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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

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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.

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Authors and Affiliations

  1. National Institute for Biotechnology and Genetic Engineering, P.O. Box 577, Jhang Road, Faisalabad, Pakistan

    Ahmed Jawaad Afzal, Salim Ahmed Bokhari, Waseem Ahmad, Mohammad Hamid Rashid & Mohammad Ibrahim Rajoka

  2. Pharmaceuticals Research Center, King Abdul Aziz City of Science and Technology, P.O. Box 6086, Riyadh, 11442, Kingdom of Saudi Arabia

    Khawar Sohail Siddiqui

Authors
  1. Ahmed Jawaad Afzal
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  2. Salim Ahmed Bokhari
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  3. Waseem Ahmad
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  4. Mohammad Hamid Rashid
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  5. Mohammad Ibrahim Rajoka
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  6. Khawar Sohail Siddiqui
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Afzal, A.J., Bokhari, S.A., Ahmad, W. et al. 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. Biotechnology Letters 22, 957–960 (2000). https://doi.org/10.1023/A:1005676824462

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  • DOI: https://doi.org/10.1023/A:1005676824462

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