In the course of screening tests of Basidiomycete proteolytic enzymes, it was observed that some strains produced milk clotting enzymes with fairly weak proteolytic activities.
When sucrose-polypeptone and sucrose-corn steep liquor media were used, only 6 strains out of 44 strains tested showed weak milk clotting activities. Cheddar cheese making with culture filtrates of these 6 strains revealed that the culture filtrates of 2 strains, Irpex lacteus Fr. and Fomitopsis pinicola (Fr.) Karst., were able to produce Cheddar cheese of good quality.
On the other hand, when sucrose-distillers solubles media were used, a lot of strains showed high proteolytic activity in addition to high milk clotting activity. The ratio of milk clotting to proteolytic activities (MCA/PA) was assumed to be an important index for the selection of organism, and F. pinicola and Coriolus consors (Berk.) Imaz. were selected as the strain with high MCA/PA ratio.
As the investigation on culture conditions of 3 strains mentioned above showed that F. pinicola and I. lacteus, were richly productive of milk clotting enzymes, the 2 strains except C. consors were used for further studies on cheese making.
Cheddar cheese making with crude enzymes revealed that cheese products produced by the enzyme of F. pinicola had a slightly bitter taste after 5 months' ripening but that those produced by the enzyme of I. lacteus had good quality.

View full abstract

In the previous paper it was reported that three strains of Basidiomycetes, Coriolus consors (Berk.) Imaz., Fomitopsis pinicola (Fr.) Karst. and Irpex lacteus Fr., produced milk clotting enzymes into culture media.
These three strains were cultivated in a liquid medium and crude enzymes were obtained from the culture filtrates. Some properties of the crude enzymes thus obtained were investigated.
These enzymes are a sort of acid proteases with optimum pH of 2.5 for casein digestion. Different from a calf rennet, the proteolytic activity of the enzymes increased along the prolongation of reaction time.
The clotting activity of the enzyme of F. pinicola was much more influenced by pH of milk and Ca ion concentrations in milk than that of the calf rennet. But the enzyme of I. lacteus is very like the calf rennet in these properties.
Optimum temperatures of the enzymes lie at 55_??_60°C.
When the ratio of milk clotting activity to proteolytic activity (MCA/PA) is measured, the ratio of the calf rennet is remarkably higher than those of microbial rennets tested. Among microbial rennets, Mucor rennet and the enzyme of I. lacteus have rather higher values than others.

View full abstract

1) Rice grains contain ferulic acid and sinapic acid in the free form and those bound with sugar such as glucose and amino acid such as glutamic acid and those bound with lipid such as palmitic acid.
2) Fungi decomposed the bound forms of these phenolic acids to produce ferulic acid and sinapic acid, which are consumed by yeast and fungi. Some of these substances are transferred into saké.
3) The used yeast and fungus cannot transfer the methyl radical to catecholic compounds to form methoxy-phenolic compounds such as ferulic acid.
4) A fairly large part of ferulic acid found in moromi mash and saké is considered to be formed from these forms of ferulic acid.
5) The bound forms of these phenolic acids were isolated and examined for their nature. Compound 111-3, one of the major compounds among them is composed of 2 ferulic acid, 3 glucose, 1 glutamic acid and 2 calcium. Its nature was examined precisely.

View full abstract

The sweetness of various sweet substances, such as sugars, synthetic sweetners, and amino acids were evaluated by sensory analysis. The relative sweetness varied in many ways as the concentration increased and the quantitative relationships between the sweetness and the concentration were established. The substances were clasified according to their patterns of the taste intensity curves.

View full abstract

Some clear and precise definitions of the interactions of tastes, i.e. additive, mixing suppressing, counteracting, and synergistic effects, are given. Interrelationships among various sweet substances, such as sugars, synthetic sweetners, and amino acids, were measured as exactly as possible and the experimental observations were systematically explained according to the given definitions. In the present experiments, additive, mixing, and synergistic effects were observed, while suppressing and counteracting effects were not found. A mathematical expression on the synergistic effect was applied to some substances which caused this phenomenon.

View full abstract

Methyl α-cyclocitrylideneacetate was successively oxidized with selenium dioxide and chromium trioxide-pyridine complex to give methyl 1'-hydroxy-α-cyclocitrylideneacetate and a mixture of methyl 3'-keto-β-cyclocitrylideneacetate and methyl 4'-keto-α-cyclocitrylideneacetate. Further, oxidation of methyl α-cyclocitrylideneacetate with tert-butyl chromate afforded methyl 4'-keto-α-cyclocitrylideneacetate and methyl 1'-hydroxy-4'-keto-α-cyclocitrylineacetate. Similarly, methyl α-cyclogeranate was oxidized to methyl 3-keto-β-cyclogeranate and methyl 4-keto-α-cyclogeranate. Methyl 1'-hydroxy-4'-keto-α-cyclocitrylideneacetate, methyl 1-hydroxy-4-keto-α-cyclogeranate and their related compounds did not show growth inhibitory activities on rice seedlings.

