Supernatant protein and cellulase activities of the anaerobic ruminal fungus Neocallimastix frontalis EB188.

Protein and cellulose activities were measured in culture supernatants of the anaerobic ruminal fungus Neocallimastix frontalis EB188 established in glucose medium and switched to either glucose, cellobiose, or cellulose media. Polyacrylamide gel electrophoresis was used to show differences caused by changing medium carbon source. Culture supernatants contained proteins with molecular weights ranging from greater than 116,000 to about 19,000. Low levels of cellulose activity were evident in glucose-grown cultures. Increased amounts of slowly migrating cellulase activities appeared in the supernatants of glucose-grown cultures switched to cellulose. Cellulase activities which reacted differentially during colorimetric and in situ assays were produced. Isoelectric points of cellulase activities varied from 3.7 to 8.3, and activities possessed optimal pHs of between 5.9 and 6.5.     

Supernatant protein and cellulase activities of the anaerobic ruminal fungus Neocallimastix frontalis EB188

Protein and cellulose activities were measured in culture supernatants of the anaerobic ruminal fungus Neocallimastix frontalis EB188 established in glucose medium and switched to either glucose, cellobiose, or cellulose media. Polyacrylamide gel electrophoresis was used to show differences caused by changing medium carbon source. Culture supernatants contained proteins with molecular weights ranging from greater than 116,000 to about 19,000. Low levels of cellulose activity were evident in glucose-grown cultures. Increased amounts of slowly migrating cellulase activities appeared in the supernatants of glucose-grown cultures switched to cellulose. Cellulase activities which reacted differentially during colorimetric and in situ assays were produced. Isoelectric points of cellulase activities varied from 3.7 to 8.3, and activities possessed optimal pHs of between 5.9 and 6.5.     

Carbon source,cyclic nucleotide,and protein inhibitor effects on protein and cellulase secretions inNeocallimastix frontalis EB188

Protein and cellulase secretion inNeocallimastix frontalis EB188 was studied. Induction of secretion in this ruminal fungus was dependent on the amount of medium cellulose and on de novo protein synthesis. Medium glucose repressed secretions and diminished the utilization of medium cellulose. Exogenously added cyclic nucleotides failed to derepress glucose repression. Medium glycerol had no effect on protein and cellulase secretion, would not support cell growth, and was nontoxic.     

Effects of folic acid on growth performance,ruminal fermentation,nutrient digestibility and urinary excretion of purine derivatives in post-weaned dairy calves

This study evaluated the effects of folic acid (FA) supplementation on growth performance, ruminal fermentation, nutrient digestibility and urinary purine derivatives (PD) excretion in dairy calves. Forty-eight Chinese Holstein male dairy calves at 60 ± 3.2 d of age and 89 ± 5.9 kg body weight (mean ± standard error) were assigned to one of four groups in a randomised block design. Calves in control group were fed basal diet, calves in low FA, medium FA and high FA groups with 3.6, 7.2 and 10.8 mg FA per kg basal diet, respectively. The dietary corn silage to concentrate ratio was 50:50 (dry matter [DM] basis). DM intake and average daily gain (ADG) quadratically increased, and feed conversion ratio quadratically decreased with increasing FA supplementation. Ruminal pH linearly decreased, whereas total volatile fatty acids quadratically increased. The unchanged acetate-to-propionate ratio was due to the similar change in acetate and propionate concentration. Ammonia N content quadratically decreased. Digestibility of DM, organic matter, crude protein, ether extract, neutral detergent fibre and acid detergent fibre linearly increased. Activities of carboxymethyl cellulase, cellobiase, xylanase and pectinase linearly increased, but α-amylase and protease quadratically increased. Abundance of Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes linearly increased, but Butyrivibrio fibrisolvens and Prevotella ruminicola quadratically increased. Urinary total PD excretion quadratically increased. The results indicated that FA supplementation increased ADG, ruminal fermentation and nutrient digestibility with promoted ruminal microbial growth and enzyme activity, and the optimum dose was 7.2 mg FA per kg basal diet for calves.     

