Characterization of a novel, ultra-large xylanolytic complex (xylanosome) from Streptomyces olivaceoviridis E-86

A novel, ultra-large xylanolytic complex (xylanosome) from Streptomyces olivaceoviridis E-86 was purified to homogeneity by ammonium sulfate precipitation and Sephacryl S-300 gel filtration chromatography. The purified xylanosome appeared as a single protein band on the non-denaturing (native) polyacrylamide gel electrophoresis (PAGE) gel with a molecular mass of approximately 1200 kDa. The optimal temperature and pH for xylanase activity was 60 °C and pH 6.0, respectively. The xylanase activity was stable within pH 4.1–10.3. It was stable up to 60 °C at pH 6.0. The xylanosome was highly specific towards oat-spelt xylan, and showed low activity towards corncob powder, but exhibited very low activity towards lichenan, CMC and p-nitrophenyl derivatives. Apparent K_m values of the xylansosome for birchwood, beechwood, soluble oat-spelt and insoluble oat-spelt xylans were 2.5, 3.6, 1.7 and 4.9 mg ml⁻¹, respectively. The main hydrolysis products of birchwood xylan were xylotriose, xylobiose and xylose. Analysis of the products from wheat arabinoxylan degradation by xylanosome confirmed that the enzyme had endoxylanase and debranching activities, with xylotriose, xylobiose, xylose and arabinose as the main degradation products. These unique properties of the purified xylanosome from Streptomyces olivaceoviridis E-86 make this enzymatic complex attractive for biotechnological applications.     

Variation of xylanosomal subunit composition of <Emphasis Type="Italic">Streptomyces olivaceoviridis</Emphasis> by nitrogen sources

Besides affecting the xylanases production, different nitrogen sources present in the media also caused changes in the xylanosomal subunit composition of Streptomyces olivaceoviridis E-86. Four xylanosome fractions, purified from the culture supernatant of S. olivaceoviridis E-86 grown on different nitrogen sources, exhibited high specificity towards different xylans and were composed of different subunits. Thus, S. olivaceoviridis E-86 regulates the expression of xylanase activity and varies the xylanosome composition according to the nitrogen sources possibly through the action of the secreted proteases.     

Subunit composition of a large xylanolytic complex (xylanosome) from Streptomyces olivaceoviridis E-86

A xylanolytic complex (xylanosome) was isolated from Streptomyces olivaceoviridis E-86 grown on corncob xylan. The isolated xylanosome exhibited a high molecular mass of approximately 3.8 x 10(7) Da (weight average) using size exclusion chromatography/multi-angle laser light scattering (SEC/MALLS), and was composed of at least 8 subunits with a mass range from 12 to 60 kDa. When a SDS-polyacrylamide gel zymogram was examined, the subunits of 47, 35, 32, and 23 kDa were found to have xylanase activity, while the 30-kDa subunit had CMCase activity. According to N-terminal sequence analyses, the 47- and 23-kDa subunits were found to be identical to the two reported xylanases, namely FXYN and GXYN, of S. olivaceoviridis E-86. Both the 35- and 32-kDa subunits were found to be truncated forms of the intact FXYN xylanase that possibly resulted from the degradation by proteases. The 15-kDa subunit consisted solely the xylan-binding domain of the FXYN xylanase. The purified xylanosome appeared to bind partially to xylan and poorly to Avicel.     

Metagenomic analysis of novel lignocellulose-degrading enzymes from higher termite guts inhabiting microbes

