Monoacylglycerols: glycolipid biosurfactants produced by a thermotolerant yeast, Candida ishiwadae.

AIMS: To isolate and characterize biosurfactants produced by a thermotolerant yeast isolated in Thailand. MATERIALS AND RESULTS: Yeast strains isolated from plant material in Thailand were first screened for the ability to produce lipase and biosurfactant. A strain Y12, identified as Candida ishiwadae by physiological tests, survived at 45 degrees C and produced relatively large amounts of biosurfactants. From the culture filtrate of this strain, two glycolipid biosurfactants, a and b, were purified by solvent fractionation, silica gel and ODS column chromatographies. Compounds a and b were determined to be monoacylglycerols; 1-linoleylglycerol and 1-oleylglycerol, respectively. Both compounds exhibited higher surfactant activities tested by the drop collapse test than several artificial surfactants such as sodium dodecyl sulphate. CONCLUSIONS: Glycolipid biosurfactants produced by a thermotolerant yeast, C. ishiwadae were characterized to be monoacylglycerols which exhibited high surfactant activities. SIGNIFICANCE AND IMPACT OF THE STUDY: A thermotolerant yeast strain, C. ishiwadae, could be a potential candidate for producing monoacylglycerols which are useful in industrial applications.

Synthesis of a novel lysophosphatidylcholine.

The novel lysophosphatidylcholine (1), which naturally occurs in the marine sponge Spirastrella abata and was reported to inhibit cholesterol biosynthesis in the Chang liver cell, has been synthesized in four steps from methyl cis-11-octadecenoate (2).

Structural features of N-glycans linked to royal jelly glycoproteins: structures of high-mannose type, hybrid type, and biantennary type glycans.

The structures of N-glycans of total glycoproteins in royal jelly have been explored to clarify whether antigenic N-glycans occur in the famous health food. The structural feature of N-glycans linked to glycoproteins in royal jelly was first characterized by immunoblotting with an antiserum against plant complex type N-glycan and lectin-blotting with Con A and WGA. For the detail structural analysis of such N-glycans, the pyridylaminated (PA-) N-glycans were prepared from hydrazinolysates of total glycoproteins in royal jelly and each PA-sugar chain was purified by reverse-phase HPLC and size-fractionation HPLC. Each structure of the PA-sugar chains purified was identified by the combination of two-dimensional PA-sugar chain mapping, ESI-MS and MS/MS analyses, sequential exoglycosidase digestions, and 500 MHz 1H-NMR spectrometry. The immunoblotting and lectinblotting analyses preliminarily suggested the absence of antigenic N-glycan bearing beta1-2 xylosyl and/or alpha1-3 fucosyl residue(s) and occurrence of beta1-4GlcNAc residue in the insect glycoproteins. The detailed structural analysis of N-glycans of total royal jelly glycoproteins revealed that the antigenic N-glycans do not occur but the typical high mannose-type structure (Man(9 to approximately 4)GlcNAc2) occupies 71.6% of total N-glycan, biantennary-type structures (GlcNAc2Man3 GlcNAc2) 8.4%, and hybrid type structure (GlcNAc1 Man4GlcNAc2) 3.0%. Although the complete structures of the remaining 17% N-glycans; C4, (HexNAc3 Hex3HexNAc2: 3.0%), D2 (HexNAc2Hex5HexNAc2: 4.5%), and D3 (HexNAc3Hex4HexNAc2: 9.5%) are still obscure so far, ESI-MS analysis, exoglycosidase digestions by two kinds of beta-N-acetylglucosaminidase, and WGA blotting suggested that these N-glycans might bear a beta1-4 linkage N-acetylglucosaminyl residue.

Antinematodal activity of some tropical rainforest plants against the pinewood nematode, Bursaphelenchus xylophilus.

