Cellulases from Neocallimastix frontalis EB188 synthesized in the presence of glycosylation inhibitors: measurement of pH and temperature optima,protease and ion sensitivities

Summary The effects of protein glycosylation inhibitors were studied in Neocallimastix frontalis EB188. Low concentrations of tunicamycin and 2-deoxy-D-glucose inhibited zoospore germination, rhizoidal elongation, carbon source utilization and the production and secretion of cellulases and proteins. The carbohydrate-trimming inhibitors, deoxynojirimycin and glucono--lactone, had no measurable effect on rhizoidal growth and carbon source utilization. Cellulases (intracellular or extracellular) synthesized in the presence of glycosylation inhibitors were sensitive to -endoglycosidase H digestion, periodate modification, certain salts, changes in incubation temperature and pH, and protease. Anthrone staining of extracellular proteins confirmed the presence of glycoproteins. In N. frontalis EB188, glycosylation of protein and cellulase occurred and was important for cellular development and the production, secretion and activity of cellulases. Offprint requests to: R. E. Calza     

Optimization of protein and cellulase secretion in Neocallimastix frontalis EB188

Variations in culture feeding protocols were used to optimize the secretion of protein and cellulase in Neocallimastix frontalis EB188. High numbers (2000/ml) of zoospores, culture feeding at 55 h using a 1:3 dilution and cotton cellulose [0.25% (w/v) final] as the carbon source increased secretion. Endoglucanase reached 1.6±0.06 IU/ml, exoglucanase reached 0.032±0.006 IU/ml and -glucosidase reached 0.874±0.090 IU/ml. Medium containing twice the concentration of non-carbon-source components failed to increase secretions. Gel electrophoresis demonstrated that eleven cellulases were present. Two cellulases were secreted only in stationary cultures. rotein and cellulase secretion in N. frontalis EB188 may be dependent on the dilution of fermentation products. Correspondence to: R. E. Calza     

Nascent synthesis and secretion of cellobiase inNeocallimastix frontalis EB188

De novo synthesis and the secretion of cellobiase fromNeocallimastix frontalis EB188 were studied. Cellobiase was secreted rapidly in cellulose switch cultures. Chemical inhibition of protein synthesis and radiotracer studies suggested secretion was dependent on nascent protein synthesis. Inhibitors of protein glycosylation also inhibited secretion. An 85,000 dalton protein (and several others) represented the principal differences in de novo synthesis between cellulose- and glucose-switched cultures. Concentrations of the 85,000 daltons protein increased with culture incubation time and ultimately accounted for 7% of the total protein secreted. This protein was purified by gel electrophoresis separations and was determined to be a cellobiase. Secretion of this cellobiase was correlated with its radiolabeling. The possibility of cellobiase induction representing a specialized gene control system inNeocallimastix frontalis EB188 is discussed.     

Regulation of protein and cellulase excretion in the ruminal fungusNeocallimastix frontalis EB188

Extracellular protein and cellulase excretion secretions were studied in the ruminal fungusNeocallimastix frontalis EB188. Cellulase assay and polyacrylamide gel electrophoresis was used to characterize protein excretion patterns caused by media switches of established cultures. Glucose switch medium caused the excretion of low levels of cellulase and increased amounts of low-molecular-weight proteins. Cellulose switch medium caused the excretion of high levels of cellulase and increased amounts of high-molecular-weight proteins. Several proteins were excreted uniquely or in greater amounts in cellulose cultures than in glucose cultures. Distinct protein excretion patterns suggested that regulation of cellulase was a closely controlled process in ruminal fungi.     

The effect of Aspergillus oryzae fermentation extract on the anaerobic fungi Neocallimastix frontalis EB 188, Piromyces communis DC 193 and Orpinomyces ssp. RW 206: generalized effects and component analysis

