Liberated Amino Acids Research Articles

Studies on the Utilization of Single Cell Proteins

To evaluate single cell proteins as food resources, both the baker's yeast (Saccharomyces) and the yeast of Candida family grown on n-paraffin were comparably subjected to the study. Extraction with heated alkali and successive precipitation at about pH 4.5 were tested to isolate protein from the cells of the above stated yeast. From the evidences experimentally obtained here, conditions in question in the present study, regarding the strength of applied alkali and its tempeature to obtain the hignest recovery of protein from the cell, were found to be as follows; extraction at 80° to 90°C for 30 to 60 minutes with 0.1 to 0.25N sodium hydroxide (approximately 100mg per g of the dried cell) was found to be the most favorable when the resulted liquid was examined in the soluble nitrogen, liberated amino acid potency, ammoniacal nitrogen level, and the total nitrogen of trichloracetic acid precipitates, of tungstate precipitates or of isoionic precipitates as the criteria. Conditions for extraction from Candida yeast were slightly milder than those conditions required for baker's yeast. Inferior extraction yield was observed in harder dehydrating conditions at 130°C of cell. Over 80% crude protein and crude ash less than 0.1% were recorded with the pH 4.5 isoionic precipitates from the supernatant liquid treated at 80°C for 60 minutes with 20 folds of 0.125N sodium hydroxide solution.

麹酵素による蛋白質の分解(第1報)

Oryzenin, the principal protein of milled rice, was fractionated into 3 fractions by Sepharose 4B gel filtration. The hydrolysis of oryzenin by the acid protease prepared from Taka-diastase was examined at several pH; 3.0, 4.0 and 6.0. (1) Optimum pH of the acid protease for the hydrolysis of oryzenin was found to be near 3.0. (2) Ratios of lower molecular peptides (phosphotungstic acid-soluble fraction) to higher molecular peptides (trichloroacetic acid-soluble fraction) liberated from oryzenin by the enzyme were determined occasionally during the long time reactions and it was found that the ratios were higher when reacted at pH 4.0 or 6.0 than at pH 3.0. (3) Elution patterns of the protein fractions of the reactants from Sepharose 4B column were obtained at times during the reactions. The gel filtration patterns were somewhat different depending on the reaction pH; Fraction I (eluted at void volume and considered to be the largest molecular fraction) disappeared rapidly and completely when reacted at pH 4.0 and the disappearance of Fraction II (eluted succesive to Fraction I) was comparatively rapid at pH 3.0. (4) Lower molecular fractions of the reactants were occasionally fractionated by Sephadex G-15 gel filtration during the reactions. It was also found that the gel filtration patterns were somewhat different depending on the reaction pH; When the protein was reacted at pH 3.0, there appeared several peaks in the gel filtration pattern between the high molecular fraction (void volume) and the small molecular fraction (eluting out volume)mainly consisted of free amino acids presumably, while these peaks were not appeared at all when the protein was reacted at pH 4.0 or 6.0. (3) Amino acid liberation patterns were again different depending on the reaction pH; Amounts of the liberated amino acids were larger when reacted at pH 4.0 or 6.0 than at pH 3.0.

Electrophoretic separation of some amino acids from their copper complexes

Mixtures of free amino acids and their copper complexes were fractionated by paper electrophoresis in borate buffer (pH 10, μ 0.05), potential gradient 6.6–10.2 V/cm on Whatman No. 4 paper strips into three major zones; acidic, neutral and alkaline. Within these zones free amino acids were separated from their copper complexes. Below pH 5–6 the complexes decomposed to a considerable extent. The amino acid composition of the common zone of complexes may be determined by the removal of copper with H 2S followed by paper chromatography of liberated amino acids.

