Fatty Acid Metabolism in Microorganisms

The production of pimelic acid from azelaic acid by microorganisms was studied. About 100 strains of bacteria which were able to utilize azelaic acid as a sole carbon source were isolated from soil and other natural materials. Among these bacteria, several strains produced a large quantity of an organic acid (pimelic acid) from azelaic acid in their culture fluids during the cultivation. The acid was isolated from the culture fluid of strain A133 in crystalline form. The crystal was identified as pimelic acid by physicochemical and biological methods.

From the results of investigations on the morphological and physiological characters, the bacterial strain A133 was assumed to be Micrococcus sp.     

Lipase from Candida paralipolytica

The extracellular lipase from Candida paralipolytica required essential activators* (usually bile- or calcium salts) for the in vitro hydrolysis of triglycerides. The reaction systems emulsified with gum arabic, gelatin, lecithin, methyl cellulose, pectin, polyvinyl alcohol, sodium cholate, or without emulsifier were compared concerning requirement for essential activator, inhibition with sodium chloride and maximum reaction rate, and the following findings have been obtained. (1) The emulsions used can be classified into five groups by the essential activator requirement. (2) The inhibition with sodium chloride depended on reaction system. (3) Each reaction system gave a similar reaction rate at pH 8.2. (4) Long-chain fatty acid dissolved in substrate was necessary to the activation with calcium salts.     

Lipids and Related Substances Inducing the Lipase Production by Candida paralipolytica

Properties of autolytic breakdown of rat skeletal muscle proteins in the alkaline pH range have been reported. The activity is almost exclusively localized in the myofibrillar fraction, but is not solubilized with Triton X-100. The activity is affected by the KCI concentration in the reaction mixture. In 0.6 M and the more concentrated KCI solutions, the maximum activity is attained. The optimum pH of the activity is in the range of pH 7.5～9.5, and the optimum temperature is between 47～57°C.

This autolytic activity seems to be different from catheptic activity which shows its optimum pH in the acid pH range. Moreover, though more than half of the catheptic activity of rat skeletal muscle is recovered in the myofibrillar fraction, the catheptic activity in the myofibrillar fraction can be removed from the fraction by the extraction with dilute saline solution containing Triton X-100.     

Purification and Characterization of Aminopeptidase from Euphausia superba

Krill aminopeptidase was purified about, 1,100-fold from an extract of Euphausia superba with DEAE-cellulose column chromatography, Toyopearl HW55, and hydroxyapatite column chromatography. The final preparation was electrophoretically homogeneous. The molecular weight was determined to be 140,000 by gel filtration and SDS-polyacrylamide disc gel electrophoresis. The optimum pH and optimum temperature were 8.4 and 45°C respectively. Krill aminopeptidase was inhibited by EDTA, Hg^{+ +} and amastatin, and partially by bestatin, and was activated by Co ^{+ +}. Alanyl-p-nitroanilide was hydrolyzed faster than leucyl-p-nitroanilide. Alanyl peptides (di-, tri-, tetra- and hexa-alanyl peptide) were hydrolyzed very fast.

These results suggest that krill aminopeptidase is an alanine aminopeptidase which is activated by cobaltous ion.     

Structures of Gibberellins A39, A48, A49 and a New Kaurenolide in Cucurbita pepo L.

The structures of three new gibberellins A₃₀, A₄₈ and A₄₉ and a new kaurenolide, isolated from seeds of Cucurbita pepo L., were elucidated. The structures of GA₃₉, GA₄₈ and GA₄₉ were shown to be ent-3α,12β-dihydroxygibberell-16-ene-7,19,20-trioic acid (1), ent-2α,3α,10,12α-tetrahydroxy-20-norgibberell-16-ene-7,19-dioic acid 19,10-lactone (5) and the epimer at C–12 of GA₄₈ (8), respectively. The kaurenolide was shown to have the structure: ent-6β,7α,12β-trihydroxykaur-16-en-19-oic acid 19,6-lactone (14).     

Isolation and Structure of Gibberellin A18 from Immature Seeds of Lupinus luteus

A new gibberellin, tentatively called Lupinus gibberellin I, was isolated from young yellow lupin seeds. It has been shown to have structure (X), and now named gibberellin A₁₈.     

