Lipid modification through lipase -based bioreactors for oil production systems
Abstract
This work describes the initial steps in exploring in vivo vegetable oil modification by a novel, post-synthetic approach. First the in vitro interactions between a lipase and oil bodies from soybeans with respect to enzymatic activity are explored. Next a series of recombinant fusions linking oil body membrane and reporter components to a lipase to examine their impact on enzyme activity is developed. In the first part, the reaction kinetics of oil body vesicles vs. oil emulsions as substrates for lipolytic reactions were measured. The initial rate of hydrolysis for the oil body system was comparatively very low due to a brief latency period. However, the complete activation of the lipase at the interface resulted in an enzyme-membrane protein complex that was catalytically enhanced 3- to 15-fold over the emulsion system for substrate concentrations in the range measured of approximately 1–5.5 mM. This disparity is explained by the availability of substrate to the enzyme active site (defined as the availability parameter “A”) which varies between the two substrates by 40-fold. A simple hyperbolic kinetic mechanism is proposed with K m replaced by the parameter, A, to account for this phenomenon, leading to a maximum rate of approximately 1450 IU/mg and a catalytic turnover number of 825/s. The interaction is verified through separation of the enzyme-membrane protein complex which shows nearly double the activity for the same emulsified substrate (activity ratio of 5:3) when compared to the native enzyme. Second, carboxy-terminus extensions containing oleosin components and/or the GFP reporter gene were recombinantly linked to a lipase from G. candidum. All of the fusions are functionally inactive as a result of the 3′ peptidyl attachments that alter overall structure. Fusions containing the central hydrophobic region of oleosin are intracellularly segregated to the membrane fraction, while those fusions without this region are strongly secreted. The P. pastoris host combined with the GFP reporter provided an effective strategy to study the complete translation of the fusions and subsequent localization.
Degree
Ph.D.
Advisors
Tao, Purdue University.
Subject Area
Agricultural engineering
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