Over-expression, purification and characterization of a β-mannanase BoMan26B from Bacteroides ovatus

Abstract: Hemicelluloses account for 25-30% of total wood dry weight. Mannan-based polysaccharides are the major component of softwood hemicelluloses. They are characterized by their β-1,4-glycosidic linked backbone composed of mannose or a combination of mannose and glucose residues. The backbone can be substituted by α-1,6-linked galactose residues. β-mannanases cleave the internal β-1,4-glycosidic bonds in the backbone of mannans. They are produced by bacteria, fungi, plants and animals. Bacteroides ovatus is a gram negative bacteria capable of growing in human gut and degrading β-mannan based dietary fibers. In this thesis, two GH26 β-mannanases (BoMan26A and BoMan26B) from Bacteroides ovatus were over- expressed and successfully purified via Ni-NTA affinity chromatography. Enzyme kinetics of BoMan26B was determined using low viscosity locust bean gum. Michaelis-Menten kinetic parameters were found to be 21.2 ±4.8 mg/ml for KM and 260 ± 27 s-1 for kcat. Moreover, the effect of calcium ions on BoMan26B activity was investigated. Results show that in the presence of 1 mM CaCl2 the enzyme activity did not change considerably but the enzyme stability was increased significantly at 37°C. Furthermore, the hydrolysis of digalactosyl- mannopentaose (G2M5) by BoMan26B was attempted to be analyzed by HPAEC-PAD but hydrolysis products could not be detected. Finally, potential occurrence of processivity of BoMan26A and BoMan26B was studied via a new viscometric analysis using a GH5 β-mannanase from Trichoderma reesei (TrMan5A) as a reference enzyme. The product profiles of TrMan5A from hydrolysis of LBG together with the rapid decrease in viscosity suggested that this enzyme is an endo-acting β-mannanase in accordance with previous suggestions. BoMan26A reduced the viscosity of LBG in a similar trend as BoMan26B. The results of viscosity assay for TrMan5A demonstrated a considerable difference compared to the other two enzymes, indicating that BoMan26A and BoMan26B are processive.