A metagenomic fosmid library was constructed from genomic DNA isolated from the microbial community residing in hindguts of a wood-feeding higher termite (Microcerotermes sp.) collected in Thailand. The library was screened for clones expressing lignocellulolytic activities. Fourteen independent active clones (2 cellulases and 12 xylanases) were obtained by functional screening at pH 10.0. Analysis of shotgun-cloning and pyrosequencing data revealed six ORFs, which shared less than 59% identity and 73% similarity of their amino acid sequences with known cellulases and xylanases. Conserved domain analysis of these ORFs revealed a cellulase belonging to the glycoside hydrolase family 5, whereas the other five xylanases showed significant identity to diverse families including families 8, 10, and 11. Interestingly, one fosmid clone was isolated carrying three contiguous xylanase genes that may comprise a xylanosome operon. The enzymes with the highest activities at alkaline pH from the initial activity screening were characterized biochemically. These enzymes showed a broad range of enzyme activities from pH 5.0 to 10.0, with pH optimal of 8.0 retaining more than 70% of their respective activities at pH 9.0. The optimal temperatures of these enzymes ranged from 50 degrees C to 55 degrees C. This study provides evidence for the diversity and function of lignocellulose-degrading enzymes in the termite gut microbial community, which could be of potential use for industrial processes such as pulp biobleaching and denim biostoning.     

Functional assembly and characterization of a modular xylanosome for hemicellulose hydrolysis in yeast

Five trimeric xylanosomes were successfully assembled on the cell surface of Saccharomyces cerevisiae. Three dockerin‐tagged fungal enzymes, an endoxylanase (XynAc) from Thermomyces lanuginosus, a β‐xylosidase (XlnDt) from Aspergillus niger and an acetylxylan esterase (AwAXEf) from Aspergillus awamori, were displayed for the synergistic saccharification of birchwood xylan. The surface‐expression scaffoldins were modular constructs with or without carbohydrate binding modules from Thermotoga maritima (family 22) or Clostridium thermocellum (family 3). The synergy due to enzyme–enzyme and enzyme–substrate proximity, and the effects of binding domain choice and position on xylan hydrolysis were determined. The scaffoldin‐based enzymes (with no binding domain) showed a 1.6‐fold increase in hydrolytic activity over free enzymes; this can be attributed to enzyme–enzyme proximity within the scaffoldin. The addition of a xylan binding domain from T. maritima improved hydrolysis by 2.1‐fold relative to the scaffoldin without a binding domain (signifying enzyme–substrate synergy), and 3.3‐fold over free enzymes, with a xylose productivity of 105 mg g^?1 substrate after 72 h hydrolysis. This system was also superior to the xylanosome carrying the cellulose binding module from C. thermocellum by 1.4‐fold. Furthermore, swapping the xylan binding module position within the scaffoldin resulted in 1.5‐fold more hydrolysis when the binding domain was adjacent to the endoxylanase. These results demonstrate the applicability of designer xylanosomes toward hemicellulose saccharification in yeast, and the importance of the choice and position of the carbohydrate binding module for enhanced synergy. Biotechnol. Bioeng. 2013; 110: 275–285. © 2012 Wiley Periodicals, Inc.     

Isolation and structural studies on a galactofuranosyl-containing glycopeptide fromAscobolus furfuraceus

A galactofuranosyl-containing glycopeptide has been isolated from mycelium ofAscobolus furfuraceus by extraction with water. The glycoconjugate was purified by DEAE-cellulose chromatography followed by gel filtration. A molecular weight of about 20 000 was determined by the latter method using standard dextrans. Neutral sugars accounted for 94.5% of the glycopeptide and were characterized as mannose, galactose, and glucose. Glucosamine was estimated colorimetrically (1.8%). The molar ratio of Man:Gal:Glc:GlcNH₂ was 68:32:16:2. A trace amount of total phosphorus (0.2%) was found. The predominant amino acids were threonine and serine. The peptide moiety was labeled with [¹⁴C]formaldehyde and the elution of radioactivity was coincident with sugar on gel filtration in the presence of sodium dodecyl sulfate. The peak of radioactivity was retarded on release of galactose by mild acid hydrolysis. These results confirm the sugar-peptide linkage.     

Isoflavones Daidzein and Genistein: Preparation by Acid Hydrolysis of Their Glycosides and the Effect on Phospholipid Peroxidation

A mixture of isoflavones was obtained by acid hydrolysis of isoflavone glycosides isolated from the products of soybean processing by successive extraction with aqueous acetone and methanol. The homogeneous isoflavones daidzein and genistein were isolated from the aglycone mixture by adsorption chromatography and identified by spectral and chromatographic methods. The effect of both isoflavones on lipid peroxidation of soy phospholipids in multilamellar vesicles was studied at various concentrations. These aglycones were found to inhibit the formation of lipid hydroperoxides and malonic dialdehyde at concentrations as low as 1 mM.     

