Fig. 1

Schematic representation of the random mutagenesis method. The circular protein expression vector is amplified repeatedly by strand displacement reaction of Phi29 DNA polymerase. Mutations are introduced in the first step by adding Mn²⁺ to reduce the fidelity of the polymerase (this is known as error-prone RCA). Subsequent amplification with Phi29 DNA polymerase (MDA) provides μg amounts of mutated DNA, sufficient for transformation into P. pastoris for enzyme production