Expression, purification, and characterization of recombinant Metarhizium anisopliae acid trehalase in Pichia pastoris.
The mature peptide of Metarhizium anisopliae acid trehalase (ATM1) (EC3.2.1.28) was successfully expressed in Pichia pastoris at high levels under the control of AOX1 promoter. The recombinant ATM1 (reATM1) was secreted into culture medium. After 48-h 0.5% methanol induction, the activity of reATM1 in the culture supernatant reached the peak, 5.35 U/mg. Enzyme with a histidine sequence appended to the C terminus was still active and was purified using metal-chelate affinity chromatography. The yield of purified reATM1 was 2.5 mg from 1L supernatant. The purified reATM1 exhibited a molecular mass of approximately 170 kDa on SDS-PAGE. The optimum temperature and pH of reATM1 were 30 degrees C and 6.0, respectively, and the K(m) and V(max) values for reATM1 were 2.6 mM and 0.305 mmol/min/mg, respectively. Studies showed that the enzymatic properties of reATM1 were similar to those of the native ATM1.
Liu
Yingchun
Y
Genetic Engineering Research Center, Bioengineering College, Chongqing University, Chongqing 400030, PR China.
Wang
Zhongkang
Z
Yin
Youping
Y
Cao
Yueqing
Y
Zhao
Hua
H
Xia
Yuxian
Y
D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D052981:Metarhizium / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D013696:Temperature; D014198:Trehalase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*
D005656:Fungal Proteins; D011994:Recombinant Proteins; D014198:Trehalase