The degradation and utilization of wheat-straw cell-wall monosaccharide components by defined ruminal cellulolytic bacteria

Cell walls (CW) of untreated wheat straw and sulphur-dioxide (SO₂)-treated wheat straw were used as model substrates for the hydrolysis and utilization of CW carbohydrates by pure cultures or pair-combinations of defined rumen bacterial strains. Fibrobacter succinogenes S85 and BL2 strains and their co-cultures with D1 were the best degraders of CW among ruminal cultures, solubilizing 37.2–39.6% of CW carbohydrates of untreated straw and 62.2–74.5% of SO₂-treated straw. Complementary action between Butyrivibrio fibrisolvens D1 and the F. succinogenes strains was identified with respect to co-culture growth and carbohydrate utilization. However, the extent of CW solubilization was determined mainly by the F. succinogenes strains. In both substrates, utilization of solubilized cellulose by F. succinogenes S85 and BL2 monocultures was higher than that of xylan and hemicellulose: 96.5–98.3%, 34.4–40.5% and 33.5–36.2%, respectively. Under scanning electron microscopy visualization, S85 and BL2 cells of the co-cultures comprised the most dense layer of bacterial cell mass attached to and colonized on straw stems and leaves, whereas D1 cells were always nearby. Stems and leaves of the untreated straw were less crowded by attached bacteria than that of the SO₂-treated straw. In both materials, the cell surface topography of S85 and BL2 bacteria attached to CW particles was specified by a coat of characteristic protuberant structures, polycellulosome complexes.     

Degradation and fermentation of cellulose by the rumen anaerobic fungi in axenic cultures or in association with cellulolytic bacteria

Three rumen anaerobic fungi—Neocallinastix frontalis MCH3,Piromyces (Piromonas) communis FL, andCaecomyces (Sphaeromonas) communis FG10—were cultured on cellulose filter paper alone or in association with one of two rumen cellulolytic bacteria,Ruminococcus flavefaciens 007 andFibrobacter succinogenes S85. Cocultures ofN. frontalis orP. communis andR. flavefaciens were markedly less effective than the fungal monocultures in degrading cellulose but more effective than the bacterial monocultures.R. flavefaciens had an antagonistic effect against both of the fungal species. In contrast, no interaction was observed between the two fungal species andF. succinogenes. Cellulose was more effectively degraded by the cocultureC. communis-R. flavefaciens than by the corresponding fungal and bacterial monocultures. The effectiveness of degradation of the cocultureC. communis-F. succinogenes was comparable to that of the bacterial strains but greater than that of the fungi; no interaction was observed between these two microorganisms.     

Bacterial cell surface structures involved in lucerne cell wall degradation by pure cultures of cellulolytic rumen bacteria

Summary Pure cultures of the cellulolytic rumen bacterial strains Bacteroides succinogenes S85, Ruminococcus flavefaciens FD1 and Ruminococcus albus 7 were grown on lucerne cell walls (CW) or on cellobiose as the sole added carbohydrate substrate. Scanning electron microscopy visualization using cationized-feritin pretreatment have shown that cell surface topology of these strains grown on and attached to CW particles was specified by a dense coat of characteristic protuberant structures. In contrast, when grown on cellobiose, the surface topology of these bacterial strains was smoother, and contained fewer protuberant structures. The ability of these bacterial strains to attach to cellulose was higher for bacteria previously adapted to lucerne CW compared to cellobiose adaptation. Bacteroides succinogenes S85 was the best digester of lucerne CW (46.5%) and also had the best adhesion capability (65.6%) after adaption to grow on CW.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 2599-E, 1989 seriesOffprint requests to: J. Miron     

Molecular cloning,expression, and characterization of a new endoglucanase gene fromFibrobacter succinogenes S85

A DNA fragment coding for a carboxymethylcellulase (CMCase) ofFibrobacter succinogenes S85 was isolated from a pUC18 gene library inEscherichia coli JM109. The CMCase gene was present as a single copy in theF. succinogenes S85 genome and was found in all the otherF. succinogenes strains tested. The gene was expressed from an endogenous promoter inE. coli and was not subject to glucose repression. Most of the CMCase activity was located in the membrane ofE. coli. Zymogram analysis and³⁵S labeling of the proteins encoded by the CMCase gene-containing plasmid indicated that the enzyme has a molecular mass of 58,000. The optimal pH and temperature of activity on CMC were respectively 6.4 and 30°C. The enzyme was active on CMC, barley -glucan, and lichenan but would not hydrolyze laminarin and exhibited no exoglucanase-type activity, suggesting that it is an endo-(1,4)--d-glucanase.     

