Structural elucidation of an exopolysaccharide from mycelial fermentation of a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis

A novel polysaccharide designated EPS-1A with an average molecular weight around 40 kDa was fractionated and purified by anion-exchange and gel-filtration chromatography from the crude exopolysaccharide (EPS) isolated from fermentation broth of Cs-HK1, a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis. The structural characteristics of EPS-1A were determined with various methods (e.g. GC, GC–MS, FT-IR, ¹H NMR and ¹³C NMR) and through acid hydrolysis, methylation, periodate-oxidation and Smith degradation. The results suggested that EPS-1A was composed of glucose, mannose and galactose at 15.2:3.6:1.0 M ratio. EPS-1A was a slightly branched polysaccharide and its backbone was composed of (1 → 6)-α-d-glucose residues (77%) and (1 → 6)-α-d-mannose residues (23%). Branching occurred at O-3 position of (1 → 6)-α-d-mannose residues of the backbone with (1 → 6)-α-d-mannose residues and (1 → 6)-α-d-glucose residues, and terminated with β-d-galactose residues.     

The Role of a d-Mannosyl-Lipid as an Intermediate in the Synthesis of Polysaccharide in Phaseolus aureus Seedlings

Particulate preparations from Phaseolus aureus produce a d-mannosyl-lipid when treated with GDP-d-mannose. This lipid complex appears to be an active d-mannose donor, and some investigators have proposed that its role might be an obligatory intermediate in mannan synthesis of higher plants. When the partially purified d-mannosyl-lipids, isotopically labeled in the d-mannose moiety, were treated with particulate enzymes under a variety of conditions, a negligible amount of material was produced that behaved as a polysaccharide. Endogenous, particle-bound d-mannosyl-¹⁴C-lipid prepared from P. aureus particles readily transferred d-mannose to GDP to yield GDP-d-mannose and was hydrolyzed to free d-mannose when treated briefly with 0.01 n HCl at 100 C. The d-mannosyl-lipid, therefore, exhibits active d-mannose transfer potential in its endogenous state. When endogenous glycosyl-lipid was incubated in the absence of GDP-d-mannose-¹⁴C, little or no polysaccharide was produced. It was, instead, slowly degraded to d-mannose. Addition of several different unlabeled sugar nucleotides had no effect on the results. Our studies to date, therefore, offer no evidence that the mannosyl-lipid is an obligatory precursor of polysaccharide.     

Chemical analysis of a polysaccharide of unripe (green) tomato (Lycopersicon esculentum)

A polysaccharide (PS-I) isolated from the aqueous extract of the unripe (green) tomatoes (Lycopersicon esculentum) consists of d-galactose, d-methyl galacturonate, d-arabinose, l-arabinose, and l-rhamnose. Structural investigation of the polysaccharide was carried out using total acid hydrolysis, methylation analysis, periodate oxidation study, and NMR studies (¹H, ¹³C, DQF-COSY, TOCSY, NOESY, ROESY, HMQC, and HMBC). On the basis of above-mentioned experiments the structure of the repeating unit of the polysaccharide (PS-I) was established as:

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Glycosylated nervogenic acid derivatives from Liparis condylobulbon (Reichb.f.) leaves

Three new nervogenic acid glycosides, 1-O-α-l-rhamnopyranosyl 3,5-bis(3-methyl-but-2-enyl)-4-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl]-benzoate, 3,5-bis(3-methyl-but-2-enyl)-4-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl]-benzoic acid, and bis{3,5-bis(3-methyl-but-2-enyl)-4-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl]-benzoyl} 1,2-O-β-d-glucopyranose, which we named condobulbosides A–C, were isolated from a methanol extract of the leaves of Liparis condylobulbon together with an apigenin C-glycoside, schaftoside. Their structures were established on the basis of spectral techniques, namely, UV, IR, HR-MS spectroscopy, both 1D and 2D NMR experiments, and chemical reactions.     

Enzymatic synthesis of d-xylulose 5-phosphate from hydroxypyruvate and d-glyceraldehyde-3-phosphate

An enzymatic method for obtaining d-xylulose 5-phosphate has been developed, based on the irreversible reaction catalyzed by transketolase: hydroxypyruvate + d-glyceraldehyde-3-phosphate → d-xylulose 5-phosphate. The preparations of sodium d-xylulose 5-phosphate, obtained using this approach, were 88% pure and contained no aldehyde admixtures.     

