Aggregation and Fragmentation of Phaseolus vulgaris Lectin

The effects of temperature on 6-O-α-maltosyl cyclodextrins (G₂-CDs) production from α- maltosylfluoride (α-G₂F) and cyclodextrins (CDs) by the transfer action of debranching enzymes such as pullulanase and isoamylase were studied.

The amounts of 6-O-α-maltosyl α-cyclodextrin (G₂-α-CD) production by purified pullulanase from Aerobacter aerogenes (A-pullulanase) and from Bacillus acidopullulyticus (B-pullulanase) increased with a rise in temperature, e.g., the amounts at 60°C were about 1.5 times higher than those at 30°C. Initial transfer ratios (G₂-α-CD formed/α-G₂F consumed) of A-pullulanase and B- pullulanase were about 62% and 25% (at 40°C), and about 50% and 15% (at 20°C), respectively. The transfer ratios of both A-pullulanase and B-pullulanase in the reaction using β-CD or γ-CD as acceptor also increased with a rise in temperature.

The transfer ratios were little affected by any change in temperature or any kind of acceptor CDs, in the case of isoamylase, and were about 60%.     

Crystal structure of pullulanase: evidence for parallel binding of oligosaccharides in the active site

The crystal structures of Klebsiella pneumoniae pullulanase and its complex with glucose (G1), maltose (G2), isomaltose (isoG2), maltotriose (G3), or maltotetraose (G4), have been refined at around 1.7-1.9A resolution by using a synchrotron radiation source at SPring-8. The refined models contained 920-1052 amino acid residues, 942-1212 water molecules, four or five calcium ions, and the bound sugar moieties. The enzyme is composed of five domains (N1, N2, N3, A, and C). The N1 domain was clearly visible only in the structure of the complex with G3 or G4. The N1 and N2 domains are characteristic of pullulanase, while the N3, A, and C domains have weak similarity with those of Pseudomonas isoamylase. The N1 domain was found to be a new type of carbohydrate-binding domain with one calcium site (CBM41). One G1 bound at subsite -2, while two G2 bound at -1 approximately -2 and +2 approximately +1, two G3, -1 approximately -3 and +2 approximately 0', and two G4, -1 approximately -4 and +2 approximately -1'. The two bound G3 and G4 molecules in the active cleft are almost parallel and interact with each other. The subsites -1 approximately -4 and +1 approximately +2, including catalytic residues Glu706 and Asp677, are conserved between pullulanase and alpha-amylase, indicating that pullulanase strongly recognizes branched point and branched sugar residues, while subsites 0' and -1', which recognize the non-reducing end of main-chain alpha-1,4 glucan, are specific to pullulanase and isoamylase. The comparison suggested that the conformational difference around the active cleft, together with the domain organization, determines the different substrate specificities between pullulanase and isoamylase.     

Cloning and nucleotide sequence of the isoamylase gene from Pseudomonas amyloderamosa SB-15

The gene (iam) coding for isoamylase (glycogen 6-glucanohydrolase) of Pseudomonas amyloderamosa SB-15 was cloned. Its nucleotide sequence contained an open reading frame of 2313 nucleotides (771 amino acids) encoding a precursor of secreted isoamylase. The precursor contained a signal peptide of 26 amino acid residues at its amino terminus and three regions homologous with those conserved in alpha-amylases (1,4-alpha-D-glucan 4-glucanohydrolase) of species ranging from prokaryotes to eukaryotes. These homologous regions were also found in another debranching enzyme, pullulanase (pullulan 6-glucanohydrolase) from Klebsiella aerogenes. Sequences of the isoamylase also showed significant homology with those between positions 300 and the carboxyl terminus of pullulanase. The regions required for the specificity of isoamylase were discussed on the basis of a comparison of its amino acid sequence with those of alpha-amylases, cyclomaltodextrin glucanotransferases, and pullulanase.     

