Purification and characterization of polygalacturonase produced by thermophilic Thermoascus aurantiacus CBMAI-756 in submerged fermentation

• Published in: Antonie van Leeuwenhoek 2007-04, Vol.91 (3), p.291-299
• Main Authors: Martins, Eduardo Silva, Silva, Denis, Leite, Rodrigo S. R, Gomes, Eleni
• Format: Article
• Language: English
• Subjects: Ascomycota - classification; Ascomycota - enzymology; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Citrus; Enzymatic activity; Enzymes; Fermentation; Fermentation - physiology; Fundamental and applied biological sciences. Psychology; Microbiology; Polygalacturonase - chemistry; Polygalacturonase - isolation & purification; Polygalacturonase - metabolism; Substrate Specificity; Thermoascus aurantiacus
• ISSN: 0003-6072; 1572-9699
• DOI: 10.1007/s10482-006-9114-6

An extracellular polygalacturonase was isolated from 5-day culture filtrates of Thermoascus aurantiacus CBMAI-756 and purified by gel filtration and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60-65°C. The apparent K m with citrus pectin was 1.46 mg/ml and the V max was 2433.3 μmol/min/mg. The apparent molecular weight of the enzyme was 30 kDa. The enzyme was 100% stable at 50°C for 1 h and showed a half-life of 10 min at 60°C. Polygalacturonase was stable at pH 5.0-5.5 and maintained 33% of initial activity at pH 9.0. Metal ions, such as Zn⁺², Mn⁺², and Hg⁺², inhibited 50, 75 and 100% of enzyme activity. The purified polygalacturonase was shown to be an endo/exo-enzyme, releasing mono, di and tri-galacturonic acids within 10 min of hydrolysis.