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Heterogeneous expression and characterization of the recombinant phytase appAM8(PDF)

Chinese Journal of Applied & Environmental Biology[ISSN:1006-687X/CN:51-1482/Q]

2015 01
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Heterogeneous expression and characterization of the recombinant phytase appAM8
SONG Mali WANG Xi DU Ju HUI Dequan LIANG Aihua
Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, China
Escherichia coli phytase glycosylation thermo stability
Q78 : Q556

To compare the property of phytase produced in different host cells, the recombinant phytase appAM8 gene was expressed in Escherichia coli (E-appAM8) and Pichia pastoris (P-appAM8). The results showed that both phytases have the same optimum pH of 4.5 and reaction temperature of 65 oC. The molecular weight of E-appAM8 and P-appAM8 was estimated to be about 47 × 103 and 53 × 103 respectively by SDS-polyacrylamide gel electrophoresis. The protein expressed in P. pastoris was modified by glycosylation, showing two diffuse bands on SDS-PAGE. When heated to 60 - 80 oC, the thermo stability of E-appAM8 was?obviously lower than that of P-appAM8. After treatment at 70 oC for 15 min, the relative activity of phytase E-appAM8 remained only 4%, while that of the P-appAM8 reached about 50% under the?same?condition, suggesting that glycosylation of phytase appAM8 improved its thermo stability. The Km of E-appAM8 and P-appAM8 was 0.245 mmol/L and 0.36 mmol/L, and their Vmax was 3 196 U/mg and 3 333 U/mg, respectively. The results suggested that glycosylation plays an important role in the thermal stability of phytase appAM8. Point mutations of appAM8 also affect its stability.


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Last Update: 2015-02-15