Studies on carbohydrate-metabolising enzymes Part XXVII. A comparison of the action of yeast isoamylase and bacterial pullulanase on amylopectin

The action of purified yeast isoamylase on amylopectin, like that of bacterial pullulanase, results in the hydrolysis of the outermost inter-chain linkages with the liberation of linear maltosaccharides having an average degree of polymerisation of approximately 15 -glucose residues. This hydrolytic action distinguishes yeast isoamylase from yeast amylo-(1→6)-glucosidase, which acts by a combination of transferase and glucosidase activities. The products of enzyme action on amylopectin are discussed in relation to the probable molecular structure of the polysaccharide.     

Action of Pseudomonas isoamylase on various branched oligo- and poly-saccharides

Pseudomonas isoamylase (EC 3.2.1.68) hydrolyzes (1 → 6)-α-D-glucosidic linkages of amylopectin, glycogen, and various branched dextrins and oligosaccharides. The detailed structural requirements for the substrate are examined qualitatively and quantitatively in this paper, in comparison with the pullulanase of Klebsiella aerogenes. As with pullulanase. Ps. isoamylase is unable to cleave D-glucosyl stubs from branched saccharides. Ps. isoamylase differs from pullulanase in the following characteristics: (1) The favored substrates for Ps. isoamylase are higher-molecular-weight polysaccharides. Most of the branched oligosaccharides examined were hydrolyzed at a lower rate, 10% or less of the rate of hydrolysis of amylopectin. (2) Maltosyl branches are hydrolyzed off by Ps. isoamylase very slowly in comparison with maltotriosyl branches. (3)Pr. isoamylase requires a minimum of three D-glucose residues in the B- or C-chain.     

Molecular cloning and biochemical characterization of a heat-stable type I pullulanase from Thermotoga neapolitana

The gene encoding a type I pullulanase from the hyperthermophilic anaerobic bacterium Thermotoga neapolitana (pulA) was cloned in Escherichia coli and sequenced. The pulA gene from T. neapolitana showed 91.5% pairwise amino acid identity with pulA from Thermotoga maritima and contained the four regions conserved in all amylolytic enzymes. pulA encodes a protein of 843 amino acids with a 19-residue signal peptide. The pulA gene was subcloned and overexpressed in E. coli under the control of the T7 promoter. The purified recombinant enzyme (rPulA) produced a 93-kDa protein with pullulanase activity. rPulA was optimally active at pH 5-7 and 80°C and had a half-life of 88 min at 80°C. rPulA hydrolyzed pullulan, producing maltotriose, and hydrolytic activities were also detected with amylopectin, starch, and glycogen, but not with amylose. This substrate specificity is typical of a type I pullulanase. Thin layer chromatography of the reaction products in the reaction with pullulan and aesculin showed that the enzyme had transglycosylation activity. Analysis of the transfer product using NMR and isoamylase treatment revealed it to be α-maltotriosyl-(1,6)-aesculin, suggesting that the enzyme transferred the maltotriosyl residue of pullulan to aesculin by forming α-1,6-glucosidic linkages. Our findings suggest that the pullulanase from T. neapolitana is the first thermostable type I pullulanase which has α-1,6-transferring activity.     

Effects of reduced malto-oligosaccharides on the thermal stability of pullulanase from Bacillus acidopullulyticus.

We investigated the effects of the reduced malto-oligosaccharides, D-glucitol (G1-OH), maltitol (G2-OH), maltotriitol (G3-OH), maltotetraitol (G4-OH), and maltopentaitol (G5-OH) on the thermal stability of Bacillus acidopullulyticus pullulanase (EC 3.2.1.41). The thermal stability depended on the concentration of D-glucitol; after heat treatment for 90 min at 60 degrees in the presence of 0.56, 0.28, 0.14, or 0M G1-OH, the residual activity was 100, 80, 32, and 10% of the control, respectively. Stability increased with the number of glucosyl residues in the alditols added; the effects of G3-OH, G4-OH, and G5-OH on stability were remarkable. Addition of 30% G2-OH, G3-OH, and G4-OH also contributed to the thermal stability of the pullulanase immobilized onto chitosan beads treated with glutaraldehyde. A high concentration of G2-OH stabilized other debranching amylases, Klebsiella pneumoniae pullulanse, Bacillus sectorramus pullulanase, and Pseudomonas amyloderamosa isoamylase (EC 3.2.1.68) under heat treatment for 48 h at 60 degrees, as well as the pullullanase of B. acidopullulyticus.     