Media carbon induction of extracellular cellulase activities inNeocallimastix frontalis isolate EB188

Extracellular cellulase induction in the ruminal fungusNeocallimastix frontalis isolate EB188 was followed. Glucose media-established cultures produced cellulase when switched to a variety of cellulose-containing media. High levels of cellulase and xylanase activities were present in cultures switched to sigma cell 100, solka floc, avicel, sisal fiber, and wheat straw, but not those switched to glucose, carboxymethylcellulose, or wood chips. Several assay substrates were used to show differential cellulase induction as well as-glucosidase activity. Cellulases hydrolyzed short oligosaccharides and released glucose from insoluble cellulose. Cellobiase activity was also indicated. Cellulase activity tolerated brief exposure to high temperature, was insensitive to certain metal ions, and possessed pH optima between 5.0 and 6.5.     

Fruiting of Agaricus bisporus

Several enzymes were assayed in extracts from mycelium-colonised compost during growth and fruiting of Agaricus bisporus (Lange) Imbach. Comparison of changes of enzyme levels in axenic and nonaxenic cultures and in cultures of non-fruiting strains indicated that they were associated directly with the fungal mycelium. Large changes were found in the amounts of laccase and cellulase which were correlated with fruit body development. Laccase concentration increased during mycelial growth and then declined rapidly at the start of fruiting. Cellulase activity could be detected throughout growth but increased at fruiting. No such changes were observed in xylanase, alkaline protease, laminarinase and acid and alkaline phosphatases. Activities of laccase and cellulase were measured in axenic cultures arrested at various stages of fruiting development. Such cultures showed that the changes in concentration of laccase and cellulase were associated with the enlargement of fruit bodies.     

Extracellular recombinant protein production from an Escherichia coli lpp deletion mutant

E. coli is one of the most commonly used host strains for recombinant protein production. However, recombinant proteins are usually found intracellularly, in either cytoplasm or periplasmic space. Inadequate secretion to the extracellular environment is one of its limitations. This study addresses the outer membrane barrier for the translocation of recombinant protein directed to the periplasmic space. Specifically, using recombinant maltose binding protein (MalE), xylanase, and cellulase as model proteins, we investigated whether the lpp deletion could render the outer membrane permeable enough to allow extracellular protein production. In each case, significantly higher excretion of recombinant protein was observed with the lpp deletion mutant. Up to 90% of the recombinant xylanase activity and 70% of recombinant cellulase activity were found in the culture medium with the deletion mutant, whereas only 40-50% of the xylanase and cellulase activities were extracellular for the control strain. Despite the weakened outer membrane in the mutant strain, cell lysis did not occur, and increased excretion of periplasmic protein was not due to cell lysis. The lpp deletion is a simple method to generate an E. coli strain to effect significant extracellular protein production. The phenotype of extracellular protein production without cell lysis is useful in many biotechnological applications, such as bioremediation and plant biomass conversion.     

Effects of serum,cycloheximide and actinomycin D on protein secretion by quiescent mouse embryo fibroblasts

Quiescent secondary cultures of Swiss mouse embryo fibroblasts secrete several proteins in response to the addition of 20% fetal calf serum (FCS). Of these proteins, a polypeptide of molecular weigth (Mr) 48 000 (48 K) was identified in the medium within an hour of mitogenic stimuli. In the next hour an additional protein of Mr 26000 (26 K) appeared in the medium. These two proteins were absent in the conditioned medium of quiescent cells. A third protein of molecular weight 45,000 (45 K) was found in small quantities in the conditioned medium of quiescent cells but a 2–3 fold increase in the level of this protein was observed in the medium of stimulated cells. The level of the serum-induced 45 K protein was much higher in the medium of cells that were treated with cycloheximide (CH) and FCS than that found in the medium of cells treated with FCS alone. A 40000 dalton protein was found to be a quiescence specific protein which was observed in large amounts in the medium of quiescent cells; the level of this protein gradually declined in the conditioned medium as the cells entered into the proliferative phase. Actinomycin D specifically inhibited the level of the 45 K secreted protein and a 29 K intracellular protein when added along with CH. In contrast to the inhibition of the synthesis of mitogen induced proteins, actinomycin D super-induced the intracellular and extracellular levels of the matrix proteins fibronectin and procollagens.     

