Abstract: Relevant examples of polyglycosylating exo-glycosidases were reported among enzymes of marine origin (Aplysia fasciata, Geobacillus, and Pecten maximus). Herein we describe the enzymatic polyglucosylation of a chromane-methanol (2-hydroxymethyl-2,5,7,8-tetramethylchroman-6-ol) performed by using the α-D: -glucosidase from the sea hare Aplysia fasciata. New di-, tri-, and tetrasaccharide derivatives were synthesized and their antioxidant activities were evaluated by DPPH test. High enzymatic substrate conversion was assessed by NMR spectroscopy, and the products were easily purified. These findings suggest that the proposed procedure is an effective process both for the molecular diversity of products and for the peculiar stereochemistry of the enzyme. At the beginning of the enzymatic reaction, only (S)-diastereomer of the monoglucoside was obtained. The isomaltoside was the most abundant disaccharide obtained and showed a radical scavenging activity similar to that of the chromane-methanol. The disaccharide can be considered a new hydrosoluble antioxidant agent useful for various technological applications (cosmetics, food industry, etc.). A relationship between the interglycosidic linkage present in disaccharides and trisaccharides and their scavenging activity was also pointed out.
Abstract: In several recent reports related to biocatalysis the enormous pool of
biodiversity found in marine ecosystems is considered a profitable natural reservoir for
acquiring an inventory of useful biocatalysts. These enzymes are characterized by
well-known habitat-related features such as salt tolerance, hyperthermostability,
barophilicity and cold adaptivity. In addition, their novel chemical and stereochemical
characteristics increase the interest of biocatalysis practitioners both in academia and
research industry. In this review, starting from the analysis of these featuring
habitat-related properties, important examples of marine enzymes in biocatalysis will be
reported. Completion of this report is devoted to the analysis of novel chemical and
stereochemical biodiversity offered by marine biocatalysts with particular emphasis on
current or potential applications of these enzymes in chemical and pharmaceutical fields.
The analysis of literature cited here and the many published patent applications concerning
the use of marine enzymes supports the view that these biocatalysts are just waiting to be
discovered, reflecting the importance of the marine environment. The potential of this
habitat should be thoroughly explored and possibly the way to access useful biocatalysts
should avoid destructive large-scale collections of marine biomass for enzyme production.
These two aspects are day by day increasing in interest and a future increase in the use of marine enzymes in biocatalysis should be expected.
Abstract: Carbohydrates are important molecules in various technological fields. The regio- and stereoselective synthesis of oligosaccharides for food and pharmaceutical molecular design and the selective glycosylation of peptides, proteins and drugs, as well as the selective hydrolysis of natural abundant saccharidic materials affording low molecular weight high-value-added products, can be all considered significant aspects. Advance in the chemical synthesis of glycosidic bond represents one of the most successful topic in organic synthetic chemistry in the last half century for the intrinsic difficulties chemists have to face dealing with carbohydrates: yields and selectivtiy. Biocatalytic strategies selected for high-yield and stereospecific construction of glycosidic bonds are based principally on the action of two classes of enzymes, namely glycoside hydrolases (endo- and exo-glycosidases) and glycosyltransferases although only enzymes of the second class are devoted to synthetic action in nature.
Reviewing patents on the preparation of glycosides is hard to manage, here a five years time frame for this review is set, considering it a suitable compromise in terms of amount of innovation and numbers of patents to analyze. Interest of researchers for the natural sources of biocatalysts, with particular emphasis on modified enzymes and lists of the most innovative patents in cosmetic, fine chemicals and food fields will be commented. Biomolecular asset of selected patents related to glycosides will be expressly considered. Large number of enzymes will be required in future in order to realize more sustainable new enzymatic processes to answer human needs.
Abstract: This review is intended to give an account of the knowledge about known enzymes of marine origin described in literature thus stimulating future applications in biocatalysis that these biocatalysts can offer to a large spectra of end-users. The uniqueness of marine biocatalysts is not only based on habitat-related properties such as salt tolerance, hyperthermostability, barophilicity, cold adaptivity,
etc. A marine enzyme in fact may carry more, e.g. novel chemical and stereochemical properties. This âchemical biodiversityâ increases interest in this field; substrate specificity and affinity are evolved properties linked to the metabolic functions of the enzymes and to ecological asset related to the natural
source and this is an important aspect in the bioprospecting for new biocatalysts. The importance of all examples reported should be sufficient to trigger the attention of the biocatalytically oriented scientific community towards marine environment as source of biocatalysts, and this could in turn enhance both
new discovery and improvement of marine enzymes.
Abstract: Anomerically free acidic xylo-oligosaccharides have shown interesting biological properties when tested against Gram-positive and Gram-negative aerobically grown bacteria, as well as against Helicobacter pylori, sarcoma-180 and other tumors. We report here a structure-activity relationship study on the role of 4-O-methyl glucuronic acid (MeGlcUA) in regulating aggregation of ²-polyxylosides of (9H-fluoren-9-yl)- methanol obtained via the action of Thermotoga neapolitana xylanase. Neutral compounds from mono- to penta-²-1,4 xylosides were obtained from this biocatalyzed reaction. In addition, acidic components among products, carrying an ±-1,2 4-O-methyl glucuronic acid (MeGlcUA) were also isolated. An antiproliferative test of these compounds on human epithelial EFO 27 ovarian cancer cells indicated that the presence of MeGlcUA modulates their biological activity, while its absence induces molecular aggregation. The three-dimensional structure of the most active MeGlcUA ²-polyxyloside was investigated by resorting to NOESY experiments supported by dynamic force-field calculations with/without constraints. The 3D structure is characterized by all sugars possessing a 4C1 chair conformation. The MeGlcUA moiety, and the external and middle xyloses adopt a hairpin-shaped conformation, generating a non-planar arrangement of the molecule with the aromatic ring folding back toward the carbohydrate chain. Such a non-planar conformation may justify the lack of aggregation.
Abstract: The biological activities of a series of mono- and oligosaccharides (b-xylosides and a-glucosides) of 9-fluorenylmethanol were investigated together with mono-b-galactoside and b-glucoside of this aglycone, produced by biocatalytic routes. By using marine glycoside hydrolases and inexpensive donors such as maltose or xylan, access to mono a-glucoside or mono-b-xyloside of 9-fluorenylmethanol was obtained. Additionally, interesting polyglycoside derivatives were isolated. Biological testing indicated that in vitro treatment with these carbohydrate derivatives may influence the balance of cytokines in the environment of human peripheral blood mononuclear cells (PBMC), restricting the harmful effect of herpes simplex type 2 replication. In fact, these carbohydrate derivatives tested in WISH cells did not show any significant antiviral activity.
Abstract: In pursuing a research on the antiviral and immunomodulatory activity of tilorone congeners, two new series of compounds were prepared
and pharmacologically explored: 9-fluorenone carboxyhydroxyesters, indicated as AG, and 9-fluorenone carboxyhydroxamides, indicated as
MG. Two of them, AG17 and MG3, were used as sugar acceptors in the transglycosylation reactions performed by alfa- and beta-glucosidases
extracted from the marine mollusc Aplysia fasciata providing different alfa- and beta-, mono- and oligosaccharides. Then aglycons and saccharides
were assayed for cytotoxicity, for anti-herpes virus-2 properties on peripheral blood mononuclear cells (PBMC) and for their capability to trigger human cells to produce antiviral cytokines such as IFNa and TNFa. Some promising compounds were individuated whereas the utility of the
biocatalytic procedures in the preparation of pure anomeric material was further focused.
Abstract:
Glycosylation is considered to be an important reaction for the chemical modification of compounds with useful biological activities. Glycoside hydrolases are biotechnologically attractive enzymes which can be used in synthetic reactions for assembling glycosidic linkages with absolute stereoselectivity at an anomeric centre. Most of these enzymes are commercially available but there is great interest in the search for
new biocatalysts with original catalytic characteristics. The marine environment has shown to be a very interesting source for new glycosyl hydrolases for both hydrolytic and synthetic aspects. In particular, Aplysia fasciata a marine herbivorous mollusc has been shown to be a potent producer of a library of glycoside hydrolases applied to the synthesis of glycosidic bonds. The impressive assortment of glycosidases in marine organisms clearly indicates that the potential biodiversity of these enzymes is still largely unexplored and that potential applications of biocatalysts from the sea
will increase in the near future.
