Role of Specific Enzymes in Biosensor for Tea Analysis

Polyphenol oxidase (PPO) from tea leaves, potato, brinjal, apple and
mushroom were assessed for their selectivity towards PP (catechin-a specific
PP present in tea leaves). PPO from tea leaves was purified to homogeneity with
three isozymes. PPO from other sources also partially purified and compared
with standard tyrosinase and laccase. Among all these enzymes tyrosinase
found to have highest substrate specificity towards catechin. This was further
confirmed by enzyme kinetics studies. In the immobilized condition PPO from
tea leaves and tyrosinase were responded to catechin. For black tea sample
analysis by tea biosensor, only tyrosinase was given measurable signals, but for
green tea sample analysis both tyrosinase and PPO were found to be good.
A detector system comprising of two electrodes was fabricated in-house
and used for the analysis of PP using enzyme based biosensor. It is
programmed to detect at various concentrations of PP with good sensitivity.
Immobilized enzyme was stabilized by various PBSAs and lysozyme
observed with greater stability and enhanced the number of analysis. Other
physico-chemical parameters were optimized. With this conditioning biosensor
could analyze about 100 samples by using single immobilized enzyme
membrane. During PP analysis it was observed that immobilized enzyme
membrane was inactivated due to fouling. This fouling was attributed to protein-
PP interaction.
Protein-PP interactions were studied using various amino acids. Amino
acid residues such as lysine, proline and serine observed with maximum affinity
with PP. 10 mM concentration of PP found to be the optimum substrate
concentration to minimize the fouling. Dissociation studies were carried out with
various dissociating agents. Glycerol found to be better polyol to prevent the
complete binding of PP with proteins.
Developed tea biosensor exhibited good robustness in a number of in lab
and field trials. Commercial black tea samples and black tea grades were
analyzed for their PP content to evaluate its quality.
ii
Biosensor system was validated by using various conventional analytical
techniques. Biosensor system analysis also validated by tea taster’s score for
various grades of tea. In all these studies biosensor has shown a good positive
correlation with other conventional analytical techniques.
Tea biosensor developed as an outcome of this research work could be a
potential tool to analyze tea quality based on PP content, hence major quality
attributes of tea such as colour and astringency more or less derived from PP
present in tea. This methodology is rapid, simple, user friendly and cost
effective. It also helps in quality monitoring at different stages of processing (inline
monitoring) and could be adapted for the on field evaluation of quality of tea.