Purification and characterization of a feruloyl esterase from the fungus Penicilliumexpansum

An extracellular phenolic acid esterase produced by the fungus Penicillium expansum in solid state culture released ferulic and ρ-coumaric acid from methyl esters of theacids, and from the phenolic-carbohydrate esters O-[5-O-(trans-feruloyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose (FAXX) and O-[5-O-((E)-ρ-coumaroyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose(PAXX). The esterase was purified 360-fold in successive stepsinvolving ultrafiltration and column chromatography by gel filtration, anion exchange andhydrophobic interaction. These chromatographic methods separated the phenolic acid esterasefrom α- l -arabinofuranosidase, pectate and pectin lyase, polygalacturonase,xylanase and β- d -xylosidase activities. The phenolic acid esterase had an apparentmass of 65 kDa under non-denaturing conditions and a mass of 57·5 kDa underdenaturing conditions. Optimal pH and temperature were 5·6 and 37 °C,respectively and the metal ions Cu2+ and Fe³+ atconcentrations of 5 mmol l⁻¹ inhibited feruloyl esterase activity by 95% and44%, respectively, at the optimum pH and temperature. The apparent K_m and V_max of the purified feruloyl esterase for methyl ferulate at pH 5·6 and 37 °Cwere 2·6 mmol l⁻¹ and 27·1 μmol min⁻¹ mg⁻¹. The corresponding constants of ρ-coumaroylesterase for methyl coumarate were 2·9 mmol l⁻¹ and 18·6μmol min−1 mg⁻¹.     

Chemical Constituents of Schisandra rubriflora Rehd. et Wils.

Schisandra rubriflora Rehd. et Wils. is a traditional Chinese medicine. To search for new and bioactive components from traditional Chinese medicines and provide scientific evidence for taxonomy, the chemical constituents of the plant were investigated by various column chromatography methods (silica gel, Sephadex LH-20, and RP-18). From the aerial parts of S. rubriflora, three new megastigmane glycosides,namely (3S, 5R, 6S, 9R)-megastigmane-3, 9-diol 3-O-[α-L-arabionfuranosyl-(1→6)-β-D-glucopyranoside](1), 7-megastigmene-3-ol-9-one 3-O-[α-L-arabionfuranosyl-(1→6)-β-D-glucopyranoside] (2), and megastigmane-3α, 4β, 9ξ-tfiol 3-O-β-D-glucopyranoside (3), along with 14 known compounds, were isolated.The structures of the new compounds were elucidated by a combination of spectroscopic and chemical methods.     

A New Steroidal Glycoside from Ophiopogon japonicus （Thunb.） Ker-Gawl.

To study the chemical constituents from traditional Chinese herb Ophiopogon japonicus （Thunb.） Ker-Gawl., a new steroidal glycoside, named ophiopojaponin C （1）, together with two known ones, was isolated by column chromatography. Spectroscopic and chemical evidence revealed the structures to be ophiopogenin 3-O-[α-L-rhamnopyranosyl（1→2）]-β-D-xylopyranosyl（1→4）-β-D-glucopyranoside （1）, diosgenin 3-O-[2-O-acetyl-α-L-rhamnopyranosyl（1→2）]-β-D-xylopyranosyl（1→3）-β-D-glucopyranoside （2）, and ruscogenin 1-O-[2-O-acetyl-α-L-rhamnopyranosyl（1→2）]-β-D-xylopyranosyl（1→3）-β-D-fucopyranoside （3）.     

Two New Saponins from Lysimachia capillipes Hemsl.

To investigate the saponins from whole plants of Lysimachia capillipes Hemsl., two new saponins, named capilliposide E (1) and capilliposide F (2), were isolated. The structures of the new sa ponins were elucidated as 3 β, 22α-dihydroxy- 16α-acetat-28→ 13 -lactone-oleanane-3 -O- [β-D-glucopyranosyl(1→2)-α-L-arabinpyranoyl]-22-O-β-D-glucopyranoside (1) and 3 β, 22α-dihydroxy- 16α-acetat-28→ 13-1actone-oleanane-3-O- { [β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)]-α-L-arabinpyranoyl }-22-O-βD-glucopyranoside (2). The structures of these compounds were determined by 1D- and 2D-NMR, MS techniques, and chemical methods.     