Abstract: The enzymatic glucosylations of naringin, performed using alpha-D-glucosidase, identified in the Mediterranean mollusc Aplysia fasciata is reported. The enzyme actively operates on maltose and has an interesting transglycosylation potential using this donor. We also investigated the use of this marine alpha-glucosidase for a food-compatible glucosylation of naringin to produce new enzymatically modified carbohydrate possessing naringin derivatives. The regioselective formations of the beta-gluco-C6 alpha-glucosyl derivative and of the corresponding isomaltosyl diglucoside of naringin were obtained in high yield and efficiency of reaction. Suspensions of naringin can be used up to approximately 90 mg/mL initial acceptor concentration. In different experiments it was demonstrated that the enzyme was still active after 48 h in presence of this high amount of acceptor and that one of the diasteromers of the naringin is preferred by the enzyme from A. fasciata during glucosylation/deglucosylation enzymatic steps. Finally, the feasibility of efficient naringin glucosylation in grapefruit juice was also demonstrated at optimal pH of the enzyme and low maltose concentrations.
Abstract: The biological activities of a series of mono- and oligosaccharides (beta-xylosides and alpha-glucosides) of 9-fluorenylmethanol were investigated together with mono-beta-galactoside and beta-glucoside of this aglycone, produced by biocatalytic routes. By using marine glycoside hydrolases and inexpensive donors such as maltose or xylan, access to mono-alpha-glucoside or mono-beta-xyloside of 9-fluorenylmethanol was obtained. Additionally, interesting polyglycoside derivatives were isolated. Biological testing indicated that in vitro treatment with these carbohydrate derivatives may influence the balance of cytokines in the environment of human peripheral blood mononuclear cells (PBMC), restricting the harmful effect of herpes simplex type 2 replication. In fact, these carbohydrate derivatives tested in WISH cells did not show any significant antiviral activity.
Abstract: The use of the extremophilic marine bacterium Thermotoga neapolitana (DSM 4359) for the bioproduction of hydrogen using complex carbohydrates as feedstock has been recently described in a patent. In this growth condition glycosyl hydrolases can be well expressed. An important issue to plan economical feasibility of biological hydrogen production comprises techniques for recovering useful materials such as physiologically
active biomolecules from biomasses grown in large quantities.
The present paper describes a series of enzymatic transglycosylation reactions performed using the crude homogenate of T. neapolitana. The
study is focused on synthetic features of the transglycosylating enzymes. Xylosidase/xylanase activity seems to be the most abundant leading to
convenient syntheses of interesting series of pure (-1,4)-xylooligosaccharides of different aglycones such as 1-hexanol (producing promising candidates for new surfactants), 9-fluorene methanol (obtaining anti-HSV agents), 1,4-butanediol (for the synthesis of new glycolipids), and
geraniol (producing aroma compounds). Furthermore, the regioselectivity during galactose, fucose, glucose, and mannose enzymatic transfers is
also investigated. The knowledge of synthetic characteristics of all these enzymes will be useful in the feasibility evaluation of large-scale processes of the biohydrogen production based on T. neapolitana.
Abstract: A convenient synthesis of beta-galactosyl derivatives of antiviral and anticancer nucleosides, which utilizes the purified beta-galactosidase activity from the hapatopancreas of Aplysia fasciata, is reported. All reactions were extremely stereo- and regioselective, since only anomerically pure 5-O-ï beta-galactosyl conjugates were formed. 5-O-beta-Galactosyl-5-fluorouridine was synthesized with a 60% yield and 5-O-ï beta-galactosyl-3â-azido-3â-deoxythymidine, the derivative of the anti-HIV drug, was obtained in 43% yield.
Abstract: Glycobiology and related disciplines have received an enormous interest in recent years as they shaded new light on the functional roles of carbohydrates in biological events leading to the understanding of mechanisms of important pathologies and to the development of new therapeutics. Although carbohydrate can be isolated from natural sources, the synthetic strategy play its own role. Nowadays the bottleneck in this field is represented by some limitations of the potential of carbohydrate containing molecules due to their complex structures which make classical chemical synthesis very difficult.
In the carbohydrate field different enzymes were used for the construction of glycosidic bonds: glycosyl hydrolases (endo- and exo-glycosidases) and glycosyltransferases.
This review deals with the application of different types of glycosyl hydrolases. Elegant examples concerning the use of genetically modified representatives of glycosyl hydrolases will be also reported; some recent advances on the use of glycosyltransferases are also included.
In compiling this review we were aware of the huge amount of excellent material published in the recent years on this topic, thus we will limit the covering of literature to the last decade.
This review will put in evidence that glycosyl hydrolases and glycosyltransferases for the synthesis of the glycosidic linkages will play a relevant role in the next future as on the bench bio-catalysts for the chemists involved in the synthesis of oligosaccharides of biological interest.
Abstract: The recent successes of marine biotechnology (development of new methods for in-sea aquaculture of marine inverterbrates, advances of genetic engineering of marine organisms) have allowed, and probably will more in future, the advancement of practical and economically viable ways for the production of enzymes from sea organisms, instead of environmentally destructive large-scale natural collections of marine biomasess.
The marine environment has shown to be very interesting for the isolation of new glycosyl hydrolases. For these enzymes attention was focused mainly on the hydrolytic capability and selectivity since historically structural identification efforts faced the complexity of oligosaccharide and/or other natural products chemical structures. Glycosyl hydrolases from sea have proved to be valuable not only for their very selective hydrolytic potential but also in the synthesis of glycosidic linkage.
In the expanding world of carbohydrate-active enzymes the discovery of thousands of glycosyltransferase and glycosyl hydrolase open reading frames has been estimated for the next years. In this context biodiversity of the sea environment will surely play an important role as a source of biotechnologically relevant biocatalysts.
Abstract: The purification and characterisation of the alpha-glucosidase from the marine mollusc Aplysia fasciata are reported. Overall substrate specificity of the pure enzyme for both hydrolytic and transglycosylation reactions was studied. Remarkable characteristics of this enzyme are indicated by the results of the interesting survey of transglycosylation reactions reported: pyridoxine glucosylation, synthesis of chromophoric (pNP) di- and trisaccharides, glucosylation of cellobiose and sucrose. For these last two acceptors both the yields of reactions and the concentrations of products are comparable to those obtained using glycosyl transferases; in addition, synthesis of pyridoxine and chromophoric glycosides were still possible using a 1:1 ratio maltose:acceptor which is a very interesting characteristic from a synthetic point of view (effortless purification, productivity of each reaction batch, etc.).
Abstract: We recently succeeded in the identification and purification of an interesting marine exo-alpha-glucosidase (EC 3.2.1.20) from the anaspidean mollusc Aplysia fasciata. The enzyme was characterized by good transglycosylation activity toward different acceptors using maltose as donor. High-yielding enzymatic alpha-glycosylation of pyridoxine using this marine enzyme is reported here; the reaction has been optimized, reaching 80% molar yield of products (pyridoxine monoglucosides 24 g/l; pyridoxine isomaltoside 35 g/l). High selectivity toward the 5' position is observed for both monoglucoside and disaccharide formation. This is the first report describing the enzymatic production of pyridoxine isomaltoside.
Abstract: The marine ecosystem can be considered a rather unexplored source of biological material (e.g. natural substances with therapeutic activity) and can also be a surprising source of enzymes carrying new and interesting catalytic activities to be applied in biocatalysis. The use of glycosyl hydrolases from marine environments dates back to the end of the 1960s and was mainly focused on the development of sensitive and reliable hydrolytic methods for the analysis of sugar chains. As a result not all the benefits of a particular enzymatic activity have been investigated, especially regarding the transglycosylation potential of these enzymes for the synthesis of glycosidic bonds. In this review, the potential of marine sources will be demonstrated reporting on the few examples found in literature for the synthesis and hydrolysis of biologically relevant oligosaccharides catalyzed by glycosyl hydrolases of marine origin. Particular emphasis is given to the synthesis of glycosidic bonds, which is easy by the use of glycosyl hydrolases. Further aspects considered in this review are applications of these biocatalysts for vegetal waste treatment in recovering useful materials, for structural identification and for preparation of target materials from new purified polysaccharides, for the synthesis or modification of food-related compounds and for glycobiology related studies.
Abstract: The use of crude extract of the hepatopancreas of Aplysia fasciata, a large mollusc belonging to the order Anaspidea containing a beta-galactosidase activity, was reported for the synthesis of different galactosides. Good yields with polar acceptors and the uncommon beta-1-3 selectivity in the transgalactosylation reactions with most of the acceptors were observed. A beta-1-2 selectivity in the hydrolytic conditions was also observed and discussed.
