Trehalose phosphorylase from Pleurotus ostreatus: characterization and stabilization by covalent modification, and application for the synthesis of alpha,alpha-trehalose

Trehalose phosphorylase from the basidiomycete Pleurotus ostreatus (PoTPase) was isolated from fungal fruit bodies through approximately 500-fold purification with a yield of 44%. Combined analyses by SDS-PAGE and gelfiltration show that PoTPase is a functional monomer of approximately 55 kDa molecular mass. PoTPase catalyzes the phosphorolysis of alpha,alpha-trehalose, yielding alpha-d-glucose 1-phosphate (alphaGlc 1-P) and alpha-d-glucose as the products. The optimum pH of PoTPase for alpha,alpha-trehalose phosphorolysis and synthesis is 6.8 and 6.2, respectively. Apparent substrate binding affinities (K(m)) were determined at pH 6.8 and 30 degrees C: alpha,alpha-trehalose (79 mM); phosphate (3.5 mM); d-glucose (40 mM); alphaGlc 1-P (4.1mM). A series of structural analogues of d-glucose were tested as glucosyl acceptors for the enzymatic reaction with alphaGlc 1-P, and robust activity with d-mannose (3%), 2-deoxy d-glucose (8%), 2-fluoro d-glucose (15%) and 2-keto-d-glucose (50%) was detected. Arsenate replaces, with 30% relative activity, phosphate in the conversion of alpha,alpha-trehalose, and vanadate strongly inhibits the enzyme activity (K(i) approximately 4 microM). PoTPase has a half-life (t(0.5)) of approximately 1 h at 30 degrees C in the absence of stabilizing compounds such as alpha,alpha-trehalose (300 mM; t(0.5)=11.5 h), glycerol (20%, w/v; t(0.5)=6.5h) or polyethylenglycol (PEG) 4000 (26%, w/v; t(0.5)=70 h). Covalent modification of PoTPase with activated derivatives of PEG 5000 increases the stability by up to 600-fold. Sucrose was converted to alpha,alpha-trehalose in approximately 60% yield using a coupled enzyme system composed of sucrose phosphorylase from Leuconostoc mesenteroides, glucose isomerase from Streptomyces murinus and the appropriately stabilized PoTPase.     

Sucrose phosphorylase: a powerful transglucosylation catalyst for synthesis of α-D-glucosides as industrial fine chemicals

Abstract

Sucrose phosphorylase is a bacterial transglucosidase that catalyzes conversion of sucrose and phosphate into α-D-glucose-1-phosphate and D-fructose. The enzyme utilizes a glycoside hydrolase-like double displacement mechanism that involves a catalytically competent β-glucosyl enzyme intermediate. In addition to reaction with phosphate, glucosylated sucrose phosphorylase can undergo hydrolysis to yield α-D-glucose or it can decompose via glucosyl transfer to a hydroxy group in suitable acceptor molecules, giving new α-D-glucosidic products. The glucosyl acceptor specificity of sucrose phosphorylase is reviewed, focusing on applications of the enzyme in glucoside synthesis. Polyhydroxylated compounds such as sugars and sugar alcohols are often glucosylated efficiently. Aryl alcohols and different carboxylic acids also serve as acceptors for enzymatic transglucosylation. The natural osmolyte 2-O-(α-D-glucopyranosyl)-sn-glycerol (GG) was prepared by regioselective glucosylation of glycerol from sucrose using the phosphorylase from Leuconostoc mesenteroides. An industrial process for production of GG as active ingredient of cosmetic formulations has been recently developed. General advantages of sucrose phosphorylase as a transglucosylation catalyst lie in the use of sucrose as a high-energy glucosyl donor and the usually weak hydrolase activity of the enzyme towards substrate and product.     

Characterization of a cellobiose phosphorylase from a hyperthermophilic eubacterium,Thermotoga maritima MSB8

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80 degrees C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70 degrees C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and alpha-D-glucose-1-phosphate. The Km for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the kcat was 5.4 s(-1). In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-beta-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s(-1)) kcat followed by 6-deoxy-D-glucose (17 s(-1)) and 2-deoxy-D-glucose (16 s(-1)). The natural substrate, D-glucose with the kcat of 8.0 s(-1) had the highest (1.1 x 10(4) M(-1) s(-1)) kcat/Km compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, alpha-D-glucose-1-phosphate, at higher concentrations.     

