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

Allahgholi, Leila LU (2016) KEMR33 20162
Department of Chemistry
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... (More)
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. (Less)
Popular Abstract
Polysaccharides are biodegradable and composed of sugars linked together by glycosidic bonds to form a polymer chain. They are available in our food and utilized in our body as a potential energy source.
Hemicelluloses are plant cell wall polysaccharides, derived from the sugars xylose, galactose, mannose and glucose. Mannan-based hemicelluloses have applications in food and pharmaceutical industries. For instance, Locust Bean Gum (LBG) is a galactomannan, which has a backbone composed of mannose residues with galactose side chains, and is widely utilized as a food thickener and stabilizing agent.
Hemicelluloses cannot be digested without help of bacteria that live in the human intestine. Usually, gut bacteria secrete enzymes to help... (More)
Polysaccharides are biodegradable and composed of sugars linked together by glycosidic bonds to form a polymer chain. They are available in our food and utilized in our body as a potential energy source.
Hemicelluloses are plant cell wall polysaccharides, derived from the sugars xylose, galactose, mannose and glucose. Mannan-based hemicelluloses have applications in food and pharmaceutical industries. For instance, Locust Bean Gum (LBG) is a galactomannan, which has a backbone composed of mannose residues with galactose side chains, and is widely utilized as a food thickener and stabilizing agent.
Hemicelluloses cannot be digested without help of bacteria that live in the human intestine. Usually, gut bacteria secrete enzymes to help the human for complete digestion of complex dietary fibres. For example, Bacteroides ovatus is a common bacterium in the human gut which utilizes a wide variety of plant cell wall polysaccharides by synthesizing enzymes called glycoside hydrolases. Two of these enzymes are β-mannanases, which play an important role in mannan degradation in the human gut.
Glycoside hydrolases can have three different modes of action: endo, exo and processive. Endo-acting enzymes cleave the internal glycoside linkages, causing a significant decrease in the viscosity of the substrate, while exo- acting enzymes cleave the terminal glycoside bonds and hardly decrease the viscosity of the substrate. Processive enzymes bind to the polymer chain and cleave a series of glycosidic linkages before its dissociation so reduce the viscosity of LBG at an intermediate level between the exo- and endo- acting enzymes.
In this project, the two β-mannanses from Bacteroides ovatus (with the name of BoMan26A and BoMan26B) were studied. The results for viscosity measurements indicate the processivity of the enzymes. Finally, the dominant product of LBG hydrolysis by the enzymes was shown to be mannobiose (a molecule consisting of two mannose units) which was another indication of the processivity of the enzymes, since it is known from previous studies that some processive enzymes produce dimers as a dominant product. (Less)
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author
Allahgholi, Leila LU
supervisor
organization
course
KEMR33 20162
year
type
H2 - Master's Degree (Two Years)
subject
keywords
processivity, Enzyme kinetics, β-mannanase BoMan26B, biochemistry, biokemi
language
English
id
8895927
date added to LUP
2017-02-03 13:38:39
date last changed
2017-02-03 13:38:39
@misc{8895927,
  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.},
  author       = {Allahgholi, Leila},
  keyword      = {processivity,Enzyme kinetics,β-mannanase BoMan26B,biochemistry,biokemi},
  language     = {eng},
  note         = {Student Paper},
  title        = {Over-expression, purification and characterization of a β-mannanase BoMan26B from Bacteroides ovatus},
  year         = {2016},
}