Purification and characterization of an exo-polygalacturonase from Pycnoporus sanguineus.
The present work describes the purification and characterization of a novel extracellular polygalacturonase, PGase I, produced by Pycnoporus sanguineus when grown on citrus fruit pectin. This substrate gave enhanced enzyme production as compared to sucrose and lactose. PGase I is an exocellular enzyme releasing galacturonic acid as its principal hydrolysis product as determined by TLC and orcinol-sulphuric acid staining. Its capacity to hydrolyze digalacturonate identified PGase I as an exo-polygalacturonase. SDS-PAGE showed that PGase I is an N-glycosidated monomer. The enzyme has a molecular mass of 42kDa, optimum pH 4.8 and stability between pH 3.8 and 8.0. A temperature optimum was observed at 50-60 degrees C, with some enzyme activity retained up to 80 degrees C. Its activation energy was 5.352calmol(-1). PGase I showed a higher affinity towards PGA than citric pectin (Km=0.55+/-0.02 and 0.72+/-0.02mgml(-1), respectively). Consequently, PGase I is an exo-PGase, EC 3.2.1.82.
Cátedra de Fitoquímica, Instituto de Estudios Vegetales Dr. A.R. Sampietro, Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, Ayacucho 471, (4000) San Miguel de Tucumán, Argentina.
Emma N
EN
Quiroga
Melina A
MA
Sgariglia
César F
CF
Molina
Diego A
DA
Sampietro
José R
JR
Soberón
Marta A
MA
Vattuone
D002957:Citrus / Q000382:microbiology; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation / Q000235:genetics; D005638:Fruit / Q000382:microbiology; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006603:Hexuronic Acids / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010368:Pectins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011096:Polygalacturonase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D055494:Pycnoporus / Q000201:enzymology*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature
D003470:Culture Media; D006603:Hexuronic Acids; C007819:galacturonic acid; D010368:Pectins; D006026:Glycoside Hydrolases; D011096:Polygalacturonase; C020136:exopolygalacturonase