• Asian-Australasian Journal of Animal Sciences
• /
• v.16 no.3
• /
• pp.389-393
• /
• 2003

Present study investigate the effect of enzyme supplementation, methods (applied to rumen or enzyme treated diet) compared with no enzyme diet, on rumen fermentation and apparent nutrient digestibility in a $3{\times}3$ Latin square design with three rumen cannulated Korean Native goats. In situ rumen degradation kinetics was studied in three rumen cannulated Holstein steers. Three diets were, no enzyme, 1% enzyme in rumen and 1% enzyme in diet. The enzyme was sprayed onto forage, and the forage: concentrate ratio was 30:70. Degradation kinetics was studied with three enzyme levels (0, 1 and 2%, w/w) and four pre-treatment times (0, 1, 12 and 24 h). Results suggested that enzyme application method did not affect rumen fermentation, ruminal enzyme activity and total tract apparent digestibility. Nutrient degradation rate and effective degradability of DM, NDF and ADF increased with increasing enzyme level and pre-treatment times. Degradation of nutrients was affected by enzymes levels or pre-treatment times. Therefore, it is probable that the improved degradation may be due to the supplemented exogenous hydrolytic enzymes under a certain condition.

• BMB Reports
• /
• v.44 no.1
• /
• pp.52-57
• /
• 2011

Termites play an important role in the degradation of dead plant materials and have acquired endogenous and symbiotic cellulose digestion capabilities. The feruloyl esterase enzyme (FAE) gene amplified from the metagenomic DNA of Coptotermes formosanus gut was cloned in the TA cloning vector and subcloned into a pET32a expression vector. The Ft3-7 gene has 84% sequence identity with Clostridium saccharolyticum and shows amino acid sequence identity with predicted xylanase/chitin deacetylase and endo-1,4-beta-xylanase. The sequence analysis reveals that probably Ft3-7 could be a new gene and that its molecular mass was 18.5 kDa. The activity of the recombinant enzyme (Ft3-7) produced in Escherichia coli (E.coli) was 21.4 U with substrate ethyl ferulate and its specific activity was 24.6 U/mg protein. The optimum pH and temperature for enzyme activity were 7.0 and $37^{\circ}C$, respectively. The substrate utilization preferences and sequence similarity of the Ft3-7 place it in the type-D sub-class of FAE.

• Asian-Australasian Journal of Animal Sciences
• /
• v.12 no.6
• /
• pp.988-1001
• /
• 1999

The rumen ecosystem is increasingly being recognized as a promising source of superior polysaccharide-degrading enzymes. They contain a wide array of novel enzymes at the levels of specific activities of 1,184, 1,069, 119, 390, 327 and $946{\mu}mol$ Reducing sugar release/min/mg protein for endoglucanase, xylanase, polygalactouronase, amylase, glucanase and arabinase, respectively. These enzymes are mainly located in the surface of rumen microbes. However, glycoside-degrading enzymes (e.g. glucosidase, fucosidase, xylosidase and arabinofuranosidase, etc.) are mainly located in the rumen fluid, when detected enzyme activities according to the ruminal compartments (e.g. enzymes in whole rumen contents, feed-associated enzymes, microbial cell-associated enzymes, and enzymes in the rumen fluid). Ruminal fungi are the primary contributors to high production of novel enzymes; the bacteria and protozoa also have important functions, but less central roles. The enzyme activities of bacteria, protozoa and fungi were detected 32.26, 19.21 and 47.60 mol glucose release/min/mL mediem for cellulose; 42.56, 14.96 and 64.93 mmol xylose release/min/mL medium after 48h incubation, respectively. The polysachharide-degrading enzyme activity of ruminal anaerobic fungi (e.g. Neocallimastix patriciarum and Piromyces communis, etc.) was much higher approximately 3~6 times than that of aerobic fungi (e.g. Tricoderma reesei, T. viridae and Aspergillus oryzae, etc.) used widely in industrial process. Therefore, the rumen ecosystem could be a growing source of novel enzymes having a tremendous potential for industrial applications.

• Asian-Australasian Journal of Animal Sciences
• /
• v.16 no.3
• /
• pp.374-379
• /
• 2003

Four rumen fistulated Murrah buffaloes were used to study the effect of four diets differing in roughage to concentrate ratio on rumen biochemical changes, microbial enzyme profile and in sacco degradability of feed in a $4{\times}4$ Latin Square design. The animals were fed four diets consisting of 80:20, 70:30, 60:40 and 50:50 ratios of wheat straw and concentrate mixtures, respectively. Wheat straw and concentrate mixture were mixed with water (0.6 l/kg feed) and complete feed mixture was offered to the animals at 8:00 h and 16:00 h in two equal parts. The variation in pH of rumen liquor (difference of maximum and minimum during 0-8 h post feeding) increased with increasing level of concentrate mixture in the diet. There was no effect of diet composition on volatile fatty acids, total nitrogen and trichloro-acetic acid precipitable nitrogen in the rumen liquor, but ammonia nitrogen increased with increasing level of concentrate mixture in the ration. Major portions of all fibre degrading enzymes were present in the particulate material (PM) of the rumen contents, but protease was absent in PM fraction. The activities of micro-crystalline cellulase, acetyl esterase and protease increased with increase in the level of concentrate mixture, but the activities of other enzymes (carboxymethylcellulase, filter paper degrading activity, xylanase, $\beta$-glucosidase and $\beta$-xylosidase) were not affected. The in sacco degradability and effective degradability of feeds increased with increasing level of concentrate mixture in the ration.

