Abstract
Dibenzothiophene, a model compound for sulfiir-containing organic molecules
found in fossil fuels, can be desulfurized to 2-hydroxybiphenyl by Nocardia asteroides
sp. strain A3H1 (A3H1) using the “4S” desulfurization pathway. In the final and ratelimiting step of this pathway, the DszB enzyme catalyzes the hydrolysis of 2-(2’-
hydroxyphenyl) benzenesulfinate to form inorganic sulfur and 2-hydroxybiphenyl. The
wildtype dszB gene from A3H1 was successfully cloned into the pBAD TOPO® and
pBAD TOPO®/ThioFusion expression vectors. Cloning efficiency of the wildtype gene
into the pBAD TOPO® vector was 86%. The in vitro directed evolution of DszB was initiated using error-prone PCR. An asexual dszB mutant library was generated and sixty
mutant genes were characterized. The average mutation rate of the dszB gene was
determined to be 0.75% ± 0.84. Attempts at overexpressing the mutant genes were
unsuccessful and no measurable induction of fusion protein expression was observed
upon arabinose addition.