View full abstract

The acid cyclization product from pyrethrosin has been proved to be a (1:1)-mixture of cyclopyrethrosin acetate containing a Δ³⁽⁴⁾-double bond and isocyclopyrethrosin acetate with a Δ⁴⁽¹⁵⁾-double bond through the reinvestigation. NMR and ORD studies on their derivatives led us to assign revised stereochemistry to pyrethrosin and its related compounds.

View full abstract

The effects of manganese ion (Mn²⁺) and adenine on the accumulation of 5' inosinic acid (IMP) by Brevibacterium ammoniagenes KY 13102, were examined. Adenine regulated the accumulation of IMP in the presence of limiting amounts of Mn²⁺ and the accumulation of hypoxanthine (Hx) in the presence of excessive amounts of the ion. Manganese ion markedly affected IMP accumulations, cell growth and cellular morphology. These biological changes caused by Mn²⁺ are related to changes in the syntheses of macromolecules. The cells cultivated under limitation of Mn²⁺ showed abnormally elongated and irregular forms irrespective of adenine levels and had smaller nucleotide pools than those of the cells in the presence of excessive Mn²⁺. The Mn²⁺ limited cells showed ability to accumulate IMP directly in the cell suspension but the Mn²⁺ excessive cells did not accumulated IMP but Hx. These results indicated that adenine and Mn²⁺ affected the IMP accumulation independently each other and adenine acted as a feedback regulator on de novo synthesis of purine nucleotide and limitation of Mn²⁺ caused morphological changes, resulting in changes of permeability of the cells. The fatty acid contents of the Mn²⁺ limited cells were higher than those of the Mn²⁺ excessive cells and the ratio of unsaturated fatty acid to saturated one was higher in the former cells.

View full abstract

Tea leaves macerated with L-phenylalanine generated rose like aroma. The gas chromatogram of the essential oil obtained from these leaves showed extremely large peak of phenylacetaldehyde. The evidence for degradation of L-phenylalanine to phenylacetaldehyde and carbon dioxide was given by the radioactive tracer experiment using L-phenylalanine-U-¹⁴C. The phenylacetaldehyde was presumed to be an intermediate product in tea fermentation from the data on the changes of the compound in tea fermentation process.

View full abstract

In the reaction system containing amino acid, tea leaves extract and (-)-epicatechin, some amino acids such as glycine, alanine, valine, leucine, isoleucine, methionine and phenylalanine produced formaldehyde, acetaldehyde, isobutyraldehyde, isovaleraldehyde, 2-methylbutanal, methional and phenylacetaldehyde, respectively. The production of these aldehydes was regarded to proceed as Strecker degradation. On the production of phenyl-acetaldehyde it was revealed in the tea leaves extract-phenol-phenylalanine system that: 1) di-phenol was the most effective co-factor in comparison with mono- and tri-phenols; 2) the optimum concentration of (-)-epicatechin was 5 ×10^-4M and the production was depressed at the concentration more than 5×10^-4M; 3) the production decreased by diluting tea leaves extract.

View full abstract

Eight monoterpenic hydrocarbons, 15 sesquiterpenic hydrocarbons, 6 aliphatic aldehydes, 1 aromatic aldehyde, 3 terpenic aldehydes, 1 terpenic ketone, 1 aliphatic ketone, 1 phenol, 1 phenol ether, 8 terpenic esters, 3 aliphatic alcohols, 9 monoterpenic alcohols, 9 sesquiterpenic alcohols, 1 aromatic alcohol, 5 fatty acids and 2 coumarin compounds were identified in cold-pressed peel oil of Yuzu (Citrus junos) based on gas chromatography, infrared spectrometry, nuclear magnetic resonance spectrometry and capillary GLC connected with fast-scan mass spectrometry. Structures of the sesquiterpenic hydrocarbons, γ-elemene and bicycloelemene are discussed.

