Biotechnology Letters 22: 957–960, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands. 957 Two simple and rapid methods for the detection of polymer-degrading enzymes on high-resolution, alkaline, cold, in situ-native (HiRACIN)-PAGE and high-resolution, in situ-inhibited native (HiRISIN)-PAGE Ahmed Jawaad Afzal 1 , Salim Ahmed Bokhari 1 , Waseem Ahmad 1 , Mohammad Hamid Rashid 1 , Mohammad Ibrahim Rajoka 1 & Khawar Sohail Siddiqui 2,∗ 1 National Institute for Biotechnology and Genetic Engineering, P.O. Box 577, Jhang Road, Faisalabad, Pakistan 2 Pharmaceuticals Research Center, King Abdul Aziz City of Science and Technology, P.O. Box 6086, Riyadh 11442, Kingdom of Saudi Arabia ∗ Author for correspondence (E-mail: sohail.siddiqui@mailcity.com) Received 2 February 2000; Revisions requested 28 February 2000; Revisions received 17 April 2000; Accepted 18 April 2000 Key words: electrophoresis, endoglucanase, glucoamylase, xylanase Abstract Two sensitive, high-resolution and exceedingly versatile methods for the detection of isoenzymes of polymer- degrading enzymes on high-resolution, alkaline, cold, in situ-native (HiRACIN)-PAGE and high-resolution in situ-inhibited, native (HiRISIN)-PAGE are described. Extracellular crude extracts containing xylanases and carboxymethylcellulases from Scopulariopsis sp. and glucoamylases from Aspergillus niger were subjected to non-denaturing PAGE containing substrates in the resolving gel. In case of HiRACIN-PAGE, the enzymes were prevented from degrading their respective substrates during run by carrying out electrophoresis at 4 ◦ C and the pH of running and resolving gel buffer systems were increased from 8.5 to 10.6. In case of HiRISIN-PAGE, adding competitive inhibitor of the enzyme, cellobiose, in the resolving gel prevents the degradation of polymer during the run. These techniques were successfully applied, for the first time, to visualize four, three and four sharp and distinct bands of xylanases, glucoamylases and CMCases, respectively. Introduction Activity staining of glucoamylase (EC 3.2.1.3), β -1,4- endoxylanase (EC 3.2.1.8) and β -1,4-endoglucanase or carboxymethylcellulase, (EC 3.2.1.4) CMCase, on non-denaturing polyacrylamide gels is routinely per- formed for the visualization of different isoenzymes in native or isoelectric focusing gels (Beguin 1983, Biely & Vrsanska 1988), determination of native molecular weights of isoenzymes in crude extract by slope method (Bryan 1977), monitoring of chemical modification by native enzyme, mobility-shift assay (NEMSA) (Rashid et al. 1997) and determination of stability of different isoenzymes by transverse- urea-gradient-PAGE (Goldenberg 1989, Siddiqui et al. 1997). The most commonly used zymographic method for polymer degrading enzymes is performed by running a conventional discontinuous nondenaturing- PAGE (Goldenberg 1989), overlaying an acrylamide gel containing separated isoenzymes on top of an agarose gel-containing substrate (xylan, starch or car- boxymethylcellulose) and incubating them for 45 to 90 min so that the enzyme molecules could diffuse into the agarose gel (Beguin 1983). This method is not only lengthy but it is also very difficult to handle the brittle agarose gel. Moreover, the activity bands are of low resolution, diffused and dim. Chen & Buller (1995) have devised an in situ staining method for the visualization of isoenzymes of xylanase on SDS- denaturing-renaturing-PAGE. Magnuson & Crawford (1997) have applied this method for the detection of activity bands of xylanase from Streptomyces viri-