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The cluster of genes of capsular K5 heparosan is composed of three regions, involved in the synthesis and the exportation of the polysaccharide. The region 2 possesses all the necessary genes involved in the synthesis of heparosan, namely kfiA, encoding alpha-4-N-acetylglucosaminyltransferase, kfiD, encoding β-3-glucuronyl transferase, kfiC, encoding UDP-glucose dehydrogenase (UDP-glucuronic acid synthesis), and kfiB encoding a protein of unknown function. The cloning and expression of kfiADCB into Escherichia coli K-12 were found to be sufficient for the production of heparosan, which accumulates in the cells due to a lack of the exporting system. The concentration of recombinant heparosan reached one gram per liter under fed-batch cultivation. The cytoplasmic localization of heparosan inside the bacteria allowed subsequent enzymatic modifications such as a partial degradation with K5 lyase when expressed intracellularly. Under these conditions, the production of DP 2-10 oligosaccharides occurred intracellularly, at a concentration similar to that of recombinant intracellular heparosan.

A methyl ester of hyaluronan in which the carboxyl groups were fully esterified was prepared using trimethylsilyl diazomethane. This derivative, while not depolymerized by hyaluronan lyases or hyaluronan hydrolases, was a substrate for both chondroitin ACI lyase (EC 4.2.2.5) from Flavobacterium heparinum and chondroitin ACII lyase (EC 4.2.2.5) from Arthrobacter aurescens. The major product isolated in these depolymerization reactions was methyl alpha-L-threo-hex-4-enepyranosyluronate-(1--3)-2-acetamido-2-deoxy-alpha,beta-D-glucopyranoside as determined by 1H NMR spectroscopy and MALDITOF mass spectrometry.

Hyaluronic acid (HA) was treated with hyaluronate lyase (GBS HA lyase, E.C. 4.2.2.1, from Streptococcus agalactiae strain 4755), and the products have been analyzed by capillary electrophoresis (CE-UV and online CE-ESIMS), gel-permeation chromatography (GPC) and viscosimetric measurements. The resulting electropherograms showed that the enzyme produced a mixture of oligosaccharides with a 4,5-unsaturated uronic acid nonreducing terminus. More exhaustive degradation of HA led to increasing amounts of di-, tetra-, hexa-, octa- and decasaccharides. Using CE, linear relationships were found between peak area of the observed oligosaccharides and reaction time. Determination of viscosity at different stages of reaction yielded an initial rapid decrease following Michaelis-Menten theory. A reaction time-dependent change in the elution position of the HA peak due to partial digestion of HA with GBS hyaluronate lyase has been observed by GPC. These results indicated that the HA lyase under investigation is an eliminase that acts in a nonprocessive endolytic manner, as at all stages of digestion a mixture of oligosaccharides of different size were found. For GBS HA lyase from Streptococcus agalactiae strain 3502, previously published findings reported an action pattern that involves an initial random endolytic cleavage followed by rapid exolytic and processive release of unsaturated disaccharides. Our results suggest that differences between the two enzymes from distinct S. agalactiae strains (GBS strains 4755 and 3502) have to be considered.

An alginate lyase, termed HdAly in the present paper, was isolated from the hepatopancreas of abalone, Haliotis discus hannai , by ammonium sulfate fractionation, followed by TOYOPEARL CM-650M column chromatography. Enzymatic properties of HdAly were similar to those of previously reported Haliotis and Turbo poly(M) lyases, e.g., it preferentially degraded a poly(β- d -mannuronate)-rich substrate with an optimal pH and temperature at pH 8.0 and 45 °C, respectively. In order to determine the primary structure of abalone lyase that is still poorly understood, cDNAs for HdAly were cloned by PCR from the abalone hepatopancreas cDNA library and sequenced. From the nucleotide sequences of the cDNAs, the sequence of 909 bp in total was determined, and the amino acid sequence of 273 residues was deduced from the translational region of 822 bp locating at nucleotide positions 27–848. The N-terminal region of 16 residues, except for the initiation Met in the deduced sequence, was regarded as the signal peptide since it was absent in the HdAly protein and showed high similarity to the consensus sequence for signal peptides of eukaryote secretary proteins. This suggests that HdAly is initially produced as a precursor possessing the signal peptide in hepatopancreatic cells and then secreted into digestive tract as the mature form. Thus, the mature HdAly was regarded to consist of 256 residues with the calculated molecular mass of 28895.5 Da. The amino acid sequence of HdAly showed 85 and 28% identity to those of Turbo cornutus alginate lyase SP2 and the C-terminal region of Chlorella virus lyase-like protein CL2, respectively, while it showed no significant identity to those of any bacterial alginate lyases. In order to provide the basis for the structure–function studies and various applications of the abalone lyase, a bacterial expression system was constructed by means of the HdAly-cDNA and pET-3a expression plasmid. Although the active recombinant HdAly was hardly produced at a cultivation temperature 37 °C in Escherichia coli BL21 (DE3), a small amount of soluble and active enzyme could be produced when the temperature was lowered to 19 °C.

