IMPROVED HETEROLOGOUS EXPRESSION OF THE WHITE-ROT FUNGAL LIGNINASE H8BY CROSSOVER LINKER MUTAGENESIS

Using the crossover-linker mutagenesis method, the 5' noncoding region of the lambda ML-1 cDNA, which encodes the ligninase H8 isozyme of th e white-rot fungus, Phanerochaete chrysosporium, was deleted with the simultaneous insertion of the putative Spodoptera frugiperda ribosome- binding sequence (RES) (TATAAAT) directly in front of the translation- initiation codon of this gene. A recombinant baculovirus, pVL-Mu-H8, c arrying the ligninase-H8 gene was successfully constructed, as determi ned by both sequence analysis and dot blot hybridization. A more than 18-fold increase in the expression of ligninase H8, compared to the pr evious pEV11-1A.3 recombinant baculovirus, was detected in the Sf-21 i nsect cells. This enzyme was detected within 3 d postinfection and was biologically active, capable of oxidizing the model lignin compound, veratryl alcohol. The molecular weight of the overexpressed 42 kD prot ein was similar to that of the native fungal ligninase-H8 isozyme and it also reacted specifically with the anti-H8 monoclonal antibody (MAb 2D4.9) in Western blot analysis.