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Synthetic Reaction of Cellvibrio Gilvus Cellobiose Phosphorylase

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J. Biochem. 112,,40-44 (1992) Synthetic Reaction of Cellvibrio gilvus Cellobiose Phosphorylase Motomitsu Kitaoka,* Takashi Sasaki,** and Haj ime Taniguchi** *Nippon Petrochemicals Co., Ltd., Tsukuba, Ibaraki 300-26; and **National Food Research Institute, Tsukuba, Ibaraki 305 Received for publication, January 13, 1992 The synthetic reactions of the cellobiose phosphorylase from Cellvibrio gilvus were investigated in detail. It was found that, besides D-glucose, some sugars having substitution or deletion of the hydroxyl group at C2 or C6 of the D-glucose molecule could serve as a glucosyl acceptor, though less effectively than D-glucose. The enzyme showed higher activity with 6-D-glucose than with the ƒ¿-anomer as an acceptor. This result indicates that it recognizes the anomeric hydroxyl group not involved directly in the reaction. ƒÀ - D-Cellobiose was also phosphorolyzed faster than the ƒ¿-anomer. Substrate inhibition was observed with D-glucose, 6-deoxy-D-glucose, or D-glucosamine as an acceptor, with D-glucose being most inhibiting. This inhibition was studied in detail and it was found that D-glucose competes with a-D-glucose-l-phosphate for its binding site. A model of competi tive substrate inhibition was proposed, and the experimental data fit well to the theoretical values that were calculated in accordance with this model. Cellobiose phosphorylase [EC 2.4.1.20] is one of the formed during the enzymatic reaction. That of the synthetic enzymes phosphorolyzing glucosides. It catalyzes revers reaction was determined by measuring the amount of P, ible phosphorolysis of D-cellobiose into D-glucose and ƒ¿- liberated from G-1-P with an acceptor. D-glucose-1-phosphate (G-1-P) with inversion of the ano The amount of G-1-P was measured by using the phos meric configuration. It is present in Clostridium thermocel phoglucomutase-glucose-6-phosphate dehydrogenase sys lum (1), Ruminococcus flavefaciens (2), Cellvibrio gilvus tem (12). D-Glucose was measured by means of the glucose (3), Fomes annosus (4), and Cellulomonas (5, 6). Alexan oxidase-peroxidase method with mutarotase (13) using the der partially purified the enzyme from C. thermocellum (7) Glucose C Test Wako (Wako Pure Chemicals, Osaka). Pi in and synthesized several disaccharides from G-1-P and the presence of G-1-P was measured selectively by the acceptor sugars using the enzyme preparation (8). We method of Lowry and Lopez (14). reported a convenient synthetic method for D-glucosyl- Kinetic Parameters•\Kinetic parameters were calcu- D-xylose from G-1-P and D-xylose using C. gilvus cells as an ated from the experimental results following the Gauss- immobilized cellobiose phosphorylase (9). We purified the Newton method described by Cleland (15) using computer enzyme from C. gilvus to an electrophoretically homogene programs written in BASIC. ous state and reported its properties (10). We also found that its reaction proceeded through an ordered bi bi RESULTS AND DISCUSSION mechanism (11). In the present paper, an extensive kinetic study on the synthetic reaction of this enzyme is reported. Substrate Specificity of the Synthetic Reaction•\Table I indicates relative initial velocities obtained with various MATERIALS AND METHODS sugars as a glucosyl acceptor. Derivatives at C1 of the D-glucose molecule such as methyl-D-glucosides and Materials•\ƒ¿-D-Glucose-1-phosphate (G-1-P) dipotas 1,5-anhydro-D-glucitol did not serve as an acceptor. Iso sium salt, ƒÀ-D-glucose, and ƒÀ-D-cellobiose were purchased mers at C3 (D-allose), C4 (D-galactose), and C5 (L-idose) from Sigma (St. Louis, USA). ƒ¿-D-Cellobiose (containing did not serve as an acceptor. In contrast, some of the C2 about 