• Journal of Animal Science and Technology
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• v.46 no.5
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• pp.763-772
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• 2004

The patterns of fungal growth and fiber digestion under the microscope, and tile productions of fibrolytic enzyme were studied in an in vitro culture with Neocallimastix frontalis SA when either filter paper or rice straw was provided as sole energy source. Under the microscopic observation, active zoospores attachment, sporangium development and complex rhizoidal system were founded on the surface and at the edge of filter paper. After 7 days of incubation, a reduced fiber mass, a decreased fiber cohesion and a weakened fiber structure by fungal digestion were clearly observed. Similar fungal development was observed with rice straw, but fungal growth and digestion took place mostly on the damaged and exposed portion of rice straw. Although there were some differences in absolute concentration and pattern, the concentration of both cellulase and xylanase increased with incubation time with the higher activity being obtained with filter paper. Their differences were large especially after 48 and 96hr of incubation(P< 0.05). The filter paper was more good inducer of cellulolytic and xylanolytic enzymes compared with complex substrate, rice straw. These findings suggest that the filter paper is the better energy source for N frontalis than the complex substrate, and structural disintegration by physical process is able to help rumen fungal growth on the lignified roughage although anaerobic rumen fungi have mechanical and enzymatic functions for fiber digestion.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.11
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• pp.1555-1565
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• 2009

Anaerobic rumen fungi have been regarded as good genetic resources for enzyme production which might be useful for feed supplements, bio-energy production, bio-remediation and other industrial purposes. In this study, an expressed sequence tag (EST) library of the rumen anaerobic fungus Neocallimastix frontalis was constructed and functional genes from the EST library were analyzed to elucidate carbohydrate metabolism of anaerobic fungi. From 10,080 acquired clones, 9,569 clones with average size of 628 bp were selected for analysis. After the assembling process, 1,410 contigs were assembled and 1,369 sequences remained as singletons. 1,192 sequences were matched with proteins in the public data base with known function and 693 of them were matched with proteins isolated from fungi. One hundred and fifty four sequences were classified as genes related with biological process and 328 sequences were classified as genes related with cellular components. Most of the enzymes in the pathway of glucose metabolism were successfully isolated via construction of 10,080 ESTs. Four kinds of hemi-cellulase were isolated such as mannanase, xylose isomerase, xylan esterase, and xylanase. Five $\beta$-glucosidases with at least three different conserved domain structures were isolated. Ten cellulases with at least five different conserved domain structures were isolated. This is the first solid data supporting the expression of a multiple enzyme system in the fungus N. frontalis for polysaccharide hydrolysis.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.11
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• pp.1576-1584
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• 2016

Acetyl xylan esterase (AXE), which hydrolyzes the ester linkages of the naturally acetylated xylan and thus known to have an important role for hemicellulose degradation, was isolated from the anaerobic rumen fungus Neocallimastix frontatlis PMA02, heterologously expressed in Escherichi coli (E.coli) and characterized. The full-length cDNA encoding NfAXE1 was 1,494 bp, of which 978 bp constituted an open reading frame. The estimated molecular weight of NfAXE1 was 36.5 kDa with 326 amino acid residues, and the calculated isoelectric point was 4.54. The secondary protein structure was predicted to consist of nine ${\alpha}$-helixes and 12 ${\beta}$-strands. The enzyme expressed in E.coli had the highest activity at $40^{\circ}C$ and pH 8. The purified recombinant NfAXE1 had a specific activity of 100.1 U/mg when p-nitrophenyl acetate (p-NA) was used as a substrate at $40^{\circ}C$, optimum temperature. The amount of liberated acetic acids were the highest and the lowest when p-NA and acetylated birchwood xylan were used as substrates, respectively. The amount of xylose released from acetylated birchwod xylan was increased by 1.4 fold when NfAXE1 was mixed with xylanase in a reaction cocktail, implying a synergistic effect of NfAXE1 with xylanase on hemicellulose degradation.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.8
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• pp.1110-1117
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• 2001

