| Summary: | Pullulanase is defined as an extracellular carbohydrase enzyme responsible for the hydrolysis of pullulan into maltotriose. Pullulanase can be extracted from <em>Agaricus bisporus</em> edible mushroom and concentrated by dialysis tube using Tris-HCl buffer. This enzyme was purified by different ratios of ammonium sulphate; the optimal ratio was 30%. It can be purified by ion exchange using a DEAE-cellulose column with a final yield of 5.25 and a purification fold of 4.38. Following Sephadex G-100 gel filtration with a yield of 2.60 and purification fold of 5.0, the total activity of the enzyme reached 160IU and the specific activity was 14.5 U/mg. This study was concerned with estimating the optimum pH and temperature of pullulanase, and it recorded an optimal pH of 7, and the optimal temperature was 60<sup>°</sup>C. The enzyme was more stable at pH 8 and 70<sup>°</sup>C; the incubation period was also determined, and it appeared the most appropriate period was 30 minutes. CaCl<sub>2 </sub>and ZnCl<sub>2</sub> were activator metal ions. Mercaptoethanol in different concentrations was the greatest inhibitor of pullulanase. One of the problems arising in the surrounding area is that much of the environmental pollution consists of starchy food waste. Pullulanase can degrade the glycosidic linkage of pullulan or starch present in the starch waste. The objective of this study is to produce enzymes like pullulanases to participate in minimizing environmental pollution through the degradation of starch waste as a biotechnological application. The conclusion of this study is that pullulanase can be produced from readily available, safe, and low-cost sources such as mushrooms.
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