Optimization of the multienzyme system for sucrose biosensor by response surface methodology.

An immobilized multienzyme- and cathodic amperometry-based biosensor for sucrose was constructed for analysis of food and fermentation samples. The multienzyme system, comprising invertase (beta-fructofuranosidase; EC 3.2.1.26), mutarotase (aldose 1-epimerase; EC 5.1.3.3) and glucose oxidase (GOD; EC 1.1.3.4), was immobilized using glutaraldehyde as a cross-linking agent. Operating parameters of the biosensor for estimation of sucrose in the range 1-10% were standardized. Response surface methodology based on a three-factor, three-variable design was used to evaluate effect of enzyme activity (10-50 IU invertase, 5-105 IU mutarotase and 1-9 IU GOD) on response of the biosensor. For the range of parameters studied, response time decreased with decreasing invertase and increasing mutarotase and GOD activities. Mutarotase at >75 IU increased the response time through inhibition by its product beta-D-glucose. Optimum conditions for analysis of sucrose were: invertase 10 IU, mutarotase 40 IU and GOD 9 IU. Under these conditions, predicted and actual experimental response times were 2.26 and 2.35 min, respectively, showing good agreement.