Production of multiple ligninases by Phanerochaete chrysosporium: effect of selected growth conditions and use of a mutant strain

Two methods are described for increasing the production of ligninase by cultures of Phanerochaete chrysosporium grown in a nitrogen-limiting medium. The first method involves addition of veratryl alcohol (0.4 mM) and excess trace metals to stationary flask cultures. (Veratryl alcohol is both a substrate for ligninase and a secondary metabolite of P. chrysosporium.) The control ligninase activity (20 units l^-1; as measured by veratryl alcohol oxidation) increases approximately fivefold as a result of these additions. H.p.l.c. analyses of the extracellular proteins produced by these flask cultures revealed at least 13 proteins, ten of which absorb at 409 nm, suggestive of haemproteins; six of these have ligninase (veratryl alcohol oxidizing) activity. The second method entails scale-up using a disc fermenter with a mutant strain which adheres well to the plastic discs, in contrast to the wild type, and which in addition produces high titres of ligninase. The ligninases produced by the mutant and wild-type strains were analysed by native-gel electrophoresis and visualized by silver staining and Western blot analysis. They were also compared by V8 protease digestion analyses. Results indicate a high degree of homology between the ligninases within each strain in addition to homology between the corresponding ligninases of the two stains.