Production, characterization, cloning and sequence analysis of a monofunctional catalase from Serratia marcescens SYBC08.

A monofunctional catalase from Serratia marcescens SYBC08 produced by liquid state fermentation in 7 liter fermenter was isolated and purified by ammonium sulfate precipitation (ASP), ion exchange chromatography (IEC), and gel filtration (GF) and characterized. Its sequence was analyzed by LC-MS/MS technique and gene cloning. The highest catalase production (20,289 U · ml(-1)) was achieved after incubation for 40 h. The purified catalase had an estimated molecular mass of 230 kDa, consisting of four identical subunits of 58 kDa. High specific activity of the catalase (199,584 U · mg(-1) protein) was 3.44 times higher than that of Halomonas sp. Sk1 catalase (57,900 U · mg(-1) protein). The enzyme without peroxidase activity was found to be an atypical electronic spectrum of monofunctional catalase. The apparent K(m) and V(max) were 78 mM and 188, 212 per µM H(2) O(2) µM heme(-1) s(-1), respectivly. The enzyme displayed a broad pH activity range (pH 5.0-11.0), with optimal pH range of 7.0-9.0: It was most active at 20 °C and had 78% activity at 0 °C. Its thermo stability was slightly higher compared to that of commercial catalase from bovine liver. LC-MS/MS analysis confirmed that the deduced amino acid sequence of cloning gene was the catalase sequence from Serratia marcescens SYBC08. The sequence was compared with that of 23 related catalases. Although most of active site residues, NADPH-binding residues, proximal residues of the heme, distal residues of the heme and residues interacting with a water molecule in the enzyme were well conserved in 23 related catalases, weakly conserved residues were found. Its sequence was closely related with that of catalases from pathogenic bacterium in the family Enterobacteriaceae. This result imply that the enzyme with high specific activity plays a significant role in preventing those microorganisms of the family Enterobacteriaceae against hydrogen peroxide resulted in cellular damage. Calalase yield by Serratia marcescens SYBC08 has potential industrial application in scavenging hydrogen peroxide.

Physicochemical and antibacterial properties of fabricated ovalbumin-carvacrol gel nanoparticles.

The applications of carvacrol are limited due to its poor stability and water solubility, and high volatility; however, ovalbumin can be used to encapsulate hydrophobic molecules, improve their aqueous solubility, and reduce their volatility. In this study, we fabricated ovalbumin-carvacrol nanoparticles (OCGns) under different pH (2, 5, 7, and 9) conditions using a gel embedding method and investigated their physicochemical and antibacterial properties. Rheological experiments revealed that the G' of ovalbumin gels (OGs) prepared under different pH conditions were OG-2 > OG-7 > OG-9 > OG-5. Carvacrol addition reduced the tight structure of ovalbumin and carvacrol under pH 5 and 7 conditions, with hardness first decreasing and then increasing, but increasing under pH 2 and 9 conditions. Fluorescence and infrared spectroscopy indicated complex formation, with carvacrol increasing the average diameter of nanoparticles prepared at pH 2, 5, 7, and 9. Encapsulation reached 89.34 and 91.86% at pH 2 and 9, respectively; however, inhibition experiments revealed that the minimum inhibitory concentration of OCGn-2 against Gram-positive Bacillus cereus and Salmonella (0.08 and 0.16 mg mL-1, respectively) was lower than that of OCGn-9 (both 0.28 mg mL-1). Moreover, OCGn-2 possessed a better dense gel structure and a higher stability, encapsulation rate, and antibacterial activity, suggesting that pH affects gel microstructure and thus the encapsulation efficiency and bacteriostatic properties of the prepared nanoparticles. These results contribute to our knowledge of the design and fabrication of polymeric nanoparticle delivery systems for bioactive compounds with beneficial properties.