Substrate-induced activation of maltose phosphorylase: interaction with the anomeric hydroxyl group of alpha-maltose and alpha-D-glucose controls the enzyme's glucosyltransferase activity

Maltose phosphorylase, long considered strictly specific for beta-D-glucopyranosyl phosphate (beta-D-glucose 1-P), was found to catalyze the reaction beta-D-glucosyl fluoride + alpha-D-glucose----alpha-maltose + HF, at a rapid rate, V = 11.2 +/- 1.2 mumol/(min.mg), and K = 13.1 +/- 4.4 mM with alpha-D-glucose saturating, at 0 degrees C. This reaction is analogous to the synthesis of maltose from beta-D-glucose 1-P + D-glucose (the reverse of maltose phosphorolysis). In acting upon beta-D-glucosyl fluoride, maltose phosphorylase was found to use alpha-D-glucose as a cosubstrate but not beta-D-glucose or other close analogs (e.g., alpha-D-glucosyl fluoride) lacking an axial 1-OH group. Similarly, the enzyme was shown to use alpha-maltose as a substrate but not beta-maltose or close analogs (e.g., alpha-maltosyl fluoride) lacking an axial 1-OH group. These results indicate that interaction of the axial 1-OH group of the disaccharide donor or sugar acceptor with a particular protein group near the reaction center is required for effective catalysis. This interaction appears to be the means that leads maltose phosphorylase to promote a narrowly defined set of glucosyl transfer reactions with little hydrolysis, in contrast to other glycosylases that catalyze both hydrolytic and nonhydrolytic reactions.     

Comparison of the binding of glucose and glucose 1-phosphate derivatives to T-state glycogen phosphorylase b

The binding of T-state- and R-state-stabilizing ligands to the catalytic C site of T-state glycogen phosphorylase b has been investigated by crystallographic methods to study the interactions made and the conformational changes that occur at the C site. The compounds studied were alpha-D-glucose, 1, a T-state-stabilizing inhibitor of the enzyme, and the R-state-stabilizing phosphorylated ligands alpha-D-glucose 1-phosphate (2), 2-deoxy-2-fluoro-alpha-D-glucose 1-phosphate (3), and alpha-D-glucose 1-methylenephosphonate (4). The complexes have been refined, giving crystallographic R factors of less than 19%, for data between 8 and 2.3 A. Analysis of the refined structures shows that the glucosyl portions of the phosphorylated ligands bind in the same orientation as glucose and retain most of the interactions formed between glucose and the enzyme. However, the phosphates of the phosphorylated ligands adopt different conformations in each case; the stability of these conformations have been studied by using computational methods to rationalize the different binding modes. Binding of the phosphorylated ligands is accompanied by movement of C-site residues, most notably a shift of a loop out of the C site and toward the exterior of the protein. The C-site alterations do not include movement of Arg569, which has been observed in both the refined complex with 1-deoxy-D-gluco-heptulose 2-phosphate (5) [Johnson, L. N., et al (1990) J. Mol. Biol. 211, 645-661] and in the R-state enzyme [Barford, D. & Johnson, L. N. (1989) Nature 340, 609-616]. Refinement of the ligand complexes has also led to the observation of additional electron density for residues 10-19 at the N-terminus which had not previously been localized in the native structure. The conformation of this stretch of residues is different from that observed in glycogen phosphorylase a.     