There is a steadily increasing need to expand sustainable resources, and carbohydrates are anticipated to play an important role in this respect, both for bulk and fine chemical preparation. The enzyme α-(1→4)-glucan lyase degrades starch to 1,5-anhydro- d -fructose. This compound, which has three different functional properties, a prochiral center together with a permanent pyran ring, renders it a potential chiral building block for the synthesis of valuable and potentially biologically active compounds. 1,5-Anhydro- d -fructose is found in natural materials as a degradation product of α-(1→4)-glucans. The occurrence of lyases and the metabolism of 1,5-anhydro- d -fructose are reviewed in the biological part of this article. In the chemical part, the elucidated structure of 1,5-anhydro- d -fructose will be presented together with simple stereoselective conversions into hydroxy/amino 1,5-anhydro hexitols and a nojirimycin analogue. Synthesis of 6- O -acylated derivatives of 1,5-anhydro- d -fructose substituted with long fatty acid residues is carried out using commercially available enzymes. Those reactions lead to compounds with potential emulsifying properties. The use of protected derivatives of 1,5-anhydro- d -fructose for the synthesis of natural products is likewise reviewed. The potential utilization of this chemical building block is far from being exhausted. Since 1,5-anhydro- d -fructose now is accessible in larger amounts through a simple-enzyme catalyzed degradation of starch by α-(1→4)-glucan lyase, the application of 1,5-anhydro- d -fructose may be considered a valuable contribution to the utilization of carbohydrates as the most abundant resource of sustainable raw materials.

The structure of epitopes recognised by anti-pectin monoclonal antibodies (mAbs) has been investigated using a series of model lime-pectin samples with defined degrees and patterns of methyl esterification, a range of defined oligogalacturonides and enzymatic degradation of pectic polysaccharides. In immuno-dot-assays, the anti-homogalacturonan (HG) mAbs JIM5 and JIM7 both bound to samples with a wide range of degrees of methyl esterification in preference to fully de-esterified samples. In contrast, the anti-HG phage display mAb PAM1 bound most effectively to fully de-esterified pectin. In competitive inhibition ELISAs using fully methyl-esterified or fully de-esterified oligogalacturonides with 3-9 galacturonic acid residues, JIM5 bound weakly to a fully de-esterified nonagalacturonide but JIM7 did not bind to any of the oligogalacturonides tested. Therefore, optimal JIM5 and JIM7 binding occurs where specific but undefined methyl-esterification patterns are present on HG domains, although fully de-esterified HG samples contain sub-optimal JIM5 epitopes. The persistence of mAb binding to epitopes in pectic antigens, with 41% blockwise esterification (P41) and 43% random esterification (F43) subject to fragmentation by endo-polygalacturonase II (PG II) and endo-pectin lyase (PL), was also studied. Time course analysis of PG II digestion of P41 revealed that JIM5 epitopes were rapidly degraded, but a low level of PAM1 and JIM7 epitopes existed even after extensive digestion, indicating that some HG domains were more resistant to cleavage by PG II. The chromatographic separation of fragments produced by the complete digestion of P41 by pectin lyase indicated that a very restricted population of fragments contained the PAM1 epitope while a (1-->4)-beta-D-galactan epitope occurring on the side chains of pectic polysaccharides was recovered in a broad range of fractions.