The ftsQ1p gearbox promoter of Escherichia coli is a major sigma S-dependent promoter in the ddlB–ftsA region

We have isolated the ypfP gene (accession number P54166) from genomic DNA of Bacillus subtilis Marburg strain 60015 ( Freese and Fortnagel, 1967 ) using PCR. After cloning and expression in E. coli , SDS–PAGE showed strong expression of a protein that had the predicted size of 43.6 kDa. Chromatographic analysis of the lipids extracted from the transformed E. coli revealed several new glycolipids. These glycolipids were isolated and their structures determined by nuclear magnetic resonance (NMR) and mass spectrometry. They were identified as 3-[ O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl]-1,2-diacylglycerol, 3-[ O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl]-1,2-diacylglycerol and 3-[ O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl]-1,2-diacylglycerol. The enzymatic activity expected to catalyse the synthesis of these compounds was confirmed by in vitro assays with radioactive substrates. In these assays, one additional glycolipid was formed and tentatively identified as 3-[ O -β- D -glucopyranosyl]-1,2-diacylglycerol, which was not detected in the lipid extract of transformed cells. Experiments with some of the above-described glycolipids as ¹⁴C-labelled sugar acceptors and unlabelled UDP-glucose as glucose donor suggest that the ypfP gene codes for a new processive UDP-glucose: 1,2-diacylglycerol-3-β- D -glucosyl transferase. This glucosyltransferase can use diacylglycerol, monoglucosyl-diacylglycerol, diglucosyldiacylglycerol or triglucosyldiacylglycerol as sugar acceptor, which, apart from the first member, are formed by repetitive addition of a glucopyranosyl residue in β (1→6) linkage to the product of the preceding reaction.     

圆果雪胆中的皂甙成分

从圆果雪胆（Ｈｅｍｓｌｅｙａ ａｍａｂｉｌｉｓ）的块茎中分离到１０个化合物,其中３个雪胆皂甙是首次从该种植物中得到,它们的结构通过光谱和化学的方法鉴定为齐墩果酸－３－Ｏ－α－吡喃阿拉伯糖（１→３）－β－葡萄糖醛酸甙,２８－Ｏ－β－Ｄ－吡喃葡萄糖齐墩果酸３－Ｏ－α－吡喃阿拉伯糖（１→３）－β－Ｄ－葡萄糖醛酸,２８－Ｏ－β－Ｄ－吡喃葡萄糖齐墩果酸３－Ｏ－「β－Ｄ－吡喃葡萄糖（１→２）」－「α－吡喃阿     

Purification and characterization of an intracellular β-glucosidase from a new strain of Leuconostoc mesenteroides isolated from cassava

The lactic acid bacterium, Leuconostoc mesenteroides, when grown on an arbutin-containing medium, was found to produce an intracellular β-glucosidase. The enzyme was purified by chromatofocusing, ion-exchange chromatography and gel filtration. The molecular mass of the purified intracellular β-glucosidase, as estimated by gel filtration, was 360 kDa. The tetrameric structure of the β-glucosidase was determined following treatment of the purified enzyme with dodecyl sulphate (SDS). The intracellular β-glucosidase exhibited optimum catalytic activity at 50°C and pH 6 with citrate–phosphate buffer, and 5·5 with phosphate buffer. The enzyme was active against glycosides with (1→4)-β, (1→4)-α and (1→6)-α linkage configuration. From Lineweaver–Burk plots, K _m values of 0·07 mmol l⁻¹ and 3·7 mmol l⁻¹ were found for p -nitrophenyl-β- D -glucopyranoside and linamarin, respectively. The β-glucosidase was competitively inhibited by glucose and by D -gluconic acid–lactone and a glucosyl transferase activity was observed in the presence of ethanol. The β-glucosidase of Leuconostoc mesenteroides, with cyanogenic activity, could be of potential interest in cassava detoxification, by hydrolysing the cyanogenic glucosides present in cassava pulp.     

Effect of alfalfa saponins and flavonoids on pea aphid

We studied the development and feeding behaviour of the pea aphid, Acyrthosiphon pisum (Harris) (Homoptera: Aphididae), on the Radius and Sapko alfalfa ( Medicago sativa L.) (Fabaceae) cultivars. Three saponins and flavones were identified in the alfalfa cultivars after thin layer chromatography separation. Cultivar Radius differed from Sapko in that it had a higher level of saponins, including zanhic acid tridesmoside and 3-GlcA,28-AraRhaXyl medicagenic acid glycoside. The flavones identified, including 7- O -β-D-glucuronopyranosyl-4'- O- [2'- O- E-feruloyl- O -β-D-glucuronopyranosyl(1→2)- O -β-D-glucuronopyranoside] apigenin, 7- O -{2- O- E-feruloyl-[β-D-glucuronopyranosyl(1→3)]- O -β-D-glucuronopyranosyl(1→2)- O -β-D-glucuronopyranoside} apigenin, and 4'- O- [2'- O -E-feruloyl- O -β-D-glucuronopyranosyl(1→2)- O -β-D-glucuronopyranoside] apigenin, occurred in tissues of both alfalfa cultivars. However, cv. Radius had very low mean flavonoid concentrations in comparison to cv. Sapko. Pea aphids that fed on cv. Radius plants showed a reduction in reproduction and survival. The aphid pre-reproductive period on cv. Radius was prolonged and the reproductive and post-reproductive periods on cv. Radius were reduced, compared to those on cv. Sapko. Cultivar Radius also negatively influenced aphid probing behaviour. This was especially the case during the initial period of the pathway phase. The results suggested that alfalfa cv. Radius, which had a higher level of saponins and a lower level of flavonoids, was less accepted by the pea aphid.     