Abstract: In pursuing research on the antiviral, interferon (IFN)-inducing tilorone congeners, a new series of fluoren-carboxyhydroxyesters has been prepared and biologically explored. These esters have subsequently been used as sugar acceptors in the enzymatic transglycosylation reaction using the 'retaining' beta-glycosidase from the archaeon Sulfolobus solfataricus (Ssbeta-Gly). Both aglycones (1-6) and corresponding beta-glucosides (beta-glu 1-beta-glu 6) have been screened for cytotoxicity, interferon-stimulating and antiviral properties against HSV-2. It was found that the addition of compounds beta-glu 5, beta-glu 6 and beta-glu 4 to HSV-2 infected U937 cells downregulates viral replication and triggers cells to release IFN-alpha/beta. Taken together, the results showed improved pharmacological profiles as a consequence of glycosylation. A molecular modelling study carried out on this series of compounds completed the structural characterisation of the novel compounds.
Abstract: Transglycosylation reactions (autocondensation of the substrate or transfer of the glycon donor moiety to different acceptors) with the hyperthermophilic glycosynthase from Sulfolobus solfataricus acting in dilute sodium formate buffer at pH 4.0 are reported; the use of 4-nitrophenyl beta-glucopyranoside as both donor and acceptor in the self-transfer reaction and a highly productive reaction with 1.1 M 2-nitrophenyl beta-glucopyranoside were possible. Interesting effects, governed by the anomeric configuration and lipophilicity of heteroacceptors, on the regioselectivity and yield of reactions were found for the first time with this enzyme and are discussed. The results demonstrate the unexplored synthetic potential of this glycosynthase; the tuning of the reaction conditions and the choice of different donors/acceptors can lead to products of applicative interest.
Abstract: A beta-D-mannosidase was purified to homogeneity from visceral mass extract of Aplysia fasciata a mollusc belonging to the order Anaspidea. The purified enzyme is a homodimer with a subunit mass of 130 kDa. Temperature and pH optima of this enzyme were 45 degrees C and 4.5, respectively. Substrate specificity tests revealed that the enzyme exerts only beta-D-mannosidase activity. The K(M) and V(max) values for p-nitrophenyl beta-D-mannopyranoside were determined to be 2.4 mM and 50.3 micromol min(-1)mg(-1), respectively. The catalytic efficiency of this beta-mannosidase (11,519 min(-1)) was significantly higher than those reported for beta-mannosidases from other sources. It was verified that this is an exo-acting glycosyl hydrolase with transglycosidase activity. When the enzyme was incubated in the presence of p-nitrophenyl beta-D-mannopyranoside, self-transfer of the mannosyl group was observed, and a 10-15% yield of a beta-1-4 disaccharide was obtained. When the reaction was performed in the presence of o-nitrophenyl alpha-D-2-deoxy-N-acetyl glucopyranoside in 3:1 molar ratio with respect to the p-nitrophenyl beta-D-mannopyranoside, two regioisomers (85:15, 12% yield) due to the beta-mannosylation of the heteroacceptor in 4 and in 6 positions were formed.
Abstract: The search for glycosyl hydrolytic activities from natural sources
and the investigation of their transglycosylation potential can provide
enzymes with new interesting catalytic activities for the synthesis of
oligosaccharides. We focused our attention on the sea hare Aplysia
fasciata Poiret, 1789, a common Mediterranean opistobranch mollusc,
describing the results on the presence of different glycosyl hydrolases
found in this organism. The activities of interest are present in the
visceral mass and in the hepatopancreas extracts which were highly
active with respect to those from other marine molluscs, namely an
exo-alpha-D-glucosidase activity and a beta-galactosidase, were
investigated. The former is able to produce panose in high percentage by
transglycosylation from maltose, or alpha-D-glucosides of alpha-methyl
mannopyranoside and 2-deoxyglucose using p-nitrophenyl
alpha-D-glucopyranoside as donor. The beta-galactosidase from
hepatopancreas produced in transgalactosylation reactions the
beta-Gal-(1-4)-GlcNAc as the most abundant product and Gal-Xyl
disaccharides of biological importance using p-nitrophenyl
beta-D-galactopyranoside as donor and GlcNAc or benzyl alpha-xyloside as
acceptors, respectively.
Abstract: A number of Penicillium isolates were recovered in association to Rhizoctonia solani strains pathogenic on tobacco and from soil on plates pre-colonized by the pathogen itself. Their antagonism toward R. solani AG-2-1 was evaluated in dual cultures in vitro. Inhibition of growth was evident to some extent in most pairings, while hyphal interactions referable to mycoparasitic relationships were not observed. However, the occurrence of plasmolysis and/or vacuolisation and the induction of monilioid cells were indicative of the release of bioactive compounds. Therefore, production of fungitoxic metabolites was tested by adding concentrated culture filtrates of each Penicillium isolate to the growth medium of R. solani. Complete and lasting inhibition was incited by culture filtrates of some isolates belonging to P. brevicompactum, P. expansum, and P. pinophilum. Three purified compounds, respectively mycophenolic acid, patulin and 3-O-methylfunicone, which were extracted from culture filtrates, were able to inhibit R. solani in vitro. Their production was also detected in dual cultures of the same Penicillium strains with R. solani prepared in sterilized soil and when the Penicillium strains were cultured directly on R. solani mycelium harvested from liquid cultures. The possible role of such metabolites in antagonism of the above-mentioned Penicillium species against R. solani is discussed.
Abstract: The use of the new glycosynthase Ta-beta-GlyE386G and of the already known Ss-beta-GlyE387G for the synthesis of interesting 4-methylumbelliferyl disaccharides and for the galactosylation of alpha- and beta-xylosides of 4-penten-1-ol is reported. The results show satisfactory yields of reaction in presence of low excesses of acceptors and demonstrated that the high activity of these enzymes at pH below neutrality is applicable in the transfer of glucose as well as of galactose from the preferred 2-NP-based donors.
Abstract: The synthesis of 2-deoxyglycosides and, for the first time, of
2-deoxygalactosides is reported using a thermophilic and thermostable
beta-glycosyl hydrolase from the archeon Sulfolobus solfataricus and
glucal or galactal as donors.
The yields observed with alkyl acceptors confirmed that the robustness
of the biocatalyst is of great help in designing practical syntheses of
pure beta-anomers of 2-deoxy derivatives of 4-penten-1-ol (obtained in
80% yield at 20 fold molar excess) and 3,4-dimethoxybenzyl alcohol
(obtained in 19% yield at 3.3 fold molar excess).
The attachment of 2-deoxyglyco units was performed on various
pyranosidic acceptors (p-nitrophenyl alpha-D-glucopyranoside,
o-nitrophenyl 2-deoxy-N-acetyl-beta-D-glucosamine and p-nitrophenyl
2-deoxy-N-acetyl-beta-D-glucosamine). At low molecular excesses of the
acceptors, satisfactory yields (20-40%) of chromophoric 2-deoxy di- and
trisaccharides were obtained.
The different regioselectivity of our enzyme with respect to mesophilic
counterparts reflects the importance of biodiversity in this field for
the construction of a library of different glycosidases with different
specificity.
Abstract: We have recently reported that a functional alpha-L-fucosidase could be expressed by a single insertional mutation in the region of overlap between the ORFs SSO11867 and SSO3060 of the hyperthermophilic Archaeon Sulfolobus solfataricus [Cobucci-Ponzano et al. J. Biol. Chem. (2003) 278, 14622-14631]. This enzyme, belonging to glycoside hydrolase family 29 (GH29), showed micromolar specificity for p-nitrophenyl-alpha-L-fucoside (pNp-Fuc) and promoted transfucosylation reactions by following a reaction mechanism in which the products retained the anomeric configuration of the substrate. The active site residues in GH29 enzymes are still unknown. We describe here the identification of the catalytic nucleophile of the reaction in the alpha-L-fucosidase from S. solfataricus by reactivation with sodium azide of the mutant Asp242Gly that shows a 10(3)-fold activity reduction on pNp-Fuc. The detailed stereochemical analysis of the fucosyl-azide produced by the mutant reactivated on pNp-Fuc revealed its inverted (beta-fucosyl azide) configuration compared with the substrate. This allows for the first time the unambiguous assignment of Asp242, and its homologous residues, as the nucleophilic catalytic residues of GH29 alpha-L-fucosidases. This is the first time that this approach is used for alpha-L-glycosidases, widening the applicability of this method.
