Characterization of a Novel Esterase Belonging to Family V from Marinobacter flavimaris

Lipolytic enzymes have attracted enormous attentions because of their ability in ester hydrolysis,ester synthesis,transesterification and other biochemical reactions.Bacteria are important sources of lipolytic enzymes applied in industry.Here,a novel lipolytic enzyme encoded by esterase gene est1347 was identified in Marinobacter flavimaris WLL162,and was purified and characterized.The lipolytic enzyme Est1347 consisted of 312 amino acid residues and a 21-amino-acids N-terminal signal peptide with a predicted molecular weight of 34.2 kDa.It belongs to family V of bacterial lipolytic enzymes based on the amino acid sequence homology analysis.Est1347 is a mesophilic and alkali-resistant enzyme with the highest activity at 45℃and pH 8.5;it is stable at temperatures below 50℃and pH 7.5–11.0.Est1347 showed a preference for middle-length chain substrate p-NPC10 and a wide range of other substrates.The Km,Vmax,Kcat and Kcat/Km values of Est1347 for p-NPC10 in pH 8.5 at 45℃were 0.9411 mmol L^(−1),1285μmol min^(−1)mg^(−1),698.91 s^(−1)and 743.65 s^(−1)(mmol L^(−1))^(−1),respectively.It is also tolerant to the metal ions,organic solvents and detergents.In conclusion,the esterase Est1347 laid a foundation for further study of bacterial lipolytic enzyme family V.

Cloning, expression and characterization of a novel esterase from a South China Sea sediment metagenome

Lipolytic enzymes, including esterases and lipases, represent a group of hydrolases that catalyze the cleavage and formation of ester bonds. A novel esterase gene, scsEst01, was cloned from a South China Sea sediment metagenome. The scsEst01 gene consisted of 921 bp encoding 307 amino acid residues. The predicted amino acid sequence shared less than 90% identity with other lipolytic enzymes in the NCBI nonredundant protein database. Scs Est01 was successfully co-expressed in E scherichia coli BL21(DE3) with chaperones(dnaK-dna J-grp E) to prevent the formation of inclusion bodies. The recombinant protein was purified on an immobilized metal ion affinity column containing chelating Sepharose charged with Ni2 +. The enzyme was characterized using p-nitrophenol butyrate as a substrate. Scs Est01 had the highest lipolytic activity at 35℃ and p H 8.0, indicative of a meso-thermophilic alkaline esterase. Scs Est01 was thermostable at 20℃. The lipolytic activity of scs Est01 was strongly increased by Fe2 +, Mn 2+ and 1% Tween 80 or Tween 20.

Microorganisms Associated with Vegetable Oil Polluted Soil

Vegetable oil Spills are becoming frequent and are potentially more challenging than petroleum hydrocarbon spills. Microbial lipases occupy a place of prominence among biocatalysts are often used for remediation of vegetable oil-polluted sites. This work was carried out to isolate microorganisms from oil-polluted sites and screen them for their lipolytic activity. Microorganisms were isolated from eight experimental soil samples contaminated with different types of vegetable oil, soil from an oil mill in Ibadan, and normal uncontaminated soil as a control. The isolates were characterized, identified and those common to at least one of the experimental sites and oil mill sites were screened for their lipolytic activity. Data obtained were analysed using Duncan Multiple Range Test. Seventy three microorganisms were isolated from the polluted soil and identified as species of Bacillus (16), Pseudomonas (12), Flavobacterium (6), Alcaligenes (2), Proteus (3), Micrococcus (1), Aspergillus (9), Penicillium (6), Saccharomyces (4), Geotrichum (1), Kluveromyces (1). Bacillus subtilis, Bacillus licheniformic, Pseudomonas cepacia, Pseudomonas fluorescens, Flavobacterium sp., Alcaligenes sp. and Candida parapsilosis which were common to at least one of the experimental site and oil mill site were preliminarily screened for lipolytic activity and all nine confirmed by presence of halos around the colonies. These screened organisms have potential for the degradation of fatty waste. They could therefore be employed in environmental clean-up of vegetable oil spill site.

Biodiversity and secretion of enzymes with potential utility in wastewater treatment

The main organic contaminants in municipal wastewater are proteins, polysaccharides, and lipids, which must be hydrolyzed to smaller units. A high concentration of oil and grease in wastewater affects biological wastewater treatment processes by forming a layer on the water surface, which decreased the oxygen transfer rate into the aerobic process. Microbial proteases, lipases, amylases, and celullases should play essential roles in the biological wastewater treatment process. The present study aimed to isolate lipase- and other hydrolytic enzyme-producing microorganisms and assess their degradation capabilities of fat and oil wastewater in the laboratory. We also evaluated microbial interactions as an approach to enhance lipolytic activity. We place emphasis on lipase activity because oil and grease are not only environmental pollutants, but also form an undesirable tough crust on pipes of sewage treatment plants. Thirty-five lipolytic microorganisms from sewage were identified and assessed for hydrolytic enzyme profiles. Lipases were characterized in detail by quantification, chain length affinity, and optimal conditions for activity. The good stability of isolated lipases in the presence of chemical agents, thermal stability, wide range of pH activity and tolerance, and affinity for different lengths of ester chains indicates that some of these enzymes may be good candidates for the hydrolysis of organic compounds present in wastewater. A combination of enzymes and fermenting bacteria may facilitate the complete hydrolysis of triglycerides, proteins, and lingo-cellulose that normally occur in the wastes of industrial processes. This study identifies enzymes and microbial mixtures capable of digesting natural polymeric materials for facilitating the sewage cleaning process.