嗜热脂肪芽孢杆菌木聚糖酶A的H297F定点突变、表达及酶学性质变化
1(武汉设计工程学院食品与生物科技学院,湖北 武汉,430205)
2(湖北省农业科学院农业质量标准与检测技术研究所,湖北 武汉,430064)
基金资助
湖北省科技创新团队计划项目T201534;武汉设计工程学院校级科研项目K201303资助。
Site-directed mutagenesis, expression and enzymatic properties of H297F in xylanase XynA of Geobacillus stearothermophilus
1(CollegeofFoodand Biology Science and Technology, Wuhan Institute of Design and Sciences,Wuhan430205,China)
2(InstituteofQualityStandard and Testing Technology for Agro-Products,HubeiAcademyof Agricultural Sciences,Wuhan430064,China)
李婵娟 , 石慧, 王曼玥, 等 . 嗜热脂肪芽孢杆菌木聚糖酶A的H297F定点突变、表达及酶学性质变化[J]. 食品与发酵工业, 0 : 1 . DOI: 10.13995/j.cnki.11-1802/ts.015979
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). Both xylanases were purified and characterized. The optimum pH of XynAH297F was 5.5, similar to XynA. After treated in pH4-10 buffers over 20hs, the residual activities of XynA and XynAH297F were above 90%, which indicated both of them had good pH stability.The optimum temperature of XynAH297F was increased from 70 °C to 75 °C, relative to XynA. In addition, the XynA and XynAH297F remained about 10% and 30% residual activities after incubated at 80 °C for 20 min, respectively. 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.
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