Abstract: Carbohydrates serve as structural components and energy sources of
cells. More interestingly, however, these biomolecules are involved in a
variety of molecular recognition processes in intercellular
communication and signal transduction such as cell adhesion,
differentiation, development and regulation. For these reasons, great
interest has arisen in carbohydrate-based pharmaceuticals and on the
development of techniques for the analysis and synthesis of
oligosaccharides. In this respect, enzymes involved in carbohydrates
hydrolysis and modification are increasingly being utilised for the
bioconversion of sugars, for the synthesis of oligosaccharides with
potential application, and for the characterisation of carbohydrate
compounds of unknown structure.
In this review, the enzymology and the applications of three glycosyl
hydrolases from the archaeon Sulfolobus solfataricus are described. In
particular, we focus on the enzymological properties of a
beta-glycosidase, an alpha-xylosidase, and an alpha-fucosidase; their
exploitation in oligosaccharides synthesis will be also described.
Abstract: We have previously shown that the hyperthermophilic glycosynthase from Sulfolobus solfataricus (Ssbeta-glyE387G) can promote the synthesis of branched oligosaccharides from activated beta-glycosides, at pH 6.5, in the presence of 2 M sodium formate as an external nucleophile. In an effort to increase the synthetic potential of hyperthermophilic glycosynthases, we report a new method to reactivate the Ssbeta-glyE387G glycosynthase and two novel mutants in the nucleophile of the beta-glycosidases from the hyperthermophilic Archaea Thermosphaera aggregans (Tabeta-gly) and Pyrococcus furiosus (CelB). We describe here that, at pH 3.0 and low concentrations of sodium formate buffer, the three hyperthermophilic glycosynthases show k(cat) values similar to those of the wild-type enzymes and 17-fold higher than those observed at the usual reactivation conditions in 2 M sodium formate at pH 6.5. Moreover, at acidic pH the three reactivated mutants have wide substrate specificity and improved efficiency in the synthetic reaction. The data reported suggest that the reactivation conditions modify the ionization state of the residue acting as an acid/base catalyst. This new reactivation method can be of general applicability on hyperthermophilic glycosynthases whose intrinsic stability allows their exploitation as synthetic tools at low pH.
Abstract: The analysis of the complete genome of the thermoacidophilic Archaeon Sulfolobus solfataricus revealed two open reading frames (ORF), named SSO11867 and SSO3060, interrupted by a -1 frameshift and encoding for the N- and the C-terminal fragments, respectively, of an alpha-l-fucosidase. We report here that these ORFs are actively transcribed in vivo, and we confirm the presence of the -1 frameshift between them at the cDNA level, explaining why we could not find alpha-fucosidase activity in S. solfataricus extracts. Detailed analysis of the region of overlap between the two ORFs revealed the presence of the consensus sequence for a programmed -1 frameshifting. Two specific mutations, mimicking this regulative frameshifting event, allow the expression, in Escherichia coli, of a fully active thermophilic and thermostable alpha-l-fucosidase (EC ) with micromolar substrate specificity and showing transfucosylating activity. The analysis of the fucosylated products of this enzyme allows, for the first time, assigning a retaining reaction mechanism to family 29 of glycosyl hydrolases. The presence of an alpha-fucosidase putatively regulated by programmed -1 frameshifting is intriguing both with respect to the regulation of gene expression and, in post-genomic era, for the definition of gene function in Archaea.
Abstract: Various peracetylated oligosaccharides (di- to hepta-) are subjected
to regioselective hydrolysis by Aspergillus niger lipase hydrolyzing
1-O-acetyl group. Molecular weight, anomeric and interglycosidic linkage
configurations play a significant role in the hydrolysis reaction.
Useful 1-O-deprotected material can be easily prepared for further
chemical elaboration.
Abstract: The mutation of putative acid/base and nucleophile of the active
sites of retaining glycosyl hydrolases, together with kinetic analysis
of the mutants, and stereochemical identification of products lead to
useful information for the understanding of the reaction mechanism of
these enzymes. This was the preliminary and fundamental step toward the
preparation of new enzymatic activities called glycosynthases. Direct
exploitation of this information has been possible, leading to the
design of four new enzymes for oligosaccharides synthesis. The interest
for these biocatalysts rises from the fact that the yield of the
reaction can be increased and selectivity can be interpreted as key
characteristic of the transfer reaction instead of a balance of
hydrolytic and transferring pathways followed either by substrates and
products. These new biocatalysts possess different specificities and are
promising and useful tools in the construction of oligosaccharide
molecules of great biological interest. This short review focused the
attention on different glycosynthases obtained from four glycosyl
hydrolases highlighting on the preparation and development of these new
enzymes.
Abstract: The hyperthermophilic number of family 1 of the glycosyl hydrolases,
the beta -glycosidase from the archaeon Sulfolobus solfataricus (Ss beta
-gly), has been used for,In efficient synthesis of beta
-2-deoxyglucosides and for stereochemical studies of the reactions of
glucal in the presence of alkyl and pyranosidic acceptors. Protonation
of the double bond of glucal resulting in the equatorially disposed
proton was observed and an indication of the protonating amino acid in
the active site vas obtained by the use of a mutant enzyme. The
regioselectivity in the formation of beta -2-deoxyglucosides of
pyranosidic acceptors is different from that reported for mesophilic
biocatalysts.
Abstract: The importance of carbohydrates in a variety of biological functions is the reason that interest has recently increased in these compounds as possible components of therapeutic agents. Thus, the need for a technique allowing the easy synthesis of carbohydrates and glucoconjugates is an emerging challenge for chemists and biologists involved in this field. At present, enzymatic synthesis has resulted in the most promising approach for the production of complex oligosaccharides. In this respect, the enzymological characteristics of the catalysts, in term of regioselectivity, substrate specificity, and operational stability, are of fundamental importance to improve the yields of the process and to widen the repertoire of the available products. Here, two methods of oligosaccharide synthesis performed by a glycosynthase and by an alpha-xylosidase from the hyperthermophilic archaeon Sulfolobus solfataricus are briefly reviewed. The approaches used and the biodiversity of the catalysts together are key features for their possible utilization in the synthesis of oligosaccharides.
Abstract: The first, recently identified, archaeal alpha-xylosidase from Sulfolobus solfataricus (XylS) shows high specificity for hydrolysis of isoprimeverose [alpha-D-xylopyranosyl-(1,6)-D-glucopyranose, (X)], the p-nitrophenyl-beta derivative of isoprimeverose, and xyloglucan oligosaccharides and has transxylosidic activity, forming, in a retaining mode, interesting alpha-xylosides. This article describes the synthesis of isoprimeverose, the disaccharidic repeating unit of xyloglucan, of the p-nitrophenyl-beta derivative of isoprimeverose, and of a trisaccharide based on isoprimeverose that is one of the trisaccharidic building blocks of xyloglucan. A substrate structure-activity relationship is recognized for both the hydrolysis and the synthesis reactions of XylS, it being a biocatalyst (i) active hydrolytically only on X-ending substrates liberating a xylose molecule and (ii) capable of transferring xylose only on the nonreducing end glucose of p-nitrophenyl-(PNP)-beta-D-cellobioside. The compounds synthesized by this enzyme are a starting point for enzymological studies of other new enzymes (i.e., xyloglucanases) for which suitable substrates are difficult to synthesize. This study also allows us to define the chemical characteristics of the xylose-transferring activity of this new archaeal enzyme, contributing to building up a library of different glycosidases with high specific selectivity for oligosaccharide synthesis.
Abstract: We here report the first molecular characterization of an alpha-xylosidase (XylS) from an Archaeon. Sulfolobus solfataricus is able to grow at temperatures higher than 80 degrees C on several carbohydrates at acidic pH. The isolated xylS gene encodes a monomeric enzyme homologous to alpha-glucosidases, alpha-xylosidases, glucoamylases and sucrase-isomaltases of the glycosyl hydrolase family 31. xylS belongs to a cluster of four genes in the S. solfataricus genome, including a beta-glycosidase, an hypothetical membrane protein homologous to the major facilitator superfamily of transporters, and an open reading frame of unknown function. The alpha-xylosidase was overexpressed in Escherichia coli showing optimal activity at 90 degrees C and a half-life at this temperature of 38 h. The purified enzyme follows a retaining mechanism of substrate hydrolysis, showing high hydrolytic activity on the disaccharide isoprimeverose and catalyzing the release of xylose from xyloglucan oligosaccharides. Synergy is observed in the concerted in vitro hydrolysis of xyloglucan oligosaccharides by the alpha-xylosidase and the beta-glycosidase from S. solfataricus. The analysis of the total S. solfataricus RNA revealed that all the genes of the cluster are actively transcribed and that xylS and orf3 genes are cotranscribed.