Sixty five methanolic extracts of Sumatran rainforest plants representing 63 species of 21 families were assayed in vivo for antinematodal activity against Bursaphelenchus xylophilus using our cotton ball-fungal mat method. Extracts of 27 plants species from 14 families exhibited antinematodal activity, while 37 species were inactive. Among them, three extracts of Bischofia javanica, Knema hookeriana and Areca catechu exhibited very strong activity at minimum effective dose (MED) of 0.7 mg/cotton ball (mg/bl.). Eight extracts from Allamanda cathartica, Ervatamia corymbosa, Hoya diversifolia, Bischofia javanica, Derris malacensis, Melastoma melabathricum, Ophiorriza konsteleary and Brucea sumatrana also showed strong activity (MED, 5 mg/bl.).

Two antinematodal phenolics from Knema hookeriana, a Sumatran rainforest plant.

The activity-guided chromatographic purification of the methanol extract of Knema hookeriana, using pine wood nematodes Bursaphelenchus xylophilus has successfully led to the isolation and characterization of two phenolic antinematodal compounds with minimum effective dose (MED) of 4.5 and 20 microg/cotton ball (microg/bl.) or 0.018 and 0.073 microM/cotton ball (microM/bl.), respectively. Based on their chemical and spectral properties, these compounds were determined to be 3-undecylphenol (1) and 3-(8Z-tridecenyl)-phenol (2). These compounds were isolated for the first time from this species, and 2 seems to be a novel compound.

Effective production of dehydro cyclic dipeptide albonoursin exhibiting pronuclear fusion inhibitory activity. II. Biosynthetic and bioconversion studies.

Albonoursin production was greatly enhanced when cyclo (L-Leu-L-Phe) (CFL), a tetrahydro derivative of albonoursin, was added to the 2-day culture of an albonoursin-producing actinomycete, Streptomyces albulus KO-23. The increase in albonoursin production paralleled the amount of CFL added. Furthermore, the resting cells of the strain catalyzed the bioconversion of CFL to albonoursin. The optimum pH and temperature for the conversion were found to be pH 10.0 and 50 degrees C. The feeding experiments and the resting-cell reactions revealed that albonoursin is biosynthesized by dehydrogenation of CFL in the actinomycete. This is the first report for a dehydrogenation of amino acid residues at the alpha,beta-positions in cyclic dipeptides.

Biosynthetic intermediates of the tetradehydro cyclic dipeptide albonoursin produced by Streptomyces albulus KO-23.

The cell-free extract of an albonoursin-producing strain Streptomyces albulus KO-23 catalyzes the conversion of cyclo(L-Leu-L-Phe) (1) to albonoursin (2). At the early stage of this conversion, two compounds were newly formed prior to albonoursin synthesis in the reaction mixture. These compounds were isolated and identified as (Z)-3-benzylidene-6-isobutyl-2,5-piperazinedione (4) and (Z)-3-benzyl-6-isobutylidene-2,5-piperazinedione (3). The cell-free extract also catalyzed the conversion of compound 3 or 4 to albonoursin. From these results, albonoursin was found to be biosynthesized via these compounds from cyclo(L-Leu-L-Phe). These didehydro diketopiperazines exhibited no inhibitory activity toward the first cleavage of sea urchin embryo in contrast to the higher cytotoxicity for albonoursin, indicating that dehydrogenation at alpha,beta-positions of both amino acid residues in diketopiperazines is required for cytotoxicity.

Novel diketopiperazine metabolism in a microorganism: two-step hydrolysis of cyclo(Gly-Leu) to amino acids and preliminary characterization of cyclo(Gly-Leu) hydrolase and dipeptidase.

A bacterium, strain NM 5-3, isolated from soil exhibited the highest cyclo(Gly-Leu) (CGL)-hydrolyzing activity and was identified as Agrobacterium radiobacter. The reaction products from CGL were dipeptides (Leu-Gly and Gly-Leu) and amino acids (Leu and Gly). Inhibitors for the dipeptidase of this strain did not inhibit the hydrolysis of CGL to dipeptides, indicating that two distinct enzymes, CGLase and a dipeptidase, were involved in its hydrolysis. The activities of these two enzymes were separated by anion-exchange column chromatography. The results indicated that strain NM5-3 hydrolyzed CGL via the dipeptides to the corresponding amino acids. The CGLase fraction was found to catalyze the hydrolysis of cyclo(Gly-D-Leu), cyclo(Gly-Gly), cyclo(L-Ala-Gly), and cyclo(D-Ala-Gly). On the other hand, the dipeptidase fraction exhibited L-specific substrate specificity.

