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Keywords:

  • Monofunctional catalase;
  • Gene coning and sequencing;
  • ESI-Q-TOF MS/MS;
  • Serratia marcescens

Abstract

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 Km and Vmax were 78 mM and 188, 212 per µM H2O2 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. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)