Abstract: A novel thermophilic glycosynthase that effects branching glycosylation has been obtained by mutation of the nucleophile in the active site of the glycosidase from Sulfolobus solfataricus. Two methods for the use of this mutant are reported.
Abstract: Six cyanobacteria representative of families able to form
heterocysts, other than Nostocaceae, have been examined for their
heterocyst glycolipids (HGs) content. While the HGs found in Nostocaceae
are dominated by the presence of triols and of the corresponding C-3
ketones as aglycones, those found in the ;ther families reported here
are mainly characterized by tetrols and by the corresponding ketones at
the omega-3 position. The latter were found in four out of the six
species examined, Scytonema hofmanni, Calothrix desertica,
Chlorogloeopsis fritschii and Fischerella muscicola. Triols and the
corresponding ketone glycosides were found only in a Microchaete sp. and
in Tolyporhrix tenuis, the latter showing the most complex array of HGs
found so far. In Calotrhix desertica and Chlorogloeopsis fritschii,
a-mannosides have been found for the first time. The relevance of these
data as a chemotaxonomic tool is discussed.
Abstract: The beta-glycosidase from the hyperthermophilic Archaeon Sulfolobus solfataricus hydrolyzes beta-glycosides following a retaining mechanism based upon the action of two amino acids: Glu387, which acts as the nucleophile of the reaction, and Glu206, which acts as the general acid/base catalyst. The activities of inactive mutants of the catalytic nucleophile Glu387Ala/Gly were restored by externally added nucleophiles. Sodium azide and sodium formate were used as external nucleophiles and the products of their reaction were characterized. Glu387Ala/Gly mutants were reactivated with 2, 4-DNP-beta-Glc substrate and the Glu387Gly mutant showed recovered activity, with the same nucleophiles, also on 2-NP-beta-Glc. The reaction catalyzed by the Glu387Gly mutant proceeded differently depending on the type of externally added nucleophile. Sodium azide restored the catalytic activity of the mutant by attacking the alpha-side of the anomeric carbon of the substrates, thereby yielding an inverting glycosidase. Sodium formate promoted the opposite behavior (retaining) in the mutant, producing 3-O-beta-linked disaccharide derivative of the substrates. A possible role of sodium formate as a biomimicking agent in replacing the natural nucleophile Glu387 is also discussed.
Abstract: The chemoenzymatic synthesis of a group of naturally occurring
cyclohexylethanoids, rengyoside-A, -B and isorengyoside, has been
performed by enzymatic glucosidation of their chemically synthesized
aglycones, rengyol, rengyoxide and isorengyol.
Abstract: Glycosidase catalysed reversed hydrolysis and transglycosidations in
dry media under focused microwave irradiation and classical heating
conditions are described.
Abstract: The synthesis of (R)- and (S)-3-(4-hydroxyphenyl)-1-methylpropyl-beta-D-glucopyranosides has been
achieved by two enzymatic steps, namely an oxide-reduction step
involving alcohol dehydrogenases from different origin for the
preparation of both aglycones in enantiomeric pure form, and a
transglycosidation step involving a thermophilic beta-glucosidase from
the archaeon Sulfolobus solfataricus.
Abstract: The heterocyst glycolipids (HGs),which have been reported to take
part in the protection against the penetration of O-2 in the specialized
cyanobacterial cells capable of nitrogen fixation, have been isolated
from Anabaena sphaerica, Anabaena cylindrica Lemm., Anabaena sp. 7120,
Anabaena sp. WSAF (new) and Nostoc linckia. In all the species, but one,
the HGs are alpha-glucosides of polyhydroxylated aglycones of different
chain length, some of which were previously described. In Anabaena sp.
WSAF only alpha-galactosides and a beta-glucoside of the same aglycones
have been isolated.
Abstract: The synthesis of glucosyl derivatives of 2,3-oxirane-dimethanol has
been accomplished chemo-enzymatically by the use of glycosidases and
lipases. High yields using thermophilic glycosidases coupled to the
diastereoselectivity and regioselectivity of lipases lead to selectively
deprotected products, useful materials for further elaboration.
Abstract: beta-Glycosidases catalyze the synthesis of glycosides when a nucleophilic acceptor other than water is present in the reaction medium. We describe the enzymatyc synthesis of 2-beta-D-galactopyranosyloxyethyl methacrylate (GalEMA) starting from 2-hydroxyethyl methacrylate (HEMA) and p-nitrophenyl-beta-D-galactopyranoside (pNPG) using a beta-glycosidase activity present in the thermophilic archaeon Sulfolobus solfataricus. This thermophilic enzyme catalyzes the transfer of the galactopyranosyl unit from pNPG to the HEMA hydroxyl group by the formation of a new beta-glycosidic bond. The conditions of the biocatalytic system have been optimized to obtain high yield of GalEMA. (c) 1996 John Wiley & Sons, Inc.
Abstract: Diastereomeric O-beta-glucosides at the primary carbons of glycerol
and erythritol have been subjected as their peracetates to enzymatic
hydrolysis by PFL. Only acetyl groups of the aglycones are hydrolyzed,
the diastereoselectivity being very high on the erythritol derivatives
and fair on the glycerol derivatives. The chemical synthesis of
(2S,3R)-1-(O-beta-D-glucopyranosyl)-butane-2,3,4-triol heptaacetate is
also reported.
Abstract: Incorporation of [1-C-13] acetate into the heterocyst glycolipids of
the cyanobacterium Anabaena cylindrica indicated that (i) the long chain
of the aglycones originates from a de novo biosynthesis and not by
elongation of a preformed shorter molecule and that (ii) there is no
apparent interconversion between the hydroxyketone and alcohol glucosides.
Abstract: The previously proposed structures for the heterocyst glycolipids of
the nitrogen-fixing cyanobacterium Anabaena cylindrica have been
revised. C-13-enriched acetonides have been utilized for the
determination of the relative stereochemistry of the 1,3-diol moiety in
the aglycone of two glycolipids. The same glycolipids, which are thought
to protect the nitrogenase from O-2, have also been detected in A.
torulosa.
Abstract:
A comparison of different systems for the beta-glycosidase-catalyzed
synthesis of 3,4'-dihydroxypropiophenone 3-O-beta-D-glucoside is
reported including various enzymatic sources and different reaction
conditions. The best yield was obtained using thermophilic
beta-glycosidase from Sulfolobus solfataricus.
Abstract: The use of commercially available mesophilic glycosidases in the
enzymatic synthesis of glycosides of different types is a well
established method suffering from some drawbacks such as a poor yield.
Substrates with three or four hydroxyl groups have been subjected to
enzymatic glucosylation using crude homogenate of the thermophilic
archaeon Sulfolobus solfataricus containing a beta-glycosidase activity
able to transfer glucose, galactose and fucose from different donors.
The stereochemistry of this reaction was interpreted in terms of
interaction with a possible ''glucose'' active site of the enzyme. In
addition masked or protected derivatives of tetritols and some simple
unsaturated alcohols were glycosylated yielding glycosides in yields
very competitive with those obtained using mesophilic enzymes, examples
of further chemical manipulation of these compounds were reported. When
using a scarce amount of acceptor, a reasonable amount of products could
be obtained by adding different aliquots of donor at time intervals.
Abstract: The lipids extracted from the membrane of the thermophilic archaeobacterium Sulfolobus solfataricus have an unusual bipolar structure. Each molecule is formed by two isoprenoid chains (with up to four cyclopentane groups per chain) ether-linked at both ends to glycerol or nonitol groups. These groups can be variably substituted, mainly with complex sugars. Fluorescence resonance energy transfer, aqueous contents mixing and calcein release assays were employed to assess whether bipolar lipid vesicles were able to undergo a calcium-induced fusion process. The possibility of getting fusion depends strongly on the phase behaviour of the lipids. With vesicles formed by the natural polar lipid extract (PLE), a mixture showing a complex polymorphic behaviour, the fusion process was observed above the temperature T congruent to 60 degrees C at 15 mM Ca2+. By contrast, no fusion was observed in vesicles of P2, a fraction displaying only the lamellar phase. A dramatic change of the fusion process was observed when egg PC or P2 was added to PLE. In this case only lipid mixing, but not a real fusion process occurred at T > or = 60 degrees C. The dependence of such a process on ionic conditions has also been studied. Additional experiments involving surface tension measurements on monolayers have been performed to assess the importance of a surface tension increase to get fusion. In contrast to other monopolar lipid systems, no detectable change in surface tension has been observed in our bipolar lipids even in cases in which the fusion process is present.
