کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
25776 | 43599 | 2006 | 10 صفحه PDF | دانلود رایگان |
An extracellular pectinase (PECI) was purified to apparent homogeneity from liquid state cultures of the thermophilic fungus Acrophialophora nainiana by ultrafiltration and a combination of gel filtration and ion-exchange chromatographic procedures. The molecular masses of PECI were 35,500 and 30,749 Da, as determined by SDS-PAGE and mass spectrometry, respectively. It was more active at 60 °C and pH 8.0 and showed high stability at 50 °C with half-life of 7 days. However at 60 and 70 °C, PECI was much less stable with half lives of approximately 20 and 3 min, respectively. The thermostability of purified PECI was also investigated by fluorescence and circular dichroism spectroscopy. Fluorescence revealed that the unfolding transition region was observed between 45 and 70 °C. A major decrease in the stability was found at 70 °C. Circular dichroism measurements at pH between 5.0 and 9.0 showed a transition temperature (Tm) range of 50–55°. The thermodynamic analysis of these results showed that EPGI is thermal stable protein exhibiting maximum stability (ΔG25) of 22.65 and 19.19 kcal/mol at pH 8.0 and 9.0, respectively. The apparent Km value on pectin from citrus fruits was 4.22 mg ml−1. PECI exhibited no detectable activity of pectin methylesterase, endo-polygalacturonase, mannanase, xylanase and cellulase. However, it showed exo-polygalacturonase and pectin lyase activities. The presence of carbohydrate was detected in the pure PECI. It was activated by l-tryptophan, DEPC, DTT, DTNB, DTP, l-cystein and β-mercaptoethanol and inhibited by NBS, Fe2+, Cu2+, Zn2+, Mn2+, Al3+ and Ca2+. The enzyme showed homology with a pectin lyases from Xanthomonas campestris and Bacillus licheniformis.
Journal: Journal of Biotechnology - Volume 123, Issue 1, 3 May 2006, Pages 33–42