Characterization of Aspergillus oryzae fermentation extract effects on the rumen fungus Neocallimastix frontalis,EB 188. Part 2. Carbon source utilization and effects on zoospore production

The effect of a commercial Aspergillus oryzae fermentation extract on the utilization of carbon source and zoospore production by the rumen fungus Neocallimastix frontalis EB 188 was determined. In addition, the composition of a soluble extract prepared from the commercial product was analyzed. This extract was added to N. frontalis EB 188 cultures grown on a variety of substrates and periodically assayed for protein, enzymes, zoospore production, and carbon source utilization. The powdered product contained 93% dry matter, more than 3,000 A. oryzaespores per gram, and did not contain strong buffers or high concentrations of salt. Measurable concentrations of DNA, protein, carbohydrate and several enzymes including cellulase and amylase were also found. Soluble extract increased fungal physiology and treated cultures produced significantly higher levels of supernatant protein and enzymes including amylase, cellulase and beta-glucosidase. The fungal response depended on culture carbon source. However, culture zoospore production was increased regardless of substrate provided. Culture utilization of glucose was more rapid in treated cultures, yet high levels of the extract greatly inhibited glucose utilization.     

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     

Fermentation extract effects on the morphology and metabolism of the rumen fungus Neocallimastix frontalis EB188

The effects of Aspergillus oryzae fermentation extract, Amaferm, on the rumen fungus Neocallimastix frontalis EB188 were studied. The secretion of cellulase was increased by 67% and rhyzoid development was increased 3.8-fold in the presence of extract. Strength of fungal response increased in a dose-dependent manner and demonstrated a positive correlation between cell surface area and enzyme secretion. Above certain concentrations of extract, however, the development of the fungus and enzyme secretions remained at control values or slightly diminished. Supernatant fluid appearance of the intracellular enzyme, malate dehydrogenase, paralleled the secretion of cellulase both in the presence and absence of extract. Ether solubilization of extract demonstrated that the active component(s) possessed a moderately polar value between 2.7 and 2.8. Thin layer chromatography separated extract into inert, inhibitory and intensely stimulating fractions. These results support the idea that by accelerating fungal growth and metabolism, Amaferm increases the rate (or extent) of fibre degradation caused by rumen fungi and that this, in turn, may contribute to enhanced animal performance.     

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.     

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.     

Inhibition of the rumen anaerobic fungus Neocallimastix frontalis by fermentation products

The effects of bacterial fermentation products on cellulose degradation by the rumen fungus Neocallimastix frontalis have been investigated. H₂, formate, lactate and ethanol were strong inhibitors, particularly at high concentrations. Acetate and malate also inhibited, whereas succinate had a variable effect. Butyrate and propionate had no inhibitory effects.     

Kinetic study of a cellobiase purified from Neocallimastix frontalis EB188

A cellobiase was purified from the culture supernatant of Neocallimastix frontalis EB188. This enzyme possessed a molecular weight of 85,000 and an isoelectric point of 6.95. The enzyme rapidly hydrolyzed cellobiose, p-nitrophenyl (pNP) beta-D-glucopyranoside (pNPG) and cellotriose and slowly hydrolyzed cellopentaose and salicin. The enzyme did not hydrolyze pNP alpha-D-glucopyranoside or pNP beta-D-cellobioside. Substrate inhibition was observed when cellobiose or pNPG were used as the substrates and glucose production was measured. The kinetic parameters were: K = 0.053 mM, V = 5.88 U/mg of protein and Ki = 0.95 mM for cellobiose; K = 0.36 mM, V = 1.05 U/mg and Ki = 8.86 mM for pNPG. Substrate inhibition was not detected during the hydrolysis of pNPG when pNP production was measured. The kinetic parameters for pNPG were: K = 0.67 mM and V = 1.49 U/mg of protein. The presence of an enzyme.glucose.substrate complex and transglucosylation was evident during the catalysis. Glucose, cellobiose, glucono-delta-lactone, galactose, lactose, maltose and salicin acted as competitive inhibitors during the hydrolysis of pNPG with the apparent inhibition constants (Kis) of 4.8 mM, 0.035 mM, 0.062 mM, 28.5 mM, 0.38 mM, 15.0 mm and 31.0 mM, respectively.     

