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.     

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.     

Coleoptile growth-inducing capacities of exo-β-(1→3)-glucanases from fungi

An exo-β-(1β3)-glucanase derived from Selerotinia libertiana induced growth of Avena sativa coleoptiles and degraded hemicelluloses and β-(1→4):(1→3) mixed linked glucan. However, neither endo-β-(1→4)- nor endo-β-(1→3)-glucanase activity could be detected in the enzyme preparation. Nojirimycin inhibited the glucan degradation caused by the enzyme but glucono-1,5-lactone did not. Another exo-β-(1→3)-glucanase derived from Basidiomycete QM 806 did not induce coleoptile growth and did not degrade the glucan. Growth-inducing properties of exo-β-(1→3)-glucanases are discussed.     

Genetic and structural analyses of Escherichia coli O107 and O117 O-antigens

The O-antigen, consisting of many repeats of an oligosaccharide, is an essential component of the lipopolysaccharide on the surface of Gram-negative bacteria. The O-antigen is one of the most variable cell constituents, and different O-antigen forms are almost entirely due to genetic variations in O-antigen gene clusters. In this paper, we present structural and genetic evidence for a close relationship between Escherichia coli O107 and E. coli O117 O antigens. The O-antigen of E. coli O107 has a pentasaccharide repeating unit with the following structure: →4)-β- d -Gal p NAc-(1→3)-α- l -Rha p -(1→4)-α- d -Glc p NAc-(1→4)-β- d -Gal p -(1→3)-α- d -Gal p NAc-(1→, which differs from the known repeating unit of E. coli O117 only in the substitution of d -GlcNAc for d -Glc. The O-antigen gene clusters of E. coli O107 and O117 share 98.6% overall DNA identity and contain the same set of genes in the same organization. It is proposed that one cluster was evolved from another via mutations, and the substitution of a few amino acids residues in predicted glycosyltransferases resulted in the functional change of one such protein for transferring different sugars in O107 ( d -GlcNAc) and O117 ( d -Glc), leading to different O-antigen structures. This is an example of the O-antigen alteration caused by nucleotide mutations, which is less commonly reported for O-antigen variations.     

Structure and expression of a barley acidic β-glucanase gene

A barley acidic -1,3-glucanase gene was recovered from a barley genomic library by homology with a partial cDNA of barley basic -1,3-glucanase isoenzyme GII. The gene, Abg2, is homologous to the PR2 family of pathogenesis-related -1,3-glucanase genes. The ABG2 protein has 81% amino acid similarity to barley basic -1,3-glucanase GII. The ABG2 protein is encoded as a preprotein of 336 amino acids including a 28 amino acid signal peptide. A 299 bp intron occurs within codon 25. The mature ABG2 protein has a predicted mass of 32642 Da and a calculated isoelectric point of 4.9. The second exon of the Abg2 gene shows a strong preference for G+C in the third position of degenerate codons. The Abg2 gene was functionally expressed in Escherichia coli. Abg2 mRNA is constitutively expressed in barley root; leaf expression of Abg2 mRNA is induced by mercuric chloride and infection by Erysiphe graminis f. sp. hordei. Southern blot analysis indicates that Abg2 is a member of a small gene family.     

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）.     

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.     

Seven members of the (1→3)-β-glucanase gene family in barley (Hordeum vulgare) are clustered on the long arm of chromosome 3 (3HL)

Members of the (13)--glucan glucanohydrolase (EC 3.2.1.39) gene family have been mapped on the barley genome using three doubled haploid populations and seven wheat-barley addition lines. Specific probes or polymerase chain reaction (PCR) primers were generated for the seven barley (13)--glucanase genes for which cDNA or genomic clones are currently available. The seven genes are all located on the long arm of chromosome 3 (3HL), and genes encoding isoenzymes GI, GII, GIII, GIV, GV and GVII (ABG2) are clustered in a region less than 20 cM in length. The region is flanked by the RFLP marker MWG2099 on the proximal side and the Barley Yellow Mosaic Virus (BYMV) resistance gene ym4 at the distal end. The gene encoding isoenzyme GVI lies approximately 50 cM outside this cluster, towards the centromere. With the exception of the gene encoding isoenzyme GIV, all of the (13)--glucanase genes are represented by single copies on the barley genome. The probe for the isoenzyme GIV gene hybridized with four DNA bands during Southern blot analysis, only one of which could be incorporated into the consensus linkage map.     

