Biochemical and molecular characterisation of xyloglucan endotransglycosylase from ripe kiwifruit

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 puri®ed 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 á mL A1 for kiwifruit xyloglucan and 100 lM for [ 3 H]XXXG-ol, a reduced heptasaccharide derived from kiwifruit xyloglucan. Kiwifruit core XET was capable of depolymerising xyloglucan in the absence of [ 3 H]XXXGol by hydrolysis, and in the presence of [ 3 H]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 ®rst 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.The Genbank accession number of the AdXET5 clone reported in this paper is L46792 Abbreviations: Con A = concanavalin A; XET = xyloglucan endotransglycosylase; FPLC = fast protein liquid chromatography; IPTG = isopropyl b-D-thiogalactopyranoside; kgf = kilogram ®rmness; PCR = polymerase chain reaction; SSC = 150 mM NaCl, 15 mM tri-sodium citrate; XXXG-ol = reduced xyloglucan-derived heptasaccharide

A Barley Xyloglucan Xyloglucosyl Transferase Covalently Links Xyloglucan, Cellulosic Substrates, and (1,3;1,4)-β-D-Glucans

“…However, after the HvXET5 enzyme was purified, its activity remained more or less constant for at least a year at Ϫ20°C. The difficulties with enzyme purification might also explain why so few XTHs have been purified from plant tissue extracts (24,38). Most of the known enzymic properties of XTHs have been determined following heterologous expression of the corresponding cDNAs (25, 26, 39 -41).…”

A xyloglucan oligosaccharide‐active, transglycosylating‐D‐glucosidase from the cotyledons of nasturtium (Tropaeolum majus L) seedlings – purification, properties and characterization of a cDNA clone

“…It is becoming increasingly clear that related enzymes are associated with xyloglucan metabolism in primary cell walls. XETs are involved in developmentally regulated xyloglucan metabolism (Nishitani and Tominaga, 1992;Potter and Fry, 1994;Redgwell and Fry, 1993;Schroder et al, 1998;de Silva et al, 1994), and a xyloglucan oligosaccharide-specific α-xylosidase is associated with xyloglucan turnover in elongation growth (Koyama et al, 1983;O'Neil et al, 1989).…”

Heterologous expression of diverse barley XTH genes in the yeast Pichia pastoris

“…For instance, XTH genes from fruits have been heterologously expressed in Escherichia coli, but subsequent protein solubilization and refolding steps were required to yield low amounts of active enzymes (Arrowsmith and de Silva 1995;Schröder et al 1998). Indeed, the majority of XTH gene products contain a structurally-important N-glycosylation site (Johansson et al 2004;Kallas et al 2005), which rules out the use of bacterial systems such as E. coli as general production hosts.…”