Effects of the supplementation of distillers’ grape residues on ruminal degradability,whole tract digestibility and nitrogen metabolism in sheep

ABSTRACT

Five ruminally and duodenally cannulated Kazakh male lambs (30 ± 2.75 kg) maintained singly in a metabolic cage were used in a 5 × 5 Latin square experiment to investigate the effect of supplementing a ration with five different levels of distillers’ grape residue (DGR) on ruminal degradability, whole tract digestibility and nitrogen (N) metabolism of growing lambs. The rations were isoenergetic and isonitrogenous and contained 0, 3.85, 7.70, 11.55 and 15.41% DGR (DM basis). Each experimental period lasted for 18 d: 10 d for adaptation to the dietary treatment and 8 d for faecal, urinary, ruminal and duodenal digesta sample collections. The outflow rate of ruminal digesta increased (p = 0.032) linearly with the increased level of dietary neutral detergent fibre content, caused by the supplementation of DGR. As a result, the effective degradability of dry matter and crude protein decreased significantly with the treatments. Although the dietary intake of N, duodenal flow of total N, and the endogenous N at the duodenum were not affected by experimental treatments, N fractions in the digesta were altered. Ruminal microbial N decreased (p < 0.01) linearly; in contrast, ruminal un-degradable protein increased linearly (p < 0.01) in response to the increased addition of DGR. Although there was no significant difference in faecal N among treatments, N retention was increased linearly (p = 0.014), owing to the remarkable reduction (p = 0.016) of urinary N excretion with an increasing level of DGR. The results indicate that the DGR has some potential benefits of increasing the supply of bypass protein and of improving the utilisation efficiency of N for sheep. Therefore, the supplementation of DGR in ruminant feeding is recommended at levels not exceeding 10% of the diet.     

Biosynthesis of the sesquiterpenic phytoalexin capsidiol in elicited root cultures of chili pepper (Capsicum annuum)

The biosynthesis of the sesquiterpenic phytoalexin capsidiol was investigated using in vitro root cultures of chili pepper (Capsicum annuum) elicited with cellulase. Optimal concentrations of cellulase and sucrose for capsidiol production were established. A simple spectrophotometric procedure to quantify capsidiol was improved. Monoclonal antibodies against a tobacco sesquiterpene cyclase were used to detect a similar protein in pepper root extracts. We found that capsidiol was secreted to the medium and the maximal production was achieved at 24 h after elicitation. In contrast, the maximal amount of the elicitor inducible sesquiterpene cyclase was found between 6 and 8 h. Addition of small amounts of polyvinylpyrrolidone was necessary for sesquiterpene cyclase enzyme activity assays.Abbreviations AP alkaline phosphatase - BCIP 5-bromo-4-chloro-3-indolylphosphate - DMF dimethyl-formamide - FPP farnesyl pyrophosphate - MAb monoclonal antibodies - NBT nitro blue tetrazolium - PVP polyvinylpyrrolidone - SC sesquiterpene cyclase     

Identification of extracellular proteins from actinomycetes responsible for the solubilisation of lignocellulose

Summary We have utilized strains of three actinomycete species, Actinomadura sp, Streptomyces cyaneus and Thermomonospora mesophila, to study the solubilisation of lignocellulose. The production of extracellular proteins, was measured for each of the organisms during 17 days growth using medium containing either glucose or ball-milled straw. Some of the extracellular proteins (as identified by SDS gel electrophoresis) were present under both growth conditions, but others were specific to the type of medium or the period of incubation. The levels of proteins were compared with the abilities of the extracellular protein preparations to solubilise a substrate of ¹⁴C-labelled lignocellulose. About 6% of the radioactive material were solubilised when the extracellular proteins from the cultures grown on glucose were incubated with the substrate, compared to 20–30% that were solubilised by the extracellular proteins from the cultures grown on ball-milled straw. Partial characterisation of an enzyme from S. cyaneus responsible for the solubilisation of lignocellulose was achieved by gel filtration of the extracellular proteins, using Superose 12. Material that eluted from the column with an apparent molecular weight of about 20 000 accounted for all of the solubilisation of ¹⁴C-labelled (i.e. lignin-derived) moieties. In contrast, when the eluate was tested for the presence of cellulases and xylanases most of the activities were found in fractions containing material with an apparent molecular weight of about 45 000. We conclude that in cultures of S. cyaneus grown on ball-milled straw, a single extracellular enzyme is responsible for the solubilisation of lignin in lignocellulose, and that this enzyme is unlikely to be a cellulase or a xylanase.     