Enhanced continuous production of lovastatin using pellets and siran supported growth of Aspergillus terreus in an airlift reactor

Lovastatin, a hypocholesterolemic agent, is a secondary metabolite produced by filamentous microorganism Aspergillus terreus in submerged batch cultivation. Lovastatin production by pellets and immobilized siran cells was investigated in an airlift reactor. The process was carried out by submerged cultivation in continuous mode with the objective of increasing productivity using pellet and siran supported growth of A terreus. The continuous mode of fermentation improves the rate of lovastatin production. The effect of dilution rate and aeration rate were studied in continuous culture. The optimum dilution rate for pellet was 0.02 h⁻¹ and for siran carrier was 0.025 h⁻¹. Lovastatin productivity using immobilized siran carrier (0.0255 g/L/h) was found to be greater than pellets (0.022 g/L/h). The productivity by both modes of fermentation was found higher than that of batch process which suggests that continuous cultivation is a promising strategy for lovastatin production.     

Nystatin and killer toxin sensitivity of free and immobilizedSaccharomyces cerevisiae

The inhibitory effect of nystatin and killer toxin on the growth of free and covalently-immobilizedSaccharomyces cerevisiae cells was studied. The resistance of immobilized cells to both agents was accompanied by increased amounts of phospholipids and sterols. The possible relationship between these changes in the membrane composition and the transduction of a signal across the cytoplasmic membrane is discussed.     

Synthesis and Pharmacological Activity of Betulin Dinicotinate

The assignment of NMR resonances of lupane triterpenoids was refined by the example of 3O,28O-dinicotinoylbetulin, obtained by acylation of betulin. Hepatoprotective, antiulcer, antiinflammatory, reparative, and anti-HIV activities were found for the compound. In addition, it was demonstrated to have immunomodulatory activity, for the first time detected among lupane triterpenoids.     

Characterisation of cerebroside 3-sulphate dispersions

Galactosyl ceramide 3-sulphate (cerebroside 3-sulphate) was tritiated using [³H]NaBH₄ with PdCl₂ as catalyst. Quantitative purification of the three components of the product was achieved by chromatography on Florisil. Dispersions, prepared by either low energy sonication or by dilution from organic solvent, were compared by controlled pore glass chromatography, ultra-centrifugation and electron microscopy. Both dispersion techniques were shown to form unilamellar vesicles, the average size of vesicles produced by sonication being far larger than those produced by solvent dilution. The diameter of isolated vesicles produced by solvent dilution was in the range 10–80 nm.     

Hydrophobic Nitroxyl Radicals Inhibit Linoleyl Alcohol Oxidation by 5-Lipoxygenase

The linoleyl alcohol oxidation catalyzed by potato tuber 5-lipoxygenase was found to be efficiently inhibited by stable nitroxyl radicals: 1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl bicyclo[2,2,2]octane-1-carboxylate, 1-adamantylacetate, dodecanoate, and octadecanoate. The dependence of apparent IC₅₀ values on the rotational correlation times of 4-hydroxy-1-oxyl-2,2,6,6-tetramethylpiperidine and its derivatives in model micellar systems was analyzed. An inhibition mechanism was proposed; it involves the interaction of a hydrophobic nitroxyl radical with the intermediate radical enzyme–substrate complex.     

Fungal Thyroiditis: An Overview

The authors review the epidemiology, clinical manifestations, diagnosis, and treatment of fungal thyroiditis cases previously reported in the medical literature. Aspergillus was by far the most common cause of fungal thyroiditis. Immunocompromised patients, such as those with leukemia, lymphoma, autoimmune diseases, and organ-transplant patients on pharmacological immunosuppression were particularly at risk. Fungal thyroiditis was diagnosed at autopsy as part of disseminated infection in a substantial number of patients without clinical manifestations and laboratory evidence of thyroid dysfunction. Local signs and symptoms of infection were indistinguishable from other infectious thyroiditis and included fever, anterior cervical pain, thyroid enlargement sometimes associated with dysphagia and dysphonia, and clinical and laboratory features of transient hyperthyroidism due to the release of thyroid hormone from follicular cell damage, followed by residual hypothyroidism. Antemortem diagnosis of fungal thyroiditis was made by direct microscopy and culture of a fine-needle aspirate, or/and biopsy in most cases. Since most patients with fungal thyroiditis had disseminated fungal infection with delay in diagnosis and treatment, the overall mortality was high.     

