Biochemical and molecular characterisation of xyloglucan endotransglycosylase from ripe kiwifruit |
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Authors: | R Schröder R G Atkinson G Langenkämper R J Redgwell |
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Institution: | (1) The Horticulture and Food Research Institute of New Zealand, Mt. Albert Research Centre, Private Bag 92169, Auckland, New Zealand, NZ;(2) School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand, NZ |
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Abstract: | Xyloglucan endotransglycosylase (XET) from the core tissue of ripe kiwifruit (Actinidia deliciosa A. Chev.] C.F. Liang et A.R. Ferguson var. deliciosa cv. Hayward) was purified 3000-fold to homogeneity. The enzyme has a molecular weight of 34 kDa, is N-glycosylated, and is
active 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 μM for 3H]XXXG-ol, a reduced heptasaccharide derived from kiwifruit xyloglucan. Kiwifruit core XET was capable of depolymerising xyloglucan
in the absence of 3H]XXXG-ol by hydrolysis, and in the presence of 3H]XXXG-ol by hydrolysis and endotransglycosylation. Six cDNA clones (AdXET1-6) with homology to other reported XETs were isolated
from ripe kiwifruit mRNA. The six cDNA clones share 93–99% nucleotide identity and appear to belong to a family of closely
related genes. Peptide sequencing indicated that ripe kiwifruit XET was encoded by AdXET6. Northern analysis indicated that
expression of the AdXET1-6 gene family was induced in ripening kiwifruit when endogenous ethylene production could first be
detected, and peaked in climacteric samples when fruit were soft. A full-length cDNA clone (AdXET5) was overexpressed in E. coli to produce a recombinant protein that showed endotransglycosylase activity when refolded.
Received: 2 June 1997 / Accepted: 17 June 1997 |
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Keywords: | :Actinidia Cell wall Endotransglycosylase Fruit Hydrolase Ripening |
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