Uptake and metabolism of sucrose by Streptococcus lactis

Transport and metabolism of sucrose in Streptococcus lactis K1 have been examined. Starved cells of S. lactis K1 grown previously on sucrose accumulated [14C]sucrose by a phosphoenolpyruvate-dependent phosphotransferase system (PTS) (sucrose-PTS; Km, 22 microM; Vmax, 191 mumol transported min-1 g of dry weight of cells-1). The product of group translocation was sucrose 6-phosphate (6-O-phosphoryl-D-glucopyranosyl-1-alpha-beta-2-D-fructofuranoside). A specific sucrose 6-phosphate hydrolase was identified which cleaved the disaccharide phosphate (Km, 0.10 mM) to glucose 6-phosphate and fructose. The enzyme did not cleave sucrose 6'-phosphate(D-glucopyranosyl-1-alpha-beta-2-D-fructofuranoside-6'-phosphate). Extracts prepared from sucrose-grown cells also contained an ATP-dependent mannofructokinase which catalyzed the conversion of fructose to fructose 6-phosphate (Km, 0.33 mM). The sucrose-PTS and sucrose 6-phosphate hydrolase activities were coordinately induced during growth on sucrose. Mannofructokinase appeared to be regulated independently of the sucrose-PTS and sucrose 6-phosphate hydrolase, since expression also occurred when S. lactis K1 was grown on non-PTS sugars. Expression of the mannofructokinase may be negatively regulated by a component (or a derivative) of the PTS.     

Practical preparation of lacto-N-biose I, a candidate for the bifidus factor in human milk

A one-pot enzymatic reaction to produce lacto-N-biose I (LNB), which is supposed to represent the bifidus factor in human milk oligosaccharides, was demonstrated. Approximately 500 mM of LNB was generated in 10-liter of reaction mixture initially containing 660 mM of sucrose and 600 mM of GlcNAc by the concurrent actions of four enzymes, sucrose phosphorylase, UDP-glucose-hexose-1-phospate uridylyltransferase, UDP-glucose 4-epimerase, and lacto-N-biose phosphorylase, in the presence of UDP-Glc and phosphate, indicating a reaction yield of 83%. LNB was isolated from the mixture by crystallization after yeast treatment. Finally, 1.4 kg of LNB of 99.6% purity was recovered after recrystallization.     

Transport and phosphorylation of disaccharides by the ruminal bacterium Streptococcus bovis

Toluene-treated cells of Streptococcus bovis JB1 phosphorylated cellobiose, glucose, maltose, and sucrose by the phosphoenolpyruvate-dependent phosphotransferase system. Glucose phosphorylation was constitutive, while all three disaccharide systems were inducible. Competition experiments indicated that separate phosphotransferase systems (enzymes II) existed for glucose, maltose, and sucrose. [14C]maltose transport was inhibited by excess (10 mM) glucose and to a lesser extent by sucrose (90 and 46%, respectively). [14C]glucose and [14C]sucrose transports were not inhibited by an excess of maltose. Since [14C]maltose phosphorylation in triethanolamine buffer was increased 160-fold as the concentration of Pi was increased from 0 to 100 mM, a maltose phosphorylase (Km for Pi, 9.5 mM) was present, and this activity was inducible. Maltose was also hydrolyzed by an inducible maltase. Glucose 1-phosphate arising from the maltose phosphorylase was metabolized by a constitutive phosphoglucomutase that was specific for alpha-glucose 1-phosphate (Km, 0.8 mM). Only sucrose-grown cells possessed sucrose hydrolase activity (Km, 3.1 mM), and this activity was much lower than the sucrose phosphotransferase system and sucrose-phosphate hydrolase activities.     

Transport and phosphorylation of disaccharides by the ruminal bacterium Streptococcus bovis.

Toluene-treated cells of Streptococcus bovis JB1 phosphorylated cellobiose, glucose, maltose, and sucrose by the phosphoenolpyruvate-dependent phosphotransferase system. Glucose phosphorylation was constitutive, while all three disaccharide systems were inducible. Competition experiments indicated that separate phosphotransferase systems (enzymes II) existed for glucose, maltose, and sucrose. [14C]maltose transport was inhibited by excess (10 mM) glucose and to a lesser extent by sucrose (90 and 46%, respectively). [14C]glucose and [14C]sucrose transports were not inhibited by an excess of maltose. Since [14C]maltose phosphorylation in triethanolamine buffer was increased 160-fold as the concentration of Pi was increased from 0 to 100 mM, a maltose phosphorylase (Km for Pi, 9.5 mM) was present, and this activity was inducible. Maltose was also hydrolyzed by an inducible maltase. Glucose 1-phosphate arising from the maltose phosphorylase was metabolized by a constitutive phosphoglucomutase that was specific for alpha-glucose 1-phosphate (Km, 0.8 mM). Only sucrose-grown cells possessed sucrose hydrolase activity (Km, 3.1 mM), and this activity was much lower than the sucrose phosphotransferase system and sucrose-phosphate hydrolase activities.     

