Comparative study of the effect of L-lysine-L-oxidase from Trichoderma harzianum Rifai and Trichoderma viride on nucleic acid synthesis in human tumor cells in vitro

L-lysine-alpha-oxidase, a new fungal enzyme catalyzing oxidative deamination of L-lysine, exerts an inhibitory effect on DNA, RNA and protein synthesis in human cells of carcinoma ovarius (CaOv) in vitro.     

Flow microcalorimetry investigation of the influence of surfactants on a heterogeneous aerobic culture

The influence of various surfactants on the biological activity of a mixed aerobic culture has been investigated by using flow microcalorimetry. The response of the culture to the addition of homologous n-alkylcarboxylates (C2 to C16) and n-alkylpyridinium bromides (C11 to C14) has been examined under endogenous and substrate saturation conditions, and inhibitory concentrations (MIC or the concentration which decreased the initial activity (heat flux) of the culture by 50%) were determined for each state. Under both conditions, the n-alkylpyridinium bromides were found to be more toxic than the n-alkylcarboxylates of identical chain length, thus confirming that the head group of the amphiphiles plays an important role in the microbial toxicity of surfactants. The relationship observed between the concentration at which 50% of the activity is lost and the chain length of the surfactant further confirms that cellular toxicity is also dependent on surfactant hydrophobicity. In relation to the biodegradability of surfactants in mixed aerobic cultures, the low concentration effects of n-alkylcarboxylates on endogenous culture were investigated in some detail. There appear to be compounded indications that these surfactants are rapidly metabolized by the microorganisms of the mixed culture, at least for homologs lower than C10.     

Specific interaction of arabinose residue in ginsenoside with egg phosphatidylcholine vesicles

The interaction of the specific sugar residue in ginsenosides with egg phosphatidylcholine vesicles was investigated by ESR spectrometry using phosphatidic acid spin-labeled at the polar head groups. Ginsenoside-Rc, which has an alpha-L-arabinofuranose residue and agglutinability toward egg yolk phosphatidylcholine vesicles (Fukuda, K. et al. (1985) Biochim. Biophys. Acta 820, 199-206), caused the restriction of the segmental motion of spin-labeled phosphatidic acid in egg phosphatidylcholine vesicles, indicating that the saponin interacted with the polar head groups of vesicles. Other ginsenosides-Rb2, Rb1, Rd and p-nitrophenyl glycoside derivatives which have less or no agglutinability were also investigated in the same manner. Only ginsenoside-Rb2 and p-nitrophenyl alpha-L-arabinofuranoside which have the specific sugar residue (arabinose) showed a strong interaction with the polar head groups of vesicles. To gain an insight into the mechanism of agglutination by ginsenoside-Rc, the interaction with the fatty acyl groups was also studied by using phosphatidylcholine spin-labeled at the fatty acyl groups. Ginsenoside-Rc increased the order parameter of the spin-labeled phosphatidylcholine, indicating that the saponin was inserted into lipid bilayers. In other saponins investigated, only ginsenoside-Rb2 interacted with the fatty acyl part of vesicles. The process of expression of agglutination by ginsenoside-Rc was discussed on the basis of the ESR studies.     

Gene cloning and sequence determination of leucine dehydrogenase from Bacillus stearothermophilus and structural comparison with other NAD(P)+-dependent dehydrogenases

The gene for leucine dehydrogenase (EC 1.4.1.9) from Bacillus stearothermophilus was cloned and expressed in Escherichia coli. The selection for the cloned gene was based upon activity staining of the replica printed E. coli cells. A transformant showing high leucine dehydrogenase activity was found to carry an about 9 kilobase pair plasmid, which contained 4.6 kilobase pairs of B. stearothermophilus DNA. The nucleotide sequence including the 1287 base pair coding region of the leucine dehydrogenase gene was determined by the dideoxy chain termination method. The translated amino acid sequence was confirmed by automated Edman degradation of several peptide fragments produced from the purified enzyme by trypsin digestion. The polypeptide contained 429 amino acid residues corresponding to the subunit (Mr 49,000) of the hexameric enzyme. Comparison of the amino acid sequence of leucine dehydrogenase with those of other pyridine nucleotide dependent oxidoreductases registered in a protein data bank revealed significant sequence similarity, particularly between leucine and glutamate dehydrogenases, in the regions containing the coenzyme binding domain and certain specific residues with catalytic importance.     