Metabolism of the reserve polysaccharide of Streptococcus mitis. Some properties of a pullulanase

1. A pullulanase has been separated from cell extracts of Streptococcus mitis. The enzyme was freed from transglucosylase by fractionation with ammonium sulphate. 2. Pullulanase was produced in the absence of inducers, and addition of glucose or maltose to the broth did not increase the yield of enzyme. 3. The pullulanase acted rapidly on alpha-(1-->6)-bonds in substrates having the structure alpha-maltodextrinyl-(1-->6)-maltodextrin, but had no action on isomaltose, 6-alpha-glucosylmaltodextrins or 6-alpha-maltodextrinylglucoses. 4. 6-alpha-Maltotriosylmaltodextrins were hydrolysed over 10 times faster than 6-alpha-maltosylmaltodextrins. 5. The branch linkages of amylopectin phosphorylase limit dextrin, glycogen phosphorylase limit dextrin and glycogen beta-amylase limit dextrin were hydrolysed. The action of pullulanase on amylopectin and glycogen was accompanied by a rise in the iodine stain of 50% and 30% respectively. 6. A reversal of pullulanase action occurred on incubation with high concentrations of maltotriose. Condensation of maltosyl units to form a branched tetrasaccharide occurred less readily. 7. S. mitis pullulanase was rapidly inactivated at temperatures higher than 40 degrees , and the enzyme did not recover activity on storage at room temperature.     

Structural Uniformity of Pullulan Produced by Several Strains of Pullularia pullulans

Extracellular polysaccharides produced by 3 strains of Pullularia pullulans were fractionated by treating with cetyl trimethyl ammonium hydroxide into soluble and insoluble fractions, and the structure of the former fraction, i.e., pullulan, was studied. The yield and the ratio of 2 fractions varied widely according to the strains. But the structure of pullulan was found to be uniform irrespective of the strains used. All 3 samples of pullulan gave only glucose on complete acid hydrolysis, and 93~95% maltotriose and 5~7% maltotetraose after isoamylase (pullulanase) action. The ratio of α-1,4- to α-1,6-glucosidic linkages calculated from periodate oxidation data coincided very well with the value expected from the ratio of maltotriose to maltotetraose units. An evidence for the complete absence of branch structure in pullulan was presented from the results of hydrolysis by pullulan 4-glucanohydrolase.     

Purification and Properties of a Thermostable Pullulanase from a Newly Isolated Thermophilic Anaerobic Bacterium, Fervidobacterium pennavorans Ven5

Extremely thermophilic anaerobic fermentative bacteria growing at temperatures between 50 and 80(deg)C (optimum, 65 to 70(deg)C) were isolated from mud samples collected at Abano Terme spa (Italy). The cells were gram-negative motile rods, about 1.8 (mu)m in length and 0.6 (mu)m in width, occurring singly and in pairs. Cells commonly formed spheroids at one end similar to Fervidobacterium islandicum and Fervidobacterium nodosum. The new isolate differs from F. nodosum by the 7% higher G+C content of its DNA (40.6 mol%) but is similar to Fervidobacterium pennavorans and F. islandicum in its G+C content and phenotypic properties. The phylogenetic dendrogram indicates that strain Ven5 belongs to the order Thermotogales and shows the highest 16S ribosomal DNA sequence similarity to F. pennavorans, F. islandicum, and F. nodosum, with similarities of 99.0, 98.6, and 96.0%, respectively. During growth on starch the strain produced a thermostable pullulanase of type I which preferentially hydrolyzed (alpha)-1,6 glucosidic linkages. The enzyme was purified 65-fold by anion-exchange, gel permeation, and hydrophobic chromatography. The native pullulanase has a molecular mass of 240,000 Da and is composed of three subunits, each with a molecular mass of 77,600 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Optimal conditions for the activity and stability of the purified pullulanase were pH 6.0 and 85(deg)C. At pH 6.0, the half-life of the enzyme was over 2 h at 80(deg)C and 5 min at 90(deg)C. This is the first report on the presence of pullulanase type I in an anaerobic bacterium.     

