Bacillus subtilis JS-17이 생산하는 Collagenase의 정제 및 특성 = Purification and Characterization of Bacillus subtilis JS-17 Collagenase.

국문 초록 (Abstract)

Collagnase는 천연 collagen의 triple-stranded helix를 분해할 수 있는 protease로서 조직의 수복과 재생 과정에서 collagen의 재형성과 세포의 이동에 아주 중요한 역할을 하고, 숙주 감염시에는 collagen 기질을 빠르게 분해함으로써 감염을 돕는다. 본 연구에서는 일반가정에서 식용하는 김치로부터 collagenase를 생산하는 균주를 분리하여 Bacillus subtilis로 동정하였으며 이를 Bacillus subtilis JS-17이라 명명하였다. Bacillus subtilis JS-17이 생산하는 collagenase의 최적 생산 조건은 $1.5\%$ fructose, $1\%$ yeast extract, $0.5\%\;K_2HPO_4,\;0.4\%\;KH_2PO_4,\;0.01\%\;MgSO_4{\cdot}4H_2O,\;0.1\%\;citrate,\;0.1\%\;CaCl_2(pH\;7.0)$의 배지에서 $30^{\circ}C$, 200 rpm으로 72시간 동안 배양하는 것이다. 최적 조건에서 Bacillus subtilis JS-17이 생산하는 collagenase를 Amberlite IRA-900 column chromatography, Sephacryl S-300 HR column chromatography, DEAE-Sephadex A-30 column chromatography를 거쳐 분리 정제하고, 얻어진 정제 효소의 특성에 대하여 검토하였다. 정제된 collagenase의 비활성은 growth medium에서 192.1 units/mg였고, $1.1\%$의 수율로 얻어졌으며 분자량은 28 kDa이었다. 정제된 collagenase는 $55^{\circ}C$까지는 $100\%$의 활성을 유지하였고 $65^{\circ}C$에서도 $60\%$ 정도의 활성을 유지하였다. 또한 pH $6.0\~9.8$에서 $60\%$ 이상의 활성을 유지하였다. 정제된 collagenase는 metalloprotease inhibitor인 EDTA와 O-phenanthroline에 의해 효소 활성이 감소하였을 뿐만 아니라 Ammoninum persulfate, L-cysteine, N-ethylmaleimide, SDS, $NaN_3$, NaF, $KMnO_4$, PMSF에 대해서도 활성이 감소하였다. 정제된 collagenase를 여러 가지 기질에 대해 효소 활성을 비교한 결과 collagen (type I)에 대해 기질 특이성을 가지고 있었다.

다국어 초록 (Multilingual Abstract)

Collagenases are generally defined as enzymes that are capable of degrading the polypeptide backbone of native collagen under conditions that do not denature the protein. An extracellular collagenase-producing bacterial strain was isolated from kimchi and identified to be Bacillus subtilis JS-17 through morphological, cultural, biochemical characteristics and 16S rDNA sequence analysis. Optimum culture condition of Bacillus subtilis JS-17 for the production of collagenase was $1.5\%$ fructose, $1\%$ yeast extract, $0.5\%\;K_2HPO_4,\;0.4\%\;KH_2PO_4,\;0.01\%\;MgSO_4\cdot7H_2O,\;0.01\%\; MnSO_4\cdot4H_2O,\;,0.1\%$ citrate and $0.1\%\;CaCl_2$. The production of collagenase was optimal at $30^{\circ}C$ for 72 hr. A collagenase was isolated from the culture filtrate of Bacillus subtilis JS-17. The enzyme was purified using Amberlite IRA-900 column chromatography, Sephacryl S-300 HR column chromatography and DEAE-Sephadex A-50 column chromatography The purified collagenase has an specific activity 192.1 units/mg. The molecular weight of the purified enzyme was estimated to be 28 kDa by SDS-PACE. The purified collagenase has $100\%$ activity up to $55^{\circ}C$.

참고문헌 (Reference)

1 "Thermostable collagenolytic activity of a novel thermophilic isolate" nishi : 612-614, 2000

2 "Structure and function of the matrix metalloproteinase" 1-4, 2001

3 "Rapid determination of substrate specificity of Clostridium histolyticum β-collagenase using an immobilized peptide library"

4 "Purification of collagenase and specificity of its related enzyme from Bacillus subtilis FS-2" 1999181-183

5 "Purification and separation of individual collagenase of Clostridium histolyticum using red dye ligand chromatography" 3077-3085, 1984

6 "Purification and properties of collagenase from Cytophaga sp" 19931894-1898

7 "Purification and characterization of three forms of collagenase from Clostridium histolyticum" 5175-5181, 1984

8 "Purification and characterization of an elastolytic protease of Vibrio vulnificus" 1783-1791, 1987

9 "Proteolytic activity of the Gymnorhynchus gigas plerocercoid purification and properties of a collagenase from the crude extract" Caabeiro 64-70, 1999

10 "Modifiec colorimetric ninhydrin methods for peptidase assay" 173-184, 1981

11 "Isolation and characterization of collagenase Ⅰ and Ⅱ from Clostridium histolyticum" 562-574, 1965

12 "Isolation and characterization of a collagenolytic enzyme from Bacillus licheniformis N22" 283-287, 1994

13 "Isolation and Properties of a Collagenase with Caseinolytic Activity from a Pseudomonas sp" hisa : 399-403, 1989

14 "Inhibition of the activities of matrix metalloproteinases 2 and 9 by chlorhexidine" immu (immu): 437-439, 1999

15 "Hydrolysis of synthetic peptides by collagenase" 567-569, 1960

16 "Differences in the degradation of native collagen by two microbial collagenase" 53-58, 1979

17 "Continuously recording fluorescent assays optimized for five human matrix metalloproteinases" 86-92, 1991

18 "Cleavage of structural proteins during the assembly of the head of bacteriophage T4" 680-227 685, 1970

19 "Characterization of the individual collagenase from Clostridium histolyticum" 3085-3091, 1984

20 "An assay for matrix metalloproteinases and proteases acting on proteoglycans casein or gelatin" 171-179, 1993

21 "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding" 248-254, 1976

22 "A modified ninhydrin colorimetric analysis for amino acids" 10-15, 1957

23 "A continuous spectrophotometric assay for Clostridium histolyticum collagenase" 356-365, 1981