Induction of xylan-degrading enzymes inButyrivibrio fibrisolvens

The formation of xylanolytic enzymes byButyrivibrio fibrisolvens NCFB 2249 was induced by xylan, xylo-oligosaccharides, and xylobiose. Inhibition of RNA or protein synthesis prevented inducetion, and enzyme formation occured only when anaerobiosis was maintained. The rate of enzyme inducetion by xylan was affected by pH and inducer concentration, and highest levels of activity occurred when the initial pH and xylan concentration were pH 6.5–7 and 2 mg/ml respectively. The ability of the cells to respond to the inducer was reduced in slowly growing cells, although cells that were grown at dilution rates that appertain in the rumen ecosystem responded rapidly to the inducer.Butyrivibrio fibrisolvens also exhibited diauxic characteristics of carbohydrate utilization, and in consequence enzyme induction and xylanolysis were delayed until readily metabolized sugars (e. g., glucose, arabinose) had been consumed.     

Regulation of anaerobic respiratory pathways in Wolinella succinogenes by the presence of electron acceptors

In Wolinella succinogenes ATP synthesis and consequently bacterial growth can be driven by the reduction of either nitrate (E₀=+0.42 V), nitrite (E₀=+0.36 V), fumarate (E₀=+0.03 V) or sulphur (E₀=-0.27 V) with formate as the electron donor. Bacteria growing in the presence of nitrate and fumarate were found to reduce both acceptors simultaneously, while the reduction of both nitrate and fumarate is blocked during growth with sulphur. These observations were paralleled by the presence and absence of the corresponding bacterial reductase activities. Using a specific antiserum, fumarate reductase was shown to be present in bacteria grown with fumarate and nitrate, and to be nearly absent from bacteria grown in the presence of sulphur. The contents of polysulphide reductase, too, corresponded to the enzyme activities found in the bacteria. This suggests that the activities of anaerobic respiration are regulated at the biosynthetic level in W. succinogenes. Thus nitrate and fumarate reduction are repressed by the most electronegative acceptor of anacrobic respiration, sulphur. By contrast, in Escherichia coli a similar effect is exerted by the most electropositive acceptor, O₂. W. succinogenes also differs from E. coli in that fumarate reductase is not repressed by nitrate.Abbreviations BV benzyl viologen - DMN 2,3-dimethyl-1,4-naphthoquinone - DMSO dimethylsulfoxide - TMAO trimethylamine-N-oxide     

Solubilization and partial purification of the GABA receptor from mouse brain and a binding assay for the solubilized receptor.

Abstract— The GABA receptor from mouse brain was solubilized with lysolecithin. A 56-fold overall purification and activation were achieved by discontinuous sucrose gradient centrifugation and solubilization. Activation of binding by both procedures was observed. The solubilized receptor has the following binding constants: K_D1= 3.5 _nM, K_D2= 52 _nM, B_{max 1}= 2.8 pmol/mg protein and B_{max 2}= 14 pmol/mg protein for muscimol; K_D1= 12 nM, K_D2= 470 nM, B_{max 1}= 1.4 pmol/mg protein and B_{max 2}= 17 pmol/mg protein for GABA. Specific GABA binding was inhibited by imidazoleacetic acid and bicuculline with IC₅₀ values of 250_nM and 1 _μM respectively. A rapid and sensitive filtration binding assay for the solubilized receptor has been developed. Lysolecithin was also found suitable for the solubilization of acetylcholine receptor from T. californica electroplaques.     

