BIOTECHNOLOGY TECHNIQUES Volume 10 No. 10 (October 1996) pp.731-734 Received as revised 30 July AN ULTRASENSITIVE COLORIMETRIC METHOD FOR THE ASSAY OF ENDO-1,4-P-D-GLUCANASE Sergej Sestak and Vladimir FarkaS* Institute of Chemistry, Slovak Academy of Sciences, Dubravska cesta 9, 8423 8 Bratislava, Slovakia Summary A calorimetric method for the assay of endo-1,4-P-D-glucanase from fungal cellulolytic systems which utilizes xyloglucan as substrate is described. The substrate forms a blue- green complex with I2 which is lost upon its breakdown with the enzyme. The new assay is a simple one-step procedure and its sensitivity is comparable with that of viscometry. Introduction Endo- 1,4-/3-D-glucanase (endoglucanase, EG, EC 3.2.1.4) is an important component of most microbial cellulolytic systems. Its role consists in random splitting 1,4+glycosidic bonds in cellulose molecules rendering them more susceptible to other enzymes of the cellulolytic system (Ryu and Mandels, 1980). According to principle used, the methods for assessmentof endoglucanase activity can be generally divided into following categories: 1) determination of the decrease of viscosity of water solutions of soluble cellulose derivatives such as Na- carboxymethylcellulose (CMC) or hydroxyethylcellulose (HEC); 2) measuring the release of reducing sugar equivalents from CMC or HEC; 3) determining the amounts of chromophores and/or fluorescent groups released from dyed water-soluble cellulose derivatives. For reviews on these methods see e. g. articles by Canevascini and Gatten (198 l), Buchholz et al. (1984), Ghose (1987) and Sharrock (1988). To date, the viscometric assay of endoglucanase with CMC as substrate has been unsurpassed in sensitivity but the serious disadvantage of viscometric method is that it requires special equipment, is quite time-consuming and therefore not suited for determination of many samples in parallel. On the other hand, the calorimetric methods based on determining released reducing equivalents from the soluble cellulose derivatives, are time-efficient but less sensitive than viscometry and suffer from the interference by reducing sugars present in the enzyme sample. The latter does not apply to methods using chromogenic substrates (e. 731