Comparative study of extracellular and intracellular β-glucosidases of a new strain of Zygosaccharomyces bailii isolated from fermenting agave juice

A yeast strain isolated in the laboratory was studied and classified as a Zygosaccharomyces bailii. Both intracellular and extracellular β-glucosidases of this yeast were purified by ion-exchange chromatography, gel filtration and hydroxylapatite (only for the intracellular enzyme). The tetrameric structure of the two β-glucosidases was determined following treatment of the purified enzyme with dodecyl sulphate. The intracellular β-glucosidase exhibited optimum activity at 65°C and pH 5.5. The extracellular enzyme exhibited optimum catalytic activity at 55°C and pH 5. The molecular mass of purified intracellular and extracellular β-glucosidases, estimated by gel filtration, was 440 and 360 kDa, respectively. Both enzymes are active against glycosides with (1 → 4)-β, (1 → 6)-β and (1 → 4)-α linkage configuration. The intracellular enzyme possesses (1 → 6)-α-arabinofuranosidase activity and extracellular enzyme (1 → 6)-α-rhamno-pyranosidase activity. The two β-glucosidases are competitively inhibited by glucose and by D-gluconic-acid-lactone and a slight glucosyl transferase activity is observed in the presence of ethanol. Since the glycosides present in wine and fruit juices represent a potential source of aromatic flavour, the possible use of the yeast β-glucosidases for the liberation of the bound aroma is discussed.     

Regulation of genes encoding β-d-glucan glucohydrolases in barley (Hordeum vulgare)

Expression patterns of barley β - d -glucan glucohydrolase genes were monitored using cDNAs encoding isoenzymes ExoI and ExoII. The cDNAs were isolated from 5-day-old seedling libraries. The enzymes are encoded by a small gene family, in which marked differences in codon usage are evident. The cDNAs can be used as specific probes for two subfamilies of β - d -glucan glucohydrolase genes. Genes of both subfamilies are transcribed in the scutellum of germinated grain, in elongating coleoptiles, and in young roots and leaves. Low levels of mRNA for the isoenzyme ExoI gene subfamily could be detected in aleurone layers of germinated grain. Most of the β - d -glucan glucohydrolase activity can be extracted from tissues with dilute aqueous buffers. Enzyme activity is highest in young leaves and elongating coleoptiles, but is not well-correlated with mRNA levels. The expression patterns are consistent with proposed roles for β -glucan glucohydrolases in the turnover or modification of cell-wall (1→3,1→4)- β - d -glucans in elongating coleoptiles and in young vegetative tissues.     

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.     

Glycosides from Roots of Cyathula officinalis Kuan

Abstract: To search for new and bioactive compounds from traditional Chinese medicines, a new glycoside, 3-O-[α- L -rhamnopyranosyl-(1→3)-( n -butyl-β- D -glucopyranosiduronate)]-28-O-β- D -glucopyranosyloleanolic acid ( 1 ), was isolated from the roots of Cyathula officinalis Kuan, along with 3-O-(methyl-β- D -glucopyranosiduronate)-28-O-β- D -glucopyranosyl oleanolic acid ( 2 ), 3-O-β- D -glucopyranosyl oleanolic acid ( 3 ), 3-O-β- D -glucuronopyranosyl oleanolic acid ( 4 ), 3-O-[β- L -rhamnopyranosyl-(1→3)-(β- D -glucuronopyranosyl)] oleanolic acid ( 5 ), 3-O-(β- D -glucuronopyranosyl)-28-O-β- D -glucopyranosyl oleanolic acid ( 6 ), 28-O-β- D -glucuronopyranosyl-(1→4)-β- D -glucopyranosyl hederagenin ( 7 ), 3-O-[β- L -rhamnopyranosyl-(1→3)-β- D -glucuronopyranosyl]-28-O-β- D -glucopyranosyl oleanolic acid ( 8 ), and 3-O-[β- D -glucopyranosyl-(1→2)-α- L -rhamnopyranosyl-(1→3)-β- D -glucuronopyranosyl]-28-O-β- D -glucopyranosyl oleanolic acid ( 9 ). The structures of these compounds were determined based on spectral and chemical evidence. The 50 per cent growth-inhibiting (GI₅₀) of compounds 1 and 5 against MDA-MB-231 (a human breast cancer cell line) was 3.44 × 10^-4 and 4.66 × 10^-4 mol/L, respectively.
(Managing editor: Wei WANG)     

Proteolytic inactivation of an extracellular (1 → 3)-β-glucanase from the fungus Acremonium persicinum is associated with growth at neutral or alkaline medium pH

Abstract The filamentous fungus Acremonium persicinum released high levels of proteolytic enzyme activity into the culture fluid during growth at pH 7 or above. Almost total inhibition of this crude activity by phenylmethylsulfonyl fluoride suggested that it was mainly due to the presence of a serine protease. This protease inactivated one of three extracellular (1 → 3)- β -glucanases produced by this fungus, although the activities of the remaining two (1 → 3)- β -glucanases did not appear to be affected. Growth of A. persicinum in acidic conditions resulted in the presence of much lower extracellular proteolytic activity and no apparent (1 → 3)- β -glucanase inactivation.     

