Open AccessTHE BIOCATALYTIC DESULFURIZATION PROJECT
Author(s) -
Steven E. Bonde,
David S. Nunn
Publication year - 2003
Language(s) - English
Resource type - Reports
DOI - 10.2172/822311
Subject(s) - saturated mutagenesis , directed evolution , biocatalysis , computational biology , cofactor , chemistry , enzyme , biochemistry , gene , biology , biochemical engineering , combinatorial chemistry , engineering , catalysis , ionic liquid , mutant
Research activities in the second quarter have largely been a continuation of efforts previously described in the first quarterly report as well as a degree of redirection of effort as a result of discussions during the first quarterly meeting held in San Diego. Chemical synthesis efforts have been refined and are currently being used to support generation of substrates for evaluation and evolution of enzymes for their oxidation. Analysis of the sulfur species in Petro Star diesel, CED extract and refinement of the speciation data is nearly complete. Molecular biology efforts continue with the cloning, expression and characterization of the DszA and DszC proteins as well as the flavin reductases to support regeneration of the essential FMN cofactors. In addition, we have initiated an evolution effort for the extension and improvement of DszA enzyme activity using Diversa's Gene Site Saturation Mutagenesis (GSSM{trademark}) technology. To support the evolution effort as well as of characterization of enzyme activities on a variety of substrates, a high-throughput mass spectroscopy-based assay has been developed. Two selection/screen strategies for the discovery and evolution of biocatalyst enzyme have been developed and are being evaluated for performance using gene libraries constructed from known biodesulfurization strains and environmental libraries