• Asian-Australasian Journal of Animal Sciences
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• v.14 no.11
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• pp.1542-1548
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• 2001

Wheat straw was treated on laboratory scale with 4% urea at a moisture level of 50% along with different amount of HC1 to fix various levels of ammonia (30, 40, 50 and 60%) and stored for 4 weeks. Result, revealed a significant (p<0.01) increase in CP content of the samples where HC1 was added. The CP content of the straw was only 7.8%, which increased to 14.1, 16.0, 15.0 and 15.2% with the addition of acid. Similarly the concentrations of NDF, ADF and hemicellulose was significantly different due to HC1 addition. The level of HC1 recommended was to trap 30% ammonia as there was not significant difference in CP content of straw due to addition of 4 levels of acids. Results of in vivo experiment conducted on nine buffaloes divided randomly into three groups of three animals in each revealed no significant difference in the intake of DM, OM, NDF, ADF, cellulose and hemicellulose in group I (ammoniated straw), group II (HC1 treated ammoniated straw) and group III (HC1 treated ammoniated straw + 1 kg barley grain), but the intake of CP was significantly (p<0.01) more in group III as compared to other 2 groups. The digestibility of DM, OM and CP was significantly (p<0.01) more in groups where HCI treated straw was fed as compared to only ammoniated straw fed group, whereas there was no significant difference in the digestibility of NDF, ADF and cellulose in 3 groups. Intake was significantly higher of nitrogen (p<0.05), calcium (p<0.01) and phosphorus (p<0.01) in group III as compared to other two groups. Animals in all the 3 groups showed positive nitrogen, calcium and phosphorus balance, though the balances of all the 3 nutrients were significantly higher in group III as compared to other 2 groups. Rumen fermentation study conducted in 3 rumen fistulated buffaloes in $3{\times}3$ latin square design offering the same 3 diets as in group I to III revealed that rumen pH was alike statistically in 3 groups and at various time intervals. The mean ammonia-N concentration was significantly (p<0.01) more in group II and III as compared to group I. The mean TVFA concentration (mM/100 ml SRL) were 6.46, 7.84 and 8.47 in 3 groups respectively and different statistically (p<0.01). Results revealed no significant difference in the activities of carboxy methyl cellulase, urease or protease at both the time of sampling (0 h and 4 h) in all the 3 groups of animals.

• Journal of Life Science
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• v.22 no.4
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• pp.437-446
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• 2012

A metagenomic library of cow rumen in the pCC1FOS phage vector was screened in $E.$ $coli$ EPI300 for cellulase activity on carboxymethyl cellulose agar plates. One clone was partially digested with $Sau$3AI, ligated into the $Bam$HI site of the pBluescript II SK+ vector, and transformed into $E.$ $coli$ $DH5{\alpha}$. We obtained a 1.5 kb insert DNA, designated $cel$5C, which hydrolyzes carboxymethyl cellulose. The cel5C gene has an open reading frame (ORF) of 1,125 bp encoding 374 amino acids. It belongs to the glycosyl hydrolase family 5 with the conserved domain LIMEGFNEIN. The molecular mass of the Cel5C protein induced from $E.$ $coli$ $DH5{\alpha}$, as analyzed by CMC SDS-PAGE, appeared to be approximately 42 kDa. The enzyme showed optimum cellulase activity at pH 4.0, and $50^{\circ}C$. We examined whether the $cel$5C gene comes from the 49 identified cow rumen bacteria using PCR. No PCR bands were identified, suggesting that the $cel$5C gene came from the unidentified cow rumen bacteria.

• 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.

• Journal of Animal Science and Technology
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• v.48 no.1
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• pp.77-90
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• 2006