Effects of dietary supplementation of rumen-protected folic acid on rumen fermentation,degradability and excretion of urinary purine derivatives in growing steers

The present experiment was undertaken to determine the effects of dietary addition of rumen-protected folic acid (RPFA) on ruminal fermentation, nutrient degradability, enzyme activity and the relative quantity of ruminal cellulolytic bacteria in growing beef steers. Eight rumen-cannulated Jinnan beef steers averaging 2.5 years of age and 419 ± 1.9 kg body weight were used in a replicated 4 × 4 Latin square design. The four treatments comprised supplementation levels of 0 (Control), 70, 140 and 210 mg RPFA/kg dietary dry matter (DM). On DM basis, the ration consisted of 50% corn silage, 47% concentrate and 3% soybean oil. The DM intake (averaged 8.5 kg/d) was restricted to 95% of ad libitum intake. The intake of DM, crude protein (CP) and net energy for growth was not affected by treatments. In contrast, increasing RPFA supplementation increased average daily gain and the concentration of total volatile fatty acid and reduced ruminal pH linearly. Furthermore, increasing RPFA supplementation enhanced the acetate to propionate ratio and reduced the ruminal ammonia N content linearly. The ruminal effective degradability of neutral detergent fibre from corn silage and CP from concentrate improved linearly and was highest for the highest supplementation levels. The activities of cellobiase, xylanase, pectinase and α-amylase linearly increased, but carboxymethyl-cellulase and protease were not affected by the addition of RPFA. The relative quantities of Butyrivibrio fibrisolvens, Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes increased linearly. With increasing RPFA supplementation levels, the excretion of urinary purine derivatives was also increased linearly. The present results indicated that the supplementation of RPFA improved ruminal fermentation, nutrient degradability, activities of microbial enzymes and the relative quantity of the ruminal cellulolytic bacteria in a dose-dependent manner. According to the conditions of this experiment, the optimum supplementation level of RPFA was 140 mg/kg DM.     

ATP-levels and cellulase formation in batch and fed-batch cultures ofTrichoderma viride grown on lactose

In the partially catabolically-derepressed mutant ofTrichoderma viride grown on lactose the excretion of cellulase into the medium started when the concentration of sugar in the medium decreased below 0.4 % and was preceded by a rapid transient increase of total ATP concentration in the culture. After reaching the maximum, the ATP level started to decrease while the excretion of cellulase continued until the lactose was practically exhausted. Repeated additions of lactose at the times of ATP decrease caused the continuation of cellulase formation for prolonged periods.     

Adaptation of renal function to hypotonic medium in the winter flounder (Pseudopleuronectes americanus)

Summary The kidneys of winter flounders transferred to hypotonic medium were investigated for glomerular and tubular handling of fluid and electrolytes and for the urinary excretion of proteins. Media were sea water (925 mosm·kg^–1) and brackish water (70 mosm·kg^–1).In sea water, the urine was hypertonic to the plasma in 7 fish of this study. Urine flow rate was correlated with the GFR. After adaptation to brackish water a delay of 1 to 3 days was observed until the kidneys switched from fluid retention to the excretion of large amounts of dilute urine. GFR and urine flow rate were increased from 0.61±0.08 to 1.58±0.29 ml·h^–1·kg^–1 and from 0.14±0.02 to 0.68±0.08 ml·h^–1·kg^–1, respectively . With increased filtered load the tubular reabsorption of fluid decreased from 74±2.4% to 45±11.2%. The excretion rates of sodium and potassium were increased due to decreased fractional sodium and potassium reabsorption. The urinary excretion of divalent cations, however, was reduced because the net tubular reabsorption of calcium was increased and the net secretion of magnesium was diminished.Both the urinary total protein concentration and the protein pattern showed no significant change, but the rate of protein excretion was increased from 0.21±0.04 to 0.60±0.05 mg·h^–1·kg^–1. The comparison of protein patterns obtained from urine and serum samples revealed that high molecular weight (HMW) proteins prevail in the serum whereas low molecular weight (LMW) proteins dominate in the urine. The diminished quantity of the HMW-protein fraction in the urine thus may reflect size selectivity of the glomerular filtration barrier for serum proteins also in the winter flounder.Abbreviations BW brackish water - SW sea water - GFR glomerular filtration rate - HMW heigh molecular weight - LMW low molecular weight     

