Abstract:
The reusability of active enzyme is important in industrial bio-catalysis applications. This is not
readily accomplished with free enzymes. The project investigated a new enzyme immobilization
matrix, ReSynTM, and compared it to three widely used matrixes, Eugergit C, Eupergit C 250 L
and DEAE Sephadex A-25. Initial comparative immobilization performed with BSA, determined
ReSynTM (covalent immobilization) to have an approximate 8 fold higher protein loading
efficiency than the other support. The enzymes selected for immobilization were polyphenol
oxidase (PPO) (first reported purification of PPO from Forelle pear) and laccase
(Novozyme (Pty) Ltd) based upon their application potential. From the comparative assessment
of immobilization based on BSA and the activity (units/mg support) attained by ReSynTM
immobilized laccase, it was deduced that ReSynTM demonstrated performance superiority over the
other supports.
The project aim was the application of immobilized PPO in the ice tea industry to increase the
value of black iced tea based upon an increased theaflavin content. The unusually rich sub-group
of polyphenols known as flavanols or catechins, may constitute up to 30% of the dry tea leaf
weight. The oxidation of green leaf polyphenols is precluded in the preparation of green tea in
contrast to the promotion of oxidation in the production of black tea. PPO catalyzes the
controlled aerobic oxidation of catechins producing various quinones which undergo condensation
reactions resulting in dimeric compounds known as theaflavins, whereas a peroxidase (PO)
enzyme produces polymeric substances, known as thearubigins, during the fermentation stage of
black tea production (Graham 1992). The production of theaflavins from flavan-3-ols (catechins),
is catalysed by PPO (Bonnely et al. 2003). The black tea theaflavins are a mixture of theaflavin,
theaflavin-3-gallate, theaflavin-3'-gallate and theaflavin-3,3'-digallate.
The project focussed on the bioconversion of green tea leaf catechins into theaflavins, as the
original method employed to acquire theaflavins, the extraction of theaflavins from black tea
(only 0.4-1.85% of the dry weight of black tea), doesn’t provide a cost-effective manner of
recovering theaflavins, and amounts to an industrially unfeasible approach (Sharma et al. 2009,
Wang & Li 2006). Preliminary exploratory investigation into the theaflavin synthesis capacity of
Forelle pear and Yacon (Smallanthus sonchifolius) leaf homogenates, respectively determined a
136% and 98% theaflavin content to that present in Lipton® Yellow label black tea, after an hour
of fermentation utilizing Lipton® Green tea as starting material. The homogenates of Forelle