Modification of optimal pH in l-arabinose isomerase from Geobacillus stearothermophilus for d-galactose isomerization

l-Arabinose isomerase from Geobacillus stearothermophilus (GSAI; EC 5.3.1.4) has been genetically evolved to increase the reaction rate toward d-galactose, which is not a natural substrate. To change the optimal pH of GSAI for d-galactose isomerization (pH optimum at 8.5), we investigated the single point mutations influencing the activity based on the sequences of the previously evolved enzymes. Among the seven point mutations found in the evolved enzymes, mutations at Val⁴⁰⁸ and Asn⁴⁷⁵ were determined to be highly influential mutation points for d-galactose isomerization activity. A random mutation was introduced into sites Val⁴⁰⁸ and Asn⁴⁷⁵ (X408V and X475N), and candidates were screened based on non-optimal pH conditions. Among the mutations of X408V and X475N, mutations of Q408V and R408V were selected. The optimal pH of the both mutations Q408V and R408V was shifted to pH 7.5. At the shifted optimal pH, the d-galactose isomerization activities of Q408V and R408V were 60 and 30% higher than that of the wild type at pH 8.5, respectively.     

Encapsulation of an Agrobacterium radiobacter extract containing d-hydantoinase and d-carbamoylase activities into alginate–chitosan polyelectrolyte complexes: Preparation of the biocatalyst

Alginate–chitosan polyelectrolyte complexes (PECs) have been used for the first time as a suitable matrix for coimmobilisation of enzymes to reproduce a multistep enzymatic route for production of d-amino acids. Encapsulation of a crude cell extract from Agrobacterium radiobacter containing d-hydantoinase and d-carbamoylase activities into the PECs with negligible leakage from the formed capsules was accomplished. All results in this study indicate that the preparation of the biocatalyst (preparation method and chitosan characteristics) play a key role in the biocatalyst's properties. The most suitable biocatalysts were prepared using a chitosan with a medium molecular weight (600 kDa) and a degree of deacetylation of 0.9. For all of the preparation conditions under study, an encapsulation yield of around 60% was achieved and the enzymatic activity yields ranged from 30 to 80% for d-hydantoinase activity and from 40 to 128% for d-carbamoylase activity relative to the activities of the soluble extract. All of the biocatalysts were able to hydrolyze l,d-hydroxyphenylhydantoin into p-hydroxyphenylglycine with yields ranging from 30 to 80%.     

Activity of yeast d-amino acid oxidase on aromatic unnatural amino acids

d-Amino acid oxidase is a FAD-dependent enzyme that catalyses the conversion of the d-enantiomer of amino acids into the corresponding α-keto acid. Substrate specificity of the enzyme from the yeast Rhodotorula gracilis was investigated towards aromatic amino acids, and particularly synthetic α-amino acids.A significant improvement of the activity (V_max,app) and of the specificity constant (the V_max,app/K_m,app ratio) on a number of the substrates tested was obtained using a single-point mutant enzyme designed by a rational approach. With R. gracilis d-amino acid oxidase the complete resolution of d,l-homo-phenylalanine was obtained with the aim to produce the corresponding pure l-isomer and to use the corresponding α-keto acid as a precursor of the amino acid in the l-form.     

8,14-Secopregnane glycosides from the aerial parts of Asclepias tuberosa

Twenty pregnane glycosides, tuberoside A₁–L₅, were isolated from the diethyl ether-soluble fraction of the MeOH extract from the aerial parts of Asclepias tuberosa (Asclepiadaceae). The pregnane glycosides were composed of 8,12;8,20-diepoxy-8,14-secopregnane as aglycon, and d-cymarose, d-oleandrose, d-digitoxose and/or d-glucose as the component sugars. Their structures were established using NMR spectroscopic analysis and chemical methodologies.     

