Revealing biases inherent in recombination protocols

Background  

The recombination of homologous genes is an effective protein engineering tool to evolve proteins. DNA shuffling by gene fragmentation and reassembly has dominated the literature since its first publication, but this fragmentation-based method is labor intensive. Recently, a fragmentation-free PCR based protocol has been published, termed recombination-dependent PCR, which is easy to perform. However, a detailed comparison of both methods is still missing.     

Purification and characterization of fumarase from the syntrophic propionate-oxidizing bacterium strain MPOB

Fumarase from the syntrophic propionate-oxidizing bacterium strain MPOB was purified 130-fold under anoxic conditions. The native enzyme had an apparent molecular mass of 114 kDa and was composed of two subunits of 60 kDa. The enzyme exhibited maximum activity at pH 8.5 and approximately 54° C. The K _m values for fumarate and l-malate were 0.25 mM and 2.38 mM, respectively. Fumarase was inactivated by oxygen, but the activity could be restored by addition of Fe²⁺ and β-mercaptoethanol under anoxic conditions. EPR spectroscopy of the purified enzyme revealed the presence of a [3Fe-4S] cluster. Under reducing conditions, only a trace amount of a [4Fe-4S] cluster was detected. Addition of fumarate resulted in a significant increase of this [4Fe-4S] signal. The N-terminal amino acid sequence showed similarity to the sequences of fumarase A and B of Escherichia coli (56%) and fumarase A of Salmonella typhimurium (63%). Received: 15 September 1995 / Accepted: 13 November 1995