The purpose of this study was to isolate cellulolytic and hemicellulolytic anaerobic bacteria inhabiting from gut of ruminants and investigate their hydrolytic enzyme activities. Extracellular CMCase activities of H-strains isolated from the rumen of a Holstein dairy cow were higher than those of D- and DC- strains from the rumen and large intestine of Korean spotted deer. Most isolated bacteria utilized more efficiently Dehority's artificial medium containing starch, glucose and cellobiose (DAS) than those in Dehority's artificial medium containing cellulose only (DAC). The results of biochemical reactions and sugar fermentation indicated that the isolated bacteria belong to one of bacterial strains of Peptostreptococcus spp., Bifidobacterium spp., Prevotela ruminicola/buccae, Clostridium beijer/butyricum and Streptococcus intermedis which are not highly cellulolytic. Activities of Avicelase, xylanase, β-D-glucosidase, α-L-arabinofuranosidase and β-xylosidase of the isolated anaerobic bacteria in DAS were higher than those in DAC. In conclusion, the results indicated the higher enzyme activities of the isolated strains cultured in DAS medium were mainly caused by their specific carbohydrate utilization for enzyme production and growth rate. The highly cellulolytic bacteria were not isolated in the present experiment. Thus further research is required to investigate characteristics of gut bacteria from Korean spotted deer.

• Journal of Animal Science and Technology
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• v.51 no.5
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• pp.379-386
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• 2009

The aim of this study was tofind out the effects of supplementation of vitamin A to the diets of high or low amounts of concentrates for ruminants. In the first experiment, ruminal fermentation patterns with the data of pH, VFA production and cellulose disappearance rates in the rumen in vitro were investigated. In the second experiment, enzyme activities, gas production and dry matter degradabilities using cellulolytic bacteria, Ruminococcus flavefaciens were investigated. Ruminal pH was higher in low amounts of concentrates than in high amounts of concentrates as expected, however, no significant differences were found. Cellulose disappearance rates improved in vitamin A addition particularly in early incubation time (before 24h) and also the production of volatile fatty acids increased in vitamin A addition. These trends were more evident in diets containing high amounts of concentrates than in low amounts of concentrates and it may indicate that vitamin A is more required in the diets of high amounts of concentrates. In the second experiment, gas production, enzyme activities and dry matter degradabilities using cellulolytic bacteria, Ruminococcus flavefaciens were not different between vitamin A added and non-added diets. Ruminococcus flavefaciens may not require additional vitamin A for its own growth.

• Journal of The Korean Society of Grassland and Forage Science
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• v.36 no.1
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• pp.7-14
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• 2016

The objective of this study was to determine the effect of bacterial inoculation (Lactobacillus plantarum or combo inoculant mixed with Lactobacillus plantarum and Lactobacillus buchneri) and addition of fibrolytic enzyme on chemical compositions and fermentation characteristics of whole crop barley (WCB) and triticale (TRT) silage, their ruminal in vitro fermentation, and digestibility. In TRT silage, enzyme addition significantly (p<0.01) decreased NDF content compared to no enzyme addition treatment. Organic acids such as lactate and acetate contents in WCB and TRT silages were significantly (p<0.01) higher compared to those in the control. Particularly, lactate content was the highest in L. plantarum treatment. Fibrolytic enzyme treatment on both silages had relatively higher lactic acid bacteria content, while mold content was lower in both treatments compared to that in the control. In vitro dry matter digestibility was generally improved in WCB silages. It was higher (p<0.01) in TRT with mixed treatment of L. plantarum, L. buchneri, and enzyme compared to others. In vitro ruminal acetate production was relatively higher in treatments with both enzyme and inoculant additions compared to that in the control. Therefore, the quality of silage and rumen fermentation could be improved by inoculants (L. plantarum and L. buchneri) regardless whether whole crop barley (WCB) or triticale (TRT) silage was used. Although it was found that fibrolytic enzyme addition to both silages had various quality and rumen fermentation values, further study is needed.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.4
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• pp.567-586
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• 2001

Gastrointestinal tract of ruminants as well as monogastric animals are colonised by a variety of microorganisms including bacteria, fungi and protozoa. Gastrointestinal ecosystem, especially the rumen is emerging as an important source for enrichment and natural selection of microbes adapted to specific conditions. It represents a virtually untapped source of novel products (e.g. enzymes, antibiotics, bacteriocins, detoxificants and aromatic compounds) for industrial and therapeutic applications. Several gastrointestinal bacteria and fungi implicated in detoxification of anti-nutritional factors (ANFs) can be modified and manipulated into promising system for detoxifying feed stuffs and enhancing fibre fermentation both naturally by adaptation or through genetic engineering techniques. Intestinal lactobacilli, bifidobacteria and butyrivibrios are being thoroughly investigated and widely recommended as probiotics. Restriction endonucleases and native plasmids, as stable vectors and efficient DNA delivery systems of ruminal and intestinal bacteria, are increasingly recognised as promising tools for genetic manipulation and development of industrially useful recombinant microbes. Enzymes can improve the nutrient availability from feed stuffs, lower feed costs and reduce release of wastes into the environment. Characterization of genes encoding a variety of commercially important enzymes such as cellulases, xylanases, $\beta$-glucanases, pectinases, amylases and phytases will foster the development of more efficacious and viable enzyme supplements and enzyme expression systems for enhancing livestock production.