5% of the ƒÀ-anomer) was obtained by ethanol derivatives (D-mannose, 2-deoxy-D-glucose, and D-gluco precipitation from a D-cellobiose solution. All other chemi samine) and C6 ones (6-deoxy-D-glucose and D-xylose) cals used in the experiments were of reagent grade. could act as an acceptor, although less effectively than Cellobiose phosphorylase was purified from C. gilvus cells D-glucose. These results indicate that the configurations of by the method described by Kitaoka et al. (11). the D-glucose molecule at C1, C3, C4, and C5 are strictly Assay Methods-Reactions of the cellobiose phospho required. Apart from D-glucose derivatives, ketose, pen rylase were carried out at 37•Ž in 50mM Tris-HCl buffer tose (except D-xylose), D-glucono-ƒÂ-lactone, and sugar (pH 7.0) containing 5mM MgCl, and 0.02% bovine serum alcohols had no acceptor activity. Essentially the same albumin as a stabilizer of the enzyme. One unit of the specificity for the acceptor molecule was reported with the activity was defined as the amount of the enzyme which partially purified enzyme from C. thermocellum (7). produces 1ƒÊmol of D-glucose or G-1-P per min with 10mM The acceptor specificity for the C2 derivatives is signifi D-cellobiose and 10mM inorganic phosphate (Pi) under the cantly different from that reported for a maltose phospho above conditions. The initial rate of the phosphorolysis was rylase (16). The cellobiose phosphorylase can accept an assayed by measuring the amount of G-1-P or D-glucose axial hydroxyl group at C2 (D-mannose) whereas it cannot 40 J. Biochem. Synthetic Reaction of Cellobiose Phosphorylase 41 accept bulky substitution at the C2 hydroxyl group (N-ace the case of the maltose phosphorylase, deletion (2-deoxy- tyl-D-glucosamine). On the other hand, the maltose phos D-glucose), substitution with an amino group (D-gluco phorylase cannot accept the former whereas it can accept samine) or substitution with a bulky group (N-acetyl- the latter. Furthermore, the deletion of the C2 hydroxyl D-glucosamine) did not decrease the initial rates. Cellobiose group (2-deoxy-D-glucose) or substitution with an amino phosphorylase, therefore, must have a recognition site for group (D-glucosamine) resulted in a significant decrease in the initial rate in the case of cellobiose phosphorylase. In TABLE I. Substrate specificity in the synthetic reaction. Values are indicated as ƒÊmol/min•EU. For experimental details, see the text. •\, under 0.03. TABLE 11. Apparent kinetic parameters of various substrates. Values were calculated at the following concentrations: a2-10mM, b5-100mM, c5-20mM, and d2.5-50mM. Fig. 2. The ƒÒ-[s] plots of the various substrates. •œ, D-glucose; •› , 6-deoxy-D-glucose; •£, D-mannose; •¢, 2-deoxy-D-glucose; •¡, D-glucosamine; • , D-xylose. Solid lines are calculated curves using the Michaelis-Menten equation. Fig. 1. Time course of the reactions with the substrates of both anomeric types. •›, ƒ¿-D-glucose; •œ, ƒÀ-D-glucose; •¢, ƒ¿-D-cellobiose; •£ , ƒÀ-D-cellobiose. Solid and broken lines indicate synthetic and phosphorolytic reactions, respectively. The synthetic reaction was carried out using 1mM ƒ¿- or ƒÀ-D-glucose with 10mM G-1-P, and was Fig. 3. The ƒÒ-[Glc] plot at various [G-1-P]. Initial concentra followed by measuring the amount of liberated P,. The phosphorolytic tions of G-1-P are: •£, 1mM; • , 2mM; •¡, 5mM; •›, 10mM; •œ, 20 reaction was done using 1mM ƒ¿- or ƒÀ-D-cellobiose with 10mM Pi, mM. Lines are theoretical ones. and was followed by measuring the amount of D-glucose produced. Vol. 112, No. 1, 1992 42 M. Kitaoka et al. the equatorial hydroxyl group at C2. On the other hand, (2-deoxy-D-glucose) fold, but substitution with an amino maltose phosphorylase does not appear to have this type of group increased the value only 6-fold (D-glucosamine). This recognition site, but has a structure which