木聚糖酶XYNB的N46D突变、表达及酶学性质变化

对来源于Streptomyces olivaceoviridis的高比活木聚糖酶XYNB进行同源建模和同源序列比较,发现第11族木聚糖酶的催化结构域在β折叠股A3和B3之间存的一个保守的氨基酸位点，该位点与木聚糖酶的pH特性有关.据此设计了XYNB的N46D定点突变.将突变酶XYNBN46D在毕赤酵母中表达,表达的XYNBN46D经纯化后与原酶XYNB(同样经毕赤酵母表达后纯化)进行酶学性质比较,结果表明, XYNBN46D的最适pH值由5.2下降到4.2，pH稳定性也向酸性pH偏移，同时，热稳定性和最适温度也有一定的提高, 但酶的比活性显著下降.结果证实，木聚糖酶XYNB的第46位Asn与其最适pH值相关.对导致酶学性质改变的可能因素进行了分析，结果为进一步的结构与功能研究提供了资料.

N46D Mutagenesis of Xylanase XYNB and Enzymatic Changes of Expression Product

A homology modeling of xylanase XYNB from Streptomyces olivaceoviridis A1 was constructed by Swiss-Model and BLAST. A conserved amino acid had been found in the catalytic domain between β-sheet A3 and B3 in the tertiary structure, which influence the pH properties of enzyme. Then a N46D mutation was introduced in XYNB by site-dirrected mutagenesis. The XYNB and mutant xylanase (XYNBN46D) expressed in Pichia pastoris were purified to homogeneity by Superdex 75-chromatograph and their enzymatic properties were determined The result revealed that the optimal pH of XYNBN46D was reduced from5.2 to 4.2. The pH stability of XYNBN46D was changed to acidophilic pH. The optimal temperature and thermostability of XYNBN46D was also changed. The specific activity of XYNBN46D was decreased significantly.A very good correlation is shown between Asn46 and the optimum pH. The mutant xylanase XYNBN46D is a good material for further research in the relationship between structure and function of xylanase.