Isolation of a β-glucosidase binding and activating polysaccharide from cell walls of Trichoderma reesei

The extracellular -glucosidase from the filamentous fungus Trichoderma reesei QM 9414 is mainly bound to the cell wall of the fungus and only partially released into the medium. Isolation of the cell walls and its hydrolysis by enzymatic treatment with Aspergillus niger cellulase released -glucosidase, which appeared tightly associated with a cell wall polysaccharide. This polysaccharide was purified by gel filtration and ion exchange chromatography and was shown to consist of mannose, galactose, glucose, galacturonic acid and glucuronic acid. It was devoid of protein and phosphate. It reassociated both with extracellular -glucosidase as well as -glucosidase released from the fungus' cell wall. Addition of the polysaccharide to the -glucosidase in vitro increased the enzyme's activity against 4-nitrophenyl--glucoside twofold. These findings suggest, that the isolated polysaccharide functions as an anchor glycan for the -glucosidase in Trichoderma reesei.     

Cell wall localization of dihydroflavonol-glucoside β-glucosidase in flowers of Petunia hybrida

Limbs of flower buds from Petunia hybrida were investigated for -glucosidase activity with dihydroflavonol-glucosides and 4-methyl-umbelliferyl--D-glucoside as substrates. Dihydroflavonol-glucoside -glucosidase is localized in the cell wall. This activity has an acid pH optimum and is also active toward 4-methyl-umbelliferyl--glucoside. Besides this activity a neutral -glucosidase is present. This activity is soluble and is not active toward dihydroflavonol-glucosides. Using starch gel electrophoresis it was shown that no difference in -glucosidase activity is present between mutants able to convert dihydroflavonols into anthocyanins and mutants accumulating dihydroflavonol-glucosides. It is concluded that -glucosidase activity is not involved in anthocyanin synthesis.Abbreviations 4MU--glc 4-methylumbelliferyl--D-glucopyranoside - dHQ-7-g dihydroquercetin-7-glucoside - dHQ-4-g dihydroquercetin-4-glucoside - dHM-4-g dihydromyricetin-4-glucoside Deceased     

Transglycosylation reactions by exoglycosidases from the termite Macrotermes subhyalinus

The ability of four exoglycosidases (-galactosidase, -glucosidase, -glucosidase and invertase) from the termite Macrotermes subhyalinus to catalyse tranglycosylation reactions was tested using lactose, cellobiose, maltose and sucrose as glycosyl donors and 2-phenylethanol as glycosyl acceptor. The experimental conditions were optimized in relation to the time course of the reaction, pH and concentrations of glycosyl donor and acceptor. Whereas the hydrolytic activity was largely predominant over the transferase activity with -galactosidase and -glucosidase, the transglycosylation activity represented 68% with -glucosidase. In addition, as demonstrated by the transglycosylation product formed, the hydrolysis of sucrose was catalysed by -glucosidase and not by invertase. On the basis of this work, -glucosidase from M. subhyalinus appears to be a valuable tool for the preparation of neoglycoconjugates.     

Ein fluoreszenzmikroskopischer Nachweis von β-Glucosidase in Wurzeln vonCicer arietinum (L.)

Zusammenfassung Die Lokalisierung von -Glucosidasen in Wurzeln der Kichererbse [Cicer arietinum (L.)] wurde in einem einstufigen fluoreszenz-optischen Verfahren mit 4-Methyl-umbelliferyl-D-glucosid untersucht. Am Beispiel der Kichererbse wird gezeigt, daß in polyphenolhaltigen Pflanzen herkömmliche Azokupplungsverfahren zur Glucosidaselokalisierung nicht anwendbar sind. Die mit der neuen Methode erhaltenen Ergebnisse decken sich im wesentlichen mit denen aus vergleichbaren Untersuchungen und zeigen, daß -Glucosidasen nicht vorhanden sind. Aryl--Glucosidasen wurden vorwiegend im äußeren Cortexbereich gefunden und nehmen mengenmäßig zur Wurzelspitze hin zu.

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Localization of -glucosidase in roots ofCicer arietinum (L.) by a fluorescence technique

Summary The localization of -glucosidases in roots of garbanzo beans [Cicer arietinum (L.)] has been investigated by a one-step fluorescence optical procedure using 4-methylumbelliferyl--D-glucoside. It is shown that the well known azo-coupling test for the localization of glucosidases cannot be applied in polyphenol containing plants. The results obtained with the new method are comparable with those described in other investigations and further show that -glucosidases are absent from the root tissues investigated. The aryl--glucosidases were mainly detected in the outer region of the cortex and quantitatively increase towards the root tip.

