Makoto Kawabata

Purification of ginger proteases by DEAE-Sepharose and isoelectric focusing

Ginger proteases in ginger rhizome (Zingiber officinale roscoe) were extracted from the ginger acetone powder and purified on DEAE-Sepharose and Sephadex G-75 columns. Before the purification, excess p-chloromercuribenzoate was added to the enzymes to prevent their autodigestion. The mercuribenzoate-proteases were further purified and fractionated by isoelectric focusing in Ampholine of pH 3-10 or pH 4-6. The proteases were fractionated into three components by the isoelectric focusing, having pI value of 4.5, 4.6 and 4.8 respectively. All these proteases had a molecular mass of 29,000 as measured by SDS-polyacrylamide gel electrophoresis and by TSK G2000SW XL gel chromatography. The Ampholine in the purified enzymes can quickly be removed by the gel chromatography of TSK G2000SW. Some divalent metal ions, such as Hg2+, Cu2+, Cd2+, and Zn2+, strongly inhibited these purified enzymes.

Oxidative Stability of Sardine Oil Embedded in Spray-dried Egg White Powder and Its Use for n-3 Unsaturated Fatty Acid Fortification of Cookies

Fine droplets of sardine oil either embedded in or covered with egg white powder, which had been prepared from an oil-in-water emulsion containing 10% protein-oil (9:1) mixture by spray-drying and freeze-drying, were examined for their oxidative stability during storage under moderate conditions (RH = 45-55%, 40°C). The antioxidative effect was estimated by measuring the peroxide value as well as the residual unsaturated fatty acid (mainly eicosapentaenoic and docosahexaenoic acids). Microencapsulation of the oil droplets with albumen by freeze-drying was not so effective in stabilizing against oxidation as that by spray-drying, probably because of the porosity of a protein coating on oil or leakage of oil through crevices. As a practical application of powdered sardine oil (i.e., entrapped in spray-dried albumen particles), plain cookies and those enriched with the oil were baked, and any difference in taste between them was evaluated by a paired preference test assessed by 32 amateur panelists. Sardine oil fortification of the cookies was judged not to affect their quality from the results of the sensory test. Spray-dried egg white powder inclusive of sardine oil was stable during prolonged storage, so that its use would be favorable for supplying an n-3 series of polyunsaturated fatty acids.

Improved chromatographic purification of human and bovine type V collagen sub-molecular species and their subunit chains from conventional crude preparations Application to cell-substratum adhesion assay for human umbilical vien endothelial cell

Two human type V collagen sub-molecular species, designated [alpha 1(V)]2 alpha 2(V) and alpha 1(V)alpha 2(V)alpha 3(V), were purified chromatographically from a commercially available preparation, in which cystine-rich collagenous contaminants were contained, with a column packed with Fractogel EMD SO3-. From bovine crude preparations, the [alpha 1(V)]2 alpha 2(V) form free from the collagenous contaminants was purified. Type V collagen subunit chains were isolated from each type V collagen molecule by anion-exchange HPLC with a Bakerbond PEI Scout column. The highly purified human type V collagen molecules and their subunit chains were used to examine the inhibitory effect on human umbilical vein endothelial cell proliferation. It was confirmed that the alpha 1(V) chain has inhibitory activity and it was found that the inhibitory effect of the [alpha 1(V)]2 alpha 2(V) form is stronger than that of the alpha 1(V)alpha 2(V)alpha 3(V) form and that the alpha 3(V) chain has no inhibitory activity.

Simple and rapid chromatographic purification of type V collagen from a pepsin digest of porcine intestinal connective tissue, an unmanageable starting material for conventional column chromatography

A chromatographic method is described for purification of type V collagen, a minor constituent in extracellular matrix, from a pepsin digest of porcine intestinal connective tissue. The starting material was a viscous and turbid solution even after centrifugation. Direct application of the sample to a commercially available DEAE-cellulose column resulted in clogging. On the other hand, type V collagen, [alpha1(V)](2)alpha2(V) form, was successfully captured by a filter paper-based DEAE-cellulose column chromatography and purified by a subsequent commercially available cation-exchange medium without clogging. This is a vast improvement over previously described salt fractionation methods.

Direct fractionation of proteins in particle-containing feedstocks by a filter paper pieces-based DEAE-cellulose column chromatography: Rapid, robust and low-cost capturing procedure for protein

Filter paper pieces-based (FPB) DEAE-celluloses was prepared for direct fractionation of proteins in particle-containing feedstocks. FPB DEAE-cellulose has a protein binding capacity equivalent to that of commercially available DEAE-cellulose. Crude extracts from porcine intestine and kiwi fruit pulp, which were unmanageable by commercially available chromatographic media due to rapid clothing, could be directly fractionated with FPB DEAE-cellulose column. In addition, effluents from an FPB DEAE-cellulose column were extensively clarified. The present approach can be used as a rapid, robust and low-cost capturing step for protein from particle-containing feedstocks.

Studies on Egg White Protein: Heterogeneity of Ovomucoid

The carbohydrate of ovomucoid was analyzed for components I, II, III and IV which were, fractionated by CMC-column chromatography. The total hexose content and the molar ratio of d-mannose to d-galactose (4:1) were identical in each component, but the d-glucosamine and sialic acid contents were found to be higher in components I and II (both are trypsin inhibitors) compared with components III and IV (both are apo-proteins of flavomucoid). The amino acid composition of each component of ovomucoid varied considerably. There were remarkable differences in the amino acid composition between components I and II, both had an antitryptic activity. The N-terminal amino acid of components I and II was alanine and in the case of components III and IV, threonine was found on the N-terminal. The free carboxylic residue of sialic acid was found to be responsible for the negative charge of ovomucoid, and its electrophoretic heterogeneity was reaffirmed by paper electrophoresis. It is evident from the ultracentrifugal ...