l-Asparaginase from E. coli

l-Asparaginase [EC 3.5.1.1], antitumor enzyme, was purified to a crystalline form from the cell free extract of Escherichia coli A-l-3 KY3598, by ethanol fractionation and chromatographies on DEAE cellulose and CM Sephadex. The crystalline enzyme was homogeneous by the criteria of ultracentrifugation: s_{20, w} was 7.87S.

The molecular weight was estimated to be 141,000 by the short column method. The pI of the enzyme protein was 4.75 according to isoelectric electrofocusing.

Amino acid analysis revealed the absence of cysteine or cystine residues in the molecule.

The enzyme exhibited optimal activity between pH 6 and 8. It was stable in the pH range 5.5 ~ 9.0.

The enzyme activity was cleared very slowly in the plasma of dog. Intravenous administration of the enzyme caused a complete regression of the Gardner lymphoma implanted in the C3H mice.     

Relationships between Binding Quality of Meat and Myofibrillar Proteins

The viscosity change of myosin A concentrated solution with or without other components was measured as the incubation time elapsed at 30°C.

The viscosity of myosin A solution increased, but that of F-actin solution did not. The shear stress at 0.04 sec^?1 was not increased to 1.0 dyne/cm² in the former, but in the latter was below 0.5 dyne/cm².

The viscosity of myosin B solution increased slightly, but that of native tropomyosin-free myosin B solution decreased remarkably. In both the shear stress at 0.04 sec^?1 was greater than or equal to 15 dynes/cm².

The speed of the viscosity increase in the presence of 3 mm pyrophosphate and 3 mm MgCl₂ was higher in concentrated solution of myosin B than in that of native tropomysin-free myosin B. The shear stress at 0.04 sec^?1 after 6 hr at 30°C was 11.5 and 8.2 dynes/cm², respectively.

The effect of native tropomyosin and actin on the viscosity change was discussed.     

Stereoselective Synthesis of Ectocarpene and Its Antipode via Microbiological Asymmetric Hydrolysis

Biological asymmetric hydrolysis of ethyl (±)-cycloheptadienecarboxylate with Rhodotorula minuta var. texensis IFO 1102 and chemical resolution of the corresponding carboxylic acid with (?)-quinine provided (R)-(+)-ethyl 2,5-cycloheptadienecarboxylate (78% e.e.) and (S)-(+)-2,5-cycloheptadienecarboxylic acid (95% e.e.), respectively. The (R)-(+)-carboxylate was converted to (R)-(?)-2,5-cycloheptadienylcarbaldehyde and the (S)-(+)-carboxylic acid to (S)-(+)-2,5-cycloheptadienylcarbaldehyde. Ectocarpene (78% e.e.), male-gamete attractant of marine brown alga, and its antipode (95% e.e.) were synthesized by stereoselective Wittig reaction between the (R)-(?)- and (S)-(+)-aldehydes and propyltriphenylphosphonium bromide in a liquid-solid two phase system using 18-crown ether-t-BuOK, respectively.     

Purification and Properties of Formyltetrahydrofolate Synthetase from Spinach

Formyltetrahydrofolate synthetase (E. C. 6. 3. 4. 3) was found in fresh spinach leaves and purified about 60-fold by treatments of ammonium sulfate, protamine sulfate, dialysis, and DEAE-cellulose column chromatography. Some properties of the enzyme were investigated. Optimum pH was found to be 7.5, and optimum temperature was observed to be at 37°C. In the enzyme reaction, FAH₄ and formate were required specifically as the substrates, and Mg⁺⁺ and ATP were essential components. The Michaelis constants for dl-FAH₄, formate, ATP and magnesium chloride were 1.7×10^?3 m, 1.7×10^?2 m, 4.1×10^?4 m and 3.3×10^?3 m, respectively. The primary product formed in the reaction catalyzed by the enzyme was suggested as N¹⁰-formyl-FAH₄ spectrophotometrically. It was observed that the enzyme also catalyzed the reverse reaction. The possible role of the enzyme in plants was discussed.     