New Triterpenic Glucoside from Beesia calthaefolia  (Maxim.) Ulbr. Native to China

Five compounds (1-5) were isolated from the rhizome of Beesia calthaefolia (Maxim.) Ulbr. Based on chemical and spectral evidence, their structures were determined as beesic acid (9-phenyl-2E, 4E, 6E, 8E-nontetraenoic acid, 1), vanillic acid (2), oleanolic acid-3-O-α-L-arabinopyranosyl-28-O-α-L -rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (3), hederasaponin B (oleanolicacid-3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl-28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester, 4) and beesioside Q (oleanolic acid-3-O-β-D -glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl-28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester, 5), respectively. Compound 1 was isolated from natural sources for the first time and compound 5 was a new compound.     

A β-(1→4)-xylosyltransferase involved in the synthesis of arabinoxylans in developing barley endosperms

A β-(1→4)-xylosyltransferase (XylTase; EC 2.4.2.24) participating in the synthesis of arabinoxylans was investigated using microsomal membranes prepared from developing barley ( Hordeum vulgare L.) endosperms. The microsomal fraction transferred Xyl from uridine 5'-diphosphoxylose (UDP-Xyl) into exogenous β-(1→4)-xylooligosaccharides derivatized at their reducing ends with 2-aminopyridine. HPLC analysis showed chain elongation of pyridylaminated β-(1→4)-xylotriose (Xyl₃-PA) by repeated attachment of one to five single xylosyl residues depending on the reaction time, leading to the formation of Xyl₄₋₈-PA. Methylation analysis and enzymatic digestions with β-xylosidase (EC 3.2.1.37) and endo -β-(1→4)-xylanase (EC 3.2.1.8) confirmed that the transfer of xylosyl residues into the newly synthesized products occurred through β-(1→4)-linkages. The activity of the XylTase was maximal at pH 6.8 and 20°C and most enhanced in the presence of 0.5% Triton X-100 and 5 m M MnCl₂. The apparent Michaelis constant and maximal velocity of the enzyme for Xyl₃-PA were 2.1 m M and 25 400 pmol min⁻¹ mg protein⁻¹, respectively. The enzyme also transferred [¹⁴C]Xyl from UDP-[¹⁴C]Xyl into higher β-(1→4)-xylooligosaccharides and birchwood xylans through β-(1→4)-linkages. The enzyme activity varied according to the stage of development (7–35 days after flowering) of the endosperms. Maximal activity occurred at 13–16 days; no activity was detectable in mature seeds. A comparison of endosperms from 10 different cultivars of barley harvested 11–22 days after flowering showed no correlation between enzyme activity and the amount of Xyl in the cell walls.     

Three New Flavone Glycosides from Drymaria diandra B1.

In order to find new structural and biologically active compounds, the constituents from the whole plant of Drymaria diandra B1. （Caryophyllaceae） were investigated and three new flavone glycosides, named drymariatins B （1）, C （2）, and D （3）, were isolated by solvent partition, Si gel, sephadex LH-20, and Rp- 18 column chromatography. Using spectroscopic methods, including two-dimensional nuclear magnetic resonance analysis, the structures of these compounds were elucidated as 6-C-（2-deoxy-β-D-fucopyranosyl）- 5,7,4＇-trihydroxyl-flavone, 6-C-（2-deoxy-β-D-fucopyranosyl）-7-O-（β-D-glucopyranosyl）-5,4＇-dibydroxyl- flavone, and 6-C-（3-keto-β-digitoxopyranosyl）-7-O-（β-D-glucopyranosyl）-5,4＇-dihydroxyl-flavone.     