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     

The effect of the presence of glucose on the fermentation of mannose by the anaerobic fungus Neocallimastix frontalis strain RE1

Abstract The disappearance of mannose and the formation of formate, acetate, lactate, ethanol and succinate by Neocallimastix frontalis strain RE1 occurred slowly when mannose was the only substrate present. When an equal quantity of glucose was present, the fermentation of mannose increased. Incubations with ¹³C-labelled mannose and glucose confirmed that the presence of both substrates resulted in increased product formation from mannose and reduced product formation from glucose. The relative proportions of products formed from the two substrates varied, possibly in part due to differences in the rates of growth of the fungus. The strains of N. frontalis able to utilize mannose may have a competitive advantage in the rumen ecosystem.     

Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis

Xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) production was investigated in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released principally into the culture fluid and had pH and temperature optima of 5.5 and 55 degrees C, respectively. In the presence of low concentrations of substrate, the enzyme was stabilized at 50 degrees C. Xylobiose was the principal product of xylanase action, with lesser amounts of longer-chained xylooligosaccharides. No xylose was detected, indicating that xylobiase activity was absent. Activities of xylanase up to 27 U ml-1 (1 U represents 1 micromol of xylose equivalents released min-1) were obtained for cultures grown on xylan (from oat spelt) at 2.5 mg ml-1 in shaken cultures. No growth occurred in unshaken cultures. Xylanase production declined with elevated concentrations of xylan (less than 2.5 mg ml-1), and this was accompanied by an accumulation of xylose and, to a lesser extent, arabinose. Addition of either pentose to cultures grown on low levels of xylan in which neither sugar accumulated suppressed xylanase production, and in growth studies with the paired substrates xylan-xylose, active production of the enzyme occurred during growth on xylan only after xylose had been preferentially utilized. When cellobiose, glucose, and xylose were tested as growth substrates for the production of xylanase (each initially at 2.5 mg ml-1), they were found to be less effective than xylan, and use of xylan from different origins (birch wood or larch wood) as the growth substrate or in the assay system resulted in only marginal differences in enzyme activity. However, elevated production of xylanase occurred during growth on crude hemicellulose (barley straw leaf). The results are discussed in relation to the role of the anaerobic fungi in the ruminal ecosystem, and the possible application of the enzyme in bioconversion processes is also considered.     

Glycosidases of the rumen anaerobic fungus Neocallimastix frontalis grown on cellulosic substrates

The rumen anaerobic fungus Neocallimastix frontalis was grown on cellulosic substrates, and the cellular distribution and types of glycosidases produced by the organism were studied. Fungal cultures were fractionated into extracellular, insoluble (membrane), and intracellular fractions and assayed for glycosidase activity by using Avicel, carboxymethylcellulose, xylan, starch, polygalacturonic acid, and the p-nitrophenyl derivatives of galactose, glucose, and xylose as substrates. Enzymic activity was highest in the extracellular fraction; however, the membrane fraction also displayed appreciable activity. The intracellular fraction was inactive towards all substrates. Polygalacturonic acid was the only substrate not hydrolyzed by the active fractions, indicating that pectinase was absent. The results show that N. frontalis, a common rumen anaerobic fungus, produces enzymes for degrading cellulose and hemicellulose, key components of plant fiber.     

Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis.

Xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) production was investigated in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released principally into the culture fluid and had pH and temperature optima of 5.5 and 55 degrees C, respectively. In the presence of low concentrations of substrate, the enzyme was stabilized at 50 degrees C. Xylobiose was the principal product of xylanase action, with lesser amounts of longer-chained xylooligosaccharides. No xylose was detected, indicating that xylobiase activity was absent. Activities of xylanase up to 27 U ml-1 (1 U represents 1 micromol of xylose equivalents released min-1) were obtained for cultures grown on xylan (from oat spelt) at 2.5 mg ml-1 in shaken cultures. No growth occurred in unshaken cultures. Xylanase production declined with elevated concentrations of xylan (less than 2.5 mg ml-1), and this was accompanied by an accumulation of xylose and, to a lesser extent, arabinose. Addition of either pentose to cultures grown on low levels of xylan in which neither sugar accumulated suppressed xylanase production, and in growth studies with the paired substrates xylan-xylose, active production of the enzyme occurred during growth on xylan only after xylose had been preferentially utilized. When cellobiose, glucose, and xylose were tested as growth substrates for the production of xylanase (each initially at 2.5 mg ml-1), they were found to be less effective than xylan, and use of xylan from different origins (birch wood or larch wood) as the growth substrate or in the assay system resulted in only marginal differences in enzyme activity. However, elevated production of xylanase occurred during growth on crude hemicellulose (barley straw leaf). The results are discussed in relation to the role of the anaerobic fungi in the ruminal ecosystem, and the possible application of the enzyme in bioconversion processes is also considered.     