Three fungi Neocallimastix frontalis EB 188, Piromyces communis DC 193 and Orpinomyces ssp. RW 206, representing the predominant cultures isolated from cattle, were shown to respond to the addition of Aspergillus oryzae fermentation extract (i.e., Amaferm; BioZyme Inc., St. Joseph, Mo.) stimulation. Growth rates, protein and cellulase secretion and fungal mass production were all accelerated in the presence of the extract. Analysis of volatile fatty acids produced by these three species suggested that extract addition increased and altered gas production. Fractionation and preliminary analysis of the components present in the soluble extract, which stimulated the growth of the cellulolytic fungus N. frontalis EB 188, were also attempted. Soluble and filtered, sterilized extract was treated prior to use as a stimulant. Pretreatments included dialysis, ultraviolet irradiation, freeze thaw cycling, boiling, autoclaving, digestion with protease, autodigestion, organic extraction, decolorizing-carbon binding and polyethylene glycol concentration. Boiling, protease treatment, organic extraction, freeze thaw cycling and decolorizing-carbon binding reduced the ability of the extract to stimulate fungal cultures. Gel electrophoresis methods demonstrated that protein- and cellulasesecretion profiles were not identical in control and stimulated cultures. High-performance liquid chromatography methods allowed the separation of the extract into a limited number of ultraviolet-absorbing peaks, of which several stimulated the physiology of the fungus. Received: 6 December 1995/Received revision: 7 February 1996/Accepted: 4 March 1996     

Fractionation of cellulases from the ruminal fungus Neocallimastix frontalis EB188.

Cellulases from the ruminal fungus Neocallimastix frontalis EB188 were separated by using hydroxylapatite column chromatography. Seven carboxymethylcellulases, six avicelases, and four beta-glucosidases accounted for the majority of the activities. The separation of enzymes was confirmed by using polyacrylamide gel electrophoresis. Electrophoretic migration, analysis of hydrolysis products, and substrate specificity measurements suggested that several different cellulases were secreted in N. frontalis EB188. The possible relationship of cellulase diversity to protein glycosylation is discussed.     

Fractionation of cellulases from the ruminal fungus Neocallimastix frontalis EB188.

Cellulases from the ruminal fungus Neocallimastix frontalis EB188 were separated by using hydroxylapatite column chromatography. Seven carboxymethylcellulases, six avicelases, and four beta-glucosidases accounted for the majority of the activities. The separation of enzymes was confirmed by using polyacrylamide gel electrophoresis. Electrophoretic migration, analysis of hydrolysis products, and substrate specificity measurements suggested that several different cellulases were secreted in N. frontalis EB188. The possible relationship of cellulase diversity to protein glycosylation is discussed.     

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.     

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.     

Engineering ‘designer’ glycomodules for boosting recombinant protein secretion in tobacco hairy root culture and studying hydroxyproline‐O‐glycosylation process in plants

The key technical bottleneck for exploiting plant hairy root cultures as a robust bioproduction platform for therapeutic proteins has been low protein productivity, particularly low secreted protein yields. To address this, we engineered novel hydroxyproline (Hyp)‐O‐glycosylated peptides (HypGPs) into tobacco hairy roots to boost the extracellular secretion of fused proteins and to elucidate Hyp‐O‐glycosylation process of plant cell wall Hyp‐rich glycoproteins. HypGPs representing two major types of cell wall glycoproteins were examined: an extensin module consisting of 18 tandem repeats of ‘Ser‐Hyp‐Hyp‐Hyp‐Hyp’ motif or (SP4)₁₈ and an arabinogalactan protein module consisting of 32 tandem repeats of ‘Ser‐Hyp’ motif or (SP)₃₂. Each module was expressed in tobacco hairy roots as a fusion to the enhanced green fluorescence protein (EGFP). Hairy root cultures engineered with a HypGP module secreted up to 56‐fold greater levels of EGFP, compared with an EGFP control lacking any HypGP module, supporting the function of HypGP modules as a molecular carrier in promoting efficient transport of fused proteins into the culture media. The engineered (SP4)₁₈ and (SP)₃₂ modules underwent Hyp‐O‐glycosylation with arabino‐oligosaccharides and arabinogalactan polysaccharides, respectively, which were essential in facilitating secretion of the fused EGFP protein. Distinct non‐Hyp‐O‐glycosylated (SP4)₁₈‐EGFP and (SP)₃₂‐EGFP intermediates were consistently accumulated within the root tissues, indicating a rate‐limiting trafficking and/or glycosylation of the engineered HypGP modules. An updated model depicting the intracellular trafficking, Hyp‐O‐glycosylation and extracellular secretion of extensin‐styled (SP4)₁₈ module and AGP‐styled (SP)₃₂ module is proposed.     

