Enzymic synthesis of lacto-N-triose II and its positional analogues

N-acetylhexosaminidase fromNocardia orientalis catalysed the synthesis of lacto-N-triose II glycoside (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OMe,3) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OMe (4) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OMe (5) throughN-acetylglucosaminyl transfer fromN,N-diacetylchitobiose (GlcNAc₂) to methyl -lactoside. The enzyme formed the mixture of trisac-charides3, 4 and5 in 17% overall yield based on GlcNAc₂, in a ratio of 20:21:59. Withp-nitrophenyl -lactoside as an acceptor, the enzyme also producedp-nitrophenyl -lacto-N-trioside II (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OC₆H₄NO₂-p,6) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OC₆H₄NO₂-p (7) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OC₆H₄NO₂-p (8). In this case, when an inclusion complex ofp-nitrophenyl lactoside acceptor with -cyclodextrin was used, the regioselectivity of glycosidase-catalysed formation of trisaccharide glycoside was substantially changed. It resulted not only in a significant increase of the overall yield of transfer products, but also in the proportion of the desired compound6.Abbreviations GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1-4)-2-acetamido-2-deoxy-d-glucose - NAHase N-acetylhexosaminidase - -CD -cyclodextrin     

Glycosylation with thioglycosides activated by dimethyl(methylthio)sulfonium tetrafluoroborate: synthesis of two trisaccharide glycosides corresponding to the blood group A and B determinants

Two trisaccharide glycosides,p-trifluoroacetamidophenylethyl 3-O-(2-acetamido-2-deoxy--d-galactopyranosyl)-2-O-(-l-fucopyranosyl)--d-galactopyranoside andp-trifluoroa-cetamidophenylethyl 2-O-(-l-fucopyranosyl)-3-O-(-d-galactopyranosyl)--d-galactopyranoside, corresponding to the human blood group A and B determinants, were synthesized. A key fucosylgalactosyl disaccharide derivative was glycosylated with galactosaminyl or galactosyl donors, respectively. Dimethyl (thiomethyl)sulfonium tetrafluoroborate was used for thioglycoside activation in coupling reactions.     

Production of β-fructofuranosidase byAspergillus japonicus

A newly isolated strain, MU-2, which produces very high -fructofuranosidase activity, was identified asAspergillus japonicus. For enzyme production by the strain, sucrose at 20% (w/v) was the best carbon source and yeast extract at 1.5 to 3% (w/v) the best nitrogen source. Total enzymatic activity and cell growth were at maximum after 48 h, at 1.57×10⁴ U/flask and 0.81 g dry cells/flask, respectively. The optimum pH value of the enzymatic reaction was between 5.0 and 5.5 and the optimum temperature 60 to 65°C. The enzyme produced 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose by fructosyl-transferring activity. The strain was found to be very useful for industrial production of -fructofuranosidase.     

d-Alanine dehydrogenase

Pseudomonas aeruginosa PA01 was found to utilise both thed- andl-isomers of -alanine and also -alanine as sole sources of carbon and energy for growth. Enzymological studies of wild-type cultures and comparison with mutants deficient in growth upon one or more isomers of alanine led to the following conclusions: (i) utilisation ofd-alanine involved its direct oxidation by an inducible, membrane-bound, cytochrome-linked dehydrogenase; (ii) utilisation ofl-alanine required its conversion to the directly oxidisabled-form by a soluble racemase; (iii) utilisation of -alanine, likel-alanine, involves both the racemase andd-alanine dehydrogenase enzymes, but in addition must involve other enzymes the identity, of which is still speculative; (iv)P. aeruginosa, likeEscherichia coli, appears to take upd-alanine andl-alanine by means of two specific permeases.Abbreviation DCPIP 2,6-dichlorophenol-indophenol     

Biosynthesis of the storage polysaccharide from the snail Biomphalaria glabrata,identification and specificity of a branching β1→6 galactosyltransferase

