Production of a heterologous endo-1,4-beta-xylanase in the yeast Pichia stipitis with an O(2)-regulated promoter

The Cryptococcus albidus XLN-gene (encoding endo-1,4-β-xylanase) was expressed in the yeast Pichia stipitis under the control of the PsADH2-promoter, which is activated under O₂ limitation. The resulting transformant produced endo-1,4-β-xylanase after a shift to anoxic conditions. Endo-1,4-β-xylanase production was enhanced by limited aeration after the shift.     

根霉TC1653产纤维素酶提纯及酶学性质研究

对根霉所产纤维素酶酶系进行了分析并研究了部分酶学性质。实验选择超滤和凝胶柱分离相结合的方式提纯纤维素酶,结果显示根霉TC1653纤维素酶系是一个完全酶系,具有一个较为明显的内切葡聚糖酶组分。β-葡萄糖苷酶组分的最适反应温度为70℃,温度高于70℃时,活性迅速下降,但在这种高温下具有最高反应活性的酶很少见,很可能又是一种新的β-葡萄糖苷酶。     

Simultaneous saccharification and fermentation of cellulose: effect of β- -glucosidase activity and ethanol inhibition of cellulases

Compared with saccharification in the absence of yeast, simultaneous saccharification and fermentation (SSF) using Trichoderma cellulases and Saccharomyces cerevisiae enhanced cellulose hydrolysis rates by 13–30%. The optimum temperature for SSF was 35°C. The requirement for β- -glucosidase (β- -glucoside glucohydrolase, EC 3.2.1.21) in SSF was lower than for saccharification: maximal ethanol production was attained when the ratio of the activity of β- -glucosidase to filter paper activity was 1.0. Ethanol inhibited cellulases uncompetitively, with an inhibition constant of 30.5 gl ⁻¹, but its effect was less severe than that of an equivalent concentration of cellobiose or glucose. No irreversible denaturation of cellulases [1,4-(1,3;1,4)-β- -glucan 4-glucanohydrolase, EC 3.2.1.4] by ethanol was observed.     

Study on the use of soya bean polysaccharide degrading enzymes in broiler nutrition

Several non-starch polysaccharide (NSP)-degrading enzymes were studied for their suitability as feed enzymes in combination with soya bean meal (SBM) in broiler nutrition. In vitro experiments with SBM and several enzymes isolated from an Aspergillus aculeatus multi-enzyme preparation or respective enzyme specificities showed that only enzymes of the 1,4-β-arabinogalactanase type led to effective solubilization of dry matter from SBM, while other enzymes needed very high concentrations to invoke this effect. 1,4-β-galactomannanase activity also showed a high rate of sugar release from SBM, but the release of sugars from treated SBM (simulated stomach digestion with pepsin) was reduced, while an 1,4-β-arabinogalactanase from Humicola insolens still led to a high release of sugars on the same substrate. Synergistic effects on the release of sugars were observed when both galactanase and galactomannanase enzymes were used. Synergistic effects of galactanase and mannanase were also noted for two different endopolygalacturonases, partly purified from the multi-enzyme preparation.

In feeding trials with broilers, increasing concentrations of the multi-enzyme preparation with numerous NSP-degrading enzymes showed beneficial effects on feed intake, but high in feed concentrations were needed. Further feeding trials with the galactanase preparation from H. insolens alone and in combination with a mannanase preparation (Gammanase) were conducted. However, due to weak in vivo stability of the galactanase, only a small effect on feed conversion was noticed. Additional supplementation of the mannanase reduced live weight gain and feed intake.

It is concluded that enzymes of the 1,4-β-arabinogalactanase type are the most likely candidates for a SBM-specific feed enzyme in broiler nutrition, possibly in combination with endopolygalacturonases. However, while a partial degradation of soya NSP may enhance digestibility, a high degree of hydrolysis may produce oligo- and monomers with anti-nutritive effects such as osmotic pressure or microbial fermentation in the small intestine.     

