Characterization and properties of acid phosphatases with phytase activity produced by Aspergillus caespitosus

• Published in: Biotechnology and applied biochemistry Vol. 40; no. Pt 2; p. 201
• Main Authors: Guimarães, Luis Henrique S, Terenzi, Héctor F, Jorge, João A, Leone, Francisco A, Polizeli, Maria de Lourdes T M
• Format: Journal Article
• Language: English
• Published: United States 01.10.2004
• Subjects: 6-Phytase - chemistry; Acid Phosphatase - biosynthesis; Acid Phosphatase - chemistry; Acid Phosphatase - isolation & purification; Aspergillus - classification; Aspergillus - enzymology; Catalysis; Enzyme Activation; Enzyme Stability; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Molecular Weight; Species Specificity; Temperature
• ISSN: 0885-4513
• DOI: 10.1042/BA20030208

High levels of thermostable acid phosphatases were produced by Aspergillus caespitosus in culture media supplemented with xylan birchwood or agricultural residues, as carbon sources. The optimal culture conditions for production of phosphatases were 40 degrees C and pH 6.0. Extra- and intra-cellular acid phosphatases were purified by chromatography on DEAE-cellulose, followed by concanavalin A-Sepharose affinity separation. Both extra- and intra-cellular enzymes were glycoproteins showing 63.0 and 58.3% of carbohydrate content respectively. Molecular masses estimated on Sepharose CL-6B column were 186 and 190+/-15 kDa, and 84 and 74+/-5 kDa according to SDS/PAGE, for extra- and intra-cellular acid phosphatases respectively. Taken together, these results suggest that both native enzymes were homodimers. Optimum temperature and pH for both phosphatase activities were 80 degrees C and 5.5 respectively. The extra- and intra-cellular acid phosphatases were stable for more than 60 min at 60 degrees C. The extracellular acid phosphatase was slightly inhibited by NaF, in contrast with the significant inhibition of the intracellular form. KH(2)PO(4) inhibited both activities equally. Both extra- and intracellular acid phosphatases were tartarate-resistant. Among several phosphorylated substrates used, the extracellular enzyme preferentially hydrolysed p-nitrophenyl phosphate. Kinetic parameters calculated for the hydrolysis of p-nitrophenyl phosphate by extracellular acid phosphatase were h (Hill coefficient)=1.2, K(0.5)=0.082 mM and V(max)=4.43 units/mg, whereas the intracellular enzyme exhibited Michaelian kinetics with K(m)=0.029 mM and V(max)=0.082 unit/mg. Phytase activity was also observed for both the enzymes, suggesting that they could be useful for biotechnological applications.