Adult snails synthesize in their albumen glands a storage polysaccharide called galactan which is utilized by the developing embryos. With [6-³H]-uridine 5diphosphogalactose the incorporation of labelled d-galactose into the polysaccharide can be traeed in freshly removed glands maintained in a bathing buffer. After centrifugation of homogenized glands, galactosyltrasferase activity is only found in the insoluble fraction. Chaps extracts of this material retain almost all of their activity and can be used for comparison of the incorporation rates into different native galactans or in various oligosaccharides. A highly efficient -(16) galactosyltransferase was detected when methyl 3-O-(-d-galactopyranosyl)--d-galactopyranoside was offered as acceptor. The substitution at the penultimate residue resulted in a branched trisaccharide as demonstrated by ¹H-NMR-spectroscopy and permethylation analysis of the reaction product. Comparable results were obtained with various oligosaccharides containing an internal galactose unit glycosidically linked 13. Attempts to separate and purify the various enzymes involved resulted in the isolation of a fraction which is able to transfer d-Gal exclusively to native galactan, but not to oligosaccharides. A further fraction was obtained from a different resin with activity for native galactan and 6-O-(-d-galactopyranosyl)-d-galactopyranose. but without any for methyl-3-O-(-d-galactopyranosyl)--d-galactopyranose. It is thus concluded that at least three different enzymes are involved in the biosynthesis of this snail galactan.Abbreviation Gal galactose - glc gas-liquid chromatography - Gro glycerol - tlc thin layer chromatography     

Partial purification and properties of γ-glutamyltranspeptidase from mycelia of Morchella esculenta

Three -glutamyltranspeptidase (enzymes I, II and III) were partially purified from the cell free extracts of the cultured mycelia of Morchella esculenta Fr. The molecular masses of enzymes were 155,000 (I), 219,000 (II) and 102,000 (III). All of them catalyzed both hydrolysis and transpeptidation of various -glutamyl compounds. -l-Glutamyl-cis-3-amino-l-proline occurring in the cultured mycelia of this fungus was a good substrate for both reactions. K _m values for hydrolysis were in the order of 10^-4 to 10^-5 M, and those for transpeptidation were in the order of 10^-2 to 10^-4 M. The enzymes were inhibited by a -glutamyltranspeptidase inhibitor, l-serine plus borate.Abbreviations -GTP -glutamyltranspeptidase - HPLC High-performance liquid chromatography     

Synthesis ofNeoglycoproteins containing the 3,6-di-O-methyl-β-d-glucopyranosyl epitope and their use in serodiagnosis of leprosy

A stratagem for the synthesis ofneoglycoproteins suitable for the selective serodiagnosis of leprosy is described in which synthetic 3,6-di-O-methyl--d-glucopyranose, the epitope of phenolic glycolipid I fromMycobacterium leprae, was used. Condensation of 8-methoxycarbonyloctanol with the acetobromo derivative of 3,6-di-O-methylglucose gave 8-methoxycarbonyloctyl 2,4-di-O-acetyl-3,6-di-O-methyl--d-glucopyranoside in 65% yield, and with absolute stereospecificity for the anomer. The deacylated product was converted to the crystalline hydrazide and coupled to bovine gamma globulin, bovine serum albumin and poly-d-lysinevia intermediate acyl azide formation to produce the 8-carbonyloctyl 3,6-di-O-methyl--d-glucopyranosyl polypeptides. Theneoglycoproteins were highly sensitive in ELISA and emulated the specificity of the native glycolipid in analysis of sera from patients throughout the spectrum of leprosy and from different geographical regions. The 8-carbonyloctyl 3,6-di-O-methyl--d-glucopyranoside-bovine serum albumin was also synthesized and shown to have about one-half the activity of the -linkedneoglycoprotein. A different synthetic approach produced the 8-carbonyloctyl 4-O-(3,6-di-O-methyl--d-glucopyranosyl)--l-rhamnopyranoside-bovine serum albumin which was also highly sensitive and specific for the serodiagnosis of leprosy. The presence of the second sugar unit, similar to that in the native glycolipid but for the absence ofO-methyl groups, seemed to provide a probe with greater felicity for the serological detection of tuberculoid leprosy.Thus, the results indicate that highly sensitive and specific antigen probes for the serodiagnosis of leprosy can be constructed based only on the terminal one or two sugars of phenolic glycolipid I, and the synthetic approach leads to the formation of haptens with absolute stereospecificity.Nomenclature BGG bovine gamma globulin - PGL-I phenolic glycolipid I - PDL poly-d-lysine - PBS phophate-buffered saline - 3,6-Me₂-Glc-Link-BSA 8-carbonyloctyl 3,6-di-O-methyl-glucopyranoside-bovine senalbumin - 3,6-Me₂-Glc-Rha-Link-BSA 8-carbonyloctyl 4-O-(3,6-di-O-methyl--d-glucopyranosyl)--l-rhan pyranoside-BSA     