The mode of action of RG-hydrolase and RG-lyase toward purified linear rhamnogalacturonan (RG) oligomers has been studied. Major tools in the characterization of the degradation products were the exo-acting RG-rhamnohydrolase and RG-galacturonohydrolase. They were used to prepare a series of standards of RG oligomers for HPAEC. 1H NMR spectroscopy confirmed the structure assignment made using HPAEC for a selection of isolated degradation products. Identification of degradation products from purified RG oligomers was then performed by comparing retention times of HPAEC peaks with those of standards. RG-hydrolase was able to cleave RG oligomers which contained five Rha units or more, i.e. DP 9 with a Rha unit at both nonreducing and reducing end. Its preferential cleavage site was at four units from the first nonreducing Rha. RG-lyase was active toward oligomers that contained at least six GalA units, i.e. DP 12 with a GalA at the nonreducing and a Rha at the reducing end. The preferential cleavage site was for the smaller oligomers four residues, and for the largest oligomer six residues from the reducing Rha. From the observed cleavage patterns it can be speculated that in hairy regions, the RG stretches have to be at least 13 residues long for RG-hydrolase and 16 residues long for RG-lyase in order to produce one tetramer.

N-Acetylneuraminate lyase and N-acyl-D-glucosamine 2-epimerase had been cloned and overexpressed in Escherichia coli. Simultaneous use of these two enzymes and feeding of appropriate amounts of pyruvate to the reaction mixture made possible the high conversion of N-acetylneuraminic acid (Neu5Ac) from N-acetyl-D-glucosamine (GlcNAc) with a 77% conversion rate on a molar basis. As a result, 29 kg of Neu5Ac was obtained from 27 kg of GlcNAc. The product was recovered by direct crystallization, and verified as identical to authentic Neu5Ac.

1,5-Anhydro- d - arabino -hex-2-ulose (1,5-anhydro- d -fructose, 1,5AnFru), produced by α-1,4-glucan lyase (EC 4.2.2.13) acting on starch, glycogen, or related d -glucose oligo- and polysaccharides as substrate, reacts with alkaline 3,5-dinitrosalicylic acid reagent (DNS) at room temperature (22 °C) within 10 min. The absorbance of the reaction mixture at 550 nm at the end of the reaction was proportional to a 1,5AnFru content in the range of 0.5 to 16 μmol (80 μg to 2.6 mg) mL −1 . 1,5AnFru determined by this colorimetric, one test tube one reagent method, was in good agreement with that found by 1 H-NMR spectroscopy and HPLC. The DNS method is also specific as other reducing sugars, such as glucose, maltose maltosaccharides, starch and glycogen do not give a colour with DNS at 22 °C; therefore, they do not interfere in the determination. The DNS method is applicable to lyase assay for both cell-free extracts and purified enzyme. Methods for reducing sugar analyses, based on the reduction of ferric and cupric ions, were examined for 1,5AnFru and they proved to be quantitative but in contrast to the DNS method, they were not specific. Instead of assaying 1,5AnFru, the activity of α-1,4-glucan lyase was analysed enzymatically by quantifying glucose or 4-nitrophenol released using maltose and 4-nitrophenyl α-maltopentaoside as substrate, respectively.

An intracellular alginate lyase was purified from Enterobacter cloacae M-1 by successive fractionation on Q Sepharose FF, SP Sepharose FF, and Sephacryl S-200 HR. The purified enzyme gave a single band on SDS-PAGE and isoelectric focusing. The enzyme easily degraded polyguluronate and produced unsaturated oligoguluronic acids with a wide range of dp. The major end product of the enzyme reaction on polyguluronate was unsaturated triuronic acid. The pattern of oligoguluronic acids (dp 2–9) generated with the enzyme was investigated by fluorophore-assisted carbohydrate electrophoresis. The enzyme was not capable of degrading oligoguluronic acids having a dp < 4. The degradation rate of heptaguluronic acid by this enzyme remarkably increased, compared with that of hexaguluronic acid, and heptaguluronic acid had a single preferential point of cleavage by this enzyme. On the basis of the cleavage pattern of oligoguluronic acids, the number of subsites was estimated to be seven for this enzyme. The catalytic site of the enzyme is located between the second and the third subsites from the non-reducing end.

