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

展开
  • 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)

李婵娟(1983-),女,硕士,讲师,研究方向:酶分子工程研究。E.mail:lichanjuan@foxmail.com

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

Expand
  • 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)

摘要

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XynAH297F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;XynAH297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。

本文引用格式

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

Abstract

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.

文章导航

/