• Asian-Australasian Journal of Animal Sciences
• /
• v.14 no.6
• /
• pp.880-884
• /
• 2001

Rumen microbiology research has undergone several evolutionary steps: the isolation and nutritional characterization of readily cultivated microbes; followed by the cloning and sequence analysis of individual genes relevant to key digestive processes; through to the use of small subunit ribosomal RNA (SSU rRNA) sequences for a cultivation-independent examination of microbial diversity. Our knowledge of rumen microbiology has expanded as a result, but the translation of this information into productive alterations of ruminal function has been rather limited. For instance, the cloning and characterization of cellulase genes in Escherichia coli has yielded some valuable information about this complex enzyme system in ruminal bacteria. SSU rRNA analyses have also confirmed that a considerable amount of the microbial diversity in the rumen is not represented in existing culture collections. However, we still have little idea of whether the key, and potentially rate-limiting, gene products and (or) microbial interactions have been identified. Technologies allowing high throughput nucleotide and protein sequence analysis have led to the emergence of two new fields of investigation, genomics and proteomics. Both disciplines can be further subdivided into functional and comparative lines of investigation. The massive accumulation of microbial DNA and protein sequence data, including complete genome sequences, is revolutionizing the way we examine microbial physiology and diversity. We describe here some examples of our use of genomics- and proteomics-based methods, to analyze the cellulase system of Ruminococcus flavefaciens FD-1 and explore the genome of Ruminococcus albus 8. At Illinois, we are using bacterial artificial chromosome (BAC) vectors to create libraries containing large (>75 kbases), contiguous segments of DNA from R. flavefaciens FD-1. Considering that every bacterium is not a candidate for whole genome sequencing, BAC libraries offer an attractive, alternative method to perform physical and functional analyses of a bacterium's genome. Our first plan is to use these BAC clones to determine whether or not cellulases and accessory genes in R. flavefaciens exist in clusters of orthologous genes (COGs). Proteomics is also being used to complement the BAC library/DNA sequencing approach. Proteins differentially expressed in response to carbon source are being identified by 2-D SDS-PAGE, followed by in-gel-digests and peptide mass mapping by MALDI-TOF Mass Spectrometry, as well as peptide sequencing by Edman degradation. At Ohio State, we have used a combination of functional proteomics, mutational analysis and differential display RT-PCR to obtain evidence suggesting that in addition to a cellulosome-like mechanism, R. albus 8 possesses other mechanisms for adhesion to plant surfaces. Genome walking on either side of these differentially expressed transcripts has also resulted in two interesting observations: i) a relatively large number of genes with no matches in the current databases and; ii) the identification of genes with a high level of sequence identity to those identified, until now, in the archaebacteria. Genomics and proteomics will also accelerate our understanding of microbial interactions, and allow a greater degree of in situ analyses in the future. The challenge is to utilize genomics and proteomics to improve our fundamental understanding of microbial physiology, diversity and ecology, and overcome constraints to ruminal function.

• Asian-Australasian Journal of Animal Sciences
• /
• v.11 no.3
• /
• pp.277-284
• /
• 1998

