Expression, Purification, and Characterization of HalB in biocatalysis
Biocatalysts can perform a variety of challenging reactions under mild conditions in a stereoselective and regioselective manner, making them a more environmentally friendly option than conventional synthetic materials. Non-heme iron/α-ketoglutarate-dependent (NHFe-αKG) halases are powerful biocatalysts capable of catalyzing the insertion of halide ions into unactivated sp3 heterocaryon centers with high stereoselectivity and regioselectivity.NHFe-αKG halases are part of a larger family of α-ketoglutarate-dependent oxygenase that contain many structurally similar subspecies.Among them, HalB, a member of the non-heme iron halogenate family, is a stand-alone halogenate and is also capable of chlorinating the δ-position of the stand-alone lysine.3 In addition to its chlorination, this enzyme family is characterized by bromides, azides and nitrate compounds.4,5 In this project I will focus on learning the protocols for the expression and purification of HalB and the characterization of its halogenation ability. I aim to overexpress HalB in E.coli cells and then purify this halogenase from other cellular components using nickel affinity chromatography. After obtaining pure HalB, I will also characterize its halogenation ability using Ultra Performance Liquid Chromatography tandem mass spec.