A simple synthesis of octyl 3,6-di-O-(α-d-mannopyranosyl)-β-d-mannopyranoside and its use as an acceptor for the assay ofN-acetylglucosaminyltransferase-I activity

A simple synthesis of octyl 3,6-di-O-(-d-mannopyranosyl)--d-mannopyranoside is described. The key features of the synthetic scheme are the formation of the -mannosidic linkage by 1-O-alkylation of 2,3,4,6-tetra-O-acetyl-,-d-mannopyranose with octyl iodide and glycosylation of unprotected octyl -d-mannopyranoside using limiting acetobromomannose. The trisaccharide is shown to be an acceptor forN-acetylglucosaminyltransferase-I with aK _M of 585 µm.     

Variant forms ofα-2-l-fucosyltransferase in human submaxillary glands from blood group ABH “secretor” and “non-secretor” individuals

The properties of the -2-l-fucosyltransferases in submaxillary gland preparations from blood group ABH secrefors and non-secretors were compared. The level of activity in the non-secretor gland homogenates amounted to about 5% only of that found in the secretor gland preparations. The enzymes from the two sources differed in solubility properties, charge and affinities for donor and acceptor substrates. The enzyme from secretor glands showed a preference for acceptors with Type 1 [d-galactosyl(1–3)-N-acetyl-d-glucosamine] structures whereas the enzyme from non-secretor glands had a preference for Type 2 [d-galactosyl(1–4)-N-acetyl-d-glucosamine] structures.These results demonstrate that expression of the secretor gene (Se) is associated with a molecular form of the -2-l-fucosyltransferase that is different from the species present in the same tissue when theSe gene is not expressed.     

Contributions of aerobic and anaerobic energy production during swimming in the bivalve molluscLimaria fragilis (family limidae)

Summary Oxygen consumption rates ( ) and a number of possible anaerobic end products were determined forLimaria fragilis at rest, and following active swimming. increased up to 8-fold (=4) during swimming. Swimming did not change the concentrations ofl-lactate, alanine or arginine phosphate in the single striated fast adductor muscle. Octopine, succinate andd-lactate were not detected in the adductor muscles of resting or active animals (<0.2 moles/g wet weight).It is concluded that the slow sustained swimming displayed byLimaria utilises predominantly aerobic mechanisms of ATP production.     

Artemisia annua L.: dedifferentiated and differentiated cultures

Dedifferentiated and differentiated tissue cultures ofArtemisia annua L. for artemisinin production were carried out. The calluses were initiated on MS medium supplemented with sucrose (30 g l^-1), myoinositol (100 mg l^-1) and RT vitamins. The auxins used were naphtaleneacetic acid (NAA), indoleacetic acid (IAA), indolebutyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-d). These were added to the basal medium either singly or in combination. The best results were obtained with 2.4-d (4.5 M : 0.02 d^-1) and NAA (5.4 M : 0.06 d^-1). Cell suspensions were established on the same media without agar. Suspension cultures showed different morphological characteristics according to the plant growth regulator supplied. Organized cultures were initiated from callus obtained on 2,4-d (4.5 M) and from bud cultures. Medium containing 6-benzylaminepurine (BA) (8.9 M)+NAA (0.54 M); Zeatin (45.62 M)+NAA (5.37 M) or BA (8.9 M) stimulated both organogenesis in callus (frequency of induction =50%) and semi-organized tissue in shoot buds. BA (13.32 M)+NAA (1.08 M) or BA (13.32 M) only stimulated multiple shoot cultures (frequency of induction =80%). Regarding artemisinin content, while the values obtained were 1.13 and 0.78 mg gDW^-1 in primary callus, artemisinin was not detected in cell suspension and only traces of it were found in multiple shoot cultures.     