This experiment was carried out to study the effects of lactic acid bacteria (LAB) inoculation and addition of cell wall degrading enzymes on the fermentation characteristics and chemical compositions of Italian ryegrass silage. An inoculant LAB with or without a cell wall degrading enzyme of Acremoniumcellulase (A), or Meicellulase (M) or a mixture of both (AM), was applied to 1 kg of fresh Italian ryegrass sample. The treatments were control untreated, LAB-treated (application rate $10^5$ cfu/g fresh sample), LAB+A 0.005%, LAB + A 0.01%, LAB+A 0.02%, LAB + M 0.005%, LAB + M 0.01%, LAB + M 0.02%, LAB+AM 0.005%, LAB + AM 0.01% and LAB+AM 0.02%. The sample was ensiled into 2-L vinyl bottle silo, with 9 silages of each treatment were made (a total of 99 silages). Three silages of each treatment were incubated at 20, 30 and $40{^{\circ}C}$ for an approximately 2-months storage period. All silages were well preserved as evidenced by their low pH values (3.79-4.20) and high lactic acid concentrations (7.71-11.34% DM). The fermentation quality and chemical composition of the control untreated and the LAB-treated silages were similar, except that for volatile basic nitrogen (VBN) content was lower (p < 0.05) in the LAB-treated silages. LAB + cellulase treatments improved the fermentation quality of silages by decreasing (p < 0.01) pH values and increasing (p<0.01) lactic acid concentrations, in all of cellulase types and incubation temperatures. Increasing amount of cellulase addition resulted in further decrease (p < 0.01) of pH value and increases (p < 0.01) of lactic acid and residual water soluble carbohydrate (WSC) concentrations. LAB + cellulase treatments reduced (p<0.01) NDF, ADF, hemicellulose and cellulose contents of silages compared with both the control untreated and LAB-treated silages. LAB + cellulase treatments did not affect the silage digestibility due to fact of in vitro dry matter digestibility (IVDMD) was similar in all silages. The silages treated with cellulase A resulted in a better fermentation quality and a higher rate of cell wall reduction losses than those of the silages treated with cellulases M and AM. Incubation temperature of $30{^{\circ}C}$ seemed to be more suitable for the fermentation of Italian ryegrass silages than those of 20 and $40{^{\circ}C}$.

• Microbiology and Biotechnology Letters
• /
• v.30 no.2
• /
• pp.172-176
• /
• 2002

A filamentous microorganism, strain FJ1, was isolated from completely rotten wood for the production of cellulolytic enzymes. For the production of the enzymes, cellulolsic wastes were used as carbon sources of strain FJ1 and rice straw showed higher enzyme activities than sawdust and pulp. The activities of CMCase, xylanase, $\beta$-glucosidase, and avicelase were 2.95, 5.89, 0.45, and 0.12 unit/ml by use of rice straw, respectively. To enhance production of the enzymes, the mixture substrate of rice straw and cellulosic materials were investigated as carbon sources. The highest activities of CMCase, $\beta$-glucosidase, and avicelase were found in the mixture of rice straw (0.5%, w/v) and avicel (0.5%, w/v), and the highest xylanase was obtained at the mixture ratio of 0.71%(w/v) and 0.29%(w/v). Addition of 0.1%(w/v) peptone showed enhanced production of the cellulolytic enzymes in which the activities of CMCase, xylanase, $\beta$-glucosidase, and avicelase were 19.23, 27.18, 1.28, and 0.53 unit/ml, respectively. The production of the enzymes using rice straw was efficiently induced in the presence of avicel and pulp containing cellulose. In particular, a medium composed of rice straw (0.5%, w/v) and pulp (0.5%, w/v) yielded larger cellulolytic enzymes: CMCase 24.3 unit/ml, xylanase 38.7 unit/ml, $\beta$-glucosidase 1.5 unit/ml, and avicelase 0.6 unit/ml. The filamentous microorganism, strain FJ1 utilized various cellulosic wastes as carbon sources and will be expected as a favorable candidate for biological saccharification of cellulosic wastes.

• Asian-Australasian Journal of Animal Sciences
• /
• v.17 no.5
• /
• pp.678-683
• /
• 2004

An experiment was carried out to study the effects of exogenous enzymes (cellulases and proteases), yeast culture and effective micro-organism (EM) culture on feed digestibility and the performance of rabbits fed rice bran rich diets over a period of ten weeks. Twenty four, 8 to 9 weeks old male and female New Zealand White rabbits were allotted to 4 dietary treatments; a basal (control) feed containing 43% rice bran, basal feed supplemented with either enzymes, yeast culture or EM. Individual feed intake, body weight gain, nutrient digestibility, carcass characteristics and feed cost were studied. Sex of the rabbits had no significant (p<0.05) influence on the parameters studied. The control group showed the lowest daily feed intake (104.8 g), body weight gain (12.8 g) and the highest feed/gain ratio (8.20 g/g). The highest daily feed intake (114.3 g), body weight gain (20.42 g) and the lowest feed/gain ratio (5.60) were observed with enzymes. Compared to the control, yeast significantly (p<0.05) improved the feed intake, body weight gain and feed/gain ratio by 4.9, 34.4 and 22.0%, respectively, while EM improved (p<0.05) them by 4.0, 32.6 and 21.6%, respectively. All the additives improved (p<0.05) the digestibility of dry matter, crude protein, crude fiber and energy by 4.9-8.7, 3.6-10.7, 5.9-8.3 and 4.3-6.4%, respectively. Higher weights of pancreas (by 38.5-56.4%) and caecum (by 13.1-26.8%, compared to the control) were recorded with all additives but liver weight was increased only by yeast (24.5%) and enzymes (26.7%). Significantly (p<0.05) higher carcass recovery percentages were observed with enzymes (60.55), yeast (60.47) and EM (56.60) as compared to the control (48.52). Enzymes, yeast and EM reduced (p<0.05) the feed cost per kg live weight by 23.8, 15.9 and 15.5%, respectively. Results revealed that enzymes, yeast culture and EM can be used to improve the feeding value of agro-industrial by-products for rabbits in Sri Lanka and thereby to reduce the feed cost. Under the present feeding system, enzyme supplement was the best.

