The apparent ileal and faecal digestibilities of amino acids and carbohydrates of rye, barley and triticum grains in pigs.

Six pigs of initial weight 40 +/- 1.7 kg with a T-piece cannula fitted at the terminal ileum were given diets composed of 96.3% of rye, barley or triticum grains ground to pass the 2.6 mm mesh, 3.3% mineral and vitamin mixture and 0.4% Cr2O3. Each diet was given to all pigs in a Latin square design, and faeces and ileal digesta were collected on 7, 8, 9 and 10, 11 and 12 day of feeding each diet, respectively. The grains varied widely in the content of pentosans, polyuroids, beta-glucan and monosaccharides. The antitrypsin activities were 0.84, 2.73 and 1.62 TUI/g in barley, rye and triticum, respectively. Ileal and faecal apparent digestibilities of nitrogen and amino acids were higher (P less than or equal to 0.01) in Triticum than in barley and rye. The digestibility of carbohydrates (pentosans, beta-glucan, polyuroids and non-structural carbohydrates) were lower in pigs fed a rye diet than of those fed a Triticum diet. The results are discussed with respect of the role to non starch polysaccharides, especially pentosans in digestibility of nitrogen and energy in animals receiving a rye diet.

Tricarboxylic acid-cycle enzymes and ATP pool in facultative and obligate methylotrophs: Pseudomonas J26 and Methylomonas Pl1.

1. No essential differences were found in the activities of tricarboxylic acid-cycle enzymes in the newly isolated facultative methylotroph Pseudomonas J26 and obligate methylotroph Methylomonas Pl1. 2-Oxoglutarate dehydrogenase and succinate dehydrogenase were absent in Methylomonas Pl1; in Pseudomonas J26 the functioning of the cycle was imparied only on the methanol medium. Citrate synthase of both organisms showed low sensitivity to 2-oxoglutarate, NADH and ATP. 2. In both methylotrophs, methanol dehydrogenase was inhibited non-competitively by ATP: the activity was reduced by half by ATP at a concentration of 5 mM. 3. Concentration of ATP in the log-phase cultures of Methylomonas Pl1 was about twice as high as in Pseudomonas J26 (4.7 and 1.7 mumol/g dry wt., respectively). 4. Differences between the energy state of Methylomonas Pl1 and Pseudomonas J26 might be due to the higher ability of the former to oxidize methanol and/or lower energy requirement for C1 assimilation by the hexulose pathway in the obligate methylotroph.

Fluorescent pigments in the newly isolated methylotrophs: Pseudomonas J16 and Methylomonas Pl1.

The pigments showing fluorescence maxima at 390, 366, 450-460 and 520 nm at excitation wavelength 254, 366 and 450 nm respectively, were detected in the cells and culture media of the obligate methylotroph Methylomonas Pl1 and facultative methylotroph Pseudomonas J26. The maximum at 520 nm is associated with the occurrence of a flavin pigment enabling growth of Lactobacillus casei E ATCC-7469 on the vitamin B2 deficient medium. The remaining fluorescence maxima are related to the prosthetic group of methanol dehydrogenase.

Compartmentation of the amino acid pool in Saccharomycopsis lipolytica.

1. The size of the free amino acid pool in S. lipolytica varies from 250 to 350 micronmoles/g dry wt., and it accounts for about 10% of the total amino acid content; 80-90% of free amino acids, including methionine, is compartmentized in vacuole. S-Adenosylmethionine (SAM) occurs in equal proportions in vacuole and cytoplasm while aspartate and glutamate are mainly cytosolic. 2. The bulk of the methionine overproduced in the ethionine-resistant mutant Etr-13, and of the exogenous methionine derived from the methionine-enriched medium, is stored in vacuole. The amount and distribution of SAM is not affected. 3. Overloading with endogenous methionine results in a significant increase in the total cytosolic amino acid pool and is associated with the increased concentration of arginine, glutamine and lysin; on overloading with exogenous methionine, only lysine is elevated.

Methionine overproduction by Saccharomycopsis lipolytica.

Six ethionine-resistant (Etr) regulatory mutants of Saccharomycopsis lipolytica Sl/1 overproducing methionine have been isolated. Five of them are also resistant to seleno-methionine. The activity of homocysteine synthase (O-acetyl-L-hormoserine-acetate lyase, adding hydrogen sulfide) is derepressed in these mutants and is not susceptible to the methionine-mediated repression. The pool of free methionine in Etr mutants is enhanced 1.5 to 18 times, and incorporation of 35S into methionine is 1.5 to 50 times higher than that in the wild strain. Neither accumulation of endogenous free methionine in Etr mutants nor the uptake of exogenous methionine is accompanied by an increase in the S-adenosylmethionine pool. This implies compartmentation of methionine metabolism in S. lipolytica.

Oxidation of methanol by facultative and obligate methylotrophs.