• Microbiology and Biotechnology Letters
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• v.21 no.3
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• pp.263-268
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• 1993

Seventeen strains were isolated from soil, cattle rumen, cereal sewage dregs, insect on agar plate containing insoluble glucan as a sole carbon source from immobilized Streptococcus mutans, which produced alpha-1,3 glucanase for lysis of dental plaque. Among these strains isolated from soil, SW-522 and SW-713 that had appeared to produce the high level of alpha-1,3 glucanase, degraded insoluble glucan from S. mutans 97.6% and 49.4%, respectively in 5 hours. The activity of crude alpha-1,3 glucanase from SW-522 was 1.3mg insoluble glucan/min.mg protein. This enzyme was entirely degraded insoluble glucan on glass tube which produced by S. mutans in TH medium with 5% sucrose.

• Journal of Microbiology and Biotechnology
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• v.25 no.11
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• pp.1835-1841
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• 2015

A cell surface display system for heterologous expression of the multifunctional cellulase, CelEx-BR12, in Escherichia coli was developed using truncated E. coli outer membrane protein C (OmpC) as an anchor motif. Cell surface expression of CelEx-BR12 cellulase in E. coli harboring OmpC-fused CelEx-BR12, designated MC4100 (pTOCBR12), was confirmed by fluorescence-activated cell sorting and analysis of outer membrane fractions by western blotting, which verified the expected molecular mass of OmpC-fused CelEx-BR12 (~72 kDa). Functional evidence for exocellulase activity was provided by enzymatic assays of whole cells and outer membrane protein fractions from E. coli MC4100 (pTOCBR12). The stability of E. coli MC4100 (pTOCBR12) cellulase activity was tested by carrying out repeated reaction cycles, which demonstrated the reusability of recombinant cells. Finally, we showed that recombinant E. coli cells displaying the CelEx-BR12 enzyme on the cell surface were capable of growth using carboxymethyl cellulose as the sole carbon source.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.3
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• pp.379-385
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• 2011

Ferulic acid esterase (FAE) and acetyl esterase (AE) cleave feruloyl groups substituted at the 5'-OH group of arabinosyl residues and acetyl groups substituted at O-2/O-3 of the xylan backbone, respectively, of arabinoxylans in the cell wall of grasses. In this study, the enzyme profiles of FAE, AE and polysaccharide hydrolases of the anaerobic rumen fungus Neocallimastix sp. YQ1 grown on Chinese wildrye grass hay (CW) or alfalfa hay (AH) were investigated by two $2{\times}4$ factorial experiments, each in 10-day pure cultures. The treatments consisted of two glucose levels ($G^+$: glucose at 1.0 g/L, $G^-$: no glucose) and four N sources (N1: 1.0 g/L yeast extract, 1.0 g/L tryptone and 0.5 g/L $(NH_4)_2SO_4$; N2: 2.8 g/L yeast extract and 0.5 g/L $(NH_4)_2SO_4$; N3: 1.6 g/L tryptone and 0.5 g/L $(NH_4)_2SO_4$; N4: 1.4 g/L tryptone and 1.7 g/L yeast extract) in defined media. The optimal combinations of glucose level and N source for the fungus on CW, instead of AH, were $G^-N4$ and $G^-N3$ for maximum production of FAE and AE, respectively. Xylanase activity peaked on day 4 and day 6 for the fungus grown on CW and AH, respectively. The activities of esterases were positively correlated with those of xylanase and carboxymethyl cellulase. The fungus grown on CW exhibited a greater volatile fatty acid production than on AH with a greater release of ferulic acid from plant cell wall.