained without NaCI was reheated with 50mM NaCI, it produced a firm and transparent gel. This gel was at least 6 times harder than the transparent lysozyme gel obtained by the one-step heating method. High-resolution scanning electron microscopy showed that the transparent gel produced by the two-step heating method had a fine network of linear polymers composed of heat-denatured lysozyme molecules.

α′ Subunit of soybean β-conglycinin forms complex with rice glutelin via a disulphide bond in transgenic rice seeds.

The α′ and β subunits of soybean β-conglycinin were expressed in rice seeds in order to improve the nutritional and physiological properties of rice as a food. The α′ subunit accumulated in rice seeds at a higher level than the β subunit, but no detectable difference in mRNA transcription level between subunits was observed. Sequential extraction results indicate that the α′ subunit formed one or more disulphide bonds with glutelin. Electron microscopic analysis showed that the α′ subunit and the β subunit were transported to PB-II together with glutelin. In mature transgenic seeds, the β subunit accumulated in low electron density regions in the periphery of PB-II, whereas the α′ subunit accumulated together with glutelin in high-density regions of the periphery. The subcellular localization of mutated α′ subunits lacking one cysteine residue in the N-terminal mature region (α′ΔCys1) or five cysteine residues in the pro and N-terminal mature regions (α′ΔCys5) were also examined. Low-density regions were formed in PB-II in mature seeds of transgenic rice expressing α′ΔCys 5 and α′ΔCys1. α′ΔCys5 was localized only in the low-density regions, whereas α′ΔCys1 was found in both low- and high-density regions. These results suggest that the α′ subunit could make a complex via one or more disulphide bonds with glutelin and accumulate together in PB-II of transgenic rice seeds.

Aggregation behaviour of bovine serum albumin as a cause of sauce liquid separation by heating

Heating white sauce with bovine serum albumin (BSA) at 90 °C caused the sauce to separate into aggregates and liquid phase, although this did not occur at 75 °C. Data from differential scanning calorimetry of BSA solution suggested that heat denaturation of BSA was insufficient at 75 °C but was complete at about 90 °C. Larger aggregates of BSA were formed by heating at 90 °C compared with 75 °C, as shown by gel permeation chromatography combined with a multi-angle laser light-scattering detector. Dynamic viscoelasticity measurement showed a higher storage modulus of BSA solution formed by heating at 90 °C than at 75 °C. Scanning electron microscopy revealed a random agglomerate structure of aggregates (spherical aggregates) obtained by 75 °C heating, while a well-developed network structure containing voids was observed after heating at 90 °C. These findings suggest that sauce liquid separation induced by heating at 90 °C is due to encapsulation and restriction of white sauce components by large structured aggregates of BSA containing voids. Aggregates generated at 75 °C were not sufficiently developed and structured; therefore there was no sauce liquid separation due to encapsulation and restriction of components. © 2000 Society of Chemical Industry

Proteinase Inhibitors in Plant Seed:Part II. Purification and Some Properties of Three Different Types of Inhibitors from Japanese Radish Seed ( Raphanus sativus )

Three different types of proteinase inhibitors, I, II and III, were fractionated from Japanese radish seed by repeated column chromatographies on SE- and CM-cellulose. The finally purified preparation of inhibitor III was found to be homogeneous by both chromatographic and electrophoretic analyses. All three of them strongly and stoichiometrically inhibited trypsin whereas they showed weak inhibition on other proteinases, such as chymotrypsin, Nagarse and Pronase. From nitrogen content and ultraviolet absorption spectra, each of the inhibitors I and III was confirmed to be a protein. The molecular weights of inhibitors I and III were calculated to be 8000 and 12,000, respectively. These inhibitors were stable at temperatures above 90°C in an acidic pH.