Cloning and functional characterization of a cDNA encoding barley soluble acid invertase (HvINV1)

摘要

We have a long-standing interest in the fructosyltransferases (FTs) of barley, and in particular in sucrose: fructan 6-fructosyltransferase (6-SFr) and sucrose: sucrose 1-fructosyltransferase (1-SST), two key enzymes of fructan synthesis. Cloning of the genes encoding barley 6-SFT and 1-SST revealed their close homology to the beta-fructosidases (invertases), raising questions about the relationship between 1-SST, 6-SFT and invertases. Here we describe the cloning of a cDNA encoding soluble acid invertase from barley (HvINV1) and we characterize its product functionally by heterologous expression in Pichia pastoris. The recombinant HvINV1 cleaves sucrose efficiently, but despite its very high amino acid sequence similarity to FTs it is devoid of FT or fructan hydrolase like side activities. Compared to the FTs, the activity of the recombinant HvINV1 is relatively easily saturable (K-m of 14 mM) and possesses a higher temperature optimum (10 degreesC more that 1-SST). Quantitative analysis of RNA levels demonstrate that HvINV1 is constitutively expressed and not affected much by enhanced sugar levels in excised leaves and roots by sucrose supply or continuous illumination of cut leaves. We hope to use HvINV1 in further studies to investigate the structure-function relationship between FTs and invertases. (C) 2004 Published by Elsevier Ireland Ltd.