Assimilation of γ-butyrobetaine,and d-and l-carnitine by resting cell suspensions of Acinetobacter calcoaceticus and Pseudomonas putida

The metabolic pattern of utilization of [1,2,3,4-¹⁴C, methyl-³H] -butyrobetaine and d-and l-[1-¹⁴C, methyl-³H]carnitine has been examined with variously grown resting cell suspensions of Acinetobacter calcoaceticus and Pseudomonas putida. Ps. putida grown on d, l-carnitine as the sole source of carbon, degraded only l-carnitine with stoichiometric accumulation of glycinebetaine. Alternatively, when grown on -butyrobetaine, Ps. putida rapidly metabolized -butyrobetaine, and to a lesser but significant extent, both d-and l-carnitine with equivalent formation of trimethylamine and degradation of the betaine carbon skeleton. Ac. calcoaceticus grown on either d,l-carnitine or -butyrobetaine, effectively utilized all three betaines at nearly the same rates. Disappearance of each of these quarternary ammonium compounds was accompanied by stoichiometric formation of trimethylamine and degradation of the carbon backbone. Utilization of the betaines and corresponding formation of trimethylamine by resting cell suspensions of appropriately grown Ac. calcoaceticus and Ps. putida, was essentially abolished under conditions of anaerobiosis and severely impaired in the presence of sodium cyanide, sodium azide, 2,4-dinitrophenol or 2,2-bipyridine. The results of the present investigations with resting cell suspensions of both Ac. calcoaceticus and Ps. putida do not support an earlier suggestion that -butyrobetaine degradation in these organisms proceeds by its prior hydroxylation to l-carnitine. Indeed, disrupted cell-free preparations of Ac. calcoaceticus and Ps. putida grown on either d,l-carnitine or -butyrobetaine showed no detectable -butyrobetaine hydroxylase activity.     

Agaroids from New Zealand members of the Gracilariaceae (Gracilariales,Rhodophyta) — a novel dimethylated agar

Polysaccharide extracts from four New Zealand members of the Gracilariaceae have been characterized by ¹³C-NMR spectroscopy and GLC analysis of alditol acetate derivatives prepared using a new double hydrolysis-reduction procedure. All were based on variously substituted repeating disaccharide units of agarobiose and 20% of its precursor containing l-galactose-6-sulfate. Gracilaria truncata yielded a firm gelling agar with 67% methylation on the 6-position of the d-galactose residues. The other extracts belong to a new class of agar molecules having methylation on both the 6-position of the d-galactose units and the 2-position of the l-sugar units. The Curdiea coriacea polysaccharide displayed this double methylation almost completely ( 96 %); the alkali-modified polymer thus had only two free hydroxy-groups per disaccharide repeat unit, yet still gave a firm gel. The Curdiea flabellata and Melanthalia abscissa extracts had this double methylation pattern but to a lesser extent, and additional xylosyl branch units on up to 18% of the repeating disaccharide units.     

Peptidoglycan types and cytochrome patterns of strains of Oerskovia turbata and O. xanthineolytica

