Antimicrobial and genotoxic activity of novel ruthenium(III) complex with n-phenyl-5-nitrosalicylideneimine

In this study, novel hexa coordinated ruthenium(III) complex of the type Na[RuCl2L2)] (where L = monobasic bidentate Schiff base derived from the condensation of 5-nitrosalicyladehyde with aniline) has been synthesized and characterized by electrospray ionization time-of-flight mass spectrometry, infrared spectroscopy and ultraviolet/visible spectrophotometry. Schiff base N-phenyl-5-nitrosalicylideneimine is coordinated to the ruthenium via imine nitrogen and phenolic oxygen. Mass spectra showed molecular ion (M-) at m/z 653.9641 which corresponds to [C26H18Cl2N4O6Ru]-. The in vitro antimicrobial properties of the Schiff base and the complex were tested by micro-dilution technique and agar plate assay for determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The compounds showed a higher antibacterial activity against tested Gram-positive bacteria (Staphylococcus aureus ATCC 33591 and ATCC 29213), whereas against the Gram-negative bacteria (Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 700603) were ineffective. The genotoxic effects of Ru(III) complex were investigated using the Cytokinesis Block Micronucleus (CBMN) assay in human lymphocytes cultures. The cell culture treated with the complex at a concentration of 3.7 µg/mL exhibit the most prominent effect of decreasing the frequency of micronucleus for 44%, while at the concentrations of 1.5 and 7.4 µg/mL effect is slightly lower (40%), compared to the control cell culture

High-level production and covalent immobilization of Providencia rettgeri penicillin G acylase (PAC) from recombinant Pichia pastoris for the development of a novel and stable biocatalyst of industrial applicability

A complete, integrated process for the production of an innovative formulation of penicillin G acylase from Providencia rettgeri (rPAC(P).(rett)) of industrial applicability is reported. In order to improve the yield of rPAC, the clone LN5.5, carrying four copies of pac gene integrated into the genome of Pichia pastoris, was constructed. The proteinase activity of the recombinant strain was reduced by knockout of the PEN gene encoding for proteinase A, resulting in an increased rPAC(P.rett) activity of approximately 40% (3.8 U/mL vs. 2.7 U/mL produced by LN5.5 in flask). A high cell density fermentation process was established with a 5-day methanol induction phase and a final PAC activity of up to 27 U/mL. A single step rPAC(P.rett) purification was also developed with an enzyme activity yield of approximately 95%. The novel features of the rPAC(P.rett) expressed in P. pastoris were fully exploited and emphasized through the covalent immobilization of rPAC(P.rett). The enzyme was immobilized on a series of structurally correlated methacrylic polymers, specifically designed and produced for optimizing rPAC(P.rett) performances in both hydrolytic and synthetic processes. Polymers presenting aminic functionalities were the most efficient, leading to formulations with higher activity and stability (half time stability gt 3 years and specific activity ranging from 237 to 477 U/g (dry) based on benzylpenicillin hydrolysis). The efficiency of the immobilized rPAC(P.rett) was finally evaluated by studying the kinetically controlled synthesis of P-lactam antibiotics (cephalexin) and estimating the synthesis/hydrolysis ratio (S/H), which is a crucial parameter for the feasibility of the process