Studies on Browning Reactions between Sugars and Amino Compounds

N-d-Glucosyl-p-aminobenzoic acid has been found to form melanoidins in methanol solution acidified with hydrogen chloride at 25°. From the reaction mixture 5-hydroxymethyl-furfural (HMF) has been isolated and identified as its 2,4-dinitrophenylhydrazone. So, comparisons between the browning reaction of HMF or furfural with aromatic amine and that of the corresponding n-glycosides have been made under the same condition. From the results obtained, it has been shown that, under the described condition, furfural is almost inactive for browning, while on the contrary, HMF is active and plays an important role in the browning reaction.     

Ribonuclease in Bovine Milk

The RNase (EC 2.7.7.16) in bovine milk was partially purified from milk whey. The basic properties and the hydrolyzing specificity of this enzyme were studied. This enzyme was heat stable. When RNA was used as the substrate, the pH optimum was 7.5 and 3′- nucleotides were produced, but the extent of the hydrolysis stopped at 31 per cent and core was left. C! and U! were hydrolyzed but purine cyclic nucleotides were not. Poly U was digested to 3′-uridylic acid passing through U! but poly A was not. These properties are quite similar to pancreatic RNase.     

Purification and Properties of Methanol Dehydrogenase and Aldehyde Dehydrogenase from Methylobacillus glycogenes

Methanol dehydrogenase (MDH) and aldehyde dehydrogenase (ALDH) were purified to a homogenous state from Methylobacillus glycogenes, an obligate methylotroph. MDH (M_r 140,000) was composed of two different subunits (M_r 60,000 and 9,000) forming an α₂β₂ structure. MDH was indicated as a metalloquinoprotein containing one atom of calcium (Ca) per enzyme molecule. Binding of Ca was so tight that it was hard to remove Ca completely without denaturation of enzyme protein. A partially resolved enzyme resumed its original enzyme activity upon exogenous addition of Ca. Purified ALDH (M_r 144,000) was composed of two identical subunits of molecular mass of 72,000. ALDH was proved to be a quinoprotein in which PQQ is bound covalently.     

Applying Proteolytic Enzymes on Soybean

An indole derivative having blue fluorescence was produced in cooked soybean digested at 37°C for 24 hr with an acid proteinase Molsin (optimum pH: 2.8) from Aspergillus saitoi or a usual acid proteinase pepsin (optimum pH: 1.6) from beef stomach. This indole derivative was identical with a condensation product from l-tryptophan and n-hexanal. Based on MS, NMR, IR and UV spectrometry, the condensation product was identified as l-pentyl-2, 3, 4, 9-tetrahydro-lH-pyrido [3, 4-b]-indole-3-carboxylic acid [trivial name: 1-pentyl-l, 2, 3, 4-tetrahydro-2-carboline carboxylic acid-(3)].

Data were presented of the formation of the above indole derivative and of the resulting consumption of l-tryptophan and n-hexanal.

The possible ocurrence of the formation of Harmala alkaloids, i.e. 2-carboline derivatives, through in vitro digestion of soybean with acid proteinases was discussed.

A carbonyl-trapping ability of l-tryptophan was suggested.     

Applying Proteolytic Enzymes on Soybean

Soy proteins were incubated with a microbial acid protease (Molsin) under the following condition: substrate concentration, 1%; enzyme-substrate ratio (by weight), 1/100; pH, 2.8; and temperature, 40°C—flavor components and related impurities are removable from crude soy-protein concentrates by their incubation for 2 hr under the above condition. The acid-precipitated fraction of soy protein incubated for 2 hr with Molsin (i.e. 2 hr-proteolyzate) showed the following composition: 10% trichloroacetic acid (TCA) insoluble fraction, 47.52%; 10% TCA soluble peptide fraction, 52.02%; and free amino acid fraction, 0.46%. Gel filtration of the 2 hr-proteolyzate gave an elution pattern showing its molecular weight distribution.

In the process of the incubation of the acid-precipitated protein, the 10% TCA insoluble fraction showed increase in amino nitrogen content, its solubility in a phosphate buffer increased to change at 6 hr, and a hydrophobic amino acid share in this fraction increased gradually.