causes steric suggests that the amino group of D-glucosamine plays a hindrance to the axial hydroxyl group at C2. certain role in binding with the enzyme. As was already The apparent kinetic parameters for the sugars which shown in Table I, 6-deoxy-D-glucose is a good acceptor, had an acceptor activity were determined at 10mM G-1-P, after D-glucose. Table II indicates that it has a slightly as shown in Table II. These values were calculated from the higher Vmax value than that of D-glucose though its Km value experimental data using the concentration ranges indicated is about 10 times as high as that of D-glucose. Thus, the C6 in the legend, i. e. the ranges where substrate inhibition is hydroxyl group of the D-glucose molecule is thought to be negligible. It is clear that D-glucose has the lowest Km value involved only in binding of the substrate. However, loss of among the tested sugars. Compared to D-glucose, the other the hydroxymethyl group (D-xylose) resulted in a signifi five sugars have generally lower Vmaxvalues and remark- cant increase in Km and decrease in Vmax, indicating that the ably higher Km values. This indicates that the hydroxyl hydroxymethyl group is important for both binding and groups at C2 and C6 of the D-glucose molecule play more of catalysis. a role in binding of D-glucose to the active site of the enzyme Recognition of the Reducing End-Neither methyl-D- than in catalysis. Comparison of the parameters for 2- glucosides nor 1,5-anhydro-D-glucitol (1-deoxy-D-gluco deoxy- and for 6-deoxy-D-glucose revealed that the hydrox pyranose) served as an acceptor, suggesting that the yl group at C2 is much more important than that of C6. enzyme recognizes the reducing hydroxyl group of the Substitution of a proton for the C2 equatorial hydroxyl acceptor D-glucose molecule. So the reaction rates were group increased the Km values 50- (D-mannose) or 80- followed in the presence of 10mM G-1-P and 1mM of either ƒ¿- or ƒÀ-D-glucose.
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    Europäisches Patentamt (19) European Patent Office & Office européen des brevets (11) EP 1 117 822 B1 (12) EUROPÄISCHE PATENTSCHRIFT (45) Veröffentlichungstag und Bekanntmachung des (51) Int Cl.: Hinweises auf die Patenterteilung: C12P 19/18 (2006.01) C12N 9/10 (2006.01) 03.05.2006 Patentblatt 2006/18 C12N 15/54 (2006.01) C08B 30/00 (2006.01) A61K 47/36 (2006.01) (21) Anmeldenummer: 99950660.3 (86) Internationale Anmeldenummer: (22) Anmeldetag: 07.10.1999 PCT/EP1999/007518 (87) Internationale Veröffentlichungsnummer: WO 2000/022155 (20.04.2000 Gazette 2000/16) (54) HERSTELLUNG VON POLYGLUCANEN DURCH AMYLOSUCRASE IN GEGENWART EINER TRANSFERASE PREPARATION OF POLYGLUCANS BY AMYLOSUCRASE IN THE PRESENCE OF A TRANSFERASE PREPARATION DES POLYGLUCANES PAR AMYLOSUCRASE EN PRESENCE D’UNE TRANSFERASE (84) Benannte Vertragsstaaten: (56) Entgegenhaltungen: AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU WO-A-00/14249 WO-A-00/22140 MC NL PT SE WO-A-95/31553 (30) Priorität: 09.10.1998 DE 19846492 • OKADA, GENTARO ET AL: "New studies on amylosucrase, a bacterial.alpha.-D-glucosylase (43) Veröffentlichungstag der Anmeldung: that directly converts sucrose to a glycogen- 25.07.2001 Patentblatt 2001/30 like.alpha.-glucan" J. BIOL. CHEM. (1974), 249(1), 126-35, XP000867741 (73) Patentinhaber: Südzucker AG Mannheim/ • BUTTCHER, VOLKER ET AL: "Cloning and Ochsenfurt characterization of the gene for amylosucrase 68165 Mannheim (DE) from Neisseria polysaccharea: production of a linear.alpha.-1,4-glucan" J. BACTERIOL. (1997), (72) Erfinder: 179(10), 3324-3330, XP002129879 • GALLERT, Karl-Christian • DE MONTALK, G. POTOCKI ET AL: "Sequence D-61184 Karben (DE) analysis of the gene encoding amylosucrase • BENGS, Holger from Neisseria polysaccharea and D-60598 Frankfurt am Main (DE) characterization of the recombinant enzyme" J.