     

α-and β-Glycosidases in maize roots

Four glycosidases were analyzed in 10 mm apical segments prepared from growing roots (15 mm) of Zea mays L. The pH optima were found to be 5.8 for -glucosidase, 4.4 for -galactosidase, 6.4 for -glucosidase and 6.0 for -galactosidase. The -glucosidase showed 4-fold higher activity than the -galactosidase. The distribution of the -glucosidase activity was signifcantly different from that of the -galactosidase, -glucosidase and -galactosidase.Abbreviations -Glu -glucosidase - -Gal -galactosidase - -Glu -glucosidase - -Gal -galactosidase     

Isolation and characterization of β-glucosidases from Aspergillus nidulans mutant USDB 1183

Two extracellular -glucosidases (cellobiase, EC 3.2.1.21), I and II, from Aspergillus nidulans USDB 1183 were purified to homogeneity with molecular weights of 240,000 and 78,000, respectively. Both hydrolysed laminaribiose, -gentiobiose, cellobiose, p-nitrophenyl--L-glucoside, phenyl--L-glucoside, o-nitrophenyl--L-glucoside, salicin and methyl--L-glucoside but not -linked disaccharides. Both were competitively inhibited by glucose and non-competitively (mixed) inhibited by glucono-1,5-lactone. -Glucosidase I was more susceptible to inhibition by Ag⁺ and less inhibited by Fe²⁺ and Fe³⁺ than -glucosidase II.     

Comparison of the ability of β-glucosidases from almonds and Fusarium oxysporum to produce n-alkyl-β-glucosides in organic solvents

Summary The effect of water activity on the synthesis of n-alkyl-D-glucosides through condensation of glucose and n-alcohols has been studied using the commercially available almond -glucosidase and -glucosidase isolated from Fusarium oxysporum. The two enzymes exhibited a different water activity optimum. The specificity and alcohol reactivity of the two enzymes have also been investigated. Both enzymes prefer primary alcohols. -Glucosidase from F. oxysporum presents a higher affinity for primary alcohols with alkyl chain length of 4–6, whereas in the case of almond -glucosidase both initial velocity and yield decrease when the carbon chain length increases.     

Cloning and expression ofClostridium stercorarium cellulase genes inEscherichia coli

Summary A genomic library of the cellulolytic thermophileClostridium stercorarium NCIB 11745 was constructed inEscherichia coli using the cosmid vector pHC79. Recombinant clones expressing cellulase components were identified by screening for hydrolysis of carboxymethyl cellulose (CMC), 4-methylumbelliferyl--D-cellobioside, and 4-methylumbelliferyl--glucoside. Analysis of substrate specificity and restriction endonuclease mapping revealed three groups of clones carrying genes for an endo--1,4-glucanase (celZ), a -cellobiosidase (celX), and a -glucosidase (bglZ), respectively. All three enzymes were capable of degrading cellodextrins. Furthermore, thecelZ gene product was active on microcrystalline cellulose (Avicel), while thebglZ -glucosidase showed cellobiase activity.     

Production of cellulases and hemicellulases by an anaerobic mixed culture from lignocellulosic biomass

A comparison of different habitats, biogas plant, rumen fluid and sewage sludge, for cellulolytic organisms indicated sewage studge was the best source. Enrichment cultura gave a mixed culture which exhibited CMCase activity as well as extracellular Avicelase, xylanase, -glucosidase, -xylosidase activities and cell-bound -glucosidase, and -xylosidase production in a synthetic medium with eleven different cellulosic and lignocellulosic substrates. The activity of extracellular -glucosidase and -xylosidase production was significantly higher than endogenous activities. Hemicellulases were induced better than cellulases. The anzyme system was stable under aerobic conditions. Of the different lignocellulosic substrates, kallar grass was the best inducer of extracellular enzymes.

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Résumé La comparaison de différents habitats: digesteur méthanique, fluide du rumen ou boue de station d'épuration, pour leur contenu en organismes cellulolytiques, indiquent que la boue de station d'épuration est la meilleure source. Une culture par enrichissement a produit une culture mixte qui a exhibé aussi bien une activité CMCase que des activitiés extracellulaires avicelasique, xylanasique, -glucosidasique et -xylosidasique et qu'une production de -glucosidase et de -xylosidase liées à la cellule, dans un milieu synthétique et pour onze substrats cellulosiques et lignocellulosiques différents. L'activité de la -glucosidase extracellulaire et la production de -xylosidase sont significativement plus élevées que les activitiés endogènes. Les hemicellulases sont mieux induites que les cellulases. Le système enzymatique est stable dans des conditions aérobies. Parmi les divers substrats lignocellulosiques, l'herbe Kallar est le meilleur inducteur d'enzymes extracellufaires.

