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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.

Project description:A hypothetic gene (THA_1941) encoding a putative cellobiose phosphorylase (CBP) from Thermosipho africanus TCF52B has very low amino acid identities (less than 12%) to all known GH94 enzymes. This gene was cloned and over-expressed in Escherichia coli BL21(DE3). The recombinant protein was hypothesized to be a CBP enzyme and it showed an optimum temperature of 75?°C and an optimum pH of 7.5. Beyond its CBP activity, this enzyme can use cellobiose and long-chain cellodextrins with a degree of polymerization of greater than two as a glucose acceptor, releasing phosphate from glucose 1-phosphate. The catalytic efficiencies (k cat/K m) indicated that cellotetraose and cellopentaose were the best substrates for the phosphorolytic and reverse synthetic reactions, respectively. These results suggested that this enzyme was the first enzyme having both cellodextrin and cellobiose phosphorylases activities. Because it preferred cellobiose and cellodextrins to glucose in the synthetic direction, it was categorized as a cellodextrin phosphorylase (CDP). Due to its unique ability of the reverse synthetic reaction, this enzyme could be a potential catalyst for the synthesis of various oligosaccharides. The speculative function of this CDP in the carbohydrate metabolism of T. africanus TCF52B was also discussed.

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

Project description:BACKGROUND:Cellobiose and xylose co-fermentation holds promise for efficiently producing biofuels from plant biomass. Cellobiose phosphorylase (CBP), an intracellular enzyme generally found in anaerobic bacteria, cleaves cellobiose to glucose and glucose-1-phosphate, providing energetic advantages under the anaerobic conditions required for large-scale biofuel production. However, the efficiency of CBP to cleave cellobiose in the presence of xylose is unknown. This study investigated the effect of xylose on anaerobic CBP-mediated cellobiose fermentation by Saccharomyces cerevisiae. RESULTS:Yeast capable of fermenting cellobiose by the CBP pathway consumed cellobiose and produced ethanol at rates 61% and 42% slower, respectively, in the presence of xylose than in its absence. The system generated significant amounts of the byproduct 4-O-?-d-glucopyranosyl-d-xylose (GX), produced by CBP from glucose-1-phosphate and xylose. In vitro competition assays identified xylose as a mixed-inhibitor for cellobiose phosphorylase activity. The negative effects of xylose were effectively relieved by efficient cellobiose and xylose co-utilization. GX was also shown to be a substrate for cleavage by an intracellular ?-glucosidase. CONCLUSIONS:Xylose exerted negative impacts on CBP-mediated cellobiose fermentation by acting as a substrate for GX byproduct formation and a mixed-inhibitor for cellobiose phosphorylase activity. Future efforts will require efficient xylose utilization, GX cleavage by a ?-glucosidase, and/or a CBP with improved substrate specificity to overcome the negative impacts of xylose on CBP in cellobiose and xylose co-fermentation.

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Structural dissection of the reaction mechanism of cellobiose phosphorylase.

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Engineering of cellobiose phosphorylase for the defined synthesis of cellotriose.

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

Project description:Clostridium thermocellum is a cellulosome-producing bacterium that is able to efficiently degrade and utilize cellulose as a sole carbon source. Cellobiose phosphorylase (CBP) plays a critical role in cellulose degradation by catalyzing the reversible phosphate-dependent hydrolysis of cellobiose, the major product of cellulose degradation, into ?-D-glucose 1-phosphate and D-glucose. CBP from C. thermocellum is a modular enzyme composed of four domains [N-terminal domain, helical linker, (?/?)(6)-barrel domain and C-terminal domain] and is a member of glycoside hydrolase family 94. The 2.4 Å resolution X-ray crystal structure of C. thermocellum CBP reveals the residues involved in coordinating the catalytic phosphate as well as the residues that are likely to be involved in substrate binding and discrimination.

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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 ?-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 ?-d-glucose 1-phosphate (?Glc1-P), required optimization and control at two main points. First, kinetic and thermodynamic restrictions upon ?Glc1-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/?Glc1-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%; ?Glc1-P: ?80%) after 1?hr of reaction. We also showed that, by keeping the glucose concentration low (i.e., 1-10?mM; 200?mM ?Glc1-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 rat liver mitochondrial polynucleotide phosphorylase.

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

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