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)
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.
LI Chan-juan et al.
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Site-directed mutagenesis, expression and enzymatic
properties of H297F in xylanase XynA of Geobacillus stearothermophilus[J]. Food and Fermentation Industries, 0
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