Synthesis of extracellular proteins in embryogenic and non-embryogenic cell cultures of alfalfa

Extracellular proteins, released into the culture medium from alfalfa cells grown in embryogenic and non-embryogenic conditions, were ³⁵S-methionine labelled at different days of culture. SDS-PAGE analysis showed significant differences between the patterns of extracellular proteins secreted into the medium devoid of 2,4-d, in which cells formed somatic embryos, or in presence of 2,4-d, in which undifferentiated cell proliferation took place. Some proteins, evident in 2,4-d-supplied cultures, disappeared when cells were subcultured in the embryogenic conditions. Western analysis with antibodies against the carrot extracellular proteins EP1 and EP2 showed the presence of homologous alfalfa proteins. In 2,4-d depleted alfalfa cells, an EP1-like protein disappeared and another one was reduced, while the presence of the EP2-like protein was, in the same conditions, strongly enhanced.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - EP extracellular proteins - ns-LTP non specific lipid transfer protein - SDS-PAGE Sodium dodecyl sulphate polyacrilamide gel electrophoresis     

Production and Biochemical Characterization of Insecticidal Enzymes from Aspergillus fumigatus Toward Callosobruchus maculatus

In the present work, Aspergillus fumigatus is described as a higher producer of hydrolytic enzymes secreted in response to the presence of the Callosobruchus maculatus bruchid pest. This fungus was able to grow over cowpea weevil shells as a unique carbon source, secreting alkaline proteolytic and chitinolytic enzymes. Enzyme secretion in A. fumigatus was induced by both C. maculatus exoskeleton as well as commercial chitin, and alkaline proteolytic and chitinolytic activities were detected after 48 hours of growth. Furthermore, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the production of specific proteins. Among them, two extracellular alkaline proteinases from culture enriched with C. maculatus exoskeleton were purified after chromatographic procedures using ion exchange and affinity columns. These proteins, named AP15 and AP30, had apparent molecular masses of 15,500 and 30,000 Da, respectively, as estimated by SDS-PAGE electrophoresis and mass spectrometry. AP30 was classified as a serine proteinase because it was inhibited by 5 mM phenylmethylsulfonyl fluoride (100%) and 50 μM leupeptin (67.94%).     

Effects of glycoprotein and basement membrane synthesis inhibitors on the growth of cultured renal collecting duct epithelium

Summary The biochemical and morphological extent of glycoprotein synthesis inhibition of cellular and extracellular proteins was studied on cultured renal collecting duct (CD) epithelium. We found that tunicamycin (4 g/ml) inhibits the glycosylation of a 150,000 d glycoprotein (gp_CDI). A 85,000 d glycoprotein (gp_CDII) was not affected. The inhibition by tunicamycin demonstrates that gp_CDI has characteristics of a N-glycan, whereas gp_CDII seems to be an O-glycan. 6-diazo-5-oxo-norleucine (4×10^–5M) which was used as glutamine analogue, did not show a comparable inhibitory effect as seen with tunicamycin. The lack of effect of norleucine demonstrates that glutamine is not the locus of glycosylation in both proteins. However, because of the tunicamycin inhibition it points to asparagine as the site of glycosylation in the gp_CDI. Long term cultures of the tissue up to 15 days in the presence of tunicamycin and norleucine and of substances usually used as basement membrane inhibitors, such as hydroxy-d-proline (1 mM), l-azetidine-2-carboxylic acid (1 mM) and o- and p-nitrophenyl-xylopyranoside (1 mM), revealed that it is possible to eliminate completely the fibroblasts beneath the cultured epithelium and within the degenerating corematerial. Experiments with hydroxy-d-proline showed the most striking effect. Experiments with l-azetidine-2-carboxylic acid and nitrophenyl-xylopyranoside resulted in the elimination of fibroblasts and dedifferentiation of the collecting duct epithelium.     