Purified preparations of alginate (mannuronate) lyase from Haliotis tuberculata have been investigated by recording the progress of product formation when acting on well-characterised alginates and alginate fragments. We have earlier shown that maximal conversion is affected by enzyme dose as well as by initial substrate concentration. This is related to a strong, reversible product inhibition, dominant in the enzymatic breakdown of the heteropolymeric (MG) fraction of the alginate compared to that of polymannuronic (MM) blocks. Polyguluronic (GG) structures were found to be inactive both as substrate and as inhibitor. A numerical two-substrate model is now presented in an attempt to analyse data by simply assigning different kinetic constants to each block fraction of the accessible substrate. Including reversible product inhibition, the general model is expressed by a set of six coupled nonlinear differential equations. If the steady-state assumption of the Michaelis-Menten initial rate analysis is accepted, these equations may be integrated directly. This approach could only adequately describe data for poly-M substrates showing no significant product inhibition. The steady-state assumption could not be maintained for results obtained with substrates rich in heteropolymeric MG blocks. Instead, the kinetic parameters were estimated by a nonlinear minimalisation of the sum squares of residuals. In this form, the model could fully describe our experimental observations.

Herbivorous marine snails like Littorina species are known to possess alginate lyases in their digestive tracts. The Littorina enzymes have been identified as endolytic polymannuronate (poly(M)) lyases (EC 4.2.2.3); however, it is still unclear which polysaccharide-lyase family (PL) the Littorina enzymes belong to, since no complete primary structure of Littorina enzymes has been determined. Thus, in the present study, we analyzed the primary structure of LbAly28, a 28 kDa alginate lyase isozyme of Littorina brevicula, by the cDNA method. LbAly28 cDNAs were amplified by PCR followed by 5′- and 3′-RACE PCRs from the L. brevicula hepatopancreas cDNA. A cDNA covering entire coding region of LbAly28 consisted of 1129 bp and encoded an amino-acid sequence of 291 residues. The deduced amino-acid sequence comprised an initiation methionine, a putative signal peptide of 14 residues, a propeptide-like region of 16 residues, and a mature LbAly28 domain of 260 residues. The mature LbAly28 domain showed 43–53% amino-acid identities with other molluscan PL-14 enzymes. The catalytically important residues in PL-14 enzymes, which were identified in the Chlorella virus glucuronate-specific lyase vAL-1 and Aplysia poly(M) lyase AkAly30, were also conserved in LbAly28. Site-directed mutagenesis regarding these residues, that is, replacements of Lys94, Lys97, Thr121, Arg 123, Tyr135, and Tyr137 to Ala, decreased the activity of recombinant LbAly28 to various degrees. From these results we concluded that LbAly28 is a member of PL-14 alginate lyases. Besides the effects of above mutations, we noticed that the replacement of T121 by Ala changed the substrate preference of LbAly28. Namely, the activities toward sodium alginate and poly(MG)-block substrate increased and became comparable with the activity toward poly(M)-block substrate. This suggests that the region including T121 of LbAly28 closely relates to the recognition of poly(MG) region of alginate.

Alginate subfractions that approach homopolymers in mannuronate or guluronate have been end-labelled with the fluorescent dyes fluorescein and tetramethylrhodamine linked via putrescine. The putrescine-alginate intermediates were obtained in high yield and the labels were attached via the terminal amino group. The fluorescent alginates are suitable as substrates for the assay (simultaneous if necessary) or l -guluronate and d -mannuronate lyases by spectrophotometry or fluorimetry.

1,5-Anhydro-D-fructose (1,5AnFru) is a monoketosaccharide that can be prepared enzymatically from starch by alpha-1,4-glucan lyase or chemically from D-glucose or D-fructose in a few steps with high yields. The formed 1,5AnFru can be derivatized both enzymatically and chemically to interesting new carbohydrate derivatives, some with biological activities. For example dehydratases, isomerases and reductases can convert 1,5AnFru to enolones (as Ascopyrone P) and sugar alcohols with antimicrobial and antioxidant properties, while chemical modifications can give similar compounds as well as natural products like 1-deoxymannonojirimycin and Clavulazine. 1,5AnFru disaccharides (glycosyl 1-->4 1,5AnFru) have been prepared as well as glycosyl 1-->4 1,5-anhydro-D-tagatose.

