Preparation and characterization of maltosyl-sucrose isomers produced by transglycosylation of maltogenic amylase from Bacillus stearothermophilus

To develop a new transfer product of sucrose, sucrose was modified to maltosyl-sucrose using the transglycosylation activity of maltogenic amylase from Bacillus stearothermophilus (BSMA). The transglycosylation reaction was conducted with maltotriose and sucrose as the donor and acceptor, respectively. The presence of various sucrose transfer products was confirmed by thin layer chromatography (TLC) and high performance anion exchange chromatography (HPAEC). The sucrose transfer products were isolated by alkali-degradation followed by charcoal column chromatography using 20% (v/v) ethanol, then purified by ion exchange and Biogel P-2 gel permeation chromatographies. The structures of the major transfer products were determined to be 6^G--maltosyl-sucrose (maltosyl-sucrose 1) and 6^F--maltosyl-sucrose (maltosyl-sucrose 2) by LC-MS and ¹³C NMR. The mixture of maltosyl-sucrose 1 and 2 showed low sweetness, high hygroscopicity, low Maillard reactivity, and high acid and heat stability. Furthermore, it had an inhibitory effect on mutansucrase and water-insoluble glucan formation. These results indicated that the mixture of maltosyl-sucrose 1 and 2 is a suitable sugar substitute useful for various food products.     

Kinetics of substrate transglycosylation by glycoside hydrolase family 3 glucan (1-->3)-beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium

To elucidate the interaction between substrate inhibition and substrate transglycosylation of retaining glycoside hydrolases (GHs), a steady-state kinetic study was performed for the GH family 3 glucan (1-->3)-beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium, using laminarioligosaccharides as substrates. When laminaribiose was incubated with the enzyme, a transglycosylation product was detected by thin-layer chromatography. The product was purified by size-exclusion chromatography, and was identified as a 6-O-glucosyl-laminaribiose (beta-D-Glcp-(1-->6)-beta-D-Glcp-(1-->3)-D-Glc) by 1H NMR spectroscopy and electrospray ionization mass spectrometry analysis. In steady-state kinetic studies, an apparent decrease of laminaribiose hydrolysis was observed at high concentrations of the substrate, and the plots of glucose production versus substrate concentration were thus fitted to a modified Michaelis-Menten equation including hydrolytic and transglycosylation parameters (K(m), K(m2), k(cat), k(cat2)). The rate of 6-O-glucosyl-laminaribiose production estimated by high-performance anion-exchange chromatography coincided with the theoretical rate calculated using these parameters, clearly indicating that substrate inhibition of this enzyme is fully explained by substrate transglycosylation. Moreover, when K(m), k(cat), and affinity for glucosyl-enzyme intermediates (K(m2)) were estimated for laminarioligosaccharides (DP=3-5), the K(m) value of laminaribiose was approximately 5-9 times higher than those of the other oligosaccharides (DP=3-5), whereas the K(m2) values were independent of the DP of the substrates. The kinetics of transglycosylation by the enzyme could be well interpreted in terms of the subsite affinities estimated from the hydrolytic parameters (K(m) and k(cat)), and a possible mechanism of transglycosylation is proposed.     

1-O-Acetyl-beta-D-galactopyranose: a novel substrate for the transglycosylation reaction catalyzed by the beta-galactosidase from Penicillium sp

1-O-Acetyl-beta-D-galactopyranose (AcGal), a new substrate for beta-galactosidase, was synthesized in a stereoselective manner by the trichloroacetimidate procedure. Kinetic parameters (K(M) and k(cat)) for the hydrolysis of 1-O-acetyl-beta-D-galactopyranose catalyzed by the beta-D-galactosidase from Penicillium sp. were compared with similar characteristics for a number of natural and synthetic substrates. The value for k(cat) in the hydrolysis of AcGal was three orders of magnitude greater than for other known substrates. The beta-galactosidase hydrolyzes AcGal with retention of anomeric configuration. The transglycosylation activity of the beta-D-galactosidase in the reaction of AcGal and methyl beta-D-galactopyranoside (1) as substrates was investigated by 1H NMR spectroscopy and HPLC techniques. The transglycosylation product using AcGal as a substrate was beta-D-galactopyranosyl-(1-->6)-1-O-acetyl-beta-D-galactopyranose (with a yield of approximately 70%). In the case of 1 as a substrate, the main transglycosylation product was methyl beta-D-galactopyranosyl-(1-->6)-beta-D-galactopyranoside. Methyl beta-D-galactopyranosyl-(1-->3)-beta-D-galactopyranoside was found to be minor product in the latter reaction.     

