Phenylalanine ammonia-lyase (PAL) was purified from bamboo shoots (Bambusa oldhami Munro) through steps of DEAE-Sephacel column chromatography, Sepharose CL-6B gel filtration, and a second DEAE-Sephacel column chromatography. Polyacrylamide gel electrophoresis showed that the purified enzyme (M_r 360, 000) still contained two minor bands with much slower mobilities (M_r ?? 660, 000) than the major one. The minor band could be the aggregated forms of native PAL, based on the fact that only a single band (M_r 86, 000) was observed upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focusing, and all bands shared common antigenic determinants as revealed by immunoblotting done with an antiserum raised against the major band. There was a sharp rise of PAL activity of bamboo shoots after harvest, and this was only partly due to the de novo synthesis of this enzyme as measured by Laurell's rocket immunoelectrophoresis.

View full abstract

EDF (Erythroid Differentiation Factor), which exhibits extensive differentiation inducing activity toward mouse Friend leukemia (MEL) cells, is produced by human THP-1 cells stimulated with TPA. The protein is a homo-dimer linked by disulfide bonds, the subunit of which has a molecular weight of 15.5 kd. We isolated cDNA clones encoding the 15.5 kd subunit of EDF and succeeded in the latter's expression in Chinese Hamster Ovary (CHO) cells. That is, the CHO cells secreted a protein possessing differentiation inducing activity. This paper reports the identity of this protein, termed recombinant EDF (r-EDF), with the natural EDF from THP-1 cells as to molecular weight, isoelectric point, amino acid sequence of the N-terminal, amino acid composition, peptide map and the specific biological activity toward MEL cells.

View full abstract

The chemical forms of manganese compounds in soybeans were studied. About 55% of the total manganese in soybeans was extracted into the aqueous supernatant. Separation of the aqueous supernatant by gel chromatography revealed that the ligand-binding manganese localized in three molecular weight fractions (> 100, 000, about 60, 000, and < 1, 000). The largest molecular weight manganese-binding compound consisted of protein, calcium, and phosphorus compound-binding manganese. Phosphorus compound-binding manganese easily dissociated from the macromolecule by gel chromatography. Manganese in the 60, 000 molecular weight fraction tightly bound to protein. This bond was affected neither by 2-mercaptoethanol, by sodium dodecyl sulfate, nor by EDTA. The lowest molecular weight ligand-binding manganese did not bind to DEAE-Sephadex A-25 resin.

View full abstract

The isolation and partial characterization of a tightly-bound-manganese containing protein in soybean seeds were performed. Sephadex G-100 rechromatography of the manganese fraction from the aqueous supernatant of soybeans showed one manganese peak corresponding to a molecular weight of 61, 000. Upon DEAE-Sephadex A-25 chromatography at pH 7.4, the manganese fraction was separated into two components; component I did not bind to the resin while component II did. Components I and II both contained zinc. The ratio of manganese to zinc contained was about 2 to 1 in both components. Components I and II showed identical absorption spectra with maximum absorption at 278 nm. The molecular weights of the two components were estimated to be the same, 63, 000, on SDS-polyacrylamide gel electrophoresis. Component I was found to be a dimer composed of two heterogeneous subunits, with molecular weights of 32, 000 and 30, 500, on SDS-polyacrylamide gel electrophoresis. Component II was also revealed to be a dimer composed of two heterogeneous subunits, with molecular weights of 32, 000 and 31, 000.

View full abstract

A simulated moving-bed adsorber (SMBA) consisting of only three or four columns packed with gel chromatographic materials was applied for the continuous desalting of bovine serum albumin (BSA) from ammonium sulfate. It was shown experimentally that the solutes could be continuously recovered in different streams at concentrations of over 50% of their feed concentrations. The recovery of BSA was in the range of 70-80%, while that of ammonium sulfate was over 95%. Calculation showed that BSA could be recovered at the concentration of over 90% of its concentration in the feed with a recovery of over 98%. The criteria for the flow rates in the SMBA were also shown for good separation.