Construction and co-expression of polycistronic plasmid encoding d-hydantoinase and d-carbamoylase for the production of d-amino acids

d-Hydantoinase and d-carbamoylase genes from Agrobacterium radiobacter TH572 were cloned by polymerase chain reaction (PCR). The plasmid pUCCH3 with a polycistronic structure that is controlled by the native hydantoinase promoter was constructed to co-express the two genes and transformed into Escherichia coli strain JM105. To obtain the highest level of expression of the d-carbamoylase and avoid intermediate accumulation, the d-carbamoylase gene was cloned closer to the promoter and the RBS region in the upstream of it was optimized. This resulted in high active expression of soluble d-hydantoinase and d-carbamoylase that is obtained without any inducer. Thus, by the constitutive recombinant JM105/pUCCH3, d-p-hydroxyphenylglycine (d-HPG) was obtained directly with 95.2% production yield and 96.3% conversion yield.     

The immobilization of d-hydantoinase and characterization under classic condition and microwave irradiation

d-Hydantoinase was covalently immobilized onto polystyrene anion exchange resin via glutaraldehyde. Immobilization conditions were optimized: the carrier as D-92 type polystyrene anion exchange resin, temperature as 25 °C, immobilization time as 12 h, and initial concentration of protein as 6 mg/ml. Under the optimized reaction conditions the activity of the free and immobilized d-hydantoinase was determined. The free and immobilized d-hydantoinase samples were characterized with their kinetic parameters, thermal, and storage stability. The K_m and V_max values were 14.985 mM and 0.6 mM/min for the free, and 27.030 mM and 1.187 mM/min for the immobilized, respectively. Operational stability of the immobilized d-hydantoinase was also detected in a circulating packed-bed reactor. The half-time of the immobilized d-hydantoinase was 11 days. Nearly 90% of activity of the immobilized d-hydantoinase was reserved for 100 days stored at 4 °C. The free and immobilized d-hydantoinases were also characterized under microwave irradiation. Results shown that the reactions catalyzed by both free and immobilized d-hydantoinase were accelerated under microwave irradiation. The half-time of the immobilized d-hydantoinase reduced to 16 min under microwave irradiation.     

Structure of the oligosaccharides isolated from Dalbergia sissoo Roxb. leaf polysaccharide

A water-soluble polysaccharide isolated from Dalbergia sissoo Roxb. leaves was purified and major homogeneous fraction obtained by GPC. Complete hydrolysis of the polysaccharide followed by paper chromatography and GLC analysis indicated the presence of l-rhamnose, d-glucuronic acid, d-galactose and d-glucose in molar ratio of 1:1:2:2.33, respectively. Partial hydrolysis of the polysaccharide furnished one tri-[I], one hepta-[II] and one nona-[III] saccharides. Hydrolysis of the oligosaccharide I, II and III followed by GLC analysis furnished d-glucose and l-rhamnose (2:1); l-rhamnose, d-galactose and d-glucuronic acid (1:3:3); and l-rhamnose, d-galactose and d-glucose (1:3:5), respectively. Methylation analysis and periodate oxidation of the oligosaccharide I indicated the presence of two non reducing glucose units linked to rhamnose by 1→2 and 1→4 linkages, respectively. Oligosaccharide II is a branched molecule with a main chain consisting of 1,3-linked β-d-galactopyranosyl (2 mol), 1,3,4 linked α-l-rhamnopyranosyl (1 mol) and 1,4,6 linked β-d-galactopyranosyl unit (1 mol) and non reducing β-d-glucuronic acid at the end along with side chains of β-d-glucouronopyranosyl units (2 mol). Oligosaccharide III is also a branched molecule with a main chain consisting of 1,3,4 linked α-l-rhamnopyranosyl (1 mol), 1,2,4 linked β-d-glucopyranosyl (1 mol), 1,3 and 1,4 linked β-d-galactopyranosyl (2 and 1 mol, respectively) having β-d-glucopyranosyl as a non reducing end.     

Identification of l-fucose-binding proteins from the Nile tilapia (Oreochromis niloticus L.) serum

Lectins are carbohydrate-binding proteins with many biological functions including cellular recognition and innate immunity. In this study, a major l-fucose-binding lectin from the serum of Nile tilapia (Oreochromis niloticus L.), designated as TFBP, was isolated by l-fucose-BSA Sepharose CL6B affinity chromatography. The SDS-PAGE (10%) analysis of TFBP revealed a major band of approximately 23 kDa with an N-terminal amino acid sequence of DQTETAGQQSXPQDIHAVLREL which did not give significant similarities to the protein databases using BLASTp searches. Ruthenium red staining indicate positive calcium-binding property of TFBP. The purified TFBP agglutinated human type O erythrocytes but not the type A and B fresh erythrocytes. Live Aeromonas hydrophila and Enterococcus faecalis cells were also agglutinated by the lectin. The fucose-binding proteins were detected in the soluble protein extracts from the gills, gut, head kidneys, liver, serum and spleen using a fucose-binding protein probe (l-fucose-BSA-horseradish peroxidase). The binding of TFBP with the l-fucose–BSA probe was inhibited by l-fucose but not by α-methyl-d-mannose.     

