Purification And Characterisation Of Feruloyl Esterase From Barley Malt

Feruloyl esterases (E.C.3.1.1.73, Ferulic acid esterases, FAE) also known as cinnamic acid esterases, are a subclass of carboxylic acid esterases which can release free ferulic acid through catalyze the hydrolysis of the ester bond between hydroxycinnamic acids and sugars on the plant cell walls. In this thesis, barley malt was used as plant materials to characterize the conditions in extraction and purification of feruloyl esterase, and discuss some properties of this enzyme. Several important results were obtained in these studies and were presented as follows:1 The optimized extraction condition of feruloyl esteraseThe text studied the conditions including the extracting time, the temperature, the proportion of barley malt in this buffer and the pH of extraction buffer, respectively. The text got the optimal extractive conditions: The feruloyl esterase was extracted from barley malt with 5-fold (v/w) (0.02 mol/L, pH 6.0) phosphoric acid buffer by muller for 40 min at 0℃, and the best enzyme activity of crude enzyme extract was shown after 12000 rpm centrifugation for 10 min.2 Purification and verificating purity of feruloyl esteraseAfter obtaining the crude enzyme extract of feruloyl esterase, separated and purified by ammonium sulfate precipitation, DEAE-Cellulose ion-exchange chromatography, and Sephadex G-100 gel filtration, respectively. The feruloyl esterase was purified 34-fold with a recovery of 11.6% as shown in SDS-PAGE gel electrophoresis, moreover, the molecular weight of purified feruloyl esterase estimated to be about 29.3 kD.3 Characteristics of feruloyl esteraseThe kinetics of feruloyl esterase were measured by the curve of Lineweaver-Burk with ferulic acid ethyl ester as substrates, the Km was 0.344 mmol/L, and the Vmax was 15.65μmol/mg·min. Characterization of the enzyme exhibited that the optimum pH and the optimum temperature were 5.5 and 50℃, respectively, and it lost little activity after one hour below 50℃from pH 4.0 to 7.0. It was inhibited by Zn²⁺, Cu²⁺, Fe³⁺, and significantly activated by Na⁺ and EDTA.4 Cellulose hydrolysis rate by feruloyl esteraseCorn stalk was hydrolysised by the mixture of feruloyl esterase and cellulase, which changed the total activity of mixed enzyme. The best mixed enzyme activity of feruloyl esterase and cellulase with the proportion was 3:7 (w/w), which was raised 20.22% than cellulase before the mixture, while the hydrolysis rate has rised 14.32%. The total activity of mixed enzyme and hydrolysis rate were reduced 24.28% and 4.57% separately with the proportion of feruloyl esterase and cellulase were 7:3 (w/w). The total activity of mixed enzyme and hydrolysis rate were rised 3.15% and 7.08% only with the proportion of feruloyl esterase and cellulase were 5:5 (w/w).