Highly selective biotransformation of arbutin to arbutin-α-glucoside using amylosucrase from Deinococcus geothermalis DSM 11300

Arbutin (Ab, 4-hydroxyphenyl β-glucopyranoside) is a glycosylated hydroquinone known to prevent the formation of melanin by inhibiting tyrosinase. An arbutin-α-glucoside was synthesized by the transglycosylation reaction of amylosucrase (AS) of Deinococcus geothermalis (DGAS) using arbutin and sucrose as an acceptor and a donor, respectively. The maximum yield of the arbutin transglycosylation product was determined to be over 98% with a 1:0.5 molar ratio of donor and acceptor molecules (sucrose and arbutin), in 50 mM sodium citrate buffer pH 7 at 35 °C. TLC and HPLC analyses revealed that only one transglycosylation product was observed, supporting the result that the transglycosylation reaction of DGAS was very specific. The arbutin transglycosylation product was isolated by preparative recycling HPLC. The structural analyses using ¹³C and ¹H NMR proved that the transglycosylated product was 4-hydroxyphenyl β-maltoside (Ab-α-glucoside), in which a glucose molecule was linked to arbutin via an α-(1 → 4)-glycosidic linkage.     

Functional characterization of the sucrose isomerase responsible for trehalulose production in plant-associated Pectobacterium species

Fifty-three plant-associated microorganisms were investigated for their ability to convert sucrose to its isomers. These microorganisms included one Dickeya zeae isolate and 7 Enterobacter, 3 Pantoea, and 43 Pectobacterium species. Eleven out of the 53 strains (21%) showed the ability to transform sucrose to isomaltulose and trehalulose. Among those, Pectobacterium carotovorum KKH 3-1 showed the highest bioconversion yield (97.4%) from sucrose to its isomers. In this strain, the addition of up to 14% sucrose in the medium enhanced sucrose isomerase (SIase) production. The SIase activity at 14% sucrose (47.6 U/mg dcw) was about 3.6-fold higher than that of the negative control (13.3 U/mg dcw at 0% sucrose). The gene encoding SIase, which is comprised a 1776 bp open reading frame (ORF) encoding 591 amino acids, was cloned from P. carotovorum KKH 3-1 and expressed in Escherichia coli. The recombinant SIase (PCSI) was shown to have optimum activity at pH 6.0 and 40 °C. The reaction temperature significantly affected the ratio of sucrose isomers produced by PCSI. The amount of trehalulose increased from 47.5% to 79.1% as temperature was lowered from 50 °C to 30 °C, implying that SIase activity can be controlled by reaction temperature.     

Enzymatic synthesis of gentiooligosaccharides by transglycosylation with β-glycosidases from Penicillium multicolor

A crude enzyme preparation from Penicillium multicolor efficiently produced mainly gentiotriose to gentiopentaose (d.p. 3-5) by transglycosylation using a high concentration of gentiobiose as the substrate. The resulting gentiotriose was examined in a gustatory sensation test using human volunteers, and was determined to have one-fifth of the bitterness of gentiobiose. The crude enzyme preparation was analyzed by chromatography to determine the enzyme responsible for formation of the gentiooligosaccharides. The transglycosylation was shown to take place in two stages by a combination of β-glucosidase and β-(1→6)-glucanase. In the initial stage, which was the rate-limiting step in the overall process, β-glucosidase produced mainly gentiotriose from gentiobiose. In the second step, β-(1→6)-glucanase acted on the resulting gentiotriose, which served as both donor and acceptor, to produce a series of gentiooligosaccharides (d.p. 4-9) by transglycosylation.     

Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 in fed-batch fermentation: Effects of sucrose concentration in a complex medium and process modeling

Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 was investigated in semi-aerobic, pH-controlled, batch and fed-batch fermentations using a complex medium containing sucrose as the main source of carbon. The effects of sucrose concentration were studied in fed-batch fermentations in which a sucrose solution was added at stable feeding rates (5, 7, 9 and 10 g/l/h). The results showed that pediocin is produced as a product of the primary metabolism and its titer could be greatly improved by adjusting the sucrose feeding rate in fed-batch fermentation. The maximum titer of pediocin of 145 AU/ml was obtained in the fed-batch culture with 7 g/l/h feeding rate and that was 119% higher compared to the titer obtained in batch culture. Higher feeding rates (9 and 10 g/l/h) resulted in decreased pediocin yields while biomass levels appeared to be rather unaffected. The specific rate of pediocin formation was also sensitive to sucrose concentration levels. A mathematical model developed on the basis of well-known rate equations for batch and fed-batch cultures and growth associated production, described successfully cell growth, sucrose assimilation, lactate production and pediocin production in fed-batch culture.     

Biosynthesis of (+)-catechin glycosides using recombinant amylosucrase from Deinococcus geothermalis DSM 11300

Amylosucrase (ASase, EC 2.4.1.4) is a glucosyltransferase that hydrolyzes sucrose into glucose and fructose and produces amylose-like glucan polymers from the released glucose. (+)-Catechin is a plant polyphenolic metabolite having skin-whitening and antioxidant activities. In this study, the ASase gene from Deinococcus geothermalis (dgas) was expressed in Escherichia coli, while the recombinant DGAS enzyme was purified using a glutathione S-transferase fusion system. The (+)-catechin glycoside derivatives were synthesized from (+)-catechin using DGAS transglycosylation activity. We confirmed the presence of two major transglycosylation products using TLC. The (+)-catechin transglycosylation products were isolated using silica gel open column chromatography and recycling-HPLC. Two (+)-catechin major transfer products were determined through ¹H and ¹³C NMR to be (+)-catechin-3′-O-α-d-glucopyranoside with a glucose molecule linked to (+)-catechin and (+)-catechin-3′-O-α-D-maltoside with a maltose linked to (+)-catechin. The presence of (+)-catechin maltooligosaccharides in the DGAS reaction was also confirmed via recycling-HPLC and enzymatic analysis. The effects of various reaction conditions (temperature, enzyme concentration, and molar ratio of acceptor and donor) on the yield and type of (+)-catechin glycosides were investigated.     

Direct regioselective 2-O-(p-toluenesulfonylation) of sucrose

2-O-(p-Toluenesulfonyl)sucrose was regioselectively synthesized by direct p-toluenesulfonylation of sucrose using N-(p-toluenesulfonyl)imidazole in the presence of molecular sieves at 40 degrees C. The reactivities of the sucrose hydroxy groups toward this sulfonylation increased in the order as follows: OH-2>OH-1'>OH-3'>OH-6>OH-6'. These results were diametrically opposite to the expected sulfonylation with p-toluenesulfonyl chloride in pyridine, for which the reactivity increased in the order as follows: OH-6', OH-6>OH-1'>OH-2. The desired 2-O-(p-toluenesulfonyl)sucrose was readily isolated by simple open reversed-phase column chromatography, followed by recrystallization, thus overcoming the main difficulties associated with regioselectivity, efficiency, and isolation techniques for the practical preparation.     

Enzymatic syntheses of T antigen-containing glycolipid mimicry using the transglycosylation activity of endo-alpha-N-acetylgalactosaminidase

Thomsen-Friedenreich antigen (T antigen) disaccharide, beta-D-galactose-(1-->3)-alpha-N-acetyl-D-galactosamine (beta-D-Gal-(1-->3)-alpha-D-GalNAc), containing glycolipid mimicry was synthesized using the transglycosylation activity of endo-alpha-N-acetylgalactosaminidase from Bacillus sp. This enzyme could transfer the disaccharide from a p-nitrophenyl substrate to water-soluble 1-alkanols and other alcohols at a transfer ratio of 70% or more. Although the transfer ratios were lower for water-insoluble than water-soluble alcohols, they were shown to increase by adding sodium cholate to the reaction mixtures. The enzyme also transferred the disaccharide directly from asialofetuin to 1-alkanols. The anomeric bond between the disaccharide and 1-alkanols of the transglycosylation product is in the alpha configuration as determined by sequential digestion of jack bean beta-galactosidase and Acremonium alpha-N-acetylgalactosaminidase. Since the transglycosylation product, beta-D-Gal-(1-->3)-alpha-D-GalNAc-(1-->O)-hexyl, efficiently inhibits the binding of anti-T antigen monoclonal antibody to asialofetuin, it has potential as an agent for blocking T antigen-mediated cancer metastasis.     