Distribution and Properties of NAD Phosphorylating Reaction

Distribution of NAD phosphorylating reactions, phosphorylation through NAD kinase and phosphotransferase, was investigated. NAD kinase activity was distributed rather widely in bacteria, whereas phosphotransferase activity with p-NPP and NAD was limited to a few genera. Proteus mirabilis showed strong activity of phosphotransferase besides NAD kinase activity.

Partial purification of the phosphotransferase was attempted. The enzyme preparation possessed phosphatase activity as well as phosphotransferase activity. Phosphorylation of NAD proceeded maximally under the conditions below pH 4.0. Cu²⁺ showed stimulating effect on the activity. Besides p-NPP and phenylphosphate, various nucleotides, especially 2′ (or 3′) isomers, served as excellent phosphoryl donors, and various kinds of nucleosides and nucleotides were phosphorylated to form nucleoside monophosphates and nucleoside diphosphates.     

Studies on the Induced Synthesis of Maleate cis-trans Isomerase by Malonate

Maleate cis-trans isomerase in Alcaligenes faecalis IB–14 was induced by malonate and purified about 100-fold over the crude cell-free extract by treatments of ammonium sulfate fractionation, Sephadex G–100 gel filtration, DEAE-cellulose and DEAE-Sephadex A–50 column chromatography. The preparation was shown to be monodisperse on ultracentrifugal analysis and Svedberg value was found to be 3.84 S.

The enzyme was most active at pH value around 8.3 and was stable over the range of pH 5.0 to 7.0 in the presence of dithiothreitol (DTT) for a few weeks, but in the absence of it, the enzyme activity was markedly decreased, especially in the alkaline region. The enzyme activity was inhibited by various sulfhydryl reagents and oxidizing agents, whereas it was not affected by metal chelating agents. The inhibition by Hg²⁺ and PCMB was overcome by the addition of sulfhydryl compounds such as DTT, 2-mercaptoethanol, l-cysteine and glutathione. It was observed that the enzyme did not require co-factor for its function.

Kinetic studies showed that Michaelis constant for maleate was 2.8×10^?3 m and the enzyme did not catalyze the reverse reaction.     

Studies on the Low Temperature Fermentation

An attempt has been made to isolate the bacteria capable of accumulating amino acids during the growth at low temperature from various natural sources. A psychrophilic strain P 145 forming glutamic acid at 5°C was obtained and identified as a Brevibacterium sp. The bacterium grew in the range of 0° to 37°C and exhibited the optimum growth at 15°C. The bacterium was defined as a facultative psychrophile.

The strain strictly required methionine only at above 28°C; below this temperature it grew normally without the amino acid. When methionine was added thiamine and biotin stimulated the growth of this strain at 28°C.

With the Brevibacterium sp. P 145 isolated from soil, the effect of incubation temperature on the extracellular amino acid accumulation has been examined from cultural and enzymological points of view. The strain was found to accumulate l-glutamic acid up to 5.88 mg/ml and l-alanine 0.38 mg/ml at 5°C, whereas it formed 0.21 mg/ml of l-glutamic acid and 2.54 mg/ml of l-alanine at 28°C.

The accumulation of l-alanine in the medium at 28°C seemed to be related to the thiamine requirement of the strain. In the case of thiamine deficiency, l-alanine was the main product in the culture at 28°C. When the incubation temperature was abruptly shifted from 28° to 5°C or from 5° to 28°C, the amino acid accumulation was also changed to that of the final temperature. l-Alanine dehydrogenase existed even in the cells grown at 5°C but was not active at this low temperature. These results were in accord with the informations obtained from cultural experiments.     

Fatty Acid Hydroxamate Formation by Intact Cells of Micrococcus sp.

When azelaic acid was used as a sole carbon source on the growth of Micrococcus sp. which was isolated from soil, intact cells of the organism catalyzed the enzymic condensation of fatty acids with hydroxylamine. Some of the characteristics of fatty acid hydroxamate formation were investigated.

The enzyme activity was tested with azelaic acid compared to other fatty acids. Azelylhydroxamate formation was activated with the addition of reduced glutathione or 2-mercaptoethanol. The reaction was inhibited by p-chloromercuribenzoate (PCMB), ethylene diamine tetraacetate (EDTA), NaF and benzoate.