A method was developed to selectively methyl esterify and then cleave GalA residues in pectic polysaccharides. The method was optimized using a rhamnogalacturonan (RG) from Arabidopsis mucilage as a model compound. The carboxyl group of the GalA residues in the RG was selectively methyl esterified using tetrabutylammonium fluoride and iodomethane in Me(2)SO containing 8% water. A 1D HMQC NMR method to determine the degree of methyl esterification was developed using (13)C-iodomethane as the methylating agent. The methyl-esterified pectins were fragmented by beta-elimination in 0.2M sodium borate, pH7.3, at 125 degrees C. The resulting oligoglycosyl fragments, which contain a nonreducing 4-deoxy-beta-l-threo-hex-4-enepyranosyluronic acid residue, were characterized using MALDI-TOF mass spectrometry, monosaccharide composition analysis, and 1D and 2D (1)H and (13)C NMR spectroscopy. Application of this method to branched RG from potato generated low-molecular-weight fragments containing two residues from the RG backbone and a single side chain. In contrast, the fragments obtained when RG is treated with RG lyase contain a minimum of four backbone residues. The chemical method thus facilitates the release and structural characterization of the side-chain structures of RG obtained from various plant sources. The method also provides a convenient method for generating fully or partially methyl-esterified homogalacturonans.

Lysis of alginates and of their saturated and unsaturated fragments was monitored by 1H NMR spectroscopy. AlxM(B) alginate lyase performs beta-elimination on the mannuronic acid (M) residues. It does not cleave the guluronic acid (G) sequences, nor the M-G or the G-M diads. In consequence, it is a true mannuronate lyase. The end product of the reaction is O-(4-deoxy-alpha-L-ery-thro-hex-4-enopyranosyl-uronic acid)-(1->(4)-O-(beta-D-mannopyranosyluronic acid)-(1->4)-O-beta-D-mannpyranuronic acid. Viscosity measurements made during degradation of a polymannuronate alginate showed that AlxM(B) behaves as an endo-enzyme. HPLC analysis of the degradation products of oligomannuronates and oligoalginates suggested that the beta-elimination requires the interaction of the enzyme with at least three sequential mannuronic acid residues. The catalytic site may possess 5 sub-sites and accommodate pentamers with different M/G ratio. Kinetic measurements showed that the specificity constant Vm/Km increased with the number of mannuronic acid residues. AlxM(B) may be reversibly inhibited by heteropolymeric blocks in a competitive manner.

Udgivelsesdato: 2000-Jul-24 A series of pectins with different distribution patterns of methyl ester groups was produced by treatment with either plant (p-PME) or fungal pectin methyl esterases (f-PME) and compared with those obtained by base catalysed de-esterification. The products generated by digestion of these pectins with either endopectin lyase (PL) or endopolygalacturonase II (PG II) from Aspergillus niger were analysed using matrix assisted laser desorption ionisation mass spectrometry (MALDIMS) and high-performance anion-exchange chromatography with pulsed amperometric or UV detection (HPAEC-PAD/UV). Time course analysis using MALDIMS was used to identify the most preferred substrate for each enzyme. For PL, this was shown to be fully methyl esterified HG whereas for PG II, long regions of HG without any methyl esterification, as produced by p-PME was the optimal substrate. The blockwise de-esterification caused by p-PME treatment gave a decrease of partly methylated oligomers in PL fingerprints, which did not effect the relative composition of partly methylated oligomers. PG II fingerprints showed a constant increase of monomers and oligomers without any methyl ester groups with decreasing degree of esterification (DE), but almost no change in the concentration of partly methylated compounds. PL fingerprints of f-PME and chemically treated pectins showed decreasing amounts of partly methyl esterified oligomers with decreasing DE, together with a relative shift towards longer oligomers. PG II fingerprints were characterised by an increase of partly methylated and not methylated oligomers with decreasing DE. But differences were also seen between these two forms of homogenous de-esterification. Introduction of a certain pattern of methyl ester distribution caused by selective removal of certain methyl ester groups by f-PME is the most reasonable explanation for the detected differences.