Abstract: The Sulfolobus solfataricus beta-glycosidase (S beta gly) is a
thermostable and thermophilic glycosyl-hydrolase with broad substrate
specificity. The enzyme hydrolizes beta-D-gluco-, fuco-, and
galactosides, and a large number of beta-linked glycoside dimers and
oligomers, linked beta 1-3, beta 1-4, and beta 1-6, It is able to
hydrolize oligosaccharides with up to 5 glucose residues. Furthermore,
it is also able to promote transglycosylation reactions. The
corresponding gene has been cloned and overexpressed both in yeast and
Escherichia coli. Based on sequence acid functional data, the S beta gly
has been assigned to the so-called EGA family of glycosyl-hydrolases,
including beta-glycosidases, beta-galactosidases and
phosho-beta-galactosidases from mesophilic and thermophilic organisms of
the three domains. The S beta gly has been crystallized and the
resolution of its structure is in progress. Because of its special
properties, the enzymes has considerable biotechnological potential.
Abstract: A facile preparation of C-13-enriched acetonides for the
determination of the relative stereochemistry of 1,3-diols is described.
The method allows inter alia the detection of minor diastereomers
arising from the synthesis of 1,3-diols.
Abstract: Various yeast and mould strains were tested in the reduction of
alpha-acetyl-gamma-butyrolactone.
syn-(3R,1'R)-alpha-(Hydroxyethyl)-gamma-butyrolactone 2 and
anti-(3R,1'S) isomer 3 were obtained enantiomerically pure. Good
enantiomeric excesses are described for the corresponding enantiomers.
Abstract: Lipid composition has proved particularly useful in the taxonomy of
microorganisms. The most spectacular grouping that can be made among
prokaryotes, on the basis of lipid analysis, is the Archaea whose
membranes are based on isopranoid glycerol ether lipids. This
presentation is meant to give an updated general survey of the
structural features of lipids of Archaea and the significance of lipid
composition in archaeal taxonomy.
Abstract: The stereochemistry of aleppotrioloside, a naturally occurring
glucoside, has been determined as
(3S)-3-[(1S)-1-hydroxyethyl]-4-methyl-1,4-pentanediol-1-O-beta-D-glucopy
ranoside by its synthesis based on two key enzymatic reactions:
Pseudomonas fluorescens lipase (PFL) catalyzed enantioselective
resolution of an aglycone precursor and transglucosylation of the
aglycone by thermophilic beta-glycosidase from Sulfolobus solfataricus.
Abstract: Enzymatic synthesis of different beta-D-glycosides was obtained
using as biocatalyst immobilized cells, crude homogenate, and
homogeneous native and recombinat beta-glycosidase activity of the
thermophilic archaeon Sulfolobus solfataricus. In particular our
investigation was concerned with the selectivity in the glycosylation of
hydroxybenzyl alcohols, salicin, 1,2-propanediol, and more complex
polyols as well as the use of immobilized cells for the synthesis of
hexyl-beta-D-glucoside. The aromatic glucosides obtained by
enzyme-catalyzed transglucosylation were used for kinetic studies of
purified Sulfolobus solfataricus enzyme in the hydrolysis reaction.
Abstract: Sulfolobus solfataricus, a thermoacidophilic archaeon, has been
utilized as catalyst for several enzymatic transformations such as
carbonyl-alcohol oxidoreductions, synthesis of glycosides, kinetic
resolution of aminoacid esters, etc. All these reactions have been
carried out at 75-degrees-C in accordance with the thermophilic nature
of the enzymes present in the organisms.
Abstract:
The heterocyst glycolipids of the cyanobacterium Cyanospira rippkae
have been isolated and their structures established to be
1-()-alpha-D-glucopyranosyl)-3R,27R-octacosanediol and
1-(O-alpha-D-glucopyranosyl)-27-keto-3R-octacosanol by spectroscopic and
chemical means. The absolute configuration at the two stereogenic
centres in the aglycone moiety of the former compound has been
established in a single step by Mosher's method on the triol derivative.
The akinetes of C. rippkae contain the same glycolipids.
Abstract: A novel rod-shaped hyperthermophilic archaeum has been isolated from a boiling marine water hole at Maronti Beach, Ischia, Italy. It grew optimally at 100 degrees C and pH 7.0 by aerobic respiration as well as by dissimilatory nitrate reduction, forming dinitrogen as a final product. Organic and inorganic compounds served as substrates during aerobic and anaerobic respiration. Growth was inhibited by elemental sulfur. The cell wall was composed of a surface layer of hexameric protein complexes arranged on a p6 lattice. The core lipids consisted mainly of glycerol diphytanyl glycerol tetraethers with various degrees of cyclization. The G+C content was 52 mol%. The new isolate resembled members of the genera Thermoproteus and Pyrobaculum by its ability to form characteristic terminal spherical bodies ("golf clubs"). On the basis of its 16S rRNA sequence, the new isolate exhibited a close relationship to the genus Pyrobaculum. It is described as a new species, which we name Pyrobaculum aerophilum (type strain: IM2; DSM 7523).
Abstract: The structural elucidation of the polar lipids in Halobacterium trapanicum is reported with particular emphasis on a new sulfated disaccharide derivative of 2,3-di-O-phytanyl-sn-glycerol. The full structural designation of this glycolipid is 2,3-di-O-phytanyl-1-O- (mannopyranosyl-(2-sulfate)-alpha-D-1-2-glucopyranosyl-alpha-D)-sn-glyce rol. The value of glycolipid structures in the taxonomy of halophilic Archaea is also discussed.
Abstract: Five non-motile Gram-positive cocci were isolated from saline soils
located in geothermal regions of the Antarctic continent. The organisms
were extremely halotolerant growing between 0 and 4.2 M NaCl. On the
basis of the results of phenotypic characterizations, lipid and quinone
analyses, and the amino acid composition of the cell wall the isolates
have been assigned to the genus Micrococcus.
Abstract: Two novel glycolipids with a very rare alpha(1-->4) diglucosyl structure have been isolated from the thermophilic bacterium Thermotoga maritima. The structures of these compounds, on the basis of chemical procedures and spectroscopic studies (FAB-MS and NMR), were shown to be: 1(3),2-dipalmitoyl-3(1)-[glucopyranosyl-(6-decanoyl)-alpha-D-(1-->4)- glucopyranosyl-alpha-D]-glycerol (Glycolipid 1) and 1(3),2-dipalmitoyl-3(1)-[glucopyranosyl-alpha-D-(1-->4)-glucopyranosyl- alpha-D]-glycerol (Glycolipid 2).
Abstract: The ''heterocyst glycolipids'' have been reported to take part in
the protection of the specialized cyanobacterial cells capable of N2
fixation against the penetration Of O2. For the first time such
glycolipids have been isolated in a pure form from a cyanobacterium,
Nodularia harveyana, and their structures have been established by
spectroscopic and chemical means to be
1-(O-alpha-D-glucopyranosyl)-3R,25R-hexacosanediol,
1-(O-alpha-D-glucopyranosyl)-3S,25R-hexacosanediol and
1-(O-alpha-D-glucopyranosyl)-3-keto-25R-hexacosanol.
Abstract: Core and complex lipids of several new hyperthermophilic archaeal
isolates were analyzed. The organisms belong to the Sulfolobales,
Arcbaeoglobus, Pyrobaculum, and Metbanococcus. A detailed structural
investigation of complex lipids of Pyrobaculum species is reported. The
different lipid structures are of help for a rapid and simple
phylogenetic classification of the new isolates. They are in agreement
with the classification based on other features.
Abstract: The effects of growth conditions on the quinone composition of the
thermophilic archaeon (archaebacterium, Woese et al., 1990) Sulfolobus
solfataricus were investigated. The total quinone content proved quite
constant over all the variations of physiological parameters with the
sole exception of the change of oxygen supply.
The effects of changes in temperature, medium or aeration on quinone
pattern were demonstrated.