Multinucleation in the conidia of polyploids derived fromTrichoderma reesei QM 9414 by colchicine treatment

Conidia ofTrichoderma reesei QM 9414 were treated with colchicine in order to obtain polyploids (diploids; tetraploids). Cellulase production by diploids (mononucleate conidia) was almost twice as great as that of the original strain, but that of tetraploids (binucleate conidia) was not increased. When these latter conidia were re-treated with 2.0% (w/v) colchicine, multiple nuclei were produced in each conidium, and their diameter was almost the same as that of the original nucleus. Cellulase production of the diploid was almost the same in either mononucleate or multinucleate nature. However, cellulase production by the tetraploid which produced multinucleate conidia was greater than that of the binucleate tetraploid and that of the diploid. The multinucleation technique can contribute to enhancing cellulase production.     

The cloned 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene from Sinorhizobium sp. strain BL3 in Rhizobium sp. strain TAL1145 promotes nodulation and growth of Leucaena leucocephala

The objective of this study was to determine the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase of symbionts in nodulation and growth of Leucaena leucocephala. The acdS genes encoding ACC deaminase were cloned from Rhizobium sp. strain TAL1145 and Sinorhizobium sp. BL3 in multicopy plasmids, and transferred to TAL1145. The BL3-acdS gene greatly enhanced ACC deaminase activity in TAL1145 compared to the native acdS gene. The transconjugants of TAL1145 containing the native or BL3 acdS gene could grow in minimal media containing 1.5mM ACC, whereas BL3 could tolerate up to 3mM ACC. The TAL1145 acdS gene was inducible by mimosine and not by ACC, while the BL3 acdS gene was highly inducible by ACC and not by mimosine. The transconjugants of TAL1145 containing the native- and BL3-acdS genes formed nodules with greater number and sizes, and produced higher root mass on L. leucocephala than by TAL1145. This study shows that the introduction of multiple copies of the acdS gene increased ACC deaminase activities of TAL1145 and enhanced its symbiotic efficiency on L. leucocephala.     

Improved frequency of transformation in rice and maize by treatment of immature embryos with centrifugation and heat prior to infection with Agrobacterium tumefaciens

The efficiency of transformation was improved by treating immature embryos with heat and centrifugation before infection with Agrobacterium tumefaciens in rice and maize. Because the effects were detected both in the levels of transgene expression after co-cultivation and in the number of independent transgenic plants obtained per embryo, conditions were first optimized based on the transgene expression, and then transformants were produced. The optimal conditions varied considerably depending on species and genotypes, but reasonably good parameters were identified for Japonica rice, Indica rice or maize. As a general tendency, the effect of centrifugation was greater than that of heat in Japonica rice, whereas that of heat was greater than that of centrifugation in Indica rice and maize A188, and the combination of the treatments was the most effective in all of the genotypes tested. The frequency of transformation was improved several fold in rice and maize. In addition, transformation of certain genotypes of maize, which were not transformable before, and transformation of maize with a less efficient vector, which could not transform maize before, became possible by these pre-treatments. In the highest case, 18 independent transgenic plants were obtained from a single immature embryo of Japonica rice. Although nothing is known about the mechanism, these pre-treatments seemed to render cells of rice and maize more competent for transformation mediated by A. tumefaciens.     

