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

Two sensitive, high-resolution and exceedingly versatile methods for the de tection of isoenzymes of polymer-degrading enzymes on high-resolution, alka line, cold, in situ-native (HiRACIN)-PAGE and high-resolution in situ-inhib ited, native (HiRISIN)-PAGE are described. Extracellular crude extracts con taining xylanases and carboxymethylcellulases from Scopulariopsis sp. and g lucoamylases from Aspergillus niger were subjected to non-denaturing PAGE c ontaining substrates in the resolving gel. In case of HiRACIN-PAGE, the enz ymes were prevented from degrading their respective substrates during run b y carrying out electrophoresis at 4 degrees C and the pH of running and res olving gel buffer systems were increased from 8.5 to 10.6. In case of HiRIS IN-PAGE, adding competitive inhibitor of the enzyme, cellobiose, in the res olving gel prevents the degradation of polymer during the run. These techni ques were successfully applied, for the first time, to visualize four, thre e and four sharp and distinct bands of xylanases, glucoamylases and CMCases , respectively.