Immobilization of fungus Aspergillus sp. by a novel cryogel technique for production of extracellular hydrolytic enzymes

Aerobic cells of a fungus isolate Aspergillus sp. CX-1 have been immobilized in macroporous cryoPAG and in different composite cryoPAGs — fibrous adjunct carriers. The productivity of the extracellular enzymes (exo-1.4-β-glucanase, endo-1.4-β-glucanase, β-glucosidase and xylanase), and the viability, growth and ultrastructure of the immobilized fungus have been studied. The enzyme activities and stability during long-term repeated batch cultivation in the immobilized fungus were higher than in free mycelia when batch cultivated. The fungus immobilized in the composite cryoPAG, containing polypropylene non-woven fabric, possessed the highest exo-1.4-β-glucanase activity, the longest durability of enzyme production (85 days) and the most reliable mechanical strength. The fungus immobilized in porous composite cryogel possessed a variety of advantages including easy control of cryogel porosity, improved mechanical strength and durability, simplicity of construction, high enzyme productivity and high stability.     

A Proteomic Approach Provides New Insights into the Control of Soil-Borne Plant Pathogens by Bacillus Species

Beneficial microorganisms (also known as biopesticides) are considered to be one of the most promising methods for more rational and safe crop management practices. We used Bacillus strains EU07, QST713 and FZB24, and investigated their inhibitory effect on Fusarium. Bacterial cell cultures, cell-free supernatants and volatiles displayed varying degrees of suppressive effect. Proteomic analysis of secreted proteins from EU07 and FZB24 revealed the presence of lytic enzymes, cellulases, proteases, 1,4-β-glucanase and hydrolases, all of which contribute to degradation of the pathogen cell wall. Further proteomic investigations showed that proteins involved in metabolism, protein folding, protein degradation, translation, recognition and signal transduction cascade play an important role in the control of Fusarium oxysporum. Our findings provide new knowledge on the mechanism of action of Bacillus species and insight into biocontrol mechanisms.     

Six new C21 steroidal glycosides from Asclepias curassavica L

Six new C₂₁ steroidal glycosides, named curassavosides A–F (3–8), were obtained from the aerial parts of Asclepias curassavica (Asclepiadaceae), along with two known oxypregnanes, 12-O-benzoyldeacylmetaplexigenin (1) and 12-O-benzoylsarcostin (2). By spectroscopic methods, the structures of the six new compounds were determined as 12-O-benzoyldeacylmetaplexigenin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (3), 12-O-benzoylsarcostin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (4), sarcostin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (5), sarcostin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-digitoxopyranoside (6), 12-O-benzoyldeacylmetaplexigenin 3-O-β-d-glucopyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (7), and 12-O-benzoylsarcostin 3-O-β-d-glucopyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (8), respectively. All compounds (1–8) were tested for in vitro cytotoxicity; only compound 3 showed weak inhibitory activity against Raji and AGZY cell lines.     

The structures of six urinary oligosaccharides that are characteristic for a patient with morquio syndrome type b

Morquio syndrome type B is an inherited, lysosomal storage disease characterised by a marked deficiency in acid β-d-galactosidase, while the 2-acetamido-2-deoxy-β-d-galactose 6-sulphate sulphatase activity is normal. Urinary oligosaccharides were studied in order to evaluate the effect of the diminished β-d-galactosidase activity on the catabolism of glycoconjugates and to compare their structures with those excreted by patients with GM₁-gangliosidosis. The following oligosaccharides were isolated: β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→6)-β-d-Manp-(1→4)- d-GlcpNAc (1), β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→6)-[α-d-Manp- (1→3)]-β-d-Manp-(1→4)-d-GlcpNAc (2a), β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)- α-d-Manp-(1→3)-[α-d-Manp-(1→6)]-β-d-Manp-(1→4)-d-GlcpNAc (2b), β-d-Galp- (1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→3)-[β-d-Galp-(1→4)-β-d-GlcpNAc-(1→ 2)-α-d-Manp-(1→6)]-β-d-Manp-(1→4)-d-GlcpNAc (3), β-d-Galp-(1→4)-β-d-Glcp- NAc-(1→2)-α-d-Manp-(1→3)-{β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-[β-d-Galp- (1→4)-β-d-GlcpNAc-(1→6)]-α-d-Manp-(1→6)}-β-d-Manp-(1→4)-d-GlcpNAc (4), β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→3)-[β-d-GlcpNAc-(1→4)]-[β- d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→6)]-β-d-Manp-(1→4)-d-Glcp- NAc (5). Significant differences between Morquio syndrome type B and GM₁-gangliosidosis have been observed, with regard to the excretion rate and the specific structures of urinary oligosaccharides. Compounds 2a, 2b, and 5 are novel members of the series of oligosaccharides isolated from the urine of patients with inherited, lysosomal storage diseases.     

