Characterization, Improvement and Utilization of Bacillus licheniformis 1,4-β-Endoglucanase from Korean Native Goat
한국재래산양으로부터 분리한Bacillus licheniformis 1,4-β-Endoglucanase의 특성규명, 역가 향상 및 이용에 관한 연구

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Seo, Jakyeom
농업생명과학대학 농생명공학부
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서울대학교 대학원
Bacillus licheniformisDFMcellulasexylanasegenome shufflingrumen microbial population
학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2013. 2. 하종규.
A series of experiments were conducted to isolate a proper Bacillus sp. from the rumen of Korean native goats and characterize its endoglucanase. In the subsequent experiment activity of the enzyme was improved by various DNA technologies and DFMs were prepared by solid-state-fermentation method to study effects of feeding DFM on rumen fermentation and feed digestion in Holstein steers.

Exp. 1. Characterization of Cellulolytic and Xylanolytic Enzymes of Bacillus licheniformis JK7 Isolated From the Rumen of a Native Korean Goat
A facultative bacterium producing cellulolytic and hemicellulolytic enzymes was isolated from the rumen of a native Korean goat. The bacterium was identified as a Bacillus licheniformis on the basis of biochemical and morphological characteristics and 16S rDNA sequences, and has been designated Bacillus licheniformis JK7. The optimum temperature for the enzymes of Bacillus licheniformis JK7 was 70℃ for endoglucanase (0.75Units/ml) and 50℃ for β-glucosidase and xylanase (0.63Units/ml, 0.44Units/ml, respectively). The optimal pH for the three enzymes was 5.0, at which their activity was 1.46, 1.10, and 1.08 Units/ml, respectively. Endoglucanase activity was increased 113% by K+, while K+, Zn+, and tween20 enhanced β-glucosidase activity. Xylanase showed considerable activity even in presence of selected chemical additives, with the exception of Mn2+ and Cu2+.

Exp. 2. Genome Shuffling to Improve β-1, 4-Endoglucanase Activity of Bacillus licheniformis JK7
Three strains (GS2-18, GS3-8 and GS3-20) which had higher endoglucanase production than those of wild type strain(WT) have been selected after genome shuffling of Bacillus licheniformis JK7. Genome shuffled strains showed higher growth performance than those of WT strain after 4 hrs up to final incubation. Endoglucanase production increased rapidly from hr 6 up to hr 16 in all strains including, WT. The increase in enzyme production was associated with an increase in cell growth.

Exp. 3. In situ Degradation Characteristics of Bacillus licheniformis Based DFM
The objective of this study was to develop Bacillus licheniformis sp. -based DFM using solid-state fermentation (SSF), then to evaluate the difference between non-fermented meal (NFM) and fermented meal (FM) in ruminal disappearance in an in situ study. FM had higher CP, EE and ash contents but had lower DM, CF, NDF, ADF and lignin compared to NFM. FM showed significantly higher DM, CP, NDF and ADF disappearance rate at 3, 6 and 24 hrs incubation. FM had microbial count (1×109 CFU/g) and it had cellulolytic and xylanolytic (9.90 units/g for endoglucanase
1.67 units/g for β-glucosidase
1.52 units/g for xylanase) enzymes, which were not found in NFM.

Exp. 4. Effects of Bacillus licheniformis -Based DFM on Rumen Fermentation and Microbial Population under in vitro Condition
The objective of this experiment was to investigate the effects of Bacillus sp.-based DFM on rumen fermentation and rumen microbial population in the in vitro experiment.

1. In vitro experiment 1
Solid-state-frmentation significantly increased in vitro DM digestibility of (p<0.05). There were no significant differences between NFM and FM in total VFA and butyrate at all time points except 3 hrs incubation (p>0.05). In contrast, NFM had significantly higher portion of acetate (0, 3hrs incubation and mean value) as well as propionate (12, 24hrs incubation and mean value) than FM (p<0.05). FM reduced the number of. flavefaciens, R. amylophilus and S. bovis in at 3, 6 and 12hr (p<0.05), which might be due to some antimicrobial substances produced by Bacillus licheniformis sp during solid-state-fermentation.

2. In vitro experiment 2
Second in vitro study was conducted to study effects of DFM on in vitro fermentation pattern and substrate digestion. All treatments had timothy as a main substrate. NFM (10%, DM basis) (Con) , 5% of FM (T5) or 10% of FM (T10) was added to incubation medium. The addition of FM resulted in higher DM digestibility than those of Con (p<0.05), indicating that FM gave beneficial effect on fiber digestibility. T5 or T10 showed significantly higher production of total, acetate, propionate, isobutyrate, butyrate, isovalerate and n-valerate than Con (p<0.05). Log copy numbers of fibrolytic (R. albus, R. flavefaciens, E. ruminantium), starch using bacteria (R. amylophilus, S. bovis) and proteolytic bacteria (P. ruminicola) increased significantly by addition of FM (p<0.05).

Exp. 5. Effects of Supplementation of Bacillus licheniformis Based DFM on Rumen Fermentation and Microbial Population in Holstein Steers
An in vivo study was conducted to confirm the beneficial effects of Bacillus-based DFM in steers. Total tract digestibility of DM, CP, EE, NDF and ADF were investigated but there was no significant difference between treatments (p>0.05). Ruminal pH variation, NH3-N concentration and microbial N also showed no difference between treatments (p>0.05). Total VFA and acetate concentrations were not different significantly between treatments at all incubation time points. Only R. flavefaciens at 6hrs and B. licheniformis at 0, 6, 12hrs were significantly different (p<0.05). The significant difference of B. licheniformis population was also detected in fecal samples. The endoglucanase, β-glucosidase and xylanase activity in the rumen also analyzed but concentration of all enzymes was not influenced by treatments.
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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Agricultural Biotechnology (농생명공학부)Theses (Ph.D. / Sc.D._농생명공학부)
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