Abstract: From hot marine sediments (depth: 106 m) at the Kolbeinsey Ridge,
Iceland, novel bacterial hyperthermophiles were isolated. Cells were
Gram-negative highly motile rods exhibiting a complex envelope
consisting of murein, an outer membrane and a surface protein layer.
Growth occurred between 67 and 95-degrees-C (opt.: 85-degrees-C; 75 min doubling time), pH 5.4 and 7.5 (opt.: pH 6.8), and 1 to 5% NaCl (opt.: 3% NaCl). The novel isolates were strict chemolithoautotrophs. They used molecular hydrogen, thiosulfate and elemental sulfur as electron donors
and oxygen (low concentrations) and nitrate as electron acceptors.
During growth, sulfuric acid was formed from S-degrees and thiosulfate.
In the late logarithmic growth phase, H2S was formed from S-degrees and
H-2. The core lipids consisted mainly of alkyl ethers of glycerol. The
GC content of the DNA was 40 mol%. By 16S rRNA comparisons, the new
isolates did not belong to any of the phyla known and represent the
deepest phylogenetic branch-off within the Bacteria domain. Based on the
physiological and molecular properties of the new isolates, we describe
here a new genus, which we name Aquifex (the "water-maker"). The type
species is Aquifex pyrophilus (type strain: Kol5a; DSM 6858).
Abstract: Crude homogenate of thermophilic archaebacterium Sulfolobus
solfataricus, possessing a beta-glycosidase, has been used to synthesize
different alkyl beta-D-glycosides starting from phenyl beta-D-glucoside,
phenyl beta-D-galactoside and lactose as carbohydrate donors. High
product yield (95% with respect to the carbohydrate donor) of octyl
beta-D-glucoside has been obtained in a two-phase system containing 5%
of water. The enantioselection for the galactosyl transfer to the
secondary hydroxyl group of propane-1,2-diol is higher than that found
using beta-galactosidase from E. coli.
Abstract: A rapid procedure for plating the thermo-acidophilic archaebacterium
Sulfolobus solfataricus using Gerlite as a gelling agent is reported.
The technique uses a clear, single layer in which the colonies are grown.
Abstract: A novel group of hyperthermophilic rod-shaped motile methanogens was
isolated from a hydrothermally heated deep sea sediment (Guaymas Basin,
Gulf of California) and from a shallow marine hydrothermal system
(Kolbeinsey ridge, Iceland). They grew between 84 and 110-degrees-C
(opt: 98-degrees-C) and from 0.2% to 4% NaCl (opt. 2%) and pH 5.5 to 7
(opt: 6.5). The isolates were obligate chemolithoautotrophes using H-2
CO2 as energy and carbon sources. In the presence of sulfur, H2S was
formed and cells tended to lyse. The cell wall consisted of a new type
of pseudomurein containing ornithin in addition to lysine and no
N-acetylglucosamine. The pseudomurein layer was covered by a
detergent-sensitive protein surface layer. The core lipid consisted
exclusively of phytanyl diether. The GC content of the DNA was 60 mol%.
By 16S rRNA comparisons the new organisms were not related to any of the
three methanogenic lineages. Based on the physiological and molecular
properties of the new isolates, we describe here a new genus, which we
name Methanopyrus (the "methane fire"). The type species is Methanopyrus
kandleri (type strain: AV19; DSM 6324).
Abstract: The thermophilic archaebacterium S. solfataricus converted Et 4-chloroacetoacetate to Et (R)-4-chloro-3-hydroxybutanoate in 97% yield, with 98% enantiomeric excess. Ten g wet cells were suspended in 100 mL pH 5 acetate buffer contg. 5 mL propan-2-ol and 1 g (60 mM) substrate. After 24 h at 30ï°, the supernatant was extd. with ether and the product purified by HPLC. Concns. of substrate >60 mM led to decreased yields.
Abstract: Five isolates of thermophilic bacteria, capable of growing at
65-degrees-C, were obtained from samples of soil collected from the
Cryptogam Ridge (Mount Melbourne) and in the area north of Edmonson
Point, Northern Victoria Land, Antarctica. Electron microscopy,
morphological and physiological properties, lipid analyses and GC
content of the isolates are described in this paper. On the basis of the
results presented the organisms belong to the eubacterial domain.
Abstract: Stereochemistry of beta-glycosyl transfer from phenyl
beta-D-galactoside, lactose, and phenyl beta-D-glucoside to various
1,2-, 1,3-, and 1,4-diols, a secondary-tertiary diol, a cyclic diol, and
a racemic alcohol has been studied using beta-glycosidase activity in a
crude preparation from the thermophilic archaebacterium Sulfolobus
solfataricus. Good enantioselection for the galactosyl transfer to the
secondary hydroxy group of different 1,2-diols has been observed. Good
yields in comparison with enzymes from other sources, and results
concerning the reaction's regioselectivity, have also been reported.
Abstract: On the basis of three isolates (strains FC6T [T = type strain], FC4,
and RG1) of extremely thermophilic chemolithoautotrophic archaebacteria
obtained from solfataric fields on Sao Miguel Island, Azores, the new
genus Stygiolobus is described. These isolates grow obligately
chemolithotrophically by reduction of S0 with H-2 (H-2-S0 lithotrophy)
and are the first strictly anaerobic members of the order Sulfolobales.
With a DNA G + C content of 38 mol%, the Stygiolobus isolates resemble
Sulfolobus spp., which, however, are faculatively organotrophic and
aerobic S0 oxidizers. The new isolates are also distinct from Acidianus
spp., which resemble Stygiolobus by growing by H-2-S0 lithotrophy.
However, Acidianus spp. can also grow aerobically by S0 oxidation and
have G + C contents of 31 mol%. At this time, only one species of the
genus Stygiolobus is known, Stygiolobus azoricus sp. nov.; the type
strain of S. azoricus is strain FC6 (= DSM 6296).
Abstract: The method of resting cells has been of interest in the development of biocatalysts applied to organic reactions.This article deals with the use of resting cells of a thermophilic archaebacterium Sulfolobus solfataricus, in the asymmetric reduction of acyclic, cyclic, and aromatic ketones. The system allows the continuous regeneration of endogenous coenzyme with the coupled substrate approach. The results indicate that the direction of hydride attack was equatorial on the re face of the carbonyl group of substrates producing (S)-alcohols with a good optical yield. A convenient system for the reuse of resting cells has been set out to synthesize (S)-alcohols on a preparative scale.
Abstract: Lipids from the archaebacterium Sulfolobus solfataricus are based on 72-membered macrocyclic tetraethers made up from two C40 diol units differently cyclized and either two glycerol moieties or one glycerol moiety and a unique branched-chain nonitol named calditol (glycerodialkylnonitol tetraethers, GDNTs). To elucidate the biosynthesis of calditol and related tetraethers, labelled precursors, [U-14C,1(3)-3H]glycerol, [U-14C,2-3H]glycerol, D-[1-14C,6-3H]glucose, D-[6-14C,1-3H]glucose, D-[1-14C,2-3H]glucose, D-[1-14C,6-3H]fructose and D-[1-14C]galactose, were fed to S. solfataricus. Without regard to stereochemistry or phosphorylation, incorporation experiments provided evidence that the biosynthesis of calditol occurs via an aldolic condensation between dihydroxyacetone and fructose, through a 2-oxo derivative of calditol as an intermediate. The latter is in turn reduced and then alkylated to yield the GDNTs. The biogenetic origins of both glycerol and C40 isoprenoid moieties of GDNTs are also discussed.
Abstract: This paper describes the determination of stereospecificity of hydride transfer reaction of an alcohol dehydrogenase isolated from the archaebacterium Sulfolobus solfataricus. The 1H-NMR and EI-MS data indicate that the enzyme transfers the pro-R hydrogen from coenzyme to substrate and is therefore an A-specific dehydrogenase.
Abstract: Cells of S. solfataricus trapped in Ca alginate were used to hydrolyze the Me, Et, and Bu esters of alanine, methionine, serine, valine, and phenylalanine with useful stereoselectivity for the L-enantiomers at different temps.
Abstract: Starch is hydrolyzed by Sulfolobus solfataricus strain MT-4 immobilized in Ca alginate beads at 70ï° to give glucose and trehalose as the major products with no formation of maltose, maltotriose, and maltotetraose.