Enzymatic conversion of cyclic dipeptides to dehydro derivatives that inhibit cell division.

The cell-free extract of an albonoursin-producing strain, Streptomyces albulus KO-23, was found to catalyze the conversion of several cyclic dipeptides having Phe and aliphatic side chain-containing amino acid residues to the corresponding dehydro derivatives. 3Z-Benzylidene-6S-methyl-2,5-piperazinedione, 3Z-benzylidene-2,5-piperazinedione, and 3Z, 6Z-dibenzylidene-2,5-piperazinedione were prepared by this conversion system. Among the dehydro cyclic dipeptides prepared, tetradehydro derivatives exhibited inhibitory activity toward the first cleavage of sea urchin embryo, while didehydro derivatives did not. We previously found that cyclo(Leu-Phe) and its didehydro derivatives did not show any inhibitory activity, in contrast to high activity in the case of albonoursin. Taken together, these findings indicate that dehydrogenation at the alpha,beta-positions of both amino acid residues in this type of cyclic dipeptide is required for the inhibitory activity.

Inhibition of Plant Transformation by Phytolaccoside B from Phytolacca americana Callus.

The newly established GUS expression bioassay on the callus extracts of 22 species of plants revealed that the methanol extract of Phytolacca americana callus had the most potent inhibitory activity against agrobacterial plant transformation. A triterpene glycoside phytolaccoside B was isolated from the extract as a genuine plant transformation inhibitor having neither antiagrobacterial nor phytotoxic activity. This compound is promising for use as a biochemical probe for studies on the plant transformation mechanism.

Novel bioactive oxazolomycin isomers produced by Streptomyces albus JA3453.

Two novel oxazolomycin isomers, oxazolomycins B (2) and C (3), were isolated from the fermentation broth of an oxazolomycin-producing strain, Streptomyces albus JA3453. Both compounds are geometrical isomers of oxazolomycin (1), the configurations of their triene moieties being (4'E, 6'E, 8'E) (2) and (4'Z, 6'E, 8'E) (3) while that of oxazolomycin (1) is (4'Z, 6'Z, 8'E). Compounds 2 and 3 exhibited potent inhibitory activity against crown gall formation with the same MIC (0.8 microgram/disk) as oxazolomycin. Compounds 2 and 3 showed no antibacterial activity against Agrobacterium tumefaciens, in contrast to oxazolomycin which has specific anti-A. tumefaciens activity.

Synthesis of a novel sialic acid derivative (sialylphospholipid) as an antirotaviral agent.

A novel sialylphospholipid (SPL) was synthesized from N-acetylneuraminic acid (NeuAc) and phosphatidylcholine (PC) by a chemical and enzymatic method and evaluated as an inhibitor of rotavirus. PC and 1,8-octanediol were conjugated by transesterification reaction of Streptomyces phospholipase D (PLD) under a water-chloroform biphasic system to afford phosphatidyloctanol, which was condensed with a protected 2-chloro-2-deoxyneuraminic acid derivative by using silver trifluoromethanesulfonate as an activator in chloroform and converted, after deprotection, to SPL. Rhesus monkey kidney cells (MA-104) were incubated with simian (SA-11 strain) and human (MO strain) rotaviruses in the presence of SPL, and the cells infected were detected indirectly with anti-rotavirus antibody. SPL showed dose dependent inhibition against both virus strains. The concentrations required for 50% inhibition (IC50) against SA-11 and MO were 4.35 and 16.1 microM, respectively, corresponding to 10(3)- and 10(4)-fold increases in inhibition as compared to monomeric NeuAc.