BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF XYLOGLUCAN ENDOTRANSGLYCOSYLASE FROM RIPE KIWIFRUIT

Xyloglucan endotransglycosylase (XET) from the core tissue of ripe kiw ifruit (Actinidia deliciosa [A. Chev,] C.F. Liang et A.R. Ferguson var . deliciosa cv. Hayward) was purified 3000-fold to homogeneity. The en zyme has a molecular weight of 34 kDa, is N-glycosylated, and is activ e between pH 5.0 and 8.0, with an optimum between 5.5 and 5.8. The K-m was 0.6 mg.mL(-1) for kiwifruit xyloglucan and 100 mu M for [H-3]XXXG -ol, a reduced heptasaccharide derived from kiwifruit xyloglucan. Kiwi fruit core XET was capable of depolymerising xyloglucan in the absence of [H-3]XXXG-ol by hydrolysis, and in the presence of [H-3]XXXG-ol by hydrolysis and endotransglycosylation. Six cDNA clones (AdXET1-6) wit h homology to other reported XETs were isolated from ripe kiwifruit mR NA. The six cDNA clones share 93-99% nucleotide identity and appear to belong to a family of closely related genes. Peptide sequencing indic ated that ripe kiwifruit XET was encoded by AdXET6. Northern analysis indicated that expression of the AdXET1-6 gene family was induced in r ipening kiwifruit when endogenous ethylene production could first be d etected, and peaked in climacteric samples when fruit were soft. A ful l-length cDNA clone (AdXETS) was overexpressed in E. coli to produce a recombinant protein that showed endotransglycosylase activity when re folded.