View full abstract

The enzyme preparation from Streptomyces No. 8 displayed β-1-3-glucanase, chitinase and protease activities, and attacked heat-treated cells of Candida albicans. The preparation, however, attacked native cells of the same strain and other Candida strains when supplemented with appropriate concentration of sodium dodecyl sulfate or variotin. Neither of the agents attacked native cells when used alone. Usefulness of the combinational use of cell wall lytic enzymes and antibiotics for therapeutic or preventative purpose is discussed.

View full abstract

The chemical constitutions of the pungent principle of Capsicum were investigated. These principles are assumed to consist of capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homodihydrocapsaicin and two or more analogues of these materials. Thin-layer chromatography and open tubular gas chromatography showed that the natural pungent mixture contains no cis-isomer of capsaicin. The chemical structure of nordihydrocapsaicin was determined as N-(4-hydroxy-3-methoxybenzyl)-7-methyloctanamide by gas chromatography, infrared spectroscopy, mass spectrometry and nuclear magnetic resonance spectroscopy. In addition, homodihydrocapsaicin was identified as N-(4-hydroxy-3-methoxybenzyl)-9-methyldecanamide. These identities were also proven by comparison with synthetic samples.

View full abstract

Cell free extracts of Hansenula miso IFO 0146 contained an enzyme which catalyzed acyloin condensation of acetaldehyde and α-ketoglutarate to form 5-hydroxy-4-ketohexanoic acid (HKH). The enzyme was specific for acetaldehyde and α-ketoglutarate. Condensation could not be demonstrated between α-ketoglutarate and other aldehydes tested (formaldehyde, propionaldehyde or butyraldehyde). No reaction occurred when boiled enzyme was used. The apparent Km values (at pH 7.5) for acetaldehyde and α-ketoglutarate are 24.4mM and 3.2mM, respectively. TPP and Mg²⁺ were not required for the reaction. The optimum pH of the reaction was 7.5_??_8.5. The reaction was inhibited by EDTA, PCMB and PMS. The enzyme forming HKH was different from that forming acetoin because the latter required TPP and was repressed when cells were grown in lactate medium while the former did not require TPP and was formed independently of its substrate. The product of this condensing reaction was isolated and identified as HKH from its chemical properties.

View full abstract

An alkaline proteinaseof Aspergillus sulphureus (Fresenius) Thom et Church has been purified in good yields from wheat bran culture by fractionation with ammonium sulfate, treatment with acrynol, and DEAE-Sephadex A-50 column chromatography. The crystalline preparation was homogeneous on sedimentation analysis and polyacrylamide gel zone electrophoresis. The molecular weight was calculated to be 23, 000 by gel filtration. The amino acid composition of the enzyme was determined. The enzyme did not precipitate with acrynol. Optimum pH for the hydrolysis of casein was 7 to 10 at 35°C for 15min.Optimum temperature was 50°C at pH 7 for 10min. The enzyme was highly stable at the range of pH 6 to 11 at 5°C. Whereas relatively stable at pH 6 to 7 at 35°C. Metalic salts tested did not affect activity. Chelating agents, sulfhydryl reagents, TPCK, and oxidizing or reducing reagents tested, except iodine, had no effect on the activity. Diisopro-pylfluorophosphate and N-bromosuccinimide almost completely inactivated the proteinase.

View full abstract

Ten of Nineteen methionine-requiring mutants isolated from Aerobacter aerogenes ATCC 8308 by treatment with N-methyl-N'-nitro-N-nitrosoguanidine were found to accumulate in a culture broth a large amount of O-succinyl-L-homoserine (OSH) which was an intermediate in the biosynthesis of methionine in Escherichia coli and Salmonella typhimurium. OSH was isolated from the culture broth and identified by the behavior in paper chromato-graphy, elementary analysis, melting point, optical density and infrared spectrum. Among these mutants, A. aerogenes KY 7056 which responds to methionine, homocysteine or systathionine was used to investigate culture conditions for OSH production. The amount of OSH accumulation reached a level of 8.36mg/ml with the medium containing 10% fructose and 1% ammonium sulfate. Addition of L-homoserine (10mg/ml) increased the amount of OSH accumulation to a level of 15.8mg/ml. Methionine or cystathionine suppressed the accumulation of OSH. Addition of δ-hydroxylysine to the fermentation medium almost abolished the OSH accumulation.