• Korean Journal of Microbiology
• /
• v.14 no.2
• /
• pp.75-83
• /
• 1976

Three conmmercial cellulases prepared from Penicillium notatum(cellulalse[K]), Trichoderma viride(cellulase[J]) and Aspergillus niger(cellulase[A]) were nalyzed with respect to their relative purity, activity and the effects of several metal ions on their activities. The activity of cellulase[K] was the strongest of all and that of cellulase[A] being the weaker. The purity of cellulalse[K] was the highest while that of cellulase[J] was the lowest. Under the assay conditions, the optimum concentrations of $Zn^{++}$ and $Mg^{++}$ ions for the activity of cellulase[K] was the highest while that of cellulase[J] was the lowest. Under the assay conditions, the optimum concentrations of $Zn^{++}$ and $Mg^{++}$ ions for the activity of cellulalse[K] was the highest while that of cellulase [A] being weaker. The purity of cellulase[K] was the highest while that of cellulase[J] was the lowest. Under the assay conditions, the optimum concentrations of $Zn^{++}$ and $MG^{++}$ ions for the activity of cellulase[K] were 2 and 7mM while those of cellulase[A] were 5 and 6 mM respectively and those of cellulase[J] were 3mM for both ions. Cellulase[K] and cellulase[J] were more strongly activated by $Zn^{++}$ than $Mg^{++}$ and cellulase[J] by $Mg^{++}$ than $Zn^{++}$. Both $Cu^{++}$ and $Mn^{++}$ ions inhibited by these metal ions. the inhibitory effects of $Mn^{++}$ ions for enzyme activities were stronger than $Cu^{++}$ ions. The Ki values of $Cu^{++}$ and $Mn^{++}$ for cellulase[K] were found to be 6.1 and 0.7mM, those of cellulase[J] were 2.6 and 0.32 mM, and those of cellulalse[A] were 2.0 and 0.2 mM respectively. Both $Cu^{++}$ and $Mn^{++}$ ions showed a pattrn of competitive inhibition of the enzyme activity. When Na-CMC was used as substrate, the Km and V values of celluase [K] were calculated to be $2.0{\times}10^{-4}mM$ and 3.43mmoles/hour, those of cellulase[J] were $2.4{\times}10^{-4}mM$ and 3.77mmoles/hour, and those of cellulase[A] were $4.0{\times}10^{-4}mM$ and 4.00mmoles/hour respectively.

• Journal of Life Science
• /
• v.33 no.10
• /
• pp.808-819
• /
• 2023

This study was conducted to develop a selection marker for the identification of the Bacillus licheniformis K12 strain in microbial communities. The strain not only demonstrates good growth at moderate temperatures but also contains enzymes that catalyze the decomposition of various polymer materials, such as proteases, amylases, cellulases, lipases, and xylanases. To identify molecular markers appropriate for use in a microbial community, a search was conducted to identify variable gene regions that show considerable genetic mutations, such as recombinase, integration, and transposase sites, as well as phase-related genes. As a result, five areas were identified that have potential as selection markers. The candidate markers were two recombinase sites (BLK1 and BLK2), two integration sites (BLK3 and BLK4), and one phase-related site (BLK5). A PCR analysis performed with different Bacillus species (e.g., B. licheniformis, Bacillus velezensis, Bacillus subtilis, and Bacillus cereus) confirmed that PCR products appeared at specific locations in B. licheniformis: BLK1 in recombinase, BLK2 in recombinase family protein, and BLK3 and BLK4 as site-specific integrations. In addition, BLK1 and BLK3 were identified as good candidate markers via a PCR analysis performed on subspecies of standard B. licheniformis strains. Therefore, the findings suggest that BLK1 can be used as a selection marker for B. licheniformis species and subspecies in the microbiome.