Genomic organization of four β-1,4-endoglucanase genes in plant-parasitic cyst nematodes and its evolutionary implications

The genomic organization of genes encoding β-1,4-endoglucanases (cellulases) from the plant-parasitic cyst nematodes Heterodera glycines and Globodera rostochiensis (HG-eng1, Hg-eng2, GR-eng1, and GR-eng2) was investigated. HG-eng1 and GR-eng1 both contained eight introns and structural domains of 2151 and 2492 bp, respectively. HG-eng2 and GR-eng2 both contained seven introns and structural domains of 2324 and 2388 bp, respectively. No significant similarity in intron sequence or size was observed between HG-eng1 and HG-eng2, whereas the opposite was true between GR-eng1 and GR-eng2. Intron positions among all four cyst nematode cellulase genes were conserved identically in relation to the predicted amino acid sequence. HG-eng1, GR-eng1, and GR-eng2 had several introns demarcated by 5′-GC…AG-3′ in the splice sites, and all four nematode cellulase genes had the polyadenylation and cleavage signal sequence 5′-GAUAAA-3′—both rare occurences in eukaryotic genes. The 5′- flanking regions of each nematode cellulase gene, however, had signature sequences typical of eukaryotic promoter regions, including a TATA box, bHLH-type binding sites, and putative silencer, repressor, and enhancer elements. Database searches and subsequent phylogenetic comparison of the catalytic domain of the nematode cellulases placed the nematode genes in one group, with Family 5, subfamily 2, glycosyl hydrolases from Scotobacteria and Bacilliaceae as the most homologous groups. The overall amino acid sequence identity among the four nematode cellulases was from 71 to 83%, and the amino acid sequence identity to bacterial Family 5 cellulases ranged from 33 to 44%. The eukaryotic organization of the four cyst nematode cellulases suggests that they share a common ancestor, and their strong homology to prokaryotic glycosyl hydrolases may be indicative of an ancient horizontal gene transfer.     

Changes in levels of mRNAs of transforming growth factor (TGF)-β1, -β2, -β3, TGF-β type II receptor and sulfated glycoprotein-2 during apoptosis of mouse uterine epithelium

To examine the roles played by transforming growth factors (TGF)-β1, -β2, -β3, and TGF-β type II receptors in the induction of apoptosis in the mouse uterine epithelium after estrogen deprivation, we investigated the expression of their mRNAs and the mRNA of sulfated glycoprotein-2 (SGP-2). Pellets containing 100 μg estradiol-17β (E₂) were implanted into ovariectomized mice and removed four days later. Apoptotic indices (percentage of apoptotic cells) of both luminal and glandular epithelia increased after E₂ pellets were removed, but administration of progesterone (P), 5-dihydrotestosterone (DHT), or continued implantation of E₂ pellets suppressed this increase. Levels of mRNAs of TGF-β1, -β2, and -β3, and SGP-2 did not increase after estrogen deprivation. However, estrogen deprivation caused a gradual increase in the level of TGF-β type II receptor mRNA, and its level increased about six-fold six days later. Moreover, E₂, P, and DHT markedly decreased the level of TGF-β type II receptor mRNA. In situ hybridization demonstrated that mRNAs of TGF-β1, -β2, -β3 and TGF-β type II receptor were localized to the epithelium. Exogenous administration of TGF-β1 into the uterine stroma induced apoptosis in the epithelium, a finding that suggests that signals produced by TGF-βs can induce apoptosis. Therefore, the present results suggest that increased sensitivity of uterine epithelial cells to TGF-βs, as demonstrated by an increase in TGF-β type II receptor mRNA, is involved in the induction of apoptosis after estrogen deprivation, although signals produced by TGF-βs do not appear sufficient to induce apoptosis.     

Isolation and characterization of a (1 → 3)-β-glucan endohydrolase from germinating barley (Hordeum vulgare): amino acid sequence similarity with barley (1 → 3, 1 → 4)-β-glucanases

A (1 → 3)-β-glucan 3-glucanohydrolase (EC 3.2.1.39) has been purified approx. 190-fold from extracts of germinating barley. The enzyme has an apparent M_r 32 000, a pI of 8.6, and a pH optimum of 5.6. Analysis of hydrolysis products released from the (1 → 3)-β-glucan, laminarin, shows that the enzyme is an endohydrolase. Sequence analysis of the 46 NH₂-terminal amino acids of the (1 → 3)-β-glucanase reveals 54% positional identity with barley (1 → 3,1 → 4)-β-glucanases (EC 3.2.1.73) and suggests a common evolutionary origin for these two classes of β-glucan endohydrolases. The barley (1 → 3)-β-glucanase also exhibits significant similarity with a (1 → 3)-β-glucanase from tobacco.     