Asp-196-->Ala mutant of Leuconostoc mesenteroides sucrose phosphorylase exhibits altered stereochemical course and kinetic mechanism of glucosyl transfer to and from phosphate

Mutagenesis of Asp-196 into Ala yielded an inactive variant of Leuconostoc mesenteroides sucrose phosphorylase (D196A). External azide partly complemented the catalytic defect in D196A with a second-order rate constant of 0.031 M-1 s-1 (pH 5, 30 degrees C) while formate, acetate and halides could not restore activity. The mutant utilized azide to convert alpha-D-glucose 1-phosphate into beta-D-glucose 1-azide, reflecting a change in stereochemical course of glucosyl transfer from alpha-retaining in wild-type to inverting in D196A. Phosphorolysis of beta-D-glucose 1-azide by D196A occurred through a ternary complex kinetic mechanism, in marked contrast to the wild-type whose reactions feature a common glucosyl enzyme intermediate and Ping-Pong kinetics. Therefore, Asp-196 is identified unambiguously as the catalytic nucleophile of sucrose phosphorylase, and its substitution by Ala forces the reaction to proceed via single nucleophilic displacement. D196A is not detectably active as alpha-glucosynthase.     

A novel sucrose phosphorylase from the metagenomes of sucrose-rich environment: isolation and characterization

Sucrose phosphorylase, an important enzyme mainly involved in the generic starch and sucrose pathways, has now caught the attention of researchers due to its transglycosylation activity. A novel sucrose phosphorylase, unspase, has been isolated, and its transglycosylation properties were characterized. Compared with Bisp, the sucrose phosphorylase from Bifidobacterium adolescentis, unspase had two deleted regions in its C: -terminal. These deleted regions were probably equivalent to the important five-stranded anti-parallel β-sheet domain in sucrose phosphorylase. Unspase has a k(m) of 21.12?mM, a V(max) of 69.24?μmol?min(-1)?mg(-1) and a k(cat) of 31.19?s(-1) with sucrose as substrate. In 3-(N-morpholino) propanesulfonic acid (MOPS) buffer, unspase transferred the glycosyl moiety to L: -arabinose, D: -fructose and L: -sorbose. Much to our surprise, unspase can catalyze the transglycosylation in which a glycosyl moiety was transferred to L: -arabinose in the presence of phosphate, which is an interesting exception to the generally accepted fact that transglycosylation can only occur under the condition of phosphate absence. The final yield of the transglycosylation product (37.9?%) in phosphate buffer was even higher than that (5.8?%) in MOPS buffer. This is a novel phenomenon that a sucrose phosphorylase can catalyze a transglycosylation reaction in the presence of phosphate.     

Properties of muscle phosphorylase b from a hagfish, Paramyxine atami

Phosphorylase b was purified to homogeneity from the muscle of a hagfish (Paramyxine atami), as judged by electrophoretic and immunological criteria. The purified enzyme was partially but not fully activated by AMP, and its conversion into the a form resulted in a three-fold increase in activity. The enzyme was stimulated by SO4(2-), and kinetic experiments showed that SO4(2-) markedly increased the affinity of enzyme toward substrates: in the presence and absence of 0.35 M SO4(2-), the apparent Km values of hagfish phosphorylase b were 0.04 and 1.3% for glycogen, 8.7 and 66 mM for glucose 1-phosphate, and 0.05 and 1.0 mM for AMP, respectively. Electrophoretic and immunological data indicated that the hagfish possessed a single molecular form of phosphorylase, like the lamprey. Some immunological relatedness between the hagfish enzyme and the enzyme from lamprey or skate was demonstrated.     

In vitro inhibition of stable 1,3-beta-D-glucan synthase activity from Neurospora crassa.

