Cloning, expression, immobilisation, characterization of xylanase from Thermophilic geobacillus Sp. TF16 and investigation of industrial applications

In this study, three experimental steps were performed: (1) cloning, (2) expression of the 996 bp xylanase gene coding from a thermophilic bacterium, Geobacillus sp. TF16, and (3) characterization of its product, Xylanase enzyme, aiming to improve its use in industry. For cloning (1), TF16 strain was determined, cloned into pET-28a(+) vector and expressed in Escherichia coli BL21(DE3)pLysS. Enzyme was purified by nickel affinity chromatography. (2) Expression analyses showed that molecular weight of recombinant enzyme was 39.8 kDa by a single band on SDS-PAGE . It is a member of family 10 of glycosyl hydrolases in terms of amino acid similarity. (3) Characterisation includes immobilization and activity assays. Free enzyme was immobilized with a yield of 58.8% on dowex, 68.1% on chitin and 85.6% on chitosan. Free and immobilized enzyme on chitosan showed the highest activity in the presence of xylan substrate. The optimum pH and temperature of the free enzyme were measured 8.5 and 55°C, whereas these were 6.0 and 65°C, respectively, for the immobilized enzyme. Vmax values were 30.86±0.96 µmol.min-1.mg-1 and 1.20±0.03 µmol.min-1.mg-1 for free and immobilized enzymes, respectively. K0,5 values were 1.75 mg/mL and 1.65 mg/mL for free and immobilized enzyme, respectively. Thermostability and pH stability of the immobilized enzyme was determined more constant than the free enzyme. Free enzyme was inhibited in the presence of some metal ions and chemicals at low concentrations, but the immobilized enzyme was not. The enzymes characterized were then applied for practical use in food industry and animal breeding. These are as follows: the rate of reducing sugar increased 1.03 and 1.05-fold in apple juice, 2.84 and 3.23-fold in orange juice, 2.7 and 5-fold in oven spring, 12 % and 34% in poultry feeds by the use of free and immobilized enzymes, respectively.