Alternatively, they can be accumulated by gene recombination. Both approaches were used to evolve the pNB esterase for higher activity towards an antibiotic intermediate in the presence of organic solvent. Four generations of random mutagenesis followed by two cycles of recombination yielded an enzyme that has more than 100 times the activity of wild type. This very significant increase in the thermostability of the enzyme is equivalent to the difference between proteins from mesophiles and many thermophilic organisms. Furthermore, the increased thermostability was accompanied by a significant increase in enzyme activity at elevated temperatures.
Detailed protocols are available on request.