View full abstract

A highly purified glucodextranase (EC 3.2.1.70) was simply prepared from cell-free culture broth of Arthrobacter globiformis I42 by ammonium sulfate fractionation, affinity chromatography on Sephadex G-100 and column chromatography on DEAE-cellulose. The purified enzyme was judged to be essentially homogeneous on PAGE and SDS-PAGE as well as isoelectric focusing. E^1%_1cm at 280nm was 18.6. The enzyme is an acidic protein with a pI of 4.31. The molecular weight of the enzyme was estimated to be about 120, 000 by SDS-PAGE. The enzyme is rich in alanine and glycine. No carbohydrate moiety seemed to be associated with the enzyme protein.
The optimum pH and temperature of the enzyme are pH 6.0 and 45°C, respectively. The enzyme was completely stable over the pH range of 5.5-7.5 at 4°C for 24 hr, and retained about 40% of its original activity after heating at 60°C for 10 min. Inactivation of the enzyme was found to be partial with 5nM Pb²+ or Zn²⁺, and complete with 5mM each of Cu²⁺, Hg²⁺, Fe³⁺, KMnO₄ and NBS. The enzyme split dextran or isomaltose to produce β-glucose, indicating that the α-glycosidic linkages in these substrates are inverted. The Km value of the enzyme for dextran is 0.014%. Phenyl α-D-glucoside is a potent competitive inhibitor (Ki=1.69mM).

View full abstract

The emulsification capacity of several edible oils was determined in an aqueous system of bovine serum albumin. The relative emulsification capacity of corn, soybean, sesame, palm, palm kernel and coconut oils was 1.00, 0.99, 0.93, 0.90, 0.82 and 0.77, respectively. The emulsification capacity of straight mixed oils lay close to the mean value of that of the component oils. The capacity of mixed oils was decreased by 30-60% by interesterification. Monoacid triacylglycerols had a very low capacity in comparison with edible oils. By interesterification, the emulsification capacity of a mixture of trioleoylglycerol and trioctanoylglycerol was increased and approached the level of interesterified mixed edible oils.

View full abstract

A survey of the total aminopeptidase activity of rabbit skeletal muscle at neutral pH values was performed, using the 2-naphthylamide derivatives of nine amino acids (Ala, Glu, Gly, Leu, Lys, Met, Pro, Ser and Val) as substrates. DEAE-cellulose column chromatography of the muscle extract revealed five major activity peaks that were eluted at 0, 0.03, 0.09, 0.11 and 0.18 M NaCl. The eluates around 0.18M NaCl showed high activity against all substrates and contained the major aminopeptidases. These aminopeptidases were the 160, 000-dalton aminopeptidase (aminopeptidase C) and hydrolase H purified from rabbit skeletal muscle. The values of substrate specificity were fairly compatible with the pattern of free amino acids increasing during the storage of rabbit muscle, indicating that these enzymes are responsible for the increment of free amino acids during aging of rabbit muscle.

View full abstract

The organophosphate (OP)-resistant Daisan Yumenoshima (3-Y_M) housefly strain, which had been reared under the pressure of malathion, was further selected by fenitrothion (3-Y_F) and by diethyl fenitrothion (3-Y_E). The 3-Y_F and 3-Y_E selected strains were highly resistant to the chemical used in the selection, and their acetylcholinesterases were insensitive to the corresponding OP-oxon. The 3-Y_F and 3-Y_M strains had a high activity for glutathione (GSH)-dependent degradation of fenitrothion, whereas that of the 3-Y_E strain was lower. Salithion was relatively effective against both the 3-Y_F and 3-Y_E strains as well as against 3-Y_M. K-1 and K-2, salioxon analogs as the inhibitors of malathion carboxylesterase, were synergistic with fenitrothion against these strains by inhibiting GSH-dependent detoxication.

