Purification and characterization of an acid phosphatase from Trichoderma harzianum.
An acid phosphatase from Trichoderma harzianum was purified in a single step using a phenyl-Sepharose chromatography column. A typical procedure showed 22-fold purification with 56% yield. The purified enzyme showed as a single band on SDS-PAGE with an apparent molecular weight of 57.8 kDa. The pH optimum was 4.8 and maximum activity was obtained at 55 degrees C. The enzyme retained 60% of its activity after incubation at 55 degrees C for 60 min. The K (m) and V (max) values for p-nitrophenyl phosphate (p-NPP) as a substrate were 165 nM and 237 nM min(-1), respectively. The enzyme was partially inhibited by inorganic phosphate and strongly inhibited by tungstate. Broad substrate specificity was observed with significant activities for p-NPP, ATP, ADP, AMP, fructose 6-phosphate, glucose 1-phosphate and phenyl phosphate.
Leitão
Vanessa Oliveira
VO
Universidade Federal de Goiás, Campus Samambaia, ICB II, Cx. Postal 131, Goiânia, GO 74001-970, Brazil.
de Melo Lima
Rhalcia Cristina
RC
Vainstein
Marilene Henning
MH
Ulhoa
Cirano J
CJ
D000135:Acid Phosphatase / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D013379:Substrate Specificity; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology*
D000135:Acid Phosphatase