     

Transformation of dehydroepiandrosterone and pregnenolone by Mucor piriformis

The mode of transformation of dehydroepiandrosterone (I, 3-hydroxyandrost-5-en-17-one) and pregnenolone (II, 3-hydroxypregn-5-en-20-one) was studied using Mucor piriformis. Biotransformation products formed from I were 3,17-dihydroxyandrost-5-ene (Ia), 3-hydroxyandrost-5-ene-7,17-dione (Ib), 3,17-dihydroxyandrost-5-en-7-one (Ic), 3,7-dihydroxyandrost-5-en-17-one (Ie). Biotransformation products formed from compound II were 3,7-dihydroxypregn-5-en-20-one (IIa) and 3,7,11-trihydroxypregn-5-en-20-one (IIb). The organism did not carry out isomerization of the 5-en-3-ol to a 4-en-3-one system in the steroid molecules tested. In addition, it failed to carry out 14-hydroxylation possibly because of the lack of a 4-en-3-one system in I and II, and stereospecific hydroxylation at the C-7 position in I and II.     

Comparative amino acid sequence analysis of Thermotoga maritima β-glucosidase (BglA) deduced from the nucleotide sequence of the gene indicates distant relationship between β-glucosidases of the BGA family and other families of β-1,4-glycosyl hydrolases

The primary structure of the bglA gene region encoding a -glucosidase of Thermotoga maritima strain MSB8 was determined. The bglA gene has the potential to code for a polypeptide of 446 amino acids with a predicted molecular mass of 51545 Da. The T, maritima -glucosidase (BglA) was overexpressed in E. coli at a level comprising approximately 15–20% of soluble cellular protein. Based on its amino acid sequence, as deduced from the nucleotide sequence of the gene, BglA can be classified as a broad-specificity -glucosidase and as a member of the -glucosidase family BGA, in agreement with the results of enzymatic characterization of the recombinant protein. Comparative sequence analysis revealed distant amino acid sequence similarities between BGA family -glucosidases, a -xylosidase, -1,4-glycanases of the enzyme family F (mostly xylanases), and other families of -1,4-glycosyl hydrolases. This result indicates that BGA -glucosidases may comprise one enzyme family within a large enzyme order of retaining -glycosyl hydrolases, and that the members of these enzyme groups may be inter-related at the level of active site architecture and perhaps even on the level of overall three-dimensional fold.     

O-Glycosylhydrolases of Marine Filamentous Fungi: β-1,3-Glucanases of Trichoderma aureviride

The ability to produce extracellular O-glycosylhydrolases was studied in 14 strains of marine filamentous fungi sampled from the bottom sediments of the South China Sea. The following activities were detected in the culture liquids of the fungi: N-acetyl--D-glucosaminidase, -D-glucosidase, -D-galactosidase, -1,3-glucanase, amylase, and pustulanase. -1,3-Glucanases were isolated by ultrafiltration, hydrophobic interaction chromatography, and ion exchange chromatography, and their properties were studied. Data on products of enzymatic digestion of laminaran, absence of transglycosylation activity, and the pattern of action of natural inhibitors confirmed that -1,3-glucanase belonged to the exo type. Inhibitor analysis demonstrated the role of a thiol group and tryptophan and tyrosine residues in the catalytic activity.     

Carboxyl-terminal modification alters the subunit interactions and assembly pathways of normal and sickle hemoglobins

Parallel isofocusing studies established that carboxypeptidase A removal of the His-146 (HC3) and Tyr-145 (HC2) residues of heme subunits affected the assembly properties of both Des (A) and Des (S) with heme chains, albeit to differing degrees. Indeed, the rate of Des (A) oligomer dissociation (k ₁), as determined by visible spectroscopy, was 4.3-fold faster than that of its native (A) counterpart. Furthermore, Soret spectral studies have affirmed distinct rates of normal (HbA), sickle (HbS), and Des HbA hemoglobin assembly (k₂) from their and [Des (A)] heme-containing monomers. Matching kinetic analysis of Des (A) and Des (S) chain assembly (with an identical chain) revealed 4.6- and 7.8-fold faster combination rates than those seen for (A) and (S) chains, respectively. This 3-fold disparity in rates strongly supports the critical role of the -6 (A3) residue, and its amino-terminal region, in chain partner recognition and subsequent human hemoglobin assembly.     

