THE DEVELOPMENT OF AN IMMOBILIZED LACTATE OXIDASE SYSTEM FOR LACTIC ACID ANALYSIS AND PYRUVIC ACID PRODUCTION
Abstract
An immobilized lactate oxidase packed-bed reactor was developed which could be used for both the pulse-flow analysis of lactic acid and for the continuous-flow production of pyruvic acid. The initial stages of this investigation involved the development of a purification scheme for lactate oxidase from Acetobacter peroxydans, a strain of acetic acid bacteria. This isolation procedure was based on the partition methods of separation, and included the use of a DE-52 Cellulose chromatographic column for separation of the lactate oxidase fraction. The purified lactate oxidase enzyme was found to have a molecular weight of 98,000, which corresponds to a molecular diameter of 60 angstroms. The enzyme was also determined to be a metalloprotein, with magnesium ion serving as the metal cofactor. The native enzyme was not subject to product inhibition and was found to have a pH optimum of 6.5 and a temperature optimum of 30 degrees Centigrade. The irreversible attachment of lactate oxidase to a porous cellulose bead support was accomplished by using benzoquinone as the coupling reagent. The pH optimum of the immobilized lactate oxidase was determined to be 6.0, while the temperature optimum was at 30 degrees Centigrade. The immobilized lactate oxidase packed-bed reactor was operated under isothermal conditions and was found not to be subject to external or internal diffusional limitations. The concentration of molecular oxygen was shown to have a significant effect on the reaction rate of the immobilized lactate oxidase. It was also found that at a given oxygen level, a limiting value for the reaction rate was approached which was only minimally affected by further increases in the lactic acid concentration. Molecular oxygen was determined to be a co-substrate of lactate oxidase in which a sequential reaction mechanism was being followed: lactic acid first binds to the enzyme before molecular oxygen. Following the attachment of both substrates to the enzyme, the oxidation products are released. A kinetic model based on this rapid equilibrium, ordered bi-reactant scheme was presented and the intrinsic reaction parameters for lactate oxidase were determined for both D-lactic acid and L-lactic acid. The model and experimental reaction rates were found to be in excellent agreement over a wide range of lactic acid and oxygen concentrations. Applications of the packed-bed reactor under continuous flow and pulse flow conditions were then considered. The use of this immobilized enzyme system for the continuous flow production of pyruvic acid was considered to be difficult at this time due to problems of both reactor design and low enzymic activity. The immobilized lactate oxidase column was successfully used under pulse flow conditions for the analysis of lactic acid in whole milk and in a fermentation system. The immobilized enzyme column was determined to be a sensitive analytical tool which exhibited a minimal loss of activity over extended periods of operation.
Degree
Ph.D.
Subject Area
Chemical engineering
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.