Isolation and characterization of the carbohydrate moiety bound to N-7-mercaptoheptanoyl-O-threonine phosphate

Abstract Component B ( N -7-mercaptoheptanoyl-threonine- O -3-phosphate) (HS-HTP) which is an absolute requirement in the methylcoenzyme M methylreductase reaction was found to be part of a complex UDP-disaccharide when isolated carefully from cell-free supernant of Methanobacterium thermoautotrophicum . The site of attachment of HS-HTP to the UDP-disaccharide was through a carboxylic-phosphoric anhydride linkage of the C-6 mannosaminuronic acid to the phosphate group in HS-HTP. This bond is quite labile and this may account for the fact that the intact molecule, called methyl reducing factor (MRF) was not isolated previously. The structure of MRF was determined by combined fast atom bombardment mass spectrometry and ¹H-, ¹³C-, and ³¹P-NMR spectroscopy and assigned as: uridine 5'-[ N -7-mercaptoheptanoyl- O -3-phosphothreonine(2-acetamido-2-deoxy- β -mannopyranuronosyl)acid anhydride]-(1 → 4)- O -2-acetamido-2-deoxy α -glucopyranosyl diphosphate.     

A spirostane hexaglycoside from Agave cantala fruits.

A new steroidal glycoside, agaveside D, isolated from the fruits of Agave cantata was characterized as 3β-{- -rhamnopyranosyl-(1→2), β- -glycopyranosyl-(1→3)-β- -glucopyranosyl[β- -xylopyransoyl-(1→4)-- -rhamnopyranosyl-(1→2)]-β- -glucopyranosyl}-25R-5- spirostane on the basis of chemical degradation and spectrometry.     

Triterpenoid saponins from Meryta lanceolata

Four new oleanane-type saponins and a known one were isolated from the leaves and stems of Meryta lanceolata. The new saponins were characterised by spectroscopic means and chemical hydrolysis as 3-O-[beta-D-glucopyranosyl-(1-->3)-beta-D-glucopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl]oleanolic acid 28-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl] ester, 3-O-[beta-D- glucopyranosyl-(1-->3)-beta-D-glucopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl]oleanolic acid 28-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-6-O-acetyl glucopyranosyl-(1-->6)-beta-D-glucopyranosyl] ester, 3-O-[beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->3)-beta-D-glucopyranosyl-(1-->3)-alpha-L-arabinopyranosyl]oleanolic acid 28-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl] ester and 3-O-[beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->3)-beta-D-glucopyranosyl-(1-->3)-alpha-L-arabinopyranosyl]echinocystic acid 28-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl] ester. The NMR assignments were made by means of HOHAHA, 1H-1H COSY, HMQC, HMBC and NOE difference studies.     

Endo-(1 → 3)β-D-glucanase activity secreted by Bacillus sp. no. 215

The production of an extracellular endo-(1 → 3)-β-D-glucanase by Bacillus sp. no. 215 was induced during growth with (1 → 3)-β-D-glucan (curdlan) from Cellulomonas flavigena strain KU as carbon and energy source. Maximum levels of activity (0.26 U ml^-1 resp. 1.40 U mg^-1) were detected in cell-free culture supernatant fluid after 25 h of aerobic growth at 55°C. The cells secreted an endo-(1 → 3)-β-D-glucanase with low electrophoretic mobility that used curdlan from C. flavigena strain KU and from Agrobacterium sp. (formerly Alcaligenes faecalis var. myxogenes ) as substrates. The enzyme activity was highest at pH 7.0 and 55°C. It exhibited a remarkably low thermal stability with a half-life of 14 min at 55°C in the presence of substrate. Divalent metal cations were required for enzyme activity.     

古蔺雪胆中的新三萜皂苷

从采自四川汉源县的古蔺雪胆(Hemsleya penxianensis var.gulinensiks)中分到9个三萜皂苷化合物,通过化学反应和光谱方法鉴定了它们的结构。其中7个为已知化合物,分别为齐墩果酸-28-O-β-D-比喃葡萄糖苷(1),3-O-β-D-吡喃葡萄糖醛基齐墩果酸苷(3),3-O-β-D-吡喃葡萄糖醛基—齐墩果酸—28-O-α-L-吡喃阿拉伯糖苷(4),3-O-β-D-吡喃葡萄糖醛基—齐墩果酸—28-O-β-D-吡喃葡萄糖苷(5),3-O-α-L-阿拉伯糖基—(1→3)—β—D-吡喃葡萄糖醛基—齐墩果酸—28—O—β—D—吡喃葡萄糖苷(6),3—O—(6′—丁酯)—β-D-吡喃葡萄糖醛基—齐墩果酸—28-O-α-L-阿拉伯糖苷(7),3-O-(6′-丁酯)—β—D吡喃葡萄糖醛基—齐墩果酸—28-O-β-D-吡喃葡萄糖苷(8)。两个新化合物,即雪胆皂苷A(2)和雪胆皂苷B(9)。     