View full abstract

α, ε-Diaminopimelic acid (DAP)-requiring mutants isolated from Aerobacter aerogenes ATCC 8308 and Serratia marcescens ATCC 19180 were found to accumulate N-succinyl-L-diaminopimelic acid (SDAP) which was an intermediate in the biosynthesis of lysine in Escherichia coli. SDAP was isolated from the culture broth and identified by the behavior in paper chromatography, melting point, elementary analysis, infrared spectrum, and optical rotation.
The culture conditions for SDAP production by A. aerogenes KY 7049 (DAP-) and S. marcescens KY 8921 (DAP-/Lys-) were investigated. A. aerogenes KY 7049 has an absolute requirement for DAP together with a relative requirement for L-lysine. High levels of DAP (2000_??_4000μg/ml) were proved to be favorable for SDAP accumulation, while if lysine along with DAP was added to the fermentation medium, optimal level of DAP for SDAP production was relatively low (about 200μg/ml at 200μg/m1 of lysine). A variety of compounds which may conceivably affect the course of a fermentation process, i. e., carbon source, inorganic nitrogen source, amino acids, vitamines, precursors, were screened at optimal levels of lysine and DAP. Thus, the amount of SDAP accumulation reached a level of 19.9 mg/ml with the medium containing 10% glucose and 2000μg/ml of DAP. S. marcescens KY 8921 requires either DAP or lysine for growth. Optimal level of DAP and lysine for SDAP accumulation was 50_??_100μg/ml.

View full abstract

The partial specific volume of the alkaline proteinase from Aspergillus sojae, determined from density measurement, was 0.696ml/g. This value is 4.1% lower than the value calculated from the known specific volumes of constituent amino acid residues. The difference between the two values of partial specific volume has a considerable influence on the calculation of molecular weight. The molecular weight, determined by the Yphantis' procedure and based on the experimentally determined partial specific volume, gave a value of 22, 600. The gel filtration behavior of the enzyme showed that the value of molecular weight appeared to be reasonable. The amino- and carboxyl-terminal amino acids of the enzyme were shown to be glycine and alanine, respectively, and leucine was the pen-ultimate amino acid on the carboxyl-terminal of the enzyme.

View full abstract

When paraffin wax is dispersed in medium as emulsion, some kinds of bacteria and yeasts readily grow on it. This paper presents a study on microbial cell production from solid paraffin. In this study a paraffin wax which contains 91% of normal paraffins ranging from C₂₅ to C₃₇ with the melting point of 62.5°C was used as a substate, but no solvent was used for the dispersion of the wax.
As a result of this study, the following have been found out. (1) Many strains of liquid normal paraffin assimilating bacteria and yeasts can assimilate paraffin wax. (2) Dried cell yields on added hydrocarbons of Corynebacterium hydrocarboclastus S-12-B2 and Candida tropicalis S-315-Yl are 70% and 56% respectively, when they are cultured by wax emulsion of 0.6% concentration. (3) When nonion surface-active agent (Plysurf A210G) was added as an emulsifing agent, highly concentrated wax emulsion was obtained, but the growth of microorganisms on it was slower. Further investigation is needed to obtain better strains of bacteria and yeasts and also to find out optimum culture conditions.

View full abstract

Photooxidation products of amino acid residues of lysozyme were identified as kynurenine and 3-oxykynurenine from tryptophan, alanine from histidine, methionine sulfoxide and methionine sulfone from methionine, cysteic acid from cystine, glutamic acid from arginine and serine from tyrosine by isolating peptides in tryptic hydrolysate, although the yield of each product did not always reach 100%.
Higher intensity of incident rays caused breakage of peptide chain, the mechanism of which was divided into two steps; the first predominated at the earlier stage of illumination with oxidation of tryptophan, the second predominated at the later stage of illumination accompaning the formation of amide and α-keto acid groups.
Carbonyls and peroxides liberated during illumination were also determined.

View full abstract

A strain of Serratia, isolated from an intestinal canal of a silkworm, produced a large quantity of protease. The enzyme was extracellular and was named Serratiopeptidase, tentatively. Protease production of this strain was over 3 times as much as that of Serratia marcescens which was known as a protease-producing organism. The highly purified enzyme was prepared from the culture supernatant through ammonium sulfate precipitation, acetone fractionation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-75.
The purified enzyme moved homogeneously with a sedimentation constant, s_{20, w} of 3.8S in ultracentrifugation and the molecular weight was determined to be 6.0×10⁴ by the Archibald method. Determination of the ultraviolet absorption spectrum indicated the E^1%_{280mμ, 1cm} was 13.0. Neither carbohydrate nor sulfur-containing amino acid was detected in the purified enzyme preparation.
The enzyme showed maximal activity at pH 9.0 and at 40°C, and was stable under lower temperatures over the pH range from 5 to 10, whereas it was unstable at 37°C in alkaline conditions. The enzyme was completely inactivated by heating at 55°C for 15 min.

View full abstract