Production of Cellulases by Aspergillus fumigatus and Characterization of One β-Glucosidase

Aspergillus fumigatus produces substantial extracellular cellulases on several cellulosic substrates including simple sugars. Low glucose potentiates enzyme production, but most cellulose-induced cellulases are repressed by high glucose. As production of cellulase in a wide substrate range is unusual, the cellulolytic complex of this thermophilic fungus was investigated. A β-glucosidase was separated by gel filtration and ion-exchange chromatography. It migrated in native polyacrylamide gel as a single protein (130 kDa), which split under denaturing conditions into two smaller proteins having molecular masses of 90 kDa and 45 kDa. However, only the 90-kDa protein was active. Conventional chromatographic procedures were unsuccessful for the separation of these two proteins. Therefore, the 130-kDa protein was studied for its kinetic properties. It hydrolyzed p-nitrophenyl-β-D-glucopyranoside (p-NPG) and cellobiose, but not β-glucans, laminarin, and p-nitrophenyl-β-D-xilopyranoside. The optimal pH and temperature of p-NPG and cellobiose hydrolysis were 5.0 and 4.0, and 65°C and 60°C, respectively. The K _m values, determined for cellobiose and p-NPG of hydrolysis, were 0.075 mM and 1.36 mM, respectively. Glucose competitively inhibited the hydrolysis of p-NPG. The K_i was 3.5 mM.     

Purification and some properties of an extracellular l-amino acid oxidase from Cellulomonas cellulans AM8 isolated from soil

Summary The soil isolate Cellulomonas cellulans AM8 produces an extracellular l-amino acid oxidase (L-AAO) with broad substrate specificity. The strain produced up to 0.35 unit (U)/ml of the extracellular L-AAO in a simple medium containing glycerol and yeast extract. The enzyme was easily purified up to 30 U/mg protein using Phenyl-Sepharose fast flow. The purified enzyme migrated as single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of 55 kDa. On native PAGE the molecular mass was approx. 300 000 kDa, which may be due to aggregation. With the exception of glycine, proline, and threonine, all the amino acids normally constituting proteins were oxidized. The V _max values from 0.7 to 35.2 U/mg for aspartic acid and lysine, respectively, and the K _m values from 0.007 to 7.1 mm for cysteine and valine, respectively, were obtained at 25° C and pH 7.0 in oxygen-saturated solutions. The L-AAO had a pH optimum of 6.5–7.5. It was stable for several months at — 30° C and for some days at 35° C. Ferricyanide served as an electron acceptor with a V _max of 50 U/mg and K _m for 0.3 mm with phenylalanine as the substrate. Correspondence to: R. D. Schmid     

Characterization of a glucose 3-dehydrogenase from the cultivated mushroom (Agaricus bisporus )

An extracellular enzyme with glucose dehydrogenase activity was purified from liquid cultures of the basidiomycete Agaricus bisporus after growth with d-cellobiose or d-glucose as carbon source. The molecular mass was measured as 57 kDa by gel filtration and 55 kDa by sodiumdodecyl sulphate/polyacrylamide gel electrophoresis, while the isoelectric point was at pH 3.6. By analysis of ¹H-NMR spectra in D₂O, the product of d-glucose oxidation was identified as 3-ketoglucose. The substrates oxidized included d-cellobiose, l-arabinose, d-xylose and sucrose, but the specificity parameter (k _cat/K _m) was highest for d-glucose. Two electron acceptors were identified, namely 2,6-dichloroindophenol and p-benzoquinone, but reduction of dioxygen, ferricyanide or cytochrome c was not detectable. The selective C-3 oxidation of d-glucose is well-characterized for Agrobacterium and Flavobacterium, but this is the first report for a fungus. Received: 19 June 1998 / Received revision: 15 September 1998 / Accepted: 17 September 1998     

Efficient expression of a Paenibacillus barcinonensis endoglucanase in Saccharomyces cerevisiae

The endoglucanase coded by celA (GenBank Access No. Y12512) from Paenibacillus barcinonensis, an enzyme with good characteristics for application on paper manufacture from agricultural fibers, was expressed in Saccharomyces cerevisiae by using different domains of the cell wall protein Pir4 as translational fusion partners, to achieve either secretion or cell wall retention of the recombinant enzyme. Given the presence of five potential N-glycosylation sites in the amino acid sequence coded by celA, the effect of glycosylation on the enzymatic activity of the recombinant enzyme was investigated by expressing the recombinant fusion proteins in both, standard and glycosylation-deficient strains of S. cerevisiae. Correct targeting of the recombinant fusion proteins was confirmed by Western immunoblot using Pir-specific antibodies, while enzymatic activity on carboxymethyl cellulose was demonstrated on plate assays, zymographic analysis and colorimetric assays. Hyperglycosylation of the enzyme when expressed in the standard strain of S. cerevisiae did not affect activity, and values of 1.2 U/ml were obtained in growth medium supernatants in ordinary batch cultures after 24 h. These values compare quite favorably with those described for other recombinant endoglucanases expressed in S. cerevisiae. This is one of the few reports describing the expression of Bacillus cellulases in S. cerevisiae, since yeast expressed recombinant cellulases have been mostly of fungal origin. It is also the first report of the yeast expression of this particular endoglucanase.     

