Pectinase production by Neurospora crassa: purification and biochemical characterization of extracellular polygalacturonase activity

Abstract

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].

Production, purification and biochemical characterization of an exo-polygalacturonase from Aspergillus niger MTCC 478 suitable for clarification of orange juice

“…Similar pH optimum of 4.0 has been reported for an exo-PG purified from Aspergillus sojae and Paecilomyces variotii de Lima Damasio et al 2010). Previous reports show that majority of fungal PGs have their pH optima in acidic range (Kester and Visser 1990;Polizeli et al 1991;Niture and Pant 2004;Yadav et al 2012). However, pH stability range of the enzyme was wider (3.0-11.0) as compared to fungal PGs reported from A. niger (Kant et al 2013).…”

“…An exo-PG from A. tubingensis has K m value of 3.2 mg/mL (Kester et al 1996). The K m values of most of the microbial PGs are in the range of 0.1-5.0 (Polizeli et al 1991;Zhang et al 1999;Niture et al 2001;Singh and Rao 2002;Thakur et al 2010a, b;Yadav et al 2012;Martins et al 2013;Chen et al 2014). An exo-PG from the fungus Penicillium frequentans has very low K m value of 0.059 (Barense et al 2001) while PG from pathogenic fungus Ustilago maydis revealed comparatively very high K m of 57.84 (Castruita-Domínguez et al 2014).…”

Characterization of an Exopolygalacturonase from <i>Leucoagaricus gongylophorus</i>, the Symbiotic Fungus of <i>Atta sexdens</i>

“…The calculated molecular weight of the PGaseLg was approximately 39.0 kDa. This molecular mass is in agreement with previously reported PGase from other sources, such as Rhizomucorpusillus [18] and Tricodermaharzianum [32] (about 31 kDa), Acrophialophoranainiana (31 to 35.5 kDa) [14], Saccharomyces cerevisiae strain IM1-8b (36 kDa) [33], Neurosporacrassa (37 kDa) [23] or Rhizopusoryzae (37.44 kDa) [34]. Higher molecular mass PGase is known, such as from E. carotovora (43 kDa) [24] and A. giganteus (69.9 kDa) [35].…”

“…Optimal temperature of polygalacturonases from Rhizomucor pusillus [18], Penicillium viridicatum [15], Thermoascus aurantiacus [20], Pycnoporus sanguineus [40] and A. giganteus [35] ranged from 55˚C to 65˚C. Other optimal temperatures are registered as 45˚C for Neurospora crassa [23], 40˚C for T. harzianum [32] and E. carotovora [24]. The activation energy was determined by using the Arrhenius equation from the slope of the plot of the natural logarithm of the activation rate constants of purified PGaseLg versus the reciprocal absolute temperature (˚K), and it was estimated at 45.6 kJ•mol −1 (10.90 kcal•mol −1 ).…”

Characterization of polygalacturonases produced by the endophytic fungus Penicillium brevicompactum in solid state fermentation - SSF

“…The increase in the synthesis of polygalacturonase by adding pectin (Maldonado and Calieri, 1989;Larios, Garcia and Huitron, 1989;Bailey, 1990;Baracat et al, 1991), or galactose (Polizeli, Jorge and Terenzi, 1991), to the growth medium, has been observed with other micro-organisms. This study was performed using galactose and polygalacturonic acid on pectin 2% as carbon sources in order to analyze the biomass, protein and polygalacturonase activity of P. brevicompactum.…”