IMMOBILISATION OF TYROSINASE USING CHITOSAN FOR THE DETECTION AND IDENTIFICATION OF PHENOLIC COMPOUNDS IN AQUEOUS SOLUTION

Abstract

Immobilization establishes the continual usage and efficiency of enzymes for industrial and commercial purposes. In this study the potential application of chitosan film for tyrosinase immobilization and as phenolics biosensor is been assessed. Chitosan gels were used to prepare the chitosan film and then tyrosinase was attached to it using standard methods. The immobilized enzyme activity was then confirmed and compared with that of free tyrosinase Total oxygen consumption and the absorbance of the chitosan film was determined after the reaction of phenolic compound with free and immobilized enzyme. Catalytic properties and kinetic parameters of free and immobilized tyrosinase were also determined. Findings shows the immobilized tyrosinase had comparable activity with that of free tyrosinase, likewise, oxygen consumption of the enzyme and its effect on concentration of substrates as well as on immobilization showed similar pattern for both free and immobilized tyrosinase. Also immobilized tyrosinase was more sensitive in its detection of phenolics; Km obtained for free and immobilized tyrosinase were 18.49 x 10-3 M and -85.71 x 10-3 M for L-DOPA, 29.05 x 10-3 M and 61.45 x 10-3 M for catechol, 30.47 x 10-3 M and 64.66 x 10-3 M for tyrosine and 27.52 x 10-3 M and 235.29 x 10-3 M for the model phenolic respectively, while the Vmax values obtained for free and immobilized tyrosinase was 15.408 U/ml and -19.048 U/ml for L-DOPA, 13.831 U/ml and 27.933 U/ml for catechol, 17.921 U/ml and 43.103 U/ml for tyrosine and 18.349 U/ml and 117.647 U/ml for the model phenolic respectively. In addition, there was an observed increase in colour intensity with corresponding increase in concentration of the phenolic compounds from the immobilized chitosan film, unlike the free chitosan film which showed no observable colour change when immersed in phenolic solution. Hence this study substantiates the possible use of immobilized tyrosinase on chitosan film for detection of phenolics in aqueous medium