Screening of tree leaves as annual renewable green biomass for phenol oxidase production and biochemical characterization of mulberry (Morus alba) leaf phenol oxidases

Fruit tree leaf tissues were screened in a search for determination of an alternative source(s) for commercial phenol oxidase (PO) production considering the importance of utilization of green biomass for production of value-added products. Mulberry, pear, sour cherry and apricot leaves were identified as promising PO production sources, due to their comparable enzyme activities with respect to mushroom (Agaricus bisporus), a well-known PO source. Within the scope of this research, further biochemical characterization was only performed for mulberry (Morus alba) leaf tissue due to its high PO activity (ca. 19 EU g⁻¹ tissue) and also its known non-toxic and edible nature which are important properties of an enzyme source to be used without detailed purification. In mulberry leaves, presence of three different PO activities, laccase, peroxidase and catechol oxidase of 62–64 kDa molecular weights, were identified. Since simple extraction/concentration steps without fractionation/purification was aimed as PO production process, operational parameters such as optimal temperature, pH and kinetic studies of overall PO activity were investigated using concentrated crude extract. The highest PO activity against 4-methyl catechol was observed at 45°C and pH 7. Michaelis–Menten kinetic parameters, K _m and V _max, of PO activity were determined as 6 mM 4-methyl catechol and 2.2 μmol quinone produced min⁻¹ ml⁻¹, respectively. PO activity of mulberry leaves increased up to late November. Consequently, mulberry leaves seem as a suitable PO source for industrial applications in which a wide range of substrate utilization is necessary.