Identification of α-amylase by random and specific mutagenesis of Texcoconibacillus texcoconensis 13CCT strain isolated from extreme alkaline–saline soil of the former Lake Texcoco (Mexico)

The alkaline α-amylase produced by Texcoconibacillus texcoconensis 13CC^T strain was identified by random mutagenesis and confirmed by directed mutagenesis. A transposon mutagenesis approach was taken to identify the gene responsible for the degradation of starch in T. texcoconensis 13CC^T strain. The deduced amino acids of the amy gene had a 99 % similarity with those of Bacillus selenitireducens MLS10 and 97 % with those of Paenibacillus curdlanolyticus YK9. The enzyme showed a maximum activity of 131.1 U/mL at 37 °C and pH 9.5 to 10.5. In situ activity of the enzyme determined by polyacrylamide gel electrophoresis showed only one band with amylolytic activity. This is the first report of a bacterium isolated from the extreme alkaline–saline soil of the former Lake Texcoco (Mexico) with amylolytic activity in alkaline conditions while its potential as a source of amylases for the industry is discussed.     

The intracellular proteolytic system of Yarrowia lipolytica and characterization of an aminopeptidase

Intracellular proteases of Yarrowia lipolytica have been scarcely studied. These enzymes may play an important role in nitrogen metabolism, posttranslational processing, nutritional stress, dimorphism, etc.; biochemical and genetic control of these enzymes can help in obtaining high-level expression of recombinant proteins in heterologous systems. In this study, we report the presence of three proteases: aminopeptidase yylAPE, carboxypeptidase yylCP and dipeptidyl aminopeptidase yylDAP, measured under several nutritional conditions. Yarrowia lipolytica produced the highest level of intracellular proteolytic enzymes, i.e. yylAPE, yylCP and yylDAP, in media with peptone during stationary growth phase. When soluble extracts were subjected to PAGE, and the three activities were revealed in gels with the corresponding substrates, only one band of activity was detected for each one. The three enzymes were affected by serine protease inhibitors. Chelating agents affected mainly APE activity. The aminopeptidase was purified by selective fractionation with ammonium sulfate and three chromatographic steps (anion exchange, hydrophobic interaction and gel filtration chromatography). The enzyme had a molecular mass of 97 kDa; optimal pH and temperature were 7.0 and 37 degrees C, respectively. The aminopeptidase showed a preference for lysine in the N-position. The K(m) value was 0.86 microM and V(max) value was 990.8 micromoL min(-1) mg(-1) for Lys-pNA.     

Mesophilic strains of Aeromonas spp. can acquire the multidrug resistance plasmid pRAS1 in horizontal transfer experiments at low temperatures

Both biotic and abiotic characteristics of an ecosystem play an important role in the horizontal transfer of DNA in nature. The abiotic factor temperature has a great impact on such transfers as it controls the metabolic activity of mesophilic microorganisms. Moreover, psychrophilic bacteria, which are not affected by low temperatures, are considered to be potential donors of DNA to mesophilic bacteria under temperature stress conditions. In our study, mesophilic Aeromonas spp. strains isolated from fresh fish were genotypically identified and used as recipients in in vitro conjugal transfer experiments using plasmid pRAS1 from psychrophilic strain Aeromonas salmonicida 718 at three different temperatures (8, 15 and 20 °C). The transfer of the plasmid was confirmed by identifying the elements of the integron in pRAS1. A low temperatures did not prevent the transfer of the pRAS1 plasmid to Aeromonas veronii, A. media, A. hydrophila and A. caviae strains, which showed detectable conjugation frequencies of 10–8 at 8 °C. In other strains of the same species, transconjugants were not detected, which indicated that the genetic background of each strain directly affected the ability to be a recipient of this plasmid at the temperatures tested. Our results demonstrate that mesophilic Aeromonas spp. strains are potential reservoirs of extrachromosomal genetic material. Implications of this plasmid transfer at low temperatures and its possible consequences for human health are discussed.