Relation of Cheddar Cheese Ripening to Bacterial Lipolysis

This investigation was undertaken to find the relationship between fat hydrolysis and lipolytic activities of lactic acid bacteria participated in Cheddar cheese ripening. Increases in titratable acidities due to lactic fermentation were completed at early stage of ripening. Ripening indices (ratio of water-soluble nitrogen to total nitrogen) increased rapidly until 90 days and thereafter gradually up to 150 days. Considerable amounts of free fatty acids were released from cheese fat throughout the ripening period. Cheese bacteria were enumerated on the media of tomato-glucose-agar and acetate-agar. About 70% of bacteria isolated from cheese at age of 150 days were classified into Lactobacillus casei and L. plantarum. Lipolytic activities of lactobacilli isolated were detected definitely on double-layered agar plates containing Victoria blue-stained olive oil. Lipase activities were determined in cheese extracts during ripening.     

Chemical Studies on Smelling Compounds in Hen’s Egg

Volatile smelling compounds of freezed egg white, freshness of which was kept by freezed storage, were collected by steam-distillation. After DNP-hydrazones of volatile carbonyl compounds were separated into four classes by column chromatography, DNP-hydrazones contained in each class were separated by thin layer chromatography. Rf and melting point of recrystalized compounds were compared with those of authentic compounds. Volatile basic compounds were collected as hydrochlorides and detected by paper and thin layer chromatography.

Acetone, acetaldehyde, formaldehyde, 2-pentanone, 2-butanone diacetyl except two unknown compounds as volatile carbonyl compounds, and ammonia, methylamine, dime-thylamine and putrescine as volatile basic compounds were tentatively identified.

Correlations between these compounds and smell of freezed egg white were discussed.     

Effects of the Storage in an Atomosphere of Carbon Dioxide on Ovomucin

The yolk index of newly laid egg was 0.50, while that of the egg stored at 30°C for 15 days in an atmosphere of carbon dioxide at a pressure of 2 kg/cm² and in air was 0.43 and 0.25, respectively.

The carbohydrate content of ovomucin gel (B) obtained from the eggs stored in an atmosphere of carbon dioxide was higher than that of ovomucin gel (B) obtained from the eggs stored in air.

The free boundary electrophoretic pattern of ovomucin gel (B) obtained from the eggs stored in an atmosphere of carbon dioxide consisted of two peaks, and the relative mobility of each peak showed the same value as that of the corresponding each peak of ovomucin gel (B) obtained from newly laid egg white.

The fractionation pattern obtained by density gradient column electrophoresis of ovomucin gel (B) obtained from the eggs stored in an atmosphere of carbon dioxide showed two peaks, peak-F and peak-S, while that of the eggs stored in air showed a considerable diminution in peak-F.

From these results, discussion was made about the occurrence and mechanism of egg white thinning when eggs were stored in an atmosphere of carbon dioxide.     

Volatile Acidic Compounds in Heat-Degradated Pork Fat

Pork fat was heated at 160~170°C for 3 hr under bubbling with air, and the volatile acidic compounds were extracted with 3 % aqueous sodium carbonate solution from the total volatile compounds collected during heating. Gas chromatographic identification of the acidic compounds was carried out on PEG-20M and Apiezon L columns. Some of the isolated compounds were identified by infrared and mass spectrometry. The compounds identified include; saturated acids, trans-2, trans-3, cis-3 alkenoic acids, hydroxy acids, keto acids, and dicarboxylic acids. Each of the unsaturated acid and γ-keto acid might be one of the precursors of γ- and δ-lactones reported previously.     

Lactones in the Flavor of Heated Pork Fat

Pork fat was heated at 160~170°C for 3 hr under bubbling with air, and the volatile compounds were collected in the cold trap. After the acidic compounds were removed from the volatile compounds by extraction with 3% aqueous sodium carbonate solution, lactones were obtained from the nonacidic compounds by saponification. Gas chromatographic analyses of lactones were carried out on the PEG-20M and Apiezon L packed columns, and then each lactone was fractionated by repeated gas chromatography. Each isolated lactone was identified by infrared spectrometry, and also three major lactones were identified by mass spectrometry. Consequently, γ-C₅—C₁₂ and δ-C₉, δ-C₁₀, δ-C₁₂ and δ-C₁₄ lactones were found in the flavor of heated pork fat. Gamma-lactones, especially γ-C₇, γ-C₈ and γ-C₉, were predominant in the flavor, and unsaturated lactones were not detected. Mechanisms for the formation of the lactones were discussed.     

An abundant LEA protein in the anhydrobiotic midge,PvLEA4, acts as a molecular shield by limiting growth of aggregating protein particles

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