Chemical Composition of Giant Cells Induced by Tunicamycin and Normal Mycelia of Penicillium citrinum

Growth of Penicillium citrinum was reduced in the presence of tunicamycin. Under this condition, reduction of yield of cell wall was greater than that of cellular protein.

Chitin content in the cell wall was several times higher in giant cells formed from conidia in the presence of tunicamycin than in normal mycelia, while reducing sugar content, presumably reflecting glucan content, did not significantly differ. Galactosamine, which was present in normal mycelia and absent in conidia, could not be detected in giant cells. The amino acid composition of the cell wall and whole cells of giant cells differed distinctly from that of normal mycelia.

Tunicamycin did not significantly inhibit the synthesis of DNA, RNA and protein as judged by incorporation of radioactive precursors, while cell wall synthesis, as judged by incorporation of radioactive N-acetylglucosamine, glucose and alanine into acid insoluble fraction, was inhibited by more than 40% in the presence of 10 μg/ml of tunicamycin. In fungi tunicamycin probably acts primarily as an inhibitor of cell wall glycoprotein synthesis and not of chitin synthesis.

Cyclic nucleotides level also differed distinctly between giant cells and mycelia.     

Studies on the Autolysis of Aspergillus oryzae

In was found that an intracellular ribonuclease was present as an inactive form in the fresh mycelium of Asp. oryzae. It was about 3 times activated either by 3 m urea or by the autolysis of mycelium at 30°C for 20 hr. The optimum pH of the ribonuclease activity was 8.3. It was heat sensitive (60°C, 10 min), and completely inhibited by 5 mm EDTA. It was activated by 1 mm Mg²⁺ and inhibited by Zn²⁺, Ca²⁺, Cd²⁺, Co²⁺ and Cu²⁺.     

Incorporation of Mevalonic Acid into a Lipid Intermediate in Cell Wall Synthesis and Nucleic Acid Fraction by Lactobacillus heterohiochii

It was, using the particulate enzyme from Micrococcus lysodeikticus, revealed that most of 2-¹⁴C-mevalonic acid incorporated into the cell of Lactobacillus heterohiochii H-1 was incorporated into the lipid intermediate of cell wall biosynthesis. About 10% of the radioactivities incorporated into the cells was, however, found in nucleic acid fraction which was extracted from lysozyme treated cells with phenol. Most of the radioactivities in the nucleic acid fraction was eluted at the beginning of the elution pattern from Sephadex G-200 or MAK-column. The material is different from tRNA and rRNA.     

Milk Clotting Enzyme from Microorganisms

Out of some 800 strains of microorganisms, a potent fungus for milk clotting enzyme was isolated from soil during the course of screening tests and was identified as one of strains of Mucor pusillus Lindt. Satisfactory results were obtained in cheese making experiments with this enzyme which could be produced effectively by solid culture on wheat bran at 30°C for about 70 hrs.

The balance between milk clotting activity and proteolytic activity of this enzyme resembled very much to that of rennet.

Microbial rennet from Mucor pusillus F-27 was obtained with high productivity by solid culture followed by water extraction. The enzyme could be precipitated by salting out with ammonium sulfate and also by mixing with various water-miscible organic solvents such as ethanol, methanol or acetone.

This enzyme is one of acid proteases having its optimal pH for milk casein digestion around 3.5. The ratio of milk clotting activity to proteolytic activity of this enzyme resembled that of calf rennet than those of other proteases of fungal origin. This was more heat stable and more resistant against pH changes than animal rennet. Apparent activity of milk clotting was more affected by Ca ion concentration in milk than that of calf rennet.

The liberation of 12% TCA soluble nitrogen from casein fraction was a little less specific than that of calf rennet. The optimal temperature for milk clotting lay around 56°C.

Electrophoretic patterns of α-peak of casein treated with this enzyme showed the weak proteolysis which resembled that with rennet.     

Determination of Fatty Acid in Surfactin and Elucidation of the Total Structure of Surfactin

Several properties of ATPase bound to the inner membrane of a psychrophilic marine bacterium Vibrio sp. strain ABE-1 were examined. The membrane-bound ATPase had two optimal peaks of the activity at pH 5.8 and 7.3. The ATPase activity was strongly inhibited by N,N’- dicyclohexylcarbodiimide (DCCD) and NaN₃ at pH 5.8 and 8.0, and stimulated by MgCl₂ and CaCl₂ at pH 8.0. At pH 8.0, the enzyme hydrolyzed GTP and ITP as well as ATP but not AMP or p-nitrophenylphosphate. CTP, UTP, and ADP were poor substrates. These characteristics indicate that there is a F₀F₁-type ATPase in the inner membrane of this bacterium. In addition, the ATPase activity was also significantly inhibited by Na₃ Vo₄, suggesting the coexistence of a P-type ATPase as a minor constituent. The membrane-bound ATPase activity was maximum at 50°C, but the strong DCCD-sensitivity observed at 20°C was greatly reduced at this temperature.     

Structure Determination of Fatty Acids from Tunicamycin Complex

The structures of ten fatty acids, which were obtained by the hydrolysis of tunicamycin complex, were determined. GLC-mass, ¹H NMR and IR spectra showed that the major acids were trans-α, β-unsaturated iso acids with the formula C₁₄H₂₈O₂, C₁₆H₂₈O₂, C₁₆H₃₀O₂ and C₁₇H₃₂O₂. The minor acids were α, β-unsaturated normal acids and saturated normal and iso acids.     

Distribution of ATP-inhibited Ribonuclease in Bacteria

ATP-inhibited ribonuclease was first found in Bacillus amyloliquefaciens cells and was isolated by the authors previously. The distribution of the ATP-inhibited ribonuclease was surveyed on various bacteria. ATP-inhibited ribonuclease was found in only several species of Bacillus genus, i.e., B. subtilis (i), B. amyloliquefaciens (ii), B. cereus (iii), B. cereus var. mycoides (iv), and B. thiiringiensis (v). ATP-inhibited ribonuclease of Bacillus genus was subdivided into two types. The first type of enzyme found in (i) and (ii) cells had optimum pH at 5.7 and was sensitive to 5 mM EDTA. The second type of enzyme found in (iii), (iv), and (v) cells had optimum pH at 6.8 and was partially inhibited by 5 mM EDTA.     

A Method of Fractionation of Bacterial Cytoplasmic Membrane

A study was made of thermogravimetric analyses of microcrystalline cellulose, (Avicell), over a temperature range from 240°C to 300°C under air and nitrogen by means of a thermal balance. For comparative purpose, cellobiose and glucose were also used. The volatilization rate of cellulose was related to the amount of pyrolytic residue and accelerated owing to the oxidation in the presence of atmospheric oxygen. The apparent activation energies of pyrolysis were obtained from weight loss data.

Quantities of carbonyl and carboxyl groups in pyrocellulose increased linearly against degradation stages of cellulose irrespective of pyrolytic temperatures and times.     

Studies on the Autolysis of Aspergillus oryzae

Mode of action of crystalline nuclease O obtained from autolyzed Aspergillus oryzae on RNA and synthetic homopolymers was examined. Crystalline nuclease O had no strict base specificity, although the velocity of hydrolysis was poly A > poly U > RNA > poly C. This enzyme did not degrade poly G. Digestion of high molecular weight RNA with an excess of this enzyme produced mono-, di- and trinucleotides with 5′-terminal phosphate. The amount of mono-, di- and trinucleotides was, respectively, 13.6, 70.0 and 16.4% of total degradation products. All the four bases were detected in mononucleotide fraction and 3′-terminals and 5′-terminals of oligonucleotides.