Investigation of the acetolysis products of the sulphated polysaccharide of Aeodes ulvoidea

Investigation of the acetolysis products of the sulphated polysaccharide of the seaweed Aeodes ulvoidea led to the isolation and characterization of the following oligosaccharides: 3-O-α- -galactopyranosyl- -galactose (1), 3-O-(2-O-methyl-α- -galactopyranosyl)- -galactose (2), 4-O-β- -galactopyranosyl-2-O-methyl- -galactose (3), 4-O-β- -galactopyranosyl-2-O-methyl- -galactose (4), O-β- -galactopyranosyl-(1→4)-O-α- -galactopyranosyl-(1→3)- -galactose (5), O-α- -galactopyranosyl-(1→3)-O-β- -galactopyranosyl-(1→4)- -galactose (6), O-α- -galactopyranosyl-(1→3)-O-β- -galactopyranosyl-(1→4)-2-O-methyl- -galactose (7), O-(2-O-methyl-α- -galactopyranosyl)-(1→3)-O-β- -galactopyranosyl-(1→4)-2-O-methyl- -galactose (10), and O-α- -galactopyranosyl-(1→3)-O-β- -galactopyranosyl-(1→4)-O-α- -galactopyranosyl-(1→3)- -galactose. In addition, the isolation of a tetrasaccharide possessing alternating - and -galactose residues demonstrates the hitherto unexpected presence of -galactose in the polysaccharide. The structure of the polysaccharide is discussed.     

Volume-regulated Cl- channels in human pleural mesothelioma cells

A novel xyloglucan-specific endo-β-1,4-glucanase (XEG), xyloglucanase, with a molecular mass of 80 kDa and a pI of 4.8, was isolated from the fungus Geotrichum sp. M128. It was found to be an endoglucanase active toward xyloglucan and not active toward carboxymethylcellulose, Avicel, or barley 1,3-1,4-β-glucan. Analysis of the precise substrate specificity using various xyloglucan oligosaccharide structures revealed that XEG has at least four subsites (−2 to +2) and specifically recognizes xylose branching at the +1 and +2 sites. The full-length cDNA encoding XEG was cloned and sequenced. It consists of a 2436-bp open reading frame encoding a 776-amino acid protein. From its deduced amino acid sequence, XEG can be classified as a family 74 glycosyl hydrolase. The cDNA encoding XEG was then expressed in Escherichia coli, and enzymatically active recombinant XEG was obtained.     

Purification and Characterization of Wall-bound Exo-l,3-{beta}-D-Glucanase from Barley (Hordeum vulgare L.) Seedlings

A ß-D-glucanase activity hydrolyzing 1,3:1,4-ß-D-glucanwas released from the cell walls of barley by 3M LiCl treatment.It was purified by sequential cation-exchange, gel-filtrationand hydrophobic chromatography. The molecular mass of the glucanasewas 66 kDa as determined by SDS-polyacrylamide gel electrophoresis.Sequence determination of the first thirty amino acids of theN-terminus revealed a high homology of this enzyme to the Pseudomonasl,4-ß-D-glucosidase (56.5%). The purified ß-D-glucanasehas a pH optimum at 5.0, and hydrolyzes oligosaccharides containingß-D-1,3 or ß-D-1,4 linkage. The glucanaseshowed maximum hydrolytic activity toward laminaritetraose,the rate being about two times that of cellotetraose and aboutfour times that of gentiobiose. Polysaccharides such as lichenan,l,3:l,4-ß-D-glucan (from barley), laminarin and pustulanare also hydrolyzed, but not carboxylmethyl-curdlan, carboxymethyl-cellulose,xyloglucan and maltose. The purified ß-D-glucanaseyielded monomeric glucose from laminarihexaose, and exhibitedcharacteristics of an exo-l,3-ß-D-glucanase (EC 3.2.1.58 [EC] ).The activity and biochemical characteristics of this enzymesuggest that it is an exo-l,3-ß-D-glucanase involvedin the rapid turnover of l,3:l,4-ß-D-glucan in barleycell walls during seedling growth. (Received September 24, 1996; Accepted December 9, 1996)     

Biotransformation of steviol by cultured cells of eucalyptus perriniana and Coffea arabica

A new biotransformation product, steviol 19-β-gentiobiosyl ester, together with steviol 19-β-glucopyranosyl ester and steviol-13-O-β-glucopyranoside 19-β-glucopyranosyl ester (rubusoside), was isolated from Eucalyptus perriniana jar fermentor culture following the administration of steviol. Only rubusoside was isolated as a biotransforination product of steviol from Coffea arabica cell suspension culture.     

