Tuffin, MarlaBauer, R.Cowan, D.A.Ngobeni, Rhulani2014-10-152024-05-092014-10-152024-05-092011https://hdl.handle.net/10566/13302>Magister Scientiae - MScThere has been a decline in the global fossil fuel reserves, due to an increasing demand for petroleum. Biofuels can be used as an alternative source of energy whereby biomass is converted to liquid fuels such as bioethanol. There is considerable interest in lipolytic enzymes because of their broad substrate range and for this purpose these enzymes have potential in a variety of biotechnological application. Lipolytic enzymes include esterases (E.C 3.1.1.1) and lipases (E.C3.1.1.3). Esterases preferentially hydrolyse short chain (<C10) ester-containing molecules that are partly soluble in water, while lipases hydrolyse a broad range of substrates preferably water-insoluble fatty acyl molecules (>C10). The aim of this study was to express, purify and characterise the lipolytic enzyme present on afosmid, Try 11, previously isolated from a metagenomic library of a Malawian hotspring, which conferred activity on tributyrin and ethyl ferulate. Bioinformatic analysisof the fosmid insert sequence predicted an open reading frame consisting of 951 bp,designated RHgene34, encoding a 317 amino acid protein with 41 % similarity to theα/β hydrolase fold-3 domain protein of Burkholderia sp. The RHgene34 protein contains conserved motifs of esterases/lipases, such as HGGG (residues 95-98),GxSxG (residues 167 - 171) and the putative catalytic triad composed of Ser157,Asp255 and His285. The gene was cloned and expressed in pET21a(+), and transformed into Escherichia coli Rosetta. p-Nitrophenol (p-Np) fatty acyl esters of different carbon chain lengths were used for kinetic characterisation of RHgene34. Kinetic analysis revealed that RHgene34 had a broad range activity on the p-Npesters, from C2 - C14. RHgene34 operates optimally at 45 ºC, pH 9.0 and has a half life of 30 mins at 45 ºC. This study demonstrates that functional screening combined with the sequence analysis is a useful approach for isolating novel enzymes from ametagenome.enBiofuelsLipolytic enzymeEsteraseLipaseEstersuwc