Studies on the Chemical Structure of Konjac Mannan

The electrophoretically homogeneous glucomannan isolated from konjac flour was composed of d-glucose and d-mannose residues in the approximate ratio of 1: 1.6. Controlled acid hydrolysis gave 4-O-β-d-mannopyranosyl-d-mannose, 4-O-β-d-mannopyranosyl-d-glucose_T 4-O-β-d-glucopyranosyl-d-glucose(cellobiose), 4-O-β-d-glucopyranosyl-d-mannose(epicellobiose), O-β-d-mannopyranosyl-(1→4)-O-β-d-mannopyranosyl-(1→4)-d-mannose, O-β-d-glucopyranosyl- (1→4)-O-β-d-mannopyranosyl-(1→4)-d-mannose, O-β-d-mannopyranosyl-(1→4)-O-β-d-glucopy- ranosyl-(1→4)-d-mannose and O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→4)-d-mannose.     

E versus Z geometry in β-d-arabino-hexopyranosidulose oximes

Koenigs–Knorr-type glycosidations of peracylated 2Z-benzoyloxyimino-glycopyranosyl bromides invariably proceed with retention of the Z-geometry. Accordingly, the many β-d-hexosidulose oximes in literature which were prepared in this way and for which the oxime geometry has not been addressed explicitly, are the Z-oximes throughout. By contrast, oximation of β-d-hexopyranosid-2-uloses leads to mixtures of E and Z oximes readily separable and structurally verifiable by ¹H and ¹³C NMR. Configurational assignments rested on comparative evaluation of NMR data of E and Z isomers, and, most notably on an X-ray structural analysis of the pivaloylated isopropyl 2E-benzoyloxyimino-2-deoxy-β-d-arabino-hexopyranoside revealing the unusual ¹S₅^1,4B conformation for the pyranoid ring.     

A pyrroloquinoline quinine-dependent membrane-bound d-sorbitol dehydrogenase from Gluconobacter oxydans exhibits an ordered Bi Bi reaction mechanism

A membrane-bound pyrroloquinoline quinine (PQQ)-dependent d-sorbitol dehydrogenase (mSLDH) in Gluconobacter oxydans participates in the oxidation of d-sorbitol to l-sorbose by transferring electrons to ubiquinone which links to the respiratory chain. To elucidate the kinetic mechanism, the enzyme purified was subjected to two-substrate steady-state kinetic analysis, product and substrate inhibition studies. These kinetic data indicate that the catalytic reaction follows an ordered Bi Bi mechanism, where the substrates bind to the enzyme in a defined order (first ubiquinone followed by d-sorbitol), while products are released in sequence (first l-sorbose followed by ubiquinol). From these findings, we proposed that the native mSLDH bears two different substrate-binding sites, one for ubiquinone and the other for d-sorbitol, in addition to PQQ-binding and Mg²⁺-binding sites in the catalytic center.     

The biosynthetic pathway of crucifer phytoalexins and phytoanticipins: De novo incorporation of deuterated tryptophans and quasi-natural compounds