Glucan synthase activity of Neurospora crassa was isolated by treatment of protoplast lysates with 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4, containing 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 200 mM inorganic phosphate, 10 microM GTP, 1 mM DTT, 10 mM sodium fluoride, and 600 mM glycerol. Resulting activity was partially purified by sucrose gradient density sedimentation. Approximately 70% of enzyme activity in the sucrose gradient peak fraction was soluble and enzyme activity was purified 7.3-fold. Partially purified enzyme activity had a half-life of several weeks at 4 degrees C, and a Km(app) of 1.66 +/- 0.28 mM. Inhibitors (Cilofungin, papulacandin B, aculeacin A, echinocandin B, sorbose and UDP) of 1,3-beta-D-glucan synthase activity were tested against crude particulate and detergent treated enzyme fractions and the Ki(app) of each inhibitor determined. It seems likely that this stable preparation of glucan synthase activity may be useful for in vitro enzyme screens for new glucan synthase inhibitors.     

Effect of metabolites and phosphorylase on the D to I conversion of glycogen synthase from human polymorphonuclear leukocytes.

The D to I conversion of glycogen synthase from human polymorphonuclear leukocytes was examined both in a gel-filtered homogenate and in a preparation of glycogen particles with adhering enzymes, purified by chromatography on concanavalin A bound to Sepharose. It was found that glucose 6-phosphate as well as mannose 6-phosphate, glucosamine 6-phosphate, and 2-deoxy-glucose 6-phosphate activated the reaction, whereas the corresponding sugars were without effect. Mn2+ and Ca2+ increased the conversion rate by 51% and 27%, respectively, whereas Mg2+ and inorganic phosphate were without effect. Sodium fluoride inhibited the reaction completely. Glycogen inhibited the reaction in physiological concentrations and 0.5 mM glucose 6-phosphate was able to overcome this inhibition. MgATP greatly augmented the inhibition caused by glycogen in the glycogen particle preparation. This combined effect could be overcome by glucose 6-phosphate in concentrations from 0.1 to 1 mM. Phosphorylase alpha purified from human polymorphonuclear leukocytes inhibited the D to I conversion in a glycogen particle preparation. The inhibition was counteracted by glucose 6-phosphate and to a lesser degree by AMP. Phosphorylase beta was also inhibitory, but only at higher concentrations than phosphorylase alpha. No phosphorylase phosphatase activity was found in the glycogen particle preparation, which may indicate that chromatography on concanavalin A-Sepharose separates this enzyme from the synthase phosphatase or partially destroys the activity of a hypothetical common protein phosphatase.     

Amperometric flow-injection determination of sucrose with a mediated tri-enzyme electrode based on sucrose phosphorylase and electrocatalytic oxidation of NADH

A new sucrose electrode is described for the determination of sucrose without interference from glucose or fructose. The sucrose electrode is based on the tri-enzymatic system of sucrose phosphorylase, phosphoglucomutase and glucose-6-phosphate 1-dehydrogenase, where NAD(P)H is produced from the last enzymatic reaction and recycled into NAD(P)+ through its electrocatalytic oxidation by Os(4,4'-dimethyl-2,2-bypyridine)2(1,10-phenanthroline-5,6-dione). The electrodes were optimised with respect to the various construction parameters and carrier composition in a FIA system and their response as a function of the pH and flow-rate was examined. The electrodes were suitable for operation in a FIA system and the analysis of real samples showed good agreement with the reference method. Typical optimised electrodes showed detection limits of 1 mM sucrose, response time of 5 min, sensitivity 1.010 nA mM(-1), and current density of 8.38 microA cm(-2), using 200 mM PIPES pH 7.25 with 10 mM phosphate and 5 mM MgCl2 as carrier.     

Transglucosylation potential of six sucrose phosphorylases toward different classes of acceptors

In this study, the transglucosylation potential of six sucrose phosphorylase (SP) enzymes has been compared using eighty putative acceptors from different structural classes. To increase the solubility of hydrophobic acceptors, the addition of various co-solvents was first evaluated. All enzymes were found to retain at least 50% of their activity in 25% dimethylsulfoxide, with the enzymes from Bifidobacterium adolescentis and Streptococcus mutans being the most stable. Screening of the enzymes’ specificity then revealed that the vast majority of acceptors are transglucosylated very slowly by SP, at a rate that is comparable to the contaminating hydrolytic reaction. The enzyme from S. mutans displayed the narrowest acceptor specificity and the one from Leuconostoc mesenteroides NRRL B1355 the broadest. However, high activity could only be detected on l-sorbose and l-arabinose, besides the native acceptors d-fructose and phosphate. Improving the affinity for alternative acceptors by means of enzyme engineering will, therefore, be a major challenge for the commercial exploitation of the transglucosylation potential of sucrose phosphorylase.     

