Enhancing thermostability of xylanase from rumen microbiota by molecular cyclization / 生物工程学报

The capacity for thermal tolerance is critical for industrial enzyme. In the past decade, great efforts have been made to endow wild-type enzymes with higher catalytic activity or thermostability using gene engineering and protein engineering strategies. In this study, a recently developed SpyTag/SpyCatcher system, mediated by isopeptide bond-ligation, was used to modify a rumen microbiota-derived xylanase XYN11-6 as cyclized and stable enzyme C-XYN11-6. After incubation at 60, 70 or 80 ℃ for 10 min, the residual activities of C-XYN11-6 were 81.53%, 73.98% or 64.41%, which were 1.48, 2.92 or 3.98-fold of linear enzyme L-XYN11-6, respectively. After exposure to 60-90°C for 10 min, the C-XYN11-6 remained as soluble in suspension, while L-XYN11-6 showed severely aggregation. Intrinsic and 8-anilino-1-naphthalenesulfonic acid (ANS)-binding fluorescence analysis revealed that C-XYN11-6 was more capable of maintaining its conformation during heat challenge, compared with L-XYN11-6. Interestingly, molecular cyclization also conferred C-XYN11-6 with improved resilience to 0.1-50 mmol/L Ca²⁺ or 0.1 mmol/L Cu²⁺ treatment. In summary, we generated a thermal- and ion-stable cyclized enzyme using SpyTag/SpyCatcher system, which will be of particular interest in engineering of enzymes for industrial application.

Isolation, purification of collagen from soft-shelled turtle calipash for application in biomaterial / 生物工程学报

Recently, research on collagen attracts more interests due to its good biological compatibility. The present study attempted to establish a fast and efficient method to purify collagen from soft-shelled turtle and to explore its application in biological materials. The structure and type of collagen fiber in calipash were determined by van Gieson staining and Picrosirius red staining, which could contribute to the isolation of collagen from soft-shelled turtle Calipash (STCC). Collagen fibers were in high content and the main collagen fiber was type I in STCC. The crude STCC solution was purified by dialysis with different cut-off molecular weight. SDS-PAGE demonstrated that the best purification was in applying 100 kDa dialysis bags after 48 h. The water absorbing capacity and holding capacity of STCC were up to 12.06 g/g and 98.21%, respectively. STCC can be degraded by collagenase in vitro entirely after 72 h. The hemolysis, skin sensitization, hemostatic and wound healing of STCC were determined by using SD rat model, and the collagen cross-linked by glutaric dialdehyde was set as a comparison. STCC and STCC cross-linked did not result in destructed red blood cell, inflamed and sensitized skin. Both materials exhibited good hemostatic effect. Thus, STCC improved the wound healing efficiently. This study implies a potential of STCC in the field of biomaterial.