Cloning of Candida pelliculosa beta-glucosidase gene and its expression in Saccharomyces cerevisiae

Summary Candida pelliculosa var. acetaetherius is a strain of yeast which can utilize cellobiose as the carbon source. From a gene library prepared from this yeast, the -glucosidase gene has been cloned in a S. cerevisiae host using a chromogenic substrate, 5-bromo-4-chloro-3-indolyl--glucoside as an indicator. It was proved by Southern analysis that the DNA fragment carrying the -glucosidase gene originated from C. pelliculosa. -Glucosidase produced by S. cerevisiae transformants was secreted into the periplasmic space. In Candida, -glucosidase was not induced by cellobiose but was derepressed by lowering the concentration of glucose. The regulation of -glucosidase synthesis in S. cerevisiae carrying the cloned -glucosidase was not clear compared with that in Candida, however, the enzyme activity in low glucose medium (0.05%) was reproducibly higher than in high glucose medium (2%). We have found the sequence that controls the expression of the -glucosidase gene negatively in S. cerevisiae.     

Enhancement of β-glucosidase stability and cellobiose-usage using surface-engineered recombinant Saccharomyces cerevisiae in ethanol production

To enhance the use of cellobiose by a recombinant Sachharomyces cerevisiae, the expressed -glucosidase that hydrolyzes cellobiose was stabilized using a surface-display system. The C-terminal half of -agglutinin was used as surface-display motif for the expression of -glucosidase in the cell wall. The surface-displayed -glucosidase had a half-life time (t _1/2) of 100 h in acidic culture broth conditions, while secreted -glucosidase had a t _1/2 of 60 h. With such stabilization of -glucosidase, the surface-engineered S. cerevisiae utilized 7.5 g cellobiose l^–1 over 60 h, while S. cerevisiae secreting -glucosidase into culture broth used 5.8 g cellobiose l^–1 over the same period.     

Involvement of β 1,4 galactosyltransferase 1 and Galβ 1→4GlcNAc groups in human hepatocarcinoma cell apoptosis

1,4 galactosyltransferase 1 ( 1,4GT1) synthesizes Gal 14GlcNAc groups in N-linked sugar chains of animal glycoproteins, which have been demonstrated to play an important role in many biological events, including sperm-egg interaction, cell migration and mammalian embryonic development. In this study, the mRNA level of 1,4GT1 was found to increase greatly during the 7721 hepatocarcinoma cells apoptosis induced by cycloheximide. Ricinus Communis Agglutinin-I staining indicated generous increase of Gal 14GlcNAc groups during apoptosis. Further study showed that the 7721 hepatocarcinoma cells transiently transfected with 1,4GT1 were more susceptible to the apoptosis induced by cycloheximide. The increased susceptibility was in accordance to the transfection concentration of 1,4GT1, which also led to the increased Gal 14GlcNAc groups on the transfected cell surface. All the observations suggested that 1,4GT1 and Gal 14GlcNAc groups might be associated with the apoptosis of human hepatocarcinoma cells.     

Characterization of two beta-tubulin genes from Geotrichum candidum.

Summary The -tubulin genes G1 and G2 from the phytopathogenic hemiascomycete Geotrichum candidum were found to be highly diverged in amino acid sequence from those of other filamentous fungi. G1 and G2 were also divergent from each other, with the coding regions sharing only 66% nucleotide sequence homology and 64% amino acid identity. However, the proteins shared 82% similarity and only 25 of the 161 non-identical amino acid substitutions were non-conservative. The organization of G1 is similar to other fungal -tubulin genes, but G2 has several unusual features; it has 2 amino acid additions in the N-terminal 40 residues and must employ an uncommon 5 splice junction sequence in preference to an overlapping perfect consensus. The amino acid change found to confer benomyl resistance in Neurospora crassa was also present in G2. G1 has four introns which are located similarly to those of -tubulin genes in other fungi. G2, however, has a single intron in a unique location. Translational fusions employing the 5 non-coding regions of the two Geotrichum -tubulin genes were made with the hygromycin phosphotransferase gene and shown to function in Schizosaccharomyces pombe and Trichoderma hamatum. However, G. candidum could not be transformed with these or other tested plasmids commonly used for fungal transformation.     

Erratum: Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis

The genes were cloned for the two apoprotein subunits, and ,of phycocyanin from the cyanobacterium Spirulina maxima = Arthrospiramaxima) strain F3. The - and -subunit gene-coding regionscontain 489 bp and 519 bp, respectively. The -subunit gene is upstreamfrom the -subunit gene, with a 111-bp segment separating them.Similarities between the -subunits of S. maxima and nine othercyanobacteria were between 58% and 99%, as were those between the -subunits. The maximum similarity between the - and -subunits from S. maxima was 27%.     

Enzymatic β-galactosidation of β-xylopyranosides

Summary The disaccharides formed by enzymatic transfer of the -D-galactopyranosyl residue fromo-nitrophenyl -d-galactopyranoside to -d-xylopyranosides have been identified. The influence of different factors on the yields of the disaccharides obtained was evaluated. Significant changes in selectivity were observed when -galactosidase fromE. coli was used instead of -galactosidase fromA. oryzae.