An exo-β-d-glucanase derived from Zea coleoptile walls with a capacity to elicit cell elongation

Cell wall proteins were extracted from maize coleoptiles, Zea mays L. B37 x MO 17, with high concentrations of LiCl. Ion-exchange, chromatofocusing and gel-filtration chromatography were employed extensively to purify exo-β-glucanase activity from the extract. The purified enzyme functioned as an exo-(1→3)-β-glucanase (E.C. 3.2.1.58) and as a glucosidase (E.C. 3.2.1.21) capable of extensive hydrolysis of the native Zea wall (1→3), (1→4)-β- d -glucan, yielding glucose as the final product. The exoglucanase also enhances elongation of maize coleoptile sections in both the presence and absence of exogenous IAA.     

Lupane-triterpene glycosides from leaves of Acanthopanax koreanum

Two new lupane-triterpene glycosides named acankoreosides C and D, were isolated from the leaves of Acanthopanax koreanum. Based on spectroscopic data, the chemical structures were determined as 3-O-β- -glucopyranosyl 3α,11α-dihydroxylup-20(29)-en-28-oic acid 28-O-α- -rhamnopyranosyl-(1→4)-β- -glucopyranosyl-(1→6)-β- -glucopyranosyl ester and 3α,11α-dihydroxylup-23-al-20(29)-en-28-oic acid 28-O-α- -rhamnopyranosyl-(1→4)-β- -glucopyranosyl-(1→6)-β- -glucopyranosyl ester, respectively.     

Triterpenoid saponins from Schefflera arboricola

Nine triterpenoid saponins were isolated from the leaves and stems of Schefflera arboricola. The saponins were characterised, on the basis of chemical and spectral evidence, as 3-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucuronopyranosyl] oleanolic acid, 3-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucuronopyranosyl] echinocystic acid, 3-O-[beta-D-apiofuranosyl-(1-->4)-beta-D-glucuronopyranosyl] oleanolic acid 28-O-beta-D-glucopyranosyl ester, 3-O-alpha-L-ramnopyranosyl-(1-->4)-[alpha-L-arabinopyranosyl-(1-->2)-] beta-D-glucuronopyranosyl oleanolic acid, 3-O-alpha-L-rhamnopyranosyl-(1-->4)-[alpha-L-arabinopyranosyl-(1-->2)-] beta-D-glucuronopyranosyl oleanolic acid 28-O-beta-D-glucopyranosyl ester, 3-O-alpha-L-rhamnopyranosyl-(1-->4)-[beta-D-galactopyranosyl-(1-->2)-] beta-D-glucuronopyranosyl oleanolic acid, 3-O-alpha-L-rhamnopyranosyl-(1-->4)-[beta-D-galactopyranosyl-(1-->2)-] beta-D-glucuronopyranosyl oleanolic acid 28-O-beta-D-glucopyranosyl ester, 3-O-beta-D-apiofuranosyl-(1-->4)-[alpha-L-arabinopyranosyl-(1-->2)-] beta-D-glucuronopyranosyl oleanolic acid and 3-O-beta-D-apiofuranosyl-(1-->4)-[alpha-L-arabinopyranosyl-(1-->2)-] beta-D-glucuronopyranosyl oleanolic acid 28-O-beta-D-glucopyranosyl ester.     

Xanthone O-glycosides from Polygala tenuifolia

Four xanthone O-glycosides, polygalaxanthones IV–VII were isolated from the roots of Polygala tenuifolia Willd., together with eight known compounds. The structures of the four xanthone O-glycosides were established as 6-O-[α- -rhamnopyranosyl-(1→2)-β- -glucopyranosyl]-1-hydroxy-3,7-dimethoxyxanthone (polygalaxanthone IV), 6-O-[α- -rhamnopyranosyl-(1→2)-β- -glucopyranosyl]-1,3-dihydroxy-7-methoxyxanthone (polygalaxanthone V), 6-O-(β- -glucopyranosyl)-1,2,3,7-tetramethoxyxanthone (polygalaxanthone VI), and 3-O-[α- -rhamnopyranosyl-(1→2)-β- -glucopyranosyl]-1,6-dihydroxy-2,7-dimethoxyxanthone (polygalaxanthone VII), respectively, on the basis of analysis of spectroscopic evidence.