알긴산 분해균 Bacillus licheniformis AL - 577가 생산하는 균체외 효소의 정제 및 특성

국문 초록 (Abstract)

알긴산을 선택적으로 분해하는 Bacillus licheniformis AL-577 균주가 생산하는 균체 외 효소를 정제하고 특성을 밝혔다. CM-Cellulose, DEAE-Sepharose 및 Gel 크로마토그래프 등의 순서대로 정제하여 정제도가 약 98배 정도인 효소를 얻었다. 정제효소의 활성 최적 pH 및 온도는 6.0 및 35℃이었고, pH 5.5이하와 pH 9.5이상의 반응조건에서는 불안정하였으며, 20℃이상의 온도에서는 불활성화가 쉽게 일어나는 효소였다. 얻어진 효소를 SDS-PAGE로 분자량을 측정한 결과 25,500 Da으로 추정되었다. NaCl 0.2 M 농도에서 최대의 활성을 나타내었고, 무첨가시에도 활성은 약간 나타내었다. Cu²+, Fe²+, Mg²+, Zn²+ 등과 같은 2가 금속이온에 의해서는 활성이 현저히 억제되었고, K+, Li+ 이온에 의해서 활성이 촉진됨을 알 수 있었다. 화학약품인 dithiothreitol와 O-phenanthroline의 첨가로 인하여 약간의 활성 증가를 가져 왔으며 반면 EDTA, L-cysteine는 현저하게 감소하였다. 이 효소는 알긴산에만 특이적으로 작용함으로써 본 효소는 alginase 또는 alginate lyase인 것으로 판단되었다.

다국어 초록 (Multilingual Abstract)

The extracellular enzyme alginase produced by Bacillus licheniformis AL-577 was purified by ion chromatography on CM-Cellulose column, DEAE-Sepharose column, and followed by gel filtration on Sephadx G-100 column. The optimum pH and temperature for the activity of the purified enzyme were 6.0 and 35℃, respectively. The enzyme was stable at the pH range of 6.0~9.0 and at 20℃. The molecular weight of the enzyme was estimated to be about 25,500 daltons by SDS-polyacrylamide gel electrophoresis. NaCl was required for high activity of the enzyme. The enzyme was inhibited by Ba²+, Co²+, Cu²+, Fe²+, Mg²+, Zn²+, NH₄+, EDTA, L-cysteine, and 2-mercaptoethanol, while stimulated by DTT, O-phenanthroline, K+ and Li+. This enzyme was proposed to be an alginase specifically degrading alginic acid.

목차 (Table of Contents)

• Abstract
  
• 서론
  
• 재료 및 방법
  
• 결과 및 고찰
  
• 요약
  
• 감사의 글
  
• 문헌

참고문헌 (Reference)

1 김해섭, "미생물을 이용한 해조류의 가수분해 및 이용 I. 다시마와 미역을 가수분해시키는 미생물군의 탐색" 한국수산과학회 35 (35): 438-444, 2002

2 Haug A, "Uronic acid sequence on alginate from different sources" 24 : 273-282, 1974

3 Tseng, CH, "Two types of alginate lyase from a marine bacterium Vibro sp. AL-9." 58 : 743-749, 1992

4 Tseng CH, "Two types of alginate lyase from a marine bacterium Vibrio sp. AL-9" 58 : 743-749, 1992

5 Hirst, EL, "The structure of alginic acid. Part Ⅴ. Isolation and unambiguous characterization of some hydrolysis products of the methylated polysaccharide" 7 : 1182-1187, 1965

6 Hirst EL, "The structure of alginic acid. Part Ⅳ. Partial hydrolysis of the reduced polysaccharide" 8 : 1493-1499, 1964

7 Keneko Y, "Symbiotic formation of alginate lyase in mixed culture of bacteria isolated from soil" 69 : 192-194, 1990

8 Haug A, "Studies on the sequence of uronic acid residues in alginic acid" 21 : 691-704, 1967

9 ung JY, "Studies on the efficient extraction process of alginic acid in sea tangle" 3 : 90-97, 1999

10 Eller J, "Studies on bacterial utilization of uronic acid. Ⅳ. Alginolytic and mannuronic acid oxidizing isolates" 80 : 193-199, 1960

