No abstract

The proglycinin synthesized in E. coli JM105 comprised approximately 20 % of the total bacterial proteins, with a yield of 39mg per liter of culture under the optimum cultivation conditions. The proglycinin was purified to homogeneity by salt precipitation, ion-exchange chromatography, and cryoprecipitation.The purified proglycinin self-assembled to a trimer with a secondary structure similar to that of the glycinin half-molecule from soybean seeds, and had properties of gel formation by heating and precipitation with calcium salt as the native glycinin and glycinin half-molecule do. This indicated that the E. coli expression system of glycinin CDNA may be used for the evaluation of the self-assembly and the food qualities of protein-engineered soybean proteins.

To determine which amino acid residues are essential for the catalytic activity of soybean P-amylase, deoxyoligonucleotide site-directed mutagenesis was employed against aspartyl, glutamyl, and cysteinyl residues located in highly conserved regions found in P-amylase family to date. Both substitution of aspartic acid at position 101 and that of glutamic acid at position 186 of the enzyme by neutral and acidic amino acids, respectively, led to the complete elimination of activity, but did not induce any significant changes in circular dichroic spectra or the binding affinity for cyclomaltohexaose, a substrate analogue.Taking account of the results obtained here, the above two amino acid residues are involved in the catalytic site of soybean P-amylase. The replacement of glutamic acid at position 345 decreased activity to below 6% of the non-mutant level, implying that this residue may also play a crucial role in /I-amylase activity, although it may not be involved at the catalytic site itself. In contrast, substitution of cysteinyl residue at position 95 by a serinyl residue led to a drastic reducing of the optimal temperature (from 50°C to 30"C), suggesting that this cysteinyl residue is responsible for the thermal stability of the enzyme.8-Amylase (1 ,4-a-D-glucan maltohydrolase) hydrolyzes a-l,4-glucosidic linkage of starch and glycogen with liberation of P-anomeric maltose from the non-reducing ends. This enzyme is found in many plant and bacteria species. Although the physicochemical properties of the enzymes from sweet potato, barley, and soybean have been well investigated [l], the catalytic mechanism of the enzyme has not yet been elucidated. Comparison of deduced sequences of P-amylases originating from plant and bacteria revealed three highly conserved sequences which very likely are involved in the segment assembly of the catalytic site of /I-amylase [2-41. In the case of a-amylase (1,4-a-~-glucan glucanohydrolase) that catalyzes the hydrolysis of 1,4-a-D-glucans, the aspartyl and glutamyl residues contained in highly conserved regions have been identified as catalytic residues by X-ray crystallography [5 -71 and by site-directed mutagenesis [8, 91. Although the action pattern of P-amylase differs from that of a-amylase and there is no significant similarity between the primary sequences, all of the aspartyl and glutamyl residues located in the conserved regions of P-amylase family can be considered possible catalytic residues. Recently, the results of affinity labeling of soybean P-amylase with 2,3-epoxy propyl a-D-glucopyranoside suggested that Glu186 was involved in the catalytic site [lo]. In addition, since the enzyme was inactivated by sulfhydryl reagents such as iodoacetamide, N-ethylmaleimide and mercuribenzoate [ l , 111, the cysteinyl residues located in the regions may also participate in the catalytic reaction.

Seaweed has been used in traditional cosmetics and as a herbal medicine in treatments for cough, boils, goiters, stomach ailments, and urinary diseases, and for reducing the incidence of tumors, ulcers, and headaches. Despite the fact that seaweeds are frequently used in the practice of human health, little is known about the role of seaweed in the context of inflammation. This study aimed to investigate the influence of Jeju endemic seaweed on a mouse macrophage cell line (RAW 264.7) under the stimulation of lipopolysaccharide (LPS). Ethyl acetate extracts obtained from 14 different kinds of Jeju seaweeds were screened for inhibitory effects on pro-inflammatory mediators. Our results revealed that extracts from five seaweeds, Laurencia okamurae, Grateloupia elliptica, Sargassum thunbergii, Gloiopeltis furcata, and Hizikia fusiformis, were potent inhibitors of the production of pro-inflammatory mediators such as nitric oxide (NO), prostaglandin E 2 (PGE 2 ), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Based on these results, the anti-inflammatory effects and low cell toxicity of these seaweed extracts suggest potential therapeutic applications in the regulation of the inflammatory response.