Responses of the rumen fungus, Neocallimastix frontalis RE1, to long chain fatty acid (LCFA) were evaluated by measuring gas production, filter paper (FP) cellulose digestion and polysaccharidase enzyme activities. LCFA (stearic acid, $C_{18:0}$; oleic acid, $C_{18:1}$; linoleic acid, $C_{18:2}$ and linolenic acid, $C_{18:3}$) were emulsitied by ultrasonication under anaerobic condition, and added to the medium. When N frontalis RE1 was grown in culture with stearic, oleic and linoleic acid, the cumulative gas production, gas pool size, FP cellulose digestion and enzymes activities significantly (p<0.05) increased at some incubation times(especially, exponential phases of fungal growth, 48~120 h of incubation) relative to that for control cultures. However, the addition of linolenic acid strongly inhibited all of the investigated parameters up to 120 h incubation, but not after 168 and 216 h of incubation. These results indicated that stearic, oleic and linoleic acids tended to have great stimulatory effects on fungal cellulolysis, whereas linolenic acid caused a significant (p<0.05) inhibitory effects on the cellulolysis by the rumen fungus. These results are the first report of the effect of LCFAs on the ruminal fungi. Further research is needed to identify the mode of action of LCFAs on fungal strains and to verify whether or not ruminal fungi have ability to hydrate unsaturated LCFAs to saturated FAs. There was high correlation between cumulative in vitro gas production and fungal growth (94.78%), FP cellulose degradation (96.34%), CMCase activity(90.86%) or xylanase activity (87.67%). Thus measuring of cumulative gas production could be a useful tool for evaluating fungal growth and/or enzyme production by ruminal fungi.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.5
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• pp.752-757
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• 1999

Studies were made of digestion of timothy (Pheleum pretense) hay, tall fescue (Festuca elatior) hay, and rice (Oryza sativa) straw in pure cultures of rumen anaerobic fungus, Neocallimastix frontails MCH3. The fungus was inoculated on ground forages (1%, w/v) in an anaerobic medium and incubated at $39^{\circ}C$. Incubation was continued for 24, 48, 72 and 96 h. The losses of dry matter, xylose and glucose of forage during incubation were determined at the end of these incubation periods. Xylose and glucose were considered to be released from xylan and cellulose, respectively. The digested xylan to digested cellulose (X/C) ratios of the substrate were calculated. Xylanase and carboxymethyl cellulose (CMCase) of culture supernatant and residual substrate was measured at the same time. The X/C ratios in the cultures on timothy hay and rice straw were greater than 0.5 in the first 24-h incubation period. The values were smaller than 0.3 in tall fesque. The ratio of xylanase activity to that of CMCase in the first 24-h incubation period correlated well with the traits in X/C ratio. However xylanase activity was still superior to CMCase in the following incubation period (48 to 96 h), although the glucose (designated as cellulose) was more intensively digested than xylose (designated as xylan). The production of these polysaccharidases appeared to correlate with substrate cell-wall sugar composition, xylose to glucose ratios, at the beginning of fast growing period.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.7
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• pp.941-946
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• 2001

The effects of various concentrations of saturated fatty acids (SFA; caprylic, capric and stearic acids) on the growth of the anaerobic fungus, Neocallimastix frontalis C5-1 isolated from the rumen of a Korean native goat were investigated. At higher concentrations of fatty acids (0.1%, w/v), the addition of SFA strongly decreased filter paper (FP) cellulose digestion and polysaccharide-degrading enzyme activity. The sensitivity of the rumen anaerobic fungus to the added fatty acids increased in the following order: caprylic ($C_{8:0}$)>capric($C_{10:0}$)>stearic($C_{18:0}$) acid, although stearic acid had no significant (p<0.05) inhibitory effects at any of the concentrations tested. However, the addition of SFA at lower concentrations (0.01 and 0.001% levels), did not inhibit FP cellulose degradation and enzyme activity. Furthermore, although these parameters were slightly stimulated by the addition of SFA, they were not statistically different from control values. This is the first report examining the effects of fatty acids on anaerobic gut fungi. We found that the lower levels of fatty acids used in this experiment were able to stimulate the growth and specific enzyme activities of rumen anaerobic fungi, whereas the higher levels of fatty acids were inhibitory with respect to fungal cellulolysis.