Glycosidases of the rumen anaerobic fungus Neocallimastix frontalis grown on cellulosic substrates.

The rumen anaerobic fungus Neocallimastix frontalis was grown on cellulosic substrates, and the cellular distribution and types of glycosidases produced by the organism were studied. Fungal cultures were fractionated into extracellular, insoluble (membrane), and intracellular fractions and assayed for glycosidase activity by using Avicel, carboxymethylcellulose, xylan, starch, polygalacturonic acid, and the p-nitrophenyl derivatives of galactose, glucose, and xylose as substrates. Enzymic activity was highest in the extracellular fraction; however, the membrane fraction also displayed appreciable activity. The intracellular fraction was inactive towards all substrates. Polygalacturonic acid was the only substrate not hydrolyzed by the active fractions, indicating that pectinase was absent. The results show that N. frontalis, a common rumen anaerobic fungus, produces enzymes for degrading cellulose and hemicellulose, key components of plant fiber.     

A highly active extracellular cellulase from the anaerobic rumen fungus Neocallimastix frontalis

Abstract An extracellular cellulase which was highly active in solubilizing the highly hydrogen bond-ordered cellulose in cotton fibre was found in a culture filtrate of the anaerobic fungus, Neocallimastix frontalis , isolated from the rumen of a sheep. The cellulase was several-fold more active in solubilizing cotton fibre per unit of endo-1,4-β-glucanase than the cellulase of the aerobic fungus Trichoderma reesei mutant strain C-30, which is one of the most active cellulases isolated so far.     

Production and regulation of cellulase by two strains of the rumen anaerobic fungus Neocallimastix frontalis

Cellulase production was examined in two strains of Neocallimastix frontalis, namely, PN-1 isolated from the ovine rumen, and PN-2 from the bovine rumen. For both strains, carboxymethylcellulase (CMCase) had a pH optimum of 6.0 and a temperature optimum of 50 degrees C. CMCase resided mainly in the culture fluid, and activities up to 170 U ml-1 (1 U represents 1 microgram of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of cellulose ml-1. For resting cultures of strain PN-1, the yield of CMCase increased from 9.9 X 10(3) to 10.4 X 10(4) U per g of cellulose degraded, as the initial cellulose concentration decreased from 10 to 0.58 mg ml-1. The range for PN-2 was 8.1 X 10(3) to 11 X 10(4) U g-1. Shaking cultures improved yields for strain PN-1 but not for PN-2. Decreased CMCase production at high initial cellulose concentrations concurred with accumulation of glucose, and addition of glucose (4 mg ml-1) to cultures grown on low cellulose in which none of the sugar accumulated repressed CMCase. Adsorption of CMCase was excluded as a likely explanation for decreased yields at high initial cellulose as only a low proportion (less than 20%) of the enzyme was adsorbed onto the growth substrate. Exoglucanase, measured with alkali-treated Sigmacell or Avicel, gave low levels of activity in the culture fluid (less than 2 U ml-1) and did not appear to be associated with the fungal rhizoid, as treatment with various solubilizing agents failed to give increased activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of lasalocid on glucose uptake, hydrogen production and the solubilization of straw by the anaerobic rumen fungus Neocallimastix frontalis

Three isolates of Neocallimastix frontalis grown in pure culture with glucose as substrate differed in their response to the presence of lasalocid in the growth medium. For two strains (RE1 and RK21) hydrogen production and glucose uptake were reduced to 50% or less of the control values in the presence of lasalocid at 0–25 μg/ ml. A third strain (PNK2) retained significant fermentative ability in the presence of at least four times this amount of lasalocid. Lasalocid was rather less inhibitory to the solubilization of straw by these fungi, PNK2 again providing the most resistant strain. It is concluded that the use of lasalocid in ruminant feeds is likely to have a strain selective effect on the rumen population of Neocallimastix.