  • The Decomposition of Starch Through Phosphorolysis by Streptoccus

    The Decomposition of Starch Through Phosphorolysis by Streptoccus

    The decomposition of starch through phosphorolysis by Streptoccus bovis by Richard N Ushijima A THESIS Submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of Master of Science in Bacteriology at Montana State College Montana State University © Copyright by Richard N Ushijima (1957) Abstract: The possibility of starch utilization by Streptococcus bovis through a starch phosphorylase was investigated. large quantities of cells were grown on a starch medium, and the cells were harvested by centrifugation. The cell-free enzyme extract was prepared by grinding the cells with glass beads in a micro-Waring blendor. Starch phosphorylase was detected in the reaction mixture containing soluble starch, inorganic phosphate, enzyme preparation, and sodium hydrosulfite. Since a maltose phosphorylase was also found, the formation of maltose was prevented by adjusting the mixture to pH 8.6, which inhibited alpha amylase activity. The experiments also revealed the presence of a phosphoglueomutase, which converts glucose-l-phosphate to glucose-6-phosphate, a product uti-lizable through the glycolytic pathways. I THE''DECOMPOSITION' OF' STARCH THROUGH ' ^ PHOSPHOROLYSIS BI STREPTOCOCCUS'BOVIS by RICHARD N„ USHIJHja A THESIS Submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of Master of Science in Bacteriology at Montana State College Approved^ Headj Major Department Chairman, Examining Committee Hi Bozeman, Montana <ii ‘‘il.j ' ') I' V r L u 5 A- CCg J- " 2 ' TABLE OF CONTENTS ACKNOWLEDGMENTS 3 ABSTRACT U INTRODUCTION 5 METHODS AND MATERIALS 0\ CN Os Culture Preparation of large cultures Preparation of cell-free bacterial extracts IO Phosphorus determination 11 Chromatography 12 Glucose determination 12 EXPERIMENTAL RESULTS lh Phosphorylase ill Amylase 15> Starch phosphorylase 20 Phosphoglucomutase 2h DISCUSSION 2? SUMMARY 31 LITERATURE CITED 32 123688 ACKmimEDGMEHTS The author takes this opportunity to express his gratitude to Dr, R» H.
  • Bba 65434 Seed Germination Studies Ii. Pathways for Starch Degradation

    Bba 65434 Seed Germination Studies Ii. Pathways for Starch Degradation

    BIOCHIMICA ET BIOPHYSICA ACTA 87 BBA 65434 SEED GERMINATION STUDIES II. PATHWAYS FOR STARCH DEGRADATION IN GERMINATING PEA SEEDLINGS RICHARD R. SWAIN* AND EUGENE E. DEKKER Department of Biological Chemistry, The University of Michigan, Ann Arbor, Mich. (U.S.A .) (Received January ioth, 1966) SUMMARY Amylase activity is present in extracts of the axis tissue of etiolated pea seedlings. Several lines of evidence establish that this starch-hydrolyzing activity is due to the presence of a fl-amylase (a-I,4-glucan maltohydrolase, EC 3.2.1.2); the properties of this enzyme are reported. Maltase (EC 3.2.1.2o) activity is also present in extracts of the same tissues. Since a-amylase (EC 3.2.1.1) activity appears in the germinating cotyledon, the following hydrolytic pathway for the degradation of starch to glucose is present in the pea seedling: a-amylase soluble fl-amylase a-glucosidase starch > oligosaccharides > maltose > glucose The importance of this pathway is considered in relation to the alternative route for starch catabolism, namely, the phosphorolytic pathway involving the enzyme phosphorylase (EC 2.4.1.1). INTRODUCTION In mammals, glycogen is the storage polysaccharide of liver and muscle tissue. At present, two rather different enzymic routes for glycogen breakdown, at least ill liver tissue, have been elucidated. The one involves the so-called "phosphorylase pathway" for glucose production and requires a sequential participation of the enzymes phosphorylase (EC 2.4.I.I), phosphoglucomutase (EC 2.7.5.I ), and glucose- 6-phosphatase (EC 3.1.3.9) (ref. I). More recently, RUTTER AND BROSEMER~ reported another route for glucose formation from glycogen in liver.