Determination of the primary structure of the peptidoglycan of 15 strains of Oerskovia showed that three different peptidoglycan types occur. Oerskovia xanthineolytica strains contain the l-Lys-d-Ser--d-Asp type, whereas Oerskovia turbata strains show the new peptidoglycan types l-Lys-l-Thr--d-Asp or l-Lys-l-Thr--d-Glu, respectively. Research on the cytochromes of Oerskovia revealed the presence of a, b and c types. O. turbata can be clearly distinguished from O. xanthineolytica by the occurrence of cytochrome a ₁ in cells, isolated from the stationary phase. The following conclusions were made: O. turbata and O. xanthineolytica can be clearly separated on the basis of different peptidoglycan types and cytochrome patterns. This distinction is in perfect correlation with the classical separation method of O. turbata and O. xanthineolytica on the basis of xanthine degradation. l-Lys-d-Ser--d-Asp peptidoglycan type does not only occur in O. xanthineolytica but also in some coryneform bacteria such as Corynebacterium manihot (Fiedler et al. 1970), Cellulomonas cartae (Stackebrandt et al. 1978; Stackebrandt and Kandler 1980), Brevibacterium fermentans and Nocardia cellulans.This paper is respectively dedicated to Professor Dr. O. Kandler, on the occasion of his 60th birthday     

Synthesis of chemically modified sialic acid-containing sialyl-LeX ganglioside analogues recognized by the selectin family

Sialyl Lewis X ganglioside analogues containing 5-acetamido-3,5-dideoxy-l-arabino-2-heptulopyranosylonic acid (C7-Neu5Ac), 5-acetamido-3,5-dideoxy-d-galacto-2-octulopyranosylonic acid (C8-Neu5Ac), and 5-acetamido-3,5-dideoxy-l-glycero-d-galacto-1-2-nonulopyranosylonic acid (8-epi-Neu5Ac) in place ofN-acetylneuraminic acid (Neu5Ac) have been synthesized. Glycosylation of 2-(trimethylsilyl)ethyl 6-O-benzoyl--d-galactopyranoside with the phenyl or methyl 2-thioglycoside derivatives of the respective sialic acids, usingN-iodosuccinimide (NIS)-trifluoromethanesulfonic acid as a promoter in acetonitrile, gave the three required 2-(trimethylsilyl)ethyl (2S)-sialyl-(2 3)--galactopyranosides. These were converted viaO-benzoylation, selective transformation of the 2-(trimethylsilyl)ethyl group to acetyl, and introduction of the methylthio group with methylthiotrimethylsilane into the corresponding glycosyl donors. Glycosylation of 2-(trimethylsilyl)ethylO-(2,3,4-tri-O-benzyl--l-fucopyranosyl)-(1 3)-O-(2-acetamido-6-O-benzyl-2-deoxy--d-glucopyranosyl)-(1 3)-2,4,6-tri-O-benzyl--d-galactopyranoside with these donors in the presence of dimethyl(methylthio)sulfonium triflate (DMTST) afforded the expected -glycosides, which were converted into the corresponding -trichloroacetimidates, and these, on coupling with (2S, 3R, 4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol, gave the required -glycosides. Finally, these were transformed via selective reduction of the azide group, condensation with octadecanoic acid,O-deacylation, and de-esterification into the target compounds in good yields.     

Induction of extracellular arabinases on monomeric substrates in Aspergillus niger

The induction of extracellular arabinases by pentose sugars and polyols generated by the metabolic pathway of l-arabinose and d-xylose catabolism in Aspergillus niger was investigated. Induction occurred with l-arabinose and l-arabitol but not with d-xylose or xylitol. l-arabitol in particular was found to be a good inducer for -l-arabinofuranosidase and endo-arabinase activities. Western blotting analysis showed both -l-arabinofuranosidase A and B to be present. No induction was observed using d-arabitol. Unlike the wild type A. niger N402 strain, the A. niger xylulose kinase negative mutant N572 also showed induction of -l-arabinofuranosidases A and B and endo-arabinase activity on d-xylose and xylitol. This is due to metabolic conversion of these compounds leading to the accumulation of both xylitol and l-arabitol in this mutant, the latter of which then acts as inducer. The induction of the two -l-arabinofuranosidases and endo-arabinase is under the control of two regulatory systems namely pathway specific induction and carbon catabolite repression. Under derepressing conditions in the wild type only -l-arabinofuranosidase B could be detected by Western blotting analysis. This indicates that -l-arabinofuranosidase B is of importance in the initiation of specific induction of the various arabinose activities in A. niger grown on arabinose containing structural polysaccharides.Abbreviations PNA p-nitrophenyl--l-arabinofuranoside     