Purification of (1→3)-β-glucan endohydrolase isoenzyme II from germinated barley and determination of its primary structure from a cDNA clone

A (13)--D-glucan 3-glucanonydrolase (EC 3.2.1.39) of apparent M _r 32 000, designated GII, has been purified from germinated barley grain and characterized. The isoenzyme is resolved from a previously purified isoenzyme (GI) on the basis of differences in their isoelectric points; (13)--glucanases GI and GII have pI values of 8.6 and 10.0, respectively. Comparison of the sequences of their 40 NH₂-terminal amino acids reveals 68% positional identity. A 1265 nucleotide pair cDNA encoding (13)--glucanase isoenzyme GII has been isolated from a library prepared with mRNA of 2-day germinated barley scutella. Nucleotide sequence analysis of the cDNA has enabled the complete primary structure of the 306 amino acid (13)--glucanase to be deduced, together with that of a putative NH₂-terminal signal peptide of 28 amino acid residues. The (13)--glucanase cDNA is characterized by a high (G+C) content, which reflects a strong bias for the use of G or C in the wobble base position of codons. The amino acid sequence of the (13)--glucanase shows highly conserved internal domains and 52% overall positional identity with barley (13, 14)--glucanase isoenzyme EII, an enzyme of related but quite distinct substrate specificity. Thus, the (13)--glucanases, which may provide a degree of protection against microbial invasion of germinated barley grain through their ability to degrade fungal cell wall polysaccharides, appear to share a common evolutionary origin with the (13, 14)--glucanases, which function to depolymerize endosperm cell walls in the germinated grain.     

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.     

Sequential cell wall transformations in response to the induction of a pedicel abscission event in Euphorbia pulcherrima (poinsettia)

Alterations in the detection of cell wall polysaccharides during an induced abscission event in the pedicel of Euphorbia pulcherrima (poinsettia) have been determined using monoclonal antibodies and Fourier transform infrared (FT-IR) microspectroscopy. Concurrent with the appearance of a morphologically distinct abscission zone (AZ) on day 5 after induction, a reduction in the detection of the LM5 (1→4)-β- d -galactan and LM6 (1→5)-α- l -arabinan epitopes in AZ cell walls was observed. Prior to AZ activation, a loss of the (1→4)-β- d -galactan and (1→5)-α- l -arabinan epitopes was detected in cell walls distal to the AZ, i.e. in the to-be-shed organ. The earliest detected change, on day 2 after induction, was a specific loss of the LM5 (1→4)-β- d -galactan epitope from epidermal cells distal to the region where the AZ would form. Such alteration in the cell walls was an early, pre-AZ activation event. An AZ-associated de-esterification of homogalacturonan (HG) was detected in the AZ and distal area on day 7 after induction. The FT-IR analysis indicated that lignin and xylan were abundant in the AZ and that lower levels of cellulose, arabinose and pectin were present. Xylan and xyloglucan epitopes were detected in the cell walls of both the AZ and also the primary cell walls of the distal region at a late stage of the abscission process, on day 7 after induction. These observations indicate that the induction of an abscission event results in a temporal sequence of cell wall modifications involving the spatially regulated loss, appearance and/or remodelling of distinct sets of cell wall polymers.     

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.     

Regulation of nitrate reduction in a cyanobacterium Phormidium uncinatum: Distinctive modes of ammonium-repression of nitrate and nitrite reductases

Abstract A 5.8 kbp DNA fragment from Clostridium cellulovorans (ATCC 35296) containing endo-β-1,4-glucanase (1,4-β- d -glucan glucanohydrolase, carboxymethylcellulase, CMCase; EC 3.2.1.4) gene, engD was cloned in Escherichia coli . The clone harboring a subcloned 3.8 kb fragment in plasmid, pEQ52V, produced an enzyme that showed both endo-β-1,4-glucanase activity as well as cellobiosidase activity. Zymograms with the engD encoded enzyme with carboxymethyl-cellulose as the substrate indicated that the molecular mass of the active protein was 50 000.     

Structure of the genes encoding Hordeum vulgare (1→3,1→4)-β-glucanase isoenzymes I and II and functional analysis of their promoters in barley aleurone protoplasts

Summary Barley (1 3,1 4)--glucanase isoenzyme II is synthesized in the aleurone cells during germination and secreted into the endosperm for hydrolysis of the cell walls. Its synthesis is stimulated by gibberellic acid (GA₃) and repressed by abscisic acid. The gene for isoenzyme I is expressed in the aleurone, scutellum and prominently in young leaves. Close functional relatedness between the two enzymes is attested by 92 % identity at the level of the amino acid sequence. The structural genes for the two enzymes each contain a large intron of 2505 by and 2952 bp, respectively, in the codon for amino acid 25 of the 28-residue signal peptide. During evolution, homologous regions of the two introns have changed position and orientation. Furthermore, a large palindromic sequence of 327 by in the 5 end of the intron is present only in the gene for isoenzyme II. In transient expression assays using barley aleurone protoplasts and chloramphenicol acetyl transferase as reporter the promoter of the isoenzyme I gene showed no response to GA₃. However, removal of a unique 151 by region extending from positions –402 to –552 upstream of the TATA box permitted low levels of GA₃-induced expression of the reporter gene, suggesting a silencer function for this domain. High levels of GA₃-responsive expression were obtained in aleurone protoplasts using the promoter of the gene encoding isoenzyme II. Truncation of this promoter revealed that sequences located within 253 bp upstream from the TATA box are sufficient to direct GA₃-stimulated expression. Using the homologous barley aleurone protoplast transfection assay, it was possible to reproduce the in vivo expression characteristics of the genes for the barley (1 3,1 4)--glucanase isoenzymes I and II with reporter gene constructs.