In vitro digestibility of the acid-precipitated fraction were improved and the lipoxygenase activity in this fraction decreased through the Molsin treatment.

Ultracentrifugal analysis showed a decreasing tendency of the cold-insoluble fraction of soy protein during its incubation with Molsin. Optical rotatory dispersion and circular dichroism study elucidated conformational changes in this fraction during its incubation either with or without Molsin.     

A New Enzymatic Microdetermination Procedure for Ethanol with Particulate Alcohol Dehydrogenase from Acetic Acid Bacteria

A new enzymatic method for microdetermination of ethanol has been established with particulate alcohol dehydrogenase from acetic acid bacteria and applied to the practical purposes. The enzyme had an optimum pH for ethanol oxidation at a fairly acidic region. Trace amounts of ethanol could be assayed by measuring the initial reaction rate as successful as by reading the end point of the reaction. Some advantages in using this enzyme for ethanol determination were pointed out comparing with NAD-linked alcohol dehydrogenase from yeast or horse liver. Impurity in the enzyme preparations, stability of reagents and coexistence of other substances in the assay mixture were not as critical as in NAD-linked enzyme. Acidic samples could also be directly determined for ethanol without preadjustment of sample pH.     

Crystalline 2-Ketogluconate Reductase from Acetobacter ascendens,the Second Instance of Crystalline Enzyme in Genus Acetobacter

Crystalline 2-ketogluconate reductase in genus Acetobacter was prepared from cell free extract of Acetobacter ascendens. Crystalline enzyme was purified 13,000-fold with a yield of 15%. Affinity chromatography on blue-dextran Sepharose 4B column successfully purified the enzyme. The enzyme was composed of three identical subunits with a molecular weight of 40,000. Substrate specificity of 2-ketogluconate reductase from two genera of acetic acid bacteria was compared using highly purified enzyme preparations, and it was confirmed that gluconate oxidation activity of the enzyme was intrinsically weak or absent in genus Acetobacter and intense in Gluconobacter. This fact must be a useful criterion for classification of acetic acid bacteria.     

High Performance Liquid Chromatography of Hydroperoxides Formed by Autoxidation of Vegetable Oils

Trilinoleoylglycerol (TL) was autoxidized at 37°C in the dark. Monohydroperoxides (MHP) obtained from the oxidized products were analyzed by high performance liquid chromatography (HPLC). Several peaks which appeared in the chromatogram were identified by infrared (IR), gas chromatography mass spectrometry (GC-MS) and enzymatic hydrolysis. Some positional and geometrical isomers of their hydroperoxy fatty acid components were separated using both absorption and reversed phase systems. Furthermore, 1-hydroperoxylinoleoyl-2,3-dilinoleoyl-glycerol and 1,3-dilinoleoyl-2-hydroperoxylinoleoylglycerol were partly separated by HPLC using an absorption system. MHP obtained from autoxidized corn oil, safflower oil and soybean oil were separated into some peaks by HPLC, although resolution into the individual isomers was incomplete. When oxidized oils were subjected to HPLC analysis directly, a linear relationship was observed between the peak areas of MHP and peroxide value in the range of 10 ~ 50 meq/kg.     

γ-Irradiation Effects on the Antioxidative Activity Developing in the Amino Acid-Sugar Reaction

The effects of γ-irradiation on the antioxidative activity developing in the amino acid-sugar reaction were investigated. The antioxidative activity of the nondialyzable melanoidin prepared from glycine and d-glucose was not much affected by γ-irradiation. However, the development of the antioxidative activity of an l-leucine-d-glucose solution on heating was markedly accelerated when the mixture had been preirradiated with γ-rays, and the development of the activity was more prominent than that of the brown color. The irradiation of a glucose solution alone accelerated the antioxidative activity development when heated with leucine, but the irradiation of a leucine solution alone did not cause a similar effect when heated with glucose. Except an l-cysteine-glucose combination, all combinations of amino acids and sugars tested gave rise to almost similar antioxidative effects.