In this short technical report, we present a fast and simple procedure for sample preparation and a single-run Reversed Phase High Performance Liquid Chromatography (RP-HPLC) determination of seven indoles (indole-3-acetic acid, indole-3-acetamide, indole-3-acetonitrile, indole-3-ethanol, indole-3-lactic acid, tryptamine and tryptophan) in bacterial culture supernatants. The separation of the analytes, after a single centrifugal filtration clean-up step, was performed using a gradient elution on a symmetry C8 column followed by fluorimetric detection (λ_ex = 280/λ_em = 350 nm). The calibration curves were linear for all of the studied compounds over the concentration range of 0.0625–125 μg mL⁻¹ (r ² ≥ 0.998) and the limits of detection were below 0.015 μg mL⁻¹. The applicability of the method was confirmed by analysis of Pseudomonas putida culture supernatants.

This study describes the development of a new colorimetric assay to determine aromatic amino acid aminotransferase (ArAT) activity. The assay is based on the transamination of l-tryptophan in the presence of 2-oxoglutarate, which yields indole-3-pyruvate (IPyA). The amount of IPyA formed was quantified by reaction with the Salkowski reagent. Optimized assay conditions are presented for ArAT isozymes isolated from Pseudomonas putida. For comparative purposes, ArAT activity was also determined by high-performance liquid chromatography. ArAT activity staining in polyacrylamide gels with the Salkowski reagent is also presented.

Chitosan has become a promising biological agent for disease control and plant growth promotion. The objective of this study was to assess the effects of chitosan, applied as an active ingredient of Beta-chikol (Poli-Farm, Łowicz, Poland), to control damping-off and Lophodermium needle cast on Scots pine seedlings growing in field conditions. Beta-chikol was used for seed treatment and as a foliar spray at recommended rates and concentrations. For each experimental variant (chitosan, fungicides, unprotected), inventories of seedlings were performed, after germination and again after six weeks. In the aboveground parts of seedlings, the concentration of endogenous salicylic acid was determined by HPLC. At the end of the growing season, seedling growth parameters were determined. Beta-chikol used as foliar spray limited infection by the damping-off fungi but was ineffective when used as a seed treatment. Lophodermium needle cast was not observed during the study period. After the application of Beta-chikol, the concentration of salicylic acid did not increase. The application of Beta-chikol enhanced all growth parameters under investigation. Our results indicate the possibility of using chitosan in the form of Beta-chikol to stimulate plant growth and protect pine seedlings against parasitic damping-off in forest nurseries.

Two Pseudomonas putida aminotransferases (ArAT I and ArAT II) that exhibit activity toward l-tryptophan were purified 104- and 395-fold using a six-stage purification procedure involving ammonium sulfate fractionation and chromatographic separation on phenyl-Sepharose, Sephadex G-100 superfine, DEAE-cellulose and Protein-Pack Q8 HR columns. Mass spectrometry analysis resulted in the identification of 27 and 20 % of the total ArAT I and ArAT II amino acid sequences. In addition, N-terminal sequence fragments of ArAT I and ArAT II were determined using the Edman degradation method. Based on the analyses performed, the studied proteins were identified as products of the tyrB-2 and phhC genes, and the presence of these genes in the investigated bacterial strain was confirmed using molecular biology methods. Extensive analysis of the substrate specificities of ArAT I and ArAT II revealed that both enzymes most efficiently catalyzed reactions involving aromatic amino acids and 2-oxoacids followed by dicarboxylic compounds. The best substrates for ArAT I and ArAT II were l-phenylalanine and phenylpyruvate. Based on these results, the studied proteins were classified as aromatic amino acid aminotransferase isozymes.

Six allozymes of aspartate aminotransferase (AAT, EC 2.6.1.1): three plastidial (AAT-2 zone) and three cytosolic (AAT-3 zone) were isolated from common wheat (Triticum aestivum) seedlings and highly purified by a five-step purification procedure. The identity of the studied proteins was confirmed by mass spectrometry. The molecular weight of AAT allozymes determined by gel filtration was 72.4±3.6 kDa. The molecular weights of plastidial and cytosolic allozymes estimated by SDS-PAGE were 45.3 and 43.7 kDa, respectively. The apparent Michaelis constant (K m) values determined for four substrates appeared to be very similar for each allozyme. The values of the turnover number (k cat) and the k cat/K m ratio calculated for allozymes with L-aspartate as a leading substrate were in the range of 88.5–103.8 s−1/10,412–10,795 s−1 M−1 for AAT-2 zone and 4.6–7.0 s−1/527–700 s−1 M−1 for AAT-3 zone. These results clearly demonstrated much higher catalytic efficiency of AAT-2 allozymes. Therefore, partial sequences of cDNA encoding AATs from different zones were obtained using the RT-PCR technique. Comparison of the AAT-2 and AAT-3 amino acid sequences from active site regions revealed five non-conservative substitutions, which impact on the observed differences in the isozymes catalytic efficiency is discussed.

Chromosomal localization of the three homoeologous genes encoding cytosolic aspartate aminotransferase in common wheat (Triticum aestivum cv. Chinese Spring, 2n = 6x = 42, AABBDD) was specified to: 3AL (0.42÷0.61), 3BL (0.38÷0.41) and 3DL (0.23÷0.81) by a comparative zymographic analysis of the enzymatic activities in deletion lines. It was also attempted to precisely explain the nature of the relationship between a number of genes encoding α and β subunits and a distribution of staining intensity of cytosolic aspartate aminotransferase allozyme activity bands using aneuploid lines of common wheat with modified third pair of homoeologous chromosomes from genomes A, B and D, on which the genes encoding subunit α (genome A) and β (genome B and D) are localized. The highest consistency between the experimental results and the theoretical distributions was achieved by substituting values of α = 0.57 and β = 0.43 in a theoretical model. These results demonstrate that the individual participation of the diploid genome A in the biosynthesis of the cytosolic aspartate aminotransferase allozymes subunits is greater than the individual participation of the diploid genomes B and D.