View full abstract

The enzymatic synthesis of alkyl β-xylosides from xylobiose and alcohols through the transxylosyl reaction of Aspergillus niger IFO 6662 enzyme was studied. Various alkyl β-xylosides were effectively synthesized from xylobiose and water miscible alcohols such as methanol, ethanol, 1-propanol and 2-propanol. The molar ratios of the products, respective β-xylosides and xylose, were approximately 1:1, indicating a theoretical yield for the transfer reaction. Various water insoluble alcohols, such as 1-butanol, 1-pentanol, 1-hexanol, benzyl alcohol and 2-butanol, were also acted as effective acceptors for the transxylosyl reaction, where a great part of the synthesized β-xylosides was found in the insoluble alcohol layer, only xylose and a trace of xylobiose being found in the water layer. Therefore, the synthesized β-xylosides, such as 1-hexyl β-xyloside, could be readily separated from the reaction mixture and crystallized after evaporation of the insoluble alcohol.

View full abstract

Four isomers of chiral 3'-hydroxy-γ-ionylideneacetic acid (3'-hydroxy-γ-acid), acidic metabolites of Cercospora cruenta, were synthesized from (+)-(S)-3-hydroxy-2, 2-dimethyl-1-cyclohexanone as the chiral starting material. A [2.3]-sigmatropic rearrangement of (+)-(S)-3, 3-dimethyl-4-tetrahydropyranyloxy-1-cyclohexenemethanol, which was followed by two steps of the Wittig reaction, gave (+)-(1'S, 3'S)-(2E, 4E)- and (+)-(1'S, 3'S)-(2Z, 4E)-3'-hydroxy-y-acids. Oxidation and subsequent reduction of (+)-(1'S, 3'S)-(2E, 4E)-3'-hydroxy-γ-acid methyl ester gave (-)-(1'S, 3'R)-(2E, 4E)-ester, which was isomerized to (-)-(1'S, 3'R)-(2Z, 4E)-ester by ultra violet irradiation.

View full abstract

The modified coefficient of consolidation, C_e, of soybean protein coagulated was not significantly affected by the applied expression pressure but was greatly affected by the coagulation condition. The value of C_e was high when soybean protein was coagulated at a high temperature, with the calcium concentration to give about half of maximum amount of bound calcium, and with moderate stirring. The value of C_e was analyzed in terms of the average specific resistance to liquid permeation, and the coefficient of volume change, of the soybean protein coagulate.

View full abstract

Two polyphenoloxidases (PPO I and PPO III, EC 1.10.3.1) were extracted and partially purified from sago palm pith by hydroxylapatite chromatography, DEAE-cellulose chromatography and gel filtration. Both purified isozymes gave a single activity band on polyacrylamide gel electrophoresis. The molecular weights of both enzymes were estimated to be 40, 000. They had the same pH optima of 6.5 but different temperature optima, 35°C for PPO I and 45°C for PPO III. PPO I was stable at neutral to alkaline pH and PPO III at acidic pH. PPO III was somewhat more stable than PPO I when incubated at various temperatures for 15 min. PPO I and PPO III oxidized well DL-epicatechin and D-catechin, respectively. Both enzymes were strongly inhibited by KCN, Na-diethyldithiocarbamate and NaHSO₃.

View full abstract

Watermelon fruit was found to be rich in α-mannosidase (EC 3.2.1.24) and β-N-acetylhexosaminidase (EC 3.2.1.52). A simple procedure has been devised for the simultaneous isolation of α-mannosidase and β-N-acetylhexosaminidase from watermelon fruit. The purified enzymes were electrophoretically homogeneous and sufficiently free from other glycosidases. The molecular weights of α-mannosidase and β-N-acetylhexosaminidase were estimated to be 270, 000 and 150, 000 by Sephadex G-200 gel filtration. The enzymes had their maximum activities at pHs 4.0-4.5 and 5.0-5.5, and their Km values for the corresponding p-nitrophenyl glycoside were 2.3 and 0.65mM, respectively. α-Mannosidase required Zn²⁺ to retain its activity and liberated 60% of the mannose from ovalbumin glycopeptide.