Catalytic properties and mode of action of endo-(1-->3)-beta-D-glucanase and beta-D-glucosidase from the marine mollusk Littorina kurila

A complex of the enzymes from the liver of the marine mollusk Littorina kurila that hydrolyzes laminaran was investigated. Two (1-->3)-beta-d-glucanases (G-I and G-II) were isolated. The molecular mass of G-I as estimated by gel-permeation chromatography and SDS-PAGE analysis was 32 and 40kDa, respectively. The G-II molecular mass according to SDS-PAGE analysis was about 200kDa. The pH optimum for both G-I and G-II was pH 5.4. The G-I had narrow substrate specificity and hydrolyzed only the (1-->3)-beta-d-glucosidic bonds in the mixed (1-->3),(1-->6)- and (1-->3),(1-->4)-beta-d-glucans down to glucose and glucooligosaccharides. This enzyme acted with retention of the anomeric configuration and catalyzed a transglycosylation reaction. G-I was classified as the glucan endo-(1-->3)-beta-d-glucosidase (EC 3.2.1.39). G-II exhibited both exo-glucanase and beta-d-glucoside activities. This enzyme released from the laminaran glucose as a single product, but retained the anomeric center configuration and possessed transglycosylation activity. The hydrolysis rate of glucooligosaccharides by G-I decreased with an increase of the substrate's degree of polymerization. In addition to (1-->3)-beta-d-glucanase activity, the enzyme had the ability to hydrolyze p-nitrophenyl beta-d-glucoside and beta-d-glucobioses: laminaribiose, gentiobiose, and cellobiose, with the rate ratio of 50:12:1. G-II may correspond to beta-d-glucoside glucohydrolase (EC 3.2.1.21).     

A novel sucrose phosphorylase from the metagenomes of sucrose-rich environment: isolation and characterization

Sucrose phosphorylase, an important enzyme mainly involved in the generic starch and sucrose pathways, has now caught the attention of researchers due to its transglycosylation activity. A novel sucrose phosphorylase, unspase, has been isolated, and its transglycosylation properties were characterized. Compared with Bisp, the sucrose phosphorylase from Bifidobacterium adolescentis, unspase had two deleted regions in its C: -terminal. These deleted regions were probably equivalent to the important five-stranded anti-parallel β-sheet domain in sucrose phosphorylase. Unspase has a k(m) of 21.12?mM, a V(max) of 69.24?μmol?min(-1)?mg(-1) and a k(cat) of 31.19?s(-1) with sucrose as substrate. In 3-(N-morpholino) propanesulfonic acid (MOPS) buffer, unspase transferred the glycosyl moiety to L: -arabinose, D: -fructose and L: -sorbose. Much to our surprise, unspase can catalyze the transglycosylation in which a glycosyl moiety was transferred to L: -arabinose in the presence of phosphate, which is an interesting exception to the generally accepted fact that transglycosylation can only occur under the condition of phosphate absence. The final yield of the transglycosylation product (37.9?%) in phosphate buffer was even higher than that (5.8?%) in MOPS buffer. This is a novel phenomenon that a sucrose phosphorylase can catalyze a transglycosylation reaction in the presence of phosphate.     

Transglycosylation reactions of Bacillus stearothermophilus maltogenic amylase with acarbose and various acceptors

