Study Of A Thermophilic Bacteria Chitin-binding Domain And Its Application In Immobilized Enzyme

Immobilization of biological affinity purification technology makes purification and immobilization of protein in one step, under mild conditions with high activity yield. Preparation of the immobilized enzyme using this technology gets more and more attention. Chitin-binding domain (ChBD) has great value in enzyme immobilization field, because of its small molecular weight, little effect on enzyme structure, high adsorption efficiency with cheap and nontoxic chitin.In this study, chitin binding domain (Tt-ChBD) from Thermoanaerobacterium thermosaccharolyticum DSM 571 was obtained by gene synthesis and the amino acid of Tt-ChBD had 72% amino acid identity with chitin-binding domain (Bc-ChBD) from mesophilic bacteria Bacillus circulans WL-12. Fusion enzyme XAR-L-Tt-ChBD (XC) was derived from fusing Tt-ChBD with XAR. According to the same methods fusion enzyme XAR-L-Bc-ChBD (XBC) was obtained. Experimental results showed that Tt-ChBD had strong adsorption specificity with colloidal and swelling chitin. The absorption rate of Tt-ChBD with colloidal chitin was above 70%, in the range of 1～6 nmol enzyme quantity. The absorption quantity and rate of Tt-ChBD were 6.6 nmol and 55%, slightly higher than that of Bc-ChBD. The absorption rate of Tt-ChBD (74%) was higher than that of Bc-ChBD (53.8%) in a higher temperature (75℃). The optimal adsorption pH of Tt-ChBD and Bc-ChBD was 4.0. The absorption rate of Tt-ChBD was higher than that of Tt-ChBD in the pH of 3.0,9.0 and 10.0. The absorption rate of Tt-ChBD to colloidal chitin was higher than that of Tt-ChBD in the range of 0-3 mol/L of ion concentration. Higher ionic strength increased the amount of adsorbed ChBD.Effects of pH and ionic strength on Tt-ChBD and Bc-ChBD were similar. Absorption rate of these affinity tags was above 60% in the pH range of 3.0 and 11.0, while that was above 80% in the range of 0 and 3 mol/L of ion concentration.Immobilized fusion enzyme XAR-L-Tt-ChBD was used hydrolyzing xylo-oilgosaccharides in batchwise. Xylose yield rates of the first 5 batch were up to 80% while production rate was 60% in the 30th batch. Using gene recombinant technology Tt-ChBD was fused to Tm-BglA and Te-BglA, respectively, and fusion protein Tm-BglA-Tt-ChBD and Te-BglA-Tt-ChBD were obtained.5% lactose was hydrolyzed by immobilized Tm-BglA-Tt-ChBD, with convertion rate of above 90% after continuous 20 batches. The yield of galacto-oligosaccharides (GOS) reached 30% under the condition of the lactose concentration of 30%. The yield GOS almost unchanged after 10 continuous operations. These immobilized enzymes were quite pure by SDS-PAGE. The above results showed that Tt-ChBD could be firmly combined with chitin and favorably applied in application.Tt-ChBD-L-XAR-L-XynA (CXX) and XAR-L-XynA-L-Tt-ChBD (XXC) were synthesized with changing the position of Tt-ChBD in fusion protein. The expression quantity of XXC was higher than that of CXX in the same expression conditions. The absorption rate of CXX to colloidal chitin was higher than that of XXC in the enzyme amount of 0.3-1.8 U. Free and immobilized fusion enzymes had similar temperature and pH profiles to XAR-L-XynA. However, the temperature and pH stabilities of immobilized CXX and XXC were higher than that of free enzymes. Xylan was hydrolyzed by immobilized XXC in batchwise. Xylose yield was 2.23 mg/mL in the first batch, and production rate was 50% after 19 batches.