View full abstract

A rapid and convenient quantitative determination of furfural (FUR) and 5-hydroxymethylfurfural (HMF) in citrus juices was accomplished by HPLC. The aqueous solution of citrus juices passed through a filter membrane (0.45 μm) was used directly for HPLC analysis. Tetrahydrofuranwater (0.3:99.7) was used isocratically for a ODS column. The calibration curves for FUR and HMF gave good linearity (r = 0.9999) over the wide range of 0, 5-1000 μg/100ml. The recovery of FUR and HMF by the standard addition method was 99.6% in average. These data from HPLC were compared with those by the colorimetric method.

View full abstract

A simple method for overproduction of a target protein by genetic engineering techniques has been established. This method involves rearranging the target gene, which contains a ribosome binding sequence for expression, in plurally repeated form, and inserting it in a 3' lower part of promoters.
The chloramphenicol acetyltransferase (CAT) structural gene was used to demonstrate the validity of this method. E. coli harboring a CAT expression plasmid, pUS(CAT)₁, which had one inserted CAT gene, was able to produce CAT at the level of only 4% of the total cellular protein according to densitometric scanning on Coomassie-blue-stained SDS-polyacrylamide gel and had the CAT activity of 3.9 × 10³ units/mg protein. However, E. coli harboring a CAT expression plasmid, pUS(CAT)₄, which had inserted four directly repeated copies of the CAT gene, could synthesize CAT up to 16% of the total cellular protein and had the CAT activity of 2.8 × 10⁴ units/mg protein. This suggests that this method should be useful for overproducing many important peptides or proteins in bacteria.

View full abstract

The nucleotide sequence of the glyoxalase I gene of Pseudomonas putida IFO 3738 was determined. The glyoxalase I gene coded for a polypeptide of 164-amino acids with a calculated molecular weight of 18, 442, which was closely similar to that obtained with purified glyoxalase I (M.W. 19, 500) from P. putida. The gene for glyoxalase I was expressed in Escherichia coli cells. Comparison of the nucleotide sequence of the gene with the N-terminal amino acid sequence of the enzyme synthesized in E. coli cells revealed that the N-terminal methionine residue was removed after translation, possibly by methionine aminopeptidase in E. coli. The glyoxalase I contained 1 zinc atom per enzyme and its pi was 4.0.

View full abstract

The growth of a mFP-resistant Brevibacterium flavum mutant, No. 221-43, having PDTS^R was synergistically and completely inhibited by mFP plus Tyr-Glu, but not by mFP plus tyrosine or pFP plus Tyr-Glu, whereas that of a mutant having DS^R was only partially inhibited by mFP plus Tyr-Glu. Tyr-Glu could replace tyrosine required for the growth of a tyrosine auxotroph. The phenylalanine uptake was competitively inhibited by tyrosine and the tyrosine uptake by phenylalanine. The phenylalanine uptake was also inhibited by mFP, but not by Tyr-Glu. Mutants having both PDTS^R and DS^R derived from strain No. 221-43 were effectively selected by the resistance to mFP plus Tyr-Glu, and produced much larger amounts of phenylalanine, with small amounts of tyrosine, than the parent. By the same method, mutants having DS^R and PDTS^R, which produced 23.4g/l of phenylalanine at maximum, were obtained from a pFP-resistant tyrosine auxotroph having PDTS^R which produced 18g/l. Similar mutants were also obtained from a tryptophan-producing strain, but produced smaller amounts of tryptophan than the parent, whereas the total amounts of tryptophan and phenylalanine produced were increased.

View full abstract

An amine dehydrogenase was purified and crystallized from the cell free extract of a Pseudomonas sp., isolated from soil by means of the enrichment technique. The crystalline enzyme gave a single band on polyacrylamide gel electrophoresis and the molecular weight of the enzyme was estimated to be 100, 000 by gel filtration on a Sephadex column. Upon SDS-gel electrophoresis, the enzyme was dissociated into two nonidentical subunits having molecular weights of 60, 000 (dehydrogenase) and 39, 000 (cytochrome c). The absorption spectrum of the enzyme showed absorption maxima at 550 nm, 524 nm, 411nm and 280 nm, and a broad shoulder at around 350 nm, indicating that the enzyme was purified as a dehydrogenase-cytochrome c complex. The prosthetic group of the dehydrogenase was identified as covalently bound pyrroloquinoline quinone. The enzyme showed a broad substrate specificity toward various amines including aliphatic monoamines, aliphatic diamines, aromatic amines and polyamines.

