Allyl rhodanine azo dye derivatives: Potential antimicrobials target d-alanyl carrier protein ligase and nucleoside diphosphate kinase.

3-Allyl-5-(4-arylazo)-2-thioxothiazolidine-4-one (HLn ) ligands (where n = 1 to 3) were hypothesized to have antimicrobial activities mediated through inhibition of new antimicrobial targets. The ligands (HLn ) were synthesized and characterized by infrared (IR) and 1 H nuclear magnetic resonance (1 H NMR) spectra. The ligands (HLn ) were in silico screened to their potential inhibition to models of d-alanyl carrier protein ligase (DltA) (from Bacillus cereus, PDB code 3FCE) and nucleoside diphosphate kinase (NDK) (from Staphylococcus aureus; PDB code 3Q8U). HL3 ligand has the best energy and mode of binding to both NDK and DltA, even though its binding to DltA was stronger than that to NDK. In antimicrobial activity of HL3 ligand, morphological and cytological changes in HL3 -treated bacteria agreed with the in silico results. The HL3 ligand showed significant antimicrobial activity against B. cereus, S. aureus, and Fusarium oxysporium. The HL3 -treated bacterial cells appeared malformed and incompletely separated. Its cell walls appeared electron-lucent and ruptured. They contained more mesosomes than normal cells. It was found that the HL3 ligand represented as a bactericide against B. cereus and S. aureusby blocking target DltA, and may target NDK.

Heterologous expression, purification, immobilization and characterization of recombinant α-amylase AmyLa from Laceyella sp. DS3.

AmyLa α-amylase gene from Laceyella sp. DS3 was heterologously expressed in E. coli BL21. E. coli BL21 maximally expressed AmyLa after 4 h of adding 0.02 mM IPTG at 37 °C. The recombinant AmyLa α-amylase was purified 2.19-fold through gel filtration and ion exchange chromatography. We immobilized the purified recombinant AmyLa α-amylase on four carriers; chitosan had the best efficiency. The recombinant free and the immobilized AmyLa α-amylase showed optimum activity in the pH ranges of 6.0-7.0 and 4.0-7.0, respectively and possessed an optimum temperature of 55 °C. The free enzyme had activation energy, Km, and Vmax of 291.5 kJ, 1.5 mg/ml, and 6.06 mg/min, respectively. The immobilized enzyme had activation energy, Km, and Vmax of 309.74 kJ, 6.67 mg/ml, and 50 mg/min, respectively. The immobilized enzyme was calcium-independent and insensitive (relative to the free enzyme) to metals. It could also be reused for seven cycles.

Purification, sequencing, and biochemical characterization of a novel calcium-independent α-amylase AmyTVE from Thermoactinomyces vulgaris.

α-Amylase from Thermoactinomyces vulgaris was highly purified 48.9-fold by ammonium sulfate precipitation, gel filtration on Sephadex G-50 column, and ion exchange chromatography column of DEAE-cellulose. The molecular weight of the enzyme was estimated to be 135 and 145 kDa by SDS-PAGE. Its high molecular weight is due to high glycosylation. The purified amylase exhibited maximal activity at pH 6.0 to 7.0 and was stable in the range of pH 4.0 to 9.0. The optimum temperature for its activity was 50 °C. The enzyme half-life time was 120 min at 50 °C, suggesting intermediate temperature stable α-amylase. The enzyme was sensitive to different metal ions, including NaCl, CoCl(2), and CaCl(2), and to different concentrations of EDTA. The enzyme activity was inhibited in the presence of 1 mM CaCl(2), suggesting that it is a calcium-independent α-amylase. The TLC showed that the amylase hydrolyzed starch to produce large maltooligosaccharides as the main products. A 1.1-kb DNA fragment of the putative α-amylase gene (amy TVE) from T. vulgaris was amplified by using two specific newly designed primers. Sequencing analysis showed 56.2 % similarity to other Thermoactinomyces α-amylases with two conserved active sites confirming its function.