Chen Nancy Jung, Paull Robert E
Department of Tropical Plant and Soil Sciences, College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa, 3190 Maile Way, Honolulu, HI 96822-2279, USA.
Department of Tropical Plant and Soil Sciences, College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa, 3190 Maile Way, Honolulu, HI 96822-2279, USA. Corresponding author; email:
Funct Plant Biol. 2003 May;30(4):433-441. doi: 10.1071/FP02208.
Papaya (Carica papaya L.) softening during fruit ripening is correlated with the activities of an endoxylanase (EC 3.2.1.8). A 32.5-kDa xylanase (CpaEXY1) from ripening fruit mesocarp was purified 45 871-fold on enzymatic activity and to homogeneity by SDS electrophoresis. The enzyme had endo- and not exo-xylanase activity, a pH optimum of 5-7 and was inhibited by Ca, Co, and Zn. Degenerate primers were constructed from five peptides obtained from the purified enzyme, and a full-length cDNA clone (AY138968) was isolated from a library constructed from ripening mesocarp. CpaEXY1 coded for a 64.96-kDa protein that had up to 61% identity with the 12 predicted Arabidopsis Family 10 endoxylanase-like sequences and 40% to the barley aleurone xylanase. The peptide sequences, obtained from the trypsin-digested purified protein, were all found between amino acid 267and 426 out of the predicted 584 amino acids. The N-terminal 27 amino acids were hydrophobic and formed a predicted secretory signal peptide. A predicted carbohydrate-binding module was located between amino acids 60and 182, distinct from the C-terminal endoxylanase catalytic center. CpaEXY1 was developmentally expressed during fruit ripening and the expression correlated with the variation in softening patterns of different varieties. The findings are consistent with the hypothesis that CpaEXY1 was expressed during fruit ripening; the expression was correlated with softening and was modified by post-translational proteolysis. This modification may take place in the cell wall, and regulate enzyme activity and cell-wall-microdomain-specific hydrolysis.
番木瓜（番木瓜属番木瓜）果实成熟过程中的软化与一种内切木聚糖酶（EC 3.2.1.8）的活性相关。从成熟果实中果皮纯化出一种32.5 kDa的木聚糖酶（CpaEXY1），其酶活性提高了45871倍，经SDS电泳达到了均一性。该酶具有内切木聚糖酶活性而非外切木聚糖酶活性，最适pH为5 - 7，受Ca、Co和Zn抑制。根据从纯化酶中获得的五个肽段构建了简并引物，并从成熟中果皮构建的文库中分离出一个全长cDNA克隆（AY138968）。CpaEXY1编码一种64.96 kDa的蛋白质，与12个预测的拟南芥第10家族内切木聚糖酶样序列具有高达61%的同一性，与大麦糊粉层木聚糖酶具有40%的同一性。从胰蛋白酶消化的纯化蛋白中获得的肽序列，均位于预测的584个氨基酸中的第267至426位氨基酸之间。N端的27个氨基酸具有疏水性，形成一个预测的分泌信号肽。一个预测的碳水化合物结合模块位于第60至182位氨基酸之间，与C端内切木聚糖酶催化中心不同。CpaEXY1在果实成熟过程中呈发育性表达，其表达与不同品种软化模式的变化相关。这些发现与以下假设一致：CpaEXY1在果实成熟过程中表达；其表达与软化相关，并通过翻译后蛋白水解进行修饰。这种修饰可能发生在细胞壁中，并调节酶活性和细胞壁微区特异性水解。