The Starch-Debranching Enzymes Isoamylase and Pullulanase Are Both Involved in Amylopectin Biosynthesis in Rice Endosperm

The activities of the two types of starch debranching enzymes, isoamylase and pullulanase, were greatly reduced in endosperms of allelic sugary-1 mutants of rice (Oryza sativa), with the decrease more pronounced for isoamylase than for pullulanase. However, the decrease in isoamylase activity was not related to the magnitude of the sugary phenotype (the proportion of the phytoglycogen region of the endosperm), as observed with pullulanase. In the moderately mutated line EM-5, the pullulanase activity was markedly lower in the phytoglycogen region than in the starch region, and isoamylase activity was extremely low or completely lost in the whole endosperm tissue. These results suggest that both debranching enzymes are involved in amylopectin biosynthesis in rice endosperm. We presume that isoamylase plays a predominant role in amylopectin synthesis, but pullulanase is also essential or can compensate for the role of isoamylase in the construction of the amylopectin multiple-cluster structure. It is highly possible that isoamylase was modified in some sugary-1 mutants such as EM-273 and EM-5, since it was present in significant and trace amounts, respectively, in these mutants but was apparently inactive. The results show that the Sugary-1 gene encodes the isoamylase gene of the rice genome.     

Opposite variations of two epididymal components and blood plasma testosterone in two breeds of rams.

1. Testicular volume (T Vol), blood plasma testosterone (T) concentration, seminal plasma alpha-glucosidase (alpha-G) specific activity, L-carnitine (L-C) concentration as well as semen characteristics were compared in eight Finnish Landrace (F) and eight Suffolk (S) rams throughout 21 months. 2. Only T Vol and T exhibited a typical seasonal variation in both breeds, whereas L-C, alpha-G and live sperm output presented a seasonal profile only in S rams. 3. L-C and alpha-G variations were opposite to those of T in S rams, while they fluctuated in F rams throughout the entire experiment, as did live sperm output. 4. Only the number of ejaculates and T were significantly higher in F rams (3.50 +/- 0.08 in 5 min and 7.62 +/- 0.40 ng/ml) than in S rams (2.30 +/- 0.05 in 5 min and 5.5 +/- 0.30 ng/ml); these two characteristics might therefore be considered as two indexes of sexual activity in rams. 5. By contrast, among all characteristics measured, only alpha-G was significantly higher in S rams than in F rams (1.33 +/- 0.04 vs 0.77 +/- 0.03 mU/mg proteins); this result, as well as seasonal alpha-G profile present in only S rams, allowed us to conclude that alpha-G might be considered as an additional index of seasonal reproduction in rams.     

Multiple branching in glycogen and amylopectin

The β-amylase limit dextrins of glycogen and amylopectin are completely debranched by joint action of isoamylase and pullulanase. Action of isoamylase alone results in incomplete debranching as a consequence of the inability of this enzyme to hydrolyze those A-chains that are two glucose units in length (half the total number of A-chains). From the reducing powers released by isoamylase acting (a) alone and (b) in conjunction with pullulanase, the relative numbers of A- (unsubstituted) and B- (substituted) chains in the β-dextrins, and therefore in the native polysaccharides themselves, can be calculated. Examination of a series of glycogens and amylopectins in this way showed that the ratio of A-chains: B-chains is markedly higher in amylopectins (1.5–2.6:1) than in glycogens (0.6–1.2:1). Glycogen typically contains A-chains and B-chains in approximately equal numbers; amylopectin typically contains approximately twice as many A-chains as B-chains. These polysaccharides therefore differ in degree of multiple branching as well as in average chain length. A consequence of these findings is that amylopectin cannot be formed in vivo by debranching of a glycogen precursor, as proposed by Erlander, since it is impossible to increase the A:B chain ratio by action of a debranching enzyme.     

A simple and sensitive method for detection of pullulanase and isoamylase activities in polyacrylamide gels

Summary Pullulanase and isoamylase activities in PAGE bands have been detected and distinguished by using a two-step, replica-gel revealing assay. The de-branching activity is first revealed as a bluish-purple band by incubating an amylopectin-agar replica gel and then exposing this to iodine vapour. In the second step, pullulanase can be distinguished from isoamylase by a similar procedure using pullulan-agar replica gel and revealing hydrolysis by flooding the plate with ethanol; pullulanase activity shows a colorless band. The procedure exclude other amylases activities. The sensitivity is such that 0.0015 unit of pullulanase and 0.0004 unit of isoamylase activities can be detected easily.     