It was observed that Bacillus stearothermophilus maltogenic amylase cleaved the first glycosidic bond of acarbose to produce glucose and a pseudotrisaccharide (PTS) that was transferred to C-6 of the glucose to give an alpha-(1-->6) glycosidic linkage and the formation of isoacarbose. The addition of a number of different carbohydrates to the digest gave transfer products in which PTS was primarily attached alpha-(1-->6) to D-glucose, D-mannose, D-galactose, and methyl alpha-D-glucopyranoside. With D-fructopyranose and D-xylopyranose, PTS was linked alpha-(1-->5) and alpha-(1-->4), respectively. PTS was primarily transferred to C-6 of the nonreducing residue of maltose, cellobiose, lactose, and gentiobiose. Lesser amounts of alpha-(1-->3) and/or alpha-(1-->4) transfer products were also observed for these carbohydrate acceptors. The major transfer product to sucrose gave PTS linked alpha-(1-->4) to the glucose residue. alpha,alpha-Trehalose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4). Maltitol gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the glucopyranose residue. Raffinose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the D-galactopyranose residue. Maltotriose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the nonreducing end glucopyranose residue. Xylitol gave PTS linked alpha-(1-->5) as the major product and D-glucitol gave PTS linked alpha-(1-->6) as the only product. The structures of the transfer products were determined using thin-layer chromatography, high-performance ion chromatography, enzyme hydrolysis, methylation analysis and 13C NMR spectroscopy. The best acceptor was gentiobiose, followed closely by maltose and cellobiose, and the weakest acceptor was D-glucitol.     

Glucosylation of alpha-butyl- and alpha-octyl-D-glucopyranosides by dextransucrase and alternansucrase from Leuconostoc mesenteroides

For the first time, glucosylation of alpha-butyl- and alpha-octylglucopyranoside was achieved using dextransucrase (DS) of various specificities, and alternansucrase (AS) from Leuconostoc mesenteroides. All the glucansucrases (GS) tested used alpha-butylglucopyranoside as acceptor; in particular, DS produced alpha-D-glucopyranosyl-(1-->6)-O-butyl-alpha-D-glucopyranoside and alpha-D-glucopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1-->6)-O-butyl-alpha-D-glucopyranoside. In contrast, alpha-octylglucopyranoside was glucosylated only by AS which was shown to be the most efficient catalyst. The conversion rates, obtained with this enzyme at sucrose to acceptor molar ratio of 2:1 reached 81 and 61% for alpha-butylglucopyranoside and alpha-octylglucopyranoside, respectively. Analyses obtained from liquid chromatography coupled with mass spectrometry revealed that different series of alpha-alkylpolyglucopyranosides regioisomers of increasing polymerization degree can be formed depending on the specificity of the catalyst.     

Bioactive sucrose esters from Bidens parviflora

An investigation on Bidens parviflora led to the isolation of three sucrose esters and a substituted truxillate. Their structures were elucidated as (6-O-(E)-p-coumaroyl)-beta-D-fructofuranosyl-(2-->1)-alpha-D-glucopyranoside, (6-O-(E)-p-coumaroyl)-beta-D-fructofuranosyl-(2-->1)-(6-O-(E)-p-coumaroyl)-alpha-D-glucopyranoside II, 6,6'-sucrose ester of (1alpha,2alpha,3beta,4beta)-3,4-bis(4-hydroxyphenyl)-1,2-cyclobutanedicarboxylic acid, dimethyl ester of (1alpha,2alpha,3alpha,4alpha)-2,4-bis(3,4-dihydroxyphenyl)-1,3-cyclobutanedicarboxylic acid on the basis of spectral and chemical evidence. These compounds were subjected to the following bioassays: the histamine release inhibition of rat mast cells induced by antigen-antibody reaction and the inhibitory activity of PGE(2) production by macrophages.     

Penta-, hexa-, and heptasaccharide acceptor products of alternansucrase

In the presence of suitable acceptor molecules, dextransucrase makes a homologous series of oligosaccharides in which the isomers differ by a single glucosyl unit, whereas alternansucrase synthesizes one trisaccharide, two tetrasaccharides, etc. For the example of maltose as the acceptor, if one considers only the linear, unbranched possibilities for alternansucrase, the hypothetical number of potential products increases exponentially as a function of the degree of polymerization (DP). Experimental evidence indicates that far fewer products are actually formed. We show that only certain isomers of DP >4 are formed from maltose in measurable amounts, and that these oligosaccharides belong to the oligoalternan series rather than the oligodextran series. When the oligosaccharide acceptor products from maltose were separated by size-exclusion chromatography and HPLC, only one pentasaccharide was isolated. Its structure was alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. Two hexasaccharides were formed in approximately equal quantities: alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc and alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. Just one heptasaccharide was isolated from the reaction mixture, alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. We conclude that the enzyme is incapable of forming two consecutive alpha-(1-->3) linkages, and does not form products with more than two consecutive alpha-(1-->6) linkages. The distribution of products may be kinetically determined.     