Although several biosynthetic intermediates in pathways to cruciferous phytoalexins and phytoanticipins are common, questions regarding the introduction of substituents at N-1 of the indole moiety remain unanswered. Toward this end, we investigated the potential incorporations of several perdeuterated d- and l-1′-methoxytryptophans, d- and l-tryptophans and other indol-3-yl derivatives into pertinent phytoalexins and phytoanticipins (indolyl glucosinolates) produced in rutabaga (Brassica napus L. ssp. rapifera) roots. In addition, we probed the potential transformations of quasi-natural compounds, these being analogues of biosynthetic intermediates that might lead to “quasi-natural” products (products similar to natural products but not produced under natural conditions). No detectable incorporations of deuterium labeled 1′-methoxytryptophans into phytoalexins or glucobrassicin were detected. l-tryptophan was incorporated in a higher percentage than d-tryptophan into both phytoalexins and phytoanticipins. However, in the case of the phytoalexin rapalexin A, both d- and l-tryptophan were incorporated to the same extent. Furthermore, the transformations of both 1′-methylindolyl-3′-acetaldoxime and 1′-methylindolyl-3′-acetothiohydroxamic acid (quasi-natural products) into 1′-methylglucobrassicin but not into phytoalexins suggested that post-aldoxime enzymes in the biosynthetic pathway of indolyl glucosinolates are not substrate-specific. Hence, it would appear that the 1-methoxy substituent of the indole moiety is introduced downstream from tryptophan and that the post-aldoxime enzymes of the glucosinolate pathway are different from the enzymes of the phytoalexin pathway. A higher substrate specificity of some enzymes of the phytoalexin pathway might explain the relatively lower structural diversity among phytoalexins than among glucosinolates.     

Synthesis of monosaccharide derivatives and polymeric prodrugs of 5-fluorouridine via two-step enzymatic or chemo-enzymatic highly regioselective strategy

Efficient protocols for the selective synthesis of monosaccharide derivatives and polymeric prodrugs of 5-fluorouridine (5-FUR) have been developed. Firstly, transesterification of 5-FUR and divinyl dicarboxylates ranging from 4 to 10 carbon atoms were performed under the catalysis of Candida antarctica lipase acrylic resin in anhydrous THF at 50 °C, respectively. A series of vinyl 5-FUR esters were prepared, with high acylation regioselectivity at 5′-OH. The influences of reaction parameters including enzyme, solvents, molar ratio of substrates, reaction time, the carbon length of acyl donor and reaction temperature were investigated in details. And then, protease-catalyzed highly regioselective acylation of d-glucose, d-mannose and d-galactose with vinyl esters of 5-FUR gave 5-FUR-saccharide derivatives successfully. Moreover, a series of polymeric prodrugs of 5-FUR with the different linker lengths were prepared by the chemo-polymerization of vinyl 5-FUR esters in DMF initiated by azobisisobutyronitrile (AIBN).     

Antioxidative defense organization in retroperitoneal white adipose tissue during acclimation to cold—The involvement of l-arginine/NO pathway

1. Retroperitoneal white adipose tissue (RpWAT) antioxidative defense was investigated in untreated, l-arginine-treated and N^ω-nitro-l-arginine methyl ester (l-NAME)-treated rats kept at 4±1 °C (1, 3, 7, 12, 21 and 45 days) and compared to control rats at 22±1 °C.

2. Cold-acclimation-induced RpWAT weight decrease was accompanied by a decline in glutathione level and increased activity of manganese superoxide dismutase (MnSOD), glutathione S-transferase (GST), catalase, glutathione peroxidase and glutathione reductase at different time-points.

3. l-arginine accelerated RpWAT weight decrease, the increase in MnSOD and GST activities and the prolonged increase of catalase, MnSOD and GST activities. l-NAME delayed cold-induced catalase activity increase and tissue weight decrease. Prolonged l-NAME-treatment had a similar effect on RpWAT as l-arginine.

4. Results suggest the involvement of l-arginine/NO pathway in RpWAT oxidative metabolic augmentation induced by cold-acclimation.

The Acceptor Specificity of Amylomaltase from Escherichia coli IFO 3806

The acceptor specificity of amylomaltase from Escherichia coli IFO 3806 was investigated using various sugars and sugar alcohols. d-Mannose, d-glucosamine, N-acetyl- d-glucosamine, d-xylose, d- allose, isomaltose, and cellobiose were efficient acceptors in the transglycosylation reaction of this enzyme. It was shown by chemical and enzymic methods that this enzyme could transfer glycosyl residues only to the C4-hydroxyl groups of d-mannose, iY-acetyl- d-glucosamine, d-allose, and d-xylose, producing oligosaccharides terminated by 4–0-α-d-glucopyranosyl-d-mannose, 4–0-α-d-glucopyranosyl-yV-acetyl-d-glucosamine, 4-O-α-d-glucopyranosyl-d-allose, and 4–0-α-d-gluco- pyranosyl-d-xylose at the reducing ends, respectively.