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Purification and properties of Cellvibrio gilvus cellobiose phosphorylase.

ABSTRACT: The cellobiose phosphorylase (EC 2.4.1.20) of Cellvibrio gilvus, which is an endocellular enzyme, has been purified 196-fold with a recovery of 11% and a specific activity of 27.4 mumol of glucose 1-phosphate formed/min per mg of protein. The purification procedure includes fractionation with protamine sulphate, and hydroxyapatite and DEAE-Sephadex A-50 chromatography. The enzyme appears homogeneous on polyacrylamide-gel electrophoresis, and a molecular weight of 280 000 was determined by molecular-sieve chromatography. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed a single band and mol.wt. 72 000, indicating that cellobiose phosphorylase consists of four subunits. The enzyme had a specificity for cellobiose, requiring Pi and Mg2+ for phosphorylation, but not for cellodextrin, gentibiose, laminaribiose, lactose, maltose, kojibiose and sucrose. The enzyme showed low thermostability, an optimum pH of 7.6 and a high stability in the presence of 2-mercaptoethanol or dithiothreitol. The Km values for cellobiose and Pi were 1.25 mM and 0.77 mM respectively. Nojirimycin acted as a powerful pure competitive inhibitor (with respect to cellobiose) of the enzyme (Ki = 45 microM). Addition of thiol-blocking agents to the enzyme caused 56% inhibition at 500 microM-N-ethylmaleimide and 100% at 20 microM-p-chloromercuribenzoate.

SUBMITTER: Sasaki T

PROVIDER: S-EPMC1154160 | biostudies-other | 1983 Mar

REPOSITORIES: biostudies-other

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Biochemical properties of GH94 cellodextrin phosphorylase THA_1941 from a thermophilic eubacterium Thermosipho africanus TCF52B with cellobiose phosphorylase activity.

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Overcoming inefficient cellobiose fermentation by cellobiose phosphorylase in the presence of xylose.

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Structure of cellobiose phosphorylase from Clostridium thermocellum in complex with phosphate.

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Cellobiose phosphorylase from Caldicellulosiruptor bescii catalyzes reversible phosphorolysis via different kinetic mechanisms.

Project description:In the process of yielding biofuels from cellulose degradation, traditional enzymatic hydrolysis, such as β-glucosidase catalyzing cellobiose, can barely resolve the contradiction between cellulose degradation and bioenergy conservation. However, it has been shown that cellobiose phosphorylase provides energetic advantages for cellobiose degradation through a phosphorolytic pathway, which has attracted wide attention. Here, the cellobiose phosphorylase gene from Caldicellulosiruptor bescii (CbCBP) was cloned, expressed, and purified. Analysis of the enzymatic properties and kinetic mechanisms indicated that CbCBP catalyzed reversible phosphorolysis and had good thermal stability and broad substrate selectivity. In addition, the phosphorolytic reaction of cellobiose by CbCBP proceeded via an ordered Bi Bi mechanism, while the synthetic reaction proceeded via a ping pong Bi Bi mechanism. The present study lays the foundation for optimizing the degradation of cellulose and the synthesis of functional oligosaccharides.

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Crystallization and X-ray diffraction studies of cellobiose phosphorylase from Cellulomonas uda.

Project description:Disaccharide phosphorylases are able to catalyze both the synthesis and the breakdown of disaccharides and have thus emerged as attractive platforms for tailor-made sugar synthesis. Cellobiose phosphorylase from Cellulomonas uda (CPCuda) is an enzyme that belongs to glycoside hydrolase family 94 and catalyzes the reversible breakdown of cellobiose [beta-D-glucopyranosyl-(1,4)-D-glucopyranose] to alpha-D-glucose-1-phosphate and D-glucose. Crystals of ligand-free recombinant CPCuda and of its complexes with substrates and reaction products yielded complete X-ray diffraction data sets to high resolution using synchrotron radiation but suffered from significant variability in diffraction quality. In at least one case an intriguing space-group transition from a primitive monoclinic to a primitive orthorhombic lattice was observed during data collection. The structure of CPCuda was determined by maximum-likelihood molecular replacement, thus establishing a starting point for an investigation of the structural and mechanistic determinants of disaccharide phosphorylase activity.

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Product solubility control in cellooligosaccharide production by coupled cellobiose and cellodextrin phosphorylase.

Project description:Soluble cellodextrins (linear β-1,4-d-gluco-oligosaccharides) have interesting applications as ingredients for human and animal nutrition. Their bottom-up synthesis from glucose is promising for bulk production, but to ensure a completely water-soluble product via degree of polymerization (DP) control (DP ≤ 6) is challenging. Here, we show biocatalytic production of cellodextrins with DP centered at 3 to 6 (~96 wt.% of total product) using coupled cellobiose and cellodextrin phosphorylase. The cascade reaction, wherein glucose was elongated sequentially from α-d-glucose 1-phosphate (αGlc1-P), required optimization and control at two main points. First, kinetic and thermodynamic restrictions upon αGlc1-P utilization (200 mM; 45°C, pH 7.0) were effectively overcome (53% → ≥90% conversion after 10 hrs of reaction) by in situ removal of the phosphate released via precipitation with Mg2+ . Second, the product DP was controlled by the molar ratio of glucose/αGlc1-P (∼0.25; 50 mM glucose) used in the reaction. In optimized conversion, soluble cellodextrins in a total product concentration of 36 g/L were obtained through efficient utilization of the substrates used (glucose: 98%; αGlc1-P: ∼80%) after 1 hr of reaction. We also showed that, by keeping the glucose concentration low (i.e., 1-10 mM; 200 mM αGlc1-P), the reaction was shifted completely towards insoluble product formation (DP ∼9-10). In summary, this study provides the basis for an efficient and product DP-controlled biocatalytic synthesis of cellodextrins from expedient substrates.

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Partial purification and properties of guinea-pig liver polynucleotide phosphorylase.

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