A stable and efficient transformation system for Butyrivibrio fibrisolvens OB156

A 9.5-kb shuttle vector capable of replication and selection in both Escherichia coli and Butyrivibrio fibrisolvens was constructed. Plasmid pUC118 provided replication functions and ampicillin resistance selection in E. coli. In B. fibrisolvens, replication was controlled by the native plasmid pRJF1 from strain OB156, and selectability was provided by a 3.5-kb fragment of plasmid pAM1 containing the erythromycin resistance gene. Optimum conditions for transformation were 15 kV/cm, 2 h recovery, and plating in an agar overlay on medium containing 10 g erythromycin/ml. Maximum efficiency was 1.1×10⁵ transformants per g plasmid DNA (average 3×10⁴), and restriction mechanisms reduced efficiency by a factor of 2×10². Nonselective growth for 200 generations gave no measurable loss of plasmid.     

Cloning and sequencing of an endoglucanase (end1) gene from Butyrivibrio fibrisolvens H17c

Summary The nucleotide sequence of a 2.8 kb DNA segment containing an endoglucanase gene (end1) from Butyrivibrio fibrisolvens H17c was determined. The B. fibrisolvens H17c gene was expressed from its own regulatory region in Escherichia coli and three putative consensus promoter sequences were identified upstream of a ribosome binding site and an ATG start codon. The complete amino acid sequence (547 residues) was deduced and homology with the Clostridium thermocellum ME gene product (EGE) was demonstrated. The endoglucanase contained a typical amino-terminal signal sequence and five repeated sequences (PDPTPVD) between amino acids 412–447. The endoglucanase showed relatively high endoglucanase activity against endoglucanase-specific substrates with 1-4 linkages but low activity against xylan and an exoglucanasespecific substrate, p-nitrophenyl--d-cellobioside.Abbreviations CMCase carboxymethylcellulase - DNS dinitrosalicylic acid - end1 gene coding for End1 - End1 endo-1,4--glucanase - nt nucleotide - ORF open reading frame     

Biochemical and Domain Analyses of FSUAxe6B,a Modular Acetyl Xylan Esterase,Identify a Unique Carbohydrate Binding Module in Fibrobacter succinogenes S85