Maltosyl-erythritol,a major transglycosylation product of erythritol by Bacillus stearothermophilus maltogenic amylase

This study was done to modify erythritol to change its physicochemical and sensory properties. Erythritol, a four-carbon sugar alcohol, was transglycosylated by Bacillus stearothermophilus maltogenic amylase with maltotriose as a donor molecule. The presence of various transglycosylation products of erythritol was confirmed by TLC and high performance ion exchange chromatography (HPIC). The major transfer product was purified by gel filtration chromatography on Bio-Gel P-2. Examination by LC-MS, matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF-MS), and 13C NMR showed that the major transfer product was maltosyl-erythritol. Results of 13C NMR of maltosyl-erythritol suggested that linkage was formed between the C1 carbon of glucose unit in maltose and either one of the two carbon atoms of the terminal hydroxyl groups of erythritol, so that a mixture of 1-O- and 4-O-alpha-maltosyl-erythritol was produced. The sweetness of maltosyl-erythritol was about 40% that of sucrose, and its negative sensory properties were less than those of erythritol.     

Regioselective sulfonylation of 6,1',6'-tri-O-tritylsucrose through dibutylstannylation: synthesis of 4'-O-sulfonyl derivatives of sucrose

3-O-Mesyl-1,6-di-O-trityl-beta-D-fructofuranosyl-(2-->1)-6-O-trityl-alpha-D-glucopyranoside (3) was synthesized via stannylation of 6,1',6'-tri-O-tritylsucrose with dibutyltin oxide in benzene, followed by treatment of the crude product with methanesulfonyl chloride in the presence of triethylamine in dichloromethane at 0 degrees C. A similar treatment of the tri-tritylsucrose in toluene, instead of benzene, yielded 4-O-mesyl-1,6-di-O-trityl-beta-D-fructofuranosyl-(2-->1)-6-O-trityl-alpha-D-glucopyranoside (4) as the major product. The X-ray crystal structure of the corresponding acetyl derivative, 3-O-acetyl-4-O-mesyl-1,6-di-O-trityl-beta-D-fructofuranosyl-(2-->1)-2,3,4-tri-O-acetyl-6-O-trityl-alpha-D-glucopyranoside (5), confirms the position and stereochemistry of the methanesulfonyl group at C-4 of the fructofuranosyl ring.     

Isolation and structural analysis of ajugose from Vigna mungo L

The hexasaccharide ajugose, alpha-D-galactopyranosyl-(1-->6)-alpha-D-galactopyranosyl-(1-->6)-O-alpha-D-galactopyranosyl-(1-->6)-alpha-D-galactopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1<-->2)-beta-D-fructofuranoside, generally uncommon in legumes, was detected in the seeds of Vigna mungo L. by TLC and paper chromatography. Ajugose was then isolated by silica gel chromatography and its structure was established by acid and enzymatic hydrolysis, fast atom bombardment mass spectrometry and both one- and two-dimensional 1H and 13C NMR techniques.     

Production of fructooligosaccharides by mycelium-bound transfructosylation activity present in Cladosporium cladosporioides and Penicilium sizovae

Different filamentous fungi isolated from molasses and jams (kiwi and fig) were screened for fructooligosaccharides (FOS) producing activity. Two strains, identified as Penicilium sizovae (CK1) and Cladosporium cladosporioides (CF₂15), were selected on the basis of the FOS yield and kestose/nystose ratio. In both strains the activity was mostly mycelium-bound. Starting from 600 g/L of sucrose, maximum FOS yield was 184 and 339 g/L for P. sizovae and C. cladosporioides, respectively. Interestingly, the highest FOS concentration with C. cladosporioides was reached at 93% sucrose conversion, which indicated a notable transglycosylation to hydrolysis ratio. The main FOS in the reaction mixtures were identified by HPAEC–PAD chromatography. C. cladosporioides synthesized mainly 1-kestose (158 g/L), nystose (97 g/L), ¹F-fructosylnystose (19 g/L), 6-kestose (12 g/L), neokestose (10 g/L) and a disaccharide (34 g/L) that after its purification and NMR analysis was identified as blastose [Fru-β(2 → 6)-Glc]. P. sizovae was very selective for the formation of ¹F-FOS (in particular 1-kestose) with minor contribution of neoFOS and negligible of levan-type FOS.