Overexpression and characterisation of heterologous esterases from a metagenomic library

Ziki, Rutendo Eugenia
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Esterases are hydrolytic enzymes that have many industrial applications. They are used in food, pharmaceutical, pulp and paper, cosmetics, biofuels and many other industries. This gives research of these enzymes major importance. Esterase genes received from CSIR Biosciences were cloned in E. coli DH5α cells. The plasmids carrying these genes were pET20b(+) for genes named Est1, Est2, Est3, Est4, Est5, Est6, Est7, Est8, Est9, Est10, Est12, Est13, Est14 and pET28a(+) for Est11. These plasmids were extracted from the cloning host and transformed into the expression host which was E. coli BL21. The cells were then induced for expression and the presence of the protein bands representing the products of expression were confirmed by running the crude enzyme extract on SDS-Page. The enzyme extracts were tested for activity using pNp-acetate. All 14 esterases were active and they were characterised in terms of pH optima, temperature optima and kinetics. The enzymes showed a pH range of 6.0 to 9.0 and temperature range of 30°C to 50°C. The enzymes were investigated for substrate specificity and they showed a greater preference for short acyl chain substrates over long acyl chain substrates. Further testing was done for activity of the enzymes using α-naphthylbutyrate and naphthol AS-D chloroacetate alongside lipases. A total of 87 enzymes were tested using these colorimetric assays and 36 of the enzymes were found to be active including all 14 esterases. These 36 enzymes were tested for use in enzymatic resolution of three different chemical compounds available as racemic mixtures. No success was observed for two of the compounds but one of them showed some enantioselectivity. This research will be furthered on at large scale to allow continued synthesis of potential HIV-1 protease inhibitors.
Submitted in fulfilment of the academic requirements for the degree of Master of Science in School of Molecular and Cell biology University of the Witwatersrand Johannesburg, South Africa