The enzyme α-Galactosidase (α-D-galactoside galactohydrolase EC 3.2.1.22) is an exoglycosidase that hydrolyzes the terminal α-galactosyl moieties of glycolipids and glycoproteins. It is ubiquitous in ...nature and possesses extensive applications in the food, pharma, and biotechnology industries. The present study aimed to purify α-galactosidase from Klebsiella pneumoniae, a bacterium isolated from the human oral cavity. The purification steps involved ammonium sulfate precipitation (70 %), dialysis, ion exchange chromatography using a DEAE-cellulose column, and affinity monolith chromatography. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was used to determine the molecular weight of the purified enzyme. The kinetic constants, Michaelis constant (Km) and maximal velocity (Vmax), for this enzyme were determined by using p-nitrophenyl-α-D-galactopyranoside as substrate. The results showed that the purification fold, specific activity, and yield were 126.52, 138.58 units/mg, and 21.5 %, respectively. The SDS-PAGE showed that the molecular weight of the purified enzyme was 75 kDa. The optimum pH and temperature of the purified α-galactosidase were detected at pH 6.0 and 50 °C, respectively. The kinetic constants, Michaelis constant (Km) and maximal velocity (Vmax), for this enzyme were 4.6 mM and 769.23 U/ml, respectively. α-galactosidase from Klebsiella pneumoniae was purified and characterized. (SDS-PAGE) analysis showed that the purified enzyme appeared as single band with a molecular weight of 75 kDa.
Paenibacillus barcinonensis is a soil bacterium bearing a complex set of enzymes for xylan degradation, including several secreted enzymes and Xyn10B, one of the few intracellular xylanases reported ...to date. The crystal structure of Xyn10B has been determined by x-ray analysis. The enzyme folds into the typical (β/α)8 barrel of family 10 glycosyl hydrolases (GH10), with additional secondary structure elements within the β/α motifs. One of these loops -L7- located at the β7 C terminus, was essential for xylanase activity as its partial deletion yielded an inactive enzyme. The loop contains residues His249–Glu250, which shape a pocket opened to solvent in close proximity to the +2 subsite, which has not been described in other GH10 enzymes. This wide cavity at the +2 subsite, where methyl-2,4-pentanediol from the crystallization medium was found, is a noteworthy feature of Xyn10B, as compared with the narrow crevice described for other GH10 xylanases. Docking analysis showed that this open cavity can accommodate glucuronic acid decorations of xylo-oligosaccharides. Co-crystallization experiments with conduramine derivative inhibitors supported the importance of this open cavity at the +2 subsite for Xyn10B activity. Several mutant derivatives of Xyn10B with improved thermal stability were obtained by forced evolution. Among them, mutant xylanases S15L and M93V showed increased half-life, whereas the double mutant S15L/M93V exhibited a further increase in stability, showing a 20-fold higher heat resistance than the wild type xylanase. All the mutations obtained were located on the surface of Xyn10B. Replacement of a Ser by a Leu residue in mutant xylanase S15L can increase hydrophobic packing efficiency and fill a superficial indentation of the protein, giving rise to a more compact structure of the enzyme.
Nucleotide sugars are building blocks for carbohydrate polymers in plant cell walls. They are synthesized from sugar-1-phosphates or epimerized as nucleotide sugars. The main precursor for primary ...cell walls is UDP-glucuronic acid, which can be synthesized via two independent pathways. One starts with the ring cleavage of myo-inositol into glucuronic acid, which requires a glucuronokinase and a pyrophosphorylase for activation into UDP-glucuronate. Here we report on the purification of glucuronokinase from Lilium pollen. A 40-kDa protein was purified combining six chromatographic steps and peptides were de novo sequenced. This allowed the cloning of the gene from Arabidopsis thaliana and the expression of the recombinant protein in Escherichia coli for biochemical characterization. Glucuronokinase is a novel member of the GHMP-kinase superfamily having an unique substrate specificity for d-glucuronic acid with a Km of 0.7 mm. It requires ATP as phosphate donor (Km 0.56 mm). In Arabidopsis, the gene is expressed in all plant tissues with a preference for pollen. Genes for glucuronokinase are present in (all) plants, some algae, and a few bacteria as well as in some lower animals.
In this study, an approach to prepare immobilized metal affinity membrane (IMAM) with high metal ions and protein adsorption capacities was developed. In the process of coupling epichlorohydrin (EPI) ...to the regenerated cellulose membrane (RC membrane), NaOH concentration is found to be the most critical. With a lower NaOH concentration, only a minimal amount of EPI reacted to the RC membrane. When NaOH concentration was higher, the membrane was distorted, which caused a significant pressure drop in flow-through operation. To optimize the IMAM performance, an objective function was defined as the ratio of the model protein, penicillin G acylase (PGA), activity adsorbed on the membrane to the transmembrane pressure drop. According to the criterion, the optimal reaction conditions were found as follows: one RC membrane immersed in 20
ml, 1.4
M NaOH, 5
ml EPI and operated at 24
°C, 150
rpm for 14
h. Under this condition, the copper ions and PGA in IMAM were significantly increased to 75.5
±
0.25
μmol/disc and 1.8
U/disc respectively. The adsorption for lysozyme on the prepared IMAM reached 1044
μg/cm
2, the highest in the literature.
Immobilization and purification of enzymes are usual requirements for their industrial use. Both purification and immobilization have a common factor: they use a solid activated support. Using a ...support for enzyme purification means having mild conditions for enzyme release and a selective enzyme–support interaction is interesting. When using a support for immobilization, however, enzyme desorption is a problem. The improvement of enzyme features through immobilization is a usual objective (e.g., stability, selectivity). Thus, a support designed for enzyme purification and a support designed for enzyme immobilization may differ significantly. In this review, we will focus our attention on the requirements of a support surface to produce the desired objectives. The ideal physical properties of the matrix, the properties of the introduced reactive groups, the best surface activation degree to reach the desired objective, and the properties of the reactive groups will be discussed.
The key to enzymes: The support surface and its activation (number and kind of groups) is a key point in the design of a matrix for enzyme purification (determining the selectivity of enzyme adsorption) and immobilization (controlling the stabilization, enzyme orientation and activity). This paper reviews the most relevant features of a support to determine their feasibility in both techniques.
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