Purification of a Protease Produced by Bacillus subtilis PCA 20-3 Isolated from Korean Traditional Meju

전통 메주로부터 분리한 Bacillus subtilis PCA 20-3 유래 Protease 의 정제

Bacillus subtilis PCA20-3 was isolated from meju and was found to produce a protease. The strain produced the maximum amount of enzyme in the medium containing soytone (0.2%), soluble starch (2%), $(NH_4)_2SO_4\;(0.1%),\;CaCl_2(0.1%),\;yeast\;extract\;(0.01%),\;K_2HPO_4\;(0.1%),\;and\;KH_2PO_4\;(0.1%)$. Protease was first concentrated by ammonium sulfate (80% saturation, w/v) precipitation of culture supernatant. Then the enzyme was purified by column chromatography using CM Sephadex C-50. The collected proteins were rechromatographed using Sephadex G-100 gel filtration column. The fraction with protease active from Sephadex G-100 gel chromatography was found to be pure when examined by SDS-polyacrylamide gel electrophoresis and YMC-pak reverse phase chromatography. Specific activity, yield and purity were 76 U/mg. 2.7%, and 7.6 fold, respectively. The molecular weight of the enzyme was estimated to be 31.5 kDa by SDS-PAGE. The number of amino acids calculated from molecular weight was evaluated about 321 residues. N-terminal sequence of the enzyme was $Val^1-Pro^2-Tyr^3-Gly^4-Val^5-Ser^6-Gln^7-Gly^8-Lys^9-Ala^{10}$.

재래식 메주로부터 분리한 Bacillus subtilis PCA203이 생산하는 protease를 분리 정제하였다. 먼저 효소 생산용 배지$(0.2%\;soytone,\;2%\;soluble\;starch,\;0.1%\;(NH_4)_2SO_4,\;0.1%\;CaCl_2,\;0.01%\;yeast\;extract,\;0.1%\;K_2HPO_4,\;0.1%\;KH_2PO_4)$를 이용하여 $30^{\circ}C$에서 20시간 배양한 다음, 원심분리하여 상징액을 분획한 후, 80% 포화 황산암모늄에 의한 염석과 CM Sephdex C-50 및 Sephadex G-100을 이용하여 비활성도 76.0 unit/mg, 수율 2.7%, 정제배수 7.6배로 효소를 정제하였다. 정제 단백질의 YMC-pack protein-RP column chromatography에 의한 순도검증에서 순도가 95% 이상인 것으로 나타났다. SDS-PAGE 분석에서 주 밴드의 분자량은 약 31.5 kDa이었고 아미노산 조성은 alanine, glycine, serine, valine의 함량이 많았으며 분자량 31,500 Da를 기준으로 하였을 경우 본 protease의 잔기수는 321잔기였다. RP-HPLC로 분획한 main peak의 N-terminal amino acid sequence를 확인한 결과 $Val^1-Pro^2-Tyr^3-Gly^4-Val^5-Ser^6-Gln^7-Gly^8-Lys^9-Ala^{10}$인 것으로 밝혀졌다.