Generation and Characterization of dszB Mutants from Nocardia asteroides A3H1

Dibenzothiophene, a model compound for sulfur-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 rate-limiting 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.

Name thesis:degree_name

Master of Science

Level thesis:degree_level

Masters

Grantor

Southwest Texas State University

Year dc:date.issued

2003

Author dc:creator

• Scott, Christopher A.

Advisor dc:contributor.advisor

• Watkins, Linette M.

• desulfurization
• Nocardia
• petroleum products

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/10877/12523

OAI identifier oai:identifier

oai:digital.library.txst.edu:10877/12523