Pectinase production by Neurospora crassa: purification and biochemical characterization of extracellular polygalacturonase activity.
The production of pectinase was studied in Neurospora crassa, using the hyperproducer mutant exo-1, which synthesized and secreted five to six times more enzyme than the wild-type. Polygalacturonase, pectin lyase and pectate lyase were induced by pectin, and this induction was glucose-repressible. Polygalacturonase was induced by galactose four times more efficiently than by pectin; in contrast the activity of lyases was not affected by galactose. The inducing effect of galactose on polygalacturonase was not glucose-repressible. Extracellular pectinases were separated by ion exchange chromatography. Pectate and pectin lyases eluted into three main fractions containing both activities; polygalacturonase eluted as a single, symmetrical peak, apparently free of other protein contaminants, and was purified 56-fold. The purified polygalacturonase was a monomeric glycoprotein (38% carbohydrate content) of apparent molecular mass 36.6-37.0 kDa (Sephadex G-100 and urea-SDS-PAGE, respectively). The enzyme hydrolysed predominantly polypectate. Pectin was also hydrolysed, but at 7% of the rate for polypectate. Km and Vmax for polypectate hydrolysis were 5.0 mg ml-1 and 357 mumol min-1 (mg protein)-1, respectively. Temperature and pH optima were 45 degrees C and 6.0, respectively. The purified polygalacturonase reduced the viscosity of a sodium polypectate solution by 50% with an increase of 7% in reducing sugar groups. The products of hydrolysis at initial reaction times consisted of oligogalacturonates without detectable monomer. Thus, the purified Neurospora crassa enzyme was classified as an endopolygalacturonase [poly(1,4-alpha-D-galacturonide) glycanohydrolase; EC 3.2.1.15].
Polizeli
M de L
Mde L
Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Brazil.
Jorge
J A
JA
Terenzi
H F
HF
D002845:Chromatography; D004790:Enzyme Induction; D005690:Galactose / Q000494:pharmacology; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009492:Neurospora crassa / Q000201:enzymology*; D010368:Pectins / Q000378:metabolism / Q000494:pharmacology; D011096:Polygalacturonase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011133:Polysaccharide-Lyases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
D010368:Pectins; D011096:Polygalacturonase; D011133:Polysaccharide-Lyases; C027349:pectin lyase; C023911:pectate lyase; D005947:Glucose; D005690:Galactose