The proportion of calcium-bound pectin in plant cell walls

The amount of pectin held in cell walls by ionic bonds only was determined by extraction with cyclohexanediamine tetraacetic acid (CDTA) at room temperature, to remove calcium ions without degrading the galacturonan chains. Enzymic degradation was avoided by extracting the cell walls with phenol-acetic acid-water during preparation. From cell walls of celery petioles, cress hypocotyls and tomato and cucumber pericarp CDTA extracted 64–100 mg g^-1 pectin, leaving 80–167 mg g^-1 uronic acid in the residue. An additional extraction at high ionic strength was used to make the galacturonan chains more flexible and thus detach any pectins held by steric interactions, but the amount released in this way was small. Most of the residual uronic acid polymers could be extracted by cold alkali and remained soluble on neutralisation, showing that it was not water-insolubility that prevented their extraction with CDTA. Covalent bonding was thought more likely.     

Mapping of the multiple regulatory sites for putP and putA expression in the putC region of Escherichia coli

Summary The effects of regulatory proteins on the expression of putP and putA were studied using put-lacZ fusion genes. The expression of the putP-lacZ gene was activated by the glnG gene product and the catabolite gene activator protein (CAP). The putA gene product inhibited activation of putP-lacZ gene expression by CAP or the glnG gene product and its inhibition was greater in the absence of proline. The expression of the putA-lacZ gene was activated by CAP and repressed by the glnG gene product. The putA gene product acted as a repressor in the absence of proline, but not in its presence. Studies using put-lacZ fusion genes with upstream deletions showed that the region required for the activation of putP by CAP was within 234 bp upstream of the translational initiation site and that that for the activation of putP was within 107 bp upstream of the translational initiation site of the putA gene. This supported the suggested locations of CAP binding sites. The region required for induction of putP and putA expression by proline was located at the Hpal site 182 bp upstream of the translational starting site of putA, suggesting that a sequence of dyad symmetry located 1 bp to the left of the HpaI site is a candidate for the binding site of the putA gene product.Abbreviations AC L-azetidine-2-carboxylic acid - Ap ampicillin - CAP catabolite gene activator protein - NR_I nitrogen regulator I - RF DNA DNA replicative form - Str streptomycin - Tc tetracycline - TTC 2,3,5-triphenyl tetrazolium chloride - UV ultraviolet     

Purification and characterization of veratryl alcohol oxidase from Comamonas sp. UVS and its role in decolorization of textile dyes

In the present work, we have purified veratryl alcohol oxidase (VAO) enzyme from Comamonas UVS to evaluate its potential to decolorize textile dyes. VAO was purified (13.9 fold) by an ion exchange followed by the size exclusion chromatography. Molecular weight of the VAO was estimated to be about 66 kDa by SDS-PAGE. The optimum pH and temperature of oxidase were 30°C and 65°C, respectively. VAO showed maximum activity with n-propanol among the various substrates (n-propanol, veratryl alcohol, L-dopa, tryptophan, etc.). Under standard assay conditions, K_m value of the enzyme was 2.5 mM towards veratrole. The enzyme activity was completely inhibited by 0.5 mM sodium azide. L-cysteine, dithiothreitol, and the metal chelator, EDTA had a slight inhibitory effect. The purified enzyme was able to decolorize textile dyes, Red HE7B (57.5%) and Direct Blue GLL (51.09%) within 15 h at 40 μg/mL concentration. GC-MS analysis of the metabolites suggested oxidative cleavage and desulphonation of these dyes.     

Effects of surfactants on cell growth and pigment production in suspension cultures ofPerilla frutescens

The effects of surfactants, adecanol LG-294 and silicone A, on anthocyanin accumulation and the growth ofPerilla frutescens cells in suspension cultures were studied. Production of the red pigment was remarkably reduced from about 1.9 g/l to 0.4 g/l by adecanol LG-294 at 0.06 ml/l but not by silicone A up to 0.4 ml/l. Several repeated shake-flask cultures also demonstrated no adverse effects of silicone A on the metabolite accumulation by the suspended cells. Furthermore, the addition of silicone A to a culture in a stirred bioreactor produced a three-fold higher growth rate and a seven-fold increase in anthocyanin compared with surfactant-free cultures. The improvement was due to the substantial reduction or prevention of foaming and of cell adhesion to the bioreactor wall.