Purification and properties of Cellvibrio gilvus cellobiose phosphorylase

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.     

Trehalase immobilization on aminopropyl glass for analytical use

Trehalase is the enzyme which hydrolyzes the disaccharide trehalose into two alpha-D-glucose molecules. In this article, we present the immobilization of trehalase on aminopropyl glass particles. The enzyme was extracted from Escherichia coli Mph2, a strain harboring the pTRE11 plasmid, which contains the trehalase gene. The partially purified enzyme had a specific activity of 356 U/mg and could be used for quantifying trehalose in the presence of sucrose, maltose, lactose, starch, and glycogen. Partially purified trehalase was immobilized by covalent coupling with retention of its catalytic activity. The support chosen for the majority of the experiments reported was aminopropyl glass, although spherisorb-5NH(2) and chitin were also tested. The immobilized enzyme was assayed continuously for 40 h, at pH 6.0 and 30 degrees C, and no release of enzyme molecules was detected during this procedure. The best condition found for storing the enzyme-support complex was at 4 degrees C in the presence of 25 mM sodium maleate, containing 7 mM beta-mercaptoethanol, 1 mM ethylenediaminetetraacetic acid (EDTA), and 50% glycerol. The enzyme under these conditions was stable, retaining approximately 100% of its initial activity for at least 28 days. The immobilized enzyme can be employed to detect trehalose molecules in micromolar concentration. The optimum pH value found was 4.5 and the K(m) app. 4.9 x 10(-3) M trehalose at pH 4.6 and 30 degrees C, with V(max) of 5.88 mumol glucose . min.(-1), as calculated by a Lineweaver-Burk plot. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 33-39, 1997.     

Phosphorolytic cleavage of sucrose by sucrose-grown ruminal bacterium <Emphasis Type="Italic">Pseudobutyrivibrio ruminis</Emphasis> strain k3

Rumen bacterium Pseudobutyrivibrio ruminis strain k3 utilized over 90 % sucrose added to the growth medium as a sole carbon source. Zymographic studies of the bacterial cell extract revealed the presence of a single enzyme involved in sucrose digestion. Thin layer chromatography showed fructose and glucose-1-phosphate (Glc1P) as end products of the digestion of sucrose by identified enzyme. The activity of the enzyme depended on the presence of inorganic phosphate and was the highest at the concentration of phosphate 56 mmol/L. The enzyme was identified as the sucrose phosphorylase (EC 2.4.1.7) of molar mass ≈54 kDa and maximum activity at pH 6.0 and 45 °C. The calculated Michaelis constant (K _m) for Glc1P formation and release of fructose by partially purified enzyme were 4.4 and 8.56 mmol/L while the maximum velocities of the reaction (v _lim) were 1.19 and 0.64 μmol/L per mg protein per min, respectively.     

Structural dissection of the reaction mechanism of cellobiose phosphorylase

Cellobiose phosphorylase, a member of the glycoside hydrolase family 94, catalyses the reversible phosphorolysis of cellobiose into alpha-D-glucose 1-phosphate and D-glucose with inversion of the anomeric configuration. The substrate specificity and reaction mechanism of cellobiose phosphorylase from Cellvibrio gilvus have been investigated in detail. We have determined the crystal structure of the glucose-sulphate and glucose-phosphate complexes of this enzyme at a maximal resolution of 2.0 A (1 A=0.1 nm). The phosphate ion is strongly held through several hydrogen bonds, and the configuration appears to be suitable for direct nucleophilic attack to an anomeric centre. Structural features around the sugar-donor and sugar-acceptor sites were consistent with the results of extensive kinetic studies. When we compared this structure with that of homologous chitobiose phosphorylase, we identified key residues for substrate discrimination between glucose and N-acetylglucosamine in both the sugar-donor and sugar-acceptor sites. We found that the active site pocket of cellobiose phosphorylase was covered by an additional loop, indicating that some conformational change is required upon substrate binding. Information on the three-dimensional structure of cellobiose phosphorylase will facilitate engineering of this enzyme, the application of which to practical oligosaccharide synthesis has already been established.