Purification and properties ofl-serine dehydratase fromLactobacillus fermentum ATCC 14931

l-Serine dehydratase fromLactobacillus fermentum was purified 100-fold. It was stabilized by the presence of 1 mM l-cysteine in 50 mM phosphate buffer. M_r=150,000 was determined by gel filtration. The enzyme consists of four apparently identical subunits (M_r=40,000) that were observed after treatment with sodium dodecyl sulfate. The apparent K_m forl-serine was 65 mM. Fe⁺⁺ was required for the enzymatic activity, and the apparent K_m value for this reaction was 0.55 mM. Maximum enzymatic activity was observed at 45°C and pH 8.0 in 50 mM phosphate buffer. At pH values different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 11,400 and 22,800 cal × mol^–1 for temperature values more than and less than 35°C respectively. The purified enzyme showed a maximum absorption between 400 and 420 nm, indicating the presence of pyridoxal-5-phosphate (PLP) as a prosthetic group. The PLP concentration was 0.027 µmoles per milligram of protein. The data suggest that there is 1 mol of PLP for each protein subunit.     

Microflora participating in the decomposition of carboxymethyl cellulose continuously added to the soil

The microbial community in the soil was analyzed during four weeks of a continuous enrichment of structural chernozem soil samples with a 0.1% solution of carboxymethyl cellulose (CMC) under aerobic and semianaerobic conditions. During the first 14 d, the total amount of the aerobic and anaerobic, cellulose-degrading microorganisms increased significantly. Various metabolic pathways were u‘ed te decompose the substrate: diverse metabolic systems were activated and different groups of microorganisms preferred in dependence on the presence of oxygen or the source of mineral nitrogen. In the later phases of cultivation, a decrease in the concentration of zymogenous microflora and in the level of substrate mineralization was observed ovon though CM-cellulase activity remained high. During the fourth week of cultivation, a conspicuous increase in the numbers of oligothropic bacteria occurring in the colcnies of the microorganisms degrading cellulose was found. The representatives of prosthecobacteria (Caulobacter, Hyphomicrobium, Prosthecomicrobium spp.) andSeliberia sp. were thus identified. This “microflora of dispersion” attends the zymogenous microbes degrading CMC and indicates later phases of the process of decomposition.     

Mycobacteria in the light of modern genetics development

It is generally assumed that genetic research of mycobacteria is delayed as compared with other, more commonly used, bacterial models, particularly in the field of genetic transfers. In the field of mutagenesis the problems have been studied to such an extent that replication maps of the chromosome of M. phlei and M. tuberculosis H37 Rv have already been constructed and a new model of the cell cycle of bacteria exhibiting a slow growth rate has been worked out. When the problems of mycobacterial genetics are looked upon in the light of gene manipulations it may be concluded that mycobacteria belong to a few models whose genes are used for cloning and that problems of practical significance will be studied by means of the most modern approaches.     

Effect of detergents on aspartate ammonia-lyase activity inEscherichia alcalescens

The effect of twelve detergents on aspartate ammonia-lyase activity of Escherichia alcalescens used for the production of L-aspartic acid was tested. Best permeabilization was found with Triton X-100, Slovafol 910 and Corona, a mixed commercial preparation. In contrast to Triton X-100 and Slovafol 910, a much narrower range of suitable concentrations was observed with Corona.     

Pigmentation changes in Brevibecteria induced by mutagenic treatment

Inducible pigmentation changes were observed in pigmented strains of Brevibacterium sp. M27 and B. flavum treated with N-methyl-N'-nitro-N-nitrosoguanidine. The highest frequency of induction was reached already at a survival of 30-40% with the maximal yield of 6-10%. As compared with the initial yellow colour, three new pigmentation types, viz. white, pink and orange, were observed. The yellow pigmented parent strains are most resistant to the lethal effects of UV radiation. By selecting pigmented mutants of all types on media containing antibiotics it was possible to obtain strains that were resistant either to tetracycline or to streptomycin. Auxotrophic pigmented mutants were also isolated. In multiple mutant strains of Brevibacterium sp. M27 a number of strainsexhibited a changed L-lysine production. In some strains the production was variable, whereasother strains did not produce L-lysine at all and stains with a limited production of other amino acids were also detected.     

