Christine J. Kelly

A gene encoding manganese peroxidase (mnp1) from Phanerochaete chrysosporium was cloned downstream of a constitutive glyceraldehyde‐3‐phosphate dehydrogenase promoter in the methylotrophic yeast Pichia pastoris. Three different expression vectors were constructed: pZBMNP contains the native P. chrysosporium fungal secretion signal, pαAMNP contains an α‐factor secretion signal derived from Saccharomyces cerevisiae, and pZBIMNP has no secretion signal and was used for intracellular expression. Both the native fungal secretion signal sequence and α‐factor secretion signal sequence directed the secretion of active recombinant manganese peroxidase (rMnP) from P. pastoris transformants. The majority of the rMnP produced by P. pastoris exhibited a molecular mass (55–100 kDa) considerably larger than that of the wild‐type manganese peroxidase (wtMnP, 46 kDa). Deletion of the native fungal secretion signal yielded a molecular mass of 39 kDa for intracellular rMnP in P. pastoris. Treatment of the secreted rMnP with endoglycosidase H (Endo H) resulted in a considerable decrease in the mass of rMnP, indicating N‐linked hyperglycosylation. Partially purified rMnP showed kinetic characteristics similar to those of wtMnP. Both enzymes also had similar pH stability profiles. Addition of exogenous Mn^II, Ca^II, and Fe^III conferred additional thermal stability to both enzymes. However, rMnP was slightly less thermostable than wtMnP, which demonstrated an extended half‐life at 55 °C.

Bioluminescence from the lux‐based bacterial reporter Pseudomonas fluorescens HK44 was experimentally investigated under growth substrate‐rich and limiting conditions in batch, continuous stirred tank (CSTR), and turbidostat reactors. A mechanistically based, mathematical model was developed to describe bioluminescence based on 1) production and decay of catalytic enzymes, and 2) reactant cofactor availability. In the model, bioluminescence was a function of inducer, growth substrate, and biomass concentration. A saturational dependence on growth substrate concentration accommodated dependence on cofactor availability and inducer concentration to accommodate enzyme production was incorporated in the model. Under growth substrate and inducer limiting conditions in the batch reactor and CSTR, bioluminescence was found to decrease in response to cellular energy limitations. The effective lux system enzyme decay rate was determined in independent measurements to be 0.35 hr⁻¹ and the model captured most of the bioluminescent behavior, except at long growth times and high cell density. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 370–378, 2003.

Influent toxicity can be a critical problem for publicly owned treatment works that use the activated sludge process as part of their treatment regime....