Production of recombinant nitrate reductase in Pichia

Period of Performance: 04/01/2001 - 03/31/2002

$364K

Phase 2 SBIR

Recipient Firm

Nitrate Elimination Company, Inc.
Lake Linden, MI 49945
Principal Investigator

Abstract

DESCRIPTION (provided by applicant): Clean, safe water is the base of good public health. NECi develops and commercializes enzyme-based products for water testing and remediation. Currently, NECi is focused on the application of higher plant Nitrate Reductase (NaR) to problems caused by excess nitrate in the environment, a pollution and human health issue worldwide. In Phase I, we demonstrated that recombinant, catalytically active NaR from Arabidopsis thaliana (AtNaR) can be produced economically in the yeast Pichia pastoris, to make NaR-based products more cost-competitive. AtNaR was purified using affinity chromatography, then performance tested in NECi nitrate test kit products. We expressed a second recombinant NaR form, YNR 1, from the yeast Pichia angusta, a key step to increase expression of fully active NaR enzyme. Efforts to engineer the NaR gene were begun using YNRI, and we developed a unique down-sized" form with nitrate-reducing ability intact. In Phase II, we propose to continue to optimize corn and yeast NaR cDNA clones for improved expression and stability, and for efficient purification. Fermentation parameters and optimal gene construction for maximal NaR production will be investigated. We will further develop and characterize down-sized forms of NaR for testing and use in NECi 's electrically-driven immobilized enzyme biosensor and water treatment prototypes. PROPOSED COMMERCIAL APPLICATION: Not Available