Acetyl xylan esterase (EC 3.1.1.72) is a member of a set of enzymes required to depolymerize hemicellulose, especially xylan that is composed of a main chain of β-1,4-linked xylopyranoside residues decorated with acetyl side groups. Fibrobacter succinogenes S85 Axe6B (FSUAxe6B) is an acetyl xylan esterase encoded in the genome of this rumen bacterium. The enzyme is a modular protein comprised of an esterase domain, a carbohydrate-binding module, and a region of unknown function. Sequences that are homologous to the region of unknown function are paralogously distributed, thus far, only in F. succinogenes. Therefore, the sequences were designated Fibrobacter succinogenes-specific paralogous module 1 (FPm-1). The FPm-1s are associated with at least 24 polypeptides in the genome of F. succinogenes S85. A bioinformatics search showed that most of the FPm-1-appended polypeptides are putative carbohydrate-active enzymes, suggesting a potential role in carbohydrate metabolism. Truncational analysis of FSUAxe6B, together with catalytic and substrate binding studies, has allowed us to delineate the functional modules in the polypeptide. The N-terminal half of FSUAxe6B harbors the activity that cleaves side chain acetyl groups from xylan-like substrates, and the binding of insoluble xylan was determined to originate from FPm-1. Site-directed mutagenesis studies of highly conserved active-site residues in the esterase domain suggested that the esterase activity is derived from a tetrad composed of Ser₄₄, His₂₇₃, Glu₁₉₄, and Asp₂₇₀, with both Glu₁₉₄ and Asp₂₇₀ functioning as helper acids, instead of a single carboxylate residue proposed to initiate catalysis.The development of strategies for biomass conversion to fuels (biofuels) is a subject of keen interest as we search for energy resources alternative to fossil fuels (39). Plant cell matter accounts for 150 to 200 billion tons of biomass on our planet annually (31). It is technically possible, but economically far from realization, to convert plant cell wall to biofuels (41). Thus, currently, plant cell wall utilization as a source of biofuels is mostly at the laboratory scale, although there is a great impetus to move production to the industrial scale.The main components of the plant cell wall are cellulose, hemicellulose, and lignin. These components form complex structures that provide the plant with physical strength (42). Biologically, there are two major steps in the production of alcohols from plant-based feedstock. The first step is an enzymatic hydrolysis of the plant cell wall components to fermentable sugars, and the second step is fermentation of the resultant sugars into alcohols. A major limitation of the process is the lack of highly efficient biocatalysts required for the first step. However, it is known that microbes, either as individuals or consortia, that harbor genes encoding enzymes that hydrolyze plant cell wall polysaccharides abound in nature. Research efforts directed at deepening knowledge of how multiple enzymes participate synergistically to degrade the plant cell wall will accelerate the capacity to achieve the goal of converting biomass to biofuels on a large scale (12, 27). However, improvement of “enzyme cocktails” developed for depolymerization of lignocellulosic biomass will be dependent on a better understanding of the structure/function of individual enzymes that together constitute the arsenal of enzymes (hydrolyzome) used by naturally occurring organisms known to be highly efficient in plant cell wall degradation.Ruminant animals harbor a variety of plant cell wall-degrading bacteria in their first stomach or rumen (26). These animals digest forages with the aid of a microbial consortium that is able to metabolize plant cell wall polysaccharides to short-chain fatty acids, the main energy source for the ruminant host. Fibrobacter succinogenes is a ubiquitous rumen bacterium and has been estimated in previous reports to occupy 0.1% to 1.0% of the microbial population in the cattle rumen, based on the quantification of 16S rRNA genes as a marker (25, 43). F. succinogenes is a significant cellulolytic rumen bacterium, and it has the ability to grow on crystalline cellulose as a sole source of carbon and energy (17). Additionally, it has been demonstrated that this bacterium can solubilize hemicelluloses, although it only partially utilized the constituent monosaccharides released (34). As further evidence, F. succinogenes failed to grow on xylose (33), a constituent of most hemicelluloses. Since F. succinogenes is a highly versatile microbe capable of degrading both cellulose and hemicellulose, strains of this bacterium are attractive models to study natural strategies for efficient deconstruction of plant cell wall polysaccharides.Through analysis of the genome sequence of F. succinogenes S85, a gene cluster that encodes more than 10 hemicellulose-targeting enzymes was identified. Most of the enzymes in the cluster are modular polypeptides, a common feature in many carbohydrate-active enzymes. Kam and coworkers (23) previously identified two acetyl xylan esterases (Axe6A and Axe6B) in this cluster and predicted that each gene encoded a polypeptide composed of two domains: an esterase catalytic domain and a family 6 carbohydrate-binding module (CBM6). Whereas Axe6A was fairly well characterized, difficulties in expression of recombinant Axe6B restricted its characterization (23). In this report, overproduction of recombinant F. succinogenes S85 Axe6B (FSUAxe6B) is demonstrated, and furthermore, it is shown that rather than having two domains, the polypeptide harbors three domains composed of an esterase, CBM6, and a region of unknown function. Bioinformatics analysis suggested that the unknown domain observed in FSUAxe6B is, so far, distributed only in F. succinogenes S85; thus, it was designated F. succinogenes-specific paralogous module 1 or FPm-1. Twenty-four polypeptides, with the majority containing glycoside hydrolase family motifs and CBMs, were found to harbor this peptide at the extreme C-terminal region. In addition to assigning a carbohydrate binding function to FPm-1, critical residues that confer esterase activity to the N-terminal half of FSUAxe6B were also identified through site-directed mutagenesis.     

