Overlapping extension PCR was used to replace the amino acid residue His 297 (H) by Phe (F) near the activity center of XynA from Geobacillus stearothermophilus.XynA and XynAH297F were separately expressed in Escherichia coli BL21 (DE3) and were both purified and characterized.The optimum pH for XynAH297F was 5.5,which was similar to that for XynA.After treatment in pH4-10 buffers over 20hs,the residual activities of XynA and XynAH97F were both above 90％,which indicated that they had good pH stability.The optimum temperature for XynAH297F (75 ℃) was increased compared with XynA (70 ℃).In addition,the XynA and XynAH297F respectively remained about 10％ and 30％ residual activities after incubated at 80 ℃ for 20 min.It proved that the thermal stability of XynAH297F was improved at certain degree.Compared with XynA,XynAH297F displayed increases in Km,Vmax and Kcat.The results showed that the affinity of enzyme to substrate was decreased,but the catalytic rate was increased.%利用重叠延伸PCR技术对嗜热脂肪芽孢杆菌木聚糖酶XynA活性中心附近的H297位点突变为F.将XynA及XynA H297F在大肠杆菌BL21(DE3)中实施了表达、纯化以及酶学性质研究.比较研究野生型木聚糖酶XynA和突变体XynAH297P的酶学性质发现,突变前后木聚糖酶的最适反应pH值均为5.5,在pH 4 ～ 10的缓冲液中处理20 h后残余酶活均在90％以上,均具有较好的pH稳定性.XynA最适反应温度为70℃,80℃处理20min后只有10％的残余酶活;XynA H297最适反应温度为75℃,且相同处理条件下XynAH297仍具有30％左右的残余活性,说明突变体的热稳定性有一定程度提高.与XynA相比,XynAH297F的动力学参数Km、Vmax和Kcat均增大,说明突变后酶对底物的亲和力降低,但催化速率有所提高.
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