Abstract: Three new strains of eubacterial hyperthermophiles were isolated from continental solfataric springs at Lac Abbe (Djibouti, Africa). Due to their morphol., lipids, and RNA polymerases they belong to the genus Thermotoga. Strains LA4 and LA10 are closely related to T. neopolitana found up to now only in the marine environment. Strain LA3 differs from T. maritima and T. neapolitana in significant physiol. and mol. properties. It is described as the new species T. thermarum.
Abstract: Examples of halophilic archaebacteria contain low levels of 1-20 mM tri-Me glycine (glycine betaine). In disrupted cell prepns., the glycine betaine is assocd. with the membrane fraction and is not detectable in cell supernatants. Cells of Natronococcus occultus grown in different salt concns. show an increase in cell-assocd. glycine betaine along with an increase in the ratio of phosphatidylglycerophosphate to phosphatidylglycerol in the cell membrane.
Abstract: The lipid compn. of two archaebacteria belonging to Thermococcales has been examd. The major complex lipid present in the Pyrococcus genus is 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-1'-myo-L-inositol (90% of total lipids). In the AN1 isolate, this lipid (40% of total lipids) and a novel 2,3-di-O-phytanyl-sn-glycero-1-(ï¡-D-glucopyranosyl 3-phosphate) (45%) were identified.
Abstract: Lipids from D. ambivalens grown under aerobic and anaerobic conditions were analyzed and compared with those of Sulfolobus solfataricus, a related microorganism. The ether lipids of aerobically anaerobically grown D. ambivalens, as well as those of S. solfataricus, had the same general features except for the degree of cyclization of the C40 isopranic chains. The quinone content of D. ambivalens was strongly affected by growth conditions. Aerobically grown cells contained caldariellaquinone, 6-(3,7,11,15,19,23-hexamethyltetracosyl)-5-methylthiobenzo[b]thiophen-4,7-quinone (83% of the quinone pool), sulfolobusquinone, 6-(3,7,11,15,19,23-hexamethyltetracosyl)-5-methylbenzo[b]thiophen-4,7-quinone (16%) and the tricyclic quinone benzo[1,2-b; 4,5-b']dithiophen-4,8-quinone (trace amts.). In anaerobically grown D. ambivalens sulfolobusquinone was the only quinone present.
Abstract: Polar lipid ext. from a halophilic archaebacterium isolated from a salt mine contains C20,C20 diether forms of phosphatidylglycerol (PG), phosphatidylglycerophosphate (PGP), phosphatidylglycerosulfate (PGS), 2,3-di-O-phytanyl-1-O-[mannopyranosyl-(6-sulfate)-ï¡-D-1 ï® 2-glucopyranosyl-ï¡-D]-sn-glycerol (S-DGD-1) and an analog of phosphatidic acid (2,3-di-O-phytanyl-sn-glycero-1-phosphate).
Abstract: Polar lipid ext. from the haloalkaliphilic archaebacterium N. occultus contains a new type of phospholipid. Spectroscopic methods establish the structure as a phosphatidylglycerol phosphate deriv. with a cyclic phosphate (2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycero-1',2'-cyclic phosphate). A 2-O-sesterterpanyl-3-O-phytanyl (C25,C20) glycerol diether form of the novel phospholipid is also present.
Abstract: Archaebacterial glycerol-bisdiphytanyl-glycerol tetraether core lipids containing from one to eight cyclopentane rings could be resolved from each other and from the parent uncyclized C40, C40 lipid by TLC. The core lipids of examples from the genera Methanobacterium, Methanobrevibacter, Methanogenium and Methanoplanus did not contain cyclized forms of glycerol-bisdiphytanyl-glycerol tetraethers, whereas the core lipids of Methanosarcina barkeri contained glycerol-bisdiphytanyl-glycerol tetraethers with from one to three cyclopentane rings in each C40 isopranoid chain.
Abstract: The polar lipids of the halophilic archaebacterium H. saccharovorum have been analyzed by spectroscopic methods, including 13C NMR, to establish structural detail. C20, C20 diether forms of phosphatidylglycerol (PG), phosphatidylglycerol phosphate (PGP), and phosphatidylglycerol sulfate (PGS) and the major polar lipids, together with the sulfated diglycosyl diether, 2,3-di-O-phytanyl-1-O-[mannopyranosyl-(6-sulfate)-ï¡-D-1ï®2-glucopyranosyl-ï¡-D]-sn-glycerol (S-DGD-1) characteristic of members of the genus Haloferax.
Abstract: The lipids of the thermophilic anaerobic eubacterium T. maritima are mainly based on n-C14, n-C16, and n-C18 fatty acids, 15,16-dimethyltriacontanedioic acid (diabolic acid), and a new compd., identified by chem. and degradative evidence as 15,16-dimethyl-30-glyceryloxytriacontanoic acid.
Abstract: On the basis of spectral data and chem. procedures, the structures of 3 complex lipids from the thermophilic archaebacterium D. mobilis were elucidated. These lipids are derivs. of glycerol-bisdiphytanylglycerol tetraether (GDGT). The least polar compd., comprising 22% of the total lipids, is a glycolipid in which 1 of the free hydroxyl groups of GDGT is linked glycosidically to a disaccharide with structure ï¡-D-Glcp(1ï®4)-ï¢-D-Galp. The 2nd compd. (21% of all lipids) is a phosphoglycolipid in which 1 of the free hydroxyls of GDGT is linked glycosidically to ï¢-D-Galp and the other is esterified to p-myo-inositol. The most polar compd. (32% of all lipids) is a phosphoglycolipid derived from ï¡-D-Glcp(1ï®4)-ï¢-D-Galp-GDGT, in which the free hydroxyl of GDGT is esterified with p-myo-inositol.
Abstract: The title compd. (I) was isolated from S. solfataricus, a thermophilic member of archaebacteria, and its structure was detd. by std. chem. and phys. methods.
Abstract: The core ether lipids of M. barkeri consisted of C20,C20 glycerol diether and trace amts. of C20,C25 glycerol diether, C20 glycerol monoether, C40,C40 diglycerol tetraethers with 1-3 cyclopentane rings, and a novel C20,C20 tetritol diether.
Abstract: The lipids of T. celer, an extremely thermophilic anaerobic sulfur-respiring archaebacterium, are characterized. On the basis of spectroscopic (1H and 13C-NMR), chromatog., and degrdn. studies, the most abundant polar lipid (about 80% of total lipid ext.) was identified as 2,3-di-O-phytanyl-sn-glycerol ester of phosphatidyl-myo-inositol. Its biosynthesis from acetate was shown by the incorporation of 14C labeled acetate.
Abstract: From Sulfolobus solfataricus, a sulfur-oxidizing thermophilic member of archaebacteria, three unusual benzothiophenquinones were isolated. Detailed NMR studies on these quinones, including multipulse mono-dimensional and two-dimensional techniques, were performed to obtain carbon and proton assignments, one-bond, geminal and vicinal coupling constants and T1 relaxation times. This report extends the known quinone composition of thermophilic archaebacteria and further supports the concept that these biomolecules can serve as a useful chemotaxonomic tool.
Abstract: An NAD+-dependent alc. dehydrogenase has been obtained in homogeneous form from the archaebacterium S. solfataricus grown at 87ï°. The enzyme, a dimer, is thermophilic, thermostable, resistant to org. solvents, and has a very broad substrate specificity. Redn. of 3-methylbutan-2-one involves a hydride attack at the re face of the carbonyl to produce the corresponding S-alc.
Abstract: An important issue to plan economical feasibility of biological hydrogen production comprises techniques for recovering useful materials such as physiologically active biomolecules from biomasses grown in large quantities. The present paper describes a series of enzymatic transglycosylation reactions performed using the crude homogenate of Thermotoga neapolitana (DSM 4359). The use of this extremophilic marine bacterium for the bioproduction of hydrogen using complex carbohydrates as feedstock has been recently described in a patent. In this growth condition glycosyl hydrolases can be well expressed. The study is focused on synthetic features of the transglycosylating enzymes. Xylosidase/xylanase activity seems to be the most abundant leading to convenient syntheses of interesting series of pure (β-1,4)-xylooligosaccharides of different aglycones such as 1-hexanol (producing promising candidates for new surfactants), 9-fluorene methanol (obtaining anti-HSV agents), 1,4-butanediol (for the synthesis of new glycolipids), and geraniol (producing aroma compounds). Furthermore, the regioselectivity during galactose, fucose, glucose and mannose enzymatic transfers is also investigated. The knowledge of synthetic characteristic of all these enzymes will be useful in the feasibility evaluation of large scale processes for the biohydrogen production.