Effects of dilution rate and pH on the ruminal cellulolytic bacterium Fibrobacter succinogenes S85 in cellulose-fed continuous culture

The ruminal cellulolytic bacterium Fibrobacter succinogenes S85 was grown in cellulose-fed continuous culture at 22 different combinations of dilution rate (D, 0.014–0.076 h^-1) and extracellular pH (6.11–6.84). Effects of pH and D on the fermentation were determined by subjecting data on cellulose consumption, cell yield, product yield (succinate, acetate, formate), and soluble sugar concentrationto response surface analysis. The extent of cellulose conversion decreased with increasing D. First-order rate constants at rapid growth rates were estimated as 0.07–0.11 h^-1, and decreased with decreasing pH. Apparent decreases in the rate constant with increasing D was not due to inadequate mixing or preferential utilization of the more amorphous regions of the cellulose. Significant quantities of soluble sugars (0.04–0.18 g/l, primarily glucose) were detected in all cultures, suggesting that glucose uptake was rather inefficient. Cell yields (0.11–0.24 g cells/g cellulose consumed) increased with increasing D. Pirt plots of the predicted yield data were used to determined that maintenance coefficient (0.04–0.06 g cellulose/g cells · h) and true growth yield (0.23–0.25 g cells/g cellulose consumed) varied slightly with pH. Yields of succinate, the major fermentation endproduct, were as high as 1.15 mol/mol anhydroglucose fermented, and were slightly affected by dilution rate but were not affected by pH. Comparison of the fermentation data with that of other ruminal cellulolytic bacteria indicates that F. succinogenes S85 is capable of rapid hydrolysis of crystalline cellulose and efficient growth, despite a lower _max on microcrystalline cellulose.     

Isolation of the sulphur reductase and reconstitution of the sulphur respiration of Wolinella succinogenes

Wolinella succinogenes can grow at the expense of sulphur reduction by formate. The enzymes involved in the catalysis of this catabolic reaction have been investigated. From the results the following conclusions are drawn: 1. The enzyme isolated as a sulphide dehydrogenase from the cytoplasmic membrane of W. succinogenes is the functional sulphur reductase that operates in the electron transport from formate to sulphur. 2. The enzyme (M_r 200,000) consists essentially of one type of subunit with the M_r 85,000 and contains equal amounts of free iron and sulphide (120 mol/g protein), but no heme. It represents the first functional sulphur reductase ever isolated. 3. The electron transport chain catalyzing sulphur reduction by formate consists merely of formate dehydrogenase and sulphur reductase. A lipophilic quinone which mediates the transfer of electrons between enzymes in other chains, is apparently not involved. This is the first known example of a phosphorylative electron transport chain that operates without a quinone. 4. The same formate dehydrogenase appears to operate in the electron transport both with sulphur and with fumarate as the terminal electron acceptor in W. succinogenes.Abbreviations DMN 2,3-Dimethyl-1,4-naphthoquinone - DTT dithiothreitol - MK menaquinone (vitamin K₂) - PMSF phenylmethane sulfonylfluoride - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-glycine - Tea triethanolamine - Hepes 4-(2-hydroxyethyl)-1-piperazineethane sulfonate Dedicated to Professor F. Schneider (Philipps-Universität Marburg) on the occasion of his 60th birthday     

Mitochondrial proline dehydrogenase deficiency in hyperprolinemic PRO/Re mice: Genetic and enzymatic analyses

Genetic analyses, involving backcross and F₂ matings, demonstrate that the type I hyperprolinemia of PRO/Re mice is caused by an abnormal allele at a single locus designated pro-1. Mice homozygous for this allele (pro-1 ^b /pro-1^b) possess a deficiency in the activity of component 1 of mitochondrial proline dehydrogenase. In liver mitochondria of normal C57BL/6J mice, two proline dehydrogenase activity components are demonstrable by electrophoretic resolution of Triton X-100 solubilized extracts. In mitochondria of PRO/Re mice, the activity of component 1 is not readily detectable. Residual proline dehydrogenase activity in PRO/Re mitochondria appears, therefore, to be due in large measure to activity component 2 which is more stable to incubation at 40 C, exhibits slower electrophoretic mobility, and is less reactive to menadione. Kinetic analyses demonstrate a K _m (proline) for the Triton X-100 solubilized enzyme activities of PRO/Re and C57BL/6J liver mitochondria of 0.4 M and 2.9×10^?3 M, respectively. C57BL/6J enzyme activity is inhibited by high substrate concentration. The activity of component 1 was not detected in other subcellular fractions of PRO/Re liver obtained by differential centrifugation. Abnormal control of respiratory chain function in PRO/Re mitochondria appears to involve primarily proline oxidation, as indicated by the level of activity of several inner membrane enzymes.     

