Immobilization of selected enriched polyphenol oxidases and their biocatalysis in organic solvent media.

摘要

Polyphenol oxidase (PPO) enzymatic extracts were recovered from apple fruit and potato tubers and enriched by an acetone precipitation. The enriched PPO extracts were immobilized by adsorption onto a wide range of inorganic supports, including chitin, alumina oxide, glass beads, Celite, Dowex and Silica gel using selected media, including water, sodium phosphate buffer and a ternary micellar system. The highest immobilization efficiencies and specific activities were obtained when the PPO extracts were suspended in sodium phosphate buffer and adsorbed onto alumina oxide. Biocatalysis of the free and immobilized PPO extracts was investigated in selected organic solvent media, including hexane, heptane, toluene and dichloromethane, using chlorogenic acid, catechin, and the endogenous phenolic compounds from apple fruit and potato tubers as substrate models. In the organic solvent media, the free PPO extracts from apple and potato demonstrated optimal enzymatic activities at 28°C and between 25 to 35°C, respectively, whereas the immobilized extracts both showed optimal enzymatic activities at 30°C. The free and immobilized extracts from apple and potato also showed similar pH values for optimal enzymatic activity in the range of 6.0 to 6.5. The immobilized apple and potato PPO extracts demonstrated a 1.5 to 1.8 and 2.1 to 3.2-fold increases in PPO activity, respectively, compared to those observed with their free counterparts, and the lowest Km values were obtained with chlorogenic acid followed by catechin and the endogenous phenolic compounds. The immobilized and free PPOs from apple and potato also showed higher Vmax values in the hexane medium followed the heptane, toluene and dichloromethane media. The end products of PPO biocatalysis were purified by size-exclusion chromatography and detected at 280 nm for the residual catechin and endogenous phenolic compounds, and at 320 nm for the PPO-catalyzed end products. Spectroscopic scanning of the end products showed absorbance maxima between 280 to 410 nm, depending on the substrate and organic solvent medium used for PPO biocatalysis. The end products from catechin as substrate appeared yellowish in color with arctan (h*) values between 80 and 86°, whereas those obtained with the endogenous phenolic compounds were more pinkish and reddish and had h* values between 50 and 65°. The melting points of the end products obtained from the endogenous potato phenolic compounds were higher than those from apple, while the molecular weights of the apple and potato endogenous phenolic compounds were in the range of 2.9 to 3.6 and 2.6 to 3.6 kDa, respectively. Mass spectra of the pyrolyzed end products showed different degrees of fragmentation, depending on the nature of the enzyme, substrates and reaction environments.