Export,purification, and activities of affinity tagged <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> levansucrase produced by <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Fructosyltransferases, like the Lactobacillus reteri levansucrase, are important for the production of new fructosyloligosaccharides. Various His₆- and Strep-tagged variants of this enzyme were recombinantly produced and exported into the growth medium using the Gram-positive bacterium Bacillus megaterium. Nutrient-rich growth medium significantly enhanced levansucrase production and export. The B. megaterium signal peptide of the extracellular esterase LipA mediated better levansucrase export compared to the one of the penicillin amidase Pac. The combination of protein export via the LipA signal peptide with the coexpression of the signal peptidase gene sipM further increased the levansucrase secretion. Fused affinity tags allowed the efficient one-step purification of the recombinant proteins from the growth medium. However, fused peptide tags led to slightly decreased secretion of tested fusion proteins. After upscaling 2 to 3 mg affinity tagged levansucrase per liter culture medium was produced and exported. Up to 1 mg of His₆-tagged and 0.7 mg of Strep-tagged levansucrase per liter were recovered by affinity chromatography. Finally, the purified levansucrase was shown to synthesize new fructosyloligosaccharides from the novel donor substrates d-Gal-Fru, d-Xyl-Fru, d-Man-Fru, and d-Fuc-Fru. R. Biedendieck and R. Beine contributed equally to this work.     

Differential expression of cellulases and xylanases by <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> grown on different carbon sources

The diversity of cellulases and xylanases secreted by Cellulomonas flavigena cultured on sugar cane bagasse, Solka-floc, xylan, or glucose was explored by two-dimensional gel electrophoresis. C. flavigena produced the largest variety of cellulases and xylanases on sugar cane bagasse. Multiple extracellular proteins were expressed with these growth substrates, and a limited set of them coincided in all substrates. Thirteen proteins with carboxymethyl cellulase or xylanase activity were liquid chromatography/mass spectrometry sequenced. Proteins SP4 and SP18 were identified as products of celA and celB genes, respectively, while SP20 and SP33 were isoforms of the bifunctional cellulase/xylanase Cxo recently sequenced and characterized in C. flavigena. The rest of the detected proteins were unknown enzymes with either carboxymethyl cellulase or xylanase activities. All proteins aligned with glycosyl hydrolases listed in National Center for Biotechnology Information database, mainly with cellulase and xylanase enzymes. One of these unknown enzymes, protein SP6, was cross-induced by sugar cane bagasse, Solka-floc, and xylan. The differences in the expression maps of the presently induced cultures revealed that C. flavigena produces and secretes multiple enzymes to use a wide range of lignocellulosic substrates as carbon sources. The expression of these proteins depends on the nature of the cellulosic substrate.     

Effects of tunicamycin on secretion and enzymatic activities of cellulase fromTrichoderma reesei

Summary The effects of tunicamycin, an inhibi-tor of N-asparagine linked glycosylation, on the synthesis, secretion, and activities of the cellulases produced byTrichoderma reesei wild type QM6a and hypersecrefing mutant RL-P37 were studied. Neither the level of secreted cellutase nor the total amount of secreted protein was affected by the drug at a concentration (5 μg/ml) that slightly in-hibited growth. SDS-polyacrylamide gel electro-phoretic mobilities of proteins secreted during growth in tunicamycin were similar to those of proteins from control cultures that had their N-linked oligosaccharides removed by endoglycosi-dase H. Isoelectric focusing patterns of secreted proteins were also altered by growth in the pres-ence of tunicamycin. All of the bands stained with Schiff’s reagent, indicating that the secreted cellu-lases contained O-linked oligosaccharides in ad-dition to N-linked sugars. Endoglucanase activity in culture broths from tunicamycin grown mycelia was more thermolabile and protease-sensitive than the same activity from control cultures. Thus, N-asparagine linked oligosaccharides do not appear to be necessary forT. reesei cellulase secretion or activity, but do seem to contribute to the stability of the enzymes. The role of O-finked oligosaccharides is being investigated.