The hydrolysis of lucerne cell-wall monosaccharide components by monocultures or pair combinations of defined ruminal bacteria

The defined ruminal bacterial strains Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD1, Ruminococcus albus 7, Butyrivibrio fibrisolvens D1, and Bacteroides ruminicola GA33 were grown, in monocultures or as combinations of pair strains, on isolated lucerne cell-walls (CW) as the sole carbohydrate substrate. Fibrobacter succinogenes S85 was the dominant strain determining extent of CW hydrolysis in all combinations with S85. The hydrolysis of cellulose, xylan, hemicellulose side-sugars, and total CW monosaccharides by pure S85 were: 58·8, 47·3, 66·9 and 57·0%, respectively. The strains combination S85 plus D1 comprised the highest complementary effect, increasing significantly the hydrolysis of cellulose and total CW monosaccharides by 16% and 13%, respectively, above the values obtained by pure S85. This complementation was expressed also in growth pattern of bacteria.
The monocultures of FD1, D1 and GA33 had very little hydrolytic effect on lucerne cellulose, but higher effects on xylan and hemicellulose side-sugars. The combinations D1 plus GA33 and 7 plus GA33 were complementary in the hydrolysis of all CW polysaccharides. The combinations FD1 plus D1, FD1 plus GA33, and 7 plus D1 were complementary only with respect to hemicellulose hydrolysis. On the other hand, the cellulolytic combinations S85 plus FD1, S85 plus 7 and FD1 plus 7 demonstrated negative interactions in lucerne CW polysaccharides hydrolysis.
Under scanning electron microscopy (SEM), S85 comprised the most dense layer of bacterial cell mass attached to and colonized on CW particles. The cell surface topology of the cellulolytic strains S85, FD1 and 7 attached to CW particles was specified by a coat of characteristic protuberant structures.     

Brain lysosomal hydrolases: I. Solubilization and electrophoretic behavior of acid hydrolases in nerve-ending and mitochondrial-lysosomal fractions from rat brain. Effects of autolysis,neuraminidase, and storage

In solubility studies of 7 acid hydrolases, the extent of solubilization by sonic disruption varied with the enzyme species and increased with increasing pH and Triton X-100 concentration of the suspension medium. Hydrolases in the nerve-ending (NE) fraction were more resistant to solubilization than those in the mitochondrial-lysosomal (M-L) fraction, but nearly quantitative solubilization was attained by sonication in an alkaline buffer containing 0,5% Triton X-100. Polyacrylamide gel electrophoresis of extracts revealed multiple components of acid phosphatase, acid esterase, arylsulfatase,-glucuronidase, and-N-acetyl-hexosaminidase. The enzyme patterns varied with the subcellular fraction and the composition of the medium. In general, the acidic (anodic) forms of these hydrolases were more readily solubilized by sonication in acidic buffer, alkaline pH and Triton X-100 being required to solubilize the basic (cationic) components. The acidic forms of these enzymes were converted to less anodic or cathodic forms, or both, during autolysis at pH 6 at 0 and 37°C, and during storage at –20°C.     

Use of a cellulase-encoding gene probe to reveal restriction fragment length polymorphisms among ruminal strains ofBacteroides succinogenes

A cloned DNA fragment specifying an endoglucanase fromBacteroides succinogenes strain BL2 was shown to hybridize under nonstringent conditions to different BamH1 fragments of chromosomal DNA from each of five rumen strains ofB. succinogenes. Direct binding of BL2 total chromosomal DNA to DNA from the other four strains was between 16% and 42% of homologous binding, confirming a high degree of interstrain divergence.Bacteroides succinogenes BL2 chromosomal DNA did not show detectable hybridization with DNA from any of 15 strains of 11 other species of rumen bacteria, in tests carried out with NaOH-lysed cells on filters.     