Formation of Isoamylase by Pseudomonas

We have isolated a Pseudomonas sp. (strain SB15) which produces an isoamylase (EC 3.2.1.9). Highest yields of this enzyme were obtained when the bacterium was grown in shaken culture in a medium containing maltose, dextrin, starch, or isomaltose. Specific carbon and nitrogen sources were required for growth. The most satisfactory medium consisted of 2% maltose, 0.4% sodium glutamate, 0.3% diammonium hydrogen phosphate, and other inorganic salts. The optimal pH for enzyme production was 5 to 6. The enzyme is stable between pH 3 and 6 but is extremely labile above pH 7. It splits amylopectin completely by combined action with beta-amylase but does not attack pullulan.     

Action of Cyclodextrin Glycosyltransferase from Bacillus megaterium Strain No. 5 on Starch

The amounts of the cyclodextrins G6, G7 and G8 produced by the action of the enzyme from Bacillus megaterium (No. 5 enzyme) and Bacillus macerans amylase (BMA) on starch-¹⁴C (U) were determined by the calculation of radioactivity. Both fractions of No. 5 enzyme produced the cyclodextrin G6, G7 and G8 in the proportion of 1: 2.4: 1. On the other hand, BMA produced the cyclodextrin G6, G7 and G8 in the proportion of 2.7: 1:1. The cyclodextrin G6 and G8 which are smaller parts of the reaction products by both fractions of No. 5 enzyme were found to be produced directly from starch, not from the redecomposition of cyclodextrin G7. The ratio of the cyclodextrin G6, G7 and G8 were almost constant, regardless of the pH range of the reaction system.

By using the maltooligosaccharides terminated at the reducing end by radioactive glucose, the action of both fractions of No. 5 enzyme and BMA on the maltooligosaccharides were compared with each other. The results showed that both fractions of No. 5 enzyme acted on oligosaccharides larger than maltose, producing the radioactive glucose as the major product from each maltooligosaccharide (G2~G8). On the other hand, BMA acted on oligosaccharides larger than maltotriose, producing the radioactive maltose as the major product.     

Anti-renal cell carcinoma chimeric antibody G250: cytokine enhancement of in vitro antibody-dependent cellular cytotoxicity

The chimeric monoclonal antibody cG250 targets the G250 antigen, a transmembrane protein which is expressed on renal carcinoma cells and is identical to the MN/CAIX antigen. In vitro studies have previously demonstrated that cG250 induces antibody-dependent cellular cytotoxicity (ADCC) of G250-positive targets. In order to investigate the upregulation of ADCC mediated by cG250, ADCC was examined using effector cells cultured in the presence or absence of the cytokines interferon-gamma (IFN-gamma), interferon-alpha isoforms IFN-alpha (2a) and IFN-alpha (2b) and interleukin-2 (IL-2), and the time course of effects over a 7-day period was determined. Renal cell carcinoma lines expressing high (SK-RC-52) and low (SK-RC-09) G250 antigen levels were used as target cells, and freshly isolated peripheral blood mononuclear cells (PBMC) from a healthy donor were used as the effector cells. PBMC were incubated with the respective cytokine at a range of concentrations or with a media alone control for a period of 7 days. The ADCC activity mediated by cG250 or control isotype matched huA33 with the different PBMC treatment groups was assessed in triplicate daily. Corresponding lymphokine activated killing (LAK) activity was measured concurrently for each treatment group. Chimeric G250 specifically recognised G250 antigen on high and low expressing cell lines SK-RC-52 and SK-RC-09, and mediated specific in vitro ADCC of both lines. In the absence of cytokine stimulation, the specific ADCC of cG250 declined rapidly within three days. IL-2 strongly enhanced and maintained cG250-mediated ADCC activity and K562 cytotoxicity when applied to PBMC in culture for seven days. IFN-gamma also enhanced the ADCC of cG250 throughout the study period, but was not as effective as the IL-2 treatment, and the SK-RC-09 line displayed lower specific cytotoxicity than the SK-RC-52 cell line. In contrast, IFN-alpha 2a and 2b increased cG250-mediated ADCC and K562 cytotoxicity for only three days of the study period. The potent and sustained immune effector activity observed with cG250 and cytokines in this in vitro study suggests that the combination immunotherapy of cG250 with cytokines such as IL-2 shows promise in the treatment of renal cell carcinoma (RCC).     