View full abstract

The yellow mutagen, 3, 3'-di-tert-butyl-biphenyldiquinone-(2, 5, 2', 5'), which was formed from butylated hydroxyanisole when reacted with sodium nitrite under acidic conditions, was transformed into a red crystal by irradiation with sunlight in an ether solution. The photolytic product was identified as 2, 6-di-tert-butyl-8-hydroxy-dibenzofuran-l, 4-quinone from instrumental analyses. During the irradiation, two kinds of free radicals were detected by electron spin resonance spectroscopy. A photolytic reaction mechanism, in which radical intermediates participate, is proposed on the basis of the data obtained. The photolytic product showed potent desmutagenic activity to frameshift-type reverse mutations induced by Trp-P-1 and Trp-P-2 for a strain of Salmonella typhimurium TA 98.

View full abstract

The effects of the state of mixing on total absorbed energy (P) in microwave cooking were investigated. When water and oil were heated independently, both of P were almost equal and the effect of the dielectric loss factor (ε") on P was negligible. P of a two-layer sample was almost equal to that of the water component alone and was affected by ε". P of an emulsion sample was almost constant and equal to that of water alone whose volume was the same as the volume of the sample. The difference between P of a two-layer sample and that of an emulsion sample was thought to be affected by the increase in the water-oil interface area, and it was found that P of the water-oil mixture is influenced by the state of mixing.

View full abstract

Calorimetric analysis was performed of the effects of penicillin G, ampicillin and polymyxin B on Escherichia coli grown on bouillon medium at pH 6.2 and 37°C. The presence of any one of these drugs shifted the patterns of growth thermograms toward longer incubation periods, although the growth rate constant remained almost unchanged. The changes in the thermogram patterns were analyzed by means of a simple mathematical model and parameters needed to characterize the actions of the drugs on the growth of E. coli were determined. The drug concentrations at which the number of viable cells at the start of incubation decreased by half during the incubation were 6.94, 0.46 and 0.04μmoldm^-3 for penicillin G, ampicillin and polymyxin B, respectively. Drug potency curves were drawn for the three drugs using the parameters determined.

View full abstract

The effects of fasting on the blood NAD level, and the urinary excretion of tryptophan metabolites, such as 5-hydroxyindole-3-acetic acid (5-HIAA), nicotinamide, N¹-methylnicotinamide (MNA), N¹-methyl-2-pyridone-5-carboxamide (2-py) and N¹-methyl-4-pyridone-3-carboxamide (4-py), in rats were investigated. Rats, weighing about 430 g, were fasted for 5 days. The body weight loss during the fasting period was about 10 g per day. The blood NAD levels during the fasting period did not decrease compared with the values during the feeding period. The urinary excretion of 5-HIAA abruptly decreased by about 1/2 on day 1 of fasting, compared with the value during the feeding period, and then remained constant. The level of urinary excretion of nicotinamide during the fasting period was almost the same as the value during the feeding peiod. However, the urinary excretion of nicotinamide metabolites, such as MNA, 2-py and 4-py, gradually decreased with the fasting time and the values had decreased by 1/2-1/3 on day 3 of fasting, compared with the values during the fasting period, and then remained constant. On the basis of these findings, the tryptophan metabolism during the fasting period was discussed.