Purification and properties ofβ-fructofuranosidase from Aspergillus japonicus

-Fructofuranosidase fromAspergillus japonicus, which produces 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose, was purified to homogeneity by fractionation with calcium acetate and ammonium sulphate and chromatography with DEAE-Cellulofine and Sephadex G-200. Its molecular size was estimated to be about 304,000 Da by gel filtration. The enzyme was a glycoprotein which contained about 20% (w/w) carbohydrate. Optimum pH for the enzymatic reaction was 5.5 to 6. The enzyme was stable over a wide pH range, from pH 4 to 9. Optimum reaction temperature for the enzyme was 60 to 65°C and it was stable below 60°C. The K_m value for sucrose was 0.21m. The enzyme was inhibited by metal ions, such as those of silver, lead and iron, and also byp-chloromercuribenzoate.     

Regulation of biosynthesis of aspartate family amino acids in the photosynthetic bacterium Rhodopseudomonas palustris

The four amino acids of the aspartate family (l-lysine, l-methionine, l-threonine, and l-isoleucine) are produced in bacteria by a branched biosynthetic pathway. Regulation of synthesis of early common intermediates and of carbon flow through distal branches of the pathway requires operation of a number of subtle feedback controls, which are integrated so as to ensure balanced synthesis of the several end products. Earlier studies with nonsulfur purple photosynthetic bacteria were instrumental in revealing the existence of alternative regulatory schemes, and in this communication we report on the control pattern of a representative of this physiological group not previously investigated, Rhodopseudomonas palustris. The results obtained from study of the properties of four key regulatory enzymes of the aspartate family pathway (-aspartokinase, homoserine dehydrogenase, homoserine kinase, and threonine deaminase) and of the effects of exogenous amino acids (i. e., the end products) on growth of the bacterium indicate that the control schema in Rps. palustris differs substantially from the schemes described for other Rhodopseudomonas species, but resembles the regulatory pattern observed in Rhodospirillum rubrum.Abbreviations A absorbancy - AK -aspartokinase - ASA aspartate -semialdehyde - DTT dithiothreitol - HS l-homoserine - HSDH homoserine dehydrogenase - HSK homoserine kinase - I l-isoleucine - KU Klett-Summerson photometer units - L l-lysine - M l-isoleucine - KU Klett-Summerson photometer units - L l-lysine - M l-methionine - ME -mercaptoethanol - PABA p-aminobenzoic acid - T l-threonine - TD threonine deaminase - RCV synthetic growth medium (see text) - YP agar medium containing 0.3% yeast extract, 0.3% peptone, and 1.5% agar - Y2T synthetic growth medium (see text)     

Methylobacterium rhodesianum MB 126 possesses two acetoacetyl-CoA reductases

Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Methylobacterium rhodesianum MB 126, an NADPH-linked d(-)--hydroxybutyryl-CoA forming reductase (enzyme A) and an NADH-and NADPH-linked l(+)--hydroxybutyryl-CoA forming reductase (enzyme B). Enzyme A and B give apparent K _m values of 15 M and 30 M for AcAc-CoA, 18 M for NADPH and 30 M for NADH, respectively. They are inhibited by AcAc-CoA at concentrations higher than 25 M and 50 M, respectively. The contribution of the two reductases to poly--hydroxybutyrate synthesis is discussed.     

Phosphate repressible and inhibitable β-lactam synthetases inCephalosporium acremonium strain C-10

Summary -Lactam production by a high producer,Cephalosporium acremonium C-10, was reduced by high concentrations of phosphate (100 mM to 215 mM). The optimal concentration for antibiotic production by this strain was 25 mM. High concentrations of phosphate did not act by increasing the rate of glucose assimilation as previously concluded (Küenzi 1980). The three -lactam synthetases tested, i.e., -(l--aminoadipyl)-l-cysteinyl-d-valine synthetase, cyclase and expandase, were all subject to phosphate repression as well as inhibition. The most susceptible to both kinds of regulation was the expandase.