1. The newly isolated methanol obligate Methylomonas sp. and the methanol facultative Pseudomonas sp. oxidize methanol at an unchanged rate over concentration range from 0.1 to 600 mM; the oxidation rate by the obligate methylotroph is 2.5 times higher (300 nmoles O2/min/mg dry wt.). Low-molecular alcohols, formaldehyde and formate serve as respiratory substrates for the intact cells of both methylotrophs. 2. Methanol dehydrogenase of both methylotrophs isolated should be classified as the phenazine methosulphate-dependent pteridine-type enzyme of double methanol-and formaldehyde-dehydrogenase function. This soluble enzyme is stimulated about 10-fold by NH+4, which results in enhancement of V max, and shows the same specificity and the same affinity toward methanol and formaldehyde (K m about 5 X 10(-5) M). Heat-inactivation of the 10-fold purified enzyme is associated with the release of a watersoluble pigment with maximum fluorescence at 420-430 nm. 3. NAD-deendent formate dehydrogenase was found to catalyse the third step of methanol oxidation in both methylotrophs.

Monophenol monooxygenase and lincomysin biosynthesis in Streptomyces lincolnensis.

Monophenol monooxygenase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase EC 1.14.18.1) was studied in melanin-positive and melanin-negative mutants of Streptomyces lincolnensis NCIB 9413, varying in the lincomycin synthesizing ability. The activities of laccase and tyrosine phenol lyase (EC 4.1.99.2) are absent in this organism. The monophenol monooxygenase catalyzes hydroxylation of monophenols (K(m) and V(max) for l-tyrosine, 2 x 10(-4) M and 8.0 nmol of O(2)/min per ml, respectively) at a slower rate than it dehydrogenates diphenols to o-quinones (K(m) and V(max) for l-3,4-dihydroxyphenylalanine, 7 x 10(-5) M and 51.7 nmol of O(2)/min per ml, respectively. It is inhibited by KCN, beta-mercaptoethanol, ethylenediaminetetraacetate, dipyridyl, thiourea, p-aminobenzoic acids and by some tryptophan metabolites. Changes in the activity of monophenol monooxygenase caused by mutation or by inhibitors are reflected in the synthesis of the antibiotic. Its participation in the biogenesis of the propylhygric moiety of lincomycin is discussed.

Non-specific acetyl-CoA carboxylase and methylmalonyl-CoA carboxyltransferase in Streptomyces noursei var. polifungini.

1. Acetyl-CoA carboxylase (EC 6.4.1.2) and methylmalonyl-CoA carboxyltransferase (EC 2.1.3.1) have been isolated from mycelia of Streptomyces noursei var. polifungini, and purified about 50-fold. 2. Both enzymes carboxylate acetyl-CoA and propionyl-CoA; the respective Km values are 1.1 and 1.6 mM with acetyl-CoA carboxylase and 2.5 and 1.25 mM with carboxyltransferase. 3. The activities of both enzymes are inhibited by free fatty acids. Almost total inhibition of methylmalonyl-CoA carboxyltransferase was observed by 0.1 mM-butyrate or 0.1 mM-C14-C18 acids. Acetyl-CoA carobxylase was affected to the same extent by these compounds at concentration of about 1 mM. 4. The role of both carboxylating enzymes is biosynthesis of the antibiotic is discussed.

Acyl-CoA pool and acyl-CoA thioesterase in Streptomyces noursei var. polifungini.

The size of acyl-CoA pool in S. noursei var. polifungini was found to be associated with the antibiotic-synthesizing ability, and was negatively correlated with both the thio-esterase activity (acetyl-CoA hydrolase, EC 3.1.2.1) and its affinity towards acetyl-CoA and propionyl-CoA. The apparent Michaelis constants with acetyl-CoA and propionyl-CoA were 33 times 10(-5) and 6.6 times 10(-5) M in the low producing strain, and 8.5 times 10(-4) and 1.2 times 10(-4) M in the high producing strain, respectively.

Limiting reactions in activation of acyl units in biosynthesis of macrolide antibiotics.

Formation of propionyl phosphate in Streptomyces erythreus synthesizing a polypropionate erythronolide ring of erythromycin was found to be catalyzed by a specific propionate kinase. The isolated and 100-fold purified kinase was devoid of activity towards acetate and other monocarboxylic acids. The selection for higher antibiotic-synthesizing ability was associated with higher kinase activity and lower K(m) values towards propionate. This relation did not apply to the mutants of S. noursei var. polifungini producing polyene tetraene antibiotics of the nystatin type, composed of acetate and propionate units. Instead, the antibiotic-synthesizing ability was correlated with the activity of acetyl- and propionyl-coenzyme A carboxylase, responsible for the formation of malonyl- and methyl-malonyl-coenzyme A intermediates in the polymerization process.