Schröder R, Atkinson R G, Langenkämper G, Redgwell R J
Food Research Institute of New Zealand, Mt. Albert Research Centre, Auckland, New Zealand.
Planta. 1998 Feb;204(2):242-51. doi: 10.1007/s004250050253.
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 Km was 0.6 mg.mL-1 for kiwifruit xyloglucan and 100 microM 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.
从成熟猕猴桃（中华猕猴桃美味变种海沃德品种，Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson var. deliciosa cv. Hayward）的核心组织中纯化出木葡聚糖内转糖基酶（XET），纯化倍数达3000倍，达到同质。该酶分子量为34 kDa，为N - 糖基化，在pH 5.0至8.0之间有活性，最适pH在5.5至5.8之间。对于猕猴桃木葡聚糖，Km为0.6 mg/mL，对于[3H]XXXG - ol（一种源自猕猴桃木葡聚糖的还原七糖），Km为100 μM。猕猴桃核心XET在无[3H]XXXG - ol时能够通过水解使木葡聚糖解聚，在有[3H]XXXG - ol时则通过水解和内转糖基化作用使其解聚。从成熟猕猴桃mRNA中分离出六个与其他已报道XET具有同源性的cDNA克隆（AdXET1 - 6）。这六个cDNA克隆的核苷酸同一性为93 - 99%，似乎属于一个紧密相关的基因家族。肽序列分析表明成熟猕猴桃XET由AdXET6编码。Northern分析表明，AdXET1 - 6基因家族的表达在成熟猕猴桃中内源性乙烯产生首次可检测到时被诱导，并在果实变软的呼吸跃变期样本中达到峰值。一个全长cDNA克隆（AdXET5）在大肠杆菌中过表达，以产生一种重组蛋白，该重组蛋白复性后表现出内转糖基酶活性。
