Heterologous production of the stain solving peptidase PPP1 from Pleurotus pulmonarius.

A novel stain solving subtilisin-like peptidase (PPP1) was identified from the culture supernatant of the agaricomycete Pleurotus pulmonarius. It was purified to homogeneity using a sequence of preparative isoelectric focusing, anion exchange and size exclusion chromatography. Peptides were identified by ab initio sequencing (nLC-ESI-QTOF-MS/MS), characterizing the enzyme as a member of the subtilase family (EC 3.4.21.X). An expression system was established featuring the pPIC9K vector, an alternative Kozak sequence, the codon optimized gene ppp1 gene without the native signal sequence with C-terminal hexa-histidine tag, and Pichia pastoris GS115 as expression host. Intracellular active enzyme was obtained from cultivations in shake flasks and in a five liter bioreactor. With reaction optima of 40 °C and a pH > 8.5, considerable bleaching of pre-stained fabrics (blood, milk and India ink), and the possibility of larger-scale production, the heterologous enzyme is well suitable for detergent applications, especially at lower temperatures as part of a more energy- and cost-efficient washing process. Showing little sequence similarity to other subtilases, this unique peptidase is the first subtilisin-like peptidase from Basidiomycota, which has been functionally produced in Pichia pastoris.

Nootkatone.

The continuing interest in the sesquiterpene ketone (+)-nootkatone is stimulated by its strong grapefruit-like odor and numerous further bioactivities. Also numerous were the attempts to chemosynthesize or biotechnologically produce the compound. Cytochrome P450 enzymes from bacteria and fungi were intensively studied and expressed in Escherichia coli and in more food compatible hosts, such as Saccharomyces cerevisiae. The lipoxygenase-catalyzed generation was demonstrated using an enzyme from several Pleurotus species. Laccases required artificial mediators for an efficient catalysis. More recently, plant valencene synthases were expressed in microbial hosts. Combined with an endogenous farnesyl diphosphate delivery pathway and a valencene oxidase, this approach opened access to high yields of nootkatone possessing the appreciated attribute of "natural" according to present food legislation. Little biochemical engineering was carried out on the novel recombinant strains, leaving many options for future improved bioprocesses.