Studies on Amino Acids. XIII

Screening experiments for e-lysine acylase activity in microorganisms have been carried out. Namely, a number of bacteria, streptomyces, molds and yeasts were cultured on the medium containing e-acyllysines, and e-lysine acylase activity was estimated by liberated amino acid detected with paper chromatography of the cultural broth. As a result, the occurrence of e-lysine acylase activity was observed in molds, especially in Aspergillus oryzae. In other organisms such as bacteria, streptomyces and yeasts, the activity was scarcely found under the conditions employed by the authors. By the same procedure employing acetylmethionine and e-benzoyl-α-acetyllysine, acylase activity, that is, hydrolytic activity toward α-N-acyl amino acids, of the microorganisms was investigated.

Studies on Amino Acids XIII–XIV.

Screening experiments for ε-lysine acylase activity in microorganisms have been carried out. Namely, a number of bacteria, streptomyces, molds and yeasts were cultured on the medium containing ε-acyllysines, and ε-lysine acylase activity was estimated by liberated amino acid detected with paper chromatography of the cultural broth. As a result, the occurrence of ε-lysine acylase activity was observed in molds, especially in Aspergillus oryzae. In other organisms such as bacteria, streptomyces and yeasts, the activity was scarcely found under the conditions employed by the authors. By the same procedure employing acetylmethionine and ε-benzoyl-α-acetyllysine, acylase activity, that is, hydrolytic activity toward α-N-acyl amino acids, of the microorganisms was investigated. For the purpose of investigating the formation of ε-lysine activity in molds, especially, Aspergillus oryzae was studied, from which it was found to be an inducible enzyme system whose activity is undetectable without induction. The respective activities inducted with ε-benzoyl-l-, -d-, and -dl-lysines are capable of hydrolyzing both optical isomers of ε-benzoyllysine regardless of the optical nature of the inducers employed. Several enzymatic properties of this fungal ε-lysine acylase are also presented.

A proteolytic system in growing and non-growing cells ofEscherichia coli

(1) Escherichia coli cells contain a protease system splitting its own proteins as well as casein labelled with131I at a slightly alkaline pH. The rate of proteolysis is not markedly influenced by ethylenediaminotetraacetic acid or by iodoacetic acid, p-chlormercuribenzoate diminishes the rate of hydrolysis of131I casein but its effect cannot be reversed by cysteine. (2) The proteolytic system is not present in the cells in the form of an enzymogen and its activity is not diminished even after 55 passages on a synthetic medium containing ammonium chloride as the sole nitrogen source. (3) In living cells proteins labelled with36S methionine are degraded at a rate not exceeding 0.2%/hr. at 29–30°, both in a growing culture and in the normal stationary phase. Similarly, the protease activity in cell-free extracts does not fundamentally change in the course of development of the culture. (4) Proteins in extracts are split at a rate of 1.8–2.3%/hr. at 32° according to results based on colorimetric estimations with tyrosine, or at a rate of 1.1–1.3%/hr, at 32° as shown by the course of degradation of35S proteins. No resynthesis of proteins from liberated amino acids took place in the extracts under the described experimental conditions. (5) The decomposition of labelled proteins in cell-free systems was stimulated by dinitrophenol and sodium azide.

DL-アミノ酸の酵素による光学的分割

A survey of mold strains for the acylase property of asymmetric hydrolysis of N-acylated DL-amino acids was undertaken. Hundred and nineteen strains of Aspergillus and twenty strains of Penicillium were cultured on solid medium, wheat bran, and the extracts of the culture medium were assayed for the acylase activity. Acetyl-DL-glutamic acid hydrolysis test with the acylase was conducted by 24 hrs. incubation at pH 5.8 and 37° The procedure of determination of the liberated amino acid was modified from the method of STEIN and MOORE and the activity was expressed by the amount of the liberated amino acid (Table 2). A number of strains of Aspergillus and several strains of Penicillium produced sufficient acylase activity. The greatest activity was obtained by five strains of A. tamarii and each strain of A. cryzae, P. vinaceous, P. oxalicum var. pectinopcrum nov. and P. corymbiferum. The presence of calcium carbonate in the survey medium for Penicillium was found to be necessary for enzyme production to prevent the pH drop of the medium and to result the greatest enzyme yields (Table 1).