View full abstract

Two active forms (F1 and F2) of Bacillus acidopullulyticus pullulanase (EC 3.2.1.41 pullulan 6-glucanohydrolase) from Promozyme 200L (Novo Industri A/S) were purified using 50% (v/v) acetone precipitation and chromatographies on CM-Toyopearl 650S, Toyopearl HW-55S, and Butyl-Toyopearl 650S columns. The F1 and F2 forms showed single bands in polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate. The optimum pHs and specific activities of F1 and F2 were about 5.0 and 200 - 220 units/mg, respectively. The molecular weights of F1 and F2 were estimated as 115, 000 and 116, 000, and their isoelectric points were 5.0 and 5.2. The Km values of F1 and F2 for pullulan were the same, 2.5mM glucose equivalents, and their k₀ values were calculated as 2.9 × 10³ and 3.6× 10³s^-1, respectively, from the Lineweaver-Burk plots. These K₀ values of F1 and F2 for pullulan are larger than that (2.9 × 10²sec^-1) of Klebsiella pneumoniae pullulanase. The activities of F1 and F2 on glycogen β-limit dextrins were 26-46% of those on pullulan, but the activity of K. pneumoniae pullulanase on glycogen β-limit dextrins was 3 -6% of that on pullulan.

View full abstract

Methyl epijasmonate has a 400 times stronger aroma than methyl jasmonate, which is known to be one of the key compounds in the aroma of tea, and the former is easily isomerized on heating. Reinvestigation of the separation and identification of tea aroma compounds indicated that steam distillation should be performed under reduced pressure, and that the gas chromatographic conditions should be controlled at below 170°C using an open tubular column coated with a non-polar liquid phase to avoid the isomerization or decomposition of methyl epijasmonate. Under the conditions, ten types of teas were analyzed, and methyl epijasmonate was shown to contribute to the aroma of tea, particularly, to that of Chinese semi-fermented teas.

View full abstract

The thermostable α-galactosidase purified from Pycnoporus cinnabarinus catalyzed the galactosyltransfer reaction in the hydrolysis of raffinose and produced several kinds of transfer products. The main transfer products were isolated and the structures of the six kinds of transfer products (oligosaccharides A, B, C, D, E, and F) were investigated by acid and enzymatic hydrolysis, and methylation analysis. Oligosaccharides A, B, C, D, E, and F were identified as O-α-D-galactopyranosyl-(1→3)-O-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside, O-α-D-galactopyranosyl-(1→3)-O-α-D-galactopyranosyl-(1→6)-O-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside, stachyose, O-α-D-galactopyranosyl-(1→3)-O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→6)-O-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside, verbascose, and ajugose, respectively.

View full abstract

The interaction of α₂-macroglobulin (α₂M) with a major fraction of serine proteinase (SepII) from Aspergillus sojae was investigated. The stoichiometry of the reaction of α₂M with SepII showed that SepII bound to α₂M in a molar ratio of about 2:1. The complex (α₂M-SepII) was separated by PAGE and chromatograph. About 80% inhibition of the proteinase activity as to the hydrolysis of casein and succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosyl-7-amino-4-methylcoumarin (Suc-LLVY-MCA) was observed. The residual activity of the complex was completely abolished by chymostatin and antipain, which were powerful inhibitors with Ki values of 3 × 10^-8 and 5 × 10^-8, respectively. Immunoelectrophoresis of the complex showed its weak cross-reactivity with antiserum to SepII (anti-SepII). The activity of the complex was partially abolished by the anti-SepII. Examination of electron micrographs left little doubt that a conformational change in shape took place. The entrapment reaction with α₂M was completed within 30 sec. When the α₂M-SepII complex was incubated at pH 3.6, a triphasic course was observed. Activation of the complex was observed within 10-17min, while inactivation was observed after 10-17min incubation. And then the activity remained at several % of the initial level. The complex was found to exhibit enhanced pH stability at pH 3.6, while alkali and heat denaturation were observed not to have drastic effects on its stability.

View full abstract

The changes in both taste and taste components of beef, pork, and chicken during storage were examined.
The brothy taste intensity of pork and chicken was significantly stronger after conditioning than before. On the other hand, for beef, there was no significant difference in the brothy taste intensity before or after conditioning. The analysis of major taste components showed that the levels of free amino acids in all meats were higher after conditioning than before. The differences in the levels of free amino acids before versus after conditioning were large in pork and chicken and very small in beef. Oligopeptide levels were lower in beef after conditioning than before, but they were higher in pork and chicken after conditioning than before. These results corresponded to results of the sensory evaluation studies described above, indicating that free amino acids and oligopeptides contributed to the improvement of meat taste during storage.

View full abstract