Sterol composition of a δ5,7-sterol-rich strain ofSaccharomyces cerevisiae during batch growth

Sterol composition was examined during batch growth on complex media containing ethanol, molasses or glucose as the carbon source. The molasses-grown cells exhibited a balanced sterol composition throughout growth, maintaining the proportion of ergosterol to 24:28-dehydroergosterol equal to 1.4. The negative effect of glucose on sterol synthesis manifested itself by decreasing the accumulation of 24:28-dehydroergosterol and total sterols but not of ergosterol. Using ethanol as the sole carbon source, a large amount of 24:28-dehydroergosterol accumulated, partly at the expense of other sterols. The gradual addition of nitrogen source during growth significantly decreased the accumulation of ergosterol, 24:28-dehydroergosterol and of total sterols. A general scheme of regulation of sterol synthesis in baker's yeast is presented.     

Studies of electron-transfer properties of salicylate hydroxylase from Pseudomonas cepacia and effects of salicylate and benzoate binding

The pH dependence of the redox behavior of salicylate hydroxylase from Pseudomonas cepacia as well as the effects of salicylate, benzoate, and chloride binding is described. At pH 7.6 in 0.02 M potassium phosphate buffer E1(0')(EFl ox/EFl.-) is -0.150 V and E2(0')(EFl.-/EFl red H-) is -0.040 V versus the standard hydrogen electrode (SHE). A maximum of 5% of FAD anion semiquinone is thermodynamically stabilized under these conditions. However, in coulometric and dithionite titrations more semiquinone is kinetically formed, indicating slow transfer of the second electron. The potential/pH dependence is consistent with a two-electron, one-proton transfer. Upon salicylate binding the midpoint potential is shifted 0.020 V negative from -0.094 to -0.114 V vs SHE at pH 7.6. A maximum of 7% of the neutral semiquinone is stabilized both in potentiometric and coulometric titrations. This small potential shift indicates that the substrate is bound nearly to the same extent to all three oxidation states of the enzyme. It is clear that the substrate binding does not make the reduction of the flavin thermodynamically more favorable. In contrast to salicylate, the potential shift caused by the effector, benzoate, is much more significant. (A maximum potential shift of -0.07 V is calculated.) Benzoate binds most tightly to the oxidized form and is least tightly bound to the two-electron-reduced form of the enzyme. For the reduction of the free enzyme the transfer of the second electron or the transfer of the proton is rate limiting, as is shown by the kinetic formation of the anionic semiquinone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Osmotic adjustment in tobacco plantlets

UsingNicotiana tabacum L. plantlets cultivatedin vitro as a model system it was proved that osmotic adjustment may be caused by a decrease in water potential of substrate as well as by a decrease in air humidity.     

Content of phenolic acids in callus culture of alfalfa (Medicago sativa): The effect of age and biochemical differentiation

Phenolic acids were separated into three fractions and determined by HPLC inMedicago sativa callus culture at the age of two, three and four weeks. The contents of free and especially of predominating ester-bound phenolic acids decreased with callus age to approx. 80 % while the content of phenolic acids nonextractable by methanol increased byca. 90 %. The proportion of benzoic acid derivatives rose from 15 to 21 % within four weeks. The determined difference in the contents of phenolic acids in the upper and lower parts of callus diminished with age. The content of bound forms was higher in the lower part regardless of the callus age. The content of free acids in two weeks old callus was half as high as in the upper part.     

Evidence for two distinct active sites on aldehyde dehydrogenase

Aldehyde dehydrogenase can catalyze the hydrolysis of esters such as p-nitrophenyl acetate as well as oxidize aldehydes to acids. It has not been proven unequivocally that the two reactions occur at the same active site. In the accompanying paper (Tu, G. C., and Weiner, H. (1988) J. Biol. Chem. 263, 1212-1217) evidence was presented which showed that cysteine at position 49 was at the active site for the dehydrogenase reaction. Evidence also was presented which showed that cysteine located at position 162 was susceptible to modification by N-ethylmaleimide. It was shown here that the two activities of the enzyme can be differently protected from inactivation by substrate analogs. Furthermore, aldehydes were found to be poor inhibitors against the esterase reaction while ester was a good inhibitor against the dehydrogenase reaction. In addition, it was possible to modify cysteine 49 with N-ethylmaleimide but not find inhibition of the esterase reactivity until cysteine 162 was modified. It appears that horse liver aldehyde dehydrogenase has two separate active sites per subunit. The data fit a model where ester can be hydrolyzed at both sites but that aldehyde oxidation occurred only at position 49.