11 Boyd J, "Structural studies of alginic acid, using a bacteria poly-α-L-guluronate lyase" 66 : 187-194, 1978

12 Ahn SJ, "Storage of waste-brown seaweed and degradation of alginate using microorganism" 13 : 313-318, 2004

13 Davidson IW,, "Purification and properties of an alginate lyalse from marine bacterium" 159 : 707-713, 1976

14 Joo DS,, "Purification and characterization of the intracellular alginase from Vibrio sp. AL-145." 23 : 432-438, 1995

15 Lowry OH, "Protein measurement with the Folin phenol reagent" 193 : 265-275, 1951

16 Joo DS, "Preparation of oligosaccharides from alginic acid enzymeic hydrolysis" 28 : 146-151, 1996

17 Sutherland IW., "Polysaccharide lyases." 16 : 323-347, 1995

18 Somogyi, M, "Notes on sugar determination" 195 : 19-23, 1952

19 Min KH, "Multiple components of endo-polyguluronide lyase of Pseudomonas sp" 81 : 539-546, 1977

20 Kim HS, "Isolation of seaweed hydrolytic strains from microfloras in rice field ditch water" 36 : 536-567, 2003

21 Tseng CH, "Isolation and some properties of alginate lyase from a marine bacterium Vibro sp. AL-128" 58 : 533-538, 1992

22 Muranmatsu T, "Isolation and properties of alginate lyase from the mid-gut gland of wreath shell Turbo cornutus" 41 : 1939-1946, 1977

23 Joo DS, "Isolatin of alginate-degrading bacteria and production of alginate-degrading activities by bacteria" 21 : 207-213, 1993

24 Alonso S, "Functional replacements for sugars in foods" 5 : 139-146, 1994

25 Lee SR, "Extraction yield and quality attributes of agar from domestic seaweeds according to various pretreatment" 7 : 109-114, 1975

26 Cho, HO, "Effectiveness of gamma irradiation of the extraction of algal polysaccharides" 6 : 36-41, 1974

27 Yang JS, "Effect of ionizing radiation on the extraction yield and viscosity of alginate" 9 : 194-198, 1997

28 Hidaka H, "Effect of fructooligoscchariedes on human intestinal flora. In Intestinal Microflora and Dietary Factors" Center for Academic Publications, Tokyo 39-64, 1984

29 Davis, BJ., "Disc-electrophoresis Ⅱ. Method and application to human serum protein" 121 : 404-427, 1964

30 Cho M, "Degradation of alginate solution and power by γ-irradiation" 7 : 141-145, 2003

31 Laemmli UK., "Cleavage of structure proteins during the assembly of the bacteriophage T." 227 : 680-686, 1970

32 Sawabe T, "Characterization of an alginolytic marine bacterium from decaying Rishiri- kombu Laminaria japonica var ochotensis" 58 : 141-145, 1992

33 Yonemoto Y, "Bacterial alginate lyase; characterization of alginate lyase producing bacteria and purification of the enzyme." 72 : 152-157, 1991

34 Guven KC, "Anticoagulant, fibrinotic and anti aggregant activity of carrageenans and alginic acid" 34 : 429-435, 1991

35 Baxter, RM, "An interpretation of the effect of salts on the lactic dehydrogenase of Halobacterium salinarium" 5 : 47-57, 1959

36 Miyake O, "An exotype alginate lyase in Sphingomonas sp. A1: overexpression in Escherichia Coli, purification and characterization of alginate lyase" 27 : 33-41, 2003

37 Gacesa, P., "Alginates" 8 : 161-182, 1988

38 ,Kashiwabara Y, "Alginate lyases of Pseudomonas sp." 66 : 503-512, 1969

39 Wong TY, "Alginate lyase: review of major sources and enzyme characteristics, structure-fuction alalysis, biological roles, and applications" 54 : 289-340, 2000

40 Haug A, Larsen B, Smidsröd O., "A study of constitution of alginic acid by partial acid hydrolysis" 20 : 183-190, 1966

41 Penman A, "A method for the determination of uronic acid sequence in alginates" 25 : 273-282, 1972