• Asian-Australasian Journal of Animal Sciences
• /
• v.15 no.12
• /
• pp.1725-1731
• /
• 2002

The distribution and activities of hydrolytic enzymes (cellulolyti, hemicellulolytic,pectinolytic and others) in the rumen compartments of Hereford bulls fed 100% alfalfa hay based diets were evaluated. The alfalfa proportion in the diet was gradually increased for two weeks. Whole rumen contents were processed into four fractions: Rumen contents including both the liquid and solid fractions were homogenized and centrifuged, and the supernatant was assayed for enzymes located in whole rumen contents (WRE); rumen contents were centrifuged and the supernatant was assayed for enzymes located in rumen fluids (RFE); feed particles in rumen contents were separated manually, washed with buffer, resuspended in an equal volume of buffer, homogenized and centrifuged and supernatant was assayed for enzymes associated with feed particles (FAE); and rumen microbial cell fraction was separated by centrifugation, suspended in an equal volume of buffer, sonicated and centrifuged, and the supernatant was assayed for enzymes bound with microbial cells (CBE). It was found that polysaccharide-degrading proteins such as $\beta$-1,4-D-endoglucanase, $\beta$-1,4-D-exoglucanase, xylanase and pectinase enzymes were located mainly with the cell bound (CBE) fraction. However, $\beta$-D-glucosidase, $\beta$-D-fucosidase, acetylesterase, and $\alpha$-L-arabinofuranosidase were located in the rumen fluids (RFE) fraction. Protease activity distributions were 37.7, 22.1 and 40.2%, and amylase activity distributions were 51.6, 18.2 and 30.2% for the RFE, FAE and CBE fractions, respectively. These results indicated that protease is located mainly in rumen fluid and with microbial cells, whereas amylase was located mainly in the rumen fluid.

• Asian-Australasian Journal of Animal Sciences
• /
• v.3 no.2
• /
• pp.115-120
• /
• 1990

Extracellular nuclease(s) in buffalo rumen fluid were purified from strained rumen fluid by a procedure involving Seitz filtration, acetone fractionation and gel filtration on Sephadex G-100. The enzyme resolved into two peaks exhibiting both DNase and RNase activities. The molecular weight of enzyme corresponding to peaks I and II were approximately 30,000 and 12,000 respectively. The properties of enzymes from the two peaks, however, were same. Optimum temperature for both DNase and RNase activities was at $50^{\circ}C$. Whereas DNase activity was stable upto $60^{\circ}C$, RNase activity was stable only up to $50^{\circ}C$. DNase activity recorded two pH optima, one at pH 5.5 and the other at pH 7.0. RNase activity recorded a broad pH optimum between pH 6.0-8.0. pH stability of the enzyme coincided with pH optima for both the activities. DNase activity was stimulated by $Mg^{2+}$ and $Mn^{2+}$ and inhibited by $Fe^{2+}$, $Zn^{2+}$, $Hg^{2+}$ and $Ag^+$. RNase activity was also stimulated by $Mg^{2+}$ and $Mn^{2+}$ and inhibited by $Cu^{2+}$, $Fe^{2+}$, $Zn^{2+}$, $Hg^{2+}$ and $Ag^+$. Reducing agents stimulated both the activities.

• Asian-Australasian Journal of Animal Sciences
• /
• v.16 no.1
• /
• pp.104-115
• /
• 2003

A series of experiments was carried out to determine the possibility for the non-ionic surfactant (NIS) as a feed additive for ruminant animals. The effect of the NIS on (1) the enzyme distribution in the rumen fluids of Hereford bulls, (2) the growth of pure culture of rumen bacteria and (3) rumen anaerobic fungi, (4) the ruminal fermentation characteristics of Korean native cattle (Hanwoo), and (5) the performances of Holstein dairy cows were investigated. When NIS was added to rumen fluid at the level of 0.05 and 0.1% (v/v), the total and specific activities of cell-free enzymes were significantly (p<0.01) increased, but those of cell-bound enzymes were slightly decreased, but not statistically significant. The growth rates of ruminal noncellulolytic species (Ruminobacter amylophilus, Megasphaera elsdenii, Prevotella ruminicola and Selenomonas ruminantium) were significantly (p<0.01) increased by the addition of NIS at both concentrations tested. However, the growth rate of ruminal cellulolytic bacteria (Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens and Butyrivibrio fibrisolvens) were slightly increased or not affected by the NIS. In general, NIS appears to effect Gram-negative bacteria more than Gram-positive bacteria; and non-cellulolytic bacteria more than cellulolytic bacteria. The growth rates of ruminal monocentric fungi (Neocallimastix patriciarum and Piromyces communis) and polycentric fungi (Orpinomyces joyonii and Anaeromyces mucronatus) were also significantly (p<0.01) increased by the addition of NIS at all concentrations tested. When NIS was administrated to the rumen of Hanwoo, Total VFA and ammonia-N concentrations, the microbial cell growth rate, CMCase and xylanase activities in the rumen increased with statistical difference (p<0.01), but NIS administration did not affect at the time of 0 and 9 h post-feeding. Addition of NIS to TMR resulted in increased TMR intake and increased milk production by Holstein cows and decreased body condition scores. The NEFA and corticoid concentrations in the blood were lowered by the addition of NIS. These results indicated that the addition of NIS may greatly stimulate the release of some kinds of enzymes from microbial cells, and stimulate the growth rates of a range of anaerobic ruminal microorganisms, and also stimulate the rumen fermentation characteristics and animal performances. Our data indicates potential uses of the NIS as a feed additive for ruminant animals.