A marine bacterium degrading the water-soluble cell wall polysaccharides from Ulva sp. (ulvan) has been isolated. The good correlation between ulvan degradation monitored by reducing-power, UV absorbance and viscosimetry, indicated that the crude enzymatic extract contains essentially an endo-ulvan lyase activity. This activity was rapidly inhibited by the reaction products which consisted of a series of ulvanobiouronic acid A 3-sulfate [-->4)-beta-D-GlcpA-(1-->4)-alpha-L-Rhap 3-sulfate-(1-->]n with 4-deoxy-L-threo-hex-4-enopyranosiduronic acid at the non-reducing end. Other deviant repeating structures with beta-D-Xylp or alpha-IdopA replacing beta-D-GlcpA in the repeating ulvanobiouronic acid disaccharide and the presence of two consecutive (1-->4) linked beta-D-Glc pA demonstrated the great variability and complexity of ulvan chemical structure.

The chromatographic behaviour of various saturated and unsaturated oligouronates obtained by acid or enzymatic degradation of homopolymeric blocks of alginates was investigated by isocratic anion exchange liquid chromatography. This approach was then applied to the determination of the catalytic properties of Haliotis tuberculata alginate lyase. This enzyme presents a high affinity for poly-beta-D-mannuronate blocks, leading to the release of O-(4-deoxy-alpha-L-erythro-hex-4-enopyranosyluronic acid)-(1-->4)-O-(beta-D-mannopyranosyluronic acid)-(1-->4)-O-beta-D-mannopyranuronic acid as the main end reaction product. Kinetic analysis with oligomannuronates of various sizes indicate that the catalytic site of Haliotis tuberculata lyase (abalone) best accommodates an oligomannuronate pentamer. The abalone lyase, however, is also capable of cleaving the G-M linkages of alginate heteropolymeric sequences. In contrast, it does not degrade the G-G nor the M-G diads. This lyase should therefore be referred to as a mannuronate beta-eliminase, indicating that the enzyme performs beta-elimination on mannuronate residues only, from both the M-M and G-M diads of alginates.

The complete structure of the acidic, extracellular, capsular polysaccharide of Rhizobium trifolii 843 has been elucidated by a combination of chemical, enzymic, and spectroscopic methods, confirming an earlier proposed sugar sequence and assigning the locations of the acyl substituents. The polysaccharide was depolymerized by a lyase into octasaccharide units which were uniform in carbohydrate composition and linkage. These units also contained a uniform distribution of acetyl and pyruvic acetal [ O -(1-carboxyethylidene)] groups, and half of them were further acylated with d -3-hydroxybutanoyl groups. A much smaller proportion ( d -3-hydroxy-butanoyl group. The locations of the substituents were determined chemically and by J -correlated, 1 H-n.m.r. spectroscopy, proton nuclear Overhauser effect (n.O.e.)_ measurements, doubie-resonance 1 H-n.m.r. spectroscopy, and 13 C-n.m.r. spectroscopy. The composition and structure of the carbohydrate chain were determined by methylation analysis using g.l.c.-m.s. fast-atom-bombardment mass spectrometry, and n.m.r. studies on the reduced, deacylated oligomer. Structural studies were supplemented by n.m.r. analyses on the original polymer. The oligosaccharides were found to be branched octasaccharides with four sugar residues in each branch, and the carbohydrate sequence agreed well with that expected from earlier work. In the abbreviated sequence and structure ( 1a ), the sugar residues are labelled “ a ” through “ h ”. The main chain ( a–d ) is composed of a 4-deoxy-α- l - threo -hex-4-enopyranosyluronic acid group ( a ) that is linked to O-4 of a 3- O -acetyl- d -glucosyluronic acid residue ( b ) which is β-linked to O-4 of a d -glucosyl residue ( c ). Residue c is β-linked to O-4 of the branching d -linked to O-4 of a d -glucosyl residue ( d ). The side chain consists of a substituted d -galactosyl group ( h ) which is β-linked to O-3 of residue 9 of a β-(1→4)-linked d -glucose trisaccharide (fragment e–f–g ). The reducing end of the resulting tetrasaccharide ( e–f–g–h ) is β-linked to O-6 of the branching d -glucose residue ( d ). In the native polymer, this branching residue is α-linked to O-4 of the modified d -glucuronic acid residue ( a ) which is the unsaturated sugar in the oligomer. A small proportion of the O-2 atoms of the acetylated d -glucosyluronic acid residues is acetylated because of ester migration. The two terminal sugars ( g and h ) of the branch chain bear 4,6- O -(1-carboxyethylidene) groups. The d -galactosyl groups of half of the oligomers are acylated by d -3-hydroxybutanoyl groups at O-3. About 5% of the oligomers bear a second d -3-hydroxybutanoyl group at O-2 of the d -galactosyl group ( h ).

