MsrA Efflux Pump Inhibitory Activity of Piper cubeba L.f. and its Phytoconstituents against Staphylococcus aureus RN4220.

MsrA, an efflux pump belonging to ATP-binding cassette (ABC) transporter family that conferred resistance to macrolides, was detected in Staphylococcus aureus strains. Herein, we report the isolation of phytoconstituents from Piper cubeba fruit methanol extract and investigated their efflux pump inhibitory potential against S. aureus MsrA pump. Four isolated compounds, viz. pellitorine, sesamin, piperic acid and tetrahydropiperine studied in combination with erythromycin in S. aureus RN4220, exhibited 2-8-fold reduction in minimum inhibitory concentration (MIC) of erythromycin. Pellitorine and sesamin decreased MIC of erythromycin by 8-fold. The real-time fluorometry-based efflux and accumulation studies of ethidium bromide (EtBr) on S. aureus RN4220 in the presence of these compounds showed reduced efflux and enhanced uptake, thus indicating inhibition of the efflux pump. Pellitorine showed significant post-antibiotic effect of erythromycin. The results revealed that the primary mechanism of action of these compounds involves steady ATP production impairment.

Enhanced stability of newly isolated trimeric l-methionine-N-carbamoylase from Brevibacillus reuszeri HSN1 by covalent immobilization.

Newly isolated and partially purified trimeric l-methionine-N-carbamoylase from Brevibacillus reuszeri HSN1 was immobilized by covalent coupling to a well-known support material, Eupergit® C. Approximately 80% enzyme activity yield was achieved with ≈61% binding of a soluble protein from a solution containing 5 mg/mL protein. The immobilized preparation was found to be quite unstable due to a poor multisubunit covalent interaction of trimeric enzyme. Additional cross-linking with polyaldehyde-dextran was done to sustain the biotechnological application of immobilized enzyme. The temperature and pH optima of immobilized enzyme were increased by 10°C and 0.5 unit, respectively. The enzyme was significantly stabilized and retained ≈93% enzyme activity when incubated at 60°C for 60 Min, whereas free enzyme lost ≈50% activity. It was recycled nine times with ≈100% conversion efficiency when batch experiments were carried out at 35°C, pH 7.5, for the 180 Min cycle, using 5% N-carbamoyl-l-methionine as the substrate. The half-life of the immobilized preparation was determined as 23 cycles and is significant. Approximately 50% of enzyme activity was retained even after 5 months of storage at 4°C, whereas free enzyme lost complete enzyme activity. Hence, we could enhance the stability of l-methionine-N-carbamoylase to make it a potential biocatalyst for biotechnological production of α-amino acids.

Trimeric l-N-carbamoylase from newly isolated Brevibacillus reuszeri HSN1: a potential biocatalyst for production of l-α-amino acids.

l-N-carbamoylase was isolated from Brevibacillus reuszeri HSN1 and purified to homogeneity in three steps, which is a reasonably short protocol for native l-N-carbamoylase. The enzyme purification protocol resulted in ≈60-fold purification of l-N-carbamoylase with specific activity of 145 µmol/Min/mg. The subunit and native molecular mass were found to be 44.3 and 132 kDa, respectively. Temperature and pH optima were determined as 50°C and 8.5, respectively. The enzyme had retained ≈86% activity at 50°C when incubated for 60 Min and the half-life was determined as 180 Min at 50°C. N-carbamoyl-l-methionine (l-N-CMet) was found to be a preferred substrate with Km and Vmax values of ≈13.5 mM and ≈103 µmol/Min/mg, respectively. The broad substrate specificity with derivatives of N-carbamoyl amino acids is advantageous to be a better biocatalyst for production of corresponding l-α-amino acids. The enzyme activity was enhanced by 73% in the presence of 0.8 mM Mn(2+) ion during the biotransformation. In the batch experiment, ≈97% conversion of 5.0% l-N-CMet into enantiomerically pure l-methionine was achieved in 10 H when carried out at pH 8.0, 45°C, and 15% wet (w/v) cell loading, under controlled conditions. The overall merits of this enzyme show promise as a potential biocatalyst for l-α-amino acid production.