Purification and partial characterization of an exo-polygalacturonase from Paecilomyces variotii liquid cultures.
An extracellular polygalacturonase (PG) produced from Paecilomyces variotii was purified to homogeneity through two chromatography steps using DEAE-Fractogel and Sephadex G-100. The molecular weight of P. variotii PG was 77,300 Da by gel filtration and SDS-PAGE. PG had isoelectric point of 4.37 and optimum pH 4.0. PG was very stable from pH 3.0 to 6.0. The extent of hydrolysis of different pectins by the purified enzyme was decreased with an increase in the degree of esterification. PG had no activity toward non-pectic polysaccharides. The apparent K(m) and V(max) values for hydrolyzing sodium polypectate were 1.84 mg/mL and 432 micromol/min/mg, respectively. PG was found to have temperature optimum at 65 degrees Celsius and was totally stable at 45 degrees Celsius for 90 min. Half-life at 55 degrees Celsius was 50.6 min. Almost all the examined metal cations showed partial inhibitory effects under enzymatic activity, except for Na(+1), K(+1), and Co(+2) (1 mM) and Cu(+2) (1 and 10 mM).
de Lima Damásio
Andre Ricardo
AR
Departamento de Bioquímica e Imunologia--Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
da Silva
Tony Márcio
TM
Maller
Alexandre
A
Jorge
João Atílio
JA
Terenzi
Hector Francisco
HF
Polizeli
Maria de Lourdes Teixeira de Moraes
Mde L
D002412:Cations; D018929:Cell Culture Techniques / Q000379:methods*; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D005110:Extracellular Space / Q000187:drug effects / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D006868:Hydrolysis / Q000187:drug effects; D007526:Isoelectric Point; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D010143:Paecilomyces / Q000187:drug effects / Q000201:enzymology*; D010368:Pectins / Q000378:metabolism; D011096:Polygalacturonase / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature
D002412:Cations; D008670:Metals; D010368:Pectins; D011096:Polygalacturonase; C003181:polygalacturonic acid