Synthesis of novel heterobranched beta-cyclodextrins having beta-D-N-acetylglucosaminyl-maltotriose on the side chain

From a mixture of N-acetylglucosaminyl-beta-cyclodextrin (GlcNAc-betaCD) and lactose, beta-D-galactosyl-GlcNAc-betaCD (Gal-GlcNAc-betaCD) was synthesized by the transfer action of beta-galactosidase. GlcNAc-maltotriose (Glc3) and Gal-GlcNAc-Glc3 were produced with hydrolysis of GlcNAc-betaCD by cyclodextrin glycosyltransferase, and Gal-GlcNAc-betaCD by bacterial saccharifying alpha-amylase respectively. Finally, GlcNAc-Glc3-betaCD and Gal-GlcNAc-Glc3-betaCD were synthesized in 5.2% and 3.5% yield when Klebsiella pneumoniae pullulanase was incubated with the mixture of GlcNAc-Glc(3) and betaCD, or Gal-GlcNAc-Glc3 and betaCD respectively. The structures of GlcNAc-Glc3-betaCD and Gal-GlcNAc-Glc3-betaCD were analyzed by FAB-MS and NMR spectroscopy and identified as 6-O-alpha-(6(3)-O-beta-D-N-acetylglucosaminyl-maltotriosyl)-betaCD, and 6-O-alpha-(4-O-beta-D-galactopyranosyl-6(3)-O-beta-D-N-acetylglucosaminyl-maltotriosyl)-betaCD respectively.     

Selective procedure for isolation of microorganisms producing pullulanase and isoamylase

Selective isolation of microorganisms producing pullulanase and isoamylase was accomplished using a two plate detection assay which distinguished both activities and excluded microorganisms producing other extracellular amylases. Over 115,000 colonies tested, 190 strains producing pullulanase and 57 strains producing isoamylase were isolated; extracellular activities of isolated strains were 0.2 to 4.2 and 0.5 to 2.1 nkat/ml of culture, respectively.This paper is dedicated to Prof. Dr. Raúl E. Trucco, in occasion of his 75th anniversary.     

Chemoenzymatic synthesis of 2-chloro-4-nitrophenyl beta-maltoheptaoside acceptor-products using glycogen phosphorylase b.

In the present work, we aimed at developing a chemoenzymatic procedure for the synthesis of beta-maltooligosaccharide glycosides. The primer in the enzymatic reaction was 2-chloro-4-nitrophenyl beta-maltoheptaoside (G(7)-CNP), synthesised from beta-cyclodextrin using a convenient chemical method. CNP-maltooligosaccharides of longer chain length, in the range of DP 8-11, were obtained by a transglycosylation reaction using alpha-D-glucopyranosyl-phosphate (G-1-P) as a donor. Detailed enzymological studies revealed that the conversion of G(7)-CNP catalysed by rabbit skeletal muscle glycogen phosphorylase b (EC 2.4.1.1) could be controlled by acarbose and was highly dependent on the conditions of transglycosylation. More than 90% conversion of G(7)-CNP was achieved through a 10:1 donor-acceptor ratio. Tranglycosylation at 37 degrees C for 30 min with 10 U enzyme resulted in G(8-->12)-CNP oligomers in the ratio of 22.8, 26.6, 23.2, 16.5, and 6.8%, respectively. The reaction pattern was investigated using an HPLC system. The preparative scale isolation of G(8-->11)-CNP glycosides was achieved on a semipreparative HPLC column. The productivity of the synthesis was improved by yields up to 70-75%. The structures of the oligomers were confirmed by their chromatographic behaviours and MALDI-TOF MS data.