• Applied Biological Chemistry
• /
• v.14 no.1
• /
• pp.59-97
• /
• 1971

As a study on the cellulase of Myriococcum albomyces the culture media for enzyme formation and properties of its crude preparation were investigated and the crude enzyme preparation was further fractionated. The results are summarized as follows: 1. Wheat bran solid culture produced stronger activities of cellulase than rice bran or defatted soy bean meal solid culture. 2. Shaking culture with wheat bran, rice bran or defatted soy bean meal produced higher cellulase activities than solid culture with the corresponding media. 3. The enzyme formation was higher at $45^{\circ}C$ than at $37^{\circ}C$ or $50^{\circ}C$ regardless of the kind of culture medium. 4. The formation of CMCase activity was more promoted by organic nitrogen source than inorganic nitrogen source. 5. The formation of cellulase activities were increased 1.5 to 3.0-fold by adding CMC, Avicel or cellulose powder as an inducer into 5% wheat bran basal medium. 6. Cellulase production using a tank culture procedure with addition of CMC or Avicel as an inducer was the highest at fifth day and thereafter decreased slightly. 7. The crude enzyme preparation showed pH optimum in 4.0 to 4.5, and pH stability in the range of 3.5 to 8.0. Optimum temperature for the activity was $65^{\circ}C$ which was higher than among other cellulases and it was stable at $60^{\circ}C$ for 120 minutes. 8. Dialyzed crude enzyme was activated by $Ca^{++}$ and $Mg^{++}$, but inhibited by $Hg^{++}$, $Cu^{++}$ and $Ag^{+}$. 9. Four different types of cellulase, i. e., fraction I, fraction II-a, fraction II-b, and fraction III were purified from the culture filtrate of Myriococcum albomyces through a sequence of ammonium sulfate fractionation, and elution chromatography on DEAE-Sephadex A-25, Amberlite CG-25 type 2 and hydroxyapatite columns. 10. These four cellulase fractions were showed to be homogenous by electrophoresis and ultracentrifugation and also gave a typical ultraviolet absorption spectrum of protein. 11. Four purified fraction showed different specificity toward substrates, fraction I has a stronger activity toward Avicel, cellulose powder, and gauze than that of other cellulase fractions. Fraction II-a had a powerful activity toward cellobiose but it was almost inactive agaisnt fibrous cellulose contrary to fraction I. On the contrary, the main component fraction II-b had a fairly higher activity on CMC and Avicel. Activity of fraction II-b toward cellobiose was about one-third of that of fraction II-a and activity on Avicel was lower than that of fraction I. Fraction III had a more powerful activity in decreasing viscosity of CMC. 12. Final hydrolysis products of fibrous cellulose by each fraction were cellobiose and glucose. Whereas oligosaccharides were predominant in the early stage of hydrolysis, prolonged reaction produced more glucose than cellobiose. Fraction I and fraction II-a acted synergically on Avicel. 13. Optimum pH for the activities of cellulase fraction I, fraction II-a, fraction II-b and fraction III were found to be 5.5, 5.0, 4.0 and $4.0{\sim}4.5$, respectively. These fractions were found to be stable in the range of pH $3.0{\sim}7.5$. 14. Optimum temperature for the activities of fraction I, fraction II-a, fraction II-b, and fraction III were $50^{\circ}C$, $55^{\circ}C$, $60^{\circ}C$ and $55^{\circ}C$, respectively. No less of activity was found by heating 120 minutes at $55^{\circ}C$ and fraction II-a was more stable than the others at $60^{\circ}C$. 15. Fraction I and fraction II-b were activated by $Ca^{++}$ and $Mg^{++}$ but inhibited by $Hg^{++}$ and $Ag^{+}$.