• Journal of agriculture & life science
• /
• v.46 no.3
• /
• pp.109-118
• /
• 2012

The gene encoding an esterase enzyme was cloned from a metagenomic library of cow rumen bacteria. The esterase gene (est2R) was 2,120 bp in length, encoding a protein of 516 amino acid residues with a calculated molecular weight of 57,286 Da. The molecular weight of the enzyme was estimated to be 57,000 Da by SDS-PAGE. Est2R shared 35.6% amino acid identity with esterase (CAH19079) of uncultured prokaryote. The Est2R was most active at $20-40^{\circ}C$, and showed optimum at $30^{\circ}C$ and pH 8.0. The most activity of Est2R for the different chain length of p-nitrophenyl ester group as substrate was p-nitrophenyl acetate. Moreover, the enzyme was found to be most active without organic solvent, followed by 98% active with ethanol, and the enzyme activity was highly affected by the acetonitrile. The enzyme was significantly inhibited by $Zn^{2+}$ but stimulated by $Ca^{2+}$. So, novel esterase gene est2R is likely to obtain from cow rumen metagenome and supposed to use for industrial purpose.

• Journal of Animal Science and Technology
• /
• v.45 no.1
• /
• pp.131-142
• /
• 2003

We evaluated the effects of adding fibrolytic enzyme into ruminant diets on ruminal fermentation (in vitro) and lactational performances of dairy cows (in vivo). Through the in vitro experiment that was carried out with different contents of NDF (34, 38, 43%) in diets, digestibilities of NDF in the rumen appeared not significantly different by the addition of enzyme but were different by NDF content in diets showing higher digestibility in NDF 43% diet. It could be attributed by the relatively higher amount of hemicellulose in the current experimental diets than in conventional diets that might have been digested easily by the addition of fibrolytic enzyme in the rumen. The addition of fibrolytic enzyme tended to increase NDF digestibilities to a little extent both in 0.05 and 0.1% enzyme levels. Ruminal pH, NH3-N concentrations and VFA production in the rumen were not affected by the addition of fibrolytic enzyme. Activities of CMCase and xylanase were higher in enzyme treated diets of both NDF 34 and 38%. In particular, the activities of xylanase that slowly decreased from 0 to 12 hr but rapidly after 24 hr indicates that the major action of the enzyme in the rumen occurs in early period of incubation. Through an in vivo experiment, fibrolytic enzyme addition into the diets of dairy cows indeed affected lactational performance of milk yield. The cows fed enzyme treated diets produced 8% (1.9kg/d) more amounts of milk than with no enzyme addition. Milk composition of milk fat and protein was not affected by enzyme addition. Overall, the results of this in vivo study indicates that fibrolytic enzyme can be used to improve milk production in lactating cows. In respect that animals in different treatments of this study had the same amounts of intake, the increased milk yield with enzyme addition may be attributed to the improved utilization of nutrients in the digestive tract.

• Asian-Australasian Journal of Animal Sciences
• /
• v.14 no.9
• /
• pp.1216-1220
• /
• 2001

Effect of hydroquinone (HQ) on rumen urease activity was studied. Hydroquinone at concentrations of 0.01 ppm, 0.1 ppm, 1 ppm, and 10 ppm inhibited urease activity of intact rumen microbes in vitro by 25%, 34%, 55% and 64% respectively. In the presence of low concentrations of $\beta$-mercaptoethanol, rumen urease could be solubilized and partially purified. The Km for the enzyme was $2{\times}10^{-3}$ M with Vmax of $319.4{\mu}moles/mg$ min. The kinetics of inhibition with partially purified rumen urease was investigated. The result showed that the inhibitory effect was not eliminated by increasing urea concentrations indicating a noncompetitive effect in nature with an inhibition constant $1.2{\times}10^{-5}$ M. Hydroquinone at the concentration of 10 ppm produced 64% urease inhibition, did not affect ruminal total dehydrogenase and proteolytic enzyme (p>0.05), but increased cellulase activity by 28% (p<0.05) in vitro. These results indicated that hydroquinone was a effective inhibitor of rumen urease and could effectively delay urea hydrolysis without a negative effect. The inhibitor appeared to offer a potential to improve nitrogen utilization by ruminants fed diets containing urea.