An extracellular poly-α- L -guluronate lyase from Klebsiella aerogenes degrades those blocks from alginate which contain both mannuronic and guluronic acid residues (poly-MG blocks) to a mixture of oligosaccharides. From an analysis of these products, it is concluded that poly-MG blocks do not have a strictly alternating sequence of the two uronic acid residues. Enzymic degradation of various samples of algal alginate to leave the poly-M blocks intact has shown that these blocks have a uniform chain-length, estimated at 24 residues.

Pectins sequentially extracted from sugar-beet pulp with water (WSP), oxalate (OXP), hot acid (HP), and cold alkali (OHP) have been degraded variously by base-catalysed β-elimination, de-esterification, endopectin lyase, pectin methyl esterase, endopolygalacturonase, and endopectate lyase. The products were studied mainly by chromatography on Sephadex G-100. The pectins contain various amounts of degradation-resistant (hairy) fragments in which the molar ratios of neutral sugar residues to galacturonic acid residues were 4.8, 4.6, 3.8, and 3.7 for WSP, OXP, HP, and OHP, respectively. The molar ratios of rhamnose residues to galacturonic acid residues in these fragments were 0.15, 0.20, 0.38 and 0.35, respectively. The pectins also contained sequences of galacturonic acid residues with relatively little neutral sugar residues attached (smooth fragments). Methyl ester and acetyl groups were distributed fairly regularly along the smooth fragments. Evidence is presented for an association of oligogalacturonic acids with the hairy fragments under the conditions of gel chromatography. Feruloyl groups are located in the hairy fragments. Other phenolic compounds, associated with the purified pectins, appear not to be covalently linked.

The cell-wall polysaccharides of carrot roots were degraded by an endopectin lyase and an endopolygalacturonase from Aspergillus niger, a cellobiohydrolase from Trichoderma reesei, and an endoglucanase and a xylanase from Dichomitus squalens used individually, in combination, or in sequence. The endopectin lyase released > 80% of the galacturonic acid, but no glucose, whereas a mixture of the cellulases removed ∼40% of the glucans and ∼15% of the pectic polysaccharides. The extraction of the pectic substances by the endopectin lyase greatly increased hydrolysis of the cellulose by the cellulases. The pectic polysaccharides were highly branched mainly through the rhamnose residues, but some parts of the homogalacturonans carried short chains of arabinose. The association of pectic polysaccharides and cellulose through ester linkages or via side chains of xylose is discussed.

The rapid preparation of multimilligram quantities of five heparin-derived oligosaccharides (1–5) is described. These oligosaccharides are the final products obtained from the action of heparin lyase (heparinase, E.C. 4.2.2.7) at its primary sites in the heparin polymer. Five oligosaccharides comprise from 75–85 wt% of commercial porcine mucosal heparins and are recovered in good yield and high purity. Four of these five oligosaccharides were further acted upon at much lower rates by prolonged treatment with heparin lyase or heparan monosulfate lyase (heparitinase, E.C. 4.2.2.8), revealing the subspecificities of these enzymes. These oligosaccharides were used as defined substrates for heparin lyase and heparan monosulfate lyase and their kinetic constants were obtained. Potential applications for these oligosaccharides include their use as defined substrates for purification of heparin monosulfate lyases, and for establishing the catalytic purity of enzyme preparations.

