Original scientific paper
Isolation and Kinetic Characterization of Fumarase from Baker's Yeast
Z. Findrik
; University of Zagreb, Faculty of Chemical Engineering and Technology,Zagreb, Croatia
E. Smailbegović
; University of Zagreb, Faculty of Chemical Engineering and Technology, Zagreb, Croatia
M. Prkačin
; University of Zagreb, Faculty of Chemical Engineering and Technology, Zagreb, Croatia
D. Vasić-Rački
; University of Zagreb, Faculty of Chemical Engineering and Technology, Zagreb, Croatia
Abstract
Isolation and purification of fumarase (fumarate hydratase EC 4.2.1.2) from baker’s yeast was carried out. Yeast cells were disrupted by three methods: glass beads, ultrasound, and the combination of these two methods. Cell disruption methods were compared in their efficiency in Fig. 1. Protein fractionation was carried out by precipitation with ammonium sulphate. The concentrations of ammonium sulphate necessary for fumarase precipitation were found experimentally and are presented in Fig. 2. After precipitation, fumarase samples were purified by gel filtration chromatography on columns filled with Sephadex G50 and Sephadex G100. Examples of the elution curve of one protein suspension sample on both columns are presented in Fig. 3 and Fig. 4. Only the samples having high fumarase activity were used in the next purifying step. Table 1 presents the collective results of the fumarase purification procedure. The techniques used enabled purification of fumarase with a yield of 25 %. The purified enzyme was employed in the hydration of fumaric acid to L-malic acid. Kinetic constants of fumarase were estimated and are presented in Table 2. They were determined from the experimental data measured by the initial reaction rate method. The hydration of fumaric acid to L-malic acid was carried out in a batch reactor and the results are presented in Fig. 5. The kinetic model was developed on the basis of kinetic data and reaction scheme, as presented by equations 1 and 2. It was combined with the mass balances in the batch reactor presented by equations 3 and 4. Considering that fumarase deactivation occurs, it was proposed that the activity loss could be described by a first-order kinetic model (equation 5). Fumarase activity was followed during the batch experiment by the enzyme assay and it was found that activity decay occurs. Deactivation constant was estimated from the independent experimental results and found to be 0.0031 min–1.
Keywords
fumarase; baker's yeast; isolation
Hrčak ID:
81507
URI
Publication date:
22.5.2012.
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