Enzymatic characterization of a glycoside hydrolase family 5 subfamily 7 (GH5_7) mannanase from Arabidopsis thaliana

Yang Wang 0 1 2 4 Francisco Vilaplana 0 1 2 4 Harry Brumer 0 1 2 4 Henrik Aspeborg 0 1 2 4 0 F. Vilaplana Wallenberg Wood Science Centre, Royal Institute of Technology (KTH) , 100 44 Stockholm, Sweden 1 Y. Wang H. Aspeborg ( 2 Y. Wang F. Vilaplana H. Brumer H. Aspeborg Division of Glycoscience, School of Biotechnology, KTH Royal Institute of Technology, AlbaNova University Centre , 106 91 Stockholm, Sweden 3 ) Division of Industrial Biotechnology, School of Biotechnology, KTH Royal Institute of Technology, AlbaNova University Centre , 106 91 Stockholm, Sweden 4 H. Brumer Michael Smith Laboratories and Department of Chemistry, University of British Columbia , 2185 East Mall, Vancouver V6T 1Z4, Canada Each plant genome contains a repertoire of -mannanase genes belonging to glycoside hydrolase family 5 subfamily 7 (GH5_7), putatively involved in the degradation and modification of various plant mannan polysaccharides, but very few have been characterized at the gene product level. The current study presents recombinant production and in vitro characterization of AtMan5-1 as a first step towards the exploration of the catalytic capacity of Arabidopsis thaliana -mannanase. The target enzyme was expressed in both E. coli (AtMan5-1e) and P. pastoris (AtMan5-1p). The main difference between the two forms was a higher observed thermal stability for AtMan5-1p, presumably due to glycosylation of that particular variant. AtMan5-1 displayed optimal activity at pH 5 and 35 C and hydrolyzed polymeric carob galactomannan, konjac glucomannan, and spruce galactoglucomannan as well as oligomeric mannopentaose and mannohexaose. However, the galactose-rich and highly branched guar gum was not as efficiently degraded. AtMan5-1 activity was enhanced by Co2+ and inhibited by Mn2+. The catalytic efficiency values for carob galactomannan were 426.8 and 368.1 min1 mg1 mL for AtMan5-1e and AtMan5-1p, respectively. Product analysis of AtMan5-1p suggested that at least five substrate-binding sites were required for manno-oligosaccharide hydrolysis, and that the enzyme also can act as a transglycosylase. - Plants can create diverse cell wall composites with different properties by varying the arrangement of the four main cell wall components cellulose, hemicelluloses, lignin and pectins (Gibson 2012). Hemicelluloses, sometimes referred to as cross-linking or matrix polysaccharides, are a class of structurally heterogeneous polymers closely interacting with cellulose and lignin. Xyloglucans, xylans, mannans and mixed-linkage -glucans are generally included in hemicelluloses category (Scheller and Ulvskov 2010). -1,4-Mannans show a widespread distribution in plant tissues and cell wall types, and have been an important plant carbohydrate since the green algae moved out of the water and began the colonization of land. In certain cell walls, i.e., the secondary cell wall of gymnosperms and the Type III primary cell wall of ferns, mannan-type polysaccharides are the main hemicelluloses (Rodriguez-Gacio Mdel et al. 2012; Silva et al. 2011). Based on the backbone monomer composition and the presence of side chains, the mannans can be divided into four groups: mannans, glucomannans, galactomannans and galactoglucomannans. Mannans and glucomannans are linear polymers with either a backbone composed exclusively of -1,4-linked mannosyl residues or a main chain with a varying distribution of glucose and mannose units joined together with -1,4 glycosidic bonds. Decorating these two types of backbones with -1,6-linked galactose side chains results in the branched galactomannans and galactoglucomannans. A further layer of complexity is the occurrence of acetylations of mannans, a modification that may mask the actual mannan polysaccharide distribution in planta (Marcus et al. 2010). Mannans are structural components of the cell wall, but can also function as energy-storage compounds in seeds, bulbs and tubers (Schroder et al. 2009). In the model plant Arabidopsis thaliana, mannan-type polysaccharides are detected in low levels in most tissues, but appear to be more abundant in flowers, siliques and inflorescence stems. Secondary cell walls of xylem elements, xylem parenchyma and interfascicular fibers contain higher amounts of mannans (Handford et al. 2003; Liepman et al. 2007). A detailed mannan labeling pattern in the stem was recently described showing similar features of mannan detection between Arabidopsis and poplar (Kim and Daniel 2012). The principal enzymes involved in the degradation and/ or modification of mannan-based polysaccharides are endo-mannanases (E.C. 3.2.1.78). These enzymes are responsible for catalyzing hydrolysis of the -1,4-linked backbone within different mannans. In the CAZy (carbohydrate-active enzymes) database (Cantarel et al. 2009), enzymes with this activity are classified into three different glycoside hydrolase families: GH5, GH26 and GH113. Endo--mannanases in these GH families share a (/)8 barrel fold struct (...truncated)