Protein secretion in phosphate-limited cultures of Bacillus subtilis 168

The secretion of proteins from Bacillus subtilis was studied under physiologically well-defined conditions in continuous cultures at a range of specific growth rates. The kinetics of secretion was analysed by using pulse-chase and immunoprecipitation techniques that allowed both processing and release to be monitored. Growth conditions were selected that were known to lead to significant changes in the anionic polymer composition of the cell wall. Under magnesium limitation only low levels of native proteins were released into the growth medium. In contrast, much higher amounts of released protein were observed under phosphate limitation. Although synthesis of native secretory proteins appeared to be highly regulated, only minor changes in the secretion of heterologous proteins were detected. Comparable kinetics of protein release of cells grown under different conditions indicated similar cell wall permeabilities. The large changes in the amounts of released proteins were not reflected in the production of chaperones and components required for protein secretion. The data suggest that the capacity of the secretion machinery is not a major limiting step in the export of native secretory proteins. Received: 23 September 1997 / Received revision: 10 November 1997 / Accepted: 16 November 1997     

Cellulase production and activity in a species ofCladosporium

ACladosporium species produced large amounts of cellulase enzyme components when grown in shake-culture with medium containing carboxymethylcellulose. There was significantly less activity when Avicel, filter paper or cotton were used as substrates. KNO₃ was better than NH₄Cl or urea for the production of cellulase. Tween 80 at 0.1% (w/v) increased the production of cellulase by 1.5 to 4.5-fold. All the cellulase components were optimally active in the assay at pH 5.0 and 60°C.     

Optimal nitrogen supply as a key to increased and sustained production of a monoclonal full‐size antibody in BY‐2 suspension culture

Plant cell cultures have been used as expression hosts for recombinant proteins for over two decades. The quality of plant cell culture‐produced proteins such as full‐size monoclonal antibodies has been shown to be excellent in terms of protein folding and binding activity, but the productivity and yield fell short of what was achieved using mammalian cell culture, in which the key to gram‐per‐liter expression levels was strain selection and medium/process optimization. We carried out an extensive media analysis and optimization for the production of the full‐size human anti‐HIV antibody 2G12 in N. tabacum cv. BY‐2. Nitrogen source and availability was found to be one key factor for the volumetric productivity of plant cell cultures. Increased amounts of nitrate in the culture medium had a dramatic impact on protein yields, resulting in a 10–20‐fold increase in product accumulation through a combination of enhanced secretion and higher stability. The results were scalable from shake flasks to stirred‐tank bioreactors, where the maximum yield per cultivation volume was 8 mg L^?1 over 7 days. During the stationary phase, antibody levels were 150‐fold higher in nitrogen‐enriched medium compared to standard medium. The enhanced medium appeared not to affect antibody quality and activity, as determined by Western blots, surface plasmon resonance binding assays and N‐glycan analysis. Biotechnol. Bioeng. 2010;107: 278–289. © 2010 Wiley Periodicals, Inc.     

Highly Basic 30- and 32-Kilodalton Proteins Associated with Synapse Formation on Polylysine-Coated Beads in Enriched Neuronal Cell Cultures

Neuronal proteins involved in axonal outgrowth and synapse formation were examined in an enriched neuronal cell culture system of the cerebellum. In rat cerebellar cell cultures, 98.9% of the cells are neurons and the remaining 1.1% of the cells are flat nonneuronal cells. These enriched neuronal cultures, examined with two-dimensional gel electrophoresis, showed protein patterns similar to those of neonatal cerebellum, but very different patterns from glial enriched cultures. High levels of a neuronal membrane acidic 29-kilodalton (kD) protein were found. It has been shown previously that neuronal cultures incubated with polylysine-coated beads will develop numerous presynaptic elements on the bead surface. We report here that isolation of the beads from enriched neuronal cell cultures incubated with [35S]methionine showed, with two-dimensional nonequilibrium pH gradient gel electrophoresis (2D-NEPHGE), levels of a basic 32-kD protein (pI 8) note detected in cultures alone, and increased levels of a 30-kD protein (pI 10). When culture medium was examined with 2D-NEPHGE, three acidic proteins were identified that were secreted by the cultured neurons. In summary, a neuronal enriched cell culture system was used with isolated polylysine-coated beads to identify basic 30-kD and 32-kD proteins that may be involved in synapse formation.