Immobilization techniques for P. aculeatum dextranase

Abstract

The dextranase purified from P. aculeatum NRRL-896 ( -I,6-glucan 6-glucano-hydrolase, EC 3.2.1.11) was immobilized on different carriers were used by different techniques including: physical adsorption, Entrapment, Covalent binding and Cross-linking. The organic carriers such as natural polymers polysaccharides (i.e. CMC, Chitosan, Alginate, Oyster mushroom stem and Activated charcoal), synthetic polymers (i.e. Polyacrylamide gel and Hydroxyapatite) and inorganic carriers (i.e. Bentonite and Silica gel) were used as matrixes for dextranase immobilization techniques.Immobilization of P. aculeatum dextranase immobilized by physical adsorption indicated that bentonite had the highest activity 263.74 units/g carrier, immobilization yield 74.88 %, specific activity 19989.44 units/100 mg protein, amount of isomaltose produced 1.12 mg/ml, and retained about 67 % of its original activity, followed by treated and untreated silica gel. While, immobilization by entrapment on alginate beads had the highest specific activity (22472.43 units/100 mg protein) followed by polyacrylamide gel (17048.46 units/100 mg protein), then hydroxyapatite treated and untreated (14549.0, 11621.40 units/100 mg protein). Highest immobilization yield 59.89 %, amount of isomaltose produced 1.14 mg/ml, and activity retained 79.0 % of its original compared to the previous carriers were also observed.Immobilization by covalent binding on chitosan had the highest activity 234.16 units g-1 carrier, immobilization yield 61.04 %, and specific activity (18143.33 units/100 mg protein) compared to the free enzyme and activated charcoal. While immobilization on chitosan retained 54.4 % of its original activity compared to that immobilized on activated charcoal 44.6 %.Immobilization by cross-linking on CMC treated via ultrasonicator had the highest activities 143.69 units/ g carrier, specific activity (18066.15 units/100mg protein) and the immobilization yield was 48.82 %, and the free ones isomaltose produced was also higher 0.51 mg/ml compared to the free enzyme 0.38 mg/ml.Oyster mushroom stem was used as a new technique for the immobilization of P. aculeatum dextranase by four different carrier forms including: native stem un-treated, glutaraldehyde, cyanogen bromide (CNBr), and carbodiimide. The low immobilization yield (30.03 %) and immobilized enzyme activity (34.88 units/g carrier) were obtained from physical adsorption on oyster stem without any treatment; the amount of isomaltose was 0.53 and 0.13 mg/ml for free and immobilized dextranase, respectively. The immobilization yield of dextranase immobilized on oyster mushroom stem by Cross-linking technique with glutaraldehyde was 76.77 % and the immobilized dextranase activity was 321.59 U/g carrier. The specific activity of the immobilized dextranase was 27051.66 units/100mg protein, and the amount librated from dextran analyzed was 1.67 mg/ml. The P. aculeatum immobilized dextranase by covalent binding on stem modified by either CNBr or carbodiimide indicated that the highest enzyme activity, yield, amount of isomaltose and activity retained were obtained with carbodiimide being 137.49 units/g carrier, 54.31 %, 1.08 mg/ml and 80.3 % compared to that immobilized with CNBr and free enzyme. Comparative studies between the immobilization techniques of P. aculeatum dextranase indicated that the immobilization technique on oyster mushroom stem by physical adsorption with cross-linking via glutaraldehyde had the higher overall performance and was used for further investigation.