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.     

Synthesis of part of the antigenic repeating-unit of streptococcus pneumoniae type II

Condensation of methyl 4-O-acetyl-3-O-(2,3,4-tri-O-acetyl-α- -rhamnopyranosyl)-α- -rhamnopyranoside with 2,3,4,6-tetra-O-benzyl-α- -glucopyranosyl chloride gave a mixture of methyl O-[2,3,4,6-tetra-O-benzyl-α- (4) and -β- -glucopyranosyl]-(1→2)-O-[(2,3,4-tri-O-acetyl-α- -rhamnopyranosyl)-(1→3)]-4-O-acetyl-α- -rhamnopyranoside (9) in 43:7 proportion in 63% yield. After chromatographic separation, removal of the benzyl and acetyl groups gave methyl O-α- -glucopyranosyl-(1→2)-[O-α- -rhamnopyranosyl-(1→3)]-α- -rhamnopyranoside and the β anomer. Removal of benzyl groups of 4 was followed by tritylation, acetylation, and detritylation of the α- -glucopyranosyl group, and finally condensation with benzyl (2,3,4-tri-O-benzyl- -glucopyranosyl chloride)uronate gave a mixture of two tetrasaccharides (15 and 16), containing the α- and β- -glucopyranosyluronic acid groups in the ratio 81:19, and an overall yield of 71%. After chromatographic separation, alkaline hydrolysis and hydrogenation of 15 gave methyl O-α- -glucopyranosyluronic acid-(1→6)-O-α- -glucopyranosyl-(1→2)-[O-α- -rhamnopyranosyl-(1→3)]-α- -rhamnopyranoside. The β- anomer was obtained by similar treatment of 16. 6-O-α- -glucopyranosyluronic acid-α,β- -glucopyranose was synthesized as a model compound.     

Cellulase, Fruit Softening and Abscission in Red RaspberryRubus idaeusL. cv Glen Clova

The ripening of raspberry fruit (Rubus ideausL. cv Glen Clova)is associated with a climacteric rise in ethylene production.As the fruit pigments change from green to red there is a progressivesoftening, loss of skin strength and a breakdown of cell wallsin the mesocarp. An increase in cellulase (endo-1,4-ß-D-glucanase)in both drupelets and receptacles accompanies these changes.The localization of cellulase in the regions of the fruit associatedwith abscission zones suggest the enzyme may be involved infruit separation as well as softening. Rubus idaeusL; raspberry; fruit ripening; ethylene; abscission; cell wall breakdown; cellulase; endo-1,4-ß-D-glucanase     

Reactivity of {(Ph3P)Pt[μ-η-H---SiH(Ar)]}2 (Ar=2-isopropyl-6-methylphenyl) with phosphines. X-ray crystal structure of trans-{(dppe)Pt[μ-SiH(Ar)]}2

The dinuclear Pt---Si complex {(Ph₃P)Pt{μ-η²-H---SiH(IMP)]}₂ (trans-1a–cis-1b=3:1; IMP=2-isopropyl-6-methylphenyl) reacted with basic phosphines such as 1,2-bis(diphenylphosphino)ethane (dppe) and dimethylphenylphosphine (PMe₂Ph) to afford different dinuclear Pt---Si complexes with loss of H₂, {(P)₂Pt[μ-SiH(IMP)]}₂ [P=dppe, trans-2a (major), cis-2b (trace); PMe₂Ph, 3 (trans only)]. Complexes 2 and 3 were characterized by multinuclear NMR spectroscopy and X-ray crystallography (2a). In contrast, the reaction of 1a,b with the sterically demanding tricyclohexylphosphine (PCy₃) afforded {(Cy₃P)Pt{μ-η²-H---SiH(IMP)]}₂ (trans-4a–cis-4b 2:1) analogous to 1a,b where the central Pt₂Si₂(μ-H)₂ core remains intact but the PPh₃ ligands have been replaced by PCy₃. Complexes 4a and 4b was characterized by multinuclear NMR and IR spectroscopies.