Isolation and Characterization of Mannanase Producing Bacillus amyloliquefaciens CS47 from Horse Feces

국문 초록 (Abstract)

진주산업대학교 농장에서 사육되고 있는 말에서 채취한 분변으로부터 mannanase 생성능이 우수한 균주를 분리하였으며 이를 CS47이라 명명하였다. 통성혐기성인 분리균 CS47의 최적 생육온도는 38℃였으며 20℃에서부터 50℃까지 다양한 온도범위에서도 생육이 가능한 것으로 확인되었다. BIOLOG를 이용하여 분리균 CS47의 생화학적 특징을 분석한 결과, 분리균은 Bacillus amyloliquefaciens 균주와 유사한 특성을 나타내었으며 DNA G+C함량 (44㏖%)도 B. amyloliquefaciens 균주의 범위(43.5-44.9)에 속하였다. 16S rDNA 염기서열을 분석한 결과에서도 분리균은 B. amyloliquefaciens FZB42 균주와 가장 높은 상동성을 나타내었으며 Bacillus속의 다른 균주들과는 93-98%의 상동성을 나타내었다. 그러나 분리균의 세포벽을 이루고 있는 주요 지방산[anteiso-15:0 (39.6%), 17:0 (7.6%), iso-15:0(37.8%)]은 Bacillus subtilis 균주와 유사한 특성을 나타내었다. 최종적으로 생화학적 특성과 16S rDNA 염기서열분석 결과를 근거로 하여 분리균 CS47은 Bacillus amyloliquefaciens CS47로 동정되었다. B. amyloliquefaciens CS47 균주가 분비하는 mannanase는 50℃, pH 6.0에서 가장 높은 활성을 보였다.

다국어 초록 (Multilingual Abstract)

The mannanase-producing bacteria, designated CS47, was isolated from the fresh feces of three horses (from a farm in Jinju National University). The isolate CS47 was facultatively anaerobic and grew at temperatures ranging from 20℃ to 50℃ with an optimal temperature of 38℃. The DNA G+C content of the isolate CS47 was 44 mlo%. The major fatty acids were anteiso-15:0 (39.6%), 17:0 (7.6%), and iso-15:0 (37.8%). The 16S rRNA gene sequence similarity between the isolate CS47 and other Bacillus strains varied from 93% to 98%. In the phylogenetic analysis based on these sequences, the isolate CS47 and Bacillus amyloliquefaciens clustered within a group and separated from other species of Bacillus. Based on the physiological and molecular properties, the isolate CS47 was classified within the genus Bacillus as Bacillus amyloliquefaciens CS47. The optimal pH and temperature for mannanase activity of B. amyloliquefaciens CS47 were pH 6.0 and 50℃, respectively. The thermal stability of mannanase from B. amyloliquefaciens CS47 is valuable when using this enzyme in industrial application.

목차 (Table of Contents)

• Introduction
• Materials and Methods
• Results and Discussion
• Acknowledgement
• References
• 초록

참고문헌 (Reference)

1 McCleary,B.V, "β-mannanase" Academic Press Inc. 596-610, 1988

2 Khanongnuch, C., "β-Mannanase and xylanase of Bacillus subtilis 5H active for bleaching of crude pulp" 86 : 461-466, 1998

3 Mandel, M, "Use of ultraviolet absorbance-temperature profile for determining the guanine plus cytosine content of DNA" 12 : 195-206, 1968

4 Miller, "Use of dinitrosalicylic acid reagent for determination of reducing sugars" 31 : 426-428, 1959

5 Slominski, B. A., "The use of enzyme technology for improved energy utilization from full-fat oilseeds. Part Ⅱ: Flaxseed" 85 : 1031-1037, 2006

6 Admark, P., "Softwood hemicellulose- degrading enzymes from Aspergillus niger: purification and properties of a β-mannanase" 63 : 199-210, 1998

7 Argenzio, R. A., "Sites of organic acid production and absorption in the equine gastrointestinal tract" 226 : 1043-1050, 1974

8 Cummings, J. H, "Role of intestinal bacteria in nutrient metabolism" 357-365, 1997

9 Ooi, T, "Purification and some properties of β-mannanase from Bacillus sp" 11 : 310-314, 1995

10 Takahashi, R., "Purification and some properties of mannanase from Streptomyces sp" 48 : 2189-2195, 1984

11 Yamaura, I, "Purification and some properties of endo-1,4-β-D-mannanase from a mud snail, Pomacea insularus (de Ordigny)" 57 : 1316-1319, 1993

12 Sachslehner A, "Purification and some properties of a thermostable acidic endo-β-1,4-d-mannanase from Sclerotium (Athelia) rolfsii" 177 : 47-55, 1999

13 Mendoza,N.S., "Purification and properties of mannanase from Bacillus subtilis" 10 : 551-555, 1994

14 Zhang, J., "Purification and characterization of β-mannanase from Bacillus licheniformis for industrial use" 22 : 1375-1378, 2000

15 Zakaria, M. M., "Purification and characterization of an endo-1,4-β-mannanase from Bacillus subtilis KU-1" 158 : 25-31, 1998

16 Arisan-Atac, I., "Purification and characterization of a β-mannanase of Trichoderma reesei C-30m" 39 : 58-62, 1993

17 Ferreira, H. M, "Purification and characterization of a β-mannanase from Trichoderma harzianum strain T4" 57 : 23-29, 2004

18 Akino, T., "Production of β-mannosidase and β-mannanase by an alkalophilic Bacillus sp" 26 : 323-327, 1987

19 Kurakake, M, "Production of β-mannanase and β-mannosidase from Aspergillus awamori K4 and their properties" 42 : 377-380, 2001

20 Mendoza, N. S., "Isolation of mannan-utilizing bacteria and the culture conditions for mannanase production" 10 : 51-54, 1994

21 Wozniewski, T., "Isolation and characterization of an endo-β-mannanase of Lilium testaceum bulbs" 31 : 3365-3370, 1992

22 Claus, D, "In Bergey's Manual of Systematic Bacteriology" Williame & Wilkins 1105-1139, 1986

23 Logan, N. A, "Identification of Bacillus strains using the API system" 130 : 1871-1882, 1984

24 Ethier, N., "Gene cloning, DNA sequencing, and expression of thermostable β-mannanase from Bacillus stearothermophilus" 64 : 4428-4432, 1998

25 Petty, L. A., "Effects of β-mannanase addition to corn-soybean meal diets on growth performance, carcass traits, and nutrient digestibility of weaning and growing-finishing pig" 80 : 1012-1019, 2002

26 Zou, X. T., "Effect of β-mannanase (Hemicell) on growth performance and immunity of broilers. Poult. Sci. 85, 2176-2179" 85 : 2176-2179, 2006

27 Stevens, C. E, "Contributions of microbes in vertebrate gastrointestinal tract to production and conservation of nutrients" 78 : 393-421, 1998

28 Mendoza, N. S., "Cloning and sequencing of β-mannanase gene from Bacillus subtilis NM-39. Biochim" 1243 : 552-554, 1995

29 YOON, KI-HONG, "Cloning and Strong Expression of a Bacillus subtilis WL-3 Mannanase Gene in B. subtilis" 한국미생물·생명공학회 17 (17): 1688-1694, 2007

30 Schaeffer, P., "Catabolic repression of bacterial sporulation" 54 : 704-711, 1965

31 Garret, L. A., "A comparative study of the intestinal microbiota of healthy horses and those suffering from equine grass sickness" 87 : 81-88, 2002