Wheat (xylanase inhibitor; TFMSA trifluoromethanesulfonic acid; TLP thaumatin-like protein; (r)TLXI (recombinant)

Wheat (xylanase inhibitor; TFMSA trifluoromethanesulfonic acid; TLP thaumatin-like protein; (r)TLXI (recombinant) thaumatin-like xylanase inhibitor; XIP xylanase inhibitor protein INTRODUCTION The flower cell is safeguarded from its surrounding environment from the cell wall which forms a structurally heterogeneous barrier. They depolymerize xylan which next to cellulose is one of the most abundant polysaccharides in the cell wall of higher vegetation. It consists of a main chain of β-1 4 Alosetron residues that Alosetron depending on the origin may be replaced with e.g. glucuronyl acetyl or arabinofuranosyl organizations to form heteroxylans. Xylanases hydrolyse the β-1 4 linkages in the xylan main chain [2]. The majority of the xylanases belong either to glycoside hydrolase family 10 Alosetron (GH10) or to the structurally unrelated glycoside hydrolase family 11 (GH11) (http://afmb.cnrs-mrs.fr/CAZY/ [3]). In both family members a pair of glutamate residues catalyses the cleavage of the glycosidic relationship one acting like a nucleophile and the additional as the acid-base catalyst. Recently a xylanase was shown to be indispensable in the infection of vegetation from the pathogen [4]. Xylanases are produced not only by micro-organisms but also by vegetation. The latter belong to GH10 and perform important physiological functions in several cells such as contribution to seed germination and fruit ripening [5]. At the same time some vegetation produce proteins which can inhibit xylanases. Over the last decade studies have exposed the presence of two types?of proteinaceous xylanase inhibitors in cereals i.e. the TAXI (xylanase inhibitor)-type [6 7 and the XIP (xylanase inhibitor protein)-type inhibitors [8 9 These proteins have been purified and characterized biochemically genetically and structurally. TAXI-type proteins occur in common wheat (and TAXI-I showed His374 of TAXI-I to be a important residue in xylanase inhibition. This histidine residue interacts in the active site with the two active glutamate residues of the xylanase clearly indicating a competitive type?of inhibition [13]. XIP-type proteins have been isolated from your above-cited cereals as well as from maize (xylanase on the one hand and with GH11 xylanase on the other hand reveal that XIP-I possesses an independent enzyme-binding site for each family of xylanases. Like TAXI XIP is definitely a competitive inhibitor interacting in the active site of the xylanases [16]. For both family members the inhibition mechanism is based on substrate mimicry. A regulatory part of TAXI and XIP in flower development is definitely disaffirmed by their lack of performance against endogenous xylanases their unique specificity towards xylanases of microbial source the ability of TAXI to inhibit two GH11 xylanases of the cereal pathogen [17] and the fact that both TAXI and XIP genes are induced by pathogens and wounding Alosetron [18]. The present study reports within the existence of a third structurally unrelated type?of xylanase inhibitor in wheat which belongs to the thaumatin family. It is further referred to as TLXI (thaumatin-like xylanase inhibitor). More particularly the purification of this protein from wheat the recognition cloning and heterologous manifestation of its related gene is explained. Additionally the biochemical characteristics and the kinetic guidelines of inhibition of both native and recombinant TLXI are discussed. EXPERIMENTAL Materials Wheat (cultivar Soissons) (from Aveve) wholemeal was prepared using a Cyclotec 1093 sample mill. All electrophoresis and chromatography press and molecular mass and pI markers were from GATA2 GE Healthcare unless specified normally. The suppliers of the packages and enzymes used in cloning and heterologous manifestation of TLXI are pointed out below. GH11 xylanase and an GH10 xylanase were supplied by Puratos (by Alosetron Ir Filip Arnaut). Two GH11 xylanases from (also known as and and were kindly made available by VTT Biotechnology (from Professor Maija Tenkanen right now at Division of Applied Chemistry and Microbiology University or college of Helsinki Helsinki Finland) and the Laboratorio de Bioquimíca (Professor Jaime Eyzaguirre Facultad de Ciencias Biológicas Pontificia Universidad Católica de Chile Santiago Chile) respectively. Dr Nathalie Juge (Institute of Food Alosetron Study Norwich U.K.) kindly offered a GH10 and a GH11 xylanase. Thermophilic GH10 and GH11 xylanases were made available by Dr Michael O’Donohue (INRA Reims France). GH10 xylanase was purified from an CBS 110.42 culture filtrate [19]. Grindamyl H 640 bakery enzyme.