Lignocellulose solubilization and conversion by extremely thermophilic Caldicellulosiruptor bescii improves by maintaining metabolic activity

The extreme thermophile Caldicellulosiruptor bescii solubilizes and metabolizes the carbohydrate content of lignocellulose, a process that ultimately ceases because of biomass recalcitrance, accumulation of fermentation products, inhibition by lignin moieties, and reduction of metabolic activity. Deconstruction of low loadings of lignocellulose (5 g/L), either natural or transgenic, whether unpretreated or subjected to hydrothermal processing, by C. bescii typically results in less than 40% carbohydrate solubilization. Mild alkali pretreatment (up to 0.09 g NaOH/g biomass) improved switchgrass carbohydrate solubilization by C. bescii to over 70% compared to less than 30% for no pretreatment, with two-thirds of the carbohydrate content in the treated switchgrass converted to acetate and lactate. C. bescii grown on high loadings of unpretreated switchgrass (50 g/L) retained in a pH-controlled bioreactor slowly purged (τ = 80 hr) with growth media without a carbon source improved carbohydrate solubilization to over 40% compared to batch culture at 29%. But more significant was the doubling of solubilized carbohydrate conversion to fermentation products, which increased from 40% in batch to over 80% in the purged system, an improvement attributed to maintaining the bioreactor culture in a metabolically active state. This strategy should be considered for optimizing solubilization and conversion of lignocellulose by C. bescii and other lignocellulolytic microorganisms.     

Purification of an LHI-RC-complex of Rhodospirillum rubrum by solubilization of chromatophores with a short-chain lecithin

Chromatophores from Rhodospirillum rubrum were solubilized using the detergent 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC). The solubilization curves are sigmoidal reaching a plateau at a detergent/protein ratio of 2–3 mol/mg corresponding to 75–90% solubilized protein. The BChl-binding proteins are stable over a large range of DHPC/protein ratios. A complex of BChl-binding proteins containing both LHI- and RC-polypeptides (LHI-RC-complex) was purified using a two step procedure. RC photochemical activity as well as absorption and near-IR CD spectra showed the complex to be active and stable after purification in presence of DHPC.Abbreviations ATPase adenosine triphosphatase - BChI bacteriochlorophyll - DHPC 1,2-diheptanoyl-sn-phosphatidylcholine - DNAse deoxyribonuclease - INT 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride - LDAO lauryl-N,N-dimethylamine-N-oxide - LHI-complex light harvesting complex - PMSF phenylmethylsulfonyl fluoride - RC-complex reaction center complex - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TCA trichloroacetic acid This work is dedicated to the memory of Prof. D. I. Arnon.     

Labdane diterpene derivatives from Holwaya mucida

Clumps of white crystals present in 40-day-old malt agar cultures of Holwaya mucida were isolated as long white needles by crystallization from ethanol following short extraction with chloroform. The levorotary compound ([] ₂₈₉ ²¹ =-193.8°) was recognized as a -lactone (C₁₇H₂₀O₅) by infrared and mass spectrometry. It was identified as 7-methoxy-3a, 10b-dimethyl-1, 2, 3, 3a, 5a, 7, 10b, 10c-octahydro-4H, 9H-furo[2, 3, 4 : 4, 5]naphtho[2, 1-c]pyran-4, 9-dione, a labdane-derived compound known as antibiotic LL-Z1271. Preparative thin-layer chromatography of the mother liquor afforded 2 minor metabolites. One was identified as LL-Z1271, the demethylated analogue of LL-Z1271. The other one named LL-Z1271, was recognized as a compound related to and : its structure could not be fully elucidated. H. mucida (anamorph: Crinula calciiformis) has no taxonomic relationship with two other LL-Z1271 producing species viz. Acrostalagmus sp. (= Acremonium cf. atrogriseum) and Oidiodendron truncatum.