The substrate specificity of heparan sulphate lyase and heparin lyase has been examined by using various oligosaccharides produced by deaminative cleavage or periodate oxidation—base-catalysed elimination of heparan sulphate. The saccharides were separated by gel and ion-exchange chromatography into fractions having high proportions of the following linkages: 2-acetamido-2-deoxy- d -glucosyl→ d -glucuronic acid (type I), 2-deoxy-2-sulphoamino- d -glucosyl→ d -glucuronic acid (type II), 2-deoxy-2-sulphoamino- d -glucosyl→ l -iduronic acid (type III), or 2-deoxy-2-sulphoamino- d -glucosyl→2- O -sulpho- l -iduronic acid (type IV). Heparan sulphate lyase cleaved heparan sulphate preparations containing high proportions of type I linkages. In contrast, oligosaccharides obtained after deaminative cleavage and rich in the type I linkage were poor substrates; longer saccharides were better substrates than shorter ones. Saccharides generated via periodate oxidation were cleaved when the type I linkage was present but resistant when the linkages were of types II-IV. The presence of ester sulphate on the 2-acetamido-2-deoxy- d -glucose residue in a type I linkage seems to hinder the enzyme. Heparan sulphate lyase was able to degrade intact chains to the same extent as did two cycles of periodate oxidation—base-catalysed elimination, suggesting that all the regular type I linkages were accessible to the enzyme. The heparin lyase could cleave saccharides which contained type IV linkages. When type II and III linkages were the only ones present, the saccharides were resistant. This enzyme also appears to require ester-sulphation of the 2-deoxy-2-sulphoamino- d -glucose residue.

The structure of the capsular polysaccharide from Escherichia coli O9:K55 (N 24c) has been studied, using methylation analysis, degradation by bacteriophage, and n.m.r. spectroscopy. Depolymerisation of the K55 polysaccharide, using the lyase enzyme borne by Klebsiella phi 5, yielded a tri- and a hexa-saccharide, analysis of which indicated the following repeating unit. (formula; see text) This structure differs from that for the repeating unit of the capsular polysaccharide of Klebsiella K5 only in the position of acetylation (position 2 of the glucose residue).

The bacterium Klebsiella aerogenes type 25 produces an extracellular alginolyase which has been partly purified. The enzyme is specific for the alpha-L-guluronosyl linkages in whole alginate and fractions therefrom. The end products of its action on polyguluronic acid blocks are mainly the unsaturated di- and tri-saccharides, with a smaller proportion of the homologous tetrasaccharide. Some general properties of the enzyme are reported.

Bacteriophage phi 64 possesses a lyase that depolymerises the capsular polysaccharide of Klebsiella K64 into a hexasaccharide having an unsaturated derivative of glucuronic acid at the non-reducing end (1). The unsaturated hex-4-enuronic acid residue generated was characterised spectroscopically (u.v. and n.m.r.) and by g.l.c.-m.s. after hydrogenation of the double bond. Partial hydrolysis, Smith degradation, methylation analysis, and n.m.r. spectroscopy have been used to establish the structures of oligosaccharides produced from the polysaccharide. Evidence from 1H-n.m.r. spectroscopy indicates that the D-Man rho residue that undergoes fission is beta. (Formula: see text).

Klebsiella K14 capsular polysaccharide was degraded by a bacteriophage-borne enzyme to afford oligosaccharides A-C which were studied by one- and two-dimensional n.m.r. spectroscopy. A and B were the repeating-unit hexasaccharide and pyruvylated hexasaccharide, respectively, while C was a dodecasaccharide. Each oligomer was terminated by a reducing mannose and a non-reducing 4-deoxy-α- l - threo -hex-4-enopyranosyluronic acid residue, indicating that the phage enzyme had cleaved the β- d -Man p -(1→4)-β- d -Glc p A linkages in the polysaccharide by a lyase, rather than the more common glycosidase, activity found with other Klebsiella bacteriophages. In this respect, the depolymerisation resembles those reported for the capsular polysaccharides of Klebsiella K5 and K64