ISOLASI, PEMURNIAN, KARAKTERISASI DAN AMOBILISASI ENZIM SELULASE DARI VOLVARIELLA VOLVACEA

• Main Author: Agustien, Anthoni
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/4479
• Institution: Institut Teknologi Bandung

<b>Abstract:<p align=\"justify\"> <br /> Cellulase is a very interesting multienzyme because it hydrolyzed cellulose substrate, the main component of agricultural wast product. In this research, cellulase enzyme isolated from Volvariella volvacea, show Cl, Cx activities and B-glucosidase activity as well. The initial purification of the enzyme from Volvariella volvacea grown in Mandels and Reese modified media using ammonium sulphate 40-60 % saturation, indicated (Cl+Cx), Cx and B-glucosidase activities, its spesific activities were 9.3; 6.5 and 2.59 unit per mg respectively. The purity of the ammonium sulphate fractions were 2; 5 and 2 fold as compared to its crude extract. Futher purification with gel filtration column chromatography using sephadex G-75 resulted three protein peaks. Peak I and peak II showed (C1+Cx), Cx and B-glucosidase activities with its specific activities 30.06; 21.09 and 14.76 unit per mg, peak III showed Cx and B-glucosidase activities with its activities 13.34 and 5.33 unit per mg. The purity of (Cl+Cx), Cx and B-glucosidase in the mixture of peak I and peak II were 6.5; 16 and 13 fold respectively. The purity of peak III which indicated only Cx and B-glucosidase activities were 10 and 5 fold. The final purification with ion exchange column chromatography using DEAE sephadex A-50 resulted nine protein peaks. Peak II and peak III both showed (C1+Cx), Cx and B-glucosidase activities; its specific activities were 61.40; 48.33; 25.87 unit per mg and 22.70; 16.50; 9.40 unit per mg respectively. The purity of (C1+Cx), Cx and B-glucosidase in peak II were 13; 37 and 23 fold, while of peak III were 5; 12.6 and 8 fold. The purified cellulase has an optimal condition : pH 6.0, temperature 50 C. The interaction time between the enzyme with (C1+Cx) and Cx activities toward its subsrate were the same, namely 45 minutes. But the interaction time between the enzyme with B-glucosidase activity was 30 minutes. Enzyme immobilization with ionic binding method, using DEAE sephadex A-50 as its support could improved the enzyme specific activity. The optimum pH of the immobilized cellulase remained constant but the optimum temperature was increased to 55 C. The interaction time between immobilized enzyme with (C1+Cx) and Cx activities toward its substrate remain unchanged; however, the interaction time between the enzyme with B-glucosidase was shifted to 45 minutes. The stability of the immobilized enzyme when operated continously indicated that the activities of Cx and B-glucosidase could be maintained for 85 hours. After operating for about 120 hours the immobilized enzyme activity was still about 50 %. The stability of the immobilized enzyme when operated repeatedly for 9 and 10 times still indicated a B-glucosidase activity of 50 % and a Cx activity of 70 %.