Thermostable alanine racemase from Bacillus stearothermophilus: molecular cloning of the gene, enzyme purification, and characterization

The alanine racemase (EC 5.1.1.1) gene of a thermophilic bacterium, Bacillus stearothermophilus, was cloned and expressed in Escherichia coli C600 with vector plasmid pICR301, which was constructed from pBR322 and the L-alanine dehydrogenase gene derived from B. stearothermophilus. A coupled assay method with L-alanine dehydrogenase and tetrazolium salts was used to detect visually the alanine racemase activity in the clones. Alanine racemase overproduced in a clone carrying the plasmid pICR4, 12 kilobases of DNA, was purified from cell extracts about 340-fold to homogeneity by five steps including heat treatment. The overproduced enzyme was confirmed to originate from B. stearothermophilus by an immunochemical cross-reaction with the enzyme of B. stearothermophilus. The purified enzyme has a molecular weight of about 78 000 and consists of two identical subunits of Mr of 39 000. At the optimum temperature (50 degrees C), the enzyme has a specific activity of 1800 units/mg (Vmax, D- to L-alanine). Resolution and reconstitution experiments together with the absorption spectrum of the enzyme clearly indicate that alanine racemase of B. stearothermophilus is a pyridoxal 5'-phosphate enzyme.     

Effect of single amino acid substitutions on the protease susceptibility of tryptophan synthase alpha subunit

In order to explore the correlation between protease susceptibility and conformational stability of a protein, the proteolytic degradation by trypsin, subtilisin and pronase P of the wild-type alpha subunit of tryptophan synthase from Escherichia coli and of its two mutant proteins was studied by measuring circular dichroism at 222 nm at various pH values at 37 degrees C. The mutant proteins are substituted by Gln or Met in place of Glu at position 49. The single amino acid substitutions at position 49 significantly affected susceptibility of this protein to the three proteases. Dependence of protease susceptibility of the wild-type and the two mutant proteins on pH was characteristic of each protein and similar for the three proteases. Comparison of the present results with the conformational stabilities of the three proteins previously measured shows that the order of resistance to the proteases among the three proteins coincides with the order of the values of unfolding Gibbs energy change, suggesting that protein degradation depends upon the conformational stability of a protein.     

Purification and characterization of thermostable leucine dehydrogenase from Bacillus stearothermophilus

Leucine dehydrogenase (l-leucine: NAD⁺ oxidoreductase, deaminating, EC 1.4.1.9) has been purified to homogeneity from a moderate thermophilic bacterium, Bacillus stearothermophilus. Am improved method of preparative slab gel electrophoresis was used effectively to purify it. The enzyme has a molecular mass of about 300,000 and consists of six subunits with identical molecular mass (Mr, 49,000). The enzyme does not lose its activity by heat treatment at 70° C for 20 min, and incubation in the pH range of 5.5–10.0 at 55° C for 5 min. It is stable in 10 mM phosphate buffer (pH 7.2) containing 0.01% 2-mercaptoethanol at over 1 month, and is resistant to detergent and ethanol treatment. The enzyme catalyzes the oxidative deamination of branched-chain l-amino acids and the reductive amination of their keto analogs in the presence of NAD⁺ and NADH, respectively, as the coenzymes. The pH optima are 11 for the deamination of l-leucine, and 9.7 and 8.8 for the amination of -ketoisocaproate and -ketoisovalerate, respectively. The Michaelis constants were determined: 4.4 mM for l-leucine, 3.3 mM for l-valine, 1.4 mM for l-isoleucine and 0.49 mM for NAD⁺ in the oxidative deamination. The B. stearothermophilus enzyme shows similar catalytic properties, but higher activities than that from Bacillus sphaericus.Dedicated to Prof. Dr. G. Drews on the occasion of his 60th birthday     

A versatile bacterial enzyme: l-methionine γ-lyase

The properties and application of l-methionine γ-lyase [methioninase, l-methionine methanethiol-lyase (deaminating), EC 4.4.1.11], a pyridoxal 5′-phosphate enzyme, purified from Pseudomonas putida and Aeromonas sp. are presented. The enzyme has multicatalytic functions: it catalyses α,γ-elimination and γ-replacement reactions of l-methionine and its analogues (e.g. ethionine, homocysteine, O-acetylhomoserine and selenomethionine), α,β-elimination and β-replacement reactions of l-cysteine and its analogues (e.g. S-methylcysteine, O-acetylserine and Se-methylselenocysteine), deamination and γ-addition of vinylglycine, and deuterium labelling at the α and β positions of l-methionine and other straight-chain l-amino acids. These reactions are applicable to the synthesis of various optically active sulphur and selenium amino acids, preparation of deuterium or tritium labelled l-amino acids, and determination of sulphur amino acids. In addition, the enzyme shows potent anti-neoplastic activity.     

Screening of thermostable leucine and alanine dehydrogenases in thermophilicBacillus strains

Summary Screening of leucine and alanine dehydrogenases in thermophilicBacillus strains was carried out to develop their utilization for industrial and analytical catalysts. Out of the 28 thermophilic strains tested, four strains,Bacillus sp. DSM 405, 730 and 1521, andB. sphaericus DSM 462, abundantly produce both the enzymes. Both the enzyme activities in these thermophiles are enhanced by addition of the substrates to a polypeptone medium.