Papaya endoxylanase biochemical characterization and isoforms expressed during fruit ripening

Papaya fruit ripening processes involve the coordinated action of several hydrolases that causes cell wall degradation. Endoxylanase participates in xylan or arabinoxylan modifications and its importance has been related to papaya softening. However, endoxylanase has been not fully characterized biochemically and kinetically. Semipurified endoxylanase from ripe 'Maradol' papaya fruit had an optimal temperature from 45 degrees C to 50 degrees C, a pH optimum of 5.5 against Remazol brilliant blue-xylan (RBB-Xylan) and enzymatic activity remained stable during 36 h at 45 degrees C. The activation energy of the enzyme was 25.5 kJ mol(-1), and the V-max at 32,37 and 42 degrees C was 788.9, 888.9 and 1085.6 mu g kg(-1) s(-1), respectively. The K-m did not change as a function of temperature and was measured as 1.8 g L-1 and was within the range reported for other xylanases. Total proteins were extracted from color-break, half-ripe and ripe fruit. A pre-endoxylanase at 63.9 kDa was identified in the color-break fruit and an active endoxylanase at 32.5 kDa that was only found in ripe fruit, when the highest enzymatic activity was obtained. Immunodetection on two-dimensional gel electrophoresis (2DE) protein blots showed three isoforms of the pre-endoxylanase at color-break and ripe stages and, four isoforms in ripe fruit that were absent in color-break fruit. The biochemical and kinetic characteristics of the endoxylanase are crucial to our understanding papaya fruit softening. (C) 2013 Elsevier B.V. All rights reserved.